Cristazine inhibits HPV-positive cervical cancer cells via inducing caspase-dependent apoptosis

Abstract

Objective: To determine whether cristazine inhibits HPV-positive cervical cancer cells and to elucidate its underlying mechanisms.
Methods: HeLa cells were treated with varying concentrations of cristazine, and cell viability, intracellular ROS, apoptosis, and apoptosis-related gene and protein expression were assessed using WST-1, DCF-DA flow cytometry, Annexin V-FITC/PI staining, quantitative RT-PCR, and Western blotting. Additionally, CaSki and SiHa cells were evaluated in viability assays, with selected ROS and apoptosis measurements performed in CaSki cells.
Results: After 24 h of treatment, cristazine reduced HeLa cell viability with an IC₅₀ of 0.223 μM and increased intracellular ROS and apoptotic populations. Caspase inhibition substantially restored viability and reduced apoptosis. Cristazine also increased TP53, CDKN1A, BAX, BCL2, and CASP3 expression, and reduced HPV18 E6/E7 expression. Additionally, Western blotting showed upregulated p53, p21 and Bax/Bcl-2 ratio, and increased cleavage of caspases-3, -8, -9, and -12 and PARP after cristazine treatment. DAPI staining showed chromatin condensation, while wound healing assays demonstrated reduced wound closure following cristazine treatment.
Conclusions: Cristazine suppresses cervical cancer cell growth via coordinated ROS-associated, p53-driven, and caspase-dependent apoptotic signaling, highlighting its potential as a marine-derived anticancer candidate that warrants further investigation.

Keywords

Cristazine / Dioxopiperazine alkaloid / Apoptosis / HPV-positive cervical cancer / ROS / p53 / Chaetomium cristatum

References

[1] Matthews HK, Bertoli C, de Bruin RAM. Cell cycle control in cancer. Nat Rev Mol Cell Biol 2022; 23(2): 74-88.

[2] Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin 2025; 75(1): 10-45.

[3] Włoszek E, Krupa K, Skrok E, Budzik MP, Deptała A, Badowska-Kozakiewicz A. HPV and cervical cancer-Biology, prevention, and treatment updates. Curr Oncol 2025; 32(3): 122. doi: 10.3390/curroncol32030122.

[4] Baba SK, Alblooshi SSE, Yaqoob R, Behl S, Al Saleem M, Rakha EA, et al. Human papilloma virus (HPV) mediated cancers: An insightful update. J Transl Med 2025; 23(1). doi: 10.1186/s12967-025-06470-x.

[5] Janiszewska J, Kostrzewska-Poczekaj M, Wierzbicka M, Brenner J, Giefing M. HPV-driven oncogenesis-much more than the E6 and E7 oncoproteins. J Appl Genet 2025; 66(1): 63-71.

[6] Chen Y, Yu D, Zhu D, Muthusamy S, Deshpande M, Kiruthiga N, et al. Exploring alkaloids and flavonoids from natural Sources: Emerging natural agents for inhibiting cervical cancer progression through apoptosis induction, anti-inflammatory effects, and oxidative stress reduction. Pathol Res Pract 2025. doi: 10.1016/j.prp.2025.156092.

[7] Gökalp F. The effective natural compounds for inhibiting cervical cancer. Med Oncol 2021; 38(2). doi: 10.1007/s12032-021-01456-3.

[8] Chuerduangphui J, Loymunkong C, Ekalaksananan T, Pientong C. Arecoline, a main alkaloid in Areca catechu, induces biological changes in human papillomavirus 16-positive cervical cancer cells via upregulation of viral oncogenes and cellular transcriptional factors. Asian Pac J Trop Biomed 2025; 15(3): 119-128.

[9] Carneiro BA, El-Deiry WS. Targeting apoptosis in cancer therapy. Nat Rev Clin Oncol 2020; 17(7): 395-417.

[10] Kashyap D, Garg VK, Goel N. Intrinsic and extrinsic pathways of apoptosis: Role in cancer development and prognosis. Adv Protein Chem Struct Biol 2021; 125: 73-120.

[11] Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochim Biophys Acta Mol Cell Res 2020; 1867(6). doi: 10.1016/j.bbamcr.2020.118688.

[12] Cheung EC, Vousden KH. The role of ROS in tumour development and progression. Nat Rev Cancer 2022; 22(5): 280-297.

[13] Nakamura H, Takada K. Reactive oxygen species in cancer: Current findings and future directions. Cancer Sci 2021; 112(10): 3945-3952.

[14] Sarkar E, Kotiya A, Bhuyan R, Raza ST, Misra A, Ahmad R, et al. Curcumin chemo-sensitizes intrinsic apoptosis through ROS-mediated mitochondrial hyperpolarization and DNA damage in breast cancer cells. Cell Signal 2025; 128. doi: 10.1016/j.cellsig.2025.111637.

[15] Evidente A. Advances on anticancer fungal metabolites: Sources, chemical and biological activities in the last decade (2012-2023). Nat Prod Bioprospect 2024; 14. doi: 10.1007/s13659-024-00452-0.

[16] Gao Y, Wang J, Meesakul P, Zhou J, Liu J, Liu S, et al. Cytotoxic compounds from marine fungi: Sources, structures, and bioactivity. Mar Drugs 2024; 22(2): 70. doi: 10.3390/md22020070.

[17] Noman E, Al-Shaibani MM, Bakhrebah MA, Almoheer R, Al-Sahari M, Al-Gheethi A, et al. Potential of anti-cancer activity of secondary metabolic products from marine fungi. J Fungi (Basel) 2021; 7(6): 436. doi: 10.3390/jof7060436.

[18] Tian Y, Li Y. A review on bioactive compounds from marine-derived Chaetomium species. J Microbiol Biotechnol 2022; 32(5): 541-550.

[19] Yun K, Khong TT, Leutou AS, Kim GD, Hong J, Lee CH, et al. Cristazine, a new cytotoxic dioxopiperazine alkaloid from the mudflat-sediment-derived fungus Chaetomium cristatum. Chem Pharm Bull 2016; 64(1): 59-62.

[20] Jo MJ, Patil MP, Jung HI, Seo YB, Lim HK, Son BW, et al. Cristazine, a novel dioxopiperazine alkaloid, induces apoptosis via the death receptor pathway in A431 cells. Drug Dev Res 2019; 80(4): 504-512.

[21] Eruslanov E, Kusmartsev S. Identification of ROS Using oxidized DCFDA and flow-cytometry. In: Armstrong D (ed.) Advanced protocols in oxidative stress II. Methods in molecular biology. Totowa, NJ: Humana Press; 2010, p. 57-72.

[22] Bordigoni A, Motte A, Tissot-Dupont H, Colson P, Desnues C. Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes. Sci Rep 2021; 11(1). doi: 10.1038/s41598-021-83489-2.

[23] Sarmiento-Salinas FL, Perez-Gonzalez A, Acosta-Casique A, Ix-Ballote A, Diaz A, Treviño S, et al. Reactive oxygen species: Role in carcinogenesis, cancer cell signaling and tumor progression. Life Sci 2021; 284. doi: 10.1016/j.lfs.2021.119942.

[24] Shah MA, Rogoff HA. Implications of reactive oxygen species on cancer formation and its treatment. Semin Oncol 2021; 48(3): 238-245.

[25] Glorieux C, Liu S, Trachootham D, Huang P. Targeting ROS in cancer: Rationale and strategies. Nat Rev Drug Discov 2024; 23(8): 583-606.

[26] Koo N, Sharma AK, Narayan S. Therapeutics targeting p53-MDM2 interaction to induce cancer cell death. Int J Mol Sci 2022; 23(9). doi: 10.3390/ijms23095005.

[27] Kung CP, Weber JD. It’s getting complicated-A fresh look at p53-MDM2-ARF triangle in tumorigenesis and cancer therapy. Front Cell Dev Biol 2022; 10. doi: 10.3389/fcell.2022.818744.

[28] Macip S, Igarashi M, Fang L, Chen A, Pan ZQ, Lee SW, et al. Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence. EMBO J 2002; 21(9): 2180-2188.

[29] Masgras I, Carrera S, De Verdier PJ, Brennan P, Majid A, Makhtar W, et al. Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21. J Biol Chem 2012; 287(13): 9845-9854.

[30] Barez SR, Atar AM, Aghaei M. Mechanism of inositol-requiring enzyme 1-alpha inhibition in endoplasmic reticulum stress and apoptosis in ovarian cancer cells. J Cell Commun Signal 2020; 14(4): 403-415.

[31] Wu H, Guo H, Liu H, Cui H, Fang J, Zuo Z, et al. Copper sulfate-induced endoplasmic reticulum stress promotes hepatic apoptosis by activating CHOP, JNK and caspase-12 signaling pathways. Ecotoxicol Environ Saf 2020; 191. doi: 10.1016/j.ecoenv.2020.110236.

[32] Liu YN, Mu YD, Wang H, Zhang M, Shi YW, Mi G, et al. Endoplasmic reticulum stress pathway mediates T-2 toxin-induced chondrocyte apoptosis. Toxicology 2021; 464. doi: 10.1016/j.tox.2021.152989.

[33] Scarth JA, Patterson MR, Morgan EL, Macdonald A. The human papillomavirus oncoproteins: A review of the host pathways targeted on the road to transformation. J Gen Virol 2021; 102(3). doi: 10.1099/jgv.0.001540.

[34] Ranasinghe V, McMillan N. Novel therapeutic strategies for targeting E6 and E7 oncoproteins in cervical cancer. Crit Rev Oncol Hematol 2025; 211. doi: 10.1016/j.critrevonc.2025.104721.

[35] Sadeghi RV, Parsania M, Sadeghizadeh M. Investigation of curcumin-loaded OA400 nanoparticle’s effect on the expression of E6 and E7 human papilloma-virus oncogenes and P53 and Rb factors in HeLa cell line. Iran J Pharm Res 2022; 21(1). doi: 10.5812/ijpr-130762.

[36] Pan M, Behmard V, Zare Z, Malekpour M, Nejadbiglari H, Yavari S, et al. Suppressing effects of green tea extract and epigallocatechin-3-gallate (EGCG) on TGF-β-induced epithelial-to-mesenchymal transition via ROS/Smad signaling in human cervical cancer cells. Gene 2021; 794. doi: 10.1016/j.gene.2021.145774.

[37] Sun X, Fu P, Xie L, Chai S, Xu Q, Zeng L, et al. Resveratrol inhibits the progression of cervical cancer by suppressing the transcription and expression of HPV E6 and E7 genes. Int J Mol Med 2021; 47(1): 335-345.

[38] Franconi R, Massa S, Paolini F, Vici P, Venuti A. Plant-derived natural compounds in genetic vaccination and therapy for HPV-associated cancers. Cancers (Basel) 2020; 12(11). doi: 10.3390/cancers12113101.

[39] Qian S, Wei Z, Yang W, Huang J, Yang Y, Wang J. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol 2022; 12. doi: 10.3389/fonc.2022.985363.

[40] Makgoo L, Mosebi S, Mbita ZA. Molecular mechanisms of HIV protease inhibitors against HPV-associated cervical cancer: Restoration of TP53 tumour suppressor activities. Front Mol Biosci 2022; 9. doi: 10.3389/fmolb.2022.875208.