Vitamin D-Induced Cytotoxicity in HT-29 Colon Cancer Cells: Apoptotic Gene Expression Study

Document Type : Original Article

Authors

Department of Biology, Ra.C., Islamic Azad University, Rasht, Iran.

10.22111/jep.2026.53863.1100

Abstract

Background: Colorectal cancer (CRC) remains a major cause of cancer deaths worldwide, emphasizing the need for new treatments. Although vitamin D is known to impact cell growth and apoptosis, its precise mechanisms in affecting CRC and regulating apoptosis-related genes are not fully understood. This study investigated the possible cytotoxicity of vitamin D on human colon adenocarcinoma HT-29 cells and examined changes in BAX and BCL-2 gene expression.
Methods: HT29 cells were cultured and exposed to vitamin D at concentrations ranging from 50 to 3750 µg/ml for 24 hours, with untreated cells as controls. Cell viability was assessed using an MTT assay, followed by RNA extraction. The RNA was reverse-transcribed into cDNA, allowing quantitative measurement of BAX and BCL-2 transcripts through real-time PCR, with data normalized to GAPDH. Statistical analysis of cell viability was performed using one-way ANOVA and Dunnett's post-hoc tests, while differences in gene expression were examined with t-tests.
Results: Vitamin D decreased HT-29 cell viability in a dose-dependent and nonlinear way (p < 0.0001), with the highest inhibition seen at concentrations between 250-500 µg/ml. However, there was no significant difference in BAX expression (p = 0.1431) or BCL-2 expression (p = 0.5943).
Conclusions: These data indicate that Vitamin D can cause cytotoxicity in HT-29 cells without altering the transcriptional targets BAX/BCL-2, and may also induce cell death through other pathways such as inhibited proliferation. Overall, this suggests that Vitamin D has potential association as a complementary therapy for CRC and warrants further mechanistic research, including studies with in vivo CRC models.

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References

Bevan, R., & Rutter, M. D. (2018). Colorectal cancer screening-who, how, and when. Clin Endosc, 51(1), 37-49. https://doi.org/10.5946/ce.2017.141
Bikle, D. D. (2015). Vitamin D: production, metabolism, and mechanisms of action. https://europepmc.org/article/nbk/nbk278935#free-full-text
Carneiro, B. A., & El-Deiry, W. S. (2020). Targeting apoptosis in cancer therapy. Nature reviews Clinical oncology, 17(7), 395-417. https://doi.org/10.1038/s41571-020-0341-y
Chiang, K. C., Kuo, S. F., Chen, C. H., Ng, S., Lin, S. F., Yeh, C. N., ... & Pang, J. H. S. (2015). MART-10, the vitamin D analog, is a potent drug for inhibiting the metastatic potential of anaplastic thyroid cancer cells. Cancer Letters, 369(1), 76-85. https://doi.org/10.1016/j.canlet.2015.07.024
Dallavalasa, S., Tulimilli, S. V., Bettada, V. G., Karnik, M., Uthaiah, C. A., Anantharaju, P. G., ... & Madhunapantula, S. V. (2024). Vitamin D in cancer prevention and treatment: A review of epidemiological, preclinical, and cellular studies. Cancers, 16(18), 3211. https://doi.org/10.3390/cancers16183211
Druliner, B. R., Rashtak, S., Ruan, X., Bae, T., Vasmatzis, N., O’Brien, D., ... & Boardman, L. A. (2016). Colorectal cancer with residual polyp of origin: a model of malignant transformation. Translational Oncology, 9(4), 280-286. https://doi.org/10.1016/j.tranon.2016.06.002
Fekete, M., Lehoczki, A., Szappanos, Á., Zábó, V., Kaposvári, C., Horváth, A., ... & Varga, J. T. (2025). Vitamin D and Colorectal Cancer Prevention: Immunological Mechanisms, Inflammatory Pathways, and Nutritional Implications. Nutrients, 17(8), 1351. https://doi.org/10.3390/nu17081351
Garland, C. F., Garland, F. C., Gorham, E. D., Lipkin, M., Newmark, H., Mohr, S. B., & Holick, M. F. (2006). The role of vitamin D in cancer prevention. American Journal of Public Health, 96(2), 252-261. https://doi.org/10.2105/AJPH.2004.045260
Gerl, R., & Vaux, D. L. (2005). Apoptosis in the development and treatment of cancer. Carcinogenesis, 26(2), 263-270. https://doi.org/10.1093/carcin/bgh283
He, Y., Zhang, X., Timofeeva, M., Farrington, S. M., Li, X., Xu, W., ... & Dunlop, M. G. (2022). Bidirectional Mendelian randomisation analysis of the relationship between circulating vitamin D concentration and colorectal cancer risk. International journal of cancer, 150(2), 303-307. https://doi.org/10.1002/ijc.33779
Ingraham, B. A., Bragdon, B., & Nohe, A. (2008). Molecular basis of the potential of vitamin D to prevent cancer. Current medical research and opinion, 24(1), 139-149. https://doi.org/10.1185/030079908X253519
Jan, R. (2019). Understanding apoptosis and apoptotic pathways targeted by cancer therapeutics. Advanced Pharmaceutical Bulletin, 9(2), 205. https://doi.org/10.15171/apb.2019.024
Jass, J. R. (2004). Hyperplastic polyps and colorectal cancer: is there a link?. Clinical Gastroenterology and Hepatology, 2(1), 1-8. https://doi.org/10.1016/S1542-3565(03)00284-2
Kandhavelu, J., Subramanian, K., Naidoo, V., Sebastianelli, G., Doan, P., Konda Mani, S., ... & Murugesan, A. (2024). A novel EGFR inhibitor, HNPMI, regulates apoptosis and oncogenesis by modulating BCL‐2/BAX and p53 in colon cancer. British Journal of Pharmacology, 181(1), 107-124. https://doi.org/10.1111/bph.16141
Klampfer, L. (2014). Vitamin D and colon cancer. World journal of gastrointestinal oncology, 6(11), 430. https://doi.org/10.4251/wjgo.v6.i11.430
Kuznia, S., Czock, D., Kopp-Schneider, A., Caspari, R., Fischer, H., Laetsch, D. C., ... & Schöttker, B. (2022). Efficacy and safety of a personalized vitamin D3 loading dose followed by daily 2000 IU in colorectal cancer patients with vitamin D insufficiency: interim analysis of a randomized controlled trial. Nutrients, 14(21), 4546. https://doi.org/10.3390/nu14214546
Milczarek, M., Filip-Psurska, B., Świętnicki, W., Kutner, A., & Wietrzyk, J. (2014). Vitamin D analogs combined with 5-fluorouracil in human HT-29 colon cancer treatment. Oncology Reports, 32(2), 491-504. https://doi.org/10.3892/or.2014.3247
Morelli, C., Rofei, M., Riondino, S., Fraboni, D., Torino, F., Orlandi, A., ... & Roselli, M. (2022). Immune response in vitamin D-deficient metastatic colorectal cancer patients: a player that should be considered for targeted vitamin D supplementation. Cancers, 14(11), 2594. https://doi.org/10.3390/cancers14112594
Morgan, E., Arnold, M., Gini, A., Lorenzoni, V., Cabasag, C. J., Laversanne, M., ... & Bray, F. (2023). Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut, 72(2), 338-344. https://doi.org/10.1136/gutjnl-2022-327736
Na, S. Y., Kim, K. B., Lim, Y. J., & Song, H. J. (2022). Vitamin D and colorectal cancer: current perspectives and future directions. Journal of Cancer Prevention, 27(3), 147. https://doi.org/10.15430/JCP.2022.27.3.147
Ng, K. (2014). Vitamin D for prevention and treatment of colorectal cancer: What is the evidence? Current colorectal cancer reports, 10(3), 339-345. http://doi.org/10.1007/s11888-014-0238-1
Ng, K., Nimeiri, H. S., McCleary, N. J., Abrams, T. A., Yurgelun, M. B., Cleary, J. M., ... & Fuchs, C. S. (2019). Effect of high-dose vs standard-dose vitamin D3 supplementation on progression-free survival among patients with advanced or metastatic colorectal cancer: the SUNSHINE randomized clinical trial. Jama, 321(14), 1370-1379. http://doi.org/10.1001/jama.2019.2402
Qian, S., Wei, Z., Yang, W., Huang, J., Yang, Y., & Wang, J. (2022). The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Frontiers in Oncology, 12, 985363. https://doi.org/10.3389/fonc.2022.985363
Siegel, R. L., Wagle, N. S., Cercek, A., Smith, R. A., & Jemal, A. (2023). Colorectal cancer statistics, 2023. CA: a cancer journal for clinicians, 73(3), 233-254. https://doi.org/10.3322/caac.21772
Srichomchey, P., Sukprasert, S., Khulasittijinda, N., Voravud, N., Sahakitrungruang, C., & Lumjiaktase, P. (2023). Vitamin D3 supplementation promotes regulatory T cells to maintain immune homeostasis after surgery for early stages of colorectal cancer. In vivo, 37(1), 286-293. DOI: https://doi.org/10.21873/invivo.13078
Tian, X., Srinivasan, P. R., Tajiknia, V., Uruchurtu, A. F. S. S., Seyhan, A. A., Carneiro, B. A., ... & El-Deiry, W. S. (2024). Targeting apoptotic pathways for cancer therapy. The Journal of Clinical Investigation, 134(14). https://doi.org/10.1172/JCI179570
Vuolo, L., Faggiano, A., & Colao, A. A. (2012). Vitamin D and cancer. Frontiers in endocrinology, 3, 23546. https://doi.org/10.3389/fendo.2012.00058
Wacker, M., & Holick, M. F. (2013). Sunlight and Vitamin D: A global perspective for health. Dermato-endocrinology, 5(1), 51-108. https://doi.org/10.4161/derm.24494
Zhan, Z. S., Zheng, Z. S., Shi, J., Chen, J., Wu, S. Y., & Zhang, S. Y. (2024). Unraveling colorectal cancer prevention: The vitamin D-gut flora-immune system nexus. World Journal of Gastrointestinal Oncology, 16(6), 2394. https://doi.org/10.4251/wjgo.v16.i6.2394
Zhang, Y., Li, Y., Wei, Y., & Cong, L. (2023). Molecular mechanism of vitamin D receptor modulating Wnt/β-catenin signaling pathway in gastric cancer. Journal of Cancer, 14(17), 3285. https://doi.org/10.7150/jca.81034
Journal of Epigenetics
Volume 7, Issue 1
April 2026
Pages 21-27

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History

  • Receive Date: 18 November 2025
  • Revise Date: 13 December 2025
  • Accept Date: 21 February 2026

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