鐵依賴型細胞死亡（Ferroptosis）近來被指出是一種計畫性的細胞壞死。其特徵為依賴鐵產生的脂質過氧化（Lipid peroxidation），可能因細胞內自由鐵（Labile iron pool）增加導致。鐵是多種細胞生理現象所需的無機元素，並與細胞增殖及生長密切相關。腫瘤細胞，如肝癌細胞，對鐵的需求較高，且鐵相關蛋白基因會過度表現。此外，近期研究發現，細胞自噬中透過選擇性攜帶蛋白Nuclear coactivator 4（NCOA4）將鐵蛋白（Ferritin）降解的鐵蛋白自噬（Ferritinophagy），可促進鐵依賴型細胞死亡發生。本研究於SRB assay中發現隨植化素Formosanin C（FC）濃度上升，肝癌細胞Hep3B與HepG2生長顯著被抑制，而加入鐵依賴型細胞死亡抑制劑Ferrostatin-1、Liproxstatin-1及同時加Vitamin E與Vitamin C後，細胞生長有部分回復。處理FC後脂質過氧化物（Reactive oxygen species）的增加，及加入抑制劑後的部分減少，皆顯示FC有引發鐵依賴型細胞死亡的效果。FC同時會增加細胞中的酸性囊泡小體比例，顯示FC在兩個細胞中皆可誘導細胞自噬。西方墨點法中觀察到LC3-II產生，並在加入細胞自噬抑制劑Bafilomycin A1後累積，也證明了FC誘導細胞自噬的效果。在細胞自噬被抑制的情況下，處理FC 24小時後，NCOA4與鐵蛋白在兩細胞中皆會堆積。免疫螢光染色法中，LC3與NCOA4或LC3與鐵蛋白的Co-localization，也顯示FC誘導鐵蛋白自噬的潛力。值得注意的是，有較高NCOA4表現量的HepG2細胞，對FC誘導的鐵依賴型細胞死亡與細胞自噬，較鐵蛋白表現量較高的Hep3B細胞敏感，表明鐵依賴型細胞死亡誘導劑在鐵蛋白自噬相關的癌症治療中的作用。

關鍵字：肝癌、皂素、鐵依賴型細胞死亡、細胞自噬、鐵蛋白自噬

Ferroptosis is a recently described form of regulated necrotic cell death. A hallmark of ferroptosis is iron-dependent lipid peroxidation, which may caused by the increased level of labile iron pool. Iron is an essential inorganic element for various cellular events and is directly associated with cell proliferation and growth. Tumor cells, such as hepatocellular carcinoma (HCC) cells, necessitated higher concentrations of iron and the genes of iron uptake proteins were highly overexpressed. Moreover, recent studies indicated that autophagy contributes to ferroptosis through ferritinophagy, a degradation of the iron-storage protein, ferritin, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Here, SRB assay showed a significant dosage-related growth inhibition of HCC HepG2 and Hep3B cells by phytochemical Formosanin C (FC), which can be partially reversed by co-administration of ferroptosis inhibitor ferrostatin-1, liproxstatin-1 and vitamin E combined with vitamin C. Elevated production of lipid reactive oxygen species by FC and partially down regulation by co-administration of ferroptosis inhibitor indicating FC-induced ferroptosis. Percentage of acidic vesicular organelles-positive cells also increased after treated with FC, representing the induction of autophagic flux in both cell lines. In western blot analysis, the production of LC3-II by FC and a further elevation of LC3-II expression by addition of autophagy inhibitor bafilomycin A1 confirmed the autophagic effect of FC. In these autophagy inhibited conditions, NCOA4 and ferritin accumulation also observed in both cell lines after treated with FC for 24 h. Co-localization of LC3 with NCOA4 and LC3 with ferritin in immunoflurescence also showing the ferritinophagy potential induced by FC. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC-induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression, suggesting the role of ferroptosis inducer in ferritinophagy-related treatment of cancers.

Keywords: hepatocellular carcinoma, saponin, ferroptosis, ferritinophagy, autophagy

財團法人台灣必安研究所。民國107年6月26日，取自
http://www.brion.org.tw/。
行政院衛生福利部統計處106年死因統計。民國107年6月10日，
取自https://dep.mohw.gov.tw/DOS/lp-3960-113.html。
Abbaspour, N., Hurrell, R., & Kelishadi, R. (2014). Review on iron and its importance for human health. J Res Med Sci, 19(2), 164-174.
Andrews, N. C., & Schmidt, P. J. (2007). Iron homeostasis. Annu Rev Physiol, 69, 69-85.
Bellelli, R., Federico, G., Matte’, A., Colecchia, D., Iolascon, A., Chiariello, M., Carlomagno, F. et al. (2016). NCOA4 Deficiency Impairs Systemic Iron Homeostasis. Cell Reports, 14(3), 411-421.
Bruix, J., Qin, S., Merle, P., Granito, A., Huang, Y. H., Bodoky, G., et al., Han, G. (2017). Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet, 389(10064), 56-66.
Campbell, J. A. (1940). Effects of Precipitated Silica and of Iron Oxide on the Incidence of Primary Lung Tumours in Mice. British Medical Journal, 2(4156), 275-280.
Chen, L., Li, X., Liu, L., Yu, B., Xue, Y., & Liu, Y. (2015). Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-gamma-lyase function. Oncol Rep, 33(3), 1465-1474.
Cheung, J. Y., Ong, R. C., Suen, Y. K., Ooi, V., Wong, H. N., Mak, T. C., Kong, S. K. et al. (2005). Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. Cancer Lett, 217(2), 203-211.
Christophe, L., Zakaria, E., Corinne, G., Jean-Claude, B., Jean-Claude, M., Bruno, C., & Antoine, G. (2013). Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib. International Journal of Cancer, 133(7), 1732-1742.
Dixon, S. J., Lemberg, Kathryn M., Lamprecht, Michael R., Skouta, R., Zaitsev, Eleina M., Gleason, Caroline E., Stockwell, Brent R. et al. (2012). Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell, 149(5), 1060-1072.
Dixon, S. J., Patel, D. N., Welsch, M., Skouta, R., Lee, E. D., Hayano, M., Stockwell, B. R. et al. (2014). Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife, 3, e02523.
Dowdle, W. E., Nyfeler, B., Nagel, J., Elling, R. A., Liu, S., Triantafellow, E., Murphy, L. O. et al. (2014). Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. Nature Cell Biology, 16, 1069.
Friedmann Angeli, J. P., Schneider, M., Proneth, B., Tyurina, Y. Y., Tyurin, V. A., Hammond, V. J., Conrad, M. et al. (2014). Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nature Cell Biology, 16, 1180.
Gao, M., Monian, P., Pan, Q., Zhang, W., Xiang, J., & Jiang, X. (2016). Ferroptosis is an autophagic cell death process. Cell Res, 26(9), 1021-1032.
Gomes, A., Fernandes, E., & Lima, J. L. F. C. (2005). Fluorescence probes used for detection of reactive oxygen species. Journal of Biochemical and Biophysical Methods, 65(2), 45-80.
Hann, H. W., Stahlhut, M. W., & Blumberg, B. S. (1988). Iron nutrition and tumor growth: decreased tumor growth in iron-deficient mice. Cancer Res, 48(15), 4168-4170.
Hou, W., Xie, Y., Song, X., Sun, X., Lotze, M. T., Zeh, H. J., 3rd, Tang, D. et al. (2016). Autophagy promotes ferroptosis by degradation of ferritin. Autophagy, 12(8), 1425-1428.
Jiang, L., Kon, N., Li, T., Wang, S. J., Su, T., Hibshoosh, H., Gu, W. et al. (2015). Ferroptosis as a p53-mediated activity during tumour suppression. Nature, 520(7545), 57-62.
Karaman, B., Battal, B., Sari, S., & Verim, S. (2014). Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World J Gastroenterol, 20(47), 18059-18060.
Kim, B. K., Kim, S. U., Park, J. Y., Kim, D. Y., Ahn, S. H., Park, M. S., Han, K. H. et al. (2012). Applicability of BCLC stage for prognostic stratification in comparison with other staging systems: single centre experience from long-term clinical outcomes of 1717 treatment-naive patients with hepatocellular carcinoma. Liver Int, 32(7), 1120-1127.
Kollara, A., & Brown, T. J. (2012). Expression and function of nuclear receptor co-activator 4: evidence of a potential role independent of co-activator activity. Cellular and Molecular Life Sciences, 69(23), 3895-3909.
Kondo, Y., Kanzawa, T., Sawaya, R., & Kondo, S. (2005). The role of autophagy in cancer development and response to therapy. Nat Rev Cancer, 5(9), 726-734.
Krstic, J., Galhuber, M., Schulz, T. J., Schupp, M., & Prokesch, A. (2018). p53 as a Dichotomous Regulator of Liver Disease: The Dose Makes the Medicine. Int J Mol Sci, 19(3).
Latunde-Dada, G. O. (2017). Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochimica et Biophysica Acta (BBA) - General Subjects, 1861(8), 1893-1900.
Lee, M. S., Yuet-Wa, J. C., Kong, S. K., Yu, B., Eng-Choon, V. O., Nai-Ching, H. W., Fung, K. P. et al. (2005). Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft. Cancer Biol Ther, 4(11), 1248-1254.
Liu, B., Fang, M., Hu, Y., Huang, B., Li, N., Chang, C., Liu, W. et al. (2014). Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation. Autophagy, 10(3), 416-430.
Lockshin, R. A., & Zakeri, Z. (2004). Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 36(12), 2405-2419.
Lu, B., Chen, X. B., Ying, M. D., He, Q. J., Cao, J., & Yang, B. (2017). The Role of Ferroptosis in Cancer Development and Treatment Response. Frontiers in Pharmacology, 8, 992.
Lay, J. Y. and Chiang, H. C. (1980) Studies on the constituents of Paris
Formosa Hayata. J. Taiwan Pharmaceu. Asso., 32, 14-27
Ma, S., Dielschneider, R. F., Henson, E. S., Xiao, W., Choquette, T. R., Blankstein, A. R., Gibson, S. B. et al. (2017). Ferroptosis and autophagy induced cell death occur independently after siramesine and lapatinib treatment in breast cancer cells. PLoS One, 12(8), e0182921.
Magarinos, M., Pulido, S., Aburto, M. R., de Iriarte Rodriguez, R., & Varela-Nieto, I. (2017). Autophagy in the Vertebrate Inner Ear. Front Cell Dev Biol, 5, 56.
Mancias, J. D., & Kimmelman, A. C. (2016). Mechanisms of Selective Autophagy in Normal Physiology and Cancer. J Mol Biol, 428(9 Pt A), 1659-1680.
Mancias, J. D., Wang, X., Gygi, S. P., Harper, J. W., & Kimmelman, A. C. (2014). Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature, 509, 105.
Mbaveng, A. T., Ndontsa, B. L., Kuete, V., Nguekeu, Y. M. M., Çelik, İ., Mbouangouere, R., Efferth, T. et al. (2018). A naturally occuring triterpene saponin ardisiacrispin B displayed cytotoxic effects in multi-factorial drug resistant cancer cells via ferroptotic and apoptotic cell death. Phytomedicine, 43, 78-85.
Orellana, E. A., & Kasinski, A. L. (2016). Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation. Bio-protocol, 6(21), e1984.
Ott, C., König, J., Höhn, A., Jung, T., & Grune, T. (2016). Reduced autophagy leads to an impaired ferritin turnover in senescent fibroblasts. Free Radical Biology and Medicine, 101, 325-333.
Ou, Y., Wang, S. J., Li, D., Chu, B., & Gu, W. (2016). Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses. Proc Natl Acad Sci U S A, 113(44), E6806-e6812.
Pap, E. H., Drummen, G. P., Winter, V. J., Kooij, T. W., Rijken, P., Wirtz, K. W., Post, J. A. et al. (1999). Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591). FEBS Lett, 453(3), 278-282.
Richmond, H. G. (1959). Induction of Sarcoma in the Rat by Iron—Dextran Complex. British Medical Journal, 1(5127), 947-940.943.
Roh, J. L., Kim, E. H., Jang, H. J., Park, J. Y., & Shin, D. (2016). Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer. Cancer Lett, 381(1), 96-103.
Sareen, S. G., Jack, L. S., & James, L. G. (2011). Advanced Nutrition and Human Metabolism(5th ed). Wadsworth.
Santoro, M., Dathan, N. A., Berlingieri, M. T., Bongarzone, I., Paulin, C., Grieco, M., Fusco, A. et al. (1994). Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma. Oncogene, 9(2), 509-516.
Shi, Y. M., Yang, L., Geng, Y. D., Zhang, C., & Kong, L. Y. (2015). Polyphyllin I induced-apoptosis is enhanced by inhibition of autophagy in human hepatocellular carcinoma cells. Phytomedicine, 22(13), 1139-1149.
Shimada, K., Skouta, R., Kaplan, A., Yang, W. S., Hayano, M., Dixon, S. J.,. Stockwell, B. R. et al. (2016). Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat Chem Biol, 12(7), 497-503.
Stevens, R. G., Jones, D. Y., Micozzi, M. S., & Taylor, P. R. (1988). Body iron stores and the risk of cancer. N Engl J Med, 319(16), 1047-1052.
Su, M., Mei, Y., & Sinha, S. (2013). Role of the Crosstalk between Autophagy and Apoptosis in Cancer. J Oncol, 2013, 102735.
Sun, X., Niu, X., Chen, R., He, W., Chen, D., Kang, R., & Tang, D. (2016). Metallothionein-1G Facilitates Sorafenib Resistance through Inhibition of Ferroptosis. Hepatology, 64(2), 488-500.
Sun, X., Ou, Z., Chen, R., Niu, X., Chen, D., Kang, R., & Tang, D. (2016). Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology, 63(1), 173-184.
Tasdemir, E., Chiara Maiuri, M., Morselli, E., Criollo, A., D'Amelio, M., Djavaheri-Mergny, M., Kroemer, G. et al. (2008). A dual role of p53 in the control of autophagy. Autophagy, 4(6), 810-814.
Tasset, I., & Cuervo, A. M. (2016). Role of chaperone-mediated autophagy in metabolism. Febs j, 283(13), 2403-2413.
Thome, M. P., Filippi-Chiela, E. C., Villodre, E. S., Migliavaca, C. B., Onzi, G. R., Felipe, K. B., & Lenz, G. (2016). Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy. J Cell Sci, 129(24), 4622-4632.
Torti, S. V., & Torti, F. M. (2013). Iron and cancer: more ore to be mined. Nat Rev Cancer, 13(5), 342-355.
van Tonder, A., Joubert, A. M., & Cromarty, A. D. (2015). Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Research Notes, 8, 47.
Wang, P., Guo, Q.-s., Wang, Z.-w., & Qian, H.-x. (2013). HBx induces HepG-2 cells autophagy through PI3K/Akt–mTOR pathway. Molecular and Cellular Biochemistry, 372(1), 161-168.
Wang, P., Wang, Z. W., Qian, H. X., & Guo, Q. S. (2013). Role of autophagy in HepG-2 cells induced by hepatitis B virus x protein. Zhonghua Yi Xue Za Zhi, 93(44), 3556-3558.
Wu, R. T., Chiang, H. C., Fu, W. C., Chien, K. Y., Chung, Y. M., & Horng, L. Y. (1990). Formosanin-C, an immunomodulator with antitumor activity. Int J Immunopharmacol, 12(7), 777-786.
Wu, T., Sempos, C. T., Freudenheim, J. L., Muti, P., & Smit, E. (2004). Serum iron, copper and zinc concentrations and risk of cancer mortality in US adults. Ann Epidemiol, 14(3), 195-201.
Xie, Y., Zhu, S., Song, X., Sun, X., Fan, Y., Liu, J., Tang, D. et al. (2017). The Tumor Suppressor p53 Limits Ferroptosis by Blocking DPP4 Activity. Cell Rep, 20(7), 1692-1704.
Yang, W. S., Kim, K. J., Gaschler, M. M., Patel, M., Shchepinov, M. S., & Stockwell, B. R. (2016). Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proceedings of the National Academy of Sciences, 113(34), E4966.
Yang, W. S., SriRamaratnam, R., Welsch, M. E., Shimada, K., Skouta, R., Viswanathan, V. S., Stockwell, B. R. et al. (2014). Regulation of ferroptotic cancer cell death by GPX4. Cell, 156(1-2), 317-331.
Yu, Y., Xie, Y., Cao, L., Yang, L., Yang, M., Lotze, M. T., Tang, D. wt al. (2015). The ferroptosis inducer erastin enhances sensitivity of acute myeloid leukemia cells to chemotherapeutic agents. Mol Cell Oncol, 2(4), e1054549.
Ying, Shao-Shun. (2000). LILIACEAE. Flora of Taiwan, second edition.
5: 57.
Zhang, C., Jia, X., Wang, K., Bao, J., Li, P., Chen, M., He, C. et al. (2016). Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells. PLoS One, 11(1), e0147405.
Zhang, J., Yang, Y., Lei, L., & Tian, M. (2015). Rhizoma Paridis Saponins Induces Cell Cycle Arrest and Apoptosis in Non-Small Cell Lung Carcinoma A549. Cells, Vol. 21.
陳玲玲. (2003). Formosanin C 抗大腸直腸癌之作用機轉. 成功大學微生物及免疫學研究所學位論文, 1-44.