Qiu Lihua, Wang Qun, Di Wen, Jiang Qin, Schefeller Erica, Derby Steve, Wanebo Harold, Yan Bingfang, Wan Yinsheng
Department of OB/GYN, Renji Hospital, Shanghai Second Medical University, P.R. China.
Int J Oncol. 2005 Sep;27(3):823-30.
Betulinic acid (BA), a pentacyclic triterpene first identified less than a decade ago, has served as a melanoma-specific cytotoxic agent, and yet its specificity is being challenged. Recently, we found that human melanoma cells exhibited less sensitivity to betulinic acid than human skin keratinocytes. This study was designed to investigate the cell signaling pathway leading human melanoma cells to increased resistance to betulinic acid treatment. In vitro experiments using cultured human melanoma cells indicated that betulinic acid transiently induced survivin expression. The expression of survivin started 30 min post-betulinic acid treatment, peaked at 2 h, remained elevated for 8 h and returned to basal level within 24 h. Similarly, epithelial growth factor (EGF) treatment induced expression of survivin in a time-dependent manner. Since epithelial growth factor receptor (EGFR) activation leads to the activation of cell signaling components that are important to cell survival, we next examined whether BA-induced survivin expression is mediated by the EGFR pathway. The results showed that BA induced EGFR tyrosine phosphorylation in a time-dependent manner. Further, BA strongly induced AKT phosphorylation in a similar pattern. AKT activation started 15 min post-treatment, peaked at approximately 1 h, remained elevated for 4 h and returned to basal level within 8 h. BA also induced ERK activation and, in contrast, weakly induced JNK and p38 activation. Pretreatment of EGFR inhibitor PD153035 blocked BA-induced EGFR phosphorylation, ERK and AKT activation, and survivin expression. Results of the MTT dye assay showed that a combination of PD153035 and BA enhanced melanoma cell death. Collectively, we conclude that betulinic acid transiently activated the EGFR/AKT cell survival pathway and induced survivin expression, contributing to less sensitivity in human melanoma cells. The data suggest that a combination of the EGFR inhibitor and betulinic acid may be a better clinical option to treat human melanoma.
桦木酸（BA）是一种不到十年前首次被鉴定出的五环三萜，曾作为一种黑色素瘤特异性细胞毒性剂，但它的特异性正受到挑战。最近，我们发现人类黑色素瘤细胞对桦木酸的敏感性低于人类皮肤角质形成细胞。本研究旨在探究导致人类黑色素瘤细胞对桦木酸治疗产生抗性增加的细胞信号通路。使用培养的人类黑色素瘤细胞进行的体外实验表明，桦木酸可短暂诱导生存素表达。生存素的表达在桦木酸处理后30分钟开始，2小时达到峰值，8小时内保持升高，24小时内恢复到基础水平。同样，表皮生长因子（EGF）处理也以时间依赖性方式诱导生存素表达。由于表皮生长因子受体（EGFR）激活会导致对细胞存活至关重要的细胞信号成分的激活，接下来我们研究了桦木酸诱导的生存素表达是否由EGFR途径介导。结果显示，桦木酸以时间依赖性方式诱导EGFR酪氨酸磷酸化。此外，桦木酸以类似模式强烈诱导AKT磷酸化。AKT激活在处理后15分钟开始，约1小时达到峰值，4小时内保持升高，8小时内恢复到基础水平。桦木酸还诱导ERK激活，相比之下，对JNK和p38的激活较弱。EGFR抑制剂PD153035预处理可阻断桦木酸诱导的EGFR磷酸化、ERK和AKT激活以及生存素表达。MTT染料测定结果表明，PD153035和桦木酸联合使用可增强黑色素瘤细胞死亡。总体而言，我们得出结论，桦木酸可短暂激活EGFR/AKT细胞存活途径并诱导生存素表达，导致人类黑色素瘤细胞敏感性降低。数据表明，EGFR抑制剂和桦木酸联合使用可能是治疗人类黑色素瘤更好的临床选择。
