肝癌（Hepatocellular carcinoma）是肝臟的原發性惡性腫瘤。肝癌的治療取決於疾病的階段，例如化療被認為用於治療晚期肝癌。這些細胞毒性藥物誘導癌細胞死亡以控制腫瘤生長。除了誘發細胞死亡之外，最近的研究顯示化療藥物也可能引發細胞老化以抑制腫瘤生長。在本研究中，我們測試了三種具潛力的臨床抗癌藥物AR-12，蕾莎瓦 (sorafenib)和順鉑 (Cisplatin)的細胞老化誘導活性。研究結果顯示，這些藥物都引發了肝癌細胞的老化反應，包括抑制細胞生長能力和增加β-半乳糖苷酶活性。再者，給予這三種藥物會導致 p16INK4a表現量增加，但卻造成p21表現量下降。這暗示AR-12、蕾莎瓦和順鉑可能會經由p16INK4a訊息傳遞路徑誘發細胞老化。此外，給予AR-12和蕾莎瓦會促使p16INK4a的負向調控因子SUV39H1在細胞內被降解，但給予順鉑則不影響SUV39H1的表現量。雖然過度表現SUV39H1可以降低AR-12和蕾莎瓦所調控的p16INK4a表現和細胞老化相關半乳糖苷酶活性，但是進一步發現，AR-12和蕾莎瓦會分別誘導細胞自噬及蛋白酶體反應來降解SUV39H1。我們觀察到在AR-12處理下，細胞質中的SUV39H1會與自噬小體有共位的現象，並且通過細胞自噬被降解。此外，在蕾莎瓦處理的細胞中也清楚地發現了在細胞質會有核蛋白SUV39H1的表現。綜合以上所述，我們闡明了在AR-12和蕾莎瓦會分別藉由細胞自噬和蛋白酶體作用來降解SUV39H1，進而活化p16INK4a而誘導肝癌細胞老化。因此，本研究提供了AR-12和蕾莎瓦抗癌作用的新機制，並且提出不同的SUV39H1蛋白水解途徑皆參與AR-12和蕾莎瓦所誘導的細胞老化。

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. The treatment of HCC varies depending on the stage of disease, such as chemotherapy considered for treating advanced HCC. These cytotoxic drugs are suggested to induce cancer cell death to control tumor growth. In addition to cell death, recent studies have indicated that chemo drugs may also trigger cell senescence responses to restrain the tumor growth. In this study, we tested senescence inducing activities of three potential clinical anti-cancer drugs, AR-12, sorafenib and cisplatin. The results showed that these drugs all triggered the senescence responses, including cell growth inhibition and increase of beta-galactosidase activity, in HCC cells. We found that these three drugs resulted in upregulation of p16INK4a, whereas the expression of p21 was downregulated. This suggests that p16INK4a signalling pathway is involved in AR-12, sorafenib and cisplatin-induced cellular senescence in hepatoma cells. Furthermore, SUV39H1, a negative regulator of p16INK4a, was down-regulated in AR-12 and sorafenib-treated HCC cells, but not in cisplatin-treated cells. Although overexpression of SUV39H1 attenuated both AR-12 and sorafenib-induced p16INK4a upregulation and beta-galactosidase activity, the degradation of SUV39H1 caused by AR-12 and sorafenib was differently mediated by autophagy and proteasome, respectively. We observed that SUV39H1 was targeted by autophagosomes in cytoplasm and degraded via autophagy under AR-12 treatment. Furthermore, the cytosolic expression of nucleus remaining protein, SUV39H1, was also clearly found in sorafenib-treated cells. Overall, we elucidated that upon AR-12 and sorafenib treatment, SUV39H1 degradation is separately mediated by autophagy and proteasome, which in turn regulates p16INK4a expression to facilitate senescence induction. Our study provides a novel mechanism to elicit senescence for the anti-cancer effect of AR-12 and sorafenib via different proteolytic regulation of SUV39H1.

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