A Naphthoquinone Compound Triggers Dna Damage-induced Apoptosis on Cholangiocarcinoma Through Upregulation of Bax and DR5

A Naphthoquinone Compound Triggers Dna Damage-induced Apoptosis on Cholangiocarcinoma Through Upregulation of Bax and DR5 | 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 A Naphthoquinone Compound Triggers Dna Damage-induced Apoptosis on Cholangiocarcinoma Through Upregulation of Bax and DR5 Gizem Bulut, Merve Kayis, Dilan Gezer, Zeliha Gokmen, Zelal Adiguzel, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6395590/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jul, 2025 Read the published version in Medical Oncology → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Cholangiocarcinoma (CCA), a devastating malignancy originating from the bile ducts, is of significant clinical importance due to its rising incidence and poor prognosis. Quinones as being naturally occurring compounds and their being highly used in anticancer drug development studies seem to be potential sources for this aim. In this study, a synthetic naphthoquinone derivative, MK13, has been tested against CCA (CCLP1 and HUCCT1) cell lines. Methods: Cell viability and proliferation rate were measured with MTT assay at the doses ranged from 1.56 to 50 µM for 48h treatment. Apoptotic cell death was showed morphologically with fluorescent double staining and biochemically with flow cytometry analysis of phosphatidylserine translocation. Oxidative stress and DNA damage were also measured with flow cytometry while gene expressions were interpreted via qPCR analysis. Results: MK13 exhibited strong antigrowth activity, especially against CCLP1 cells. Cell death resulted from apoptosis that was shown to be triggered by severe DNA damage which is independent of oxidative stress. Apoptosis was confirmed at molecular level with the upregulation of BAX , a proapoptotic BH-3 only protein, and DR5 , a cell surface death receptor. Conclusion: MK13 seems to be a promising anticancer compound against CCA and deserves further in vivo studies for proof of concept. anticancer drug bile duct cancer chemotherapy cell death quinones Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cholangiocarcinoma (CCA), a malignant tumor arising from the epithelial cells of the bile ducts, is mentioned as bile duct cancer. Carcinoma of bile duct is classified in three anatomic primary forms: intrahepatic (iCCA), peripheral and distal (together extrahepatic eCCA). The iCCA originates within the liver, peripheral occurs at the hepatic hilum where the bile ducts exit the liver, and distal CCA is found outside the liver [ 1 ]. This classification is crucial as it affects diagnosis, treatment strategies, and patient outcomes. The global and regional incidence of CCA is increasing, with epidemiological data revealing rising incidence and mortality rates, a trend that is alarming given the complexity of this disease especially for intrahepatic CCA [ 2 , 3 , 4 ]. The clinical significance of CCA is profound due to its severe impact on patient health and quality of life. The disease is often diagnosed at an advanced stage, which complicates treatment and worsens prognosis. The prognosis for CCA is generally poor, characterized by low survival rates compared to other malignancies. Current survival statistics reflect these challenges, with median survival times varying depending on the type and stage of the cancer, yet, due to the late diagnosis, the overall survival rate for CCA is dramatically lower, less than 2 years [ 5 , 6 , 7 ]. In terms of treatment, the landscape includes surgical interventions, chemotherapy, and radiation therapy. Surgical options, while potentially curative, are limited by high recurrence rates and the difficulty of achieving complete resection. Chemotherapy, a standard treatment approach, includes several drugs such as gemcitabine, cisplatin, oxaliplatin, and 5-fluorouracil. Gemcitabine combined with cisplatin is one of the most commonly used regimens, though the effectiveness varies and is often accompanied by significant side effects, including nausea, fatigue, and myelosuppression. Each of these treatments has limitations that contribute to the overall poor prognosis for patients. The need for novel therapeutic strategies is pressing, driven by the current gaps in treatment efficacy and the challenges associated with early detection [ 8 ]. The lack of effective targeted therapies for CCA, coupled with difficulties in diagnosing the disease at a manageable stage, underscores the urgent need for new research and innovative approaches. Naphthoquinones (NQs), a diverse group of compounds characterized by their naphthalene ring structure with two adjacent carbonyl groups, are gaining attention as potential therapeutics in cancer research. These compounds are secondary metabolites found naturally in various plants, fungi, and bacteria, where they often contribute to the organism’s defense mechanisms [ 9 ]. Their chemical structure, comprising a fused aromatic ring system with two carbonyl groups, imparts unique chemical reactivity and biological activity. Historically, naphthoquinones have been utilized in traditional medicine, but contemporary research has expanded their potential applications, particularly in oncology [ 10 ]. In anticancer research, naphthoquinones have shown promise due to their ability to interfere with critical cellular processes and signaling pathways. Today there are some examples of quinone derivatives used as chemotherapeutic drugs such as Adriamycin (Doxorubicin), Mitomycin C, and Daunomycin. NQs are known to exert their effects through various mechanisms, including the induction of oxidative stress, modulation of apoptosis pathways, and acting as topoisomerase II inhibitors [ 11 , 12 , 13 ]. These mechanisms disrupt the growth and survival of cancer cells, making naphthoquinones attractive candidates for targeted therapy. There are studies conducted with our research groups for investigation of anti-cancer activities of different NQ derivatives which highlights the potential of these group of compounds as potential chemotherapy drugs [ 14 , 15 , 16 ]. Recent literature highlights the growing interest in naphthoquinones as potential treatments for CCA. Studies have demonstrated that certain naphthoquinone derivatives can inhibit the proliferation of CCA cells, induce apoptosis, and interfere with critical signaling pathways involved in tumor growth and resistance. For instance, compound plumbagin has shown efficacy in preclinical models by targeting specific molecular pathways associated with CCA progression [ 17 ]. These findings suggest that naphthoquinones could play a significant role in the development of novel therapeutic strategies for CCA, offering new avenues for treatment where conventional therapies may fall short. This study aims to investigate the anticancer effect of MK13, a synthetic NQ derivative that has already been documented for its synthesis and characterization, on CCA cell lines. [ 14 ] MK13, a 1,4-naphthoquinone derivative, was obtained by the nucleophilic displacement reaction that has been shown in previous studies. [ 14 , 18 , 19 ] It has been found that MK13 induces cell death by apoptosis resulting from DNA damage independent of ROS, deserving further attention for in vivo proof-of-concept studies. 2. Materials and Methods Cell culture Human cholangiocarcinoma (CCA) cell lines HUCCT1 and CCLP1 were cultured with RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin. Cells were incubated at 37°C and 5% CO2 condition. When they reached 80% confluency cells were collected with 0.5% trypsin-EDTA (Gibco, USA) for subculturing and seeding. Cytotoxicity and growth rate measurements Cells were seeded to 96-well culture plates as 5x103 cell/well and incubated overnight for attachment. Next day, cells were treated with different doses of MK13 in the range of 1.56 to 50 µM as 2-fold dilutions. At the end of 48h treatment MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) viability assay was conducted to measure cell viability. To measure growth rate, another 96-well plate was seeded with the same condition of cells and the next day during the treatment procedure, this well was subjected to MTT assay. The absorbance values obtained from this plate, time zero (Tz), was used for the calculation of growth rate as previously explained [ 20 ]. MTT crystals were solubilized by using 0.01 M HCl 10% SDS (w/v). Absorbance was measured as OD by using LumiStar Omega Spectrophotometry, BMG Labtech. Morphological evaluations with fluorescent staining To investigate the mode of the cell death caused by MK13, cells were stained with Hoechst 33342 dye and propidium iodide as two nuclear dyes. Cells were treated with IC 90 dose of the compound and at the end of 12–24 and 48h incubation period double staining was performed as previously described [ 21 ]. The cells were visualized by using Zeiss Axio Observer fluorescent microscope and images were taken by using Zen Blue Software. Flow Cytometry Analyses : To gain deeper understanding of the cellular events triggered by MK13 treatment, flow cytometry analysis was performed. CCLP1 cells were seeded to 6-well culture plates as 3x105 cell/well and next day treated with IC 90 dose for the compound. For the flow cytometry Guava Muse Cell Analyzer device was used and the fallowing kits were performed according to producer’s guidelines: Annexin V & Dead Cell (Cat. No. MCH100105), Oxidative Stress kit (MCH100111) and γH2AX Activation (MCH200105) Kit (FCCS025153). Gene expression analysis with qPCR CCLP1 cells were seeded on 6-well plates and the next day treated with IC 90 dose and harvested for RNA isolation after 6h of treatment with the MK13. Cellular RNA was isolated by using the NucleoSpin RNA Mini kit (MN Macherey-Nagel, Germany). Isolated RNA quality and quantity was measured by Nanodrop (Invitrogen, USA) and 1 microgram of each total RNA sample was used for cDNA synthesis with the iScript cDNA Synthesis Kit (BioRad, Germany). For the gene expression analysis, qPCR method was used, 10 ng cDNA was used as template for each reaction that contains QuantiNova Syber Green Mix (Qiagen, Germany) and Applied Biosystem QuantStudio (Thermo Fisher Scientific, USA) device was used. Sample setup and analysis were accomplished using the macro file for each gene panel with the Light-Cycler 480 Software 1.5. Results were analyzed by using ΔΔCT method. GAPDH was used as a housekeeping gene and fold change of expression was calculated according to it. Selected genes were classified into three groups as prosurvival genes ( AKT2F, AURKA, BAG4, BCL2, BCL2A1, BCL2L2, BCL2L10, BCL10, BCLXL, BIRC3, BIRC4, BIRC5, BIRC6, BMI1, CDC2, CDC20, CDK4, CDC25A, CDK2, MDM2, MCL1, NEK2, GRB2, TNFRSF11B), proapoptotic genes (APAF1, BAD, BAX, BCLAF1, BAK1, BID, BIK, BIM, BMF, BNIP1, CASP2, CASP3, CASP4, CASP6, CASP8, CASP9, DR4, DR5, HRK, FAS, PUMA, XPA) and cellular stress related genes (CAT, BAG3, ERCC1, ERCC3, GADD45A, XRCC5, NOXA, MUDYH, NFKB, RAD52, LIG4, OGG1, SOD1, PRDX1, BECN1, GPX1, RAD51, GSTP1, BNIP2, BNIP3, BNIP3L, SIRT2, RIPK2 ). Statistical analysis Two-way ANOVA was used for comparing the viability results with the control group. All statistical analyses were conducted using GraphPad Prism. Data for each independent replicate is presented as mean ± standard deviation. 3. Results 3.1. MK13 has a cytotoxic effect in both CCA cell lines In order to investigate the anticancer capacity of the MK13 compound, dose response analysis was performed with differential doses ranging between 1.56 to 50 µM via MTT viability assay (Fig. 1 a). Results showed that both cell lines were responsive for the MK13 compound distinctly from each other. While HUCCT1 cells responded slowly by increased doses CCLP1 cells suddenly diminished at lower doses. Interestingly, the higher doses as 25–50 µM had the same effect on both cell line as about 80% decrease in viability. In the case of CCLP1, this could be a result of the fact that slightly higher doses could trigger adaptation to particular type of persistent stress and triggers respectively higher proliferative rates. Therefore, even in the range of 3–12 µM MK13 has highly toxic effects, the increased doses could evoke a particular adaptation mechanism. These kinds of dose response curves are named as non-monotonic dose response curves and could be concluded from different phases of responses such as bi-phasic curve of CCLP1 [ 22 ]. Indeed, this bi-phasic response was also observed in growth rate curve (Fig. 1 b). Overall, CCLP1 cell line was found to be more responsive with lower IC 50 and GI 50 value (0.9 µM and 0.5 µM) with almost 10-fold lower than that of HUCCT1 (10.1 µM and 5 µM) (Table 1 ). Table 1 Calculated doses for CCA cell lines treated with MK13 for 48h. MK13 (µM) HUCCT1 CCLP1 IC 50 10.1 0.9 IC 90 >50 3.3 GI 50 5 0.5 TGI 11.8 1.1 LD 50 >50 1.9 3.2. MK13 triggers apoptotic cell death on both cell line Observational experiment for the MK13 induced cell death was designed by using fluorescent staining methodology. For this purpose, CCLP1 and HUCCT1 cells were treated with IC 90 values of MK13 (3.3 µM and 50 µM) and fluorescently stained after 12-24-48h incubation period. Two nuclear fluorescent dye, Hoechst 33342 and propidium iodide (PI) was used to distinguish apoptotic and necrotic nuclear morphology. Hoechst 33342 dye is a membrane permeable nuclear dye while PI cannot pass through membrane therefore only stain the nuclei of the calls have their membrane integrity lost. Therefore, the nuclei stained with PI are necrotic cells. When cells are dying with apoptosis, nucleus become smaller, and chromatin get condensed then followed with chromatin fragmentation [ 23 ]. This phenomenon is known as pyknosis, and the nucleus show this morphology called pyknotic nucleus. Thus, if a cell is positive for both dye and show pyknotic morphology, this indicates another stage of apoptosis; secondary necrosis, which can be observed at cell culture where efferocytosis cannot take place [ 24 ]. MK13 treatment of CCLP1 cells resulted with increased number of pyknotic nuclei in a time dependent manner, which indicates the apoptotic cell death morphology. Nuclear fragmentation, called karyorrhexis is also observed. Moreover, the cell confluence was significantly decreased after MK13 treatment with time compared to the control (Fig. 2 and Fig. 3 ). Fluorescent images demonstrate that the treatment of these cells with MK13 triggers apoptotic cell death, evident with the pycnotic nucleus. The number of pycnotic nucleus was observed to be increased in a time dependent manner while total number of cells was significantly decreased in treatment groups. In addition to that, after 24h incubation seconder necrosis was observed as pycnotic nucleus positive for PI signal. For HUCCT1 cell line, the cell death mode is found to be a bit different from CCLP1. These cells have pyknotic nuclei but did not undergo seconder necrosis where cell membrane integrity is lost. Lots of cells are found to be in an early apoptotic stage with low number of PI positive seconder necrotic cells (Fig. 3 ). 3.3. Apoptosis triggered by MK13 is caused by DNA Damage which is independent of oxidative stress Flow cytometry analysis was performed in order to understand the cellular responses to the MK13 treatment. Thus, cells were treated with IC 90 dose of MK13 and collected after 12h and 24h of treatment. Phosphatidylserine translocation showed that one-fourth of the cells were in late apoptotic stages while total apoptotic cell number was more than half of the cell population after 12h treatment (Fig. 4 a). Even though there was no dramatic change between 12h and 24h treatment for the total apoptotic cell number, at any time point there were almost no dead cells without phosphatidylserine translocation. Which indicates that MK13 treatment does not cause random or catastrophic death such as necrosis. Most of the naphthoquinone derivatives have shown to be responsible for sudden ROS generation and followed DNA damage [ 25 , 26 , 27 ]. After 24h of treatment both early and late apoptotic populations were observed to be increased which indicates the biological time-course apoptotic cell death. Next, oxidative stress and DNA damage levels were evaluated with flow cytometry to investigate if these have a play in MK13 dependent cytotoxicity. For the oxidative stress aspect, there was no excessive ROS generation either at 12h nor 24h treatment (8.47% and 5.84% respectively) (Fig. 4 b). However, for DNA damage measurement via H2AX phosphorylation activation status, DNA damage was accumulated as the treatment duration increased 12h to 24h (22.6–36.90%) (Fig. 4 c). It can be concluded that the DNA damage caused by MK13 was not associated with ROS generation, otherwise, it could be direct interaction of MK13 to the DNA and forming DNA crosslinks. 3.4. Proapototic and prosurvival genes are differentially regulated after MK13 exposure To evaluate gene expression regulations after MK13 treatment qPCR analysis was performed. Investigated genes for qPCR were divided into three groups: prosurvival, proapoptotic and other cellular stress related genes. After 6h treatment for IC 90 dose of MK13, qPCR was performed (Fig. 5 A and Fig. 6 ). According to the results most of the prosurvival genes are observed to be downregulated. On the other hand, proapototic genes were shown to be affected from MK13 treatment even for that short period of treatment. APAF1, PUMA, NOXA, BAX and most importantly DR5 were upregulated while CASP2, CASP6, CASP8, BCLAF1, BID, BIK were downregulated (Fig. 5 b and Fig. 6 ). The oxidative stress genes tested were not significantly affected from MK13 exposure as DNA damage repair related genes ERCC3, XRCC5, RAD51, RAD52 except for ERCC1 (Fig. 5 c and Fig. 6 ). In summary, the genes were significantly affected from the MK13 treatment were found to be upregulated BAX and DR5 . 4. Discussion CCA remains a highly aggressive cancer with limited treatment options and poor patient outcomes, emphasizing the need for novel therapeutic agents. Naphthoquinone derivatives have garnered interest for their diverse anticancer mechanisms, including pro-apoptotic and oxidative stress-inducing activities. In this study, we explored the anticancer potential of MK13, a naphthoquinone derivative, in CCA. Two cell lines used for this study, CCLP1 and HUCCT1, were observed to be both responsive to the MK13 treatment, but the compound is found to be more effective on CCLP1 in terms of both cell viability and proliferation (Fig. 1 ). The selective behavior of MK13 is distinguishable especially on lower doses ranging from 1.56 µM to 12.5 µM and also reflected in the IC 50 and GI 50 values. Thus, the following experiments to understand the anti-cancer activity of MK13 were conducted on CCLP1 cell line. The Hoechst 33342 and PI double staining revealed that the cells treated with IC 90 dose of MK13 showed apoptotic nuclear morphology which are chromatin condensation and pyknosis (Fig. 2 ) [ 21 ]. Moreover, the number of apoptotic nuclei increased with the incubation time, alongside the number of PI positive nuclei indicates seconder necrosis [ 23 , 24 ]. These findings were also confirmed via flow cytometry with phosphatidylserine translocation (Fig. 4 a). Both cell lines have distinct TP53 mutations that result in different outcomes for drug responding. CCLP1 cells harbor R306X missense mutation that results in premature termination codon (PTC) thus, truncated protein will be eliminated through non-sense mediated decay [ 25 , 26 ]. On the other hand, HUCCT1 cells have R175H structural mutant classified as gain-of-function mutation [ 27 ]. This mutation increases cells susceptibility to become cancerous via different mechanisms. Cells carrying this mutant are defective for G2/M cell cycle check point and progress to mitosis bypassing the DNA damage and they become more resistant to apoptosis [ 28 , 29 , 30 ]. As a dominant-negative mutation p53 R175H mutant protein also interferes with wild-type p53 to function and regulate the expression of its target gene [ 31 ]. NQs exert their anticancer activity by triggering apoptosis through oxidative stress due to dramatic accumulation of intracellular ROS, as in the case of plumbagin and many other synthetic NQ derivatives [ 32 , 33 , 34 , 35 , 36 , 37 ]. However, the flow cytometry results counteract this general fact, because CCLP1 cells treated with IC 90 dose of MK13 were not suffered from robust ROS generation (Fig. 4 b). In an unpublished study of our research group, we reported an almost 10-fold increase of ROS in the same cell line treated with another synthetic NQ derivative. Here, the level of ROS was considered biologically non-significant in the scope of our experience to be responsible for observed apoptosis. Moreover, it should be noted that some studies have shown that quinones may act as antioxidants, so it is not unexpected to observe no oxidative stress in MK13-treated cells. Some plant extracts containing diverse quinone derivatives showed antioxidant characteristics together with strong anti-cancer activities [ 38 , 39 ]. On the other hand, CCLP1 cells shown to be suffering from DNA damage showed via flow cytometry using H2AX phosphorylation which was increased along with the incubation time (Fig. 4 c). Most naphthoquinones are known to induce DNA damage [ 40 ]. Interestingly here the damage is not caused by ROS accumulation but could be direct interaction of MK13 to the DNA itself inside the nucleus such as a quinone derivative chemotherapy drug doxorubicin [ 41 ]. This was also supported by the upregulation of GADD45A , that plays a pivotal role in the DNA damage induced cellular responses. GADD45A plays a role in drug response by modulating the cellular stress response and apoptosis. Its expression has been linked to enhanced chemo sensitivity in various types of cancer, including melanoma, where downregulation of GADD45A was associated with increased sensitivity to cisplatin [ 42 ]. Additionally, GADD45A interacts with other proteins involved in the apoptotic pathway, such as p53 and PCNA, thereby influencing the cellular response to DNA damage and stress [ 43 ]. The interplay between GADD45A and BAX is particularly noteworthy; GADD45A can influence the expression and activity of BAX, thereby affecting the apoptotic threshold of cancer cells [ 40 ]. Here, the p53 mutation status of these cell lines presents a significant insight into the prediction of a cell line to that kind of DNA intercalating agents [ 28 , 29 ]. The truncated p53 baring CCLP1 cells are more responsive to MK13 at dramatically lower doses and devastating DNA damage was observed after treatment. The responsiveness of this cell line therefore can be lined up with its DNA damage response-deficiency due to p53R306X mutation. Moreover, DR5 is the most affected gene that showed significant increase for expression after 6h treatment with MK13 together with BAX . These results imply that cells undergo intrinsic apoptosis shown by BAX upregulation (Fig. 5 ) beside BCL2 downregulation (Fig. 5 b) [ 42 , 44 ]. The relationship between the DR5 (Death Receptor 5) and BAX (Bcl-2-associated X protein) is pivotal in understanding drug response, particularly in the context of apoptosis in cancer therapy. DR5 is a receptor that mediates apoptosis through the extrinsic pathway, while BAX is a pro-apoptotic member of the Bcl-2 family that plays a crucial role in mitochondrial-mediated intrinsic apoptosis. Together with intrinsic apoptosis, cells might become more sensitive to extrinsic apoptosis due to the DR5 upregulation after MK13 treatment. A similar result was observed with a study done with doxorubicin in lung and colorectal cancer cell lines that has upregulation of DR5 after doxorubicin, and combination with DR5 agonist resulted in improved cytotoxic activity [ 45 , 46 ]. More studies have demonstrated that the upregulation of DR5 can enhance the apoptotic response of cancer cells to various treatments. For instance, gefitinib, an EGFR inhibitor, has been shown to upregulate DR5 expression, which subsequently increases the sensitivity of non-small cell lung cancer (NSCLC) cells to extrinsic apoptosis. This process is linked to BAX activation, leading to enhanced apoptosis [ 47 ]. Similarly, the combination of triterpenoid pristimerin with taxol has been reported to induce cell death in cervical cancer cells through a mechanism involving the upregulation of DR5 and the activation of BAX, highlighting the interconnected roles of these proteins in mediating drug responses [ 48 ]. Moreover, the expression of DR5 has been correlated with the efficacy of chemotherapy in NSCLC patients, where high levels of DR5 and its interaction with c-FLIP proteins were associated with improved drug-induced apoptosis [ 49 , 50 ]. This suggests that DR5 not only serves as a marker for drug response but also plays an active role in mediating the apoptotic effects of chemotherapeutic agents through pathways involving BAX. Thus, the MK13 not only triggers intrinsic apoptosis pathway and cause cell death but also sensitize CCLP1 cells to possible external stimuli to induce extrinsic apoptosis pathway. Therefore, it enables new possible combinational therapy strategies. This study underlines the anticancer therapy potential of MK13 as an inducer of intrinsic apoptosis and demonstrates that MK13 makes cells more vulnerable for DR5-associated extrinsic apoptosis on CCA. Future studies should be conducted as co-treatment or sequential treatment strategies with DR5 agonist. Declarations Funding This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa (Project numbers: FBA-2024-37858). Conflicts of Interest : The authors declare no conflicts of interest. Author Contribution Conceptualization, Zeliha GOKMEN and Engin ULUKAYA; Data curation, Gizem BULUT, Merve KAYIS and Dilan GEZEN; Investigation, Chiara RAGGI and Engin ULUKAYA; Methodology, Zelal ADIGUZEL, Chiara RAGGI and Engin ULUKAYA; Resources, Zelal ADIGUZEL and Engin ULUKAYA; Supervision, Engin ULUKAYA; Visualization, Gizem BULUT; Writing – original draft, Gizem BULUT; Writing – review & editing, Gizem BULUT, Zelal ADIGUZEL, Chiara RAGGI and Engin ULUKAYA. Acknowledgement This publication is based upon work from the European Network for the Study of Cholangiocarcinoma and the COST Action Precision-BTC-Network CA22125, supported by COST (European Cooperation in Science and Technology; www.cost.eu). 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Jabbar AA, Ibrahim IAA, Abdullah FO, Aziz KF, Alzahrani AR, Abdulla MA. Chemopreventive effects of Onosma mutabilis against azoxymethane-induced colon cancer in rats via amendment of Bax/Bcl-2 and NF-κB signaling pathways. Curr Issues Mol Biol. 2023;45:885–902. https://doi.org/10.3390/cimb45020057 . Artykov AA, Belov DA, Shipunova VO, Trushina DB, Deyev SM, Dolgikh DA, et al. Chemotherapeutic agents sensitize resistant cancer cells to the DR5-specific variant DR5-B more efficiently than to TRAIL by modulating the surface expression of death and decoy receptors. Cancers. 2020;12(5):1129. https://doi.org/10.3390/cancers12051129 . Yang PY, Hu DN, Kao YH, Lin IC, Chou CY, Wu YC. Norcantharidin induces apoptosis in human prostate cancer cells through both intrinsic and extrinsic pathways. Pharmacol Rep. 2016;68(5):874–80. https://doi.org/10.1016/j.pharep.2016.04.010 . Dong Y, Yang G, Deng H, Chen W, An G. Gefitinib upregulates death receptor 5 expression to mediate rmhtrail-induced apoptosis in gefitinib-sensitive nsclc cell line. Oncotargets Ther. 2015;8:1603–10. https://doi.org/10.2147/ott.s73731 . Eum D, Byun J, Yoon C, Seo W, Park K, Lee J, et al. Triterpenoid pristimerin synergizes with taxol to induce cervical cancer cell death through reactive oxygen species-mediated mitochondrial dysfunction. Anticancer Drugs. 2011;22(8):763–73. https://doi.org/10.1097/CAD.0b013e328347181a . Zheng H, Zhang Y, Zhan Y, Liu S, Lu J, Wen Q, et al. Expression of DR5 and c flip proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy. Oncol Rep. 2019;42(6):2363–70. https://doi.org/10.3892/or.2019.7355 . Surapally S, Jayaprakasam M, Verma RS. Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia. Pharmacol Rep. 2020;72(4):1032–46. https://doi.org/10.1007/s43440-020-00073-7 . Additional Declarations No competing interests reported. Supplementary Files GraficalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 20 Jul, 2025 Read the published version in Medical Oncology → Version 1 posted Editorial decision: Revision requested 03 Jun, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers invited by journal 30 Apr, 2025 Editor assigned by journal 08 Apr, 2025 Submission checks completed at journal 08 Apr, 2025 First submitted to journal 07 Apr, 2025 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-6395590","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452072832,"identity":"869a6c95-2f21-4458-ab41-acb56d34ab7b","order_by":0,"name":"Gizem Bulut","email":"","orcid":"","institution":"Istinye University","correspondingAuthor":false,"prefix":"","firstName":"Gizem","middleName":"","lastName":"Bulut","suffix":""},{"id":452072833,"identity":"4fdf01f5-5ac2-4e91-acfa-df1379a6ef76","order_by":1,"name":"Merve Kayis","email":"","orcid":"","institution":"Koc University, KUTTAM","correspondingAuthor":false,"prefix":"","firstName":"Merve","middleName":"","lastName":"Kayis","suffix":""},{"id":452072834,"identity":"4d60b478-cede-41fd-8cae-2d09df1c4085","order_by":2,"name":"Dilan Gezer","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Dilan","middleName":"","lastName":"Gezer","suffix":""},{"id":452072835,"identity":"b9f2a13b-a094-477d-bd41-0d66a1c04aec","order_by":3,"name":"Zeliha Gokmen","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Zeliha","middleName":"","lastName":"Gokmen","suffix":""},{"id":452072836,"identity":"b8a86477-4fc6-499d-a962-fb749ec982d2","order_by":4,"name":"Zelal Adiguzel","email":"","orcid":"","institution":"Koc University, KUTTAM","correspondingAuthor":false,"prefix":"","firstName":"Zelal","middleName":"","lastName":"Adiguzel","suffix":""},{"id":452072837,"identity":"a42d74c5-898c-4ac7-8ee7-026b646bbba5","order_by":5,"name":"Chiara Raggi","email":"","orcid":"","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Raggi","suffix":""},{"id":452072838,"identity":"7c4e9c89-bdbb-4b71-88f0-3b677fcbf1f2","order_by":6,"name":"Engin Ulukaya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYFAC5gbGBiDFzwwkEiBCBgS0MEK0SDYDWQgtCURoMTjAAKYJa+GXSGyTnFFzWN74OPvzBw93HJZjYG/eJsH44x5OLZIzgFo2HDtsuO0wj2FD4pnDxgw8x8okGBKKcWoxuAHU8oDtMCNQC2NDYtvhxAaJHDOgFtwuswdr+XfYfnMz+0OQlvoG+Tf4tRiA/LIRaPgGZgZDkJYEBgke/FokzjxstpzZl548A+gXoL/SDdt40ootEtJwa+FvTz54s+ebtW1///EHH3+2Wcvzsx/eeOODDW4tUNCMYLCBKIIaGBjqMBijYBSMglEwCuAAADQmV7lYevd8AAAAAElFTkSuQmCC","orcid":"","institution":"Istinye University, Istinye University","correspondingAuthor":true,"prefix":"","firstName":"Engin","middleName":"","lastName":"Ulukaya","suffix":""}],"badges":[],"createdAt":"2025-04-07 15:08:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6395590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6395590/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12032-025-02927-7","type":"published","date":"2025-07-20T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82042798,"identity":"31bafd3a-4850-488e-a082-157b0ead69ff","added_by":"auto","created_at":"2025-05-06 09:22:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46807,"visible":true,"origin":"","legend":"\u003cp\u003eMK13 has a cytotoxic effect on both cell lines as dose dependent manner. MTT viability (a) and growth rate (b) results after 48h of treatment with different doses for HUCCT1 and CCLP1 cell lines. Results are shown as the means of two independent experiments and error bars indicate standard deviation. Single asterisk (*) denotes the significance of comparison between untreated and treated groups at p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/30510b1d3a9282de7b5fad65.jpg"},{"id":82042799,"identity":"b7b73c59-6a92-48d2-9f6b-7f2075b1a737","added_by":"auto","created_at":"2025-05-06 09:22:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89271,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of cell death mode of CCLP1 cells. Double Fluorescent staining of nuclei belongs to CCLP1 cells after MK13 treatment for 12-24-48h. Images taken with 10x objective, and the yellow framed images taken with 40x objective. Short arrows point early apoptotic nuclei evident with PI negativity, long arrows point late apoptotic nuclei evident with PI positivity. Blue: Hoechst 33342 dye, red: propidium iodide\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/6781491cf92fc4c4f9c2ecdd.jpg"},{"id":82042800,"identity":"18c59093-f544-4c28-af2d-78b5bbc0084b","added_by":"auto","created_at":"2025-05-06 09:22:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84687,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of cell death mode of HUCCT1 cells. Double Fluorescent staining of nuclei belongs to HUCCT1 cells after MK13 treatment for 12-24-48h. Images taken with 10x objective, and the yellow framed images taken with 40x objective. Short arrows point early apoptotic nuclei evident with PI negativity, long arrows point late apoptotic nuclei evident with PI positivity. Blue: Hoechst 33342 dye, red: propidium iodide dye\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/2f6e616798d1df444b0a864e.jpg"},{"id":82044551,"identity":"d6a3b9bf-f7ba-4499-8be6-fbf8ac4aafe9","added_by":"auto","created_at":"2025-05-06 09:30:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99712,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic cell death triggered by MK13 treatment was associated with DNA damage. Flow cytometry analysis of CCLP1 cells with IC90 dose of MK13 after 12-24h incubation for a) phosphatidylserine translocation, b) oxidative stress, c) H2AX activation\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/d93b5a8babed69b25b8f95fe.jpg"},{"id":82045906,"identity":"60d8d680-6e98-4aab-a489-27aa1815883b","added_by":"auto","created_at":"2025-05-06 09:38:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45663,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR analysis results of CCLP1 cells after MK13 IC90 treatment for 6h. a) Prosurvival genes, b) proapoptotic genes, c) cellular stress related genes. These genes were analyzed with single qPCR run. The fold change is calculated as ΔΔCT method and GAPDH was used as housekeeping gene\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/27951c752c8fcfa20fa02748.jpg"},{"id":82044557,"identity":"2006ca33-cd9a-457a-b03b-ade6e721d8ce","added_by":"auto","created_at":"2025-05-06 09:30:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31025,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR analysis results of CCLP1 cells treated with IC90 dose of MK13 for 6h. The fold change is calculated as ΔΔCT method and GAPDH was used as housekeeping gene. Results are shown as a means of two independent experiments and error bars indicate standard deviation\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/041048404d0bf8ae7c8159c2.jpg"},{"id":87219326,"identity":"0e2618de-cacd-4ea2-b0d2-b7aef3b7f20f","added_by":"auto","created_at":"2025-07-21 16:03:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1119796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/38d683b6-a1df-4fb5-b686-b2e654a48132.pdf"},{"id":82045899,"identity":"7706cd70-9070-4e5a-b85e-58625da5adf8","added_by":"auto","created_at":"2025-05-06 09:38:26","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":69269,"visible":true,"origin":"","legend":"","description":"","filename":"GraficalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6395590/v1/914b6b0138975e847c9f892a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA Naphthoquinone Compound Triggers Dna Damage-induced Apoptosis on Cholangiocarcinoma Through Upregulation of Bax and DR5\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCholangiocarcinoma (CCA), a malignant tumor arising from the epithelial cells of the bile ducts, is mentioned as bile duct cancer. Carcinoma of bile duct is classified in three anatomic primary forms: intrahepatic (iCCA), peripheral and distal (together extrahepatic eCCA). The iCCA originates within the liver, peripheral occurs at the hepatic hilum where the bile ducts exit the liver, and distal CCA is found outside the liver [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This classification is crucial as it affects diagnosis, treatment strategies, and patient outcomes. The global and regional incidence of CCA is increasing, with epidemiological data revealing rising incidence and mortality rates, a trend that is alarming given the complexity of this disease especially for intrahepatic CCA [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The clinical significance of CCA is profound due to its severe impact on patient health and quality of life. The disease is often diagnosed at an advanced stage, which complicates treatment and worsens prognosis. The prognosis for CCA is generally poor, characterized by low survival rates compared to other malignancies. Current survival statistics reflect these challenges, with median survival times varying depending on the type and stage of the cancer, yet, due to the late diagnosis, the overall survival rate for CCA is dramatically lower, less than 2 years [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn terms of treatment, the landscape includes surgical interventions, chemotherapy, and radiation therapy. Surgical options, while potentially curative, are limited by high recurrence rates and the difficulty of achieving complete resection. Chemotherapy, a standard treatment approach, includes several drugs such as gemcitabine, cisplatin, oxaliplatin, and 5-fluorouracil. Gemcitabine combined with cisplatin is one of the most commonly used regimens, though the effectiveness varies and is often accompanied by significant side effects, including nausea, fatigue, and myelosuppression. Each of these treatments has limitations that contribute to the overall poor prognosis for patients. The need for novel therapeutic strategies is pressing, driven by the current gaps in treatment efficacy and the challenges associated with early detection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The lack of effective targeted therapies for CCA, coupled with difficulties in diagnosing the disease at a manageable stage, underscores the urgent need for new research and innovative approaches.\u003c/p\u003e \u003cp\u003eNaphthoquinones (NQs), a diverse group of compounds characterized by their naphthalene ring structure with two adjacent carbonyl groups, are gaining attention as potential therapeutics in cancer research. These compounds are secondary metabolites found naturally in various plants, fungi, and bacteria, where they often contribute to the organism\u0026rsquo;s defense mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Their chemical structure, comprising a fused aromatic ring system with two carbonyl groups, imparts unique chemical reactivity and biological activity. Historically, naphthoquinones have been utilized in traditional medicine, but contemporary research has expanded their potential applications, particularly in oncology [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In anticancer research, naphthoquinones have shown promise due to their ability to interfere with critical cellular processes and signaling pathways. Today there are some examples of quinone derivatives used as chemotherapeutic drugs such as Adriamycin (Doxorubicin), Mitomycin C, and Daunomycin. NQs are known to exert their effects through various mechanisms, including the induction of oxidative stress, modulation of apoptosis pathways, and acting as topoisomerase II inhibitors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These mechanisms disrupt the growth and survival of cancer cells, making naphthoquinones attractive candidates for targeted therapy. There are studies conducted with our research groups for investigation of anti-cancer activities of different NQ derivatives which highlights the potential of these group of compounds as potential chemotherapy drugs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent literature highlights the growing interest in naphthoquinones as potential treatments for CCA. Studies have demonstrated that certain naphthoquinone derivatives can inhibit the proliferation of CCA cells, induce apoptosis, and interfere with critical signaling pathways involved in tumor growth and resistance. For instance, compound plumbagin has shown efficacy in preclinical models by targeting specific molecular pathways associated with CCA progression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These findings suggest that naphthoquinones could play a significant role in the development of novel therapeutic strategies for CCA, offering new avenues for treatment where conventional therapies may fall short.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the anticancer effect of MK13, a synthetic NQ derivative that has already been documented for its synthesis and characterization, on CCA cell lines. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] MK13, a 1,4-naphthoquinone derivative, was obtained by the nucleophilic displacement reaction that has been shown in previous studies. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] It has been found that MK13 induces cell death by apoptosis resulting from DNA damage independent of ROS, deserving further attention for \u003cem\u003ein vivo\u003c/em\u003e proof-of-concept studies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eCell culture\u003c/strong\u003e \u003cp\u003eHuman cholangiocarcinoma (CCA) cell lines HUCCT1 and CCLP1 were cultured with RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin. Cells were incubated at 37\u0026deg;C and 5% CO2 condition. When they reached 80% confluency cells were collected with 0.5% trypsin-EDTA (Gibco, USA) for subculturing and seeding.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCytotoxicity and growth rate measurements\u003c/strong\u003e \u003cp\u003eCells were seeded to 96-well culture plates as 5x103 cell/well and incubated overnight for attachment. Next day, cells were treated with different doses of MK13 in the range of 1.56 to 50 \u0026micro;M as 2-fold dilutions. At the end of 48h treatment MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) viability assay was conducted to measure cell viability. To measure growth rate, another 96-well plate was seeded with the same condition of cells and the next day during the treatment procedure, this well was subjected to MTT assay. The absorbance values obtained from this plate, time zero (Tz), was used for the calculation of growth rate as previously explained [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MTT crystals were solubilized by using 0.01 M HCl 10% SDS (w/v). Absorbance was measured as OD by using LumiStar Omega Spectrophotometry, BMG Labtech.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMorphological evaluations with fluorescent staining\u003c/strong\u003e \u003cp\u003eTo investigate the mode of the cell death caused by MK13, cells were stained with Hoechst 33342 dye and propidium iodide as two nuclear dyes. Cells were treated with IC\u003csub\u003e90\u003c/sub\u003e dose of the compound and at the end of 12\u0026ndash;24 and 48h incubation period double staining was performed as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The cells were visualized by using Zeiss Axio Observer fluorescent microscope and images were taken by using Zen Blue Software.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eFlow Cytometry Analyses\u003c/span\u003e: To gain deeper understanding of the cellular events triggered by MK13 treatment, flow cytometry analysis was performed. CCLP1 cells were seeded to 6-well culture plates as 3x105 cell/well and next day treated with IC\u003csub\u003e90\u003c/sub\u003e dose for the compound. For the flow cytometry Guava Muse Cell Analyzer device was used and the fallowing kits were performed according to producer\u0026rsquo;s guidelines: Annexin V \u0026amp; Dead Cell (Cat. No. MCH100105), Oxidative Stress kit (MCH100111) and γH2AX Activation (MCH200105) Kit (FCCS025153).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGene expression analysis with qPCR\u003c/strong\u003e \u003cp\u003eCCLP1 cells were seeded on 6-well plates and the next day treated with IC\u003csub\u003e90\u003c/sub\u003e dose and harvested for RNA isolation after 6h of treatment with the MK13. Cellular RNA was isolated by using the NucleoSpin RNA Mini kit (MN Macherey-Nagel, Germany). Isolated RNA quality and quantity was measured by Nanodrop (Invitrogen, USA) and 1 microgram of each total RNA sample was used for cDNA synthesis with the iScript cDNA Synthesis Kit (BioRad, Germany). For the gene expression analysis, qPCR method was used, 10 ng cDNA was used as template for each reaction that contains QuantiNova Syber Green Mix (Qiagen, Germany) and Applied Biosystem QuantStudio (Thermo Fisher Scientific, USA) device was used. Sample setup and analysis were accomplished using the macro file for each gene panel with the Light-Cycler 480 Software 1.5. Results were analyzed by using ΔΔCT method. GAPDH was used as a housekeeping gene and fold change of expression was calculated according to it. Selected genes were classified into three groups as prosurvival genes (\u003cem\u003eAKT2F, AURKA, BAG4, BCL2, BCL2A1, BCL2L2, BCL2L10, BCL10, BCLXL, BIRC3, BIRC4, BIRC5, BIRC6, BMI1, CDC2, CDC20, CDK4, CDC25A, CDK2, MDM2, MCL1, NEK2, GRB2, TNFRSF11B), proapoptotic genes (APAF1, BAD, BAX, BCLAF1, BAK1, BID, BIK, BIM, BMF, BNIP1, CASP2, CASP3, CASP4, CASP6, CASP8, CASP9, DR4, DR5, HRK, FAS, PUMA, XPA) and cellular stress related genes (CAT, BAG3, ERCC1, ERCC3, GADD45A, XRCC5, NOXA, MUDYH, NFKB, RAD52, LIG4, OGG1, SOD1, PRDX1, BECN1, GPX1, RAD51, GSTP1, BNIP2, BNIP3, BNIP3L, SIRT2, RIPK2\u003c/em\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eTwo-way ANOVA was used for comparing the viability results with the control group. All statistical analyses were conducted using GraphPad Prism. Data for each independent replicate is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cb\u003eMK13 has a cytotoxic effect in both CCA cell lines\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn order to investigate the anticancer capacity of the MK13 compound, dose response analysis was performed with differential doses ranging between 1.56 to 50 \u0026micro;M via MTT viability assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Results showed that both cell lines were responsive for the MK13 compound distinctly from each other. While HUCCT1 cells responded slowly by increased doses CCLP1 cells suddenly diminished at lower doses. Interestingly, the higher doses as 25\u0026ndash;50 \u0026micro;M had the same effect on both cell line as about 80% decrease in viability. In the case of CCLP1, this could be a result of the fact that slightly higher doses could trigger adaptation to particular type of persistent stress and triggers respectively higher proliferative rates. Therefore, even in the range of 3\u0026ndash;12 \u0026micro;M MK13 has highly toxic effects, the increased doses could evoke a particular adaptation mechanism. These kinds of dose response curves are named as non-monotonic dose response curves and could be concluded from different phases of responses such as bi-phasic curve of CCLP1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Indeed, this bi-phasic response was also observed in growth rate curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Overall, CCLP1 cell line was found to be more responsive with lower IC\u003csub\u003e50\u003c/sub\u003e and GI\u003csub\u003e50\u003c/sub\u003e value (0.9 \u0026micro;M and 0.5 \u0026micro;M) with almost 10-fold lower than that of HUCCT1 (10.1 \u0026micro;M and 5 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalculated doses for CCA cell lines treated with MK13 for 48h.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMK13 (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHUCCT1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCLP1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLD\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2. \u003cb\u003eMK13 triggers apoptotic cell death on both cell line\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eObservational experiment for the MK13 induced cell death was designed by using fluorescent staining methodology. For this purpose, CCLP1 and HUCCT1 cells were treated with IC\u003csub\u003e90\u003c/sub\u003e values of MK13 (3.3 \u0026micro;M and 50 \u0026micro;M) and fluorescently stained after 12-24-48h incubation period. Two nuclear fluorescent dye, Hoechst 33342 and propidium iodide (PI) was used to distinguish apoptotic and necrotic nuclear morphology. Hoechst 33342 dye is a membrane permeable nuclear dye while PI cannot pass through membrane therefore only stain the nuclei of the calls have their membrane integrity lost. Therefore, the nuclei stained with PI are necrotic cells. When cells are dying with apoptosis, nucleus become smaller, and chromatin get condensed then followed with chromatin fragmentation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This phenomenon is known as pyknosis, and the nucleus show this morphology called pyknotic nucleus. Thus, if a cell is positive for both dye and show pyknotic morphology, this indicates another stage of apoptosis; secondary necrosis, which can be observed at cell culture where efferocytosis cannot take place [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. MK13 treatment of CCLP1 cells resulted with increased number of pyknotic nuclei in a time dependent manner, which indicates the apoptotic cell death morphology. Nuclear fragmentation, called karyorrhexis is also observed. Moreover, the cell confluence was significantly decreased after MK13 treatment with time compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Fluorescent images demonstrate that the treatment of these cells with MK13 triggers apoptotic cell death, evident with the pycnotic nucleus. The number of pycnotic nucleus was observed to be increased in a time dependent manner while total number of cells was significantly decreased in treatment groups. In addition to that, after 24h incubation seconder necrosis was observed as pycnotic nucleus positive for PI signal. For HUCCT1 cell line, the cell death mode is found to be a bit different from CCLP1. These cells have pyknotic nuclei but did not undergo seconder necrosis where cell membrane integrity is lost. Lots of cells are found to be in an early apoptotic stage with low number of PI positive seconder necrotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cb\u003eApoptosis triggered by MK13 is caused by DNA Damage which is independent of oxidative stress\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis was performed in order to understand the cellular responses to the MK13 treatment. Thus, cells were treated with IC\u003csub\u003e90\u003c/sub\u003e dose of MK13 and collected after 12h and 24h of treatment. Phosphatidylserine translocation showed that one-fourth of the cells were in late apoptotic stages while total apoptotic cell number was more than half of the cell population after 12h treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Even though there was no dramatic change between 12h and 24h treatment for the total apoptotic cell number, at any time point there were almost no dead cells without phosphatidylserine translocation. Which indicates that MK13 treatment does not cause random or catastrophic death such as necrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost of the naphthoquinone derivatives have shown to be responsible for sudden ROS generation and followed DNA damage [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After 24h of treatment both early and late apoptotic populations were observed to be increased which indicates the biological time-course apoptotic cell death. Next, oxidative stress and DNA damage levels were evaluated with flow cytometry to investigate if these have a play in MK13 dependent cytotoxicity. For the oxidative stress aspect, there was no excessive ROS generation either at 12h nor 24h treatment (8.47% and 5.84% respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, for DNA damage measurement via H2AX phosphorylation activation status, DNA damage was accumulated as the treatment duration increased 12h to 24h (22.6\u0026ndash;36.90%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). It can be concluded that the DNA damage caused by MK13 was not associated with ROS generation, otherwise, it could be direct interaction of MK13 to the DNA and forming DNA crosslinks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4. \u003cb\u003eProapototic and prosurvival genes are differentially regulated after MK13 exposure\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo evaluate gene expression regulations after MK13 treatment qPCR analysis was performed. Investigated genes for qPCR were divided into three groups: prosurvival, proapoptotic and other cellular stress related genes. After 6h treatment for IC\u003csub\u003e90\u003c/sub\u003e dose of MK13, qPCR was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). According to the results most of the prosurvival genes are observed to be downregulated. On the other hand, proapototic genes were shown to be affected from MK13 treatment even for that short period of treatment. \u003cem\u003eAPAF1, PUMA, NOXA, BAX\u003c/em\u003e and most importantly \u003cem\u003eDR5\u003c/em\u003e were upregulated while \u003cem\u003eCASP2, CASP6, CASP8, BCLAF1, BID, BIK\u003c/em\u003e were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The oxidative stress genes tested were not significantly affected from MK13 exposure as DNA damage repair related genes \u003cem\u003eERCC3, XRCC5, RAD51, RAD52\u003c/em\u003e except for \u003cem\u003eERCC1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In summary, the genes were significantly affected from the MK13 treatment were found to be upregulated \u003cem\u003eBAX\u003c/em\u003e and \u003cem\u003eDR5\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCCA remains a highly aggressive cancer with limited treatment options and poor patient outcomes, emphasizing the need for novel therapeutic agents. Naphthoquinone derivatives have garnered interest for their diverse anticancer mechanisms, including pro-apoptotic and oxidative stress-inducing activities. In this study, we explored the anticancer potential of MK13, a naphthoquinone derivative, in CCA. Two cell lines used for this study, CCLP1 and HUCCT1, were observed to be both responsive to the MK13 treatment, but the compound is found to be more effective on CCLP1 in terms of both cell viability and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The selective behavior of MK13 is distinguishable especially on lower doses ranging from 1.56 \u0026micro;M to 12.5 \u0026micro;M and also reflected in the IC\u003csub\u003e50\u003c/sub\u003e and GI\u003csub\u003e50\u003c/sub\u003e values. Thus, the following experiments to understand the anti-cancer activity of MK13 were conducted on CCLP1 cell line. The Hoechst 33342 and PI double staining revealed that the cells treated with IC\u003csub\u003e90\u003c/sub\u003e dose of MK13 showed apoptotic nuclear morphology which are chromatin condensation and pyknosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, the number of apoptotic nuclei increased with the incubation time, alongside the number of PI positive nuclei indicates seconder necrosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings were also confirmed via flow cytometry with phosphatidylserine translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eBoth cell lines have distinct \u003cem\u003eTP53\u003c/em\u003e mutations that result in different outcomes for drug responding. CCLP1 cells harbor R306X missense mutation that results in premature termination codon (PTC) thus, truncated protein will be eliminated through non-sense mediated decay [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. On the other hand, HUCCT1 cells have R175H structural mutant classified as gain-of-function mutation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This mutation increases cells susceptibility to become cancerous via different mechanisms. Cells carrying this mutant are defective for G2/M cell cycle check point and progress to mitosis bypassing the DNA damage and they become more resistant to apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As a dominant-negative mutation p53\u003csup\u003eR175H\u003c/sup\u003e mutant protein also interferes with wild-type p53 to function and regulate the expression of its target gene [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNQs exert their anticancer activity by triggering apoptosis through oxidative stress due to dramatic accumulation of intracellular ROS, as in the case of plumbagin and many other synthetic NQ derivatives [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the flow cytometry results counteract this general fact, because CCLP1 cells treated with IC\u003csub\u003e90\u003c/sub\u003e dose of MK13 were not suffered from robust ROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In an unpublished study of our research group, we reported an almost 10-fold increase of ROS in the same cell line treated with another synthetic NQ derivative. Here, the level of ROS was considered biologically non-significant in the scope of our experience to be responsible for observed apoptosis. Moreover, it should be noted that some studies have shown that quinones may act as antioxidants, so it is not unexpected to observe no oxidative stress in MK13-treated cells. Some plant extracts containing diverse quinone derivatives showed antioxidant characteristics together with strong anti-cancer activities [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. On the other hand, CCLP1 cells shown to be suffering from DNA damage showed via flow cytometry using H2AX phosphorylation which was increased along with the incubation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Most naphthoquinones are known to induce DNA damage [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Interestingly here the damage is not caused by ROS accumulation but could be direct interaction of MK13 to the DNA itself inside the nucleus such as a quinone derivative chemotherapy drug doxorubicin [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This was also supported by the upregulation of \u003cem\u003eGADD45A\u003c/em\u003e, that plays a pivotal role in the DNA damage induced cellular responses. GADD45A plays a role in drug response by modulating the cellular stress response and apoptosis. Its expression has been linked to enhanced chemo sensitivity in various types of cancer, including melanoma, where downregulation of GADD45A was associated with increased sensitivity to cisplatin [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, GADD45A interacts with other proteins involved in the apoptotic pathway, such as p53 and PCNA, thereby influencing the cellular response to DNA damage and stress [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The interplay between GADD45A and BAX is particularly noteworthy; GADD45A can influence the expression and activity of BAX, thereby affecting the apoptotic threshold of cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Here, the p53 mutation status of these cell lines presents a significant insight into the prediction of a cell line to that kind of DNA intercalating agents [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The truncated p53 baring CCLP1 cells are more responsive to MK13 at dramatically lower doses and devastating DNA damage was observed after treatment. The responsiveness of this cell line therefore can be lined up with its DNA damage response-deficiency due to p53R306X mutation.\u003c/p\u003e \u003cp\u003eMoreover, \u003cem\u003eDR5\u003c/em\u003e is the most affected gene that showed significant increase for expression after 6h treatment with MK13 together with \u003cem\u003eBAX\u003c/em\u003e. These results imply that cells undergo intrinsic apoptosis shown by BAX upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) beside \u003cem\u003eBCL2\u003c/em\u003e downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The relationship between the DR5 (Death Receptor 5) and BAX (Bcl-2-associated X protein) is pivotal in understanding drug response, particularly in the context of apoptosis in cancer therapy. DR5 is a receptor that mediates apoptosis through the extrinsic pathway, while BAX is a pro-apoptotic member of the Bcl-2 family that plays a crucial role in mitochondrial-mediated intrinsic apoptosis. Together with intrinsic apoptosis, cells might become more sensitive to extrinsic apoptosis due to the DR5 upregulation after MK13 treatment. A similar result was observed with a study done with doxorubicin in lung and colorectal cancer cell lines that has upregulation of DR5 after doxorubicin, and combination with DR5 agonist resulted in improved cytotoxic activity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. More studies have demonstrated that the upregulation of DR5 can enhance the apoptotic response of cancer cells to various treatments. For instance, gefitinib, an EGFR inhibitor, has been shown to upregulate DR5 expression, which subsequently increases the sensitivity of non-small cell lung cancer (NSCLC) cells to extrinsic apoptosis. This process is linked to BAX activation, leading to enhanced apoptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Similarly, the combination of triterpenoid pristimerin with taxol has been reported to induce cell death in cervical cancer cells through a mechanism involving the upregulation of DR5 and the activation of BAX, highlighting the interconnected roles of these proteins in mediating drug responses [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Moreover, the expression of DR5 has been correlated with the efficacy of chemotherapy in NSCLC patients, where high levels of DR5 and its interaction with c-FLIP proteins were associated with improved drug-induced apoptosis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This suggests that DR5 not only serves as a marker for drug response but also plays an active role in mediating the apoptotic effects of chemotherapeutic agents through pathways involving BAX. Thus, the MK13 not only triggers intrinsic apoptosis pathway and cause cell death but also sensitize CCLP1 cells to possible external stimuli to induce extrinsic apoptosis pathway. Therefore, it enables new possible combinational therapy strategies.\u003c/p\u003e \u003cp\u003eThis study underlines the anticancer therapy potential of MK13 as an inducer of intrinsic apoptosis and demonstrates that MK13 makes cells more vulnerable for DR5-associated extrinsic apoptosis on CCA. Future studies should be conducted as co-treatment or sequential treatment strategies with DR5 agonist.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was funded by the Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa (Project numbers: FBA-2024-37858).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConflicts of Interest\u003c/b\u003e: The authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Zeliha GOKMEN and Engin ULUKAYA; Data curation, Gizem BULUT, Merve KAYIS and Dilan GEZEN; Investigation, Chiara RAGGI and Engin ULUKAYA; Methodology, Zelal ADIGUZEL, Chiara RAGGI and Engin ULUKAYA; Resources, Zelal ADIGUZEL and Engin ULUKAYA; Supervision, Engin ULUKAYA; Visualization, Gizem BULUT; Writing \u0026ndash; original draft, Gizem BULUT; Writing \u0026ndash; review \u0026amp; editing, Gizem BULUT, Zelal ADIGUZEL, Chiara RAGGI and Engin ULUKAYA.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis publication is based upon work from the European Network for the Study of Cholangiocarcinoma and the COST Action Precision-BTC-Network CA22125, supported by COST (European Cooperation in Science and Technology; www.cost.eu). The authors of Koc University gratefully acknowledge the use of the services and facilities of the Ko\u0026ccedil; University Research Center for Translational Medicine (KUTTAM)\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenavides M, Ant\u0026oacute;n A, Gallego J, G\u0026oacute;mez MA, Jim\u0026eacute;nez-Gordo A, La Casta A, et al. Biliary tract cancers: SEOM clinical guidelines. 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Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia. Pharmacol Rep. 2020;72(4):1032\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s43440-020-00073-7\u003c/span\u003e\u003cspan address=\"10.1007/s43440-020-00073-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"anticancer drug, bile duct cancer, chemotherapy, cell death, quinones","lastPublishedDoi":"10.21203/rs.3.rs-6395590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6395590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCholangiocarcinoma (CCA), a devastating malignancy originating from the bile ducts, is of significant clinical importance due to its rising incidence and poor prognosis. Quinones as being naturally occurring compounds and their being highly used in anticancer drug development studies seem to be potential sources for this aim. In this study, a synthetic naphthoquinone derivative, MK13, has been tested against CCA (CCLP1 and HUCCT1) cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Cell viability and proliferation rate were measured with MTT assay at the doses ranged from 1.56 to 50 µM for 48h treatment. Apoptotic cell death was showed morphologically with fluorescent double staining and biochemically with flow cytometry analysis of phosphatidylserine translocation. Oxidative stress and DNA damage were also measured with flow cytometry while gene expressions were interpreted via qPCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e MK13 exhibited strong antigrowth activity, especially against CCLP1 cells. Cell death resulted from apoptosis that was shown to be triggered by severe DNA damage which is independent of oxidative stress. Apoptosis was confirmed at molecular level with the upregulation of \u003cem\u003eBAX\u003c/em\u003e, a proapoptotic BH-3 only protein, and \u003cem\u003eDR5\u003c/em\u003e, a cell surface death receptor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e MK13 seems to be a promising anticancer compound against CCA and deserves further \u003cem\u003ein vivo\u003c/em\u003e studies for proof of concept.\u003c/p\u003e","manuscriptTitle":"A Naphthoquinone Compound Triggers Dna Damage-induced Apoptosis on Cholangiocarcinoma Through Upregulation of Bax and DR5","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 09:22:21","doi":"10.21203/rs.3.rs-6395590/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-03T20:06:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T13:26:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33591322164956720373140808742368201606","date":"2025-05-20T12:10:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266772407127161327492101606509188676313","date":"2025-05-15T02:51:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T15:23:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17762678065814261556952450928414981694","date":"2025-05-05T12:18:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-01T03:00:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-08T11:05:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-08T11:03:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2025-04-07T15:04:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2c6aa05a-e16c-4e42-a490-ad298afc3cd2","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T15:59:46+00:00","versionOfRecord":{"articleIdentity":"rs-6395590","link":"https://doi.org/10.1007/s12032-025-02927-7","journal":{"identity":"medical-oncology","isVorOnly":false,"title":"Medical Oncology"},"publishedOn":"2025-07-20 15:57:13","publishedOnDateReadable":"July 20th, 2025"},"versionCreatedAt":"2025-05-06 09:22:21","video":"","vorDoi":"10.1007/s12032-025-02927-7","vorDoiUrl":"https://doi.org/10.1007/s12032-025-02927-7","workflowStages":[]},"version":"v1","identity":"rs-6395590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6395590","identity":"rs-6395590","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}