Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapy-induced cytotoxicity in osteosarcoma cells
Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapy-induced cytotoxicity in osteosarcoma cells
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敲低自噬相关蛋白 6 Beclin-1 可减少细胞生长、侵袭和转移，并对骨肉瘤细胞化疗诱导的细胞毒性产生积极作用
DOI：
10.1007/s13277-014-2868-y
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发表时间：
2015-04-01
期刊：
影响因子：
--
通讯作者：
Lao, Lifeng
中科院分区：
文献类型：
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作者：
Zhang, Wei;Li, Qianyi;Lao, Lifeng
Beclin-1是一种众所周知的自噬关键调节因子，与许多疾病有关，包括癌症、衰老和退行性疾病。Beclin-1在大肠癌、原发性十二指肠腺癌和肝癌细胞中高表达，并且Beclin-1过表达可诱导白血病细胞自噬性死亡。然而，Beclin-1介导的自噬在骨肉瘤中的确切作用和分子机制至今仍不清楚。在这里，我们评估了Beclin-1在体内和体外人骨肉瘤细胞系中的作用。为了表征Beclin-1在骨肉瘤细胞系中的内源性表达，我们进行了实时PCR和Western印迹分析。我们进一步分析了化疗后骨肉瘤细胞中Beclin-1的水平，并研究了自噬抑制对化疗诱导的细胞毒性的影响。我们使用针对Beclin-1的小干扰RNA（siRNA）感染具有相对高Belcin-1表达的骨肉瘤细胞系。此外，我们确定Beclin-1敲低与骨肉瘤细胞生长、迁移和侵袭的功能相关性，并研究基质金属肽酶-2（MMP-2）、MMP-9、磷酸肌醇3-激酶p85 α（PI 3 Kp 85 α）和磷酸化AKT（p-AKT）的表达水平。结果显示，HOS骨肉瘤细胞表现出更高的Beclin-1表达。抗癌药物包括阿霉素、顺铂和甲氨蝶呤，均诱导人骨肉瘤细胞中Beclin-1的上调，siRNA介导的Beclin-1敲低抑制细胞增殖、迁移和侵袭，这由3-（4，5-二甲基噻唑基-2）-2，5-二苯基四唑溴铵、wil愈合和transwell试验表明。抗癌药物诱导的细胞凋亡明显增加。Beclin-1的敲低或3-甲基腺嘌呤（自噬和PI 3 K的抑制剂）对自噬的抑制使它们对化疗显着更敏感。加入泛半胱天冬酶抑制剂ZVAD-FMK可部分逆转顺铂诱导的细胞死亡。当Beclin-1表达被抑制时，HOS细胞中PI 3 Kp 85 α、p-AKT和MMP-9的表达下调。Beclin-1缺失的HOS细胞裸鼠皮下移植瘤体积明显小于对照组。这些结果表明，通过siRNA敲低Beclin-1可能通过阻断PI 3 K/AKT信号通路对骨肉瘤细胞的生长和迁移产生抑制作用。Beclin-1基因敲低通过激活凋亡途径使其对化疗敏感。本研究结果提示Beclin-1在骨肉瘤的增殖和肿瘤进展中起重要作用，抑制自噬可提高抗癌药物治疗的疗效。
Beclin-1, a well-known key regulator of autophagy, has been implicated in many disorders, including cancer, aging, and degenerative diseases. Previous studies demonstrated that Beclin-1 participated in tumorgenesis and was highly expressed in colorectal cancer cells, primary duodenal adenocarcinoma, and hepatocellular carcinoma cells, and overexpression of Beclin-1 could induce autophagic cell death in leukemia cells. However, the exact effects and molecular mechanisms of Beclin-1-mediated autophagy in osteosarcoma are still unknown up to now. Here, we evaluated the role of Beclin-1 in human osteosarcoma cell lines in vivo and in vitro. In order to characterize the endogenous expression of Beclin-1 in osteosarcoma cell lines, we performed real-time PCR and Western blot analysis. We further analyzed the level of Beclin-1 in osteosarcoma cells after chemotherapy and investigated the impact of autophagy inhibition on chemotherapy-induced cytotoxicity. We used the small interfering RNA (siRNA) directed against Beclin-1 to infect the osteosarcoma cell line with relatively high Belcin-1 expression. Furthermore, we determine the functional relevance of Beclin-1 knockdown to osteosarcoma cell growth, migration, and invasion, and investigate the expression levels of matrix metallopeptidase-2 (MMP-2), MMP-9, phosphoinositide 3-kinase p85 alpha (PI3Kp85 alpha), and phosphorylated AKT (p-AKT). As a result, HOS osteosarcoma cells exhibited higher Beclin-1 expression. Anticancer agents including doxorubicin, cisplatin, and methotrexate each induced Beclin-1 up-regulation in human osteosarcoma cells, and siRNA-mediated knockdown of Beclin-1 suppressed cell proliferation, migration, and invasion indicated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthetrazolium bromide, would healing, and transwell assays. Cell apoptosis induced by anticancer agents was markedly increased. Knockdown of Beclin-1 or inhibition of autophagy by 3-methyladenine (an inhibitor of autophagy and PI3K) rendered them significantly more sensitive to chemotherapy. Addition of the pan-caspase inhibitor ZVAD-FMK partly reversed the cisplatin-induced cell death. When Beclin-1 expression was inhibited, the expression of PI3Kp85 alpha, p-AKT, and MMP-9 was downregulated in HOS cells. In addition, the tumor volumes in subcutaneous nude mouse models in Beclin-1-deleted HOS cells were significantly smaller than those of control group. These results suggested that knockdown of Beclin-1 by siRNA exerts inhibitory effects on growth and migration of osteosarcoma cells possibly via blockade of the PI3K/AKT signaling. Beclin-1 knockdown rendered them significantly more sensitive to chemotherapy through activating apoptosis pathway. The results of this study suggest that Beclin-1 plays an important role in proliferation and tumor progression in osteosarcoma and inhibition autophagy can increase the efficacy of anticancer agent therapy.