Exploring the anti-proliferative and cytotoxic impact of doxycycline on C6 glioma cells

Abstract

Doxycycline is a member of the tetracycline group and is a bacteriostatic antibiotic. Therefore, it stops/slows down the reproduction and spread of pathogenic microorganisms and gives the immune system the necessary time to destroy them. In this study, cytotoxic, anti-proliferative, and apoptotic effects of doxycycline on the rat glioma cell line derived from Rattus norvegicus were observed. To show the cytotoxicity of doxycycline, MTT test was performed to obtain the IC50 value and the dosages of treatment were determined accordingly. With the colony formation test, it was observed that the determined doxycycline dosages reduced the colony formation ability of the single cells. Similarly, wound healing test also showed that doxycycline treatment reduced the ability of cells to migrate. A dose-dependent decrease in the cell number was detected by DAPI staining after doxycycline treatment and the expression levels of cancer related genes were shown by the RT-qPCR method. In conclusion, doxycycline was found to have anti-proliferative and cytotoxic effects in rat glioma cell line, and more comprehensive studies are needed before doxycycline can be used as a complementary agent in cancer treatment.

Keywords

• Cancer
• cytotoxicity
• doxycycline
• proliferation
• rat glioma
• RT-qPCR

References

1. Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A., Wong, W. W., & Yuan, J. (1996). Human ICE/CED-3 protease nomenclature. Cell, 87(2), 171.
2. Barth, R. F., & Kaur, B. (2009). Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. Journal of Neuro-Oncology, 94(3), 299-312.
3. Brodersen, D. E., Clemons, W. M., Carter, A. P., Morgan-Warren, R. J., Wimberly, B. T., & Ramakrishnan, V. (2000). The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell, 103(7), 1143-1154.
4. Chan, G. C., & Kamath, S. (2023). Pediatric primary cutaneous marginal zone lymphoma treated with doxycycline. Cutis, 112(5), E1-E4.
5. Choi, S. H., Yang, M. K., Kim, T. M., & Choi, H. J. (2024). Feasibility of oral doxycycline as first-line therapy for conjunctival mucosa-associated lymphoid tissue lymphoma. Eye, 38(1), 82-88.
6. Dugray, A., Geay, J. F., Foudi, A., Bonnet, M. L., Vainchenker, W., Wendling, F., ... & Turhan, A. G. (2001). Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. Leukemia, 15(10), 1658-1662.
7. Duncan, J. L., Ahmad, R. N., Danesi, H., Slade, D. J., Davalos, R. V, & Verbridge, S. S. (2024). Electro-antibacterial therapy (EAT) to enhance intracellular bacteria clearance in pancreatic cancer cells. Bioelectrochemistry, 157, 108669.
8. Feitosa, R. C., Ishikawa, E. S. A., da Silva, M. F. A., da Silva-Junior, A. A., & Oliveira-Nascimento, L. (2022). Five decades of doxycycline: Does nanotechnology improve its properties?. International Journal of Pharmaceutics, 618, 121655.

9. Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., & Van Bree, C. (2006). Clonogenic assay of cells in vitro. Nature Protocols, 1(5), 2315-2319.
10. Ghasemi, K., & Ghasemi, K. (2022). A Brief look at antitumor effects of doxycycline in the treatment of colorectal cancer and combination therapies. European Journal of Pharmacology, 916, 174593.
11. Grobben, B., De Deyn, P., & Slegers, H. (2002). Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion. Cell and Tissue Research, 310(3), 257-270.
12. Han, Y., Song, H., Li, Y., Li, R., Chen, L., Gao, B., Chen, Y., & Wang, S. (2024). The combination of tetracyclines effectively ameliorates liver fibrosis via inhibition of EphB1/2. International Immunopharmacology, 126, 111261.
13. Hassan, S. N., Mohamed Yusoff, A. A., Idris, Z., Mohd Redzwan, N., & Ahmad, F. (2022). Exploring the cytotoxicity and anticancer effects of doxycycline and azithromycin on human glioblastoma multiforme cells. Neurological Research, 44(3), 242-251.
14. Jonkman, J. E. N., Cathcart, J. A., Xu, F., Bartolini, M. E., Amon, J. E., Stevens, K. M., & Colarusso, P. (2014). An introduction to the wound healing assay using live-cell microscopy. Cell Adhesion & Migration, 8(5), 440-451.
15. Kelly, G. L., & Strasser, A. (2020). Toward targeting antiapoptotic MCL-1 for cancer therapy. Annual Review of Cancer Biology, 4(1), 299-313. Kumar, P., Nagarajan, A., & Uchil, P. D. (2018). Analysis of cell viability by the MTT assay. Cold Spring Harbor Protocols, 2018(6), pdb.prot095505.
16. Lamb, R., Fiorillo, M., Chadwick, A., Ozsvari, B., Reeves, K. J., Smith, D. L., ... & Lisanti, M. P. Doxycycline down-regulates DNA-PK and radiosensitizes tumor initiating cells: implications for more effective radiation therapy. Oncotarget. 2015; 6: 14005-25.
17. Li, J., Qin, Y., Zhao, C., Zhang, Z., & Zhou, Z. (2023). Tetracycline antibiotics: Potential anticancer drugs. European Journal of Pharmacology, 956, 175949.
18. Liu, F. S. (2009). Mechanisms of chemotherapeutic drug resistance in cancer therapy—a quick review. Taiwanese Journal of Obstetrics and Gynecology, 48(3), 239-244.
19. Liu, H., Tao, H., Wang, H., Yang, Y., Yang, R., Dai, X., ... & Sun, T. (2021). Doxycycline inhibits cancer stem cell-like properties via PAR1/FAK/PI3K/AKT pathway in pancreatic cancer. Frontiers in Oncology, 10, 619317.
20. Liu, S., Liu, X., Wang, H., Zhou, Q., Liang, Y., Sui, A., ... & Sun, M. (2015). Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells. Oncology Reports, 33(5), 2477-2483.
21. Luger, A. L., Sauer, B., Lorenz, N. I., Engel, A. L., Braun, Y., Voss, M., ... & Ronellenfitsch, M. W. (2018). Doxycycline impairs mitochondrial function and protects human glioma cells from hypoxia-induced cell death: implications of using tet-inducible systems. International Journal of Molecular Sciences, 19(5), 1504.
22. Mi, T., Zhang, Z., Zhanghuang, C., Jin, L., Tan, X., Liu, J., ... & He, D. (2024). Doxycycline hydrochloride inhibits the progress of malignant rhabdoid tumor of kidney by targeting MMP17 and MMP1 through PI3K-Akt signaling pathway. European Journal of Pharmacology, 964, 176291.
23. Miyashita, T., Krajewski, S., Krajewska, M., Wang, H. G., Lin, H. K., Liebermann, D. A., ... & Reed, J. C. (1994). Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene, 9(6), 1799-1805.
24. Morini, M., Mottolese, M., Ferrari, N., Ghiorzo, F., Buglioni, S., Mortarini, R., ... & Albini, A. (2000). The α3β1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (mmp‐9) activity. International Journal of Cancer, 87(3), 336-342.
25. Nassar, H., Sippl, W., Dahab, R. A., & Taha, M. (2023). Molecular docking, molecular dynamics simulations and in vitro screening reveal cefixime and ceftriaxone as GSK3β covalent inhibitors. RSC Advances, 13(17), 11278-11290.
26. Pascal, J. M. (2018). The comings and goings of PARP-1 in response to DNA damage. DNA Repair, 71, 177-182.
27. Pournajaf, S., Afsordeh, N., & Pourgholami, M. H. (2024). In vivo C6 glioma models: an update and a guide toward a more effective preclinical evaluation of potential anti-glioblastoma drugs. Reviews in the Neurosciences, 35(2), 183-195.
28. Qin, Y., Zhang, Q., Lee, S., Zhong, W. L., Liu, Y. R., Liu, H. J., ... & Zhou, H. G. (2015). Doxycycline reverses epithelial-to-mesenchymal transition and suppresses the proliferation and metastasis of lung cancer cells. Oncotarget, 6(38), 40667-40679.
29. Romanishin, A., Vasilev, A., Khasanshin, E., Evtekhov, A., Pusynin, E., Rubina, K., ... & Semina, E. (2024). Oncolytic viral therapy for gliomas: Advances in the mechanisms and approaches to delivery. Virology, 110033.
30. Shutter, M. C., & Akhondi, H. (2023). Tetracycline. In: StatPearls. Treasure Island (FL): StatPearls Publishing; Last accessed on August 20, 2024.
31. Singh, S., Khanna, D., & Kalra, S. (2021). Minocycline and doxycycline: More than antibiotics. Current Molecular Pharmacology, 14(6), 1046-1065.
32. Siregar, O., Lelo, A., Rahyussalim, A. J., Ilyas, S., Benny, Kurniawati, T., ... & Fathurrahman, I. (2023). Doxycycline as a potential MMP‐1 inhibitor for the treatment of spondylitis tuberculosis: A study in rabbit model. BioMed Research International, 2023(1), 7421325.
33. Souza, T. K. F., Nucci, M. P., Mamani, J. B., da Silva, H. R., Fantacini, D. M. C., de Souza, L. E. B., ... & Gamarra, L. F. (2018). Image and motor behavior for monitoring tumor growth in C6 glioma model. PLoS One, 13(7), e0201453.
34. Sun, T., Zhao, N., Ni, C., Zhao, X., Zhang, W., Su, X., Zhang, D., Gu, Q., & Sun, B. (2009). Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK). Cancer Letters, 285(2), 141-150.
35. Swartz, A. M., Li, Q. J., & Sampson, J. H. (2014). Rindopepimut: a promising immunotherapeutic for the treatment of glioblastoma multiforme. Immunotherapy, 6(6), 679-690.
36. Syapin, P. J., Martinez, J. M., Curtis, D. C., Marquardt, P. C., Allison, C. L., Groot, J. A., ... & Bergeson, S. E. (2016). Effective reduction in high ethanol drinking by semisynthetic tetracycline derivatives. Alcoholism: Clinical and Experimental Research, 40(12), 2482-2490.
37. Umfress, A., Speed, H. E., Tan, C., Ramezani, S., Birnbaum, S., Brekken, R. A., ... & Bibb, J. A. (2021). Neuropathological effects of chemotherapeutic drugs. ACS Chemical Neuroscience, 12(16), 3038-3048.
38. Wang, S. Q., Zhao, B. X., Liu, Y., Wang, Y. T., Liang, Q. Y., Cai, Y., ... & Li, G. F. (2016). New application of an old drug: Antitumor activity and mechanisms of doxycycline in small cell lung cancer. International Journal of Oncology, 48(4), 1353-1360.
39. Wang-Gillam, A., Siegel, E., Mayes, D. A., Hutchins, L. F., & Zhou, Y. H. (2007). Anti-tumor effect of doxycycline on glioblastoma cells. Journal of Cancer Molecules, 3(5), 147-153.
40. Warner, A. J., Hathaway-Schrader, J. D., Lubker, R., Davies, C., & Novince, C. M. (2022). Tetracyclines and bone: Unclear actions with potentially lasting effects. Bone, 159, 116377.
41. Wehrli, J. M., Xia, Y., Offenhammer, B., Kleim, B., Müller, D., & Bach, D. R. (2023). Effect of the matrix metalloproteinase inhibitor doxycycline on human trace fear memory. ENeuro, 10(2).
42. Yang, Y. L., Wang, L. X., Fei, X. M., Lei, F., Lu, W. P., Yu, X. Q., & Zhang, S. (2022). The Effect of Doxycycline on the Expression of MMP-2 and MMP-9 in Multiple Myeloma. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 30(2), 487-492.
43. Zhang, L., Xu, L., Zhang, F., & Vlashi, E. (2017). Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer. Cell Cycle, 16(8), 737-745.