探討p53與Aurora A在bortezomib誘發大腸癌細胞之G2/M期停滯與細胞凋亡的角色

Abstract

Bortezomib (也稱為Velcade)是一種蛋白酶體抑制劑，已經被臨床用於治療多發性骨髓瘤。大腸癌是世界上發病率與致死率最主要的癌症之一。p53是一個腫瘤抑制蛋白，可調控bortezomib所誘發的細胞週期停滯與細胞凋亡。Aurora A是一種serine/threonine激酶具有調控有絲分裂的功能，會大量表現在許多人類癌細胞中，包括大腸癌。然而，p53與Aurora A在bortezomib處理大腸癌後的角色與調控功能仍不清楚。在本論文中，我們發現處理bortezomib後，會降低多種人類大腸癌細胞的細胞存活率，以及增加G2/M時期細胞數與細胞凋亡。有趣地是bortezomib會明顯地增加p53和Aurora A蛋白表現，並會共同位於G2期停滯的核仁中。利用免疫沉澱法與共軛焦顯微技術分析，我們發現Aurora A蛋白會直接在核仁中與p53蛋白結合。再者，bortezomib在帶有p53功能正常的大腸癌細胞所誘發的細胞凋亡能力，會比p53基因喪失的大腸癌細胞較為敏感。此外，結合bortezomib與MLN8237 (一種Aurora A專一抑制劑)處理大腸癌細胞後，會增強p53下游蛋白的表現(p21及PUMA)和細胞凋亡蛋白(活化態caspase 3與PARP分解)，而誘導大腸癌細胞之G2/M期停滯與細胞凋亡增加。綜合以上結果，我們首次發現Aurora A與p53扮演相反的角色，調控bortezomib所誘發的人類大腸癌細胞之G2/M期停滯與細胞凋亡。

Bortezomib (also called Velcade) is a proteasome inhibitor that has been used in clinical therapy for multiple myeloma. Colorectal cancer (CRC) is one of most common cause of cancer mortality in worldwide. p53, a tumor suppressor, has been shown to mediate bortezomib-induced cell cycle arrest and apoptosis. Aurora A, a serine/threonine kinase regulated mitosis, is highly expressed in various human cancer cells including CRC. However, the role and regulation of p53 and Aurora A in the bortezomib-treated CRC remain unclear. Here, we show that bortezomib reduced the cell viability and increased the G2/M fractions and apoptosis in various human CRC cells. Interestingly, bortezomib markedly increased p53 and Aurora A proteins, which were co-located in the nucleolus of the G2-arrested cells. Using immunoprecipitation and confocal microscopy assays, we demonstrated that Aurora A proteins could directly bind with p53 proteins in the nucleolus. Moreover, the p53-wild type CRC cells displayed higher on the induction apoptosis than the p53-null CRC cells following bortezomib. Besides, the combination of bortezomib and MLN8237 (an Aurora A specific inhibitor) enhanced the expressions of p53 downstream proteins (p21 and PUMA) and apoptotic proteins (active caspase 3 and PARP cleavage) for induction of G2/M arrest or apoptosis in CRC cells. Together, these findings demonstrate for the first time that Aurora A and p53 display the opposite role on the regulation of G2/M arrest and apoptosis following bortezomib in the human CRC cells.