Table4_4-Methoxydalbergione Inhibits Bladder Cancer Cell Growth via Inducing Autophagy and Inhibiting Akt/ERK Signaling Pathway.DOCX

Bladder cancer (BC) ranks the fourth in incidence in cancers of men and is a common malignant tumor in women. 4-Methoxydalbergione (4MOD), which is purified from Dalbergia sissoo Roxb, has been shown to have anticancer capacity for osteosarcoma and astroglioma. The role of 4MOD in bladder cancer has not been investigated. This study aims to evaluate the anticancer effect of 4MOD in BC cells and its possible mechanisms. The two human bladder cancer cell lines J82 and UMUC3 were used to evaluate the proliferation inhibitory effect of 4MOD by CCK8 and clonogenic assays. The migratory and invasive ability of tumor cells was examined by scratch test and transwell assay. Apoptosis was detected by flow cytometry and TUNEL assays. The autophagy-related molecules including Beclin-1 and LC3 were examined by Western blotting analysis. Furthermore, the RT-PCR was used to detect the mRNA expression of LC3. 4MOD repressed cell proliferation, migration, invasion and induced cell apoptosis in a concentration-dependent manner. The IC50 values of J82 and UMUC3 were 8.17 and 14.50 μM respectively. The mRNA and protein expression ratio of light chain 3-II (LC3-II)/LC3-I and the protein expression of Beclin-1 were increased when the BC cells were treated with 4MOD. The treatment of 4MOD attenuated the phosphorylation of Akt and ERK in the BC cells. We revealed that the 4MOD inhibits BC cells growth by inducing autophagy and inhibiting Akt/ERK signaling pathway. Our study provides new insights into the mechanism by which 4MOD weakens the proliferation of BC cells. This study demonstrates that 4MOD provided a lead compound for the development of novel compound with potent anticancer effect on BC cells.

膀胱癌（Bladder cancer, BC）在男性恶性肿瘤发病率中位列第四，同时也是女性常见的恶性肿瘤。4-甲氧基黄檀醌（4-Methoxydalbergione, 4MOD）是从印度黄檀（Dalbergia sissoo Roxb）中纯化得到的天然活性成分，既往研究已证实其对骨肉瘤与星形胶质细胞瘤具有抗癌活性，但目前尚未有关于4MOD在膀胱癌中作用的相关研究。本研究旨在评估4MOD对膀胱癌细胞的抗癌作用及其潜在分子机制。本研究采用人膀胱癌细胞系J82与UMUC3，通过CCK-8实验与克隆形成实验检测4MOD对细胞增殖的抑制作用；采用划痕实验与Transwell实验分析肿瘤细胞的迁移与侵袭能力；通过流式细胞术与TUNEL染色检测细胞凋亡水平；采用蛋白质印迹法（Western blotting）检测自噬相关分子Beclin-1与微管相关蛋白1轻链3（Light Chain 3, LC3）的蛋白表达；此外通过逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）检测LC3的mRNA表达水平。实验结果显示，4MOD可通过浓度依赖性方式抑制膀胱癌细胞的增殖、迁移与侵袭，并诱导细胞凋亡；J82与UMUC3细胞的半最大效应浓度（IC50）分别为8.17 μM与14.50 μM。经4MOD处理后，膀胱癌细胞中LC3-II/LC3-I的mRNA与蛋白表达比值，以及Beclin-1的蛋白表达水平均显著升高；同时4MOD可减弱膀胱癌细胞中Akt（Protein kinase B）与ERK（Extracellular Signal-Regulated Kinase）的磷酸化水平。本研究证实，4MOD可通过诱导自噬并抑制Akt/ERK信号通路，从而抑制膀胱癌细胞的增殖，为阐明4MOD调控膀胱癌细胞增殖的分子机制提供了新的研究视角。本研究表明，4MOD可作为潜在先导化合物，为开发针对膀胱癌的高效新型抗肿瘤药物提供了坚实的研究基础。