肝癌(Hepatocellular carcinoma, HCC)為世界上最常見且具有高度轉移與復發性的惡性腫瘤。儘管有許多常規癌症治療方法，但肝癌細胞具有抗藥性是目前治療中最主要的問題。本實驗利用組織蛋白去乙醯?抑制劑(Histone deacetylases inhibitors, HDACis)建立抗藥性肝癌細胞株並進一步分析其抗藥性發展機制。透過二維電泳(Two-dimensional gel electrophoresis, 2-DE)與反轉錄PCR (Reverse transcription PCR, RT-PCR)分析肝癌細胞(HA22T)及抗藥性肝癌細胞(HDACis-R cells)的蛋白質及mRNA表現，並顯示鈣網蛋白(Calreticulin, CRT)在HDACis-R cells中的表現量較低。CRT為一助疊蛋白(chaperone)，在內質網中能幫助蛋白質正確的折疊；而在非內質網中，則能與mRNA結合並穩定mRNA。本研究指出，HDACis-R cells的CRT表現量不受傳統內質網壓力途徑GRP78-PERK所調節。此外，我們也發現HDACis-R cells的CRT顯示具有轉譯後修飾(泛素化-ubiquitin modification)且能被蛋白?體(proteasomal)降解。重要的是，我們在肝癌細胞中，透過處理MG132與Plasmid的轉染，能使CRT在細胞中累積。而細胞中累積的CRT，能在化療藥物的刺激下促進HDACis-R cells凋亡。期望在接下來的實驗中，能透過核質分離與螢光顯微鏡的實驗，進一步確認CRT是否是透過轉位至細胞核的方式，進而促使肝癌細胞凋亡，特別是在HDACis-R cells。
Hepatocellular carcinoma (HCC) is one of the most common fatal malignant tumors that have highly metastatic and recurrent properties in the world. Although there are many conventional therapies were improved, the major cause of chemotherapy failure is the development of drug resistance to chemotherapeutic agents in HCC cells. In this study, we used histone deacetylases inhibitors (HDACis) to establish drug-resistant liver cancer cells and further analyzed the development of drug resistance of molecular mechanisms in liver cancer cells. In the two-dimensional gel electrophoresis (2-DE) and reverse transcription PCR (RT-PCR) data showed that the calreticulin (CRT) protein and mRNA was altered in HDACis-resistant cells and was lowly expressed in HDACis-resistant cells compared with HA22T parental cells. CRT is a chaperone to help proteins fold correctly inside the ER and is an mRNA binding protein inside the non-ER to the mRNA stability. In this study, we showed that thapsigargin-induced ER stress activation not through ER stress down-stream protein GRP78-PERK expression to regulate CRT expression in HDACis-R cells. However, we observed that post-translational modifications of CRT (ubiquitin modification) that could be degraded by proteasomal degradation in HDACis-R cells. Importantly, post-translational modifications of CRT was accumulated by using MG132 or transfecting with CRT plasmid in liver cancer cells, and that induced cell apoptosis by combining with HDAC inhibitors in liver cancer cells, especially in the HDACis-R cells. In the future study, the HDAC inhibitor-mediated overexpression CRT translocation to the cell nucleus and enhances chemosensitivity in HDAC inhibitor-resistant hepatocellular carcinoma cells was measured by cytoplasmic and nuclear extraction and immunofluorescence assay.
