Blocking MAPK/ERK pathway sensitizes hepatocellular carcinoma cells to temozolomide via downregulating MGMT expression.
Blocking MAPK/ERK pathway sensitizes hepatocellular carcinoma cells to temozolomide via downregulating MGMT expression.
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DOI：
10.21037/atm-20-5478
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发表时间：
2020-10
影响因子：
--
通讯作者：
Liang R
中科院分区：
文献类型：
--
作者：
Li Q;Ren B;Gui Q;Zhao J;Wu M;Shen M;Li D;Li D;Chen K;Tao M;Liang R
肝细胞癌（HCC）是中国第四大常见恶性肿瘤。替莫唑胺（Temozolomide， TMZ）是一种常用的体外杀伤肝癌细胞的化疗药物。然而，HCC细胞可能具有对TMZ的内在抗性。TMZ耐药的一个关键机制是o6 -甲基鸟嘌呤- dna甲基转移酶（MGMT）的过表达。研究表明，MAPK可能与MGMT表达有关，U0126是MEK1和MEK2的高选择性抑制剂，而MEK1和MEK2是丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK）通路级联的关键分子。索拉非尼是另一种广泛应用于HCC的靶向药物，它可以抑制包括MAPK/ERK在内的多种激酶。本研究旨在探讨MAPK/ERK抑制剂U0126和索拉非尼联合TMZ治疗HCC的疗效。在HCC细胞中，U0126和索拉非尼阻断了MAPK/ERK信号通路。通过MTT法、流式细胞术和TUNEL法观察阻断MAPK/ERK信号通路对tmz诱导的细胞毒性的影响。Western-blot检测DNA损伤蛋白和MGMT的表达。下调MAPK/ERK信号通路后，采用实时荧光定量聚合酶链反应（RT-qPCR）和免疫荧光法分别测定MGMT mRNA表达和MGMT蛋白表达。用MGMT过表达质粒转染HepG2细胞。转染后，采用MTT和Western-Blot检测U0126对tmz诱导的MGMT OE细胞毒性的影响。MTT法检测索拉非尼对tmz诱导的肝癌细胞毒性的影响。U0126能增强肝癌细胞对TMZ的化学敏感性。同时，我们还发现U0126增加了TMZ对HepG2细胞DNA的损伤。RT-qPCR和Western blot结果显示，U0126通过阻断MAPK/ERK通路下调MGMT mRNA和MGMT蛋白的表达。此外，转染MGMT表达质粒后，MGMT过表达恢复了u0126诱导的肝癌细胞对TMZ的化学敏感性。索拉非尼还能增加肝癌细胞对TMZ的化疗敏感性。我们的研究表明U0126联合TMZ在晚期HCC患者中的应用具有巨大的临床潜力。
Hepatocellular carcinoma (HCC) is the fourth most common malignant tumor in China. Temozolomide (TMZ) is a common chemotherapy drug which can effectively kill HCC cells in vitro. However, it is possible that HCC cells possess intrinsic resistance to TMZ. A key mechanism of TMZ resistance is the overexpression of O6-methylguanine-DNA methyltransferase (MGMT). Studies have shown that MAPK may be related to MGMT expression, U0126 is a highly selective inhibitor of MEK1 and MEK2, which were crucial molecule in cascade of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway. Sorafenib was another widely applicated target drug in HCC which could inhibit multiple kinases including MAPK/ERK. This research was aimed to investigate the efficacy of MAPK/ERK inhibitor U0126 and sorafenib combine with TMZ in the treatment of HCC. In HCC cells, MAPK/ERK signaling pathway was blocked by U0126 and sorafenib. The effect of blocking MAPK/ERK signaling pathway on TMZ-induced cytotoxicity was evaluated by MTT assay, flow cytometry and TUNEL assay. DNA damage protein and the expression of MGMT were detected by Western-blot. After the downregulation of MAPK/ERK signaling pathway, MGMT mRNA expression and the protein expression of MGMT were quantified by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence assay, respectively. HepG2 cells were transfected with an MGMT over expression plasmid. After transfection, the effect of U0126 on TMZ-induced cytotoxicity was evaluated by MTT and Western-Blot in MGMT OE cells. The influence of Sorafenib on TMZ-induced cytotoxicity to HCC cells was also detected by MTT assay. U0126 can enhance the chemosensitivity of HCC cells to TMZ. At the same time, we also found that U0126 increases the damage to DNA caused by TMZ in HepG2 cells. Moreover, the results from RT-qPCR and Western blot showed that U0126 downregulated MGMT mRNA and MGMT protein expression via blocking MAPK/ERK pathway. Furthermore, after transfection with an MGMT expression plasmid, overexpression of MGMT restored U0126-induced chemosensitivity to TMZ in HCC cells. Sorafenib can also increase the chemosensitivity of HCC cells to TMZ. Our studies suggest great clinical potential for the utilization of combined U0126 and TMZ in patients with advanced HCC.
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DOI：
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影响因子：
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