Zhou Yu-Qiong, Li Dao-Xia, Zheng Xue-Lian, Yan You-Yi, Lin Yong, Wang Xia, Liao Lin-Chuan
West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Laboratory of Molecular and Translational Medicine, West China Institute of Women and Children's Health, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2013 Nov;44(6):859-64.
To investigate the effect of recombinant soluble CD40 ligand (rsCD40L) on Wogonin mediated antitumor activity in cancer cells and the underlying molecular mechanisms.
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 50 microg/mL of acridine orange and 50 microg/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 alpha (TNF-alpha) neutralizing antibody on cell death induced by rsCD40L and Wogonin co-treatment were also investigated.
rsCD40L significantly enhanced Wogonin-induced cell death of ovarian cancer cells SKOV3. A dose-dependent synergism was found with a fixed rsCD40L dose (1 microg/mL) and increased concentrations of Wogonin (5 micromol/L-15 micromol/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-alpha neutralizing antibody that blocks TNF-alpha binding to its receptor also significantly suppressed the cell death enhancing effect, indicating that autocrine TNF-alpha played a role of sensitization.
rscCD40L sensitizes cancer cells to wogonin-mediated apoptosis, which may involve autocrine of TNF-alpha, and the combination of rsCD40L and Wogonin may have a potential for cancer therapy.
研究重组可溶性CD40配体（rsCD40L）对汉黄芩素介导的癌细胞抗肿瘤活性的影响及其潜在分子机制。
使用细胞毒性检测试剂盒，基于乳酸脱氢酶（LDH）的释放检测细胞死亡。为进行细胞死亡的形态学研究，用50μg/mL吖啶橙和50μg/mL溴化乙锭对细胞染色，并在荧光显微镜下观察和拍照。通过蛋白质免疫印迹法评估凋亡途径的激活。还研究了泛半胱天冬酶抑制剂Z-VAD-FMK和肿瘤坏死因子α（TNF-α）中和抗体对rsCD40L和汉黄芩素联合处理诱导的细胞死亡的影响。
rsCD40L显著增强了汉黄芩素诱导的卵巢癌细胞SKOV3的细胞死亡。在固定的rsCD40L剂量（1μg/mL）和增加的汉黄芩素浓度（5μmol/L - 15μmol/L）下发现了剂量依赖性协同作用。rsCD40L和汉黄芩素联合处理的细胞表现出典型的凋亡形态，并增强了半胱天冬酶途径的激活。正如预期的那样，泛半胱天冬酶抑制剂Z-VAD-FMK抑制了rsCD40L和汉黄芩素联合处理的SKOV3细胞的协同细胞死亡。有趣的是，阻断TNF-α与其受体结合的TNF-α中和抗体也显著抑制了细胞死亡增强作用，表明自分泌TNF-α起到了致敏作用。
rscCD40L使癌细胞对汉黄芩素介导的凋亡敏感，这可能涉及TNF-α的自分泌，并且rsCD40L和汉黄芩素的组合可能具有癌症治疗潜力。
