細胞老化是一個細胞週期及細胞生長被長期阻滯的一個現象，最主要受到p53/p21 和p16INK4a/Rb此二訊息傳遞路徑所調控，近期的研究顯示細胞老化是細胞抵抗腫瘤發生的一個機制。細胞自噬被報導指出能調節細胞老化的發展，但細胞自噬調控老化的機制仍是不太清楚的；在本研究中，我們藉由刀豆素A (Concanavalin A)所誘發的老化細胞中發現了一個可能的機制以來解釋細胞自噬是如何調控細胞老化的。刀豆素A是能與甘露糖結合的外源凝集素(lectins)，本實驗室過去發現刀豆素A能在肝癌細胞中誘發細胞自噬進而導致細胞死亡或抑制細胞生長，而細胞生長被抑制為老化細胞的其中一個特徵，在本研究中我們發現，刀豆素A在兩種肝癌細胞株Huh7、 HepG2中皆能抑制細胞生長和增加細胞老化相關半乳糖苷酶活性 (senescence-associated β-galactosidase activity)，證實刀豆素A得以在肝腫瘤細胞中引發細胞老化，此外，給予刀豆素A的情況之下p16INK4A表現量增加但p21表現量下降，表示刀素A可能是透過p16INK4A訊息傳遞路徑誘發細胞老化。我們進一步發現，利用細胞自噬相關shRNA或加入細胞自噬抑制劑氯奎寧 (chloroquine) 可以抑制刀豆素A所調控的p16INK4A表現和細胞老化相關半乳糖苷酶活性，說明刀豆素A所誘發的老化細胞中細胞自噬可能藉由調控p16INK4A進而誘發老化。最後我們發現，在給與了刀豆素A之後，細胞內p16INK4A的負向調控因子，SUV39H1會被泛素化且與自噬小體有共位的現象，而利用細胞自噬相關shRNA或chloroquine可以回復SUV39H1的表現，再者，異位表現SUV39H1可以降低刀豆素A所調控的p16INK4A表現和細胞老化相關半乳糖苷酶活性。綜合以上所述，刀豆素A引發的細胞自噬作用能降解SUV39H1而活化p16INK4A進而誘導肝癌細胞老化。因此，本研究為刀豆素A所誘導的自噬作用提供了新的抗腫瘤機制，也提供了新的機制說明細胞自噬如何調控老化。

Senescence is a state of prolonged cell cycle arrest regulated by p53 and p16INK4a/Rb pathway which serves as a tumor suppressive mechanism. Autophagy has recently been identified as an effector of senescence. However, how autophagy regulates senescence is not fully understood. In this study, we uncover the potential regulation between autophagy and senescence in Concanavalin A (ConA)-treated hepatoma cells. Con A, a lectin with mannose binding activity, was discovered to induce autophagic cell death and growth inhibition in hepatoma cells and this effect is one feature of cellular senescence. Here we further demonstrated that Con A induces cellular senescence in two hepatoma cell lines, Huh7 and HepG2, which was characterized by cellular growth inhibition and upregulation of senescence-associated β-galactosidase activity. We found that Con A treatment resulted in upregulation of p16INK4a, whereas the expression of p21 was downregulated. This suggests that p16INK4a signalling pathway is involved in Con A-induced cellular senescence in hepatoma cells. Silencing or inhibition of autophagy by shRNA against autophagy-related gene or chloroquine (CQ) treatment reversed the Con A-induced growth arrest, senescence-associate β-galactosidase activity and p16INK4A expression, suggesting that autophagy may regulate Con A-induced senescence via controlling expression of p16INK4A. Furthermore, SUV39H1, a negative regulator of p16 and senescence, was discovered to be ubiqitinated and colocalized with autophagosome in Con A treated senescent cells. Silencing of LC3 or inhibition of autophagic degradation by CQ was able to rescue Con A-downregulated SUV39H1 expression. Furthermore, ectopic expression of SUV39H1 reversed Con A-triggered p16 induction and senescence. Taken together, SUV39H1 is degraded by Con A-induced autophagy which in turn regulates p16 INK4A expression to facilitate senescence establishment. Our study provides a novel mechanism for the anti-cancer effect of Con A and new insight into the role of autophagy in the regulation of senescence progression.

Abe, M., Koga, H., Yoshida, T., Masuda, H., Iwamoto, H., Sakata, M., Hanada, S., Nakamura, T., Taniguchi, E., Kawaguchi, T., et al. (2012). Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor-kappaB/hypoxia-inducible factor-1alpha axis under hypoxic conditions. Hepatology research : the official journal of the Japan Society of Hepatology 42, 591-600.

Acosta, J.C., Banito, A., Wuestefeld, T., Georgilis, A., Janich, P., Morton, J.P., Athineos, D., Kang, T.W., Lasitschka, F., Andrulis, M., et al. (2013). A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nature cell biology 15, 978-990.

Arzumanyan, A., Reis, H.M., and Feitelson, M.A. (2013). Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma. Nature reviews Cancer 13, 123-135.

Bhoori, S., Schiavo, M., Russo, A., and Mazzaferro, V. (2007). First-line treatment for hepatocellular carcinoma: resection or transplantation? Transplantation proceedings 39, 2271-2273.

Bosch-Presegue, L., Raurell-Vila, H., Marazuela-Duque, A., Kane-Goldsmith, N., Valle, A., Oliver, J., Serrano, L., and Vaquero, A. (2011). Stabilization of Suv39H1 by SirT1 Is Part of Oxidative Stress Response and Ensures Genome Protection. Mol Cell 42, 210-223.

Bracken, A.P., Kleine-Kohlbrecher, D., Dietrich, N., Pasini, D., Gargiulo, G., Beekman, C., Theilgaard-Monch, K., Minucci, S., Porse, B.T., Marine, J.C., et al. (2007). The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes & development 21, 525-530.

Braig, M., and Schmitt, C.A. (2006). Oncogene-induced senescence: putting the brakes on tumor development. Cancer research 66, 2881-2884.

Campisi, J. (2001). Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 11, S27-S31.

Campisi, J., and di Fagagna, F.D. (2007). Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Bio 8, 729-740.

Capparelli, C., Guido, C., Whitaker-Menezes, D., Bonuccelli, G., Balliet, R., Pestell, T.G., Goldberg, A.F., Pestell, R.G., Howell, A., Sneddon, S., et al. (2012). Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production. Cell cycle 11, 2285-2302.

Chang, C.P., Yang, M.C., Liu, H.S., Lin, Y.S., and Lei, H.Y. (2007). Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model. Hepatology 45, 286-296.

Collado, M., and Serrano, M. (2006). The power and the promise of oncogene-induced senescence markers. Nature reviews Cancer 6, 472-476.

Coppe, J.P., Desprez, P.Y., Krtolica, A., and Campisi, J. (2010). The senescence-associated secretory phenotype: the dark side of tumor suppression. Annual review of pathology 5, 99-118.

Cristofalo, V.J., and Pignolo, R.J. (1993). Replicative senescence of human fibroblast-like cells in culture. Physiological reviews 73, 617-638.

Di Bisceglie, A.M. (2009). Hepatitis B and hepatocellular carcinoma. Hepatology 49, S56-60.

Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America 92, 9363-9367.

Donato, F., Tagger, A., Gelatti, U., Parrinello, G., Boffetta, P., Albertini, A., Decarli, A., Trevisi, P., Ribero, M.L., Martelli, C., et al. (2002). Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women. American journal of epidemiology 155, 323-331.

Dou, Z.X., Xu, C.Y., Donahue, G., Shimi, T., Pan, J.A., Zhu, J.J., Ivanov, A., Capelll, B.C., Drake, A.M., Shah, P.P., et al. (2015). Autophagy mediates degradation of nuclear lamina. Nature 527, 105-109.
El-Serag, H.B. (2011). Hepatocellular carcinoma. The New England journal of medicine 365, 1118-1127.

El-Serag, H.B. (2012). Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 142, 1264-1273 e1261.

El-Serag, H.B., and Rudolph, K.L. (2007). Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132, 2557-2576.

Forner, A., Llovet, J.M., and Bruix, J. (2012). Chemoembolization for intermediate HCC: is there proof of survival benefit? Journal of hepatology 56, 984-986.

Fullgrabe, J., Klionsky, D.J., and Joseph, B. (2014). The return of the nucleus: transcriptional and epigenetic control of autophagy. Nat Rev Mol Cell Bio 15, 65-74.
Gewirtz, D.A. (2014). Autophagy and senescence in cancer therapy. J Cell Physiol 229, 6-9.

Goehe, R.W., Di, X., Sharma, K., Bristol, M.L., Henderson, S.C., Valerie, K., Rodier, F., Davalos, A.R., and Gewirtz, D.A. (2012). The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep? J Pharmacol Exp Ther 343, 763-778.

Goldstein, S. (1990). Replicative senescence: the human fibroblast comes of age. Science 249, 1129-1133.

Greten, T.F., Manns, M.P., and Malek, N. (2009). [Sorafenib for the treatment of HCC--the beginning of a new era in the treatment of HCC]. Zeitschrift fur Gastroenterologie 47, 55-60.

Gullberg, M., and Larsson, E.L. (1983). Induction of cytolytic T lymphocytes by mitogenic lectins is specifically inhibited by anti-Lyt-2 antibodies. Eur J Immunol 13, 476-480.

Hai, H., Tamori, A., and Kawada, N. (2014). Role of hepatitis B virus DNA integration in human hepatocarcinogenesis. World journal of gastroenterology 20, 6236-6243.
Hayflick, L., and Moorhead, P.S. (1961). The serial cultivation of human diploid cell strains. Experimental cell research 25, 585-621.

Heymann, F., Hamesch, K., Weiskirchen, R., and Tacke, F. (2015). The concanavalin A model of acute hepatitis in mice. Lab Anim-Uk 49, 12-20.

Ikeda, K., Saitoh, S., Suzuki, Y., Kobayashi, M., Tsubota, A., Koida, I., Arase, Y., Fukuda, M., Chayama, K., Murashima, N., et al. (1998). Disease progression and hepatocellular carcinogenesis in patients with chronic viral hepatitis: a prospective observation of 2215 patients. Journal of hepatology 28, 930-938.

Jiang, Y.F., He, B., Li, N.P., Ma, J., Gong, G.Z., and Zhang, M. (2011). The oncogenic role of NS5A of hepatitis C virus is mediated by up-regulation of survivin gene expression in the hepatocellular cell through p53 and NF-kappaB pathways. Cell biology international 35, 1225-1232.

Jin, Y., Fuller, L., Carreno, M., Zucker, K., Roth, D., Esquenazi, V., Karatzas, T., Swanson, S.J., 3rd, Tzakis, A.G., and Miller, J. (1997). The immune reactivity role of HCV-induced liver infiltrating lymphocytes in hepatocellular damage. Journal of clinical immunology 17, 140-153.

Kang, C., Xu, Q., Martin, T.D., Li, M.Z., Demaria, M., Aron, L., Lu, T., Yankner, B.A., Campisi, J., and Elledge, S.J. (2015a). The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science 349, aaa5612.

Kang, C., Xu, Q.K., Martin, T.D., Li, M.Z., Demaria, M., Aron, L., Lu, T., Yankner, B.A., Campisi, J., and Elledge, S.J. (2015b). The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science 349.
Kang, H.T., Lee, K.B., Kim, S.Y., Choi, H.R., and Park, S.C. (2011). Autophagy impairment induces premature senescence in primary human fibroblasts. PloS one 6, e23367.

Kao, C.F., Chen, S.Y., Chen, J.Y., and Wu Lee, Y.H. (2004). Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein. Oncogene 23, 2472-2483.

Kim, K.H., and Lee, M.S. (2014). Autophagy-a key player in cellular and body metabolism. Nat Rev Endocrinol 10, 322-337.

Kimbi, G.C., Kramvis, A., and Kew, M.C. (2005). Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B. World journal of gastroenterology 11, 6416-6421.

Krishnamurthty, J., Torrice, C., Ramsey, M.R., Kovalev, G.I., Al-Regaiey, K., Su, L.S., and Sharpless, N.E. (2004). Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114, 1299-1307.

Kuilman, T., Michaloglou, C., Vredeveld, L.C., Douma, S., van Doorn, R., Desmet, C.J., Aarden, L.A., Mooi, W.J., and Peeper, D.S. (2008). Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133, 1019-1031.
Laberge, R.M., Sun, Y., Orjalo, A.V., Patil, C.K., Freund, A., Zhou, L., Curran, S.C., Davalos, A.R., Wilson-Edell, K.A., Liu, S., et al. (2015). MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nature cell biology 17, 1049-1061.

Lai, Y.C., Chuang, Y.C., Chang, C.P., and Yeh, T.M. (2015). Macrophage migration inhibitory factor has a permissive role in concanavalin A-induced cell death of human hepatoma cells through autophagy. Cell Death Dis 6, e2008.

Lee, J., Giordano, S., and Zhang, J.H. (2012). Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 441, 523-540.

Lei, H.Y., and Chang, C.P. (2007). Induction of autophagy by concanavalin A and its application in anti-tumor therapy. Autophagy 3, 402-404.

Lim, J.S., Park, S.H., and Jang, K.L. (2012). Hepatitis C virus Core protein overcomes stress-induced premature senescence by down-regulating p16 expression via DNA methylation. Cancer letters 321, 154-161.

Liu, Y., and Levine, B. (2015). Autosis and autophagic cell death: the dark side of autophagy. Cell death and differentiation 22, 367-376.

Maiuri, M.C., Zalckvar, E., Kimchi, A., and Kroemer, G. (2007). Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Bio 8, 741-752.
Matteoni, C.A., Younossi, Z.M., Gramlich, T., Boparai, N., Liu, Y.C., and McCullough, A.J. (1999). Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116, 1413-1419.
Mazzaferro, V., Bhoori, S., Sposito, C., Bongini, M., Langer, M., Miceli, R., and Mariani, L. (2011). Milan criteria in liver transplantation for hepatocellular carcinoma: an evidence-based analysis of 15 years of experience. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 17 Suppl 2, S44-57.

Michaloglou, C., Vredeveld, L.C.W., Soengas, M.S., Denoyelle, C., Kuilman, T., van der Horst, C.M.A.M., Majoor, D.M., Shay, J.W., Mooi, W.J., and Peeper, D.S. (2005). BRAF(E600)-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720-724.

Mittal, S., and El-Serag, H.B. (2013). Epidemiology of hepatocellular carcinoma: consider the population. Journal of clinical gastroenterology 47 Suppl, S2-6.
Mizushima, N., Yoshimori, T., and Levine, B. (2010). Methods in Mammalian Autophagy Research. Cell 140, 313-326.

Morgan, T.R., Mandayam, S., and Jamal, M.M. (2004). Alcohol and hepatocellular carcinoma. Gastroenterology 127, S87-S96.

Munoz-Espin, D., and Serrano, M. (2014). Cellular senescence: from physiology to pathology. Nature reviews Molecular cell biology 15, 482-496.

Nagasue, N., Uchida, M., Makino, Y., Takemoto, Y., Yamanoi, A., Hayashi, T., Chang, Y.C., Kohno, H., Nakamura, T., and Yukaya, H. (1993). Incidence and factors associated with intrahepatic recurrence following resection of hepatocellular carcinoma. Gastroenterology 105, 488-494.

Nakamura, H., Aoki, H., Hino, O., and Moriyama, M. (2011). HCV core protein promotes heparin binding EGF-like growth factor expression and activates Akt. Hepatology research : the official journal of the Japan Society of Hepatology 41, 455-462.

Olsen, C.L., Gardie, G.B., Yaswen, P., and Stampfer, M.R. (2002). Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion. Oncogene 21, 6328-6339.

Park, S.H., Jung, J.K., Lim, J.S., Tiwari, I., and Jang, K.L. (2011). Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation. The Journal of general virology 92, 1309-1317.

Prieur, A., and Peeper, D.S. (2008). Cellular senescence in vivo: a barrier to tumorigenesis. Curr Opin Cell Biol 20, 150-155.

Ravikumar, B., Sarkar, S., Davies, J.E., Futter, M., Garcia-Arencibia, M., Green-Thompson, Z.W., Jimenez-Sanchez, M., Korolchuk, V.I., Lichtenberg, M., Luo, S., et al. (2010). Regulation of mammalian autophagy in physiology and pathophysiology. Physiological reviews 90, 1383-1435.

Rayess, H., Wang, M.B., and Srivatsan, E.S. (2012). Cellular senescence and tumor suppressor gene p16. International journal of cancer 130, 1715-1725.

Raza, A., and Sood, G.K. (2014). Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World journal of gastroenterology 20, 4115-4127.
Roussel, M.F. (1999). The INK4 family of cell cycle inhibitors in cancer. Oncogene 18, 5311-5317.

Rubinsztein, D.C., Shpilka, T., and Elazar, Z. (2012). Mechanisms of autophagosome biogenesis. Current biology : CB 22, R29-34.

Schnegelsberg, B., Schumacher, U., and Valentiner, U. (2011). Lectin histochemistry of metastasizing and non-metastasizing breast and colon cancer cells. Anticancer Res 31, 1589-1597.

Sherwood, S.W., Rush, D., Ellsworth, J.L., and Schimke, R.T. (1988). Defining cellular senescence in IMR-90 cells: a flow cytometric analysis. Proceedings of the National Academy of Sciences of the United States of America 85, 9086-9090.

Sidler, C., Li, D., Wang, B., Kovalchuk, I., and Kovalchuk, O. (2014a). SUV39H1 downregulation induces deheterochromatinization of satellite regions and senescence after exposure to ionizing radiation. Frontiers in genetics 5, 411.
Sidler, C., Woycicki, R., Li, D., Wang, B., Kovalchuk, I., and Kovalchuk, O. (2014b). A role for SUV39H1-mediated H3K9 trimethylation in the control of genome stability and senescence in WI38 human diploid lung fibroblasts. Aging 6, 545-563.

Stolz, A., Ernst, A., and Dikic, I. (2014). Cargo recognition and trafficking in selective autophagy. Nature cell biology 16, 495-501.

Vrancken, K., Paeshuyse, J., and Liekens, S. (2012). Angiogenic activity of hepatitis B and C viruses. Antiviral chemistry & chemotherapy 22, 159-170.

Wang, H.X., Liu, M., Weng, S.Y., Li, J.J., Xie, C., He, H.L., Guan, W., Yuan, Y.S., and Gao, J. (2012). Immune mechanisms of Concanavalin A model of autoimmune hepatitis. World journal of gastroenterology 18, 119-125.

Wu, L.P., Wang, X., Li, L., Zhao, Y., Lu, S.L., Yu, Y., Zhou, W., Liu, X.Y., Yang, J., Zheng, Z.X., et al. (2008). Histone deacetylase inhibitor depsipeptide activates silenced genes through decreasing both CpG and H3K9 methylation on the promoter. Mol Cell Biol 28, 3219-3235.

Young, A.R., and Narita, M. (2010). Connecting autophagy to senescence in pathophysiology. Curr Opin Cell Biol 22, 234-240.

Zhai, B., and Sun, X.Y. (2013). Mechanisms of resistance to sorafenib and the corresponding strategies in hepatocellular carcinoma. World journal of hepatology 5, 345-352.

Zhang, W., Li, J., Suzuki, K., Qu, J., Wang, P., Zhou, J., Liu, X., Ren, R., Xu, X., Ocampo, A., et al. (2015). Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348, 1160-1163.
Zindy, F., Quelle, D.E., Roussel, M.F., and Sherr, C.J. (1997). Expression of the p16(INK4a) tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 15, 203-211.