rigosertib联合化疗对Kirsten大鼠肉瘤病毒癌基因同源物突变型结直肠癌小鼠的疗效和不良反应

The efficacy and side effects of rigosertib combined with chemotherapy in KRAS mutant colorectal cancer mice

目的探讨rigosertib(RGS)与结直肠癌经典化疗药物联合对KRAS突变型结直肠癌的疗效及不良反应。方法通过裸鼠皮下接种构建KRAS突变型结直肠癌模型,采用随机数字表法及随机数余数法随机分为对照组、RGS组、5-氟尿嘧啶(5-FU)组、奥沙利铂(OXA)组、伊立替康(IRI)组、5-FU+RGS组、OXA+RGS组和IRI+RGS组,观察各组小鼠体重和肿瘤体积的变化,采用免疫组化染色、原位末端标记荧光染色、Western blot法检测肿瘤组织增殖抑制及凋亡情况,通过小鼠血常规、血生化及肝脏组织切片HE染色分析化疗联合RGS的不良反应情况。采用细胞计数试剂盒8检测OXA与RGS联用对KRAS突变型结直肠癌细胞DLD1和HCT116的协同作用。结果对照组、RGS组、5-FU组、OXA组、IRI组、5-FU+RGS组、OXA+RGS组和IRI+RGS组小鼠移植瘤均成瘤,肿瘤生长快慢不一,其中OXA+RGS组肿瘤增长较慢。到给药后55 d,OXA+RGS组的肿瘤体积倍数为37.019±8.634,均小于RGS组(77.571±15.387,P=0.029)和OXA组(92.500±13.279,P=0.008),具有协同效应。免疫组化染色显示,对照组、OXA组、RGS组和OXA+RGS组小鼠移植瘤组织Ki-67指数分别为(100.0±16.8)%、(35.6±11.3)%、(54.5±18.1)%和(15.4±3.9)%,OXA+RGS组低于对照组(P=0.014),但与OXA组和RGS组比较差异无统计学意义(均P>0.05)。TUNEL荧光染色结果显示,OXA+RGS组凋亡细胞荧光信号强度为3.878±0.547,高于OXA组(1.515±0.442,P=0.005)和RGS组(1.966±0.261,P=0.008)。Western blot检测结果显示,OXA+RGS组小鼠移植瘤组织中cleaved-PARP、cleaved-caspase 3和cleaved-caspase 8等凋亡相关蛋白表达量均高于对照组、OXA组和RGS组。裸鼠接受RGS、5-FU、OXA、IRI等单药和联合治疗后,未见明显肝肾功能损害,但OXA+RGS组白细胞为(0.385±0.215)×10^(9)/L,血红蛋白为(56.000±24.000)g/L,均低于对照组[分别为(5.598±0.605)×10^(9)/L和(153.333±2.231)g/L,均P<0.01]。结论RGS与5-FU、IRI联用治疗KRAS突变型结直肠癌不能增强疗效,但与OXA联用具有更强的抗肿瘤效应。RGS单药不会对小鼠造成明显的骨髓抑制和肝肾功能损伤,但与OXA联用不良反应可能会相应增加。

Objective To investigate the effect of rigosertib(RGS)combined with classic chemotherapy drugs including 5-fluorouracil,oxaliplatin,and irinotecan in colorectal cancer.Methods Explore the synergy effects of RGS and 5-fluorouracil(5-FU),oxaliplatin(OXA),and irinotecan(IRI)on colorectal cancer by subcutaneously transplanted tumor models of mice.The mice were randomly divided into control group,RGS group,5-FU group,OXA group,IRI group,5-FU+RGS group,OXA+RGS group and IRI+RGS group.The synergy effects of RGS and OXA on KRAS mutant colorectal cancer cell lines in vitro was detected by CCK-8.Ki-67 immunohistochemistry and TdT-mediated dUTP nick-end labeling(TUNEL)staining were performed on the mouse tumor tissue sections,and the extracted tumor tissue was analyzed by western blot.The blood samples of mice after chemotherapy and RGS treatment were collected,blood routine and liver and kidney function analysis were conducted,and H&E staining on liver sections was performed to observe the side effects of chemotherapy and RGS.Results The subcutaneously transplanted tumor models were established successfully in all groups.55 days after administration,the fold change of tumor size of OXA+RGS group was 37.019±8.634,which is significantly smaller than 77.571±15.387 of RGS group(P=0.029)and 92.500±13.279 of OXA group(P=0.008).Immunohistochemical staining showed that the Ki-67 index of tumor tissue in control group,OXA group,RGS group and OXA+RGS group were(100.0±16.8)%,(35.6±11.3)%,(54.5±18.1)%and(15.4±3.9)%,respectively.The Ki-67 index of OXA+RGS group was significantly lower than that in control group(P=0.014),but there was no significant difference compared to OXA group and RGS group(OXA:P=0.549;RGS:P=0.218).TUNEL fluorescence staining showed that the apoptotic level of OXA+RGS group was 3.878±0.547,which was significantly higher than 1.515±0.442 of OXA group(P=0.005)and 1.966±0.261 of RGS group(P=0.008).Western blot showed that the expressions of apoptosis related proteins such as cleaved-PARP,cleaved-caspase 3 and cleaved-caspase 8 in the tumor tissues of mice in the OXA+RGS group were higher than those in control group,OXA group and RGS group.After the mice received RGS combined with chemotherapy drugs,there was no significant effect on liver and kidney function indexes,but the combined use of oxaliplatin and RGS significantly reduced the white blood cells[(0.385±0.215)×10^(9)/L vs(5.598±0.605)×10^(9)/L,P<0.001]and hemoglobin[(56.000±24.000)g/L vs(153.333±2.231)g/L,P=0.001]of the mice.RGS,chemotherapy combined with RGS and chemotherapy alone did not significantly increase the damage to liver cells.Conclusions The combination of RGS and oxaliplatin has a stronger anti-tumor effect on KRAS mutant colorectal cancer.RGS single agent will not cause significant bone marrow suppression and hepatorenal injury in mice,but its side effects may increase correspondingly after combined with chemotherapy.