{"paper_id":"49c525a0-c1d0-4f68-9893-e85724a9b569","body_text":"Estrogen Receptor-positive Breast Cancer Cells are Sensitized by Piperine to Chemo/Radio Therapy through Lowering the expression of a NHEJ repair protein DNA-PK | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Estrogen Receptor-positive Breast Cancer Cells are Sensitized by Piperine to Chemo/Radio Therapy through Lowering the expression of a NHEJ repair protein DNA-PK Koniyan Shaheer, Swathi Prabhu, H. Shabeer Ali, Divya Lakshmanan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2401099/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Gamma radiation(γ) and other DNA targeted compounds generate highly lethal DNA double-stranded breaks (DSBs) inducing the cells to undergo apoptosis. Non-homologous end joining (NHEJ), one of the primary DSB repair pathways, plays an important role in providing cancer cells resistance against radio/chemotherapeutic agents resulting in cancer progression and relapse. Downregulating DNA-PK, a key protein in NHEJ could result in the accretion of DSBs, thereby sensitizing the cells towards radiation. Methods Cytotoxicity assays, Clonogenic assays, DNA damage assays, Flowcytometry analysis, Confocal Microscopy, immunofluorescence, and Immunoblotting were carried out. Combinatorial index calculations were done using Compusyn Analysis and data analysis was done using one-way ANOVA and two-way ANOVA, where a p-value of ≤ 0.0001 was considered significant. Results Here we found that the treatment of MCF7 cells with piperine, lead to the accumulation of DSBs induced by γ-radiation through lowering DNA-PK complex (comprising of DNA-PKcs/Ku70/Ku80), by altering the estrogen receptor (ER) α /β ratio. Piperine lowered DNA-PK mediated NHEJ repair through its transcription factor, ERα. Upregulation of ERβ, a nuclear hormone transcription factor promoting tumor suppression positively correlated with lowered expression of ERα and DNA-PK marked by the accumulation of radiation-induced DSBs and DNA damage response, cell cycle arrest leading to the intrinsic pathway of apoptosis. Conclusion Breast Cancer cells may be sensitized to radiation by altering the expression of DNA-PKc Complex, a key dsDNA repair protein machinery through selective estrogen receptor modulation. This study proposes a new strategy for combating acquired radioresistance through estrogen receptor-mediated modulation of the NHEJ pathway. Breast cancer cells Radiosensitization Piperine γ-radiation JC-1 analysis γH2AX Estrogen receptor α /β Selective estrogen receptor modulator. DNA-PK complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gamma radiation produces extremely lethal DNA double-stranded breaks (DSBs), inducing the cells to undergo apoptosis ( 1 – 4 ). These therapies mainly target genomic DNA, subsequently resulting in the formation of highly toxic DSBs inducing cell cycle arrest and cell death ( 2 , 3 ). However, these treatments are initially effective in arresting tumor growth and reducing tumor burden, but eventually, it leads to resistance and disease progression ( 3 , 5 , 6 ). Increased cellular DNA repair is one of the major mechanisms underlying this drug resistance, and various DNA repair pathways are upregulated to combat DNA damage. In mammals, DSBs are repaired mainly by the error-prone, nonhomologous end-joining (NHEJ) repair pathway ( 7 – 11 ). NHEJ initiation occurs when the ring-shaped Ku heterodimer, composed of the Ku70 and Ku80 proteins, identifies and binds to the target DSB in a sequence-independent manner and upon binding to the DSB, Ku70/ 80 recruits DNA-PKcs to the DNA ends directly to form the active DNA-PK complex, which results in the activation of the catalytic subunit, DNA-PKcs’s (kinase activity) which further processes ligation through DNA ligase IV along with additional NHEJ machinery proteins such as XRCC4, Artemis, and, XLF ( 7 ). Enhanced activity of DNA-PKcs is observed in most of the cancer types such as hepatocellular carcinoma, oral squamous cell carcinoma, colorectal cancer, etc., which results in the error-prone mediated repair of DNA resulting in the survival of cancer cells culminating in cancer progression( 12 – 14 ). Increased DNA-PKcs activity leads to enhanced DNA repair and reduced sensitivity to genotoxic and radio- or chemotherapies by the cancer cells leading to therapy resistance and poor patient survival ( 15 , 16 ). Recent studies demonstrate the existence of crosstalk mechanisms between the DNA repair machinery and estrogen hormone signaling pathways cooperating to influence cancer progression and therapeutic response ( 17 – 19 ). Estrogen signaling is a balance among two conflicting functions exerted by two different receptors (ERα & ERβ) as well as their splice variants ( 20 ). ERα is reportedly involved in oncogenesis and development of progression, invasion, and metastasis of cancer ( 21 ). On the contrary, ERβ suppresses cancer progression and induction of apoptosis( 22 ). Few studies reported that the DNA-PKcs complex component, Ku70, complexes with ERα, assisting the transcriptional function of ERα, and further it transcriptionally activates DNA-PKcs ( 23 , 24 ). The possibilities that the estrogen signaling (ERα & ERβ mediated) pathways can be selectively modulated with selective estrogen receptor modulating compounds (SERMs) from plants add up to new and promising therapeutic opportunities ( 15 , 17 ). Thus, we hypothesize that targeting the molecular machinery driving the DNA repair pathways and apoptosis in cancer cells through ER modulation may lead to a novel elevated sensitivity to DNA damaging agents like ionizing radiation, making cancer cells sensitive to radiation and other chemotherapeutic agents. Thus, incorporating a SERM along with DNA-targeted therapy in ER-positive cancer types may result in enhanced sensitization of cells to DNA-damaging agents. In addition, based on the results, we anticipate that the use of spices such a s pepper during radio/chemotherapy may enhance the sensitivity of ERα positive cancer cells towards DNA damaging agents without effecting normal cells. Thus this study offers a new stratergy for combating therapy resistance by indirectly targeting NHEJ repair pathway through direct estrogen receptor modulation. Methods The phytocompound piperine (> 97%), Bazidoxefene, and dimethyl sulphoxide (DMSO) were procured from Sigma-Aldrich, India. Cell culture reagents such as Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin solution, L-glutamine were procured from Gibco, Thermofischer. Antibodies were procured from either CST or Abcam. Trypan blue dye, MTT was procured from HiMedia, India. All other chemicals, Reagents, and other lab accessories were all analytical or molecular grade and were procured from reliable commercial sources. Cell culture To decide if the radiosensitization effects of piperine was restricted to ER positive breast cancers appropriate cell lines were used. Human breast adenocarcinoma cell lines; MCF7 ( an ER positive cell line), MDA-MB-231, and MDA-MB-468, (ER negative cell lines) were procured from National Centre for Cell Science (NCCS), Pune, India. The cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and 2 mM L-glutamine. The cell culture was maintained at 37 o C in a humidified incubator containing 5% CO 2 . Piperine, cisplatin, and γ-radiation treatment on MCF7 To optimize the dose of piperine, cisplatin, and γ-radiation to be used in combination treatment against breast cancer cell line MCF7, a cell proliferation assay with varying doses of these agents was studied For radiation experiment, MCF7 cells were seeded in T-12.5/ T-25 flasks at a density of 3x10 5 / 5x10 5 cells/ flask and cultured for 24 h. The optimum dose of piperine, cisplatin, and gamma radiation to be used for the combination experiment were determined by treating the cells at various concentrations of the compounds/ γ- radiation. The cells exposed to the vehicle (DMSO) served as the control. For combination treatment cells were exposed to the selected dose of piperine and or cisplatin for 2 h. Following 2 h of post-treatment with the compounds, the cells were exposed to γ- radiation using Low Dose gamma Irradiator-2000 (BRIT, Mumbai, India) and then incubated for 48 h. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Dose optimization was determined by MTT assay and Trypan blue dye exclusion respectively ( 51 , 52 ). Morphological and Cell proliferation analysis Cell proliferation and viability studies were done using MTT, trypan blue dye exclusion assay, and colony-forming assay. MCF7 cells were cultured in T12.5 flasks; under culture conditions for 24 h. Cells were treated with the selected concentration of the piperine and chemotherapeutic drug cisplatin alone or in combination and exposed to a single dose of γ-radiation with 60Co source as irradiator. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Images were collected using an inverted light microscope (Zeiss Primo Vert, Mumbai, India) and a ZOE imager (Bio-Rad, Mumbai, India). The cell proliferation was determined by trypan blue dye exclusion assay and MTT at 48 h of incubation after irradiation. Absorbance at 570 nm was done using a microplate reader (Fluostar Omega, BMG Labtech) and the percentage of cell growth relative to cells treated with vehicle alone was calculated. CompuSyn analysis of the combination treatment CompuSyn software (ComboSyn, Inc.USA) was used to quantitatively depict the effect of the combination treatment. The data from the cytotoxicity studies were taken to compute Combination Index (CI) values, CI plot, dose-response curve, median effect plot, and normalized isobologram. Combination Index values are less than one it indicates synergism (CI < 1), if Combination Index values are equal to one, it is an additive effect (CI = 1) and if Combination Index values are greater than one, it represents antagonism (CI > 1) of the co-treatment ( 53 , 54 ). The combination index (CI) is calculated by using the formula: CI = d1/DA1 + d2/DA2 Where DA1 is the dose of γ-radiation required to decrease the 'x' percentage of proliferation alone, and d1 is the dose of γ-radiation required to decrease ‘x’ percentage of proliferation in combination with piperine (d2). DA2 is similarly the dose of piperine required to decrease ‘x’ percentage of proliferation alone, and d2 is the dose of piperine required to decrease ‘x’ percentage of proliferation in combination with γ-radiation (d1). Clonogenic survival assays Trypsinized MCF7cells were plated in 35 mm dishes at a density of 3x10 3 cells/ well and allowed to grow overnight as described previously. Piperine and cisplatin treatment at a concentration of 75 µM and 3 µM respectively and incubated for 2 h, followed by γ-radiation at a dose of 2 Gy. For the clonogenic assay, the cells were grown for 7 days and then stained with 0.1% Crystal violet for 1h and the photographs were captured and analyzed for confluency using Image J software ( 55 , 56 ). Cell cycle analysis MCF7 cells were seeded in T-12.5 flasks at a density of 3x10 5 cells/ flask and cultured for 24 h. The cells were then exposed to piperine and cisplatin/ 2 Gy of γ-radiation and then incubated for 24 h. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Trypsinized cells were washed with PBS and then fixed with 75% ethanol and incubated overnight at -20 o C. Cells were washed twice with PBS and incubated with RNase A solution (50 µg/ mL; Himedia), for 2 h at 37 o C followed by propidium iodide (5 µg/mL; HiMedia) for 10 min in dark. Cell cycle analysis was performed using Guava EasyCyte (Millipore, India) ( 33 ). Cytokinesis-Blocked Micronucleus Cytome assay After incubation of MCF7cells with combination treatment for 48 h, Cytochalasin-B (Cyto-B) (Sigma) was added to cells and incubated for 24 h onto glass slides, air-dried for 10 min, fixed, and was further stained using Giemsa for scoring. A total of 50 cells, including mononucleated and binucleated (BN), were counted per slide. The data from these cell counts were used to determine the DNA damage biomarkers. The methodology used to score and calculate DNA damage biomarkers was previously described ( 57 ). Gamma-H2AX assay The assay was performed according to the previously described protocol ( 58 ). Briefly, Cells (1 × 10 6 cells/ dish) were seeded on poly-lysine coated 11 m glass coverslip in 35 mm culture dishes. After treatment and incubation, cells were given PBS (1X) wash, followed by using 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 30 min. The samples were blocked for 1 h with 1% BSA followed by incubation with Alexa Fluor 488-conjugated phospho-histone-γ-H2AX antibody (Ser139) (BD Biosciences) overnight at 4 o C. Samples were washed 3 times for 5 min in PBS. For confocal imaging, slides were prepared with DABCO/ PI mounting. DABCO/PI mounting medium prepared by dissolving 1.25 g DABCO powder in 45 mL glycerol at 37 o C or 8 h. Once dissolved 5 mL PBS (pH 8.6) was added, and then followed by the addition of PI at 0.00002% as the final concentration. The cells stained with DABCO/ PI were then analyzed under Zoe imager/ Confocal microscope (Carl Zeiss, Göttingen, Germany) with a 60× objective (oil immersion, aperture 1.3). Fifty nuclei from each experiment were counted and evaluated. Images were processed using Image J software and the corrected total cell fluorescence (CTCF) was evaluated using the formula. \"CTCF =(Integrated Density-(Area of selected cell X Mean fluorescence \" \"of background readings))\" Immunofluorescence assay Cells were grown on 35 mm dishes for 24 h and were fixed with 4% PFA at room temperature for 30 min after piperine, cisplatin, and radiation treatment for 48 h. Then, the slides were permeabilized with PBS containing 0.2% Triton-X at room temperature for 30 min and blocked for 30 min at 4°C with blocking buffer containing 2.5% bovine serum albumin and 0.2% Triton-X in PBS. Cells were incubated with DNA-PKcs, ERα, and β antibodies (1:500) overnight and then washed with PBS twice and incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor488 (1:1000) at room temperature for another 1 h. After staining with DABCO/PI in the dark for 30 min, cells were observed under a ZOE imager (Biorad)( 59 ). Immunoblot assay Immunoblots of MCF7 cells treated with piperine (75 µM), alone or in a combination of cisplatin (3 µM) or and γ-radiation (2 Gy) for 24 h were performed using the standard protocol. Protein lysates were prepared, post 24 h of radiation after washing the trypsinized cells with phosphate-buffered saline (PBS) and treating with extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X, 1 mM EDTA, 0.5% Tween 20, 0.1% SDS), protease inhibitor cocktail (10 µL/ mL) for 30 min, followed by centrifugation at 12000 rpm, for 20 minutes at 4 o C. The protein samples were quantified using the Bradford method where bovine serum albumin (BSA) was taken as a standard. Protein samples (30 µg) were run in a 7–12% SDS gel followed by transfer to polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membranes were blocked using 4% BSA and further incubated with specific primary antibodies such as Bax, BCL-2, Cleaved caspase 3, Cleaved caspase 9, and Cleaved PARP-1 for checking the expression of apoptosis proteins, Ku70 and Ku80, and DNA Ligase IV for checking the expression of Non-homologous end-joining DNA repair proteins and p53, Phospho p53 and ATM for checking the expression of DNA damage response and ERα and ERβ for studying the receptor-mediated signaling in various treatment groups. Primary antibody incubation was done overnight at 4 o C. The unbound antibody was washed off using PBST for 30 min with buffer change every 10 min. Further, the blots were incubated with a secondary antibody anti-rabbit-HRP) for 1 h at RT on a shaker. The unbound antibody was washed off using PBST for 30 min by changing the buffer every 10 min. The blots loaded with beta-actin served as the loading control for normalizing the protein level. The membrane was developed by ChemDoc chemiluminescence method using ECL solution to detect the signal. The bands were quantified by densitometric analysis using Image J software 2.0. The quantified bands were normalized with their respective β-actin levels ( 60 ). Measurement of reactive oxygen species MCF7cells were plated at a density of 0.35x 10 6 in 12.5 cm2 tissue culture flasks and incubated overnight. Pre-treated with piperine, cisplatin, alone or in combination for 2 h and irradiated the relevant flasks with 2 Gy. Intracellular ROS levels were detected after 24 h of radiation by flow cytometry using 2´, 7´-dichlorofluorescein diacetate (DCFDA; Sigma Aldrich, India) at a final concentration of 10 µM for 15 min at 37°C. For positive gating, cells were treated with H 2 O 2 at a concentration of 1 mM and harvested after 4 h of incubation at 37 o C. The fluorescence intensity was measured at an absorption wavelength of 480 nm and an emission wavelength of 525 nm using a flow cytometer ( 61 ). Mitochondrial membrane potential (ΔΨm) assay MCF7 cells seeded at a density of 0.35x 10 6 in 12.5 cm2 tissue culture flasks were incubated overnight at 37 o C. A pre-treatment of piperine, cisplatin alone, or in combination was given 2 h before radiation treatment and incubated for 24 h at 37 o C. Post 24 h radiation, the cells were harvested and change in the ΔΨm was assessed using JC-1 dye (Calbiochem, Cat.#. 420200, India). The cells treated with H 2 O 2 were taken for positive gating. The fluorescence intensity of both JC-1 green monomers and red JC-1 aggregates (JC- 1 fluorescence intensity falls λex = 580 nm, λem = 595) was recorded using a flow cytometer( 62 ). Multiple ligand pharmacophore modeling The pharmacophore of piperine and other standard estrogen receptor agonists/antagonists was developed using the ligand-based pharmacophore modeling in the Phase module of maestro 11.0. Hydrogen bond (HB) donors (D), HB acceptors (A), aromatic ring (R), hydrophobic/non-polar groups (H), and positive/negative ionizable groups were selected as the pharmacophore features for the modeling( 63 ). The standard agonists/antagonists with promising fitness scores and structural similarity were selected for further processes. Molecular docking studies The molecular docking studies were performed using Schrodinger suite 11.0. The three-dimensional structures of ERα in its agonistic conformation (PDB ID 1X7R) and antagonistic conformation (PDB ID 1ERR), and ERβ (1X7J) were downloaded from the protein data bank. The downloaded protein structures were processed and energy minimized using the protein preparation wizard. The protein preparation involves assigning bond orders, adding hydrogens, creating disulfide bonds, and creating zero-order bonds to metals. The HET states of the proteins were generated using ‘Epik’ at pH 7 ± 2.0 and restrained minimization was performed using OPLS3 forcefield ( 64 ). Further, a receptor grid was generated from each structure by selecting the co-crystal ligand as the centroid of the receptor. Similarly, the ligand molecules such as piperine, estradiol, genistein, and bazedoxifene were downloaded from the NCBI-Pubchem database and prepared for docking with the ligprep module. The ligand molecules were structurally optimized at near-neutral pH (7 ± 1), and subjected to energy minimization using OPLS3 force field. Eventually, the prepared ligand sets were docked against the receptor grid of the above-mentioned target proteins. Extra precision (XP) flexible docking was performed using the GLIDE module and the affinity of the ligands towards the target proteins were ascertained in terms of negative glide score (kCal/mol). 50 docking poses were collected for each ligand. The binding poses were generated using pymol software (free license). Statistical Analysis All assays were repeated three times and triplicate results are shown. Data were represented as Mean ± SD. Data analysis was done using one-way ANOVA and two-way ANOVA (for cell cycle analysis), where a p-value of < 0.0001 was considered significant. Results Piperine sensitizes breast cancer cells towards γ-radiation treatment through a synergistic mechanism Piperine, cisplatin, and γ-radiation treatment significantly inhibited cell growth in a dose-dependent manner (Figure S1). To assess the combinatory effect of piperine, cisplatin, and γ-radiation, we have selected a dose less than the IC 30 value (a dose of individual treatment which kills 25–30% of cells). Thus, 75 µM of piperine, 3 µM of cisplatin, and 2 Gy of γ-radiation were selected for the combination study. As shown in Fig. 1 , there is no significant proliferation inhibition or morphology changes in treatment with the selected doses of piperine or cisplatin (Cis) or ionization radiation (IR) irradiation alone. However, cell proliferation was severely inhibited, and morphology was affected due to a combination of piperine, cisplatin, and γ irradiation. A combination of a piperine dose of 75 µM along with a cisplatin dose of 3 µM or γ-radiation (2 Gy) resulted in cell growth inhibition rates of 45–55% at 48 h-7 days. These results demonstrate that piperine treatment, along with cisplatin and γ-radiation, could inhibit the proliferation of MCF7. Similarly, enhanced proliferation inhibition was observed in another estrogen-responsive cell line, HT-29 ( 25 ). However, in estrogen receptor-negative cell lines MDA-MB-468 and MDA- MB-231, the combination treatment did not show significant cell death (Figure S1). To check whether the observed enhanced cytotoxicity of the combination treatment is synergistic or additive, we calculated the CI values and generated a CI plot for the equi-effective dose of the piperine and cisplatin against γ-radiation using CompuSyn software. A dose-response curve for individual test agents was plotted (Fig. 1 e). As shown in Figure. 1f, the calculated CI value is less than 1 for the selected dose used in the combination treatment comprising piperine with the γ-radiation demonstrating a synergistic or sensitization effect. Taken together, these results show that pre-treatment with piperine effectively sensitized breast cancer cells to the cytotoxic effects of γ-radiation, compared to the individual treatment alone. Thus piperine may be beneficial during chemo-radiotherapy. Piperine augmented IR/Cis induced ROS accumulation leading to DNA damage. To assess whether the synergistic effect of the combination treatment was mediated through Reactive oxygen species (ROS) generation, we evaluated the intracellular generation of ROS by flow cytometry (Figs. 2 a and b). The cells were treated with all three agents either as a single agent or in combination with induced ROS. As expected, combination treatment generated a higher amount of ROS compared with that in cells treated with a single regime. The assay suggests that the enhancement in the cisplatin/radiation-induced cytotoxicity by piperine may be associated with increased ROS levels. Since the intracellular generation of ROS is associated with DNA damage, we assessed the extent of genotoxicity by performing a Cytochalasin-B-blocked micronuclei assay. We observed a significant increase in the nuclear bud, micronuclei formation, and a nucleoplasmic bridge in the combination treatment indicating that piperine augmented the radiation-induced double-stranded DNA damage (Figs. 2 c and d). Since phosphorylation of histone H2AX at Ser139 is a dominant response to DSB, resulting in the formation of an increased number of structures known as ionizing radiation-induced foci (IRIF) or γH2AX foci, we confirmed the DSBs by immunofluorescence sensing for γH2AX as well (Fig. 2 e and f). Piperine treatment modulated p53/ATM-dependent DNA damage response inducing cell cycle arrest. In response to DNA damage induced by chemo/ radiotherapy, p53 is overexpressed. While p53 was upregulated with IR and cisplatin, piperine treatment reduced its expression as a single agent and in its combination (Fig. 3 a & b). Since p53 is an ERα regulated gene, its downregulation by piperine may be mediated through ER modulation. In response to DNA damage, p53 is stabilized and activated by phosphorylation at Ser15 by ATM. Interestingly we observed treatment-dependent upregulation of phospho-p53 and ATM (Fig. 3 a, c, and d) in combination treatment with piperine confirming that piperine augmented the IR and cisplatin-induced DNA damage response. This enhanced appearance of DDR proteins in the combination treatment may be due to piperine-mediated downregulation of DNA repair. Interestingly, the expression pattern of all the DDR proteins we checked, γH2AX, phospho-p53, and ATM, was almost similar, confirming that piperine enhanced cisplatin/radiation-induced DNA damage. Cell cycle arrest is induced by p53 activation in response to DNA damage leading to DNA repair or apoptosis ( 26 ). Hence we analyzed the influence of piperine and its combination with cisplatin/ IR on the cell cycle status of MCF7 cells. As expected, cell cycle analysis showed a significant G2 M stall at 24 h for the combination of piperine, cisplatin, and γ-radiation (Fig. 3 e and f). While piperine augmented the DNA damage and IR-induced G2/ M staling of cells in ER-positive breast cancer cells (MCF7), it failed to induce similar G2/ M arrest in ER-negative breast cancer cells (MDA-MB-468 and MDA-MB-231) (Figure S2). Besides, in ER-negative cells, piperine pretreatment reduced the IR-induced G2/ M arrest pointing towards an estrogen receptor-dependent sensitization. In conclusion, piperine augmented the DSBs induced by IR/ cisplatin treatment leading the cells to undergo G2/ M arrest in ER-positive cancer cells. Identification of piperine as a Selective estrogen receptor modulator of ER-alpha Since we observed that piperine-sensitized estrogen-responsive cells like MCF7 and HT-29 towards IR compared to estrogen receptor-negative cells, we were interested to know if it exhibits any estrogen receptor modulating effects. We analyzed the expression and nuclear translocation of ER subtypes in MCF7 cells with or without piperine treatment, and interestingly we found that piperine decreased the expression and nuclear translocation of ERα (Fig. 4 a, b) and enhanced the expression and nuclear translocation of ERβ (Fig. 4 c, d) in a dose-dependent manner in MCF7 cells. These results point towards a receptor-specific modulation of signaling by piperine. To further understand the mechanism behind the observed estrogen receptor-modulating effects upon piperine treatment, we performed pharmacophore modeling of piperine with an established estrogen agonist and antagonist. Interestingly, we observed that piperine shares a common pharmacophore feature with most of the established estrogen receptor ligands. Hydrogen bond acceptors (A), aromatic ring (R), and hydrophobic/non-polar groups (H) are the predominant pharmacophore features that were found in common between piperine and several standard estrogen receptor agonists/antagonist (Table S1 and S2). From the pharmacophore modeling, two pharmacophore hypotheses, AAR and AHR (Fig. 4 e), were observed as the common feature among piperine and other estrogen receptor agonists. In Fig. 4 e, piperine, as well as the standard estrogen receptor agonists/antagonists such as bazedoxifene, -a third-generation selective estrogen receptor modulator (SERM), estradiol- the natural ligand of estrogen, and many of the established SERMs, such as genistein, coumestrol, daidzein, portfolio, repensol, and formononetin, was found to possess the common pharmacophore feature AAR which gives a fitting score of 1.825 for piperine and ranges from 1.8 to 3 for other agonists. Besides, piperine has pharmacophore features AHR that can be best fitted with that of some of the SERM such as bazedoxifene, estradiol, cyclofenil, ormeloxifen, raloxifene, tamoxifen, toremifene, ospemifene, and lasofoxifene respectively. The fitness score of piperine for AHR pharmacophore was found as 1.87. It can be concluded that some pharmacophore features of piperine that can be found in common in other estrogen receptor agonists are key to mimicking the estrogen receptor agonistic activity. The hypothesis was further supported by molecular docking studies (Fig. 4 f and g). We found that the key element of the conformational switch, the short helical region, the helix 12 (H12), which adopts a ligand-dependent conformation assumed an agonistic position for piperine (Fig. 4 f). This conformation was similar to the natural ligand estradiol and the known agonist genistein (Figure S3 b) but with lower binding energy. Piperine exhibited comparable affinity towards the ligand-binding domain (LBD) of ERα in an agonistic conformation as the reference ligands (estradiol and genistein) used for validation of the docking study (Figure S3 a). The binding of piperine and the reference ligands (estradiol and genistein) in the active site of ERα exactly where the co-crystal ligand interacts. A detailed description of the active site residues involved in the interaction also reveals the involvement of several active site residues in common for stabilizing the binding of piperine and the reference ligands (Figure S3 b). Docking studies performed against ERα (1X7R) expressed a docking score of -8.977 kCal/mol with piperine. The docking score was − 11.155 kCal/mol and − 10.758 kCal/mol for estradiol and genistein, respectively. As expected from the results of pharmacophore modeling of piperin, the key pharmacophore feature ‘aromatic ring (R)’ was found to involve in π-π (pi-pi) stacking interaction with Phe-404 present in the active site of ERα (Fig. 4 g). Similar π-π stacking interaction can also be observed in the interaction diagram of estradiol and genistein in which the aromatic pharmacophore interacts with the ERα active site residue Phe 404 (Figure S3 b ). The result of the molecular docking studies supports the pharmacophore hypothesis. In addition to the π-π stacking interaction, hydrogen bonding and salt bridge interaction can also be observed in the case of estradiol and genistein as the major stabilizing interactions. Bazedoxifene adopted only antagonistic conformation within the LBD of ERα. Interestingly due to its small structure, piperine could also adopt antagonistic conformation, which is thermodynamically stable but with higher energy (Figure S3 c) than compared with piperine in its agonistic conformation. The docking studies of piperine and bazedoxifene with ERβ (PDB ID 1X7J) failed to give any output. The automated docking program was aborted for bazedoxifene/piperine and ERβ interaction because of the poor orientation of piperine and/ or the presence of bulky side chains of bazedoxifene with the receptor. However, estradiol and genistein interacted well with the active site of ERβ (Figure S3 d). Thus we assume that piperine is a weak agonist of ERα and competed with its natural ligand, estradiol, for the receptor interaction. This resulted in the reduced translocation of the activated ERα to the nucleus to exert its downstream signaling events. Thus, in conclusion, piperine interacts only with the LBD of ERα as a weak agonist and not with ERβ. This accounts for the decreased nuclear translocation observed for ERα and an increased translocation of activated ERβ to the nucleus. The results suggest that piperine may be used as complementary medicine for estrogen-positive cancer therapy. This also emphasizes the need to judiciously consuming food rich in Phyto-estrogens during chemo/radiotherapy. Piperine down-regulates the expression of DNA PKcs, an estrogen-responsive gene, and other repair proteins of the NHEJ pathway. The role of estrogen in the development and progression of breast cancer is demonstrated in several previous studies ( 27 – 29 ). The transcriptional activation and gene expression of the DNA-PKcs- catalytic subunit, one of the key proteins of the NHEJ pathway, are directly mediated by ERα in response to estrogen ( 23 ). NHEJ, the major DSB repair pathway upregulated during cancer therapy, imparts therapy resistance in cancers by mediating the error-prone repair conferring a survival advantage to cancer cells. Since piperine modulates estrogen receptors’ expression selectively (Fig. 4 a, b, c, d), we assumed that it might modulate the expression of DNA-PKcs involved in the NHEJ machinery. To understand the role of piperine in modulating estrogen receptor expression and NHEJ proteins during chemo/radiotherapy, protein expression studies are carried out. The expression and translocation of estrogen receptors showed an increase in ERα and a decrease in the expression of ERβ in response to treatment. Interestingly enhanced translocation of activated ERα to the nucleus was observed in control and with radiation-treated cells (Figure S4 a and b). Piperine and cisplatin treatment, on the other hand, reduced the ERα nuclear translocation (Figure S4 c) and altered the ERα/ERβ ratio (Fig. 5 a, b, and c). The expression of NHEJ proteins was also altered upon combination treatment. We observed enhanced expression of DNA-PKcs, Ku70, and Ku80 in the cells treated with radiation, cisplatin, and its combination. On the other hand, piperine drastically reduced the cisplatin/ IR-induced expression of these key NHEJ proteins (Fig. 5 d-i). DNA-PKcs, along with Ku70/ Ku80 heterodimer, form the DNA-PKcs complex, which initiates NHEJ. The expression of Ligase IV was also reduced in piperine treated group and also in combination treatment compared to radiation treatment alone.Interestingly we observed the same pattern of expression for DNA-PKcs, p53, Ku70, and Ku80 under similar experimental conditions. Since DNA-PKcs ad p53 is already established to be an ERα regulated gene, we speculate that the expression of Ku70 and Ku80 is also directly or indirectly regulated by ERα. Accordingly, the observed increase in DNA damage upon combination treatment may be due to a decrease in the NHEJ-mediated DNA repair. Overexpression of NHEJ proteins is known to confer therapy resistance, and its ablation confers radio/chemosensitization. Thus these results indicate that a complementary therapy with piperine or any weak estrogen receptor agonist or antagonist along with radio/chemo agents may reduce therapy resistance.Interestingly, as observed for piperine, bazedoxifene down-regulated the IR-induced expression of DNA-PKcs (Figure S4 e and f), confirming our postulation. To reconfirm this assumption, we treated MCF7 cell lines with the third-generation SERM, bazedoxifene, which shared common pharmacophore features with piperine. As expected, bazedoxifene failed to downregulate the expression of DNA-PKcs in estrogen non-responsive cell lines, MDA-MB 231, MDA-MB 468, and A549 (Figure S4 g-i). Hence it may be assumed that piperine or any selective estrogen receptor modulators (SERMs) may sensitize radiation-resistant estrogen-responsive cancers to radiation by downregulating the NHEJ proteins. Treatment of Piperine in conjunction with radiation and cisplatin-induced mitochondrial pathway of apoptosis Elevated intracellular ROS generated during co-treatment causes damage to DNA. Cells with DNA damage are arrested at the G2/ M phase (Fig. 2 a-d), eliciting DNA repair or cellular apoptosis. If DSBs are left unrepaired, apoptosis is elicited through the mitochondria-mediated intrinsic or extrinsic pathway. ROS can activate BAX BCL2 Associated X]( 30 ), a key tumor suppressor protein involved in the intrinsic pathway of apoptosis. Interestingly co-treatment resulted in the up-regulation of BAX and down-regulation of BCL2 (Fig. 6 a-f), the main molecular players eliciting mitochondrial membrane depolarization setting off apoptosis. The mitochondrial membrane potential was analyzed using JC-1 dye which is taken up by the mitochondria of only viable cells. JC-1 dye accumulation in mitochondria of viable cells is potential-dependent, indicated by a fluorescence emission shift from green (~ 529 nm) to red (~ 590 nm), measured using a flow cytometer as shown in Fig. 6 g and h, a decrease in red fluorescent J-aggregates with an increase in green fluorescent J-monomers, upon co-treatment points towards a depolarization of the membrane potential of mitochondria, which is an important physiological feature of apoptosis. The activities of caspase 9 and 3 were also enhanced (Fig. 6 a-f) upon treatment, indicating that apoptosis was induced through intrinsic pathways. Cleavage of Poly(ADP-ribose) polymerase-1 (PARP-1) by caspases is considered as a hallmark feature of apoptosis. The analyses of PARP-1 cleavage showed that piperine enhances PARP-1 cleavage as a single regime and in combination. Together these results point towards the that piperine sensitizes estrogen-responsive cancer cells to radiation by augmenting the radiation-induced DNA damage via downregulating the DNA-PKcs complex, the key component of the NHEJ repair pathway inducing cells to G2/ M arrest leading to apoptosis. Discussion Identification of piperine as a modulator of estrogen receptor subtype (ERα/ERβ) expression and activation Estrogen signaling is a balance between two opposing functions exerted by two specific nuclear hormone receptors (ERα and ERβ) and their splice variants. They are the members of the ligand-activated transcription factors falling under the nuclear-hormone receptor (NR) superfamily ( 20 ). ERα promotes proliferation and confers invasive properties on cancer cells ( 21 ). In contrast, ERβ suppresses epithelial to mesenchymal transition and promotes apoptosis ( 22 ). Interestingly, in our study, we found that the expression of ERα/ ERβ is altered by piperine. We found a downregulation in ERα expression with an upregulation in ERβ (Fig. 4 a, b, c, d and Fig. 5 a, b), indicating that piperine might inhibit cell proliferation and induce apoptosis. In the classical or direct genomic estrogen signaling pathway, the nuclear estrogen receptors ERα and ERβ act as ligand-activated transcription factors regulating the expression of its target gene ( 31 , 32 ). ERα/ ERβ binds to its ligand in the cytoplasm, inducing a conformational change resulting in receptor dimerization and activation of ligand-bounded receptors ( 33 ). The activated receptor-ligand complex is then translocated to the nucleus, where it binds to the ERE sequences at the enhancer regions within or close to promoters and/or 3'-untranslated regions of the target genes to initiate the transcription ( 34 ). We found a decrease in the nuclear translocation of ERα and an increase in the nuclear translocation of ERβ with piperine treatment in a dose-dependent manner (Fig. 3 a, b, c, d), confirming that piperine is involved in the activation/deactivation and nuclear translocation of ERs. Our results indicate that piperine shared common pharmacophore features with estradiol and established estrogen receptor agonists and antagonists (Fig. 4 e and Table S1 and S2). The ligand-binding domain (LBD) of ERα plays a prominent role in maintaining agonistic/ antagonistic conformation. LBD comprises a beta-hairpin and twelve α-helices (H1-H12), of which H12 plays a critical role in ligand-dependent activation of the receptor ( 35 , 36 ). Agonist binding favors an active conformation, where H12 takes a position across H3 and H11 and supports co-regulator binding. Estradiol and genistein adopted only agonistic conformation (Figure S3 b), while raloxifene (the data is not shown) and bazedoxifene agreed to an antagonistic position (Figure S3 c). Piperine could interact well in the LBD of ERα in its agonistic conformation, and the interaction may be comparable to that of estradiol and genistein used for validating the docking results (Fig. 4 f). The binding of the antagonist to the LBD causes H12 to shift from its agonistic position (Figure S4 a). This disrupts co-regulator binding and favors co-repressor binding inhibiting transcriptional activation. Due to its molecular structure and flexibility, piperine could also adopt an antagonistic conformation that is thermodynamically stable but with higher energy than compared with bazedoxifene, an estrogen receptor antagonist (Figure S3 c). This points out that piperine may favor agonistic conformation over antagonistic. Hence we assume that piperine may be a weak agonist of ERα. The in silico study is supported by in-vitro studies, where we found reduced expression of ERα (with piperine treatment (Fig. 5 a and b) together with reduced nuclear translocation of ERα (Fig. 4 a and b and Figure S4 a and b) confirming the probability of piperine to be a weak estrogen receptor agonist. The ability of piperine to downregulate ERα and lower its activation may be because it is competing with the natural ligand estradiol for the LBD of ERα. On other hand, the available or displaced estradiol activates ERβ, enhancing its expression (Fig. 5 a and d) and activation (Fig. 4 c and d and Figure S4 c and d). Since ERβ regulates the apoptotic and tumor suppressor proteins, its upregulation and activation by piperine may also be considered a promising therapeutic approach. Hence the potential of piperine, a key spice used in abundance by the Asian population in downregulating and suppressing ERα together with upregulation and activation of ERβ, points towards the judicial use of spices during cancer therapy. Downregulation of ERα by SERMS as a novel strategy for sensitizing cancers to DNA-damaging therapies Though the concept of either suppressing ERα or activation of ERβ is acknowledged as a promising clinical approach towards the management and prevention of cancers, their modulation was never reported to sensitize cancers towards DNA damaging therapies( 17 – 19 ). Thus this is the first study introducing the proof of concept that ERα downregulation by SERMs sensitizes cancers to DNA damaging agents (here, platinum-based chemo drug; cisplatin and radiotherapy), and this strategy may help to overcome therapy resistance. In our study, we found that piperine downregulates the expression of p53, Ku70, Ku80, and DNA-PKcs in ER-positive breast cancer cells (Fig. 3 a and b, Fig. 5 d, e f, h and i). The p53 is a tumor suppressor gene that participates in DNA damage response, and Ku70, and Ku80, along with DNA-PKcs are DNA repair proteins that play a critical role in initiating NHEJ ( 7 , 37 ). While the expression of p53 and DNA-PKcs are transcriptionally regulated by ERα, Ku70/ 80 are stabilized by ERα binding ( 23 , 38 ). Also, we observed that while piperine treatment downregulated p53 expression, its phosphorylation/ activation was enhanced with piperine. And p53 is stabilized and activated by phosphorylation at Ser15 by ATM, in response to DNA damage ( 3 , 39 – 41 ). Thus the treatment-dependent upregulation of DDR proteins γH2AX, phospho-p53, and ATM in combination treatment with piperine (Figs. 2 and 3 ) confirmed that piperine amplified the IR/ cisplatin-induced DNA damage response. This correlated with the downregulation of ERα-regulated proteins- p53 and the NHEJ repair protein. Managing therapy resistance in estrogen-responsive cancers by Downregulating DNA-PKcs, a key component of NHEJ machinery through its transcription modulation NHEJ repair is error-prone and it confers new mutations to the cancer cells favoring their proliferation and invasion ( 42 ). NHEJ proteins, DNA-PKcs, Ku70, Ku80, and Ligase-IV were upregulated in the majority of the cancers ( 43 – 46 ). Ablation or silencing of one or more NHEJ repair proteins is reported to sensitize cancers to ionizing radiation ( 47 ). The DNA-PKcs is known to confer resistance to DNA targeted therapies such a platin-based drugs and radiation through promoting error-prone DNA repair ( 15 , 16 ). Piperine augmented the IR/ cisplatin-induced DNA damage by downregulating the NHEJ proteins; DNA PKcs, Ku70 and Ku80 (Fig. 5 d, e, f, h and i). Concurrently, we also observed a decrease in the expression and activation of ERα (Fig. 5 and b), a DNA-PKC transcription regulator. ERα is also binding partner of Ku70/ Ku80 and stabilizes the protein. Thus we assume that treatment with an ERα weak-agonist, may reduce the ligand dependent activation and function of the receptor effecting the expression and stabilization of the key NHEJ factors. This in turn sensitizes cancer cells to DNA-damaging therapeutics and also may help in overcoming therapy resistance. As proof of our concept, we used bazedoxifene a third-generation SERM, along with cisplatin/ ionizing radiation therapy, and the results were comparable to piperine. Cell cycle arrest is a prominent response to p53 activation ( 26 ). The p53 may be activated by phosphorylation or acetylation ( 48 ). Here we found that enhanced phosphorylation of phospho-p53 in combination treatment with piperine corresponded to the arrest of cells at the G2/ M phase of the cell cycle (Fig. 3 a, b, e and f). Phospho-p53 induced cell-cycle arrest is reversible if DNA breaks are repaired ( 49 ). We found a reduction in the DSB repair proteins and enhancement in DNA damage upon piperine co-treatment indicating that piperine-induced cell cycle arrest may lead to apoptosis . Apoptosis is a dynamic cellular death program induced by endogenous or exogenous factors for the removal of damaged cells. Apoptosis was in-turn confirmed by checking for apoptotic markers and important molecular events. In addition to DNA damage (Fig. 2 c, d, e and f), ROS generated during treatment (Fig. 2 a and b) causes a change in protein expression, damage to cell membranes, mitochondria, etc., initiating apoptosis. ROS activates Bax [BCL-2 Associated X], a key tumor suppressor protein involved in the intrinsic pathway of apoptosis ( 30 , 50 ). Interestingly the combination treatment altered the BAX/BCL-2 levels, eliciting mitochondrial membrane depolarization (Fig. 6 a, b, c, g and h). The mitochondrial membrane stability was measured and combination treatment was found to depolarize the membrane. Besides, there was enhanced expression of the initiator and effector caspase-9, and caspase-3, respectively, and the cleavage of PARP-1, a cellular substrate of caspases, confirming apoptosis (Fig. 6 a, d, e and f). Similar results were observed in our previous studies on HT-29 cells ( 25 ). Thus as shown in Fig. 7 we assume that piperine sensitizes ER-positive cells towards DNA damaging agents by downregulating the NHEJ-DSB repair through suppressing ERα activation and enhancing ERβ, thus inducing cells to the intrinsic pathway of apoptosis. Conclusion DNA-PK complex comprising of DNA-PKcs/ Ku70/ Ku80 are the major proteins known to be involved in initiating NHEJ pathway and repair of DSBs. Here we found that the treatment of MCF7 cells with piperine, lead to the accumulation of DSBs induced with gamma irradiation through lowering the DNA-PK complex through altering the ERα/ ERβ ratio. The elucidated mechanism of action of the piperine is summarized in the illustration (Fig. 7 ). We found that piperine shared common pharmacophore features with selective estrogen receptor modulators (SERMs) and interacted with ERα, but failed to interact with ERβ. Piperine may be a weak agonist of ERα and competed with its natural ligand, estradiol for the receptor interaction, resulting in the reduced expression and translocation of the activated ERα to the nucleus to exert its downstream signaling events. Piperine downregulated NHEJ through DNA-PKcs modulation mediated through its transcription factor, estrogen receptor alpha (ERα). On other hand, overexpression of ERβ, a nuclear hormone transcription factor promoting tumor suppression positively correlated with lowered expression of ERα and DNA-PK complex, marked by the accumulation of radiation-induced DSBs leading to cell cycle arrest inducing intrinsic pathway of apoptosis. We also used bazedoxifene a third-generation SERM, along with cisplatin/ionizing radiation therapy and the results were comparable to piperine. Thus suppression of ERα activation through SERMs may be used to target NHEJ when co-administered with platinum-based chemo/ radiotherapy and this offers a novel strategy towards management and prevention of cancers and improvement of existing regimens. Thus this is the first study introducing the concept that estrogen receptor (ERα) downregulation sensitizes cancers to DNA damaging agents and helps to overcome therapy resistance. Abbreviations CI Combination index DSB DNA double-stranded break dsDNA double stranded DNA Erα Estrogen receptor alpha ERβ Estrogen receptor NHEJ Non-homologous End Joining ROS Reactive oxygen Species Declarations Ethics approval and consent to participate: Not Applicable Consent for publication: Not Applicable Availability of data and materials : Data sharing is not applicable to this article as no datasets were generated or analysed during the current study Competing interests: Authors hereby declare that they don’t have any conflicting interests to declare Funding: DLM is supported by Start-up grant (YSS/2015/000987), SERB, DST, Govt. of India and Seed Grant (YU/Seed grant/065-2018) from Yenepoya (deemed to be University). KS is supported by ICMR-SRF, Govt. of India. Authors' contributions DLM conceptualized the study, designed the experiments, analyzed and interpreted the results. KS performed the in-vitro experiments and analyzed the data. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2401099\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":162049187,\"identity\":\"4133732e-9bbc-4e1f-aba7-65df3f6c530b\",\"order_by\":0,\"name\":\"Koniyan Shaheer\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yenepoya Research Centre, Yenepoya (Deemed to be University)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Koniyan\",\"middleName\":\"\",\"lastName\":\"Shaheer\",\"suffix\":\"\"},{\"id\":162049188,\"identity\":\"15dbcd0e-f571-4b1c-a569-36f94904ac8c\",\"order_by\":1,\"name\":\"Swathi Prabhu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yenepoya Research Centre, Yenepoya (Deemed to be University)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Swathi\",\"middleName\":\"\",\"lastName\":\"Prabhu\",\"suffix\":\"\"},{\"id\":162049189,\"identity\":\"03e2f8bd-ea2f-4642-a430-beb6f0caa48d\",\"order_by\":2,\"name\":\"H. Shabeer Ali\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kannur University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"H.\",\"middleName\":\"Shabeer\",\"lastName\":\"Ali\",\"suffix\":\"\"},{\"id\":162049190,\"identity\":\"0c54bc19-10b2-4ae1-89a8-7fb565f8b125\",\"order_by\":3,\"name\":\"Divya Lakshmanan\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"\",\"institution\":\"Yenepoya Research Centre, Yenepoya (Deemed to be University)\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Divya\",\"middleName\":\"\",\"lastName\":\"Lakshmanan\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2022-12-21 11:29:24\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2401099/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2401099/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":30892571,\"identity\":\"536c0a5b-a00c-4d53-97b3-ca8d11efc73d\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 16:56:59\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":357883,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePiperine sensitizes estrogen receptor-positive breast cancer cells towards γ-radiation treatment through a synergistic mechanism\\u003c/strong\\u003e. \\u003cstrong\\u003ea:\\u003c/strong\\u003e Relative cell proliferation assay after the co-treatment by MTT, \\u003cstrong\\u003eb\\u003c/strong\\u003e: Trypan blue dye exclusion, and\\u003cstrong\\u003e c\\u0026amp;d\\u003c/strong\\u003ecolony-forming assay in MCF7 cells. n=3, Data represent mean ± SD.\\u003cstrong\\u003ee:\\u003c/strong\\u003e Dose-response curve of piperine (Pip) and γ-radiation (IR). IRPip indicates combination o γ-radiation (2 Gy) and piperine (75 μM), \\u003cstrong\\u003ef: \\u003c/strong\\u003eCombination index (CI) plot and CI table show that CI for the selected combination is \\u0026lt;1, indicating synergism. . \\u003cstrong\\u003eg\\u003c/strong\\u003e: Representative\\u003cstrong\\u003e \\u003c/strong\\u003emicroscopic images of the cells showing a reduction in the number of cells following treatment. (See also Figure S1).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/f757531e0270fce6e2ff734f.png\"},{\"id\":30895040,\"identity\":\"ce2e045a-5422-46a3-8b1f-e0066c0a3d1c\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 17:12:59\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":333760,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePiperine augmented IR/Cis induced ROS accumulation leading to DNA damage a:\\u003c/strong\\u003e Flowcytometric analysis of the effect of co-treatment on the production of reactive oxygen species (ROS) by DCFD staining. Dot plot shows a spectral shift upon treatment indicating ROS accumulation. H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2 \\u003c/sub\\u003e(100 µM) treated cells served as a positive control. \\u003cstrong\\u003eb\\u003c/strong\\u003e: Bar diagram presents the distribution of cell population with RO,\\u0026nbsp; as the mean ± SD, n=3.\\u003cstrong\\u003ec:\\u003c/strong\\u003e Effect of piperine on chromosomal damage in MCF7 cells in combination with radiation after 24 h of co-treatment. Binucleated cells with micronuclei (MNi), nuclear buds (NBUD), and binucleated cells with the nucleoplasmic bridge (NPB) were counted by light microscopy. \\u003cstrong\\u003ed:\\u003c/strong\\u003e Representative number of binucleated cells with chromosomal damages in individual treatments is shown in the bar diagram. N=50 cells. \\u003cstrong\\u003ee: \\u003c/strong\\u003eImmunofluorescence sensing for γH2AX.\\u003cstrong\\u003e \\u003c/strong\\u003eNuclear staining was done with propidium iodide while γ-H2AX staining was done by Alexa Fluor 488-conjugated phospho-histone-γ- H2AX antibody (Ser139) appeared as green points (foci). The stained cells were then observed under a confocal microscope (Carl Zeiss, Göttingen, Germany) with a 60× objective (oil immersion, aperture 1.3). The relative quantity of cells with H2AX foci in individual treatments is shown in the bar diagram. N=50.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/2fe0156eb8cd1e3571198f2a.png\"},{\"id\":30893942,\"identity\":\"111cdb52-2c82-430a-a7ed-f909d78bef54\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 17:04:59\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":251183,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePiperine treatment modulated p53/ATM-dependent DNA damage Response inducing Cell cycle arrest. a: \\u003c/strong\\u003eRepresentative western blot showing the pattern of p53, phospho-p53, and ATM expression in MCF7 cells\\u003cstrong\\u003e. \\u003c/strong\\u003eBar graphs showing\\u003cstrong\\u003e \\u003c/strong\\u003eexpression\\u003cstrong\\u003e \\u003c/strong\\u003eof\\u003cstrong\\u003eb: \\u003c/strong\\u003ep53 \\u003cstrong\\u003ec:\\u003c/strong\\u003e phospho-p53, and \\u003cstrong\\u003ed:\\u003c/strong\\u003e ATM\\u003cstrong\\u003e \\u003c/strong\\u003ein\\u003cstrong\\u003e \\u003c/strong\\u003efold change. (Mean ± SD). \\u003cstrong\\u003ee:\\u003c/strong\\u003e Histogram representation of the cell cycle distribution, G1, S, and G2/ M phases following co-treatment of piperine and radiation on MCF7 cells. The co-treated cells were subsequently analyzed by flow cytometry using PI staining to determine the cell cycle.\\u003cstrong\\u003e f\\u003c/strong\\u003e: Quantification of the cells at different phases of the cell cycle is presented by bar diagram as the Mean ± SD, n=3.\\u003cstrong\\u003e \\u003c/strong\\u003e(See also Figure S2).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/496dd414b69086ef37e87cee.png\"},{\"id\":30895041,\"identity\":\"82b8411c-528e-472d-ad8a-e629ed112ad5\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 17:12:59\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":628125,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIdentification of piperine as a selective estrogen receptor modulator of ER-alpha.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea: \\u003c/strong\\u003eRepresentative images of the immunofluorescence analysis using anti-ERα and \\u003cstrong\\u003ec:\\u003c/strong\\u003e ERβantibodies showing nuclear localization and differential expression of ERα and Erβ with selected concentrations of piperine in MCF7 cells. Goat Anti-Rabbit IgG H and L (Alexa Fluor 488) secondary antibody was used for probing. Nuclei (red) were stained with propidium iodide. \\u003cstrong\\u003eb:\\u003c/strong\\u003e Bar diagrams present the relative expression of ERα and \\u003cstrong\\u003ed:\\u003c/strong\\u003eERβ positive cells as the mean ± SD, n=3.\\u003cstrong\\u003ee: \\u003c/strong\\u003ePharmacophore model of piperine shows it follows two hypotheses (AHR and AAR). \\u0026nbsp;Ligand pharmacophore mapping shows the best fit of bazedoxifene (grey) pharmacophores with piperine.\\u003cstrong\\u003ef:\\u003c/strong\\u003e Structures of piperine (red), estradiol (blue), and genistein (green) were superimposed in the active site cavity of estrogen receptor α (PDB ID 1X7R) to demonstrate that these ligands have same target site. \\u003cstrong\\u003eg:\\u003c/strong\\u003e Ligand interaction diagram of piperine with estrogen receptor α. (See also Figure S3).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/38aecde4c6a270efd767a2f2.png\"},{\"id\":30892578,\"identity\":\"8724b89f-5c28-4b22-865a-ba7bbef9d0a1\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 16:56:59\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":337363,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePiperine down-regulates the expression of DNA PKCs, an estrogen-responsive gene, and other repair proteins of the NHEJ pathway a:\\u003c/strong\\u003e Representative western blot showing the pattern of ERα and ERβ expression after the co-treatment in MCF7 cells\\u003cstrong\\u003e. b \\u003c/strong\\u003eand\\u003cstrong\\u003e c: \\u003c/strong\\u003eBar graphs showing\\u003cstrong\\u003e \\u003c/strong\\u003efold change of ERα and ERβ expression in MCF7 cells. (Mean ± SD)\\u003cem\\u003e. \\u003c/em\\u003e\\u003cstrong\\u003ed:\\u003c/strong\\u003e\\u003cem\\u003e \\u003c/em\\u003eRepresentative western blot showing the pattern of Ku70, Ku80, and DNA Ligase IV expression after the co-treatment in MCF7 cells\\u003cstrong\\u003e. e, f, and g c: \\u003c/strong\\u003eBar graphs showing\\u003cstrong\\u003e \\u003c/strong\\u003efold change of Ku70, Ku80, and DNA Ligase IV expression in MCF7 cells. (Mean ± SD). \\u003cstrong\\u003eh\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/em\\u003eand \\u003cstrong\\u003eI: \\u003c/strong\\u003eRepresentative images of the immunofluorescence analysis using anti-DNA PKCs antibody showing nuclear localization with selected concentrations of piperine in MCF7 cells. Goat Anti-Rabbit IgG H and L (Alexa Fluor 488) secondary antibody was used for probing. Nuclei (red) were stained with propidium iodide. \\u003cstrong\\u003eb:\\u003c/strong\\u003e Bar diagrams present the relative expression of DNA PKcs positive cells as the mean ± SD, n=3. (See also Figure S4).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/bbf4473770a1462cb1842023.png\"},{\"id\":30892577,\"identity\":\"a89d8341-5dd3-460c-b865-1cb92b087e9b\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 16:56:59\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":249840,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTreatment of Piperine in conjunction with radiation and cisplatin-induced mitochondrial pathway of Apoptosis.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;a:\\u003c/strong\\u003e Representative western blot showing the pattern of apoptosis proteins such as BAX, BCL-2, Cleaved caspase-9, caspase-3, and cleaved PARP-1 expression in MCF7 cells\\u003cstrong\\u003e. 5b-f: \\u003c/strong\\u003eBar graphs showing\\u003cstrong\\u003e \\u003c/strong\\u003efold change of BAX, BCL-2, Cleaved caspase-9, caspase-3, and cleaved PARP-1 expression in MCF7 cells. (Mean ± SD). \\u003cstrong\\u003eg:\\u003c/strong\\u003e Flowcytometric analysis of the effect of co-treatment on mitochondrial membrane potential by JC-1 staining. Dot plot shows the spectral shift from red to green upon treatment. H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2 \\u003c/sub\\u003etreated (100 µM) cells served as a positive control. JC-red represents cells with intact membrane potential and JC-green represents cells with collapsed membrane potential. \\u003cstrong\\u003eh\\u003c/strong\\u003e: Bar diagram presents the distribution of the cells with an induced change in mitochondrial membrane potential after the co-treatment as the mean ± SD, n=3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/013f828f3495137a7cf33bb6.png\"},{\"id\":30892574,\"identity\":\"6a547999-0028-4155-8c6b-82036ae46d9e\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 16:56:59\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":281333,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMechanism of action of piperine in estrogen receptor (ER) - positive cancer cell line\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePiperine sensitizes estrogen-responsive cancer cells to radiation and cisplatin therapy through lowering the DNA-PKcs complex, a key component of the NHEJ pathway through modulating the expression and translocation of ER subtypes. Estrogen binds to its intracellular receptor ERα. The binding activates ERα and it dimerizes and gets translocated to the nucleus to exert its transcription function. ERα can induce the expression of DNA-PKcs; an important component of the NHEJ mediated DNA repair mechanism. The DNA-PKcs expression is upregulated during the treatment of cancer with DNA damaging agents such as platinum drugs and irradiation. Enhanced expression of DNA-PKcs is associated with the treatment (clinical) resistance, as it can mediate error-prone DNA repair conferring a survival advantage to the cancer cells with repaired (mutated) DNA. Piperine on other hand down-regulates estrogen receptor alpha expression and function. This in turn down-regulates DNA-PKcs expression thus preventing DNA repair and sensitizes cells to DNA damaging agents (here cisplatin and ionizing radiation) and induces intrinsic pathways of apoptosis.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/31ae0c8f0f51b1d5c664770a.png\"},{\"id\":30895042,\"identity\":\"560cbea4-a423-4d63-85b5-0fc2ed8a1a1e\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 17:13:06\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3229412,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/2314229b-a0a1-4d43-96fd-58544cce5378.pdf\"},{\"id\":30893944,\"identity\":\"c2358e0a-1db5-4500-aaa5-5acc193d38e7\",\"added_by\":\"auto\",\"created_at\":\"2022-12-29 17:04:59\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":731957,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementalinformationwithfigurelegends.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2401099/v1/b6e9deee67ea490107e9bd12.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Estrogen Receptor-positive Breast Cancer Cells are Sensitized by Piperine to Chemo/Radio Therapy through Lowering the expression of a NHEJ repair protein DNA-PK\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eGamma radiation produces extremely lethal DNA double-stranded breaks (DSBs), inducing the cells to undergo apoptosis (\\u003cspan additionalcitationids=\\\"CR2 CR3\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). These therapies mainly target genomic DNA, subsequently resulting in the formation of highly toxic DSBs inducing cell cycle arrest and cell death (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). However, these treatments are initially effective in arresting tumor growth and reducing tumor burden, but eventually, it leads to resistance and disease progression (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e). Increased cellular DNA repair is one of the major mechanisms underlying this drug resistance, and various DNA repair pathways are upregulated to combat DNA damage. In mammals, DSBs are repaired mainly by the error-prone, nonhomologous end-joining (NHEJ) repair pathway (\\u003cspan additionalcitationids=\\\"CR8 CR9 CR10\\\" citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e). NHEJ initiation occurs when the ring-shaped Ku heterodimer, composed of the Ku70 and Ku80 proteins, identifies and binds to the target DSB in a sequence-independent manner and upon binding to the DSB, Ku70/ 80 recruits DNA-PKcs to the DNA ends directly to form the active DNA-PK complex, which results in the activation of the catalytic subunit, DNA-PKcs\\u0026rsquo;s (kinase activity) which further processes ligation through DNA ligase IV along with additional NHEJ machinery proteins such as XRCC4, Artemis, and, XLF (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eEnhanced activity of DNA-PKcs is observed in most of the cancer types such as hepatocellular carcinoma, oral squamous cell carcinoma, colorectal cancer, etc., which results in the error-prone mediated repair of DNA resulting in the survival of cancer cells culminating in cancer progression(\\u003cspan additionalcitationids=\\\"CR13\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e). Increased DNA-PKcs activity leads to enhanced DNA repair and reduced sensitivity to genotoxic and radio- or chemotherapies by the cancer cells leading to therapy resistance and poor patient survival (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eRecent studies demonstrate the existence of crosstalk mechanisms between the DNA repair machinery and estrogen hormone signaling pathways cooperating to influence cancer progression and therapeutic response (\\u003cspan additionalcitationids=\\\"CR18\\\" citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). Estrogen signaling is a balance among two conflicting functions exerted by two different receptors (ERα \\u0026amp; ERβ) as well as their splice variants (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). ERα is reportedly involved in oncogenesis and development of progression, invasion, and metastasis of cancer (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). On the contrary, ERβ suppresses cancer progression and induction of apoptosis(\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Few studies reported that the DNA-PKcs complex component, Ku70, complexes with ERα, assisting the transcriptional function of ERα, and further it transcriptionally activates DNA-PKcs (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe possibilities that the estrogen signaling (ERα \\u0026amp; ERβ mediated) pathways can be selectively modulated with selective estrogen receptor modulating compounds (SERMs) from plants add up to new and promising therapeutic opportunities (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). Thus, we hypothesize that targeting the molecular machinery driving the DNA repair pathways and apoptosis in cancer cells through ER modulation may lead to a novel elevated sensitivity to DNA damaging agents like ionizing radiation, making cancer cells sensitive to radiation and other chemotherapeutic agents. Thus, incorporating a SERM along with DNA-targeted therapy in ER-positive cancer types may result in enhanced sensitization of cells to DNA-damaging agents. In addition, based on the results, we anticipate that the use of spices such a s pepper during radio/chemotherapy may enhance the sensitivity of ERα positive cancer cells towards DNA damaging agents without effecting normal cells. Thus this study offers a new stratergy for combating therapy resistance by indirectly targeting NHEJ repair pathway through direct estrogen receptor modulation.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003eThe phytocompound piperine (\\u0026gt;\\u0026thinsp;97%), Bazidoxefene, and dimethyl sulphoxide (DMSO) were procured from Sigma-Aldrich, India. Cell culture reagents such as Dulbecco\\u0026rsquo;s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin solution, L-glutamine were procured from Gibco, Thermofischer. Antibodies were procured from either CST or Abcam. Trypan blue dye, MTT was procured from HiMedia, India. All other chemicals, Reagents, and other lab accessories were all analytical or molecular grade and were procured from reliable commercial sources.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell culture\\u003c/h2\\u003e \\u003cp\\u003eTo decide if the radiosensitization effects of piperine was restricted to ER positive breast cancers appropriate cell lines were used. Human breast adenocarcinoma cell lines; MCF7 ( an ER positive cell line), MDA-MB-231, and MDA-MB-468, (ER negative cell lines) were procured from National Centre for Cell Science (NCCS), Pune, India. The cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and 2 mM L-glutamine. The cell culture was maintained at 37\\u003csup\\u003eo\\u003c/sup\\u003eC in a humidified incubator containing 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePiperine, cisplatin, and γ-radiation treatment on MCF7\\u003c/h2\\u003e \\u003cp\\u003eTo optimize the dose of piperine, cisplatin, and γ-radiation to be used in combination treatment against breast cancer cell line MCF7, a cell proliferation assay with varying doses of these agents was studied For radiation experiment, MCF7 cells were seeded in T-12.5/ T-25 flasks at a density of 3x10\\u003csup\\u003e5\\u003c/sup\\u003e/ 5x10\\u003csup\\u003e5\\u003c/sup\\u003e cells/ flask and cultured for 24 h. The optimum dose of piperine, cisplatin, and gamma radiation to be used for the combination experiment were determined by treating the cells at various concentrations of the compounds/ γ- radiation. The cells exposed to the vehicle (DMSO) served as the control. For combination treatment cells were exposed to the selected dose of piperine and or cisplatin for 2 h. Following 2 h of post-treatment with the compounds, the cells were exposed to γ- radiation using Low Dose gamma Irradiator-2000 (BRIT, Mumbai, India) and then incubated for 48 h. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Dose optimization was determined by MTT assay and Trypan blue dye exclusion respectively (\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMorphological and Cell proliferation analysis\\u003c/h2\\u003e \\u003cp\\u003eCell proliferation and viability studies were done using MTT, trypan blue dye exclusion assay, and colony-forming assay. MCF7 cells were cultured in T12.5 flasks; under culture conditions for 24 h. Cells were treated with the selected concentration of the piperine and chemotherapeutic drug cisplatin alone or in combination and exposed to a single dose of γ-radiation with 60Co source as irradiator. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Images were collected using an inverted light microscope (Zeiss Primo Vert, Mumbai, India) and a ZOE imager (Bio-Rad, Mumbai, India). The cell proliferation was determined by trypan blue dye exclusion assay and MTT at 48 h of incubation after irradiation. Absorbance at 570 nm was done using a microplate reader (Fluostar Omega, BMG Labtech) and the percentage of cell growth relative to cells treated with vehicle alone was calculated.\\u003c/p\\u003e \\u003cp\\u003eCompuSyn analysis of the combination treatment\\u003c/p\\u003e \\u003cp\\u003eCompuSyn software (ComboSyn, Inc.USA) was used to quantitatively depict the effect of the combination treatment. The data from the cytotoxicity studies were taken to compute Combination Index (CI) values, CI plot, dose-response curve, median effect plot, and normalized isobologram. Combination Index values are less than one it indicates synergism (CI\\u0026thinsp;\\u0026lt;\\u0026thinsp;1), if Combination Index values are equal to one, it is an additive effect (CI\\u0026thinsp;=\\u0026thinsp;1) and if Combination Index values are greater than one, it represents antagonism (CI\\u0026thinsp;\\u0026gt;\\u0026thinsp;1) of the co-treatment (\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe combination index (CI) is calculated by using the formula: CI\\u0026thinsp;=\\u0026thinsp;d1/DA1\\u0026thinsp;+\\u0026thinsp;d2/DA2\\u003c/p\\u003e \\u003cp\\u003eWhere DA1 is the dose of γ-radiation required to decrease the 'x' percentage of proliferation alone, and d1 is the dose of γ-radiation required to decrease \\u0026lsquo;x\\u0026rsquo; percentage of proliferation in combination with piperine (d2). DA2 is similarly the dose of piperine required to decrease \\u0026lsquo;x\\u0026rsquo; percentage of proliferation alone, and d2 is the dose of piperine required to decrease \\u0026lsquo;x\\u0026rsquo; percentage of proliferation in combination with γ-radiation (d1).\\u003c/p\\u003e \\u003cp\\u003eClonogenic survival assays\\u003c/p\\u003e \\u003cp\\u003eTrypsinized MCF7cells were plated in 35 mm dishes at a density of 3x10\\u003csup\\u003e3\\u003c/sup\\u003e cells/ well and allowed to grow overnight as described previously. Piperine and cisplatin treatment at a concentration of 75 \\u0026micro;M and 3 \\u0026micro;M respectively and incubated for 2 h, followed by γ-radiation at a dose of 2 Gy. For the clonogenic assay, the cells were grown for 7 days and then stained with 0.1% Crystal violet for 1h and the photographs were captured and analyzed for confluency using Image J software (\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell cycle analysis\\u003c/h2\\u003e \\u003cp\\u003eMCF7 cells were seeded in T-12.5 flasks at a density of 3x10\\u003csup\\u003e5\\u003c/sup\\u003e cells/ flask and cultured for 24 h. The cells were then exposed to piperine and cisplatin/ 2 Gy of γ-radiation and then incubated for 24 h. Vehicle Control cells placed in the chamber but not irradiated served as a sham control. Trypsinized cells were washed with PBS and then fixed with 75% ethanol and incubated overnight at -20\\u003csup\\u003eo\\u003c/sup\\u003eC. Cells were washed twice with PBS and incubated with RNase A solution (50 \\u0026micro;g/ mL; Himedia), for 2 h at 37 \\u003csup\\u003eo\\u003c/sup\\u003eC followed by propidium iodide (5 \\u0026micro;g/mL; HiMedia) for 10 min in dark. Cell cycle analysis was performed using Guava EasyCyte (Millipore, India) (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCytokinesis-Blocked Micronucleus Cytome assay\\u003c/h2\\u003e \\u003cp\\u003eAfter incubation of MCF7cells with combination treatment for 48 h, Cytochalasin-B (Cyto-B) (Sigma) was added to cells and incubated for 24 h onto glass slides, air-dried for 10 min, fixed, and was further stained using Giemsa for scoring. A total of 50 cells, including mononucleated and binucleated (BN), were counted per slide. The data from these cell counts were used to determine the DNA damage biomarkers. The methodology used to score and calculate DNA damage biomarkers was previously described (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGamma-H2AX assay\\u003c/h2\\u003e \\u003cp\\u003eThe assay was performed according to the previously described protocol (\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e). Briefly, Cells (1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells/ dish) were seeded on poly-lysine coated 11 m glass coverslip in 35 mm culture dishes. After treatment and incubation, cells were given PBS (1X) wash, followed by using 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 30 min. The samples were blocked for 1 h with 1% BSA followed by incubation with Alexa Fluor 488-conjugated phospho-histone-γ-H2AX antibody (Ser139) (BD Biosciences) overnight at 4 \\u003csup\\u003eo\\u003c/sup\\u003eC. Samples were washed 3 times for 5 min in PBS. For confocal imaging, slides were prepared with DABCO/ PI mounting. DABCO/PI mounting medium prepared by dissolving 1.25 g DABCO powder in 45 mL glycerol at 37\\u003csup\\u003eo\\u003c/sup\\u003eC or 8 h. Once dissolved 5 mL PBS (pH 8.6) was added, and then followed by the addition of PI at 0.00002% as the final concentration. The cells stained with DABCO/ PI were then analyzed under Zoe imager/ Confocal microscope (Carl Zeiss, G\\u0026ouml;ttingen, Germany) with a 60\\u0026times; objective (oil immersion, aperture 1.3). Fifty nuclei from each experiment were counted and evaluated. Images were processed using Image J software and the corrected total cell fluorescence (CTCF) was evaluated using the formula.\\u003c/p\\u003e \\u003cp\\u003e\\\"CTCF =(Integrated Density-(Area of selected cell X Mean fluorescence \\\"\\u003c/p\\u003e \\u003cp\\u003e\\\"of background readings))\\\"\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescence assay\\u003c/h2\\u003e \\u003cp\\u003eCells were grown on 35 mm dishes for 24 h and were fixed with 4% PFA at room temperature for 30 min after piperine, cisplatin, and radiation treatment for 48 h. Then, the slides were permeabilized with PBS containing 0.2% Triton-X at room temperature for 30 min and blocked for 30 min at 4\\u0026deg;C with blocking buffer containing 2.5% bovine serum albumin and 0.2% Triton-X in PBS. Cells were incubated with DNA-PKcs, ERα, and β antibodies (1:500) overnight and then washed with PBS twice and incubated with Goat Anti-Rabbit IgG H\\u0026amp;L (Alexa Fluor488 (1:1000) at room temperature for another 1 h. After staining with DABCO/PI in the dark for 30 min, cells were observed under a ZOE imager (Biorad)(\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunoblot assay\\u003c/h2\\u003e \\u003cp\\u003eImmunoblots of MCF7 cells treated with piperine (75 \\u0026micro;M), alone or in a combination of cisplatin (3 \\u0026micro;M) or and γ-radiation (2 Gy) for 24 h were performed using the standard protocol. Protein lysates were prepared, post 24 h of radiation after washing the trypsinized cells with phosphate-buffered saline (PBS) and treating with extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X, 1 mM EDTA, 0.5% Tween 20, 0.1% SDS), protease inhibitor cocktail (10 \\u0026micro;L/ mL) for 30 min, followed by centrifugation at 12000 rpm, for 20 minutes at 4\\u003csup\\u003eo\\u003c/sup\\u003eC. The protein samples were quantified using the Bradford method where bovine serum albumin (BSA) was taken as a standard. Protein samples (30 \\u0026micro;g) were run in a 7\\u0026ndash;12% SDS gel followed by transfer to polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membranes were blocked using 4% BSA and further incubated with specific primary antibodies such as Bax, BCL-2, Cleaved caspase 3, Cleaved caspase 9, and Cleaved PARP-1 for checking the expression of apoptosis proteins, Ku70 and Ku80, and DNA Ligase IV for checking the expression of Non-homologous end-joining DNA repair proteins and p53, Phospho p53 and ATM for checking the expression of DNA damage response and ERα and ERβ for studying the receptor-mediated signaling in various treatment groups. Primary antibody incubation was done overnight at 4\\u003csup\\u003eo\\u003c/sup\\u003eC. The unbound antibody was washed off using PBST for 30 min with buffer change every 10 min. Further, the blots were incubated with a secondary antibody anti-rabbit-HRP) for 1 h at RT on a shaker. The unbound antibody was washed off using PBST for 30 min by changing the buffer every 10 min. The blots loaded with beta-actin served as the loading control for normalizing the protein level. The membrane was developed by ChemDoc chemiluminescence method using ECL solution to detect the signal. The bands were quantified by densitometric analysis using Image J software 2.0. The quantified bands were normalized with their respective β-actin levels (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMeasurement of reactive oxygen species\\u003c/h2\\u003e \\u003cp\\u003eMCF7cells were plated at a density of 0.35x 10\\u003csup\\u003e6\\u003c/sup\\u003e in 12.5 cm2 tissue culture flasks and incubated overnight. Pre-treated with piperine, cisplatin, alone or in combination for 2 h and irradiated the relevant flasks with 2 Gy. Intracellular ROS levels were detected after 24 h of radiation by flow cytometry using 2\\u0026acute;, 7\\u0026acute;-dichlorofluorescein diacetate (DCFDA; Sigma Aldrich, India) at a final concentration of 10 \\u0026micro;M for 15 min at 37\\u0026deg;C. For positive gating, cells were treated with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e at a concentration of 1 mM and harvested after 4 h of incubation at 37\\u003csup\\u003eo\\u003c/sup\\u003eC. The fluorescence intensity was measured at an absorption wavelength of 480 nm and an emission wavelength of 525 nm using a flow cytometer (\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMitochondrial membrane potential (ΔΨm) assay\\u003c/h2\\u003e \\u003cp\\u003eMCF7 cells seeded at a density of 0.35x 10\\u003csup\\u003e6\\u003c/sup\\u003e in 12.5 cm2 tissue culture flasks were incubated overnight at 37\\u003csup\\u003eo\\u003c/sup\\u003eC. A pre-treatment of piperine, cisplatin alone, or in combination was given 2 h before radiation treatment and incubated for 24 h at 37\\u003csup\\u003eo\\u003c/sup\\u003eC. Post 24 h radiation, the cells were harvested and change in the ΔΨm was assessed using JC-1 dye (Calbiochem, Cat.#. 420200, India). The cells treated with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e were taken for positive gating. The fluorescence intensity of both JC-1 green monomers and red JC-1 aggregates (JC- 1 fluorescence intensity falls λex\\u0026thinsp;=\\u0026thinsp;580 nm, λem\\u0026thinsp;=\\u0026thinsp;595) was recorded using a flow cytometer(\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMultiple ligand pharmacophore modeling\\u003c/h2\\u003e \\u003cp\\u003eThe pharmacophore of piperine and other standard estrogen receptor agonists/antagonists was developed using the ligand-based pharmacophore modeling in the Phase module of maestro 11.0. Hydrogen bond (HB) donors (D), HB acceptors (A), aromatic ring (R), hydrophobic/non-polar groups (H), and positive/negative ionizable groups were selected as the pharmacophore features for the modeling(\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e). The standard agonists/antagonists with promising fitness scores and structural similarity were selected for further processes.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular docking studies\\u003c/h2\\u003e \\u003cp\\u003eThe molecular docking studies were performed using Schrodinger suite 11.0. The three-dimensional structures of ERα in its agonistic conformation (PDB ID 1X7R) and antagonistic conformation (PDB ID 1ERR), and ERβ (1X7J) were downloaded from the protein data bank. The downloaded protein structures were processed and energy minimized using the protein preparation wizard. The protein preparation involves assigning bond orders, adding hydrogens, creating disulfide bonds, and creating zero-order bonds to metals. The HET states of the proteins were generated using \\u0026lsquo;Epik\\u0026rsquo; at pH 7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.0 and restrained minimization was performed using OPLS3 forcefield (\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e). Further, a receptor grid was generated from each structure by selecting the co-crystal ligand as the centroid of the receptor. Similarly, the ligand molecules such as piperine, estradiol, genistein, and bazedoxifene were downloaded from the NCBI-Pubchem database and prepared for docking with the ligprep module. The ligand molecules were structurally optimized at near-neutral pH (7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1), and subjected to energy minimization using OPLS3 force field.\\u003c/p\\u003e \\u003cp\\u003eEventually, the prepared ligand sets were docked against the receptor grid of the above-mentioned target proteins. Extra precision (XP) flexible docking was performed using the GLIDE module and the affinity of the ligands towards the target proteins were ascertained in terms of negative glide score (kCal/mol). 50 docking poses were collected for each ligand. The binding poses were generated using pymol software (free license).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eAll assays were repeated three times and triplicate results are shown. Data were represented as Mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. Data analysis was done using one-way ANOVA and two-way ANOVA (for cell cycle analysis), where a p-value of \\u0026lt;\\u0026thinsp;0.0001 was considered significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePiperine sensitizes breast cancer cells towards γ-radiation treatment through a synergistic mechanism\\u003c/h2\\u003e \\u003cp\\u003ePiperine, cisplatin, and γ-radiation treatment significantly inhibited cell growth in a dose-dependent manner (Figure S1). To assess the combinatory effect of piperine, cisplatin, and γ-radiation, we have selected a dose less than the IC\\u003csub\\u003e30\\u003c/sub\\u003e value (a dose of individual treatment which kills 25\\u0026ndash;30% of cells). Thus, 75 \\u0026micro;M of piperine, 3 \\u0026micro;M of cisplatin, and 2 Gy of γ-radiation were selected for the combination study. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, there is no significant proliferation inhibition or morphology changes in treatment with the selected doses of piperine or cisplatin (Cis) or ionization radiation (IR) irradiation alone. However, cell proliferation was severely inhibited, and morphology was affected due to a combination of piperine, cisplatin, and γ irradiation. A combination of a piperine dose of 75 \\u0026micro;M along with a cisplatin dose of 3 \\u0026micro;M or γ-radiation (2 Gy) resulted in cell growth inhibition rates of 45\\u0026ndash;55% at 48 h-7 days. These results demonstrate that piperine treatment, along with cisplatin and γ-radiation, could inhibit the proliferation of MCF7. Similarly, enhanced proliferation inhibition was observed in another estrogen-responsive cell line, HT-29 (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). However, in estrogen receptor-negative cell lines MDA-MB-468 and MDA- MB-231, the combination treatment did not show significant cell death (Figure S1). To check whether the observed enhanced cytotoxicity of the combination treatment is synergistic or additive, we calculated the CI values and generated a CI plot for the equi-effective dose of the piperine and cisplatin against γ-radiation using CompuSyn software. A dose-response curve for individual test agents was plotted (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). As shown in Figure. 1f, the calculated CI value is less than 1 for the selected dose used in the combination treatment comprising piperine with the γ-radiation demonstrating a synergistic or sensitization effect. Taken together, these results show that pre-treatment with piperine effectively sensitized breast cancer cells to the cytotoxic effects of γ-radiation, compared to the individual treatment alone. Thus piperine may be beneficial during chemo-radiotherapy.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003ePiperine augmented IR/Cis induced ROS accumulation leading to DNA damage.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo assess whether the synergistic effect of the combination treatment was mediated through Reactive oxygen species (ROS) generation, we evaluated the intracellular generation of ROS by flow cytometry (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea and b). The cells were treated with all three agents either as a single agent or in combination with induced ROS. As expected, combination treatment generated a higher amount of ROS compared with that in cells treated with a single regime. The assay suggests that the enhancement in the cisplatin/radiation-induced cytotoxicity by piperine may be associated with increased ROS levels.\\u003c/p\\u003e \\u003cp\\u003eSince the intracellular generation of ROS is associated with DNA damage, we assessed the extent of genotoxicity by performing a Cytochalasin-B-blocked micronuclei assay. We observed a significant increase in the nuclear bud, micronuclei formation, and a nucleoplasmic bridge in the combination treatment indicating that piperine augmented the radiation-induced double-stranded DNA damage (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec and d). Since phosphorylation of histone H2AX at Ser139 is a dominant response to DSB, resulting in the formation of an increased number of structures known as ionizing radiation-induced foci (IRIF) or γH2AX foci, we confirmed the DSBs by immunofluorescence sensing for γH2AX as well (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee and f).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003ePiperine treatment modulated p53/ATM-dependent DNA damage response inducing cell cycle arrest.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn response to DNA damage induced by chemo/ radiotherapy, p53 is overexpressed. While p53 was upregulated with IR and cisplatin, piperine treatment reduced its expression as a single agent and in its combination (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea \\u0026amp; b). Since p53 is an ERα regulated gene, its downregulation by piperine may be mediated through ER modulation. In response to DNA damage, p53 is stabilized and activated by phosphorylation at Ser15 by ATM. Interestingly we observed treatment-dependent upregulation of phospho-p53 and ATM (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, c, and d) in combination treatment with piperine confirming that piperine augmented the IR and cisplatin-induced DNA damage response. This enhanced appearance of DDR proteins in the combination treatment may be due to piperine-mediated downregulation of DNA repair. Interestingly, the expression pattern of all the DDR proteins we checked, γH2AX, phospho-p53, and ATM, was almost similar, confirming that piperine enhanced cisplatin/radiation-induced DNA damage.\\u003c/p\\u003e \\u003cp\\u003eCell cycle arrest is induced by p53 activation in response to DNA damage leading to DNA repair or apoptosis (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). Hence we analyzed the influence of piperine and its combination with cisplatin/ IR on the cell cycle status of MCF7 cells. As expected, cell cycle analysis showed a significant G2 M stall at 24 h for the combination of piperine, cisplatin, and γ-radiation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee and f). While piperine augmented the DNA damage and IR-induced G2/ M staling of cells in ER-positive breast cancer cells (MCF7), it failed to induce similar G2/ M arrest in ER-negative breast cancer cells (MDA-MB-468 and MDA-MB-231) (Figure S2). Besides, in ER-negative cells, piperine pretreatment reduced the IR-induced G2/ M arrest pointing towards an estrogen receptor-dependent sensitization. In conclusion, piperine augmented the DSBs induced by IR/ cisplatin treatment leading the cells to undergo G2/ M arrest in ER-positive cancer cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIdentification of piperine as a Selective estrogen receptor modulator of ER-alpha\\u003c/h2\\u003e \\u003cp\\u003eSince we observed that piperine-sensitized estrogen-responsive cells like MCF7 and HT-29 towards IR compared to estrogen receptor-negative cells, we were interested to know if it exhibits any estrogen receptor modulating effects. We analyzed the expression and nuclear translocation of ER subtypes in MCF7 cells with or without piperine treatment, and interestingly we found that piperine decreased the expression and nuclear translocation of ERα (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, b) and enhanced the expression and nuclear translocation of ERβ (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec, d) in a dose-dependent manner in MCF7 cells. These results point towards a receptor-specific modulation of signaling by piperine.\\u003c/p\\u003e \\u003cp\\u003eTo further understand the mechanism behind the observed estrogen receptor-modulating effects upon piperine treatment, we performed pharmacophore modeling of piperine with an established estrogen agonist and antagonist. Interestingly, we observed that piperine shares a common pharmacophore feature with most of the established estrogen receptor ligands. Hydrogen bond acceptors (A), aromatic ring (R), and hydrophobic/non-polar groups (H) are the predominant pharmacophore features that were found in common between piperine and several standard estrogen receptor agonists/antagonist (Table S1 and S2). From the pharmacophore modeling, two pharmacophore hypotheses, AAR and AHR (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee), were observed as the common feature among piperine and other estrogen receptor agonists.\\u003c/p\\u003e \\u003cp\\u003eIn Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee, piperine, as well as the standard estrogen receptor agonists/antagonists such as bazedoxifene, -a third-generation selective estrogen receptor modulator (SERM), estradiol- the natural ligand of estrogen, and many of the established SERMs, such as genistein, coumestrol, daidzein, portfolio, repensol, and formononetin, was found to possess the common pharmacophore feature AAR which gives a fitting score of 1.825 for piperine and ranges from 1.8 to 3 for other agonists. Besides, piperine has pharmacophore features AHR that can be best fitted with that of some of the SERM such as bazedoxifene, estradiol, cyclofenil, ormeloxifen, raloxifene, tamoxifen, toremifene, ospemifene, and lasofoxifene respectively. The fitness score of piperine for AHR pharmacophore was found as 1.87. It can be concluded that some pharmacophore features of piperine that can be found in common in other estrogen receptor agonists are key to mimicking the estrogen receptor agonistic activity. The hypothesis was further supported by molecular docking studies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef and g). We found that the key element of the conformational switch, the short helical region, the helix 12 (H12), which adopts a ligand-dependent conformation assumed an agonistic position for piperine (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). This conformation was similar to the natural ligand estradiol and the known agonist genistein (Figure S3 b) but with lower binding energy. Piperine exhibited comparable affinity towards the ligand-binding domain (LBD) of ERα in an agonistic conformation as the reference ligands (estradiol and genistein) used for validation of the docking study (Figure S3 a). The binding of piperine and the reference ligands (estradiol and genistein) in the active site of ERα exactly where the co-crystal ligand interacts. A detailed description of the active site residues involved in the interaction also reveals the involvement of several active site residues in common for stabilizing the binding of piperine and the reference ligands (Figure S3 b). Docking studies performed against ERα (1X7R) expressed a docking score of -8.977 kCal/mol with piperine. The docking score was \\u0026minus;\\u0026thinsp;11.155 kCal/mol and \\u0026minus;\\u0026thinsp;10.758 kCal/mol for estradiol and genistein, respectively. As expected from the results of pharmacophore modeling of piperin, the key pharmacophore feature \\u0026lsquo;aromatic ring (R)\\u0026rsquo; was found to involve in π-π (pi-pi) stacking interaction with Phe-404 present in the active site of ERα (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eg). Similar π-π stacking interaction can also be observed in the interaction diagram of estradiol and genistein in which the aromatic pharmacophore interacts with the ERα active site residue Phe 404 (Figure S3 b ). The result of the molecular docking studies supports the pharmacophore hypothesis. In addition to the π-π stacking interaction, hydrogen bonding and salt bridge interaction can also be observed in the case of estradiol and genistein as the major stabilizing interactions. Bazedoxifene adopted only antagonistic conformation within the LBD of ERα. Interestingly due to its small structure, piperine could also adopt antagonistic conformation, which is thermodynamically stable but with higher energy (Figure S3 c) than compared with piperine in its agonistic conformation.\\u003c/p\\u003e \\u003cp\\u003eThe docking studies of piperine and bazedoxifene with ERβ (PDB ID 1X7J) failed to give any output. The automated docking program was aborted for bazedoxifene/piperine and ERβ interaction because of the poor orientation of piperine and/ or the presence of bulky side chains of bazedoxifene with the receptor. However, estradiol and genistein interacted well with the active site of ERβ (Figure S3 d). Thus we assume that piperine is a weak agonist of ERα and competed with its natural ligand, estradiol, for the receptor interaction. This resulted in the reduced translocation of the activated ERα to the nucleus to exert its downstream signaling events. Thus, in conclusion, piperine interacts only with the LBD of ERα as a weak agonist and not with ERβ. This accounts for the decreased nuclear translocation observed for ERα and an increased translocation of activated ERβ to the nucleus. The results suggest that piperine may be used as complementary medicine for estrogen-positive cancer therapy. This also emphasizes the need to judiciously consuming food rich in Phyto-estrogens during chemo/radiotherapy.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003ePiperine down-regulates the expression of DNA PKcs, an estrogen-responsive gene, and other repair proteins of the NHEJ pathway.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe role of estrogen in the development and progression of breast cancer is demonstrated in several previous studies (\\u003cspan additionalcitationids=\\\"CR28\\\" citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e). The transcriptional activation and gene expression of the DNA-PKcs- catalytic subunit, one of the key proteins of the NHEJ pathway, are directly mediated by ERα in response to estrogen (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). NHEJ, the major DSB repair pathway upregulated during cancer therapy, imparts therapy resistance in cancers by mediating the error-prone repair conferring a survival advantage to cancer cells. Since piperine modulates estrogen receptors\\u0026rsquo; expression selectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, b, c, d), we assumed that it might modulate the expression of DNA-PKcs involved in the NHEJ machinery. To understand the role of piperine in modulating estrogen receptor expression and NHEJ proteins during chemo/radiotherapy, protein expression studies are carried out. The expression and translocation of estrogen receptors showed an increase in ERα and a decrease in the expression of ERβ in response to treatment. Interestingly enhanced translocation of activated ERα to the nucleus was observed in control and with radiation-treated cells (Figure S4 a and b). Piperine and cisplatin treatment, on the other hand, reduced the ERα nuclear translocation (Figure S4 c) and altered the ERα/ERβ ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea, b, and c).\\u003c/p\\u003e \\u003cp\\u003eThe expression of NHEJ proteins was also altered upon combination treatment. We observed enhanced expression of DNA-PKcs, Ku70, and Ku80 in the cells treated with radiation, cisplatin, and its combination. On the other hand, piperine drastically reduced the cisplatin/ IR-induced expression of these key NHEJ proteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed-i). DNA-PKcs, along with Ku70/ Ku80 heterodimer, form the DNA-PKcs complex, which initiates NHEJ. The expression of Ligase IV was also reduced in piperine treated group and also in combination treatment compared to radiation treatment alone.Interestingly we observed the same pattern of expression for DNA-PKcs, p53, Ku70, and Ku80 under similar experimental conditions. Since DNA-PKcs ad p53 is already established to be an ERα regulated gene, we speculate that the expression of Ku70 and Ku80 is also directly or indirectly regulated by ERα. Accordingly, the observed increase in DNA damage upon combination treatment may be due to a decrease in the NHEJ-mediated DNA repair. Overexpression of NHEJ proteins is known to confer therapy resistance, and its ablation confers radio/chemosensitization. Thus these results indicate that a complementary therapy with piperine or any weak estrogen receptor agonist or antagonist along with radio/chemo agents may reduce therapy resistance.Interestingly, as observed for piperine, bazedoxifene down-regulated the IR-induced expression of DNA-PKcs (Figure S4 e and f), confirming our postulation. To reconfirm this assumption, we treated MCF7 cell lines with the third-generation SERM, bazedoxifene, which shared common pharmacophore features with piperine. As expected, bazedoxifene failed to downregulate the expression of DNA-PKcs in estrogen non-responsive cell lines, MDA-MB 231, MDA-MB 468, and A549 (Figure S4 g-i). Hence it may be assumed that piperine or any selective estrogen receptor modulators (SERMs) may sensitize radiation-resistant estrogen-responsive cancers to radiation by downregulating the NHEJ proteins.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTreatment of Piperine in conjunction with radiation and cisplatin-induced mitochondrial pathway of apoptosis\\u003c/h2\\u003e \\u003cp\\u003eElevated intracellular ROS generated during co-treatment causes damage to DNA. Cells with DNA damage are arrested at the G2/ M phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea-d), eliciting DNA repair or cellular apoptosis. If DSBs are left unrepaired, apoptosis is elicited through the mitochondria-mediated intrinsic or extrinsic pathway. ROS can activate BAX\\u003c/p\\u003e \\u003cp\\u003eBCL2 Associated X](\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e), a key tumor suppressor protein involved in the intrinsic pathway of apoptosis. Interestingly co-treatment resulted in the up-regulation of BAX and down-regulation of BCL2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-f), the main molecular players eliciting mitochondrial membrane depolarization setting off apoptosis.\\u003c/p\\u003e \\u003cp\\u003eThe mitochondrial membrane potential was analyzed using JC-1 dye which is taken up by the mitochondria of only viable cells. JC-1 dye accumulation in mitochondria of viable cells is potential-dependent, indicated by a fluorescence emission shift from green (~\\u0026thinsp;529 nm) to red (~\\u0026thinsp;590 nm), measured using a flow cytometer as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eg and h, a decrease in red fluorescent J-aggregates with an increase in green fluorescent J-monomers, upon co-treatment points towards a depolarization of the membrane potential of mitochondria, which is an important physiological feature of apoptosis.\\u003c/p\\u003e \\u003cp\\u003eThe activities of caspase 9 and 3 were also enhanced (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-f) upon treatment, indicating that apoptosis was induced through intrinsic pathways. Cleavage of Poly(ADP-ribose) polymerase-1 (PARP-1) by caspases is considered as a hallmark feature of apoptosis. The analyses of PARP-1 cleavage showed that piperine enhances PARP-1 cleavage as a single regime and in combination. Together these results point towards the that piperine sensitizes estrogen-responsive cancer cells to radiation by augmenting the radiation-induced DNA damage via downregulating the DNA-PKcs complex, the key component of the NHEJ repair pathway inducing cells to G2/ M arrest leading to apoptosis.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIdentification of piperine as a modulator of estrogen receptor subtype (ERα/ERβ) expression and activation\\u003c/h2\\u003e \\u003cp\\u003eEstrogen signaling is a balance between two opposing functions exerted by two specific nuclear hormone receptors (ERα and ERβ) and their splice variants. They are the members of the ligand-activated transcription factors falling under the nuclear-hormone receptor (NR) superfamily (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). ERα promotes proliferation and confers invasive properties on cancer cells (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). In contrast, ERβ suppresses epithelial to mesenchymal transition and promotes apoptosis (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Interestingly, in our study, we found that the expression of ERα/ ERβ is altered by piperine. We found a downregulation in ERα expression with an upregulation in ERβ (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, b, c, d and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea, b), indicating that piperine might inhibit cell proliferation and induce apoptosis.\\u003c/p\\u003e \\u003cp\\u003eIn the classical or direct genomic estrogen signaling pathway, the nuclear estrogen receptors ERα and ERβ act as ligand-activated transcription factors regulating the expression of its target gene (\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). ERα/ ERβ binds to its ligand in the cytoplasm, inducing a conformational change resulting in receptor dimerization and activation of ligand-bounded receptors (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). The activated receptor-ligand complex is then translocated to the nucleus, where it binds to the ERE sequences at the enhancer regions within or close to promoters and/or 3'-untranslated regions of the target genes to initiate the transcription (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). We found a decrease in the nuclear translocation of ERα and an increase in the nuclear translocation of ERβ with piperine treatment in a dose-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, b, c, d), confirming that piperine is involved in the activation/deactivation and nuclear translocation of ERs.\\u003c/p\\u003e \\u003cp\\u003eOur results indicate that piperine shared common pharmacophore features with estradiol and established estrogen receptor agonists and antagonists (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee and Table S1 and S2). The ligand-binding domain (LBD) of ERα plays a prominent role in maintaining agonistic/ antagonistic conformation. LBD comprises a beta-hairpin and twelve α-helices (H1-H12), of which H12 plays a critical role in ligand-dependent activation of the receptor (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e). Agonist binding favors an active conformation, where H12 takes a position across H3 and H11 and supports co-regulator binding. Estradiol and genistein adopted only agonistic conformation (Figure S3 b), while raloxifene (the data is not shown) and bazedoxifene agreed to an antagonistic position (Figure S3 c). Piperine could interact well in the LBD of ERα in its agonistic conformation, and the interaction may be comparable to that of estradiol and genistein used for validating the docking results (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). The binding of the antagonist to the LBD causes H12 to shift from its agonistic position (Figure S4 a). This disrupts co-regulator binding and favors co-repressor binding inhibiting transcriptional activation. Due to its molecular structure and flexibility, piperine could also adopt an antagonistic conformation that is thermodynamically stable but with higher energy than compared with bazedoxifene, an estrogen receptor antagonist (Figure S3 c). This points out that piperine may favor agonistic conformation over antagonistic. Hence we assume that piperine may be a weak agonist of ERα. The in silico study is supported by in-vitro studies, where we found reduced expression of ERα (with piperine treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea and b) together with reduced nuclear translocation of ERα (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea and b and Figure S4 a and b) confirming the probability of piperine to be a weak estrogen receptor agonist. The ability of piperine to downregulate ERα and lower its activation may be because it is competing with the natural ligand estradiol for the LBD of ERα. On other hand, the available or displaced estradiol activates ERβ, enhancing its expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea and d) and activation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec and d and Figure S4 c and d). Since ERβ regulates the apoptotic and tumor suppressor proteins, its upregulation and activation by piperine may also be considered a promising therapeutic approach. Hence the potential of piperine, a key spice used in abundance by the Asian population in downregulating and suppressing ERα together with upregulation and activation of ERβ, points towards the judicial use of spices during cancer therapy.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDownregulation of ERα by SERMS as a novel strategy for sensitizing cancers to DNA-damaging therapies\\u003c/h2\\u003e \\u003cp\\u003eThough the concept of either suppressing ERα or activation of ERβ is acknowledged as a promising clinical approach towards the management and prevention of cancers, their modulation was never reported to sensitize cancers towards DNA damaging therapies(\\u003cspan additionalcitationids=\\\"CR18\\\" citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). Thus this is the first study introducing the proof of concept that ERα downregulation by SERMs sensitizes cancers to DNA damaging agents (here, platinum-based chemo drug; cisplatin and radiotherapy), and this strategy may help to overcome therapy resistance.\\u003c/p\\u003e \\u003cp\\u003eIn our study, we found that piperine downregulates the expression of p53, Ku70, Ku80, and DNA-PKcs in ER-positive breast cancer cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea and b, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed, e f, h and i). The p53 is a tumor suppressor gene that participates in DNA damage response, and Ku70, and Ku80, along with DNA-PKcs are DNA repair proteins that play a critical role in initiating NHEJ (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). While the expression of p53 and DNA-PKcs are transcriptionally regulated by ERα, Ku70/ 80 are stabilized by ERα binding (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). Also, we observed that while piperine treatment downregulated p53 expression, its phosphorylation/ activation was enhanced with piperine. And p53 is stabilized and activated by phosphorylation at Ser15 by ATM, in response to DNA damage (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR40\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). Thus the treatment-dependent upregulation of DDR proteins γH2AX, phospho-p53, and ATM in combination treatment with piperine (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e and \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) confirmed that piperine amplified the IR/ cisplatin-induced DNA damage response. This correlated with the downregulation of ERα-regulated proteins- p53 and the NHEJ repair protein.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eManaging therapy resistance in estrogen-responsive cancers by Downregulating DNA-PKcs, a key component of NHEJ machinery through its transcription modulation\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eNHEJ repair is error-prone and it confers new mutations to the cancer cells favoring their proliferation and invasion (\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e). NHEJ proteins, DNA-PKcs, Ku70, Ku80, and Ligase-IV were upregulated in the majority of the cancers (\\u003cspan additionalcitationids=\\\"CR44 CR45\\\" citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e). Ablation or silencing of one or more NHEJ repair proteins is reported to sensitize cancers to ionizing radiation (\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e). The DNA-PKcs is known to confer resistance to DNA targeted therapies such a platin-based drugs and radiation through promoting error-prone DNA repair (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Piperine augmented the IR/ cisplatin-induced DNA damage by downregulating the NHEJ proteins; DNA PKcs, Ku70 and Ku80 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed, e, f, h and i).\\u003c/p\\u003e \\u003cp\\u003eConcurrently, we also observed a decrease in the expression and activation of ERα (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e and b), a DNA-PKC transcription regulator. ERα is also binding partner of Ku70/ Ku80 and stabilizes the protein. Thus we assume that treatment with an ERα weak-agonist, may reduce the ligand dependent activation and function of the receptor effecting the expression and stabilization of the key NHEJ factors. This in turn sensitizes cancer cells to DNA-damaging therapeutics and also may help in overcoming therapy resistance. As proof of our concept, we used bazedoxifene a third-generation SERM, along with cisplatin/ ionizing radiation therapy, and the results were comparable to piperine.\\u003c/p\\u003e \\u003cp\\u003eCell cycle arrest is a prominent response to p53 activation (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). The p53 may be activated by phosphorylation or acetylation (\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e). Here we found that enhanced phosphorylation of phospho-p53 in combination treatment with piperine corresponded to the arrest of cells at the G2/ M phase of the cell cycle (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, b, e and f). Phospho-p53 induced cell-cycle arrest is reversible if DNA breaks are repaired (\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e). We found a reduction in the DSB repair proteins and enhancement in DNA damage upon piperine co-treatment indicating that piperine-induced cell cycle arrest may lead to apoptosis .\\u003c/p\\u003e \\u003cp\\u003eApoptosis is a dynamic cellular death program induced by endogenous or exogenous factors for the removal of damaged cells. Apoptosis was in-turn confirmed by checking for apoptotic markers and important molecular events. In addition to DNA damage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec, d, e and f), ROS generated during treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea and b) causes a change in protein expression, damage to cell membranes, mitochondria, etc., initiating apoptosis. ROS activates Bax [BCL-2 Associated X], a key tumor suppressor protein involved in the intrinsic pathway of apoptosis (\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e). Interestingly the combination treatment altered the BAX/BCL-2 levels, eliciting mitochondrial membrane depolarization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea, b, c, g and h). The mitochondrial membrane stability was measured and combination treatment was found to depolarize the membrane. Besides, there was enhanced expression of the initiator and effector caspase-9, and caspase-3, respectively, and the cleavage of PARP-1, a cellular substrate of caspases, confirming apoptosis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea, d, e and f). Similar results were observed in our previous studies on HT-29 cells (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). Thus as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e we assume that piperine sensitizes ER-positive cells towards DNA damaging agents by downregulating the NHEJ-DSB repair through suppressing ERα activation and enhancing ERβ, thus inducing cells to the intrinsic pathway of apoptosis.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eDNA-PK complex comprising of DNA-PKcs/ Ku70/ Ku80 are the major proteins known to be involved in initiating NHEJ pathway and repair of DSBs. Here we found that the treatment of MCF7 cells with piperine, lead to the accumulation of DSBs induced with gamma irradiation through lowering the DNA-PK complex through altering the ERα/ ERβ ratio. The elucidated mechanism of action of the piperine is summarized in the illustration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). We found that piperine shared common pharmacophore features with selective estrogen receptor modulators (SERMs) and interacted with ERα, but failed to interact with ERβ. Piperine may be a weak agonist of ERα and competed with its natural ligand, estradiol for the receptor interaction, resulting in the reduced expression and translocation of the activated ERα to the nucleus to exert its downstream signaling events. Piperine downregulated NHEJ through DNA-PKcs modulation mediated through its transcription factor, estrogen receptor alpha (ERα). On other hand, overexpression of ERβ, a nuclear hormone transcription factor promoting tumor suppression positively correlated with lowered expression of ERα and DNA-PK complex, marked by the accumulation of radiation-induced DSBs leading to cell cycle arrest inducing intrinsic pathway of apoptosis. We also used bazedoxifene a third-generation SERM, along with cisplatin/ionizing radiation therapy and the results were comparable to piperine. Thus suppression of ERα activation through SERMs may be used to target NHEJ when co-administered with platinum-based chemo/ radiotherapy and this offers a novel strategy towards management and prevention of cancers and improvement of existing regimens. Thus this is the first study introducing the concept that estrogen receptor (ERα) downregulation sensitizes cancers to DNA damaging agents and helps to overcome therapy resistance.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eCI\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eCombination index\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eDSB\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eDNA double-stranded break\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003edsDNA\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003edouble stranded DNA\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eErα\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eEstrogen receptor alpha\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eERβ\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eEstrogen receptor\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eNHEJ\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eNon-homologous End Joining\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eROS\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eReactive oxygen Species\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate:\\u0026nbsp;\\u003c/strong\\u003eNot Applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication:\\u0026nbsp;\\u003c/strong\\u003eNot Applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials :\\u0026nbsp;\\u003c/strong\\u003eData sharing is not applicable to this article as no datasets were generated or analysed during the current study\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests:\\u0026nbsp;\\u003c/strong\\u003eAuthors hereby declare that they don\\u0026rsquo;t have any conflicting interests to declare\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDLM is supported by Start-up grant (YSS/2015/000987), SERB, DST, Govt. of India and Seed Grant (YU/Seed grant/065-2018) from Yenepoya (deemed to be University). KS is supported by ICMR-SRF, Govt. of India.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDLM conceptualized the study, designed the experiments, analyzed and interpreted the results. KS performed the in-vitro experiments and analyzed the data. SP assisted KS with few \\u0026nbsp;in-vitro experiments. HSA performed the in-silico experiments. DLM and KS wrote the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors would like to thank Prof. Somashekarappa H M, Director, Centre for Application of Radioisotopes and Radiation Technology (CARRT), Mangalore University, Karnataka, India, for providing \\u0026gamma;-radiation facility.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eKeta O, Petković V, Cirrone P, Petringa G, Sakata D, Shin W, et al. DNA double-strand breaks in cancer cells as a function of proton linear energy transfer and its variation in time. Int J Radiat Biol sInternet]. Taylor \\u0026amp; Francis; 2021;0:1\\u0026ndash;12. Available from: https://doi.org/10.1080/09553002.2021.1948140\\u003c/li\\u003e\\n\\u003cli\\u003eRoos WP, Kaina B. DNA damage-induced apoptosis : From specific DNA lesions to the DNA damage response and apoptosis. CANCER Lett [Internet]. Elsevier Ireland Ltd; 2012; Available from: http://dx.doi.org/10.1016/j.canlet.2012.01.007\\u003c/li\\u003e\\n\\u003cli\\u003eSantivasi WL, Xia F. Ionizing Radiation-Induced DNA Damage, Response, and Repair. 2014;21. \\u003c/li\\u003e\\n\\u003cli\\u003eVignard J, Mirey G, Salles B. Ionizing-radiation induced DNA double-strand breaks : A direct and indirect lighting up. Radiother Oncol [Internet]. Elsevier Ireland Ltd; 2013;108:362\\u0026ndash;9. Available from: http://dx.doi.org/10.1016/j.radonc.2013.06.013\\u003c/li\\u003e\\n\\u003cli\\u003eFrosina G. DNA repair and resistance of gliomas to chemotherapy and radiotherapy. Mol Cancer Res. 2009;7:989\\u0026ndash;99. \\u003c/li\\u003e\\n\\u003cli\\u003eChen Y, Zhao Y, Yang X, Ren X, Huang S, Gong S, et al. USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma. Nat Commun. Springer US; 2022;13. \\u003c/li\\u003e\\n\\u003cli\\u003eChang HHY, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol [Internet]. Nature Publishing Group; 2017;18:495\\u0026ndash;506. Available from: http://dx.doi.org/10.1038/nrm.2017.48\\u003c/li\\u003e\\n\\u003cli\\u003eScully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol [Internet]. Springer US; Available from: http://dx.doi.org/10.1038/s41580-019-0152-0\\u003c/li\\u003e\\n\\u003cli\\u003eFu YW, Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, et al. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res. Oxford University Press; 2021;49:969\\u0026ndash;85. \\u003c/li\\u003e\\n\\u003cli\\u003eChen X, Xu X, Chen Y, Fox T, Gellert M, Yang W. Article Structure of an activated DNA-PK and its implications for NHEJ ll ll Article Structure of an activated DNA-PK and its implications for NHEJ. Mol Cell [Internet]. Elsevier Inc.; 2021;81:801-810.e3. Available from: https://doi.org/10.1016/j.molcel.2020.12.015\\u003c/li\\u003e\\n\\u003cli\\u003eBayley R, Borel V, Moss RJ, Sweatman E, Ruis P, Ormrod A, et al. H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ. Mol Cell [Internet]. The Author(s); 2022;82:1924-1939.e10. 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YSRAO [Internet]. Elsevier Inc.; 2010;20:217\\u0026ndash;22. Available from: http://dx.doi.org/10.1016/j.semradonc.2010.06.003\\u003c/li\\u003e\\n\\u003cli\\u003eShaheer K, Somashekarappa HM, Lakshmanan MD. Piperine sensitizes radiation-resistant cancer cells towards radiation and promotes intrinsic pathway of apoptosis. J Food Sci. 2020;85:4070\\u0026ndash;9. \\u003c/li\\u003e\\n\\u003cli\\u003eTanbridge ERICJS. DNA damage and p53-mediated cell cycle arrest : A reevaluation. 1996;93:15209\\u0026ndash;14. \\u003c/li\\u003e\\n\\u003cli\\u003eBurns KA, Korach KS. Estrogen receptors and human disease: An update. Arch Toxicol. 2012;86:1491\\u0026ndash;504. \\u003c/li\\u003e\\n\\u003cli\\u003eDeroo BJ, Korach KS. Estrogen receptors and human disease. J Clin Invest. 2006;116:561\\u0026ndash;70. \\u003c/li\\u003e\\n\\u003cli\\u003eSadlack B, K\\u0026uuml;hn R, Schorle H, Rajewsky K, M\\u0026uuml;ller W, Horak I. 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Cold Spring Harb Perspect Med. 2016;6:1\\u0026ndash;16. \\u003c/li\\u003e\\n\\u003cli\\u003eNaseri MH, Mahdavi M, Davoodi J, Tackallou HS, Goudarzvand M, Neishabouri SH. Up regulation of Bax and down regulation of Bcl2 during 3-NC mediated apoptosis in human cancer cells. Cancer Cell Int [Internet]. Cancer Cell International; 2015;15:1\\u0026ndash;9. Available from: http://dx.doi.org/10.1186/s12935-015-0204-2\\u003c/li\\u003e\\n\\u003cli\\u003eProtocol B. Trypan Blue Exclusion Test of Cell Viability. 1997;2\\u0026ndash;3. \\u003c/li\\u003e\\n\\u003cli\\u003eMeerloo J Van, Kaspers GJL, Cloos J. Cell Sensitivity Assays : The MTT Assay. 731:237\\u0026ndash;45. \\u003c/li\\u003e\\n\\u003cli\\u003eChou TC. Drug combination studies and their synergy quantification using the chou-talalay method. Cancer Res. 2010;70:440\\u0026ndash;6. \\u003c/li\\u003e\\n\\u003cli\\u003eChou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27\\u0026ndash;55. \\u003c/li\\u003e\\n\\u003cli\\u003eGuzm\\u0026aacute;n C, Bagga M, Kaur A, Westermarck J, Abankwa D. ColonyArea: An ImageJ plugin to automatically quantify colony formation in clonogenic assays. PLoS One. 2014;9:14\\u0026ndash;7. \\u003c/li\\u003e\\n\\u003cli\\u003eRajendran V, Jain MV. Chapter 8. 2018;1692:89\\u0026ndash;95. \\u003c/li\\u003e\\n\\u003cli\\u003eFenech M. The in vitro micronucleus technique. Mutat Res - Fundam Mol Mech Mutagen. 2000;455:81\\u0026ndash;95. \\u003c/li\\u003e\\n\\u003cli\\u003eNagelkerke A, Kuijk SJA Van, Sweep FCGJ, Nagtegaal ID, Hoogerbrugge N, Martens JWM, et al. Constitutive expression of c -H2AX has prognostic relevance in triple negative breast cancer. Radiother Oncol [Internet]. Elsevier Ireland Ltd; 2011;101:39\\u0026ndash;45. Available from: http://dx.doi.org/10.1016/j.radonc.2011.07.009\\u003c/li\\u003e\\n\\u003cli\\u003eRen G, Sha T, Guo J, Li W, Lu J. Cucurbitacin B induces DNA damage and autophagy mediated by reactive oxygen species ( ROS ) in MCF-7 breast cancer cells. J Nat Med. Springer Japan; 2015; \\u003c/li\\u003e\\n\\u003cli\\u003eGallo-Oller G, Ordo\\u0026ntilde;ez R, Dotor J. A new background subtraction method for Western blot densitometry band quantification through image analysis software. J Immunol Methods [Internet]. Elsevier B.V; 2018;457:1\\u0026ndash;5. Available from: https://doi.org/10.1016/j.jim.2018.03.004\\u003c/li\\u003e\\n\\u003cli\\u003eFigueroa D, Asaduzzaman M, Young F. Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2\\u0026prime;,7\\u0026prime;\\u0026ndash;dichlorofluorescin diacetate (DCFDA) assay. J Pharmacol Toxicol Methods [Internet]. Elsevier Inc; 2018;94:26\\u0026ndash;33. Available from: https://doi.org/10.1016/j.vascn.2018.03.007\\u003c/li\\u003e\\n\\u003cli\\u003eFauzi AN, Norazmi MN, Yaacob NS. Tualang honey induces apoptosis and disrupts the mitochondrial membrane potential of human breast and cervical cancer cell lines. Food Chem Toxicol [Internet]. Elsevier Ltd; 2011;49:871\\u0026ndash;8. Available from: http://dx.doi.org/10.1016/j.fct.2010.12.010\\u003c/li\\u003e\\n\\u003cli\\u003eGao Q, Wang Y, Hou J, Yao Q, Zhang J. Multiple receptor-ligand based pharmacophore modeling and molecular docking to screen the selective inhibitors of matrix metalloproteinase-9 from natural products. J Comput Aided Mol Des. Springer International Publishing; 2017;31:625\\u0026ndash;41. \\u003c/li\\u003e\\n\\u003cli\\u003eDhiman P, Malik N, Khatkar A. Natural based piperine derivatives as potent monoamine oxidase inhibitors: An in silico ADMET analysis and molecular docking studies. BMC Chem [Internet]. Springer International Publishing; 2020;14:1\\u0026ndash;16. Available from: https://doi.org/10.1186/s13065-020-0661-0\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Breast cancer cells, Radiosensitization, Piperine, γ-radiation, JC-1 analysis, γH2AX, Estrogen receptor α /β, Selective estrogen receptor modulator. DNA-PK complex\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2401099/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2401099/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eGamma radiation(γ) and other DNA targeted compounds generate highly lethal DNA double-stranded breaks (DSBs) inducing the cells to undergo apoptosis. Non-homologous end joining (NHEJ), one of the primary DSB repair pathways, plays an important role in providing cancer cells resistance against radio/chemotherapeutic agents resulting in cancer progression and relapse. Downregulating DNA-PK, a key protein in NHEJ could result in the accretion of DSBs, thereby sensitizing the cells towards radiation.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eCytotoxicity assays, Clonogenic assays, DNA damage assays, Flowcytometry analysis, Confocal Microscopy, immunofluorescence, and Immunoblotting were carried out. Combinatorial index calculations were done using Compusyn Analysis and data analysis was done using one-way ANOVA and two-way ANOVA, where a p-value of \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026le;\\u003c/span\\u003e\\u0026thinsp;0.0001 was considered significant.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eHere we found that the treatment of MCF7 cells with piperine, lead to the accumulation of DSBs induced by γ-radiation through lowering DNA-PK complex (comprising of DNA-PKcs/Ku70/Ku80), by altering the estrogen receptor (ER) α /β ratio. Piperine lowered DNA-PK mediated NHEJ repair through its transcription factor, ERα. Upregulation of ERβ, a nuclear hormone transcription factor promoting tumor suppression positively correlated with lowered expression of ERα and DNA-PK marked by the accumulation of radiation-induced DSBs and DNA damage response, cell cycle arrest leading to the intrinsic pathway of apoptosis.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e \\u003cp\\u003eBreast Cancer cells may be sensitized to radiation by altering the expression of DNA-PKc Complex, a key dsDNA repair protein machinery through selective estrogen receptor modulation. This study proposes a new strategy for combating acquired radioresistance through estrogen receptor-mediated modulation of the NHEJ pathway.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Estrogen Receptor-positive Breast Cancer Cells are Sensitized by Piperine to Chemo/Radio Therapy through Lowering the expression of a NHEJ repair protein DNA-PK\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2022-12-29 16:56:53\",\"doi\":\"10.21203/rs.3.rs-2401099/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"841472e1-bdfc-45d8-8555-19c55de1aade\",\"owner\":[],\"postedDate\":\"December 29th, 2022\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2022-12-29T16:56:56+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2022-12-29 16:56:53\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2401099\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2401099\",\"identity\":\"rs-2401099\",\"version\":[\"v1\"]},\"buildId\":\"_2-kVJe1T_tPrBINL-cwx\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}