Cholecalciferol Potential in the Treatment of Advanced Cervical Cancer Patients: Evidence for Autophagy and Mitochondrial Mechanisms

Cholecalciferol Potential in the Treatment of Advanced Cervical Cancer Patients: Evidence for Autophagy and Mitochondrial Mechanisms | 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 Article Cholecalciferol Potential in the Treatment of Advanced Cervical Cancer Patients: Evidence for Autophagy and Mitochondrial Mechanisms Dodi Suardi, Raden Tina Dewi Judistiani, Viko Duvadilan Wibowo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4893534/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 Introduction. Cholecalciferol, also known as vitamin D3, has been investigated in various cancer studies with conflicting results. Cervical cancer, the second most common cancer among women, has a high fatality rate in its advanced stages. Supplementation of cholecalciferol is recommended for cervical cancer patients. There is a significant need to explore potential pathways to understand how cholecalciferol affects cervical cancer, particularly in autophagy mechanisms and mitochondrial function. Methods. An observational study was carried out on 66 patients with advanced-stage cervical cancer at Hasan Sadikin Hospital. These patients received an oral dose of 10,000 IU of cholecalciferol daily during and after radiation therapy. The patients were monitored closely using RECIST criteria. Sixteen patients agreed to have their blood collected to measure cholecalciferol and autophagy biomarkers before and after radiotherapy. The biomarkers targeted for measurement were p62, LC3b1, LC3b2, TOM20, and COX4. Results. All participants exhibited clinical improvement. The average serum cholecalciferol level exceeded 30 ng/ml, indicating that the administered dose was adequate to treat and prevent deficiency. Significant changes were observed in all the studied biomarkers. While p62 and LC3b2 levels increased, LC3b1, TOM20, and COX4 levels decreased significantly. There was no correlation between TOM20 and COX4 levels before radiation; however, a trend towards correlation was noted post-radiation (R=0.488; p=0.055). Conclusion. This study demonstrated that a daily intake of 10,000 IU of cholecalciferol in advanced cervical cancer patients (stages IIB—IVA) undergoing radiotherapy was sufficient to treat and prevent deficiency. The anticipated clinical improvement was achieved. The involvement of autophagy is suggested to play a crucial role in enhancing the effectiveness of radiation treatment. Health sciences/Biomarkers Biological sciences/Cancer/Cancer metabolism Biological sciences/Cancer Biological sciences/Cancer/Gynaecological cancer Biological sciences/Cancer/Gynaecological cancer/Cervical cancer Vitamin D TOM20 COX4 P62 LC3b1 LC3b2 Introduction In 2020, Indonesia reported a high incidence of cervical cancer, with 36,633 cases and 21,003 deaths. Despite early screening programs, many new cases were diagnosed at an advanced stage (IIB-IIIB).[1] Radiation therapy, although the preferred treatment, has a long waiting list and possible resistance, contributing to high mortality rates. Radiation damages DNA and cell membranes to stop cell proliferation but can also affect surrounding healthy cells.[2, 3] The relationship between autophagy and cell death is complex and essential for cell survival regulation. p62 is a cellular protein with an LC3-interacting region (LIR) that interacts with LC3 to form autophagosomal membranes in the autophagy process. Both p62 and LC3 are autophagosome markers and indicate autophagy's activation or inhibition. Mitochondria are crucial for controlling cell death and normal physiological apoptosis processes. The anti-apoptotic oncoprotein Bcl-2 stabilizes mitochondrial membrane integrity, prevents mega channel opening, and maintains a pro-oxidant state by regulating cytochrome c oxidase (COX) activity and mitochondrial respiration. COX is the terminal complex of the electron transport chain in the inner mitochondrial membrane. Pro-apoptotic proteins Bax and Bak regulate mitochondrial outer membrane permeabilization in programmed cell death, while TOM20, an essential protein in the outer mitochondrial membrane, controls the Bcl-2/mitochondria interaction. Various diagnostic methods are used in clinical practice.[3, 4] Cholecalciferol (1,25(OH)2D3), the active form of vitamin D3, is linked to the p53 tumor suppressor, inhibition of proliferation, and an increase in pro-apoptotic proteins (BAX, BAK, BAD). It also decreases anti-apoptotic proteins (Bcl-2, Bcl-XL) and regulates caspase 12 and Ca2+/calpain in cancer growth, affecting the cell cycle, inflammation signaling, and the microenvironment. This information suggests that vitamin D supplementation could benefit cervical cancer patients.[5, 6] Vitamin D deficiency is prevalent in Indonesia, affecting 70-80 percent of the population. Vitamin D enhances radiation sensitivity by triggering cell death through autophagy and decreasing the cell's ability to reproduce.[6] Autophagy can shift from being a protective agent to a cytotoxic one, potentially improving the effects of radiation. Biomarkers involved in autophagy, such as p62, LC3, TOM20, and COX4, can be measured to assess autophagy and mitochondrial function.[3] This study will evaluate changes in autophagy biomarkers and mitochondrial functions in advanced-stage cervical cancer. Subjects and Methods An observational study was conducted on 66 patients cohort who received 10.000 IU daily cholecalciferol during and after radiation treatment. The patient was offered to have serum levels of cholecalciferol and biomarkers p62, LC3b1, LC3b2, TOM20, and COX4 measured before and after treatment. Sample size was determined for paired numerical categorical analytical research using the formula: Where: Z α = standard deviation of alpha Z β = standard deviation of beta S = combined standard deviation. X1-X2 = minimum difference in average that is considered significant Type 1 error is set at 5% for two-way hypothesis so the value of Z α = 1.96. Type 2 error is set at 5%, so the value of Z β = 1.64. The minimal sample for this study is 13 patients and 10% with the probability of sample dropout. Thus, the sample needed for this study is 13 + 1,3 = 14.3 ≈ 15. Sixteen subjects with advanced stage (IIB-IVA) cervical carcinoma consented. The diagnosis was based on clinical and histopathological examination from biopsy specimens. The serum cholecalciferol level was assessed by a competitive Enzyme-linked immunosorbent assay (ELISA) (kit reagent for 25(OH)D Euroimmune®). The patient’s clinical response was determined by RECIST criteria, which include complete response, partial response, stable disease, and progressive disease. Clinical examination included inspection of the portion, vaginal wall, fornix, and bimanual examination to evaluate the cervix size and consistency, as well as parametrium involvement. A transrectal ultrasonography examination was established to evaluate tumour size and location. The possible confounding variable was histopathological results. The histopathological results are acquired through a cervical tissue biopsy sample that is stained with haematoxylin-eosin in the Anatomical Pathology laboratory.[7, 8] Cervical cancer radiotherapy treatment was done with 20-30Gy internal radiation and continued with 40-50Gy external radiation. The autophagy and mitochondrial biomarkers were performed using Western Blot analysis (Odyssey®). The biomarkers were measured before and three months after completion of radiotherapy treatment (6-month interval). Analysis and correlation index were performed for biomarkers. Descriptive analysis was carried out on the characteristics of the subjects, histopathological typing of tax, and tumour size. Pre- and post-analysis and correlation index were performed for biomarkers. Results There were 16 out of 66 subjects in the cohort who gave consent to participate. The mean age was 50.25 ± 13.94years, while the mean parity was 3.323 ± 1.310. Histopathological typing was adenocarcinoma (18.8%), keratinizing squamous cell carcinoma (18.8%), non-keratinizing squamous cell carcinoma (43.8%), and other types (18.8%). The initial average tumor size was 139.8 ± 177.9 mm and reduced to an average size of 11.70 ± 10.9 mm after radiation. Until the end of 3 months follow up after radiation, all 66 subjects showed complete response. Biomarkers measurement results from 16 subjects are presented in Table 1 and Table 2 . Table 1 Comparison of autophagy and mitochondrial biomarkers pre and post-radiation Group Level p-Value Pre Post Difference P62 N = 14 N = 16 N = 16 0.008* Median 0.48 0.79 LC3b1 N = 11 N = 11 N = 11 0.041* Median 0.49 0.26 0.41 LC3b2 N = 14 N = 16 N = 16 0.030* Median 0.48 0.79 -0.17 TOM 20 N = 16 N = 16 N = 16 0.026* Median 0.96 0.27 0.026 COX4 N = 16 N = 16 N = 16 0.008* Median 0.91 0.29 0.008 Note: For numerical data, the p-value is tested using the paired T-test if the data is normally distributed with the alternative Wilcoxon test if the data is not normally distributed. The significance value is based on a p-value < 0.05. The variables p62, LC3b1, TOM20, and COX4 are not normally distributed. Table 2 Correlation Analysis Table between TOM20 and COX4 Variables Correlation R p-Value Correlation of TOM20 Pre with COX4 Pre Spearman 0.341 0.196 Correlation of TOM20 Post with COX4 Post Spearman 0.488 0.055 Note: significance value p < 0.05. The ** sign indicates significant or statistically significant. r: correlation coefficient. Descriptive analysis was carried out on the characteristics of the subjects, histopathological typing of cervical cancer, and tumor size. Pre- and post-significant changes before and after radiation were observed in all biomarkers. The other biomarkers showed significant reduction except for p62 and LC3b2, which increased significantly. Correlation analyses were done for TOM20 and COC4. In both before and after radiation, TOM 20 and COX4 did not show any correlation. Discussion Mitophagy is a selective degradation of mitochondria. It has been reported that mitophagy plays a regulatory role in apoptosis and maintaining cell health because it triggers mitochondria turnover and inhibits dysfunctional mitochondria accumulation that can cause cellular degeneration. Mitophagy is regulated by PINK1 protein and parkin. For the selection and disposal of impaired mitochondria, mitophagy also has a role in regulating the number of mitochondria to alter cellular metabolic needs, for mitochondrial steady-state turnover, and during phases of cellular development, such as during cell differentiation. Mitophagy can also regulate energy metabolism in the body until a certain limit and reduces damage caused by external stimuli, thereby protecting the human body from abnormal conditions.[ 9 ] Autophagy is a conserved intracellular degradation system that uses lysosomes to break down cytoplasmic components, contributing significantly to cellular homeostasis by recycling biomolecules and organelles. Its relationship with cancer is intricate and dual-faceted, acting both as a promoter and a suppressor depending on the specific stage and type of cancer involved. It transports substrates to lysosomes through various mechanisms. The most prevalent form of autophagy is macroautophagic (often just called autophagy), which involves the creation of new autophagosomes to deliver materials to lysosomes.[ 10 ] The most distinctive characteristic of autophagy is its ability to degrade nearly all cytoplasm components, including biomolecules such as proteins, nucleic acids, and lipids, as well as various organelles and invading microbes. This degradation process is often selective, making autophagy a crucial mechanism for maintaining cellular homeostasis. It recycles macromolecule precursors to provide nutrients and building blocks, thereby supporting cell survival. However, uncontrolled and persistent activation of autophagy can lead to cellular disintegration and, ultimately, cell death. Dysregulation of autophagy has been linked to various diseases, including neurodegenerative disorders, infectious diseases, and cancers such as liver, colorectal, gastric, breast, and ovarian malignancies.[ 10 ] Under normal conditions and in the early stages of cancer, autophagy acts as a protective mechanism, shielding cells from harmful stimuli and preventing malignant transformation. By mitigating the damaging effects of reactive oxygen species (ROS), autophagy helps prevent DNA damage and maintains genome integrity. During periods of starvation, the production of ROS triggers autophagy. Specifically, hydrogen peroxide (H2O2) reversibly modifies the cysteine residues of ATG4, disrupting the active site necessary for the delipidation of LC3.[ 14 ] Apoptosis, or programmed cell death, is a vital mechanism in biology, essential for developmental sculpturing, tissue homeostasis, and eliminating unwanted cells. Mitochondria play a critical role in regulating this process. Calcium ions (Ca2+) have long been recognized as participants in apoptotic pathways, with mitochondria acting as critical regulators and synchronizers of Ca2 + signalling. Excessive Ca2 + accumulation within mitochondria can trigger apoptosis. The dynamics of Ca2 + between the endoplasmic reticulum (ER) and mitochondria are influenced by the Bcl-2 family proteins, which are pivotal in apoptosis. The number and shape of mitochondria are tightly controlled through processes like mitochondrial fusion and fission, mediated by various mitochondrial-shaping proteins. During apoptotic cell death, mitochondrial fission is observed and appears crucial for advancing the apoptotic pathway.[ 11 ] Apoptotic cell death inhibits oncogenesis at various stages, from initial transformation to metastasis. Cell death also serves as a critical component of cancer treatment, acting as the primary mechanism of action for many anticancer therapies. Most stimuli induce apoptosis through the mitochondrial pathway, where the defining event is mitochondrial outer membrane permeabilization (MOMP). Most approaches inducing mitochondrial outer membrane permeabilization (MOMP) in cancer treatment focus on inhibiting anti-apoptotic BCL-2 proteins. The rationale behind this strategy is that blocking BCL-2 function should either directly initiate apoptosis or enhance sensitivity to other pro-apoptotic therapies. Cancer cells often evade apoptosis by upregulating anti-apoptotic BCL-2 proteins, thereby preventing mitochondrial outer membrane permeabilization (MOMP). The BCL-2 gene was initially identified at a chromosomal translocation breakpoint, placing it under immunoglobulin heavy chain enhancer control, leading to constitutively high BCL-2 expression. While BCL-2 expression alone is not oncogenic, it significantly enhances tumour onset when combined with growth-promoting oncogenes. Numerous studies have confirmed that upregulation of anti-apoptotic BCL-2 proteins is a common feature in various cancers, facilitated by mechanisms like copy number amplification, oncogenic signaling-driven transcriptional upregulation, or suppression of microRNAs that inhibit BCL-2 expression.[ 12 ] Cancer cells also circumvent apoptosis initiation mechanisms. For instance, many cancers exhibit loss of function mutations in the p53 tumour suppressor, which usually induces apoptosis via upregulation of BH3-only proteins such as PUMA in response to DNA damage. Consequently, loss of p53 function can disable DNA damage-induced apoptosis in specific cell types. However, therapies like chemotherapy or radiotherapy can trigger apoptosis independently of p53 through DNA damage.[ 12 ] Cancer is defined by the abnormal and uncontrolled proliferation of cells, which can invade tissues and disrupt normal physiological functions, potentially becoming life-threatening if untreated. The causes of cancer can stem from genetic mutations or environmental factors such as smoking, physical inactivity, and nutritional imbalances. Vitamin D is a pivotal nutrient involved in numerous biochemical pathways and is crucial to overall health. It is particularly significant in various disease processes. Vitamin D is essential not only in preventing malignancies but also as a complementary approach to cancer treatment. It exerts its effects through direct and indirect biochemical mechanisms, contributing to its therapeutic potential in cancer management.[ 13 ] Vitamin D exists in two forms: Vitamin D2, derived from UV irradiation of yeast sterol ergosterol and naturally found in sun-exposed mushrooms, and Vitamin D3, synthesized in the skin and abundant in oil-rich fish like salmon, mackerel, and herring. Commercially available Vitamin D3 is typically derived from 7-dehydrocholesterol, naturally present in the skin or obtained from lanolin. Both forms are used in food fortification and supplements.[ 14 ] After ingestion, Vitamin D (D2 or D3) is absorbed into chylomicrons, entering the lymphatic system and the bloodstream. Initially biologically inert, Vitamin D undergoes hydroxylation in the liver by vitamin D-25-hydroxylase (25-OHase) to form 25(OH)D. Subsequent hydroxylation in the kidneys by 25(OH)D-1α-hydroxylase (CYP27B1) converts it to the biologically active form, 1,25-dihydroxyvitamin D (1,25(OH)2D). This active form interacts with vitamin D receptors in various tissues, including the small intestine and kidneys, significantly stimulating calcium absorption and enhancing dietary calcium and phosphorus absorption efficiency. In osteoblasts, 1,25(OH)2D induces receptor activator of nuclear factor κB ligand (RANKL) expression, which promotes the maturation of osteoclasts that resorb bone matrix and release calcium and minerals into the bloodstream. In the kidneys, 1,25(OH)2D enhances calcium reabsorption from the urine.[ 14 ] The vitamin D receptor is widespread throughout the body, influencing many biological processes. 1,25(OH)2D inhibits cellular proliferation, promotes terminal differentiation, inhibits angiogenesis, stimulates insulin production, suppresses renin production, and enhances macrophage cathelicidin production. Additionally, it promotes its degradation by upregulating 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), converting 25(OH)D and 1,25(OH)2D into water-soluble inactive forms. Various tissues and cells possess 1α-hydroxylase activity, allowing for local production of 1,25(OH)2D, which regulates numerous genes, contributing to the diverse health benefits associated with vitamin D.[ 14 ] This study shows a significant increase in the autophagy biomarker levels of p62 and LC3b2. Lc3b1 and the mitochondrial function biomarkers TOM20 and COX4 were significantly reduced. Theoretically, LC3b2 remains associated with the autophagosome until the fusion of the autophagosome with the lysosome occurs. After that, LC3b2 trapped inside the autophagosome will be degraded. No single marker can likely be used as the only assay to monitor autophagy in ca cx cells.[ 15 , 16 ] The correlation between TOM20 and COX4 post-radiation tends to be moderate-positive. This suggests that radiotherapy increased the concentrations of TOM20 and COX4, indicating the degradation of these mitochondrial surface proteins as a result of radiation and treatment. The role of cholecalciferol in cancer prevention and treatment has been observed in epidemiological and preclinical studies, with various mechanisms proposed to explain its anticancer effects. Collected data suggest that cholecalciferol can regulate the entire process of tumorigenesis, from initiation to metastasis and cell-microenvironment interactions. These mechanisms include regulating cell behaviours such as proliferation, differentiation, apoptosis, autophagy, and epithelial-mesenchymal transition (EMT), as well as modulating cell-microenvironment interactions like angiogenesis, antioxidation, inflammation, and the immune system.[ 16 ] Maintaining a balance between mitophagy and mitochondrial production is crucial for cellular health. Targeting autophagy in cancer therapy is a highly intriguing strategy. A deeper understanding of how particular cancer entities can suppress autophagic mechanisms to support cancer survival and evade death could potentially reverse cancer progression. Thus, timing is crucial, given autophagy's controversial role in cancer development. Treatments targeting this mechanism must be administered precisely in the right place and time to be beneficial; otherwise, they may cause unintended harm. Despite positive outcomes from autophagy modulators, the complex nature of autophagy modulation suggests that autophagy itself may not be a critical target and might not be the best standalone approach to alter tumor evolution due to its paradoxical role unless its exact mechanisms are fully elucidated. The molecular mechanisms of autophagy remain an area for further discovery. Whether targeting autophagic mechanisms in cancer is a good approach or potentially a double-edged sword remains an open question. However, suppressing autophagic mechanisms could potentially reverse cancer progression. Conclusion This study showed that supplementation of 10.000 IU daily cholecalciferol in patients with advanced cervical cancer subjects’ stages IIB - IVA during seven weeks of radiotherapy course, followed by 12 weeks after completion, resulted in better complete response. The evidence showed that cholecalciferol levels remained at toxic levels but enabled all subjects to stay in a normal state of serum cholecalciferol level. It is highly hopeful that if cholecalciferol is supplemented in advanced-stage cervical cancer management undergoing radiotherapy treatment, the mortality rate in Indonesia will be further reduced. Limitations This study was a single-center study with a limited number of participants. In addition, this is a preliminary attempt to determine the accuracy of Cerviray's A.I. compared to expert assessment of cervical precancerous lesions. Therefore, a larger study with more participants is needed to confirm the findings of this study. Abbreviations 1,25(OH)2D3 1,25-dihydroxycholecalciferol Bax Bcl2-asocciated x Protein Bcl-2 B Cell Lymphoma-2 COX Cytochrome C Oxidase DNA Deoxyribonucleid Acid LC3 Light Chain 3 LIR LC3-interacting region ROS Reactive Oxygen Species TOM20 Translocase of Outer Membrane 20 UV Ultraviolet Declarations Ethics Approval and Informed Consent to Participate All study participants provided written informed consent prior to engaging in any study-related procedures. This study was conducted by the Declaration of Helsinki of the World Medical Association Declaration. Helsinki. All methods were performed by relevant guidelines and regulations after obtaining approval and recommendations from the Ethics Committee Review Board of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference number: LB.02.01/X.2.2.1/1919/2022). Data availability The dataset used and /or analysed during the current study available from the corresponding author on reasonable request Consent for publication All authors have contributed in this study and approved the final manuscript. Competing Interest The authors declare that they have no conflict of interest. Funding Funding for this study was provided by the Universitas Padjadjaran Research Funding Grant, awarded to Budi Setiabudiawan and Dodi Suardi. Authors’ contributions Study conception and design: DS, RTDJ, BH, RL, RS, SR Data collection: DS, RTDJ, VDW Analysis and interpretation of result: DS, RTDJ, NT, RS, SR, BS, YMH Draft manuscript preparation: DS, RTDJ, VDW, ASN Acknowledgements The authors wish to thank the research team at RSUP, Dr. Hasan Sadikin, Universitas Padjajaran Bandung, and the Biomolecular laboratory at Faculty of Medicine Universitas Padjadjaran for the hard work during the recruitment and process that occurred during the COVID-19 pandemic. A gratitude is addressed to Imedco LTD for their valuable support in the provision of Hi-D® 5000 cholecalciferol for our study while maintaining author independence in the analysis of the study result. References Sung, H., et al., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians, 2021. 71 (3): p. 209-249. Seiwert, T.Y., J.K. Salama, and E.E. Vokes, The concurrent chemoradiation paradigm—general principles. Nature clinical practice Oncology, 2007. 4 (2): p. 86-100. Bhatla, N., et al., Cancer of the cervix uteri: 2021 update. International Journal of Gynecology & Obstetrics, 2021. 155 : p. 28-44. Barakat, R.R., M. Markman, and M. Randall, Principles and practice of gynecologic oncology . 2009: Lippincott Williams & Wilkins. Jeon, S.-M. and E.-A. Shin, Exploring vitamin D metabolism and function in cancer. Experimental & molecular medicine, 2018. 50 (4): p. 1-14. Negri, M., et al., Vitamin D-induced molecular mechanisms to potentiate cancer therapy and to reverse drug-resistance in cancer cells. Nutrients, 2020. 12 (6): p. 1798. Kim, I. and J.J. Lemasters, Mitochondrial degradation by autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes during nutrient deprivation. American Journal of Physiology-Cell Physiology, 2011. Kim, I., S. Rodriguez-Enriquez, and J.J. Lemasters, Selective degradation of mitochondria by mitophagy. Archives of biochemistry and biophysics, 2007. 462 (2): p. 245-253. Bjørkøy, G., et al., Monitoring autophagic degradation of p62/SQSTM1. Methods in enzymology, 2009. 452 : p. 181-197. Hama, Y., Y. Ogasawara, and N.N. Noda, Autophagy and cancer: Basic mechanisms and inhibitor development. Cancer Science, 2023. 114 (7): p. 2699-2708. Lopez, J. and S. Tait, Mitochondrial apoptosis: killing cancer using the enemy within. British journal of cancer, 2015. 112 (6): p. 957-962. Jeong, S.-Y. and D.-W. Seol, The role of mitochondria in apoptosis. BMB reports, 2008. 41 (1): p. 11-22. Sobhi, P., et al., Vitamin D and potential effects on cancers: A review. Molecular Biology Reports, 2024. 51 (1): p. 190. Holick, M.F., et al., Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. The Journal of clinical endocrinology & metabolism, 2011. 96 (7): p. 1911-1930. Legianawati, D., et al., Profil Penatalaksanaan Kanker Serviks Stadium IIB–IIIB dengan Terapi Radiasi dan Kemoradiasi di Rumah Sakit Umum Pusat Dr. Hasan Sadikin Bandung Periode Tahun 2015–2017. J Farm Klin Indones, 2019. 8 (3). Chino, J., et al., Radiation therapy for cervical cancer: executive summary of an ASTRO clinical practice guideline. Practical radiation oncology, 2020. 10 (4): p. 220-234. Additional Declarations No competing interests reported. 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03:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4893534/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4893534/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86304090,"identity":"2b967726-8305-4da1-8377-a029c5a49607","added_by":"auto","created_at":"2025-07-09 06:54:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":625663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4893534/v1/cddc610c-c4bd-482c-92f5-2e08c58da753.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCholecalciferol Potential in the Treatment of Advanced Cervical Cancer Patients: Evidence for Autophagy and Mitochondrial Mechanisms\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2020, Indonesia reported a high incidence of cervical cancer, with 36,633 cases and 21,003 deaths. Despite early screening programs, many new cases were diagnosed at an advanced stage (IIB-IIIB).[1] Radiation therapy, although the preferred treatment, has a long waiting list and possible resistance, contributing to high mortality rates. Radiation damages DNA and cell membranes to stop cell proliferation but can also affect surrounding healthy cells.[2, 3]\u003c/p\u003e\n\u003cp\u003eThe relationship between autophagy and cell death is complex and essential for cell survival regulation. p62 is a cellular protein with an LC3-interacting region (LIR) that interacts with LC3 to form autophagosomal membranes in the autophagy process. Both p62 and LC3 are autophagosome markers and indicate autophagy\u0026apos;s activation or inhibition. Mitochondria are crucial for controlling cell death and normal physiological apoptosis processes. The anti-apoptotic oncoprotein Bcl-2 stabilizes mitochondrial membrane integrity, prevents mega channel opening, and maintains a pro-oxidant state by regulating cytochrome c oxidase (COX) activity and mitochondrial respiration. COX is the terminal complex of the electron transport chain in the inner mitochondrial membrane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePro-apoptotic proteins Bax and Bak regulate mitochondrial outer membrane permeabilization in programmed cell death, while TOM20, an essential protein in the outer mitochondrial membrane, controls the Bcl-2/mitochondria interaction. Various diagnostic methods are used in clinical practice.[3, 4]\u003c/p\u003e\n\u003cp\u003eCholecalciferol (1,25(OH)2D3), the active form of vitamin D3, is linked to the p53 tumor suppressor, inhibition of proliferation, and an increase in pro-apoptotic proteins (BAX, BAK, BAD). It also decreases anti-apoptotic proteins (Bcl-2, Bcl-XL) and regulates caspase 12 and Ca2+/calpain in cancer growth, affecting the cell cycle, inflammation signaling, and the microenvironment. This information suggests that vitamin D supplementation could benefit cervical cancer patients.[5, 6]\u0026nbsp;Vitamin D deficiency is prevalent in Indonesia, affecting 70-80 percent of the population.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVitamin D enhances radiation sensitivity by triggering cell death through autophagy and decreasing the cell\u0026apos;s ability to reproduce.[6] Autophagy can shift from being a protective agent to a cytotoxic one, potentially improving the effects of radiation. Biomarkers involved in autophagy, such as p62, LC3, TOM20, and COX4, can be measured to assess autophagy and mitochondrial function.[3] This study will evaluate changes in autophagy biomarkers and mitochondrial functions in advanced-stage cervical cancer.\u003c/p\u003e"},{"header":"Subjects and Methods","content":"\u003cp\u003eAn observational study was conducted on 66 patients cohort who received 10.000 IU daily cholecalciferol during and after radiation treatment. The patient was offered to have serum levels of cholecalciferol and biomarkers p62, LC3b1, LC3b2, TOM20, and COX4 measured before and after treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSample size was determined for paired numerical categorical analytical research using the formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"174\" height=\"62\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere:\u003c/p\u003e\n\u003cp\u003eZ\u003csub\u003e\u0026alpha;\u003c/sub\u003e = standard deviation of alpha\u003c/p\u003e\n\u003cp\u003eZ\u003csub\u003e\u0026beta;\u003c/sub\u003e = standard deviation of beta\u003c/p\u003e\n\u003cp\u003eS = combined standard deviation.\u003c/p\u003e\n\u003cp\u003eX1-X2 = minimum difference in average that is considered significant\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Type 1 error is set at 5% for two-way hypothesis so the value of Z\u003csub\u003e\u0026alpha;\u003c/sub\u003e = 1.96. Type 2 error is set at 5%, so the value of Z\u003csub\u003e\u0026beta;\u003c/sub\u003e = 1.64.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"56\" width=\"184\"\u003e\u003c/p\u003e\n\u003cp\u003eThe minimal sample for this study is 13 patients and 10% with the probability of sample dropout. Thus, the sample needed for this study is 13 + 1,3 = 14.3 \u0026asymp; 15. Sixteen subjects with advanced stage (IIB-IVA) cervical carcinoma consented. The diagnosis was based on clinical and histopathological examination from biopsy specimens.\u003c/p\u003e\n\u003cp\u003eThe serum cholecalciferol level was assessed by a competitive Enzyme-linked immunosorbent assay (ELISA) (kit reagent for 25(OH)D Euroimmune\u0026reg;).\u003c/p\u003e\n\u003cp\u003eThe patient\u0026rsquo;s clinical response was determined by RECIST criteria, which include complete response, partial response, stable disease, and progressive disease. Clinical examination included inspection of the portion, vaginal wall, fornix, and bimanual examination to evaluate the cervix size and consistency, as well as parametrium involvement. A transrectal ultrasonography examination was established to evaluate tumour size and location. \u0026nbsp;The possible confounding variable was histopathological results. The histopathological results are acquired through a cervical tissue biopsy sample that is stained with haematoxylin-eosin in the Anatomical Pathology laboratory.[7, 8]\u003c/p\u003e\n\u003cp\u003eCervical cancer radiotherapy treatment was done with 20-30Gy internal radiation and continued with 40-50Gy external radiation. The autophagy and mitochondrial biomarkers were performed using Western Blot analysis (Odyssey\u0026reg;). The biomarkers were measured before and three months after completion of radiotherapy treatment (6-month interval). \u0026nbsp;Analysis and correlation index were performed for biomarkers. Descriptive analysis was carried out on the characteristics of the subjects, histopathological typing of tax, and tumour size. Pre- and post-analysis and correlation index were performed for biomarkers.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere were 16 out of 66 subjects in the cohort who gave consent to participate. The mean age was 50.25\u0026thinsp;\u0026plusmn;\u0026thinsp;13.94years, while the mean parity was 3.323\u0026thinsp;\u0026plusmn;\u0026thinsp;1.310. Histopathological typing was adenocarcinoma (18.8%), keratinizing squamous cell carcinoma (18.8%), non-keratinizing squamous cell carcinoma (43.8%), and other types (18.8%). The initial average tumor size was 139.8\u0026thinsp;\u0026plusmn;\u0026thinsp;177.9 mm and reduced to an average size of 11.70\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9 mm after radiation. Until the end of 3 months follow up after radiation, all 66 subjects showed complete response. Biomarkers measurement results from 16 subjects are presented in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eComparison of autophagy and mitochondrial biomarkers pre and post-radiation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLevel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDifference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP62\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.008*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLC3b1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.041*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLC3b2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.030*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOM 20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.026*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCOX4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.008*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: For numerical data, the p-value is tested using the paired T-test if the data is normally distributed with the alternative Wilcoxon test if the data is not normally distributed. The significance value is based on a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The variables p62, LC3b1, TOM20, and COX4 are not normally distributed.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation Analysis Table between TOM20 and COX4\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of TOM20 Pre with COX4 Pre\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSpearman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.341\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.196\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of TOM20 Post with COX4 Post\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSpearman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.488\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.055\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: significance value p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The ** sign indicates significant or statistically significant. r: correlation coefficient.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDescriptive analysis was carried out on the characteristics of the subjects, histopathological typing of cervical cancer, and tumor size. Pre- and post-significant changes before and after radiation were observed in all biomarkers. The other biomarkers showed significant reduction except for p62 and LC3b2, which increased significantly. Correlation analyses were done for TOM20 and COC4. In both before and after radiation, TOM 20 and COX4 did not show any correlation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMitophagy is a selective degradation of mitochondria. It has been reported that mitophagy plays a regulatory role in apoptosis and maintaining cell health because it triggers mitochondria turnover and inhibits dysfunctional mitochondria accumulation that can cause cellular degeneration. Mitophagy is regulated by PINK1 protein and parkin. For the selection and disposal of impaired mitochondria, mitophagy also has a role in regulating the number of mitochondria to alter cellular metabolic needs, for mitochondrial steady-state turnover, and during phases of cellular development, such as during cell differentiation. Mitophagy can also regulate energy metabolism in the body until a certain limit and reduces damage caused by external stimuli, thereby protecting the human body from abnormal conditions.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAutophagy is a conserved intracellular degradation system that uses lysosomes to break down cytoplasmic components, contributing significantly to cellular homeostasis by recycling biomolecules and organelles. Its relationship with cancer is intricate and dual-faceted, acting both as a promoter and a suppressor depending on the specific stage and type of cancer involved. It transports substrates to lysosomes through various mechanisms. The most prevalent form of autophagy is macroautophagic (often just called autophagy), which involves the creation of new autophagosomes to deliver materials to lysosomes.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe most distinctive characteristic of autophagy is its ability to degrade nearly all cytoplasm components, including biomolecules such as proteins, nucleic acids, and lipids, as well as various organelles and invading microbes. This degradation process is often selective, making autophagy a crucial mechanism for maintaining cellular homeostasis. It recycles macromolecule precursors to provide nutrients and building blocks, thereby supporting cell survival. However, uncontrolled and persistent activation of autophagy can lead to cellular disintegration and, ultimately, cell death. Dysregulation of autophagy has been linked to various diseases, including neurodegenerative disorders, infectious diseases, and cancers such as liver, colorectal, gastric, breast, and ovarian malignancies.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eUnder normal conditions and in the early stages of cancer, autophagy acts as a protective mechanism, shielding cells from harmful stimuli and preventing malignant transformation. By mitigating the damaging effects of reactive oxygen species (ROS), autophagy helps prevent DNA damage and maintains genome integrity. During periods of starvation, the production of ROS triggers autophagy. Specifically, hydrogen peroxide (H2O2) reversibly modifies the cysteine residues of ATG4, disrupting the active site necessary for the delipidation of LC3.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eApoptosis, or programmed cell death, is a vital mechanism in biology, essential for developmental sculpturing, tissue homeostasis, and eliminating unwanted cells. Mitochondria play a critical role in regulating this process. Calcium ions (Ca2+) have long been recognized as participants in apoptotic pathways, with mitochondria acting as critical regulators and synchronizers of Ca2\u0026thinsp;+\u0026thinsp;signalling. Excessive Ca2\u0026thinsp;+\u0026thinsp;accumulation within mitochondria can trigger apoptosis. The dynamics of Ca2\u0026thinsp;+\u0026thinsp;between the endoplasmic reticulum (ER) and mitochondria are influenced by the Bcl-2 family proteins, which are pivotal in apoptosis. The number and shape of mitochondria are tightly controlled through processes like mitochondrial fusion and fission, mediated by various mitochondrial-shaping proteins. During apoptotic cell death, mitochondrial fission is observed and appears crucial for advancing the apoptotic pathway.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eApoptotic cell death inhibits oncogenesis at various stages, from initial transformation to metastasis. Cell death also serves as a critical component of cancer treatment, acting as the primary mechanism of action for many anticancer therapies. Most stimuli induce apoptosis through the mitochondrial pathway, where the defining event is mitochondrial outer membrane permeabilization (MOMP). Most approaches inducing mitochondrial outer membrane permeabilization (MOMP) in cancer treatment focus on inhibiting anti-apoptotic BCL-2 proteins. The rationale behind this strategy is that blocking BCL-2 function should either directly initiate apoptosis or enhance sensitivity to other pro-apoptotic therapies. Cancer cells often evade apoptosis by upregulating anti-apoptotic BCL-2 proteins, thereby preventing mitochondrial outer membrane permeabilization (MOMP). The BCL-2 gene was initially identified at a chromosomal translocation breakpoint, placing it under immunoglobulin heavy chain enhancer control, leading to constitutively high BCL-2 expression. While BCL-2 expression alone is not oncogenic, it significantly enhances tumour onset when combined with growth-promoting oncogenes. Numerous studies have confirmed that upregulation of anti-apoptotic BCL-2 proteins is a common feature in various cancers, facilitated by mechanisms like copy number amplification, oncogenic signaling-driven transcriptional upregulation, or suppression of microRNAs that inhibit BCL-2 expression.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCancer cells also circumvent apoptosis initiation mechanisms. For instance, many cancers exhibit loss of function mutations in the p53 tumour suppressor, which usually induces apoptosis via upregulation of BH3-only proteins such as PUMA in response to DNA damage. Consequently, loss of p53 function can disable DNA damage-induced apoptosis in specific cell types. However, therapies like chemotherapy or radiotherapy can trigger apoptosis independently of p53 through DNA damage.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCancer is defined by the abnormal and uncontrolled proliferation of cells, which can invade tissues and disrupt normal physiological functions, potentially becoming life-threatening if untreated. The causes of cancer can stem from genetic mutations or environmental factors such as smoking, physical inactivity, and nutritional imbalances. Vitamin D is a pivotal nutrient involved in numerous biochemical pathways and is crucial to overall health. It is particularly significant in various disease processes. Vitamin D is essential not only in preventing malignancies but also as a complementary approach to cancer treatment. It exerts its effects through direct and indirect biochemical mechanisms, contributing to its therapeutic potential in cancer management.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eVitamin D exists in two forms: Vitamin D2, derived from UV irradiation of yeast sterol ergosterol and naturally found in sun-exposed mushrooms, and Vitamin D3, synthesized in the skin and abundant in oil-rich fish like salmon, mackerel, and herring. Commercially available Vitamin D3 is typically derived from 7-dehydrocholesterol, naturally present in the skin or obtained from lanolin. Both forms are used in food fortification and supplements.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAfter ingestion, Vitamin D (D2 or D3) is absorbed into chylomicrons, entering the lymphatic system and the bloodstream. Initially biologically inert, Vitamin D undergoes hydroxylation in the liver by vitamin D-25-hydroxylase (25-OHase) to form 25(OH)D. Subsequent hydroxylation in the kidneys by 25(OH)D-1α-hydroxylase (CYP27B1) converts it to the biologically active form, 1,25-dihydroxyvitamin D (1,25(OH)2D). This active form interacts with vitamin D receptors in various tissues, including the small intestine and kidneys, significantly stimulating calcium absorption and enhancing dietary calcium and phosphorus absorption efficiency. In osteoblasts, 1,25(OH)2D induces receptor activator of nuclear factor κB ligand (RANKL) expression, which promotes the maturation of osteoclasts that resorb bone matrix and release calcium and minerals into the bloodstream. In the kidneys, 1,25(OH)2D enhances calcium reabsorption from the urine.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe vitamin D receptor is widespread throughout the body, influencing many biological processes. 1,25(OH)2D inhibits cellular proliferation, promotes terminal differentiation, inhibits angiogenesis, stimulates insulin production, suppresses renin production, and enhances macrophage cathelicidin production. Additionally, it promotes its degradation by upregulating 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), converting 25(OH)D and 1,25(OH)2D into water-soluble inactive forms. Various tissues and cells possess 1α-hydroxylase activity, allowing for local production of 1,25(OH)2D, which regulates numerous genes, contributing to the diverse health benefits associated with vitamin D.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] This study shows a significant increase in the autophagy biomarker levels of p62 and LC3b2. Lc3b1 and the mitochondrial function biomarkers TOM20 and COX4 were significantly reduced.\u003c/p\u003e \u003cp\u003eTheoretically, LC3b2 remains associated with the autophagosome until the fusion of the autophagosome with the lysosome occurs. After that, LC3b2 trapped inside the autophagosome will be degraded. No single marker can likely be used as the only assay to monitor autophagy in ca cx cells.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe correlation between TOM20 and COX4 post-radiation tends to be moderate-positive. This suggests that radiotherapy increased the concentrations of TOM20 and COX4, indicating the degradation of these mitochondrial surface proteins as a result of radiation and treatment. The role of cholecalciferol in cancer prevention and treatment has been observed in epidemiological and preclinical studies, with various mechanisms proposed to explain its anticancer effects. Collected data suggest that cholecalciferol can regulate the entire process of tumorigenesis, from initiation to metastasis and cell-microenvironment interactions. These mechanisms include regulating cell behaviours such as proliferation, differentiation, apoptosis, autophagy, and epithelial-mesenchymal transition (EMT), as well as modulating cell-microenvironment interactions like angiogenesis, antioxidation, inflammation, and the immune system.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eMaintaining a balance between mitophagy and mitochondrial production is crucial for cellular health. Targeting autophagy in cancer therapy is a highly intriguing strategy. A deeper understanding of how particular cancer entities can suppress autophagic mechanisms to support cancer survival and evade death could potentially reverse cancer progression. Thus, timing is crucial, given autophagy's controversial role in cancer development. Treatments targeting this mechanism must be administered precisely in the right place and time to be beneficial; otherwise, they may cause unintended harm. Despite positive outcomes from autophagy modulators, the complex nature of autophagy modulation suggests that autophagy itself may not be a critical target and might not be the best standalone approach to alter tumor evolution due to its paradoxical role unless its exact mechanisms are fully elucidated. The molecular mechanisms of autophagy remain an area for further discovery. Whether targeting autophagic mechanisms in cancer is a good approach or potentially a double-edged sword remains an open question. However, suppressing autophagic mechanisms could potentially reverse cancer progression.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed that supplementation of 10.000 IU daily cholecalciferol in patients with advanced cervical cancer subjects\u0026rsquo; stages IIB - IVA during seven weeks of radiotherapy course, followed by 12 weeks after completion, resulted in better complete response. The evidence showed that cholecalciferol levels remained at toxic levels but enabled all subjects to stay in a normal state of serum cholecalciferol level. It is highly hopeful that if cholecalciferol is supplemented in advanced-stage cervical cancer management undergoing radiotherapy treatment, the mortality rate in Indonesia will be further reduced.\u003c/p\u003e \u003cp\u003eLimitations\u003c/p\u003e \u003cp\u003eThis study was a single-center study with a limited number of participants. In addition, this is a preliminary attempt to determine the accuracy of Cerviray's A.I. compared to expert assessment of cervical precancerous lesions. Therefore, a larger study with more participants is needed to confirm the findings of this study.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e1,25(OH)2D3\u0026nbsp;1,25-dihydroxycholecalciferol\u003c/p\u003e\n\u003cp\u003eBax\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bcl2-asocciated x Protein\u003c/p\u003e\n\u003cp\u003eBcl-2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;B Cell Lymphoma-2\u003c/p\u003e\n\u003cp\u003eCOX\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cytochrome C Oxidase\u003c/p\u003e\n\u003cp\u003eDNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Deoxyribonucleid Acid\u003c/p\u003e\n\u003cp\u003eLC3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Light Chain 3\u003c/p\u003e\n\u003cp\u003eLIR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;LC3-interacting region\u003c/p\u003e\n\u003cp\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Reactive Oxygen Species\u003c/p\u003e\n\u003cp\u003eTOM20\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Translocase of Outer Membrane 20\u003c/p\u003e\n\u003cp\u003eUV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ultraviolet\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Informed Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study participants provided written informed consent prior to engaging in any study-related procedures. This study was conducted by the Declaration of Helsinki of the World Medical Association Declaration. Helsinki. All methods were performed by relevant guidelines and regulations after obtaining approval and recommendations from the Ethics Committee Review Board of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference number: LB.02.01/X.2.2.1/1919/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used and /or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have contributed in this study and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by the Universitas Padjadjaran Research Funding Grant, awarded to Budi Setiabudiawan and Dodi Suardi.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy conception and design: DS, RTDJ, BH, RL, RS, SR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection:\u003c/strong\u003e DS, RTDJ, VDW\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis and interpretation of result:\u003c/strong\u003e DS, RTDJ, NT, RS, SR, BS, YMH\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDraft manuscript preparation:\u003c/strong\u003e DS, RTDJ, VDW, ASN\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the research team at RSUP, Dr. Hasan Sadikin, Universitas Padjajaran Bandung, and the Biomolecular laboratory at Faculty of Medicine Universitas Padjadjaran for the hard work during the recruitment and process that occurred during the COVID-19 pandemic. A gratitude is addressed to \u0026nbsp;Imedco LTD for their valuable support in the provision of Hi-D\u0026reg; 5000 cholecalciferol for our study while maintaining author independence in the analysis of the study result.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H., et al., \u003cem\u003eGlobal cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.\u003c/em\u003e CA: a cancer journal for clinicians, 2021. \u003cstrong\u003e71\u003c/strong\u003e(3): p. 209-249.\u003c/li\u003e\n\u003cli\u003eSeiwert, T.Y., J.K. Salama, and E.E. 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Seol, \u003cem\u003eThe role of mitochondria in apoptosis.\u003c/em\u003e BMB reports, 2008. \u003cstrong\u003e41\u003c/strong\u003e(1): p. 11-22.\u003c/li\u003e\n\u003cli\u003eSobhi, P., et al., \u003cem\u003eVitamin D and potential effects on cancers: A review.\u003c/em\u003e Molecular Biology Reports, 2024. \u003cstrong\u003e51\u003c/strong\u003e(1): p. 190.\u003c/li\u003e\n\u003cli\u003eHolick, M.F., et al., \u003cem\u003eEvaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline.\u003c/em\u003e The Journal of clinical endocrinology \u0026amp; metabolism, 2011. \u003cstrong\u003e96\u003c/strong\u003e(7): p. 1911-1930.\u003c/li\u003e\n\u003cli\u003eLegianawati, D., et al., \u003cem\u003eProfil Penatalaksanaan Kanker Serviks Stadium IIB\u0026ndash;IIIB dengan Terapi Radiasi dan Kemoradiasi di Rumah Sakit Umum Pusat Dr. Hasan Sadikin Bandung Periode Tahun 2015\u0026ndash;2017.\u003c/em\u003e J Farm Klin Indones, 2019. \u003cstrong\u003e8\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eChino, J., et al., \u003cem\u003eRadiation therapy for cervical cancer: executive summary of an ASTRO clinical practice guideline.\u003c/em\u003e Practical radiation oncology, 2020. \u003cstrong\u003e10\u003c/strong\u003e(4): p. 220-234.\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":"Vitamin D, TOM20, COX4, P62, LC3b1, LC3b2","lastPublishedDoi":"10.21203/rs.3.rs-4893534/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4893534/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction. \u003c/strong\u003eCholecalciferol, also known as vitamin D3, has been investigated in various cancer studies with conflicting results. Cervical cancer, the second most common cancer among women, has a high fatality rate in its advanced stages. Supplementation of cholecalciferol is recommended for cervical cancer patients. There is a significant need to explore potential pathways to understand how cholecalciferol affects cervical cancer, particularly in autophagy mechanisms and mitochondrial function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e An observational study was carried out on 66 patients with advanced-stage cervical cancer at Hasan Sadikin Hospital. These patients received an oral dose of 10,000 IU of cholecalciferol daily during and after radiation therapy. The patients were monitored closely using RECIST criteria. Sixteen patients agreed to have their blood collected to measure cholecalciferol and autophagy biomarkers before and after radiotherapy. The biomarkers targeted for measurement were p62, LC3b1, LC3b2, TOM20, and COX4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults. \u003c/strong\u003eAll participants exhibited clinical improvement. The average serum cholecalciferol level exceeded 30 ng/ml, indicating that the administered dose was adequate to treat and prevent deficiency. Significant changes were observed in all the studied biomarkers. While p62 and LC3b2 levels increased, LC3b1, TOM20, and COX4 levels decreased significantly. There was no correlation between TOM20 and COX4 levels before radiation; however, a trend towards correlation was noted post-radiation (R=0.488; p=0.055).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion. \u003c/strong\u003eThis study demonstrated that a daily intake of 10,000 IU of cholecalciferol in advanced cervical cancer patients (stages IIB—IVA) undergoing radiotherapy was sufficient to treat and prevent deficiency. The anticipated clinical improvement was achieved. The involvement of autophagy is suggested to play a crucial role in enhancing the effectiveness of radiation treatment.\u003c/p\u003e","manuscriptTitle":"Cholecalciferol Potential in the Treatment of Advanced Cervical Cancer Patients: Evidence for Autophagy and Mitochondrial Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-26 17:23:04","doi":"10.21203/rs.3.rs-4893534/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":"1737866e-8399-4cd1-9917-e0ca1cdd8f11","owner":[],"postedDate":"September 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38252617,"name":"Health sciences/Biomarkers"},{"id":38252618,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":38252619,"name":"Biological sciences/Cancer"},{"id":38252620,"name":"Biological sciences/Cancer/Gynaecological cancer"},{"id":38252621,"name":"Biological sciences/Cancer/Gynaecological cancer/Cervical cancer"}],"tags":[],"updatedAt":"2025-07-09T06:54:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-26 17:23:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4893534","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4893534","identity":"rs-4893534","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}