Alpha-tomatine induces ROS-mediated mitochondrial apoptosis in laryngeal carcinoma (HEp-2) cells
Paper Details
Alpha-tomatine induces ROS-mediated mitochondrial apoptosis in laryngeal carcinoma (HEp-2) cells
Abstract
Laryngeal squamous cell carcinoma (LSCC) remains a significant global health challenge, showing minimal improvement in survival rates even with advancements in standard treatments. The growing interest in phytochemicals as potential anticancer agents has highlighted Alpha-tomatine (AT) a steroidal glycoalkaloid derived from tomato plants, because of its strong cytotoxic properties. However, the effects of AT on laryngeal cancer have not been explored previously. This study aims to evaluate the anticancer efficacy of AT in HEp-2 human laryngeal carcinoma cells and to clarify the molecular pathways involved. AT significantly inhibited cell growth in a dose-dependent manner, with an IC₅₀ of 29.6 μM. The DCFH-DA staining results showed a marked increase in intracellular reactive oxygen species (ROS), underscoring oxidative stress as a crucial factor in cytotoxicity. Cells treated with AT exhibited significant mitochondrial membrane depolarization (loss of Δψm) and displayed notable apoptotic morphological changes, as evidenced by AO/EtBr and DAPI staining. Results from Annexin V/PI flow cytometry indicated a concentration-dependent increase in both early and late apoptotic cell populations. The comet assay revealed substantial DNA fragmentation, highlighting the genotoxic effects mediated by reactive oxygen species. The findings indicate that AT induces intrinsic apoptosis in HEp-2 cells through mechanisms related to oxidative stress, mitochondrial dysfunction, and DNA damage. These results suggest that AT may be a valuable phytochemical candidate for further investigation as a treatment for laryngeal cancer.
Amalia IF, Sayyidah A, Larasati KA, Budiarti SF. 2020. Molecular docking analysis of α-tomatine and tomatidine to inhibit epidermal growth factor receptor activation in non-small-cell lung cancer (NSCLC). JSMARTech 2(1), 001–006. https://doi.org/10.21776/ub.jsmartech.2020.002.01.1
Annamalai G, Kathiresan S, Kannappan N. 2016. [6]-Shogaol, a dietary phenolic compound, induces oxidative stress-mediated mitochondrial-dependent apoptosis through activation of proapoptotic factors in Hep-2 cells. Biomedicine & Pharmacotherapy 82, 226–236. https://doi.org/10.1016/j.biopha.2016.04.044
Azhamuthu T, Kathiresan S, Senkuttuvan I, Asath NAA, Ravichandran P, Vasu R. 2024a. Usnic acid alleviates inflammatory responses and induces apoptotic signaling through inhibiting NF-κB expression in human oral carcinoma cells. Cell Biochemistry and Function 42(4), e4074. https://doi.org/10.1002/cbf.4074
Azhamuthu T, Kathiresan S, Senkuttuvan I, Asath NAA, Ravichandran P, Vasu R. 2024b. Usnic acid alleviates inflammatory responses and induces apoptotic signaling through inhibiting NF-κB expression in human oral carcinoma cells. Cell Biochemistry and Function 42(4), e4074. https://doi.org/10.1002/cbf.4074
Chao M-W, Chen C-H, Chang Y-L, Teng C-M, Pan S-L. 2012. α-Tomatine-mediated anticancer activity in vitro and in vivo through cell-cycle- and caspase-independent pathways. PLOS ONE 7, e44093. https://doi.org/10.1371/journal.pone.0044093
Chen Y, Li S, Sun F, Han H, Zhang X, Fan Y, Tai G, Zhou Y. 2010. In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants. Pharmaceutical Biology 48(9), 1018–1024. https://doi.org/10.3109/13880200903440211
De Haan LR, Reiniers MJ, Reeskamp LF, Belkouz A, Ao L, Cheng S, Ding B, Van Golen RF, Heger M. 2022. Experimental conditions that influence the utility of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA) as a fluorogenic biosensor for mitochondrial redox status. Antioxidants 11(8), 1424. https://doi.org/10.3390/antiox11081424
Echeverría C, Martin A, Simon F, Salas CO, Nazal M, Varela D, Pérez-Castro RA, Santibanez JF, Valdés-Valdés RO, Forero-Doria O, Echeverría J. 2022. In vivo and in vitro antitumor activity of tomatine in hepatocellular carcinoma. Frontiers in Pharmacology 13, 1003264. https://doi.org/10.3389/fphar.2022.1003264
Friedman M. 2013. Anticarcinogenic, cardioprotective, and other health benefits of tomato compounds lycopene, α-tomatine, and tomatidine in pure form and in fresh and processed tomatoes. Journal of Agricultural and Food Chemistry 61(40), 9534–9550. https://doi.org/10.1021/jf402654e
Fu Z, Lv J. 2024. Commentary on “Updated disease distributions, risk factors, and trends of laryngeal cancer: a global analysis of cancer registries.” International Journal of Surgery 110(7), 4435–4436. https://doi.org/10.1097/JS9.0000000000001355
Fujimaki J, Sayama N, Shiotani S, Suzuki T, Nonaka M, Uezono Y, Oyabu M, Kamei Y, Nukaya H, Wakabayashi K, Morita A, Sato T, Miura S. 2022. The steroidal alkaloid tomatidine and tomatidine-rich tomato leaf extract suppress the human gastric cancer-derived 85As2 cells in vitro and in vivo via modulation of interferon-stimulated genes. Nutrients 14(5), 1023. https://doi.org/10.3390/nu14051023
GBD 2019 Respiratory Tract Cancers Collaborators. 2021. Global, regional, and national burden of respiratory tract cancers and associated risk factors from 1990 to 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet Respiratory Medicine 9(9), 1030–1049. https://doi.org/10.1016/S2213-2600(21)00164-8
Gheena S, Ezhilarasan D. 2019. Syringic acid triggers reactive oxygen species–mediated cytotoxicity in HepG2 cells. Human & Experimental Toxicology 38(6), 694–702. https://doi.org/10.1177/0960327119839173
He Z, Hu Y, Yang X, Han B, Li S, Huang S, Chen X. 2025. Global trends and cross-country inequalities in laryngeal cancer: a systematic analysis of the 2021 Global Burden of Disease study represented by China. Tobacco Induced Diseases 23, 1–16. https://doi.org/10.18332/tid/205796
Huang H, Chen S, Van Doren J, Li D, Farichon C, He Y, Zhang Q, Zhang K, Conney AH, Goodin S, Du Z, Zheng X. 2015. α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells. Molecular Medicine Reports 11(6), 4573–4578. https://doi.org/10.3892/mmr.2015.3238
Hut A-R, Boia ER, Para D, Iovanescu G, Horhat D, Mikša L, Chiriac M, Galant R, Motofelea AC, Balica NC. 2025. Laryngeal cancer in the modern era: evolving trends in diagnosis, treatment, and survival outcomes. Journal of Clinical Medicine 14(10), 3367. https://doi.org/10.3390/jcm14103367
Kim H, Xue X. 2020. Detection of total reactive oxygen species in adherent cells by 2′,7′-dichlorodihydrofluorescein diacetate staining. Journal of Visualized Experiments 160, 60682. https://doi.org/10.3791/60682
Kúdelová J, Seifrtová M, Suchá L, Tomšík P, Havelek R, Řezáčová M. 2013. α-Tomatine activates cell cycle checkpoints in the absence of DNA damage in human leukemic MOLT-4 cells. Journal of Applied Biomedicine 11(2), 93–103. https://doi.org/10.2478/v10136-012-0033-8
Lee K-R, Kozukue N, Han J-S, Park J-H, Chang E, Baek E-J, Chang J-S, Friedman M. 2004. Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells. Journal of Agricultural and Food Chemistry 52(10), 2832–2839. https://doi.org/10.1021/jf030526d
Lee S-T, Wong P-F, Cheah S-C, Mustafa MR. 2011. α-Tomatine induces apoptosis and inhibits nuclear factor-κB activation in human prostatic adenocarcinoma PC-3 cells. PLOS ONE 6(4), e18915. https://doi.org/10.1371/journal.pone.0018915
Lekhak N, Bhattarai HK. 2024. Phytochemicals in cancer chemoprevention: preclinical and clinical studies. Cancer Control 31, 10732748241302902. https://doi.org/10.1177/10732748241302902
Lu J, Wu L, Wang X, Zhu J, Du J, Shen B. 2018. Detection of mitochondrial membrane potential to study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Journal of Visualized Experiments 137, 56317. https://doi.org/10.3791/56317
Pumiputavon K, Chaowasku T, Saenjum C, Osathanunkul M, Wungsintaweekul B, Chawansuntati K, Wipasa J, Lithanatudom P. 2017. Cell cycle arrest and apoptosis induction by methanolic leaf extracts of four Annonaceae plants. BMC Complementary and Alternative Medicine 17(1), 294. https://doi.org/10.1186/s12906-017-1811-3
Qian S, Fang H, Zheng L, Liu M. 2021. Zingerone suppresses cell proliferation via inducing cellular apoptosis and inhibition of the PI3K/AKT/mTOR signaling pathway in human prostate cancer PC-3 cells. Journal of Biochemical and Molecular Toxicology 35(1), e22611. https://doi.org/10.1002/jbt.22611
Rinkel RN, Verdonck-de Leeuw IM, Doornaert P, Buter J, De Bree R, Langendijk JA, Aaronson NK, Leemans CR. 2016. Prevalence of swallowing and speech problems in daily life after chemoradiation for head and neck cancer based on SWAL-QOL and SHI. European Archives of Oto-Rhino-Laryngology 273(7), 1849–1855. https://doi.org/10.1007/s00405-015-3680-z
Shih Y-W, Shieh J-M, Wu P-F, Lee Y-C, Chen Y-Z, Chiang T-A. 2009. α-Tomatine inactivates PI3K/Akt and ERK signaling pathways in human lung adenocarcinoma A549 cells: effect on metastasis. Food and Chemical Toxicology 47(8), 1985–1995. https://doi.org/10.1016/j.fct.2009.05.011
Silva-Beltrán NP, Ruiz-Cruz S, Cira-Chávez LA, Estrada-Alvarado MI, Ornelas-Paz JJ, López-Mata MA, Del-Toro-Sánchez CL, Ayala-Zavala JF, Márquez-Ríos E. 2015. Total phenolic, flavonoid, tomatine, and tomatidine contents and antioxidant and antimicrobial activities of tomato plant extracts. International Journal of Analytical Chemistry 2015, 284071. https://doi.org/10.1155/2015/284071
Sucha L, Hroch M, Řezáčová M, Rudolf E, Havelek R, Šípera L, Cmielová J, Kohlerová R, Bezrouk A, Tomšík P. 2013. Cytotoxic effect of α-tomatine in MCF-7 human breast cancer cells depends on cholesterol interaction and does not involve apoptosis. Oncology Reports 30(6), 2593–2602. https://doi.org/10.3892/or.2013.2778
Syed FQ, Elkady AI, Mohammed FA, Mirza MB, Hakeem KR, Alkarim S. 2018. Chloroform fraction of Foeniculum vulgare induces ROS-mediated mitochondria-caspase-dependent apoptosis in MCF-7 cells. Journal of Ethnopharmacology 218, 16–26. https://www.sciencedirect.com/science/article/pii/S0378874117346123
Tam CC, Nguyen K, Nguyen D, Hamada S, Kwon O, Kuang I, Gong S, Escobar S, Liu M, Kim J, Hou T, Tam J, Cheng LW, Kim JH, Land KM, Friedman M. 2021. Antimicrobial properties of tomato leaves, stems, and fruit and their relationship to chemical composition. BMC Complementary Medicine and Therapies 21(1), 229. https://doi.org/10.1186/s12906-021-03391-2
Venkatachalam J, Jeyadoss VS, Bose KSC, Subramanian R. 2024. Marine seaweed endophytic fungi-derived metabolites promote ROS-induced cell cycle arrest and apoptosis in human breast cancer cells. Molecular Biology Reports 51(1), 611. https://doi.org/10.1007/s11033-024-09511-8
Zhang D, Yuan H, Mo S-S, Mo X-W, Tang W-Z, Yan L-H. 2020. α-Tomatine inhibits proliferation, metastasis, drug resistance and immune infiltration through the PI3K-AKT-MAPK axis in gastric cancer. Research Square Preprint. https://doi.org/10.21203/rs.3.rs-53224/v1
Zhao B, Zhou B, Bao L, Yang Y, Guo K. 2015. α-Tomatine exhibits anti-inflammatory activity in lipopolysaccharide-activated macrophages. Inflammation 38(5), 1769–1776. https://doi.org/10.1007/s10753-015-0154-9
Zhou T, Wang X, Zhu Q, Zhou E, Zhang J, Song F, Xu C, Shen Y, Zou J, Zhu H, Su K, Lu W, Yi H, Huang W. 2025. Global trends and risk factors of laryngeal cancer: a systematic analysis for the Global Burden of Disease Study (1990–2021). BMC Cancer 25(1), 296. https://doi.org/10.1186/s12885-025-13700-4
Zorova LD, Demchenko EA, Korshunova GA, Tashlitsky VN, Zorov SD, Andrianova NV, Popkov VA, Babenko VA, Pevzner IB, Silachev DN, Plotnikov EY, Zorov DB. 2022. Is the mitochondrial membrane potential (ΔΨ) correctly assessed? intracellular and intramitochondrial modifications of the ΔΨ probe rhodamine 123. International Journal of Molecular Sciences 23(1), 482. https://doi.org/10.3390/ijms23010482
Nihal Ahamed Abul Kalam Azad, Suresh Kathiresan, Theerthu Azhamuthu, Senkuttuvan Ilanchit Chenni, Pugazhendhi Ravichandran, Maharani Jaganathan, Rajeswari Vasu, Pratheeba Veerapandiyan, 2025. Alpha-tomatine induces ROS-mediated mitochondrial apoptosis in laryngeal carcinoma (HEp-2) cells. Int. J. Biosci., 27(6), 14-24.
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