钯（II）配合物通过抑制自噬和破坏多种信号通路增强结直肠癌细胞中的 ROS 依赖性凋亡作用。
Palladium (II) Complex Enhances ROS-Dependent Apoptotic Effects via Autophagy Inhibition and Disruption of Multiple Signaling Pathways in Colorectal Cancer Cells.
机构信息
Department of Biology, Faculty of Arts and Science, Uludag University, Bursa, Turkey.
Moleculer Cancer Research Center, Istinye University, Istanbul, Turkey.
出版信息
Anticancer Agents Med Chem. 2021;21(10):1284-1291. doi: 10.2174/1871520620666200929153804.
BACKGROUND
Inhibition of autophagy is reported to be a therapeutically effective strategy in overcoming resistance that is a deadly outcome in cancer. One of the most common reasons for chemo-resistance to treatment is the patients with tumors exhibiting a KRAS mutation, which occurs in approximately 40% of colorectal cancer patients.
OBJECTIVE
Hence, we assessed whether a Palladium (Pd)(II) complex is a promising anticancer complex, compared to 5-fluorouracil in KRAS wt HT-29 and KRAS mutant HCT-15 cells.
METHODS
HCT-15 and HT-29 cells were used for colorectal cancer and Chloroquine (CQ) was used as an inhibitor of autophagy. In this context, cells were treated with Pd(II) complex and 5-FU in combination with CQ for 48h and cell viability was measured by SRB assay. Cell death mode was examined with M30 and M65 ELISA assays, using annexin V/propidium iodide. Autophagy was determined by Acridine Orange (AO) staining. Furthermore, the expressions of various autophagy and apoptosis-related proteins were evaluated with Western blotting. Luminex assay and the level of Reactive Oxygen Species (ROS) were examined.
RESULTS
Cell viability was found to decrease in a dose-dependent manner and CQ enhanced cytotoxic effect in Pd(II) and 5-FU treated cells in colorectal cancer cells. Our data showed that inhibition of autophagic flux significantly increased intrinsic apoptosis through the activation of ROS. We showed that combinatorial treatment with CQ induced apoptosis via the caspase-dependent mitochondrial pathway. Luminex analysis revealed that the combination resulted in a down-regulation of NF-κB/AKT/CREB signaling pathways in both cell lines, however, decreased Erk1/2 protein expression was only observed after treatment with CQ combination in HCT-15 cells.
CONCLUSION
We suggest that the inhibition of autophagy along with Pd(II) and 5-FU treatment has a synergistic effect on KRAS-mutant colorectal cancer cells. Autophagy inhibition by CQ promotes apoptosis via blockade of the NF-κB/AKT/CREB and activation of ROS.
背景
据报道，抑制自噬是克服癌症中致命耐药性的一种有效治疗策略。化疗耐药的最常见原因之一是肿瘤患者存在 KRAS 突变，约 40%的结直肠癌患者存在这种突变。
目的
因此，我们评估了钯（II）配合物是否比 5-氟尿嘧啶更适合治疗 KRASwtHT-29 和 KRAS 突变 HCT-15 细胞。
方法
使用 HCT-15 和 HT-29 细胞进行结直肠癌研究，并用氯喹（CQ）作为自噬抑制剂。在这种情况下，用 Pd（II）配合物和 5-FU 联合 CQ 处理细胞 48 小时，并用 SRB 法测量细胞活力。通过 M30 和 M65 ELISA 检测细胞死亡模式，用 Annexin V/碘化丙啶染色。通过吖啶橙（AO）染色确定自噬。此外，用 Western blot 评估各种自噬和凋亡相关蛋白的表达。用 Luminex 检测和活性氧（ROS）水平检测。
结果
细胞活力呈剂量依赖性降低，CQ 增强了 Pd（II）和 5-FU 处理的结直肠癌细胞中的细胞毒性作用。我们的数据表明，抑制自噬流通过激活 ROS 显著增加了内在凋亡。我们表明，CQ 联合治疗通过 caspase 依赖性线粒体途径诱导凋亡。Luminex 分析显示，组合治疗导致两种细胞系中的 NF-κB/AKT/CREB 信号通路下调，然而，只有在用 CQ 联合处理后，HCT-15 细胞中的 Erk1/2 蛋白表达才降低。
结论
我们认为，抑制自噬与 Pd（II）和 5-FU 联合治疗对 KRAS 突变型结直肠癌细胞具有协同作用。CQ 抑制自噬通过阻断 NF-κB/AKT/CREB 并激活 ROS 促进凋亡。
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