PHA-665752 和西妥昔单抗联合治疗对 KRAS 或 BRAF 突变的人结直肠癌细胞的体外和小鼠异种移植生长的影响。

Effects of PHA-665752 and Cetuximab Combination Treatment on In Vitro and Murine Xenograft Growth of Human Colorectal Cancer Cells with KRAS or BRAF Mutations.

机构信息

Department of Oncology, Hebei General Hospital, Shijiazhuang 050051, Hebei, China.

Second Department of Surgery, the Fourth Hospital of Hebei Medical University, Tianshan Road, Shijiazhuang 050011, Hebei, China.

出版信息

Curr Cancer Drug Targets. 2018;18(3):278-286. doi: 10.2174/1568009617666170330112841.

DOI:10.2174/1568009617666170330112841

PMID:28359236

Abstract

BACKGROUND

It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts.

METHODS

Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels.

RESULTS

The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05).

CONCLUSION

Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did.

摘要

背景

目前尚不清楚阻断 c-Met 信号和表皮生长因子受体信号是否能有效抑制人结直肠癌（CRC）细胞的生长。在这项研究中，我们研究了 c-Met 抑制剂 PHA-665752 单独使用以及与西妥昔单抗联合使用对体外培养的人 CRC 细胞和小鼠异种移植瘤生长的影响。

方法

用西妥昔单抗处理携带 HCT-116 异种移植瘤的人 CRC 细胞系（Caco2、HCT-116 和 HT-29）和小鼠，同时存在或不存在 PHA-665752。分别用 MTT 和 TUNEL 检测细胞活力和细胞凋亡。用免疫组化法测定波形蛋白作为上皮间质转化的标志物。用 Western blot 检测信号蛋白表达水平。

结果

MTT 法显示 PHA-665752 呈剂量依赖性抑制 Caco2、HCT-116 和 HT-29 细胞的生长，但只有 Caco2 细胞的生长受到西妥昔单抗的抑制。与 PHA-665752 单药治疗组相比，联合应用 PHA-665752 和西妥昔单抗抑制 Caco2 细胞和 RAS 突变 CRC 细胞系的增殖。然而，与 PHA-665752 单药治疗组相比，具有 BRAF 突变的 HT-29 细胞无明显作用。与单用西妥昔单抗（P=0.033）或 PHA-665752（P<0.01）治疗的小鼠相比，联合应用西妥昔单抗和 PHA-665752 治疗的小鼠的平均肿瘤体积明显较小。同样，联合应用 PHA-665752 和西妥昔单抗治疗的小鼠的波形蛋白表达明显低于单用西妥昔单抗或 PHA-665752 治疗的小鼠（每组均 P<0.05）。TUNEL 检测显示，联合应用 PHA-665752 和西妥昔单抗可显著增加 CRC 细胞凋亡。Western blot 分析信号蛋白表达显示 PHA-665752 抑制 Met 磷酸化（P<0.05）。此外，单用西妥昔单抗或联合应用 PHA-665752 均可有效抑制 EGFR 磷酸化（P<0.05）。