Norcantharidin Sensitizes Lung Adenocarcinoma Cells to Multitherapy via Targeting the Deregulated Hedgehog Cascade

Norcantharidin Sensitizes Lung Adenocarcinoma Cells to Multitherapy via Targeting the Deregulated Hedgehog Cascade | 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 Norcantharidin Sensitizes Lung Adenocarcinoma Cells to Multitherapy via Targeting the Deregulated Hedgehog Cascade Bingjie Cui, Xin Zhang, Fei Wang, Hongliang Dong, Cuilan Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4761721/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 Norcantharidin (NCTD), a demethylated analog of cantharidin, has been identified as one of potential anti-tumor drug candidates in various human neoplasms. However, the NCTD-mediated interference with multidrug-resistance development and sustenance of lung adenocarcinoma (LAD) and its underlying molecular interaction mechanisms remains undefined yet. In this study, NCTD significantly inhibited cell growth of LAD cells in a dose-dependent manner when applied alone and magnified the sensitization of LAD cells to multiple therapeutic agents. Selective repression of sonic Hedgehog (SHH) signaling pathway by NCTD dramatically arrested cancer stemness development and maintenance such as the sphere formation capacities of LAD cells. Mechanistic analysis revealed that NCTD prohibited nuclear translocation of GLI1, the key terminal transcription factor of SHH cascade in LAD cells. In vivo studies confirmed that NCTD alone reduced propagation of LAD cells and enhanced the 5-FU and Osimertinib-based cancer progression inhibition while have no side effect on body weight. Taken together, our results demonstrate that NCTD represses SHH cascade-mediated cancer stemness to overcome the intrinsic resistance of LAD cells to multi-drug treatment, which implies that NCTD might be a therapeutic drug candidate that could be a de novo option to eradicate the treatment resistance against multiple therapeutic agents if co-applied in LAD treatment clinically. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Non-small cell lung cancer (NSCLC), which accounts for 80% of lung cancers, consists mainly of two pathological subtypes - lung adenocarcinoma (LAD) and lung squamous cell carcinoma (SCC) which claim about 50% and 40% of all NSCLC patients respectively.[ 1 , 2 ] Although combined conventional chemotherapy regimens and current available targeting agents against EGFR have augmented responsive rate of LAD patients, development of primary and secondary drug resistance still leads to very low 5-year survival rate.[ 3 , 4 ] Therefore, novel anti-cancer compounds that could (re)sensitize LAD cells to chemo- and targeting-drugs with bearable side effects are urgently called for in clinic. Abundant evidence has proposed that cancer stemness cells (CSCs), characterized with unlimited proliferative property and multiple differentiation potential, played an unneglectable role in multi-drug resistance formation and tumor metastasis.[ 5 ] As one of principal signaling pathways illustrated well in stem cell proliferation and differentiation, sonic Hedgehog (SHH) cascade not only physiologically regulates cellular homeostasis during embryonic development but also contribute to the tumorigenicity of CSCs.[ 6 ] In physiological conditions, the transmembrane protein Patched (PTCH) prohibited protein Smoothened (SMO) from translocating to the primary cilium. Binding of SHH ligand to PTCH receptor releases SMO to promote the phosphorylation and translocation of the key transcription factor Glioma associated oncogene 1 (GLI1) into nucleus, leading to stimulation of its downstream targets including SHH, PTCH and stem cell transcription factor SRY-related high-mobility-group box 2 (SOX2).[ 6 – 8 ] Pivotal components of the SHH pathway including SMO and GLI1 were reported to be aberrantly overexpressed in various cancer entities including lung cancer.[ 9 , 10 ] We and other groups have demonstrated that specific inhibition of SMO or GLI1 facilitated cisplatin-based standard and targeted treatment of LAD cells.[ 11 – 13 ] Norcantharidin (NCTD), a demethylated form of cantharidin which was extracted from medicinal insect blister beetle, has been used in traditional Chinese medicine for years to treat patients suffered from cancer. Accumulating studies have proved that NCTD possessed potent anti-proliferative or pro-apoptotic effect in various cancer entities including breast cancer, gastric cancer, liver cancer, prostate cancer with tolerable side effects.[ 14 – 21 ] The analog of NCTD, cantharidin was intensively discovered to initiate DNA damage and depress expression of DNA repair-associated protein in H460, one of LAD cell lines.[ 22 – 24 ] It has been demonstrated that NCTD could not only facilitate anticancer activity of gefitinib in NSCLC cells but also re-sensitize tyrosine kinase inhibitors refractory EGFR mutant NSCLC cells to gefitinib treatment through blockade of Met/PI3k/Akt pathway.[ 25 , 26 ] Our previous study has demonstrated that NCTD promote LAD cell apoptosis and inhibit cellular invasion via Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signaling pathway. [ 27 ] Whether NCTD could coordinate with conventional chemotherapeutic drugs for LAD treatment via repressing lung CSCs and its underlying molecular mechanisms remain to be further disclosed. In the present study, we investigated effects of NCTD on LAD parental and stem-like cells. Herein, we report that NCTD sensitizes LAD cells to multi-drug treatment in vitro and vivo by repressing GLI1 mediated stemness features, implying that NCTD may serve as a potential chemosensitizer for lung cancer patients. 2 Materials and methods 2.1 Cell culture and reagents The LAD cell lines A549 and H1299 were purchased from American Type Culture Collection (ATCC) and maintained in DMEM medium supplemented with 10% fetal bovine serum (Hyclone, USA), 100 units/ml penicillin, and 100 µg/ml streptomycin under 37°C with 5% CO 2 . Norcantharidin was purchased from Sigma and dissolved in PBS to 1 mg/ml for storage. Chemo-drugs including Cisplatin (DDP), Paclitaxel (PTX), Gemcitabine (GEM), 5-Fluorouracil (5-FU) were obtained from the pharmacy of Binzhou Medical University Hospital, Gefitinib and Osimertinib were obtained from the Shandong Zengfeng Biotechnology Co., Ltd. BMS-833923 (S7138) and Purmorphamine (S3042) were purchased from Selleck Chemicals. Concentrations of agents and durations of incubation were illustrated specifically in each experiment. 2.2 Cell proliferation assay Cells were counted automatically using Countstar and seeded at a density of 2000 cells per well into 96-well plate one day before drug treatment unless specified. CCK8 (Dojindo, Japan) test was performed at various time points upon drug incubation according to manufacturer’s guide. Calculation of relative proliferation was described before. [ 28 ] The half maximal inhibitory concentration (IC50) of NCTD were calculated with GraphPad software. 2.3 EDU staining Cells were seeded into 96-well plates with 4000 cells each well and incubated overnight. NCTD was added at concentrations of 0, 4, 8, 16 µg/ml, respectively. After incubation for 48 hours, EDU staining was carried out with the EDU staining kit (Bioscience, China) according to the instructions. Finally, the fluorescent signal was imaged and documented with fluorescence microscope (Olympus Corporation, Japan). 2.4 Clonal formation assay Clonal formation assay was performed as before. [ 28 ] Briefly, cells were counted and evenly inoculated into 12-well plates with 800 cells per well and incubated overnight. NCTD was applied at doses of 0, 4, 8, 16 µg/ml, respectively followed by incubation for a week. The cells were washed twice with PBS before fixation with 4% paraformaldehyde for 15 min. Crystal violet was added and dye for 30 min. Crystal violet was removed and washed before imaging. 2.5 Cell cycle analysis Cell cycle analysis was carried out as before. [ 11 ] 2 × 10 5 cells were collected and washed with pre-cold PBS followed by resuspension with 400 µl propidium iodide (PI) solution supplemented with 200 µg/ml PI, 100 µg/ml RNase and 0.2% Triton X-100. Samples were incubated at 4°C for 30 min before Flow Cytometric analysis. Fluorescent signals were collected at FL-2 channel with by Accuri C6 (Thermofisher, USA) and analyzed with ModFit software. 2.6 Sphere formation assay Sphere formation assay was performed as described before. [ 28 ] Cells were plated into ultra-low-attachment 12-well plate (Corning, USA) at a density of 1000 cells per well and cultured in serum-free DMEM medium supplemented with 20 ng/ml EGF (Peprotech, USA), 20 ng/ml FGF (Peprotech, USA) and 2% B27 (Gibco, USA). Drugs were added at the beginning of spheroid culture and medium was half refreshed every 3 days without new drug addition. Cell spheroids were observed and photographed by an inverted microscope (Olympus, Japan) after 10 to 14 days. Diameters of at least ten representative spheroids were measured and used for calculation. 2.7 RNA interference LAD cells were transiently transfected in 12-well plate using Lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions. Chemically synthesized non-target siRNA as control and double-stranded siRNAs for Gli1 (Santa Cruz, sc-37911) were transfected in parallel at a concentration of 50 nmol. Cells were collected and counted 24 hours after transfection for sphere formation assay. 2.8 Reverse-transcriptase PCR (RT-PCR) RT-PCR were performed as described before. [ 28 ] RNA was extracted with Trizol (TransGen Biotech, China) and reversely transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA) according to manufacturer’s instructions. Polymerase chain reactions were performed with 2× Taq Master Mix (Vazyme, China). Primers for cell cycle regulators and SHH cascade components were used as published. [ 11 ] 2.9 Immunoblot Immunoblot was performed as published before. [ 11 ] Total protein from cells was extracted and protein concentrations were quantified using a BCA assay kit (Thermo Scientific, USA). For plasma and nuclear protein separation, a cytoplasmic nuclear separation kit (Beyotime Biotechnology, China) was used. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were incubated with primary and secondary antibody sequentially at 4°C overnight and room temperature for one hour respectively. Enhanced chemiluminescence signals (ECL, Amersham) were detected using the Image Lab software. Antibodies used for detecting cell cycle arrest and SHH pathway were described before. [ 11 ] 2.10 Immunofluorescence Slides covered with cells were washed twice with PBS and once with immunofluorescence PBS. Ice cold acetone was added to cover the slides and incubated for 8 min before washing again with PBS. GLI1 primary antibody (1:200; Santa Cruz) was incubated at room temperature for 2.5 h, washed twice with PBS, and stained at 37℃ for 2 h with a secondary antibody containing Alex Flour 568-Donkey anti-mouse IgG (H + L) (1:200). Finally, the slides were sealed by DAPI and observed by fluorescence microscope (Olympus Corporation, Japan). 2.11 Living and dead cell staining The cells were inoculated in 24-well plates with a density of 80%. After incubation overnight, NCTD drugs were added in different experimental groups at concentrations of 0, 4, 8, 16 µg/ml, respectively. After incubation for 48 hours, the cells were washed twice with PBS and stained with AM/PI probe respectively. Finally, the cells were photographed with fluorescence microscope (Olympus Corporation, Japan). 2.12 Drug release experiment Prepare 42 tubes NCTD@F127 and 5-FU@F127, each of which will be dissolved by mixing 0.1 mg NCTD/0.8 mg 5-FU with 100 µl F127 (20% w/v) ice bath. After incubation at 37°C to form a gel, 0.5ml normal saline was slowly added along the tube wall. The tubes were then incubated continuously at 37°C for two weeks. Three parts of the supernatant were taken every day and stored in -20. Finally, quantitative analysis was performed with high-performance liquid phase (Angilent, USA). 2.13 Mice experiment 3- to 5-week-old female BALB/c athymic (nu/nu) mice were purchased from Charles River Laboratories in Beijing. Mice were housed in a specific pathogen free (SPF) biosafety lab and maintained in accordance with institutional guidelines of Binzhou Medical University Hospital. All animal experiments were performed with the approval of Binzhou Medical University Hospital Committee for Animal Care. Mice were subcutaneously injected with 5 × 10 6 A549 cells and randomly divided into different groups (5 mice per group, a total of 30 mice) when diameter of palpable tumors reached approximately 5 mm. Xenografted mice received vehicle (50 µl 20% w/v F127), NCTD@F127 (2.5 mg/kg), 5-FU@F127 (1.5 mg/kg), Osimertinib@F127 (10mg/kg) or their combination (1.5 mg/kg 5-FU + 2.5mg/kg NCTD@F127 or 10 mg/kg Osimertinib + 2.5mg/kg NCTD@F127) injected subcutaneously once a week for three weeks. Body weight and tumor volume was measured every second day. Tumor volume was calculated as 0.5 × a 2 × b (a and b represent short and long diameter of tumor bulk respectively). Mice were euthanized after 3 weeks’ treatment. Tumor tissues were collected from xenografts at the end of treatment and stained by immunohistochemistry. All animal care and experimental protocols were approved by Binzhou Medical University Hospital Committee for Animal Care (Ethics approval number: 20231208-54). 2.14 Immunohistochemistry (IHC) Tumor tissue was fixed with 4% paraformaldehyde and paraffin-embedded sections were performed. The tissue sections were incubated with GLI1 antibody (santa cruz) and enzyme-labeled secondary antibody. The tissue was stained with diaminobenzidine and hematoxylin and imaged under light microscope (Olympus Corporation, Japan). Quantification and statistical analysis was performed by calculating the average number of GLI1-positive cells in three different fields per mouse (5 mice per group). 2.15 Statistical analysis Data were analyzed with Microsoft Excel and GraphPad Prism 7 and presented as mean ± SD. The unpaired Student’s t test was used to compare two groups and one-way ANOVA test was used when compare multiple groups. Two-way ANOVA test was used for analyzing data from drug combinational treatment. A p value < 0.05, 0.01 and 0.001 was considered statistically significant and marked by one, two and three asterisks respectively. 3 Results 3.1 Anti-proliferative effect of NCTD in LAD cell lines As a demethylated form of cantharidin (CTD), the chemical formula of NCTD was displayed (Fig. 1 A). To validate the anti-proliferative effect of NCTD in LAD cell lines, we firstly treated A549 and H1299 cells with various concentrations of NCTD. CCK8 assay disclosed that NCTD could remarkably prohibit A549 and H1299 cell expansion in a dose-dependent manner from 5 to 80 µg/ml. The half maximal inhibitory concentration (IC50) of NCTD in A549 and H1299 cells were 22.67 and 17.72 µg/ml respectively (Fig. 1 B). EDU staining (Fig. 1 C), clonal formation experiments (Fig. 1 D) and statistical results also showed that NCTD inhibited proliferation of A549 and H1299 cells in a concentration-dependent manner. 3.2 Induction of cell cycle arrest by NCTD in LAD cells To unveil how NCTD exert anti-proliferative effect in LAD cells, we applied NCTD at distict concentrations for 48 hours followed by PI cell cycle staining. Cell cycle analysis (Fig. 2 A) and histogram quantification (Fig. 2 B) demonstrated that NCTD induced G1/S cycle arrest in A549 cells when applied at 4 and 8 µg/mL while induced G2/M cycle arrest when applied 16 µg/mL. For H1299, NCTD had marginal effect on cell cycle arrest when applied at below 8 µg/mL while induced G2/M cycle arrest when applied at 16 µg/mL. Further investigation of molecular mechanisms by RT-PCR indicated that NCTD significantly decreased expression of pivotal cell cycle regulators including Cyclin A, Cyclin B1, CDK2 and CDK4 in A549 cells dose dependently (Fig. 2 C). For H1299, NCTD depressed transcription level of CDK2 and CDK4 when applied at high dose (16 µg/mL), which goes in line with PI staining data (Fig. 2 A, B). Immunoblot assay of the protein expression level of the above cell cycle regulators confirmed RT-PCR result (Fig. 2 D). 3.3 NCTD deactivates Hedgehog pathway in LAD cell lines Our previous study revealed that selective repression of Hedgehog cascade chain potentiated the anti-cancer efficacy of DDP for non-small lung cancer cells both in vitro and in vivo.11 Herein we examine whether NCTD abates the SHH signaling and employs this targeting mode to sensitize LAD cells to other chemotherapeutic drugs. RT-PCR (Fig. 3 A) and immunoblot (Fig. 3 B) assay indicated that NCTD depressed GLI1 expression at both transcriptional and translational levels, which implies that NCTD executes its function specifically through GLI1, the pivotal transcription factor of Hedgehog pathway. GLI1, as a key molecule in the Hedgehog pathway, is also its downstream molecule, enters the nucleus after dephosphorylation modification and then regulates its own expression. Therefore, we speculated that NCTD may inhibit the expression of its own target genes by blocking GLI1's entry into the nucleus. We then examined GLI1 expression and localization in cells 48 hours after NCTD treatment of LAD cells by immunoblotting and immunofluorescence assays. Immunoblotting showed that NCTD significantly reduced the expression of GlI1 in the nucleus compared with the control group (Fig. 3 C). Immunofluorescence staining also revealed that GLI1 was evenly distributed in the cells in the control group, and when LAD cells were treated with NCTD, GLI1 was mainly distributed around the nucleus in the cytoplasm, with the nuclear location apparently empty (Fig. 3 D). Therefore, NCTD can regulate the expression level of GLI1 by regulating GLI1 heterotopic in Hedgehog signaling pathway. 3.4 GLI1 can antagonize the proliferation inhibition and apoptosis induction of LAD cells by NCTD Previous experiments showed that NCTD inhibited LAD cell proliferation and GLI1 expression in a dose-dependent manner. To further verify that NCTD inhibits cell proliferation by inhibiting GLI1 expression, we screened LAD cell lines that stably overexpress GLI1 and used pcDNA3.1 as the control group (Fig. 4 A). First, the proliferation of LAD cells in pcDNA3.1 and GLI1 groups was evaluated after treatment with different concentrations of NCTD. Results of the CCK8 assay indicated that compared with pcDNA3.1, GLI1 overexpression significantly antagonized the inhibitory effect of NCTD on the growth of lung cancer cells. (Fig. 4 B). Similarly, in vitro colony formation assay displayed that overexpression of GLI1 promotes the clonogenicity of LAD cells (Fig. 4 C). The results of staining of living and dead cells corroborated that the number of apoptotic cells in the GLI1 overexpression group was significantly lower than that in the pcDNA3.1 group upon NCTD treatment (Fig. 4 D). These results indicated that the inhibitory effect of NCTD on LAD cell proliferation was mainly achieved by inhibiting the expression of GLI1. 3.5 Inhibition of DDP-resistant stemness-prone A549 spheroids by NCTD GLI1, the core transcription factor of SHH signaling cascade, has been reported to regulate tumor stemness, 28 one of its downstream targets which implies silencing SHH signaling may efficiently block the pluripotency sustenance in cancer cells. We have shown that NCTD selectively inhibits GLI1 expression in the Hedgehog signaling pathway. Next, we further explored whether NCTD affects the ability of LAD cells to form stem cell spheres by blocking the SHH signaling pathway. Our sphere formation assay disclosed that DDP at 4 and 8 µg/mL did not alter the sphere sizes and numbers from A549 cells (Fig. 5 A). In contrast, NCTD at 8 µg/mL, robustly reduced A549 sphere formation (Fig. 5 B). For specificity in interfering the cancer stemness with silencing SHH pathway, we applied siRNA against GLI1 and SMO inhibitor BMS to A549 cells and found that inhibition of either GLI1 or SMO significantly repressed cancer stemness sphere formation compared to their corresponding control samples (siRNA scramble and DMSO separately for GLI1 and SMO) (Fig. 5 C, D).The SMO agonist Purmorphamine (PUR) had marginal effect on sphere size but remarkably increased sphere numbers in our settings (Fig. 5 D). 3.6 NCTD sensitizes LAD cells to conventional chemo-drug treatments in vitro The stemness of tumor cells plays an important role in the formation of multidrug resistance and metastasis, and NCTD can regulate the stemness of LAD cells by inhibiting GLI1 expression in the Hedgehog pathway. Our previous research showed that selective inhibition of the Hedgehog cascade enhances the anti-cancer effects of DDP on non-small lung cancer cells in vitro and in vivo. Next, we investigated whether NCTD could sensitize the treatment of multiple chemotherapeutic agents in LAD cells. DDP, PTX, GEM ,5-FU, Gefitinib and Osimertinib, four conventional chemo drugs and two targeting agents commonly used in clinic for LAD therapy were tested. To explore their proper dose of anti- proliferative activity in lung cancer, we firstly applied them separately to our LAD cell lines A549 and H1299, respectively at various concentrations and detected their inhibition of cell proliferation. CCK8 assay revealed that all six chemo-drugs inhibited cell growth of A549 and H1299 in both dose-dependent manners (Fig. 6 A). Similar to other chemo-drugs, NCTD inhibited A549 growth dose-dependently but with a wider concentration range from 1 to 100 µg/mL, which indicated that NCTD is not as potent as commonly used chemotherapeutic regimens in clinic. Since NCTD alone is not a strong neoplastic cell killing agent, we further investigated whether NCTD could facilitate the anti-cancer effect of other chemo-drugs. Following co-treatment of NCTD (1 µg/ml) with these drugs in LAD cells, CCK8 assays demonstrated that NCTD significantly enhanced anti-proliferation effect of 5-FU, GEM and Gefitinib in A549 cells, while additionally aggravating the growth inhibition mediated by PTX, 5-FU, Gefitinib and Osimertinib respectively in H1299 cells (Fig. 6 B). Sequential co-application of NCTD with various chemo-drugs to LAD cells revealed that NCTD robustly sensitized both A549 and H1299 to multiple drug therapies which are commonly administered as the firstly line of chemotherapy for LAD patients (Fig. 6 C). The above experimental results suggest that NCTD can be used as a chemotherapy sensitizer and will play a better therapeutic effect on tumors when combined with other chemotherapy drugs. 3.7 NCTD magnifies the in vivo anti-tumor efficacy of 5-FU and Osimertinib In order to further explore the chemotherapy sensitization effect of NCTD in vivo, we tested 5-FU and Osimertinib as representative of chemo and targeting drug respectively in A549 subcutaneously implanted mice model. To achieve better therapeutic efficacy, F127 was used as a drug carrier to wrap different drugs prolong drug release in vivo. Mice were treated with F127 as control, NCTD@F127 (2.5 mg/kg), 5-FU@F127 (20 mg/kg), Osimertinib@F127 (10 mg/kg), or their combination (5-FU + NCTD@F127, Osimertinib + NCTD@F127). According to the results of in vitro drug release experiment, the drug was completely released in about one week, so the drug was injected into the tumor once a week for three consecutive weeks (Fig. S1 ). Tumor images and growth curves show that NCTD remarkably increases the therapeutic efficacy of 5-FU and Osimertinib (Fig. S2, Fig. 7 A and B). Osimertinib + NCTD@F127 was the most effective combinational therapy, followed by 5-FU + NCTD@F127. The single treatment group also had a certain therapeutic effect, but not as obvious as the combined treatment. There was no significant change in the body weight of mice in each treatment group, indicating that the combination of drugs significantly improved the efficiency of treatment and did not produce toxic side effects (Fig. 7 C). Immunohistochemistry showed that GLI1 expression was decreased in all treatment groups compared with F127 group. The effect of NCTD + 5-FU@F127 and NCTD + Osimertinib@F127 combined treatment group was more significant than that of NCTD@F127,5-FU@F127 and Osimertinib@F127 (Fig. 7 D, E) single administered group. The combination of NCTD with 5-FU and Osimertinib improve the efficacy of chemotherapy, indicating that NCTD can be used as a chemotherapy sensitizer for the treatment of tumors. 4 Discussion Rapid discovery and developments of novel targeting inhibitors against EGFR and ALK have not replaced conventional chemotherapy, particularly cisplatin-based regimens serving as a first line therapeutic strategy for LAD patient treatment in clinic. In the current study, we unveiled the potent role of NCTD in prohibiting LAD stem cell proliferation and sensitizing the LAD cells to those four commonly used chemo-regimens including DDP, PTX, GEM and 5-FU as well as two targeted drugs Gefitinib and Osimertinib in vitro. In vivo study further confirmed that NCTD abated tumor growth of LAD cells and significantly promoted the therapeutic efficacy of both chemo and targeting drugs. Mechanistic exploration deciphered that NCTD exert selective tumor-arresting functions in LAD cells by nuclear translocation and activation of GLI1, the critical transcription factor of Hedgehog cascade (Fig. 8 ). It has been reported that NCTD boosted therapy efficacy of conventional chemotherapy in hepatic carcinoma, breast cancer and bladder cancer cells. [ 29 – 31 ] In addition, NCTD could enhance the anticancer activity of gefitinib and cisplatin in NSCLC parental cells while the underlying molecular mechanisms remain elusive. [ 25 ] Here we further unveiled its potent efficacy in coordinating with multi-drug by inhibiting GLI1 mediated LAD cancer stemness. NCTD prohibited nuclear translocation of GLI1 and activation of its subexpression which leads to multidrug resistance. Data regarding overexpression of GLI1 counteracted anticancer efficacy of NCTD further verified that NCTD attenuated stemness related features of LAD cells by specifically targeting GLI1. It has been demonstrated that aberrant activation of GLI1 signaling augmented stemness features of lung and gastric cancer cells.[ 7 , 32 , 33 ] Our previous drug screening using well-established LAD cell lines for Hedgehog cascade inhibitors also discovered that molecules targeting GLI1 exerted robust anti-stemness capacity.[ 28 ] In epithelial ovarian cancer, GLI1 was found to modulate drug sensitivity by directly binding to the drug efflux ATP-binding cassette (ABC) transporters including ABCB1 and ABCG2,[ 34 ] reinforcing its role as a drug susceptibility upstream regulator. Our previous wok identified another Chinese herb, Scutellariabarbata D. Don extraction (SBE) as a Hedgehog inhibitor which inhibited the whole Hedgehog cascade by targeting SMO. Different from SBE, data in this article indicated that NCTD specifically downregulate expression of GLI1, the terminal executor of Hedgehog cascade that located downstream of SMO therefore may function better in SMO inhibitor resistant scenario. Furthermore, NCTD has been reported to disrupt epithelial-mesenchymal transition and metastasis in colon cancer, lung cancer and seldom developed mucoepidermoid carcinoma.[ 35 – 38 ] Our previous in vitro studies also corroborated the anti-invasive and -metastatic effect of NCTD on LAD cells mediated by transcription factor YAP.[ 27 ] Since no observable distal organ metastasis was found in subcutaneously implanted lung cancer model in all experimental groups in our setting, as an extension of in vitro findings, investigation of anti-metastatic effect of NCTD using tail injection established lung cancer model might be used in the future. Of note, although in vivo tumor growth curve and tumor weight indicated that NCTD facilitate 5-FU and Osimertinib, no additive adverse effect was observed by combinatory therapy. Additionally, pilot efforts have been made to boost anti-tumor potent of NCTD while soft its accompanying side effect by modification of the compound backbone itself or drug delivery system. [ 39 – 43 ] In brief, our study demonstrated that NCTD sensitized LAD cells to chemo-drug treatment through repressing GLI1 mediated cancer stemness properties. This adds more experimental evidence that NCTD may serve as a multi-drug sensitizer to potentially facilitate LAD therapy in clinic. Declarations Data availability The datasets generated or analyzed during the study are available from the corresponding author on reasonable request. Acknowledgements Not applicable. Funding This study was supported by National Natural Science Foundation of China (31900441, 82373097), Natural Science Foundation of Shandong Province (ZR2019MC026, ZR2023QH080, ZR2023MH126 and ZR2022QH192), Funds of Shandong traditional Chinese Medicine Science and Technology Development Project (2019-0514 and 2019WS321), Binzhou Medical University Research Program (BY2022KJ64), Qilu Outstanding Young Talents in Health Project, Taishan Scholarship and Double-Hundred Project. Author information Authors and Affiliations Department of Medical Research Center, Binzhou Medical University Hospital, Binzhou, P.R. China Bing-jie Cui, Xin Zhang, Fei Wang, Hong-liang Dong, Cuilan Liu, Wei-wei Chen, Jiong Deng, Yan Wu, Jing Du Department of Hematology Oncology, Binzhou Medical University Hospital, Binzhou, P.R. China Xin Zhang Clinical Laboratory, Binzhou Medical University Hospital, Binzhou, P.R. China Wei-wei Chen Department of Oncology, Binzhou Medical University Hospital, Binzhou, P.R. China Yan Wu, Jing Du Contributions Jing Du, Yan Wu contributed to the conception of the study; Bingjie Cui, Xin Zhang performed the experiment; Jing Du, Bingjie Cui contributed significantly to analysis and manuscript preparation; Jing Du, Bingjie Cui performed the data analyses and wrote the manuscript; Fei Wang, Hongliang Dong, Cuilan Liu, Weiwei Chen, Jiong Deng helped perform the analysis with constructive discussions. Correspondance Jing Du, Medical Research Center, Binzhou Medical University Hospital, Binzhou, China. Email: [email protected] Yan Wu, Medical Research Center, Binzhou Medical University Hospital, Binzhou, China. Email: [email protected] Ethics declarations Ethics approval and consent to participate All animal experiments were performed with the approval of Binzhou Medical University Hospital Committee for Animal Care，ethics review number is 20231208-51. Consent for publication Not applicable. 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Sun CY, Zhu Y, Li XF, Tang LP, Su ZQ, Wang XQ et al . Norcantharidin alone or in combination with crizotinib induces autophagic cell death in hepatocellular carcinoma by repressing c-Met-mTOR signaling. Oncotarget 2017; 8: 114945-114955. Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M et al . Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nature reviews Clinical oncology 2015; 12: 445-464. Wang D, Yang C, Wang Z, Yang Y, Li D, Ding X et al . Norcantharidin combined with Coix seed oil synergistically induces apoptosis and inhibits hepatocellular carcinoma growth by downregulating regulatory T cells accumulation. Scientific reports 2017; 7: 9373-9384. Wang WJ, Wu MY, Shen M, Zhi Q, Liu ZY, Gong FR et al . Cantharidin and norcantharidin impair stemness of pancreatic cancer cells by repressing the beta-catenin pathway and strengthen the cytotoxicity of gemcitabine and erlotinib. International journal of oncology 2015; 47: 1912-1922. Wang Z, You D, Lu M, He Y, Yan S. Inhibitory effect of norcantharidin on melanoma tumor growth and vasculogenic mimicry by suppressing MMP-2 expression. Oncology letters 2017; 13: 1660-1664. Wu H, Fan F, Liu Z, Shen C, Wang A, Lu Y. Norcantharidin combined with EGFR-TKIs overcomes HGF-induced resistance to EGFR-TKIs in EGFR mutant lung cancer cells via inhibition of Met/PI3k/Akt pathway. Cancer chemotherapy and pharmacology 2015; 76: 307-315. Yu BQ, Gu DS, Zhang XL, Li JF, Liu BY, Xie JW. GLI1-mediated regulation of side population is responsible for drug resistance in gastric cancer. Oncotarget 2017; 8: 27412-27427. Zhang X, Zhang B, Zhang P, Lian L, Li L, Qiu Z et al . Norcantharidin regulates ERalpha signaling and tamoxifen resistance via targeting miR-873/CDK3 in breast cancer cells. PloS one 2019; 14: e0217181. Zhang XP, Luo LL, Liu YQ, Liu XS, An FY, Sun SB et al . Norcantharidin combined with diamminedichloroplatinum inhibits tumor growth and cancerometastasis of hepatic carcinoma in murine. J Cancer Res Ther 2018; 14: S1035-S1040. Zheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC et al . Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways. Anticancer research 2016; 36: 6031-6042. Zhu Y, Mi Y, Wang Z, Jia X, Jin Z. Norcantharidin inhibits viability and induces cell cycle arrest and apoptosis in osteosarcoma. Oncology letters 2019; 17: 456-461. Additional Declarations No competing interests reported. Supplementary Files supplementfigures1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4761721","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342312072,"identity":"ed15a4f1-b5f5-4cc1-af35-f0051317f9a6","order_by":0,"name":"Bingjie Cui","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Bingjie","middleName":"","lastName":"Cui","suffix":""},{"id":342312073,"identity":"61f9a461-1cac-4426-9d8c-a91e4b72b29e","order_by":1,"name":"Xin Zhang","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":342312074,"identity":"13585810-b001-4e5f-a7ec-31cfb5322cc0","order_by":2,"name":"Fei Wang","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Wang","suffix":""},{"id":342312075,"identity":"482eefc6-dfad-4d7c-910c-527261e3458a","order_by":3,"name":"Hongliang Dong","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Dong","suffix":""},{"id":342312076,"identity":"794ca860-23d1-4082-9c80-767990eed67c","order_by":4,"name":"Cuilan Liu","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Cuilan","middleName":"","lastName":"Liu","suffix":""},{"id":342312077,"identity":"b4f1b4bf-0737-4583-88b1-5b5fef5a5de8","order_by":5,"name":"Weiwei Chen","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Chen","suffix":""},{"id":342312078,"identity":"61ef1b52-a755-4c4f-96a6-b2936b979a53","order_by":6,"name":"Jiong Deng","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Jiong","middleName":"","lastName":"Deng","suffix":""},{"id":342312079,"identity":"a531e09f-df82-4c0f-88cf-80ccbdca8765","order_by":7,"name":"Yan Wu","email":"","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wu","suffix":""},{"id":342312081,"identity":"4ae8ccb7-3115-404d-b75d-e1dd4ac09e68","order_by":8,"name":"Jing Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3PoQoCQRCA4RFBy+nVNaivsHKggj7MLIJX1GwwbDIpvooiKLY5Fq54h1XBYjFfEpO4h8W0nk1wf5gwMF8YAJvtFyMChkA1/lrzWQmS9w0BYIAklplJOVDB8XI/+1t3LxIYd4QsxmQkFQp7bcTrcCfjNYPIF9IZoZFwipr6FzVcBvMN5KZKSObwD+RwS4nPVUmTRyYSFVKCPEyJzED0L14b+6qxm5XWDEPfmzoDMykf1eV076p6y41XSTLpVBfFyEyA0duCegrme50rP57YbDbbv/cEc21QkNmfzPQAAAAASUVORK5CYII=","orcid":"","institution":"Medical Research Center, Binzhou Medical University Hospital, Binzhou, China","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2024-07-18 10:07:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4761721/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4761721/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62864243,"identity":"51de8427-6bc5-40ad-9bee-63cb8d7edda3","added_by":"auto","created_at":"2024-08-20 11:06:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583194,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-proliferative effect of NCTD in LAD cell lines. (A) Chemical formula of cantharidin (CTD) and norcantharidin (NCTD), (B) IC50 curve of NCTD in A549 and H1299 cells generated by GraphPad software, (C) EdU staining (left) and quantification (right) showed dose-dependent inhibitory effect of NCTD on lung cancer cell proliferation. EdU: 5-Ethynyl-2’-deoxyuridine. Scale=100 µM, (D) Clonal formation staining (left) and quantification (right) confirmed the inhibitory effect of NCTD on the growth and expansion of lung cancer cells. NCTD drug concentration was µg/mL. Data were analyzed by one-way ANOVA test of GraphPad software. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. control.\u003c/p\u003e","description":"","filename":"FIG.1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/822fe0ed214dbd2be2fb02c5.jpg"},{"id":62863684,"identity":"ebb90e39-06df-4496-ad4b-f556ab870680","added_by":"auto","created_at":"2024-08-20 10:58:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":627344,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of cell cycle arrest by NCTD in LAD cells. (A) Cell cycle analysis of LAD cells upon NCTD incubation, (B) Quantification of cell cycle arrest in A549 and H1299 cells. Cells were treated with NCTD at 0, 4, 8 and 16 µg/mL respectively. PI staining was performed 48 hours after drug incubation. PI: Propidiumiodide. Histograms are shown as mean ± SD, RT-PCR analysis (C) and immunoblot assay (D) of expression level of key cell cycle regulators in A549 and H1299 cells. House keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected in parallel as inner control for RT-PCR and α-tubulin was loaded as inner control for immunoblot assay. NCTD drug concentration was µg/mL. Histograms are shown as mean±SD. Data were analyzed by two-way ANOVA test of GraphPad software. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. DMSO treated control.\u003c/p\u003e","description":"","filename":"FIG.2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/c4ced9126bdacf6e056ab4a6.jpg"},{"id":62863690,"identity":"a37e1eb6-638b-4ddb-9955-7465cc41c0cc","added_by":"auto","created_at":"2024-08-20 10:58:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":376720,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD inhibits cell proliferation and regulates genes associated with stemness by inhibiting GLI1 from entering the nucleus. (A) RT-PCR analysis of Hedgehog cascade transcription level in A549 and H1299 cells upon NCTD treatment for 48 hours. The housekeeping gene GAPDH was amplified in parallel as inner control, (B) Immunoblot analysis of Hedgehog cascade expression level in A549 and H1299 cells upon NCTD treatment for 48 hours. α-tubulin was loaded as inner control, (C) NCTD significantly decreased the expression level of GLI1 protein in LAD cell nucleus. Histone H3 was loaded as inner control of nucleus, (D) immunofluorescence staining confirmed that NCTD promoted GLI1 transfer from nucleus to cytoplasm. Scale=25 µM. NCTD drug concentration was µg/mL. Data were analyzed by one-way ANOVA test of GraphPad. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001 vs. DMSO treated control.\u003c/p\u003e","description":"","filename":"FIG.3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/5bae5771af2157f0aaeb7673.jpg"},{"id":62864246,"identity":"61e0f8df-65d0-4bf6-b7fd-32efb0c089b5","added_by":"auto","created_at":"2024-08-20 11:06:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":430865,"visible":true,"origin":"","legend":"\u003cp\u003eThe compensatory experiment of overexpressing GLI1 in LAD cells. (A) RT-PCR experiments confirmed successful overexpression of GLI1 in A549 and H1299 cells. The housekeeping gene GAPDH was amplified in parallel as inner control, (B) CCK8 assay showed that GLI1 overexpression significantly antagonized the inhibitory effect of NCTD on LAD cell growth. pcDNA3.1, the empty vector backbone as control. GLI1, GLI1 overexpressing plasmid based on pcDNA3.1, (C) Clonal formation assay (left) and quantification (right) showed that GLI1 overexpression antagonistic effect of NCTD on H1299 cell proliferation, (D) the results of live and dead cell staining (right) and quantification (left) showed that GLI1 overexpression had antagonistic effect on NCTD-induced apoptosis of H1299 cells. Scale=100 µM. NCTD drug concentration was µg/mL. Data were analyzed by one-way ANOVA test of GraphPad. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. PBS treated control.\u003c/p\u003e","description":"","filename":"FIG.4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/4db9be87919b263b38e41d45.jpg"},{"id":62863686,"identity":"8077edee-efb8-4c53-8213-25ea35b47c9a","added_by":"auto","created_at":"2024-08-20 10:58:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":473458,"visible":true,"origin":"","legend":"\u003cp\u003eDisruption of Hedgehog cascade represses formation of DDP-resistant A549 spheroid in vitro. Cell sphere formation (left) and quantification (right) of A549 cells upon DDP (A) or NCTD (B) treatment, (C) Cell sphere formation (left) and quantification (right) of A549 cells upon GLI1 RNA interference, (D) Cell sphere formation (upper panel) and quantification (lower panel) of A549 cells upon SMO agonist (PUR) and inhibitor (BMS) incubation. DDP, PUR and BMS were applied at 8 µg/mL, 10 and 5 µM respectively. PUR and BMS denote Purmorphamine and BMS-833923 respectively. Histograms are shown as mean±SD. Data were analyzed by one-way ANOVA test when comparing multiple groups and Student’s t test when comparing two groups. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001 vs. control.\u003c/p\u003e","description":"","filename":"FIG.5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/bb0ec2e69ff4ba5f2a6dba91.jpg"},{"id":62864568,"identity":"d5df2d45-43d5-439a-a8cf-05c797512aca","added_by":"auto","created_at":"2024-08-20 11:14:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":485308,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD sensitizes lung adenocarcinoma cells to chemotherapeutic treatment. (A) CCK8 assay of A549 (left) and H1299 (right) upon incubation of concentration distinct drugs for 48 hours. The dosage of A549 group was NCTD (0，1，2，4，6，8 µg/mL), DDP (0，0.05，0.1，0.2，0.4，0.8 µg/mL), PTX (0，0.005，0.01，0.02，0.04，0.08 µg/mL), GEM (0，0.01，0.2，0.4，0.8，0.16 µg/mL), 5-FU (0，0.025，0.05，0.1，0.2，0.4 µg/mL), Gefitinib (0，1.375，2.75，5.5，11，22 µg/mL) and Osimertinib (0，1，1.5，2，2.5，3 µg/mL). The dosage of H1299 group was NCTD (0，0.25，0.5，1，2，4 µg/mL), DDP (0，0.05，0.1，0.2，0.4，0.8 µg/mL), PTX (0，0.005，0.01，0.02，0.04，0.08 µg/mL), GEM (0，0.05，0.1，0.2，0.4，0.5 µg/mL) and 5-FU (0，0.125，0.25，0.5，1，2 µg/mL), Gefitinib (0，1.375，2.75，5.5，11，22 µg/mL) and Osimertinib (0，1，2，3，4，5 µg/mL), (B) CCK8 assay of A549 (left) and H1299 (right) upon combinational incubation of NCTD and distinct drugs for 48 hours. NCTD, DDP, PTX, GEM, 5-FU Gefitinib and Osimertinib were applied at 1, 0.5, 0.02, 0.16, 0.4, 5.5 and 3 µg/mL respectively, (C) CCK8 assay of A549 (left) and H1299 (right) upon pre-treatment with NCTD for 48 hours followed by incubation of various chemo-drugs. NCTD, DDP, PTX, GEM, 5-FU Gefitinib and Osimertinib were applied at 1, 0.5, 0.02, 0.16, 0.4, 5.5 and 3 µg/mL correspondingly. All experiments were performed in triplicates. Histograms are shown as mean ± SD. Data were analyzed by two-way ANOVA test of GraphPad software. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001 vs. DMSO treated control.\u003c/p\u003e","description":"","filename":"FIG.6..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/e4631d002b00d68620097686.jpg"},{"id":62864979,"identity":"710a82fe-7522-4b6f-ab56-0ffa40d3d438","added_by":"auto","created_at":"2024-08-20 11:22:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":945587,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-drug sensitization of NCTD in vivo. (A) Pictures of tumors after drug treatment in each group, (B) Relative tumor growth curve. Relative tumor volume is calculated by dividing individual tumor volume with its primary volume before treatment (at day 0), (C) Body weight curve of each group after drug administration, (D) Representative images of GLI1 immunohistochemical staining of tumor tissue from A549 subcutaneously transplanted mice upon various treatment. Scale=100 µM, (E) Statistical analysis of GLI1 histochemical staining in each group. Histograms are shown as mean±SD and data were analyzed by one-way ANOVA test. **p\u0026lt;0.01 vs. control.\u003c/p\u003e","description":"","filename":"FIG.7..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/af50d1aa71b259add4620c51.jpg"},{"id":62863691,"identity":"758f7ed6-726d-403d-b153-91b0649d24e0","added_by":"auto","created_at":"2024-08-20 10:58:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":369707,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram illustrating mode of mechanisms underlying NCTD therapy. NCTD selectively inhibits GLI1 and its downstream targets of Hedgehog pathway, greatly inhibits the development and maintenance of cancer stem cells, and plays an important role in enhancing chemosensitivity to a variety of chemotherapy and targeted drugs.\u003c/p\u003e","description":"","filename":"FIG.8..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/af5ee03bae391679ff5beee3.jpg"},{"id":65665367,"identity":"39fcf7cc-e6a8-4bd3-a416-08f1299eb9ed","added_by":"auto","created_at":"2024-10-01 05:54:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4957035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/020e1197-38e5-44f9-b4e4-0ee7465c2b27.pdf"},{"id":62863692,"identity":"02e5bb19-cbd7-4259-9837-512dd09d179b","added_by":"auto","created_at":"2024-08-20 10:58:55","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":19207202,"visible":true,"origin":"","legend":"","description":"","filename":"supplementfigures1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4761721/v1/8ffe676a9918d13e7cf1df11.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Norcantharidin Sensitizes Lung Adenocarcinoma Cells to Multitherapy via Targeting the Deregulated Hedgehog Cascade","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC), which accounts for 80% of lung cancers, consists mainly of two pathological subtypes - lung adenocarcinoma (LAD) and lung squamous cell carcinoma (SCC) which claim about 50% and 40% of all NSCLC patients respectively.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Although combined conventional chemotherapy regimens and current available targeting agents against EGFR have augmented responsive rate of LAD patients, development of primary and secondary drug resistance still leads to very low 5-year survival rate.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Therefore, novel anti-cancer compounds that could (re)sensitize LAD cells to chemo- and targeting-drugs with bearable side effects are urgently called for in clinic.\u003c/p\u003e \u003cp\u003eAbundant evidence has proposed that cancer stemness cells (CSCs), characterized with unlimited proliferative property and multiple differentiation potential, played an unneglectable role in multi-drug resistance formation and tumor metastasis.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] As one of principal signaling pathways illustrated well in stem cell proliferation and differentiation, sonic Hedgehog (SHH) cascade not only physiologically regulates cellular homeostasis during embryonic development but also contribute to the tumorigenicity of CSCs.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] In physiological conditions, the transmembrane protein Patched (PTCH) prohibited protein Smoothened (SMO) from translocating to the primary cilium. Binding of SHH ligand to PTCH receptor releases SMO to promote the phosphorylation and translocation of the key transcription factor Glioma associated oncogene 1 (GLI1) into nucleus, leading to stimulation of its downstream targets including SHH, PTCH and stem cell transcription factor SRY-related high-mobility-group box 2 (SOX2).[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Pivotal components of the SHH pathway including SMO and GLI1 were reported to be aberrantly overexpressed in various cancer entities including lung cancer.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] We and other groups have demonstrated that specific inhibition of SMO or GLI1 facilitated cisplatin-based standard and targeted treatment of LAD cells.[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eNorcantharidin (NCTD), a demethylated form of cantharidin which was extracted from medicinal insect blister beetle, has been used in traditional Chinese medicine for years to treat patients suffered from cancer. Accumulating studies have proved that NCTD possessed potent anti-proliferative or pro-apoptotic effect in various cancer entities including breast cancer, gastric cancer, liver cancer, prostate cancer with tolerable side effects.[\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] The analog of NCTD, cantharidin was intensively discovered to initiate DNA damage and depress expression of DNA repair-associated protein in H460, one of LAD cell lines.[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] It has been demonstrated that NCTD could not only facilitate anticancer activity of gefitinib in NSCLC cells but also re-sensitize tyrosine kinase inhibitors refractory EGFR mutant NSCLC cells to gefitinib treatment through blockade of Met/PI3k/Akt pathway.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eOur previous study has demonstrated that NCTD promote LAD cell apoptosis and inhibit cellular invasion via Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signaling pathway. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Whether NCTD could coordinate with conventional chemotherapeutic drugs for LAD treatment via repressing lung CSCs and its underlying molecular mechanisms remain to be further disclosed. In the present study, we investigated effects of NCTD on LAD parental and stem-like cells. Herein, we report that NCTD sensitizes LAD cells to multi-drug treatment in vitro and vivo by repressing GLI1 mediated stemness features, implying that NCTD may serve as a potential chemosensitizer for lung cancer patients.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and reagents\u003c/h2\u003e \u003cp\u003eThe LAD cell lines A549 and H1299 were purchased from American Type Culture Collection (ATCC) and maintained in DMEM medium supplemented with 10% fetal bovine serum (Hyclone, USA), 100 units/ml penicillin, and 100 \u0026micro;g/ml streptomycin under 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Norcantharidin was purchased from Sigma and dissolved in PBS to 1 mg/ml for storage. Chemo-drugs including Cisplatin (DDP), Paclitaxel (PTX), Gemcitabine (GEM), 5-Fluorouracil (5-FU) were obtained from the pharmacy of Binzhou Medical University Hospital, Gefitinib and Osimertinib were obtained from the Shandong Zengfeng Biotechnology Co., Ltd. BMS-833923 (S7138) and Purmorphamine (S3042) were purchased from Selleck Chemicals. Concentrations of agents and durations of incubation were illustrated specifically in each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eCells were counted automatically using Countstar and seeded at a density of 2000 cells per well into 96-well plate one day before drug treatment unless specified. CCK8 (Dojindo, Japan) test was performed at various time points upon drug incubation according to manufacturer\u0026rsquo;s guide. Calculation of relative proliferation was described before. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] The half maximal inhibitory concentration (IC50) of NCTD were calculated with GraphPad software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 EDU staining\u003c/h2\u003e \u003cp\u003eCells were seeded into 96-well plates with 4000 cells each well and incubated overnight. NCTD was added at concentrations of 0, 4, 8, 16 \u0026micro;g/ml, respectively. After incubation for 48 hours, EDU staining was carried out with the EDU staining kit (Bioscience, China) according to the instructions. Finally, the fluorescent signal was imaged and documented with fluorescence microscope (Olympus Corporation, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Clonal formation assay\u003c/h2\u003e \u003cp\u003eClonal formation assay was performed as before. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Briefly, cells were counted and evenly inoculated into 12-well plates with 800 cells per well and incubated overnight. NCTD was applied at doses of 0, 4, 8, 16 \u0026micro;g/ml, respectively followed by incubation for a week. The cells were washed twice with PBS before fixation with 4% paraformaldehyde for 15 min. Crystal violet was added and dye for 30 min. Crystal violet was removed and washed before imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell cycle analysis\u003c/h2\u003e \u003cp\u003eCell cycle analysis was carried out as before. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were collected and washed with pre-cold PBS followed by resuspension with 400 \u0026micro;l propidium iodide (PI) solution supplemented with 200 \u0026micro;g/ml PI, 100 \u0026micro;g/ml RNase and 0.2% Triton X-100. Samples were incubated at 4\u0026deg;C for 30 min before Flow Cytometric analysis. Fluorescent signals were collected at FL-2 channel with by Accuri C6 (Thermofisher, USA) and analyzed with ModFit software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sphere formation assay\u003c/h2\u003e \u003cp\u003eSphere formation assay was performed as described before. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Cells were plated into ultra-low-attachment 12-well plate (Corning, USA) at a density of 1000 cells per well and cultured in serum-free DMEM medium supplemented with 20 ng/ml EGF (Peprotech, USA), 20 ng/ml FGF (Peprotech, USA) and 2% B27 (Gibco, USA). Drugs were added at the beginning of spheroid culture and medium was half refreshed every 3 days without new drug addition. Cell spheroids were observed and photographed by an inverted microscope (Olympus, Japan) after 10 to 14 days. Diameters of at least ten representative spheroids were measured and used for calculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA interference\u003c/h2\u003e \u003cp\u003eLAD cells were transiently transfected in 12-well plate using Lipofectamine 3000 (Invitrogen) according to manufacturer\u0026rsquo;s instructions. Chemically synthesized non-target siRNA as control and double-stranded siRNAs for Gli1 (Santa Cruz, sc-37911) were transfected in parallel at a concentration of 50 nmol. Cells were collected and counted 24 hours after transfection for sphere formation assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Reverse-transcriptase PCR (RT-PCR)\u003c/h2\u003e \u003cp\u003eRT-PCR were performed as described before. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] RNA was extracted with Trizol (TransGen Biotech, China) and reversely transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA) according to manufacturer\u0026rsquo;s instructions. Polymerase chain reactions were performed with 2\u0026times; Taq Master Mix (Vazyme, China). Primers for cell cycle regulators and SHH cascade components were used as published. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunoblot\u003c/h2\u003e \u003cp\u003eImmunoblot was performed as published before. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Total protein from cells was extracted and protein concentrations were quantified using a BCA assay kit (Thermo Scientific, USA). For plasma and nuclear protein separation, a cytoplasmic nuclear separation kit (Beyotime Biotechnology, China) was used. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were incubated with primary and secondary antibody sequentially at 4\u0026deg;C overnight and room temperature for one hour respectively. Enhanced chemiluminescence signals (ECL, Amersham) were detected using the Image Lab software. Antibodies used for detecting cell cycle arrest and SHH pathway were described before. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Immunofluorescence\u003c/h2\u003e \u003cp\u003eSlides covered with cells were washed twice with PBS and once with immunofluorescence PBS. Ice cold acetone was added to cover the slides and incubated for 8 min before washing again with PBS. GLI1 primary antibody (1:200; Santa Cruz) was incubated at room temperature for 2.5 h, washed twice with PBS, and stained at 37℃ for 2 h with a secondary antibody containing Alex Flour 568-Donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (1:200). Finally, the slides were sealed by DAPI and observed by fluorescence microscope (Olympus Corporation, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Living and dead cell staining\u003c/h2\u003e \u003cp\u003eThe cells were inoculated in 24-well plates with a density of 80%. After incubation overnight, NCTD drugs were added in different experimental groups at concentrations of 0, 4, 8, 16 \u0026micro;g/ml, respectively. After incubation for 48 hours, the cells were washed twice with PBS and stained with AM/PI probe respectively. Finally, the cells were photographed with fluorescence microscope (Olympus Corporation, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Drug release experiment\u003c/h2\u003e \u003cp\u003ePrepare 42 tubes NCTD@F127 and 5-FU@F127, each of which will be dissolved by mixing 0.1 mg NCTD/0.8 mg 5-FU with 100 \u0026micro;l F127 (20% w/v) ice bath. After incubation at 37\u0026deg;C to form a gel, 0.5ml normal saline was slowly added along the tube wall. The tubes were then incubated continuously at 37\u0026deg;C for two weeks. Three parts of the supernatant were taken every day and stored in -20. Finally, quantitative analysis was performed with high-performance liquid phase (Angilent, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Mice experiment\u003c/h2\u003e \u003cp\u003e3- to 5-week-old female BALB/c athymic (nu/nu) mice were purchased from Charles River Laboratories in Beijing. Mice were housed in a specific pathogen free (SPF) biosafety lab and maintained in accordance with institutional guidelines of Binzhou Medical University Hospital. All animal experiments were performed with the approval of Binzhou Medical University Hospital Committee for Animal Care. Mice were subcutaneously injected with 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e A549 cells and randomly divided into different groups (5 mice per group, a total of 30 mice) when diameter of palpable tumors reached approximately 5 mm. Xenografted mice received vehicle (50 \u0026micro;l 20% w/v F127), NCTD@F127 (2.5 mg/kg), 5-FU@F127 (1.5 mg/kg), Osimertinib@F127 (10mg/kg) or their combination (1.5 mg/kg 5-FU\u0026thinsp;+\u0026thinsp;2.5mg/kg NCTD@F127 or 10 mg/kg Osimertinib\u0026thinsp;+\u0026thinsp;2.5mg/kg NCTD@F127) injected subcutaneously once a week for three weeks. Body weight and tumor volume was measured every second day. Tumor volume was calculated as 0.5 \u0026times; a\u003csup\u003e2\u003c/sup\u003e \u0026times; b (a and b represent short and long diameter of tumor bulk respectively). Mice were euthanized after 3 weeks\u0026rsquo; treatment. Tumor tissues were collected from xenografts at the end of treatment and stained by immunohistochemistry.\u003c/p\u003e \u003cp\u003e All animal care and experimental protocols were approved by Binzhou Medical University Hospital Committee for Animal Care (Ethics approval number: 20231208-54).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eTumor tissue was fixed with 4% paraformaldehyde and paraffin-embedded sections were performed. The tissue sections were incubated with GLI1 antibody (santa cruz) and enzyme-labeled secondary antibody. The tissue was stained with diaminobenzidine and hematoxylin and imaged under light microscope (Olympus Corporation, Japan). Quantification and statistical analysis was performed by calculating the average number of GLI1-positive cells in three different fields per mouse (5 mice per group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed with Microsoft Excel and GraphPad Prism 7 and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The unpaired Student\u0026rsquo;s t test was used to compare two groups and one-way ANOVA test was used when compare multiple groups. Two-way ANOVA test was used for analyzing data from drug combinational treatment. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.01 and 0.001 was considered statistically significant and marked by one, two and three asterisks respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Anti-proliferative effect of NCTD in LAD cell lines\u003c/h2\u003e \u003cp\u003eAs a demethylated form of cantharidin (CTD), the chemical formula of NCTD was displayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To validate the anti-proliferative effect of NCTD in LAD cell lines, we firstly treated A549 and H1299 cells with various concentrations of NCTD. CCK8 assay disclosed that NCTD could remarkably prohibit A549 and H1299 cell expansion in a dose-dependent manner from 5 to 80 \u0026micro;g/ml. The half maximal inhibitory concentration (IC50) of NCTD in A549 and H1299 cells were 22.67 and 17.72 \u0026micro;g/ml respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). EDU staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), clonal formation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and statistical results also showed that NCTD inhibited proliferation of A549 and H1299 cells in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Induction of cell cycle arrest by NCTD in LAD cells\u003c/h2\u003e \u003cp\u003eTo unveil how NCTD exert anti-proliferative effect in LAD cells, we applied NCTD at distict concentrations for 48 hours followed by PI cell cycle staining. Cell cycle analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and histogram quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated that NCTD induced G1/S cycle arrest in A549 cells when applied at 4 and 8 \u0026micro;g/mL while induced G2/M cycle arrest when applied 16 \u0026micro;g/mL. For H1299, NCTD had marginal effect on cell cycle arrest when applied at below 8 \u0026micro;g/mL while induced G2/M cycle arrest when applied at 16 \u0026micro;g/mL. Further investigation of molecular mechanisms by RT-PCR indicated that NCTD significantly decreased expression of pivotal cell cycle regulators including Cyclin A, Cyclin B1, CDK2 and CDK4 in A549 cells dose dependently (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). For H1299, NCTD depressed transcription level of CDK2 and CDK4 when applied at high dose (16 \u0026micro;g/mL), which goes in line with PI staining data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Immunoblot assay of the protein expression level of the above cell cycle regulators confirmed RT-PCR result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 NCTD deactivates Hedgehog pathway in LAD cell lines\u003c/h2\u003e \u003cp\u003eOur previous study revealed that selective repression of Hedgehog cascade chain potentiated the anti-cancer efficacy of DDP for non-small lung cancer cells both in vitro and in vivo.11 Herein we examine whether NCTD abates the SHH signaling and employs this targeting mode to sensitize LAD cells to other chemotherapeutic drugs. RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and immunoblot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) assay indicated that NCTD depressed GLI1 expression at both transcriptional and translational levels, which implies that NCTD executes its function specifically through GLI1, the pivotal transcription factor of Hedgehog pathway. GLI1, as a key molecule in the Hedgehog pathway, is also its downstream molecule, enters the nucleus after dephosphorylation modification and then regulates its own expression. Therefore, we speculated that NCTD may inhibit the expression of its own target genes by blocking GLI1's entry into the nucleus. We then examined GLI1 expression and localization in cells 48 hours after NCTD treatment of LAD cells by immunoblotting and immunofluorescence assays. Immunoblotting showed that NCTD significantly reduced the expression of GlI1 in the nucleus compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Immunofluorescence staining also revealed that GLI1 was evenly distributed in the cells in the control group, and when LAD cells were treated with NCTD, GLI1 was mainly distributed around the nucleus in the cytoplasm, with the nuclear location apparently empty (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Therefore, NCTD can regulate the expression level of GLI1 by regulating GLI1 heterotopic in Hedgehog signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 GLI1 can antagonize the proliferation inhibition and apoptosis induction of LAD cells by NCTD\u003c/h2\u003e \u003cp\u003ePrevious experiments showed that NCTD inhibited LAD cell proliferation and GLI1 expression in a dose-dependent manner. To further verify that NCTD inhibits cell proliferation by inhibiting GLI1 expression, we screened LAD cell lines that stably overexpress GLI1 and used pcDNA3.1 as the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). First, the proliferation of LAD cells in pcDNA3.1 and GLI1 groups was evaluated after treatment with different concentrations of NCTD. Results of the CCK8 assay indicated that compared with pcDNA3.1, GLI1 overexpression significantly antagonized the inhibitory effect of NCTD on the growth of lung cancer cells. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, in vitro colony formation assay displayed that overexpression of GLI1 promotes the clonogenicity of LAD cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results of staining of living and dead cells corroborated that the number of apoptotic cells in the GLI1 overexpression group was significantly lower than that in the pcDNA3.1 group upon NCTD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicated that the inhibitory effect of NCTD on LAD cell proliferation was mainly achieved by inhibiting the expression of GLI1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Inhibition of DDP-resistant stemness-prone A549 spheroids by NCTD\u003c/h2\u003e \u003cp\u003eGLI1, the core transcription factor of SHH signaling cascade, has been reported to regulate tumor stemness, 28 one of its downstream targets which implies silencing SHH signaling may efficiently block the pluripotency sustenance in cancer cells. We have shown that NCTD selectively inhibits GLI1 expression in the Hedgehog signaling pathway. Next, we further explored whether NCTD affects the ability of LAD cells to form stem cell spheres by blocking the SHH signaling pathway. Our sphere formation assay disclosed that DDP at 4 and 8 \u0026micro;g/mL did not alter the sphere sizes and numbers from A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, NCTD at 8 \u0026micro;g/mL, robustly reduced A549 sphere formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). For specificity in interfering the cancer stemness with silencing SHH pathway, we applied siRNA against GLI1 and SMO inhibitor BMS to A549 cells and found that inhibition of either GLI1 or SMO significantly repressed cancer stemness sphere formation compared to their corresponding control samples (siRNA scramble and DMSO separately for GLI1 and SMO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).The SMO agonist Purmorphamine (PUR) had marginal effect on sphere size but remarkably increased sphere numbers in our settings (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 NCTD sensitizes LAD cells to conventional chemo-drug treatments in vitro\u003c/h2\u003e \u003cp\u003eThe stemness of tumor cells plays an important role in the formation of multidrug resistance and metastasis, and NCTD can regulate the stemness of LAD cells by inhibiting GLI1 expression in the Hedgehog pathway. Our previous research showed that selective inhibition of the Hedgehog cascade enhances the anti-cancer effects of DDP on non-small lung cancer cells in vitro and in vivo. Next, we investigated whether NCTD could sensitize the treatment of multiple chemotherapeutic agents in LAD cells. DDP, PTX, GEM ,5-FU, Gefitinib and Osimertinib, four conventional chemo drugs and two targeting agents commonly used in clinic for LAD therapy were tested. To explore their proper dose of anti- proliferative activity in lung cancer, we firstly applied them separately to our LAD cell lines A549 and H1299, respectively at various concentrations and detected their inhibition of cell proliferation. CCK8 assay revealed that all six chemo-drugs inhibited cell growth of A549 and H1299 in both dose-dependent manners (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Similar to other chemo-drugs, NCTD inhibited A549 growth dose-dependently but with a wider concentration range from 1 to 100 \u0026micro;g/mL, which indicated that NCTD is not as potent as commonly used chemotherapeutic regimens in clinic.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince NCTD alone is not a strong neoplastic cell killing agent, we further investigated whether NCTD could facilitate the anti-cancer effect of other chemo-drugs. Following co-treatment of NCTD (1 \u0026micro;g/ml) with these drugs in LAD cells, CCK8 assays demonstrated that NCTD significantly enhanced anti-proliferation effect of 5-FU, GEM and Gefitinib in A549 cells, while additionally aggravating the growth inhibition mediated by PTX, 5-FU, Gefitinib and Osimertinib respectively in H1299 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Sequential co-application of NCTD with various chemo-drugs to LAD cells revealed that NCTD robustly sensitized both A549 and H1299 to multiple drug therapies which are commonly administered as the firstly line of chemotherapy for LAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The above experimental results suggest that NCTD can be used as a chemotherapy sensitizer and will play a better therapeutic effect on tumors when combined with other chemotherapy drugs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 NCTD magnifies the in vivo anti-tumor efficacy of 5-FU and Osimertinib\u003c/h2\u003e \u003cp\u003eIn order to further explore the chemotherapy sensitization effect of NCTD in vivo, we tested 5-FU and Osimertinib as representative of chemo and targeting drug respectively in A549 subcutaneously implanted mice model. To achieve better therapeutic efficacy, F127 was used as a drug carrier to wrap different drugs prolong drug release in vivo. Mice were treated with F127 as control, NCTD@F127 (2.5 mg/kg), 5-FU@F127 (20 mg/kg), Osimertinib@F127 (10 mg/kg), or their combination (5-FU\u0026thinsp;+\u0026thinsp;NCTD@F127, Osimertinib\u0026thinsp;+\u0026thinsp;NCTD@F127). According to the results of in vitro drug release experiment, the drug was completely released in about one week, so the drug was injected into the tumor once a week for three consecutive weeks (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Tumor images and growth curves show that NCTD remarkably increases the therapeutic efficacy of 5-FU and Osimertinib (Fig. S2, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). Osimertinib\u0026thinsp;+\u0026thinsp;NCTD@F127 was the most effective combinational therapy, followed by 5-FU\u0026thinsp;+\u0026thinsp;NCTD@F127. The single treatment group also had a certain therapeutic effect, but not as obvious as the combined treatment. There was no significant change in the body weight of mice in each treatment group, indicating that the combination of drugs significantly improved the efficiency of treatment and did not produce toxic side effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Immunohistochemistry showed that GLI1 expression was decreased in all treatment groups compared with F127 group. The effect of NCTD\u0026thinsp;+\u0026thinsp;5-FU@F127 and NCTD\u0026thinsp;+\u0026thinsp;Osimertinib@F127 combined treatment group was more significant than that of NCTD@F127,5-FU@F127 and Osimertinib@F127 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E) single administered group. The combination of NCTD with 5-FU and Osimertinib improve the efficacy of chemotherapy, indicating that NCTD can be used as a chemotherapy sensitizer for the treatment of tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eRapid discovery and developments of novel targeting inhibitors against EGFR and ALK have not replaced conventional chemotherapy, particularly cisplatin-based regimens serving as a first line therapeutic strategy for LAD patient treatment in clinic. In the current study, we unveiled the potent role of NCTD in prohibiting LAD stem cell proliferation and sensitizing the LAD cells to those four commonly used chemo-regimens including DDP, PTX, GEM and 5-FU as well as two targeted drugs Gefitinib and Osimertinib in vitro. In vivo study further confirmed that NCTD abated tumor growth of LAD cells and significantly promoted the therapeutic efficacy of both chemo and targeting drugs. Mechanistic exploration deciphered that NCTD exert selective tumor-arresting functions in LAD cells by nuclear translocation and activation of GLI1, the critical transcription factor of Hedgehog cascade (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been reported that NCTD boosted therapy efficacy of conventional chemotherapy in hepatic carcinoma, breast cancer and bladder cancer cells. [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] In addition, NCTD could enhance the anticancer activity of gefitinib and cisplatin in NSCLC parental cells while the underlying molecular mechanisms remain elusive. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Here we further unveiled its potent efficacy in coordinating with multi-drug by inhibiting GLI1 mediated LAD cancer stemness. NCTD prohibited nuclear translocation of GLI1 and activation of its subexpression which leads to multidrug resistance. Data regarding overexpression of GLI1 counteracted anticancer efficacy of NCTD further verified that NCTD attenuated stemness related features of LAD cells by specifically targeting GLI1. It has been demonstrated that aberrant activation of GLI1 signaling augmented stemness features of lung and gastric cancer cells.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Our previous drug screening using well-established LAD cell lines for Hedgehog cascade inhibitors also discovered that molecules targeting GLI1 exerted robust anti-stemness capacity.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] In epithelial ovarian cancer, GLI1 was found to modulate drug sensitivity by directly binding to the drug efflux ATP-binding cassette (ABC) transporters including ABCB1 and ABCG2,[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reinforcing its role as a drug susceptibility upstream regulator. Our previous wok identified another Chinese herb, Scutellariabarbata D. Don extraction (SBE) as a Hedgehog inhibitor which inhibited the whole Hedgehog cascade by targeting SMO. Different from SBE, data in this article indicated that NCTD specifically downregulate expression of GLI1, the terminal executor of Hedgehog cascade that located downstream of SMO therefore may function better in SMO inhibitor resistant scenario.\u003c/p\u003e \u003cp\u003eFurthermore, NCTD has been reported to disrupt epithelial-mesenchymal transition and metastasis in colon cancer, lung cancer and seldom developed mucoepidermoid carcinoma.[\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Our previous in vitro studies also corroborated the anti-invasive and -metastatic effect of NCTD on LAD cells mediated by transcription factor YAP.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Since no observable distal organ metastasis was found in subcutaneously implanted lung cancer model in all experimental groups in our setting, as an extension of in vitro findings, investigation of anti-metastatic effect of NCTD using tail injection established lung cancer model might be used in the future.\u003c/p\u003e \u003cp\u003eOf note, although in vivo tumor growth curve and tumor weight indicated that NCTD facilitate 5-FU and Osimertinib, no additive adverse effect was observed by combinatory therapy. Additionally, pilot efforts have been made to boost anti-tumor potent of NCTD while soft its accompanying side effect by modification of the compound backbone itself or drug delivery system. [\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] In brief, our study demonstrated that NCTD sensitized LAD cells to chemo-drug treatment through repressing GLI1 mediated cancer stemness properties. This adds more experimental evidence that NCTD may serve as a multi-drug sensitizer to potentially facilitate LAD therapy in clinic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated or analyzed during the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (31900441, 82373097), Natural Science Foundation of Shandong Province (ZR2019MC026, ZR2023QH080, ZR2023MH126 and ZR2022QH192), Funds of Shandong traditional Chinese Medicine Science and Technology Development Project (2019-0514 and 2019WS321), Binzhou Medical University Research Program (BY2022KJ64), Qilu Outstanding Young Talents in Health Project, Taishan Scholarship and Double-Hundred Project.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Medical Research Center, Binzhou Medical University Hospital, Binzhou, P.R. China\u003c/p\u003e\n\u003cp\u003eBing-jie Cui, Xin Zhang, Fei Wang, Hong-liang Dong, Cuilan Liu, Wei-wei Chen, Jiong Deng, Yan Wu, Jing Du\u003c/p\u003e\n\u003cp\u003eDepartment of Hematology Oncology, Binzhou Medical University Hospital, Binzhou, P.R. China\u003c/p\u003e\n\u003cp\u003eXin Zhang\u003c/p\u003e\n\u003cp\u003eClinical Laboratory, Binzhou Medical University Hospital, Binzhou, P.R. China\u003c/p\u003e\n\u003cp\u003eWei-wei Chen\u003c/p\u003e\n\u003cp\u003eDepartment of Oncology, Binzhou Medical University Hospital, Binzhou, P.R. China\u003c/p\u003e\n\u003cp\u003eYan Wu, Jing Du\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Du, Yan Wu contributed to the conception of the study; Bingjie Cui, Xin Zhang performed the experiment; Jing Du, Bingjie Cui contributed significantly to analysis and manuscript preparation; Jing Du, Bingjie Cui performed the data analyses and wrote the manuscript; Fei Wang, Hongliang Dong, Cuilan Liu, Weiwei Chen, Jiong Deng helped perform the analysis with constructive discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Du, Medical Research Center, Binzhou Medical University Hospital, Binzhou, China. Email: [email protected]\u003c/p\u003e\n\u003cp\u003eYan Wu, Medical Research Center, Binzhou Medical University Hospital, Binzhou, China. Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All animal experiments were performed with the approval of Binzhou Medical University Hospital Committee for Animal Care，ethics review number is 20231208-51.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026apos;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional file 1\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdorno-Cruz V, Kibria G, Liu X, Doherty M, Junk DJ, Guan D\u003cem\u003e et al\u003c/em\u003e. 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Oncology letters 2019; 17: 456-461.\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":"[email protected]","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":"","lastPublishedDoi":"10.21203/rs.3.rs-4761721/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4761721/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNorcantharidin (NCTD), a demethylated analog of cantharidin, has been identified as one of potential anti-tumor drug candidates in various human neoplasms. However, the NCTD-mediated interference with multidrug-resistance development and sustenance of lung adenocarcinoma (LAD) and its underlying molecular interaction mechanisms remains undefined yet. In this study, NCTD significantly inhibited cell growth of LAD cells in a dose-dependent manner when applied alone and magnified the sensitization of LAD cells to multiple therapeutic agents. Selective repression of sonic Hedgehog (SHH) signaling pathway by NCTD dramatically arrested cancer stemness development and maintenance such as the sphere formation capacities of LAD cells. Mechanistic analysis revealed that NCTD prohibited nuclear translocation of GLI1, the key terminal transcription factor of SHH cascade in LAD cells. In vivo studies confirmed that NCTD alone reduced propagation of LAD cells and enhanced the 5-FU and Osimertinib-based cancer progression inhibition while have no side effect on body weight. Taken together, our results demonstrate that NCTD represses SHH cascade-mediated cancer stemness to overcome the intrinsic resistance of LAD cells to multi-drug treatment, which implies that NCTD might be a therapeutic drug candidate that could be a de novo option to eradicate the treatment resistance against multiple therapeutic agents if co-applied in LAD treatment clinically.\u003c/p\u003e","manuscriptTitle":"Norcantharidin Sensitizes Lung Adenocarcinoma Cells to Multitherapy via Targeting the Deregulated Hedgehog Cascade","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-20 10:58:49","doi":"10.21203/rs.3.rs-4761721/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"cc99351d-3a32-4c4d-891c-bb7ddaec48a9","owner":[],"postedDate":"August 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-01T05:38:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-20 10:58:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4761721","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4761721","identity":"rs-4761721","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}