化療藥物Ara-C抗藥性細胞株MV-4-11-R的建立及其特性研究

Abstract

Ara-C 是治療AML常用的藥物，大部分病人可以獲得良效，但仍有部分患者病情不能緩解或緩解後復發。目前認為導致病患復發的主要原因是患者對化療藥物產生抗藥性。已知的抗藥機制繁多，但目前為止還不足以解釋Ara-C產生抗藥性的原因。為了更了解Ara-C的抗藥性機制，本實驗室建立出對Ara-C有抗藥性的MV-4-11 AML細胞株(MV-4-11-R)，再以細胞抑殺實驗檢測Ara-C對MV-4-11-R細胞株之生長抑制情形，結果顯示其IC50為3.37μM，與母細胞MV-4-11-P (IC50為0.26 μM)相差13倍。對這兩株細胞進行一系列的生物特性分析。結果發現MV-4-11-P和MV-4-11-R在細胞生長曲線和細胞週期的分布(G1期: 63% vs. 61%；S期: 28% vs. 31%；G2/M期: 7% vs. 7%以及sub-G1期: 1.5% vs. 2.2%)沒有差異；在細胞型態上沒有顯著差異。利用流式細胞儀分析，發現MV-4-11-R表現較多的CD56(22.6% vs.37%)、CD16(5.7 % vs.8.8%)及較少的HLA-DR(12.7% vs.2.4%)。另外，也觀察到MV-4-11-R細胞中糖基化FLT3蛋白下降，其p-FLT3的磷酸化程度也較MV-4-11-P少。進一步分析FLT3-ITD的下游，結果顯示MV-4-11-R細胞中Stat5α之Tyr694、CREB之Ser133的磷酸化程度下降。關於Ara-C代謝酶的檢測，觀察到MV-4-11-P和MV-4-11-R在hENT1 mRNA (0.97 vs.0.74)(P=0.22)、DCK mRNA(0.33 vs.0.30)(P=0.48)以及NT5C2 mRNA(1.10 vs.1.45)(P=0.19)沒有顯著的差異。在多重抗藥性基因的檢測顯示，MV-4-11-P和MV-4-11-R在MDR1 mRNA(12.0x10-5 vs. 9.0 x10-5)(P=0.68)和MRP mRNA(0.18 vs.0.15)(P=0.31)表達沒有顯著的差異。接著，分別利用不同組的蛋白晶片分析這兩株細胞諸多蛋白的蛋白量或其磷酸化程度，例如MV-4-11-R中有p53之Ser15(像素密度: 672 vs. 4048)、Ser46(像素密度: 1242 vs.3725)以及Ser392(像素密度: 802 vs. 2981)的磷酸化程度增多。在磷酸酶蛋白晶片觀察到MV-4-11-R中ERK1/2之Thr202/Thr204,Thr185/Tyr187(像素密度: 956 vs. 6675)、Akt之Ser473(像素密度: 1132 vs. 1219)、MEK1/2之Ser18/Ser22,Ser22/Ser26(像素密度: 547 vs. 898)以及 p53之Ser15(像素密度: 605 vs.5896)、Ser46(像素密度: 1080 vs.4340)以及Ser392 (像素密度: 1529 vs. 4143)的磷酸化程度增多。MV-4-11-R中Stat5α之Tyr694(像素密度: 2441 vs.986)、Stat5β之Tyr699(像素密度: 4302 vs.1319)、Stat5α/β之Tyr694(像素密度: 3562 vs.1067)以及CREB之Ser133(像素密度: 5896 vs.606)的磷酸化程度減少。另外，在酪胺酸激酶蛋白晶片也發現MV-4-11-R中Axl之磷酸化蛋白量(像素密度: 5550 vs. 7110)比MV-4-11-P來的多，這些結果都以西方墨點進行確認。進一步檢測細胞中mRNA表現情況以及p53的基因序列，結果發現MV-4-11-P和MV-4-11-R在Axl mRNA(0.02 vs.0.04)(P=0.33)和Gas6 mRNA(0.45 vs. 0.57)(P=0.17)表達無顯著的差異。在基因序列，則發現這兩株細胞都具有p53基因突變，其中MV-4-11-P帶有R248W突變；MV4-11-R則帶有R248W和D281G突變。 總體來說，實驗室所建立出來MV-4-11-R細胞確實對Ara-C產生抗藥性。從我們的實驗結果顯示p53-D281G、Axl、Akt以及ERK1/2是造成MV-4-11-R對Ara-C產生抗藥性的可能原因。

Ara-C is a chemotherapeutic drug that commonly used in acute myeloid leukemia (AML).However, a substantial amount of patients suffer from relapse after remission. Drug resistance is the primary cause of leukemia chemotherapy failure. Although numerous mechanisms of drug resistance have been described, they are still not able to fully explain the development of resistance to Ara-C. To further investigate the mechanisms of resistance to Ara-C, we established Ara-C resistance leukemia cell lines from MV-4-11, which is known as MV-4-11-R. Cell viability showed that MV-4-11-R (IC50 =0.26 μM) were 13 fold resistance to Ara-C than parental cell line MV-4-11-P. There were no significant differences in the cell growth, cell cycle distribution (G1 phase: 63% vs. 61%；S phase: 28% vs. 31%；G2/M phase: 7% vs. 7% and sub-G1: 1.5% vs. 2.2%) and morphology between MV-4-11-P and MV4-11-R. The MV-4-11-R cells have more with CD56 (22.6% vs.37%) and CD16 (5.7 % vs.8.8%) expression, less with HLA-DR (12.7% vs.2.4%) expression. The expression of glycosylated FLT3 was found to be decreased in MV-4-11-R cells. Reduction of glycosylated FLT3 in MV-4-11-R was associated with decreased Stat5α and CREB activation. To investigate the mechanism underlying Ara-C resistant in MV-4-11-R, mRNA levels of Ara-C metabolizing enzymes and multidrug resistance gene were measured. There were no significant differences in hENT1 mRNA (0.97 vs.0.74)(P=0.22), DCK mRNA (0.33 vs.0.30)(P=0.48), NT5C2 mRNA (1.10 vs.1.45)(P=0.19), MDR1 mRNA (12.0x10-5 vs. 9.0 x10-5)(P=0.68) and MRP mRNA (0.18 vs.0.15)(P=0.31) between MV-4-11-P and MV-4-11-R.By using various kinds of Proteosome ProfilerTM Antibody Arrays, we found that increase of p-p53 protein Ser15 (pixel density: 672 vs. 4048), Ser46 (pixel density: 1242 vs. 3725) and Ser392 (pixel density: 802 vs. 2981) in MV-4-11-R cells. Human Phospho-kinase Antibody Array showed that p-ERK1/2 Thr202/Thr204,Thr185/Tyr187 (pixel density: 956 vs. 6675), p-Akt Ser473 (pixel density: 1132 vs. 1219), p-MEK1/2 Ser18/Ser22,Ser22/Ser26 (pixel density: 547 vs. 898) and p-p53 Ser15 (pixel density: 605 vs. 5896), Ser46 (pixel density: 1080 vs. 4340) and Ser392 (pixel density: 1529 vs. 4143) was increased in MV-4-11-R. While p-Stat5α Tyr694 (pixel density: 2441 vs. 986), p-Stat5β Tyr699 (pixel density: 4302 vs. 1319), p-Stat5α/β Tyr694 (pixel density: 3562 vs. 1067) and p-CREB Ser133 (pixel density: 5896 vs. 606) was decreased in MV-4-11-R. From the results of Human Phospho-RTK Antibody Array, we found that p-Axl (pixel density: 5550 vs. 7110) protein was increased in MV-4-11-R cells. We have confirmed these results by western blot.We also analyzed the the Axl and Gas6 mRNA expression and p53 gene status by DNA sequencing. The results showed that both Axl mRNA (0.02 vs. 0.04)(P=0.33) and Gas6 mRNA (0.45 vs. 0.57)(P=0.17) expression were no significant difference between MV-4-11-P and MV-4-11-R. The DNA sequencing results showed that MV-4-11-P cells harbored R248W mutation whereas MV-4-11-R cells harbored R248W and D281G mutations. As results, we established Ara-C resistant cell line – MV-4-11-R. Our results demonstrated the acquisition of Ara-C resistance with aberrant expression of p53-D281G, Axl, Akt and ERK1/2.