CD40配体对汉黄芩素抗肿瘤作用的影响及其机理探讨

Recombinant Soluble CD40 Ligand Enhances Wogonin-induced Antitumor Activity

目的探讨重组可溶性CD40配体(recombinant soluble CD40ligand,rsCD40L)对汉黄芩素诱导的抗肿瘤作用的影响及其作用机理。方法采用乳酸脱氢酶(LDH)释放实验检测rsCD40L与汉黄芩素共处理对卵巢癌细胞株SKOV3的细胞毒作用;吖啶橙/溴化乙锭双重染色后荧光显微镜下观察处理后细胞形态学改变;采用Western blot检测细胞凋亡相关分子caspase-8、caspase-3、多聚ADP核糖聚合酶的变化,在此基础上,加入caspase抑制剂Z-VAD-FMK和肿瘤坏死因子α(tumor necrosis factorα,TNF-α)中和抗体,采用LDH释放实验,进一步检测阻断这些信号通路对肿瘤细胞死亡的影响。结果 rsCD40L可增强汉黄芩素对SKOV3细胞的杀伤作用(P<0.05)。随着汉黄芩素剂量的增加,对肿瘤细胞的杀伤作用也随之增强(P<0.05)。经rsCD40L与汉黄芩素共处理的SKOV3细胞呈现典型的凋亡形态学改变;caspase信号通路被显著活化,且caspase抑制剂Z-VAD-FMK可抑制增强的细胞毒作用。TNF-α中和抗体也可抑制这一增强的细胞毒作用。结论 rsCD40L可增强汉黄芩素诱导的肿瘤细胞死亡,其机理与增强肿瘤细胞凋亡及诱导TNF-α的自分泌有关,提示将rsCD40L和汉黄芩素合用具有潜在的抗肿瘤作用价值。

Objective To investigate the effect of recombinant soluble CD40 ligand （rsCD40L） on Wogonin mediated antitumor activity in cancer cells and the underlying molecular mechanisms. Methods Cell death was detected based on the release of lactate dehydrogenase （LDH） Using a cytotoxicity detection kit. For morphological study of cell death, cells were stained with 50μg/mL of acridine orange and 50 μg/mL of ethidium bromide and observed and photographed under a fluorescence microscope. Activation of apoptosis pathway was evaluated by Western blot. The effects of pan-caspase inhibitor Z-VAD-FMK and tumor necrosis factor a （TNF-α） neutralizing antibody on ceil death induced by rsCD40L and Wogonin co-treatment were also investigated. Results rsCD40L significantly enhanced Wogonin-induced cell death of ovarian cancer cells SKOV3. A dose-dependent synergism was found with a fixed rsCD40L dose （1μg/mL） and increased concentrations of Wogonin （5 μmol/L-15 μmol/L）. rsCD40L and Wogonin co-treated cells showed typical apoptotic morphologies and enhanced activation of caspases pathway. As expected, the pan-caspase inhibitor Z-VAD-FMK inhibited synergistic cell death of rsCD40L and Wogonin co-treated SKOV3 cells. Interestingly, the TNF-α neutralizing antibody that blocks TNF-α binding to its receptor also significantly suppressed the cell death enhancing effect, indicating that autocrine TNF-α played a role of sensitization. Conclusion rscCD40L sensitizes cancer cells to wogonin-mediated apoptosis, which may involve autocrine of TNF-α, and the combination of rsCD40L and Wogonin may have a potential for cancer therapy.