Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

  • Cheol Park (Department Division of Basic Sciences, College of Liberal Studies, Dong-eui University) ;
  • Hee-Jae Cha (Department of Parasitology and Genetics, Kosin University College of Medicine) ;
  • Su Hyun Hong (Anti-Aging Research Center, and Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • Heui-Soo Kim (Department of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Sun-Hee Leem (Department of Biomedical Sciences, College of Natural Sciences, Dong-A University) ;
  • Jung-Hyun Shim (Departments of Health Sciences and Biomedicine, Health & Life Convergence Sciences, BK21 Four, College of Pharmacy, Mokpo National University) ;
  • Gi-Young Kim (Department of Marine Life Science, Jeju National University) ;
  • Kyoung Ah Kang (Department of Biochemistry, College of Medicine, and Jeju Research Center for Natural Medicine, Jeju National University) ;
  • Jin Won Hyun (Department of Biochemistry, College of Medicine, and Jeju Research Center for Natural Medicine, Jeju National University) ;
  • Yung Hyun Choi (Anti-Aging Research Center, and Department of Biochemistry, Dong-eui University College of Korean Medicine)
  • Received : 2026.02.04
  • Accepted : 2026.03.16
  • Published : 2026.05.01
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Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that selectively targets cancer cells and induces apoptosis. However, many cancers, including bladder cancer, develop resistance to TRAIL, limiting the efficacy of TRAIL-based therapies. This study investigated whether betulinic acid (BA), a pentacyclic triterpenoid with anticancer and chemosensitizing properties, increases TRAIL-mediated apoptosis in TRAIL-resistant human bladder cancer cells. Combination treatment with BA and TRAIL significantly increased cytotoxicity and apoptosis compared to either treatment alone. This combination treatment also increased reactive oxygen species (ROS) production, increased Bax expression and Bid cleavage (tBid formation), and downregulated Bcl-2 levels. These effects were accompanied by caspase activation via extrinsic and intrinsic pathways, leading to cytochrome c release via mitochondrial membrane destabilization, thereby contributing to increased apoptosis. Furthermore, the combination treatment inhibited phosphoinositide 3-kinase (PI3K) and Akt phosphorylation; this effect was amplified by a PI3K inhibitor but abrogated by ROS inhibition. Collectively, our results suggest that BA sensitizes bladder cancer cells to TRAIL-induced apoptosis via ROS-dependent activation of the apoptotic pathway and inhibition of PI3K/Akt signaling. Therefore, the BA and TRAIL combination exhibits potential to overcome TRAIL resistance in human bladder cancer.

Keywords

Acknowledgement

This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korean government (RS-2023-00270936).

References

  1. Agrawal, M., Agrawal, S. K. and Chopra, K. (2025) Overcoming drug resistance in ovarian cancer through PI3K/AKT signaling inhibitors. Gene 948, 149352. https://doi.org/10.1016/j.gene.2025.149352
  2. Alizadeh Zeinabad, H. and Szegezdim, E. (2022) TRAIL in the treatment of cancer: From soluble cytokine to nanosystems. Cancers (Basel) 14, 5125.
  3. An, T., Zha, W. and Zi, J. (2020) Biotechnological production of betulinic acid and derivatives and their applications. Appl. Microbiol. Biotechnol. 104, 3339-3348. https://doi.org/10.1007/s00253-020-10495-1
  4. Ataş, M. N., Ertuğrul, B., İplik, E. S., Çakmakoğlu, B. and Ergen, A. (2022) Effects of betulinic acid on AKT/mTOR pathway in renal cell carcinoma. Turk. J. Urol. 48, 58-63. https://doi.org/10.5152/tud.2022.21276
  5. Banerjee, S., Banerjee, S., Bishayee, A., Da Silva, M. N., Sukocheva, O. A., Tse, E., Casarcia, N. and Bishayee, A. (2024) Cellular and molecular mechanisms underlying the potential of betulinic acid in cancer prevention and treatment. Phytomedicine 132, 155858. https://doi.org/10.1016/j.phymed.2024.155858
  6. Chaudhry, G. E., Akim, A. M., Sung, Y. Y. and Muhammad, T. S. T. (2022) Cancer and apoptosis. Methods Mol. Biol. 2543, 191-210. https://doi.org/10.1007/978-1-0716-2553-8
  7. Chestnut, C., Subramaniam, D., Dandawate, P., Padhye, S., Taylor, J., 3rd, Weir, S. and Anant, S. (2021) Targeting major signaling pathways of bladder cancer with phytochemicals: a review. Nutr. Cancer 73, 2249-2271. https://doi.org/10.1080/01635581.2020.1856895
  8. Davoodi-Moghaddam, Z., Jafari-Raddani, F., Delshad, M., Pourbagheri-Sigaroodi, A. and Bashash, D. (2023) Inhibitors of the PI3K/AKT/mTOR pathway in human malignancies; trend of current clinical trials. J. Cancer Res. Clin. Oncol. 149, 15293-15310.
  9. Deng, Q., Chen, L., Zhang, G., Liu, L., Luo, S. M. and Gao, X. (2024) TRIAL-based combination therapies in cancers. Int. Immunopharmacol. 138, 112570.
  10. Deng, Y., Bi, R., Guo, H., Yang, J., Du, Y., Wang, C. and Wei, W. (2019) Andrographolide enhances TRAIL-induced apoptosis via p53-mediated death receptors up-regulation and suppression of the NF- κB pathway in bladder cancer cells. Int. J. Biol. Sci. 15, 688-700. https://doi.org/10.7150/ijbs.30847
  11. Fan, Z., Cui, Y., Chen, L., Liu, P. and Duan, W. (2024) 23-Hydroxybetulinic acid attenuates 5-fluorouracil resistance of colorectal cancer by modulating M2 macrophage polarization via STAT6 signaling. Cancer Immunol. Immunother. 73, 83. https://doi.org/10.1007/s00262-024-03662-0
  12. Farooqi, A. A., Venera, R., Kapanova, G., Tanbayeva, G., Akhmetova, G., Kudabayev, Y. and Turgambayeva, A. (2023) TRAIL-mediated signaling in bladder cancer: realization of clinical efficacy of TRAILbased therapeutics in medical oncology. Med. Oncol. 40, 236. https://doi.org/10.1007/s12032-023-02078-7
  13. Farzan, M., Farzan, M., Shahrani, M., Navabi, S. P., Vardanjani, H. R., Amini-Khoei, H. and Shabani, S. (2024) Neuroprotective properties of Betulin, Betulinic acid, and Ursolic acid as triterpenoids derivatives: a comprehensive review of mechanistic studies. Nutr. Neurosci. 27, 223-240. https://doi.org/10.1080/1028415X.2023.2180865
  14. Fernandes, S., Vieira, M., Prudêncio, C. and Ferraz, R. (2024) Betulinic acid for glioblastoma treatment: reality, challenges and perspectives. Int. J. Mol. Sci. 25, 2108.
  15. Fulda, S., Friesen, C., Los, M., Scaffidi, C., Mier, W., Benedict, M., Nuñez, G., Krammer, P. H., Peter, M. E. and Debatin, K. M. (1997) Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res. 57, 4956-4964.
  16. Fulda, S., Jeremias, I. and Debatin, K. M. (2004) Cooperation of betulinic acid and TRAIL to induce apoptosis in tumor cells. Oncogene 23, 7611-7620. https://doi.org/10.1038/sj.onc.1207970
  17. Gampa, S. C., Garimella, S. V. and Pandrangi, S. (2023) Nano-TRAIL: a promising path to cancer therapy. Cancer Drug Resist. 6, 78-102. https://doi.org/10.20517/cdr
  18. Gupta, J., Abed, H. S., Uthirapathy, S., Kyada, A., Rab, S. O., Shit, D., Janney, B., Nathiya, D., Kadhim, A. J. and Mustafa, Y. F. (2025) Beyond TRAIL resistance: novel strategies for potentiating TRAILinduced apoptosis in cancer. Exp. Cell Res. 450, 114619. https://doi.org/10.1016/j.yexcr.2025.114619
  19. Ha, Y. S., Kim, T. K., Heo, J., Oh, J., Kim, S. K., Kim, J., Lee, J., Yang, S. R., Hwang, S. and Kim, S. J. (2025) Rocaglamide-A mitigates LPS-induced hepatic inflammation by modulating JNK/AP-1 signaling cascade and ROS production in hepatocytes. Toxicol. Res. 41, 47-59. https://doi.org/10.1007/s43188-024-00263-y
  20. Hassan, D., Menges, C. W., Testa, J. R. and Bellacosa, A. (2024) AKT kinases as therapeutic targets. J. Exp. Clin. Cancer Res. 43, 313.
  21. He, Y., Sun, M. M., Zhang, G. G., Yang, J., Chen, K. S., Xu, W. W. and Li, B. (2021) Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct. Target. Ther. 6, 425.
  22. Heikaus, S., van den Berg, L., Kempf, T., Mahotka, C., Gabbert, H. E. and Ramp, U. (2008) HA14-1 is able to reconstitute the impaired mitochondrial pathway of apoptosis in renal cell carcinoma cell lines. Cell. Oncol. 30, 419-33. https://doi.org/10.1155/2008/693095
  23. Huan, J., Grivas, P., Birch, J. and Hansel, D. E. (2022) Emerging roles for mammalian target of Rapamycin (mTOR) complexes in bladder cancer progression and therapy. Cancers (Basel) 14, 1555. https://doi.org/10.3390/cancers14061555
  24. Huang, J., Li, Q., Wang, H., Zhang, S., Zhou, X., Huang, F., Chen, J., Xu, L. and Kang, D. (2025) Betulinic acid inhibits glioma progression by inducing ferroptosis through the PI3K/Akt and NRF2/HO-1 pathways. J. Gene Med. 27, e70011. https://doi.org/10.1002/jgm.v27.2
  25. Jan, R. and Chaudhry, G. E. (2019) Understanding apoptosis and apoptotic pathways targeted cancer therapeutics. Adv. Pharm. Bull. 9, 205-218. https://doi.org/10.15171/apb.2019.024
  26. Jiang, M., Zhang, K., Zhang, Z., Zeng, X., Huang, Z., Qin, P., Xie, Z., Cai, X., Ashrafizadeh, M., Tian, Y. and Wei, R. (2025) PI3K/AKT/mTOR axis in cancer: from pathogenesis to treatment. MedComm (2020) 6, e70295. https://doi.org/10.1002/mco2.v6.8
  27. Jiao, S., Zhu, H., He, P. and Teng, J. (2016) Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway. Biomed. Pharmacother. 84, 1533-1537. https://doi.org/10.1016/j.biopha.2016.11.028
  28. Jin, C. Y., Molagoda, I. M. N., Karunarathne, W. A. H. M., Kang, S. H., Park, C., Kim, G. Y. and Choi, Y. H. (2018) TRAIL attenuates sulforaphane-mediated Nrf2 and sustains ROS generation, leading to apoptosis of TRAIL-resistant human bladder cancer cells. Toxicol. Appl. Pharmacol. 352, 132-141. https://doi.org/10.1016/j.taap.2018.05.022
  29. Kim, S. Y., Hwangbo, H., Kim, M. Y., Ji, S. Y., Kim, D. H., Lee, H., Kim, G. Y., Moon, S. K., Leem, S. H., Yun, S. J., Kim, W. J., Cheong, J., Park, C. and Choi, Y. H. (2021) Betulinic acid restricts human bladder cancer cell proliferation in vitro by inducing caspase-dependent cell death and cell cycle arrest, and decreasing metastatic potential. Molecules 26, 1381. https://doi.org/10.3390/molecules26051381
  30. Kim, J. S., Son, H., Han, J., Cha, H., Lee, J. H., Pack, S. P. and Lee, C. S. (2025) Evaluation of mitochondrial activity via cellular interactions between adrenal and neuronal cells in a microfluidic coculture device. Biotechnol. Bioprocess Eng. 30, 502-519. https://doi.org/10.1007/s12257-025-00194-x
  31. Korkusuz, G., Oy, C., Secme, M., Yurtseven, D. G. and Kocoglu, S. S. (2026) Betulinic acid isolated from Betula platyphylla induces apoptosis and reduces the mTOR/PI3K/AKT signaling pathway in endometrial cancer cells. Histol. Histopathol. 41, 411-419.
  32. Lao, X. Y., Sun, Y. L., Zhao, Z. J., Liu, J. and Ruan, X. F. (2025) Pharmacological effects of betulinic acid and its protective mechanisms on the cardiovascular system. Fitoterapia 183, 106561. https://doi.org/10.1016/j.fitote.2025.106561
  33. Lee, J., Park, J. R., Lee, H., Hong, S. H., Kim, W. J., Eickelberg, O., Park, S. M., Ryu, S., Cho, S. J., Kim, S. J. and Yang, S. R. (2025) Fludarabine attenuates inflammation and dysregulated autophagy in alveolar macrophages via inhibition of STAT1/IRF1 pathway. Lab. Anim. Res. 41, 12.
  34. Lena, A., Rechichi, M., Salvetti, A., Bartoli, B., Vecchio, D., Scarcelli, V., Amoroso, R., Benvenuti, L., Gagliardi, R., Gremigni, V. and Rossi, L. (2009) Drugs targeting the mitochondrial pore act as cytotoxic and cytostatic agents in temozolomide-resistant glioma cells. J. Transl. Med. 7, 13.
  35. Lestari, D. Y., Mastutik, G. and Mukono, I. S. (2024) Betulinic acid and oleanolic acid modulate CD81 expression and induce apoptosis in triple-negative breast cancer cells through ROS generation. Med. Oncol. 42, 24. https://doi.org/10.1007/s12032-024-02574-4
  36. Li, D., Wang, J., Tuo, Z., Yoo, K. H., Yu, Q., Miyamoto, A., Zhang, C., Ye, X., Wei, W., Wu, R. and Feng, D. (2024) Natural products and derivatives in renal, urothelial and testicular cancers: targeting signaling pathways and therapeutic potential. Phytomedicine 127, 155503. https://doi.org/10.1016/j.phymed.2024.155503
  37. Liu, W., Li, S., Qu, Z., Luo, Y., Chen, R., Wei, S., Yang, X. and Wang, Q. (2019) Betulinic acid induces autophagy-mediated apoptosis through suppression of the PI3K/AKT/mTOR signaling pathway and inhibits hepatocellular carcinoma. Am. J. Transl. Res. 11, 6952-6964.
  38. Lou, H., Li, H., Zhang, S., Lu, H. and Chen, Q. (2021) A review on preparation of betulinic acid and its biological activities. Molecules 26, 5583. https://doi.org/10.3390/molecules26185583
  39. Lu, T., Wei, D., Yu, K., Ma, D., Xiong, J., Fang, Q. and Wang, J. (2020) Betulinic acid restores imatinib sensitivity in BCR-ABL1 kinase-independent, imatinib-resistant chronic myeloid leukemia by increasing HDAC3 ubiquitination and degradation. Ann. N. Y. Acad. Sci. 1467, 77-93. https://doi.org/10.1111/nyas.v1467.1
  40. Luiz-Ferreira, A., Pacifico, T., Cruz, Á. C., Laudisi, F., Monteleone, G. and Stolfi, C. (2023) TRAIL-sensitizing effects of flavonoids in cancer. Int. J. Mol. Sci. 24, 16596.
  41. Luongo, F., Colonna, F., Calapà, F., Vitale, S., Fiori, M. E. and De Maria, R. (2019) PTEN tumor-suppressor: the dam of stemness in cancer. Cancers (Basel) 11, 1076. https://doi.org/10.3390/cancers11081076
  42. Masum, A. A., Aoki, S., Rahman, M. M. and Hisamatsu, Y. (2024) Chemical synthetic approaches to mimic the TRAIL: promising cancer therapeutics. RSC Med. Chem. 15, 3639-3651. https://doi.org/10.1039/D4MD00183D
  43. Mizutani, Y., Yoshida, O., Miki, T. and Bonavida, B. (1999) Synergistic cytotoxicity and apoptosis by Apo-2 ligand and adriamycin against bladder cancer cells. Clin. Cancer Res. 5, 2605-2612.
  44. Mosadegh, M., Noori Goodarzi, N. and Erfani, Y. (2025) A Comprehensive insight into apoptosis: Molecular mechanisms, signaling pathways, and modulating therapeutics. Cancer Invest. 43, 33-58. https://doi.org/10.1080/07357907.2024.2445528
  45. Moyer, A., Tanaka, K. and Cheng, E. H. (2025) Apoptosis in cancer biology and therapy. Annu. Rev. Pathol. 20, 303-328. https://doi.org/10.1146/pathmechdis.2025.20.issue-1
  46. Park, C., Jeong, J. W., Han, M. H., Lee, H., Kim, G. Y., Jin, S., Park, J. H., Kwon, H. J., Kim, B. W. and Choi, Y. H. (2021) The anti-cancer effect of betulinic acid in u937 human leukemia cells is mediated through ROS-dependent cell cycle arrest and apoptosis. Anim. Cells Syst. (Seoul) 25, 119-127. https://doi.org/10.1080/19768354.2021.1915380
  47. Porfyris, O., Detopoulou, P., Adamantidi, T., Tsoupras, A., Papageorgiou, D., Ioannidis, A. and Rojas Gil, A. P. (2025) Phytochemicals as chemo-preventive and therapeutic agents against bladder cancer: a comprehensive review. Diseases 13, 103. https://doi.org/10.3390/diseases13040103
  48. Rashid, K., Kalthoff, H., Abdulkadir, S. A. and Adam, D. (2025) Death ligand receptor (DLR) signaling: its non-apoptotic functions in cancer and the consequences of DLR-directed therapies. Drug Discov. Today 30, 104299. https://doi.org/10.1016/j.drudis.2025.104299
  49. Sabeel, Z., Wang, J., Dong, J., Liu, Y., Yu, C. and Yang, Z. (2025) The duality of GSK-3beta in urinary bladder cancer: tumor suppressor and promoter roles through multiple signaling pathways. Biochim. Biophys. Acta Rev. Cancer 1880, 189324.
  50. Singh, D., Tewari, M., Singh, S. and Narayan, G. (2021) Revisiting the role of TRAIL/TRAIL-R in cancer biology and therapy. Future Oncol. 17, 581-596. https://doi.org/10.2217/fon-2020-0727
  51. Song, M., Cheon, J. and Kwon, S. (2025) Enhanced cytotoxicity of natural killer cells with Zn-alginate hydrogel microspheres. Biotechnol. Bioprocess Eng. 30, 30-41.
  52. Stutz, C., Fontão, A. P. G. A., Silva, G. W. S. E., Seito, L. N., Perdomo, R. T. and Sampaio, A. L. F. (2025) Betulinic acid acts in synergism with imatinib mesylate, triggering apoptosis in MDR leukemia cells. Planta Med. 91, 19-28. https://doi.org/10.1055/a-2440-4847
  53. Tufail, M., Wan, W. D., Jiang, C. and Li, N. (2024) Targeting PI3K/AKT/mTOR signaling to overcome drug resistance in cancer. Chem. Biol. Interact. 396, 111055. https://doi.org/10.1016/j.cbi.2024.111055
  54. Tufail, M. (2025) PTEN-mediated resistance in cancer: from foundation to future therapies. Toxicol. Rep. 14, 101987.  https://doi.org/10.1016/j.toxrep.2025.101987
  55. Voelkel-Johnson, C. (2011) TRAIL-mediated signaling in prostate, bladder and renal cancer. Nat. Rev. Urol. 8, 417-427. https://doi.org/10.1038/nrurol.2011.81
  56. Xu, T., Pang, Q., Wang, Y. and Yan, X. (2017b) Betulinic acid induces apoptosis by regulating PI3K/Akt signaling and mitochondrial pathways in human cervical cancer cells. Int. J. Mol. Med. 40, 1669-1678. https://doi.org/10.3892/ijmm
  57. Xu, Y., Li, J., Li, Q. J., Feng, Y. L. and Pan, F. (2017a) Betulinic acid promotes TRAIL function on liver cancer progression inhibition through p53/Caspase-3 signaling activation. Biomed Pharmacother. 88, 349-358. https://doi.org/10.1016/j.biopha.2017.01.034
  58. Zhang, H., Zhou, M., Ye, C., Qin, J., Lu, X., Wang, C., Wang, X. and Jin, X. (2024) Betulinic acid inhibits the proliferation of human laryngeal carcinoma cells through reactive oxygen species-mediate mitochondrial apoptotic pathway. Toxicol. In Vitro 95, 105756. https://doi.org/10.1016/j.tiv.2023.105756
  59. Zhang, Y., He, N., Zhou, X., Wang, F., Cai, H., Huang, S. H., Chen, X., Hu, Z. and Jin, X. (2021) Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells. Aging (Albany N.Y.) 13, 21251-21267.