Doxorubicin誘導MCF-7/WT轉變為抗藥性細胞株的過程中基因變化之探討

Abstract

癌細胞對多種化療藥物產生抵抗力，此種現象稱為多重抗藥性，常為造成化療失敗的原因。細胞產生抗藥性的原因一直被熱烈的探討，且也可以使用細胞毒性藥物來篩選並建立抗藥性細胞株。藉由減少、喪失或變更藥物標靶、使細胞凋亡的訊息傳遞受阻、增進細胞DNA受藥物損害後的修復能力、活化更多與細胞內解毒及藥物代謝相關的酵素、增強排除藥物至細胞外的功能、減少藥物進入細胞的量，癌細胞可對單一種藥物或者一系列藥物產生抗藥性。 MCF-7/ADR為對doxorubicin具抗藥性的人類乳癌細胞株，相較於MCF-7/WT，其過量表現P-glycoprotein減少了doxorubicin在細胞內的量並展現多重抗藥性的樣貌。本實驗室將MCF-7/WT培養在以1 nM doxorubicin為起始濃度的培養基中，待細胞生長穩定後，增加為兩倍藥物量，篩選對doxorubicin有抗藥性的細胞稱MCF-7/ADR-n，n為1 nM的倍數。目前分別建立了MCF-7/ADR-1、MCF-7/ADR-2、MCF-7/ADR-4、MCF-7/ADR-8、MCF-7/ADR-16、MCF-7/ADR-32、MCF-7/ADR-64、MCF-7/ADR-128、MCF-7/ADR-256、MCF-7/ADR-512、MCF-7/ADR-1024共11種對不同濃度的doxorubicin具有抗藥性的細胞株，細胞培養的終點止於P-glycoprotein的出現為MCF-7/ADR-1024，其與MCF-7/ADR具有相同程度的抗藥性(對doxorubicin的IC50分別為10.3 μM及12.9 μM)。 本研究利用一系列不同抗藥性程度的細胞，來探討doxorubicin對MCF-7產生抗藥性的過程中，在ABC轉運蛋白、細胞凋亡、抗氧化及藥物代謝、DNA修復、細胞增生等方面之相關基因表現有無變化。在ABC轉運蛋白方面，從MCF-7/ADR-32細胞開始MRP1運輸蛋白的mRNA表現量開始漸升，在MCF-7/ADR-256 中MRP1的mRNA表現量為最高點，然而在MCF-7/ADR-512、MCF-7/ADR-1024中下降至與MCF-7/WT相同程度，而P-gp的mRNA卻僅在MCF-7/ADR-1024才大量表現。 MRP1出現及消失與P-gp的出現兩者之間是否有相關，目前尚無法證明，而MRP1運輸蛋白mRNA的大量表現可能為細胞對doxorubicin具有較低程度抗藥性的原因。在細胞凋亡方面，Bcl-2的mRNA在MCF-7/ADR-1024時表現量減少，GCS的mRNA表現量則是增加；在DNA修補方面，BRCA1/2的mRNA表現量隨著對doxorubicin抗藥性程度增加而減少，野生型p53在MCF-7/ADR-1024及MCF-7/ADR中大量減少，突變型p53基因表現量在MCF-7/ADR-1024及MCF-7/ADR中大量增加，而總p53的基因及蛋白表現量均呈現上升的趨勢，顯示突變可能會增加p53的半衰期，而使總p53蛋白量增加。在抗氧化還原方面，GCL及轉錄因子Nrf2在基因表現量上並沒有明顯變化，GSTπ只在MCF-7/ADR-1024及MCF-7/ADR中大量表現。在EMT相關基因中，E-cadherin的mRNA表現量在MCF-7/ADR-1024及MCF-7/ADR中大量減少，N-cadherin、Vimentin及可抑制E-cadherin表現的轉錄因子ZEB1、ZEB2、Twist1、Slug表現量在MCF-7/ADR-1024及MCF-7/ADR中增加，細胞的型態也在MCF-7/ADR-1024時與MCF-7/WT(類似上皮細胞)有所不同，較類似間質細胞。另外，PKCα的基因表現量隨著對doxorubicin抗藥性程度增加而增加，ER-α的mRNA在MCF-7/ADR-1024及MCF-7/ADR中幾乎沒有表現。 總而言之，以建立的11種不同程度的doxorubicin抗藥性細胞，分析比較細胞間的基因表現差異，可使我們對MCF-7細胞對doxorubicin產生抗藥性的原因有更多了解，也提供往後其他與抗藥性相關研究的基礎。

The ability of cancer cells to become simultaneously resistant to different drugs — a trait known as multidrug resistance — remains a significant impediment to successful chemotherapy. Cellular mechanisms of drug resistance have been intensively studied, as experimental models can be easily generated by in vitro selection with cytotoxic agents. Cancer cells in culture can become resistant to a single drug, or a class of drugs with a similar mechanism of action, by altering the drug’s cellular target or by increasing repair of drug-induced damage, frequently to DNA. MCF-7/ADR is a doxorubicin-resistant cell line. In comparison with MCF-7/WT, MCF-7/ADR overexpresses P-gp that lowers the intracellular concentration of doxorubicin and exhibits the multidrug resistnace. Resistant cells were selected by doxorubicin at the starting concentration 1 nM. After the cells were tolerable, doubling the drug concentration was applied till the cells acquired tolerance. Followed by repeated treatments, 11 cell lines were established with incremental resistance to doxorubicin. The series of cells are so-called MCF-7/ADR-n and named as MCF-7/ADR-1、MCF-7/ADR-2、MCF-7/ADR-4、MCF-7/ADR-8、MCF-7/ADR-16、MCF-7/ADR-32、MCF-7/ADR-64、MCF-7/ADR-128、MCF-7/ADR-256、MCF-7/ADR-512、MCF-7/ADR-1024, respectively. To investigate the gene expression profiles during resistance induction, the expression levels were measured for genes involving in ABC transporter protein, apoptosis, antioxidant and drug metabolism, DNA repair and cell proliferation in the series of MCF-7/ADR-n. Regarding ABC transporters, MRP1 expression increased slightly in MCF-7/ADR-32 and a peak was observed in MCF-7/ADR-256, then declined afterwards. P-gp mRNA bursted only in MCF-7/ADR-1024. The association of MRP1 with P-gp is still unclear. Regarding apoptosis genes, Bcl-2 expression decreased with the elevated resistance to doxorubicin, but GCS mRNA expression was in the opposite way. Regarding DNA repair system, BRCA1/2 expression levels decreased with the elevation of doxorubicin resistance. Wild-type p53 mRNA importantly dropped in MCF-7/ADR-1024, while mutant p53 mRNA markedly appeared in MCF-7/ADR-1024. Levels of total p53 mRNA and protein were elevated with the increased doxorubicin resistance. Longer half life of mutated p53 may contribute to the elevation. Regarding antioxidant and drug metabolism, GCL and transcription factor Nrf2 showed slightly changes in mRNA expression, and GSTπ was overexpressed only in MCF-7/ADR-1024. Regarding EMT related genes, E-cadherin mRNA almost vanished in MCF-7/ADR-1024, but N-cadherin、Vimentin and E-cadherin transcription suppressors (i.e. ZEB1、ZEB2、Twist1 and Slug) were overexpressed in MCF-7/ADR-1024. MCF-7/ADR-1024 is mesenchymal-like and is different from MCF-7/WT. PKCα mRNA rised with the elevation of doxorubicin resistance, while ER-α mRNA dropped in MCF-7/ADR-1024. In conclusion, a series of MCF-7 cells with incremental resistance to doxorubicin were established. Eleven cell lines were employed for measurement of gene expression to better understand gene profiles in the induction of doxorubicin resistance in MCF-7.