抑制磷脂酰肌醇-3-羟基激酶/蛋白激酶B信号传导通路对宫颈癌的放疗增敏作用
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摘要
目的：放射增敏是肿瘤治疗研究的热点,磷脂酰肌醇-3-羟基激酶／蛋白激酶B(PI3K/AKT)信号传导通路是调节放射抵抗的主要因素,其功能失调存在于多种人体肿瘤中。PI3K阻滞剂能够增强多种肿瘤对放射治疗的敏感性,但是目前尚没有宫颈癌体内实验方面的研究报道。本实验旨在探讨PI3K阻滞剂对宫颈癌的放疗增敏作用。
方法：培养PI3K功能活性和AKT磷酸化水平稳定的人宫颈癌HeLa细胞株,皮下注射于BALB/c-nu小鼠体内(1×106／200μl),建立裸鼠宫颈癌移植瘤模型。由于LY294002对PI3K具有确切的阻滞作用,因此选作本实验的PI3K阻滞剂。将荷瘤裸鼠随机分为空白对照组(假照射+DMSO)、单纯放射治疗组(照射+DMSO)、单纯药物组(假照射+LY294002)和联合治疗组(照射+LY294002)。采用腹腔注射方法给药。各组裸鼠经治疗后部分处死,取其肿瘤组织制作病理切片,并应用Western blotting对各组裸鼠肿瘤组织AKT磷酸化(phospho-Ser 473 AKT)水平进行检测,评估LY294002对PI3K活性的抑制作用。对裸鼠的放射治疗设置0,2,4,6Gy四个剂量水平,药物LY294002设置0,100,150mg/kg三个剂量水平,计算肿瘤细胞集落形成率,评估对宫颈癌肿瘤细胞的抑制作用。应用Hoechst 33258凋亡染色对放射增敏治疗后的肿瘤细胞凋亡率进行检测。未处死部分裸鼠(n=5)用于肿瘤生长延迟实验,通过长期观察测量肿瘤体积变化,绘制肿瘤生长曲线,反映治疗对肿瘤生长的延迟作用。
结果：成功建立裸鼠宫颈癌移植瘤模型。Western blotting结果显示,单纯放疗组、单纯药物组与联合治疗组phospho-Ser 473 AKT表达均明显减少,且以联合治疗组最为显著,表明各组PI3K活性受到明显抑制。克隆形成实验分析表明,联合治疗组对宫颈癌肿瘤细胞的抑制作用最为显著,并且呈LY294002剂量依赖性和放射剂量依赖性,放治疗射与LY294002在各个实验剂量水平均具有协同作用(P<0.05)。Hoechst 33258凋亡染色检测显示,空白对照组的肿瘤细胞凋亡率为(8.67±4.37)%,单纯放射治疗组为(21.67±13.17)%,单纯药物组为(15±9.90)%,而联合治疗组为(35.5±13.67)%,较之其他治疗组显著增高(P=0.003)。肿瘤生长延迟实验表明,联合治疗组的肿瘤生长延迟最为显著(P=0.003)：肿瘤体积增长至240mm3的时间为37±9.0天,单纯放疗组为22±5.0天,单纯药物组(LY294002)为19±12.0天,空白对照组为10±6.2天。此外,单独应用LY294002 (100mg/kg)亦能降低AKT磷酸化水平,抑制宫颈癌肿瘤细胞生长,增加凋亡,延迟肿瘤生长。
结论：较单纯放射治疗,PI3K阻滞剂LY294002与放射治疗联合应用可以显著降低宫颈癌肿瘤组织的AKT磷酸化水平,增加肿瘤细胞凋亡,抑制肿瘤生长。这表明,阻滞PI3K可能通过降低AKT磷酸化水平显著增强宫颈癌对放射治疗的敏感性。同时,单独应用PI3K阻滞剂亦能抑制宫颈癌生长、增加凋亡。本实验使用荷瘤裸鼠首次证实了PI3K阻滞剂对宫颈癌具有放疗增敏作用,为进一步研究如何提高宫颈癌的放射治疗效果提供了新思路。
Objective:The research on enhancing radiosensitization is one of the highlights of tumor therapy research. Phosphoinositide-3-kinase (PI3K)/AKT pathway appears to be principal in regulating resistance to radiotherapy and is deregulated in many human cancers, and PI3K inhibition can sensitize these cancer cells to radiation. However, no research on cervical cancer in vivo has been reported. The present study further investigated whether PI3K inhibition could sensitize cervical cancer to radiation in vivo.
Methods:HeLa cells with sustained PI3K activity and AKT phosphorylation were injected subcutaneously into BALB/c nude mice to establish tumors(1×106/200μl). LY294002 was selected as PI3K inhibitor due to its confirmed effects in former studies. The tumor bearing mice were randomly assigned to control (sham radiation+DMSO), radiation alone(radiation+DMSO), LY294002 alone(sham radiation+LY294002), or combined application of LY294002 and radiation group(radiation+LY294002). LY294002 and/or control solution (DMSO) was administered intraperitoneally. Part of the mice after treatments in each group sacrificed for paraffin sections and evaluation of the blocking efficiency of LY294002 on PI3K activity (phospho-Ser 473 AKT) by Western blotting. The survival efficiency of cells after treatments with LY294002 (0,100,150mg/kg) and/or radiation (0,2,4, 6Gy) in vivo was measured by clonogenic assays and the suppression of treatments on cervical cancer was evaluated. Besides, apoptosis happened after radiosensitizing effect was tested by Hoechst 33258 staining. Part of the mice after treatments were kept for tumor regrowth assay, and the effect of tumor regrowth delay was evaluated by a long-term observation of tumor volume and tumor growth curve.
Results:Mice bearing cervical cancer cell xenografts were established successfully. Western blotting showed remarkable reduction of AKT phosphorylation in all the three treatment groups and the most of which was in the combined application group, indicating the remarkably suppressed PI3K activity. Clonogenic assay showed that combined application of radiation and LY294002 lead to the most significant suppression of the regrowth of cervical cancer cells, and the effect was radiation dose-dependent and LY294002 dose-dependent. The synergism of radiation and LY294002 was detected at each level of them by Factorial analysis (P<0.05). The rate of cell apoptosis was higher in combined group (35.5±13.67)% than that in the control group (8.67±4.37)%, radiation (21.67±13.17)% and LY294002 group (15±9.90)% (P=0.003). Tumor regrowth assay showed the lowest increase of tumor volume in combined application group (P=0.003) and the time to tumor volume of 240 mm3 Was 10±6.2 days in control and 19±12.0 days in the LY294002 alone, while it increased to 22±5.0 days in radiation alone and 37±9.0 days in combined group. Besides, LY294002 (100mg/kg) alone decreased cell survival and produced xenograft regrowth delay.
Conclusions:Compared with radiation alone, the combined application of PI3K inhibitor LY294002 and radiation can significantly enhance dephosphorylation of AKT in cervical cancer, increase tumor cell apoptosis and suppress tumor regrowth, indicating that PI3K inhibition can significantly enhance radiation efficacy via dephosphorylation of AKT in cervical cancer. Besides, PI3K inhibition alone can also suppress tumor regrowth. The present study initially proved the significant radiosensitization of PI3K inhibition in cervical cancer in vivo with nude mice bearing HeLa cell xenograft. This may provide novel therapeutic opportunities to enhance the effect of radiotherapy against cervical cancer.
方法：培养PI3K功能活性和AKT磷酸化水平稳定的人宫颈癌HeLa细胞株,皮下注射于BALB/c-nu小鼠体内(1×106／200μl),建立裸鼠宫颈癌移植瘤模型。由于LY294002对PI3K具有确切的阻滞作用,因此选作本实验的PI3K阻滞剂。将荷瘤裸鼠随机分为空白对照组(假照射+DMSO)、单纯放射治疗组(照射+DMSO)、单纯药物组(假照射+LY294002)和联合治疗组(照射+LY294002)。采用腹腔注射方法给药。各组裸鼠经治疗后部分处死,取其肿瘤组织制作病理切片,并应用Western blotting对各组裸鼠肿瘤组织AKT磷酸化(phospho-Ser 473 AKT)水平进行检测,评估LY294002对PI3K活性的抑制作用。对裸鼠的放射治疗设置0,2,4,6Gy四个剂量水平,药物LY294002设置0,100,150mg/kg三个剂量水平,计算肿瘤细胞集落形成率,评估对宫颈癌肿瘤细胞的抑制作用。应用Hoechst 33258凋亡染色对放射增敏治疗后的肿瘤细胞凋亡率进行检测。未处死部分裸鼠(n=5)用于肿瘤生长延迟实验,通过长期观察测量肿瘤体积变化,绘制肿瘤生长曲线,反映治疗对肿瘤生长的延迟作用。
结果：成功建立裸鼠宫颈癌移植瘤模型。Western blotting结果显示,单纯放疗组、单纯药物组与联合治疗组phospho-Ser 473 AKT表达均明显减少,且以联合治疗组最为显著,表明各组PI3K活性受到明显抑制。克隆形成实验分析表明,联合治疗组对宫颈癌肿瘤细胞的抑制作用最为显著,并且呈LY294002剂量依赖性和放射剂量依赖性,放治疗射与LY294002在各个实验剂量水平均具有协同作用(P<0.05)。Hoechst 33258凋亡染色检测显示,空白对照组的肿瘤细胞凋亡率为(8.67±4.37)%,单纯放射治疗组为(21.67±13.17)%,单纯药物组为(15±9.90)%,而联合治疗组为(35.5±13.67)%,较之其他治疗组显著增高(P=0.003)。肿瘤生长延迟实验表明,联合治疗组的肿瘤生长延迟最为显著(P=0.003)：肿瘤体积增长至240mm3的时间为37±9.0天,单纯放疗组为22±5.0天,单纯药物组(LY294002)为19±12.0天,空白对照组为10±6.2天。此外,单独应用LY294002 (100mg/kg)亦能降低AKT磷酸化水平,抑制宫颈癌肿瘤细胞生长,增加凋亡,延迟肿瘤生长。
结论：较单纯放射治疗,PI3K阻滞剂LY294002与放射治疗联合应用可以显著降低宫颈癌肿瘤组织的AKT磷酸化水平,增加肿瘤细胞凋亡,抑制肿瘤生长。这表明,阻滞PI3K可能通过降低AKT磷酸化水平显著增强宫颈癌对放射治疗的敏感性。同时,单独应用PI3K阻滞剂亦能抑制宫颈癌生长、增加凋亡。本实验使用荷瘤裸鼠首次证实了PI3K阻滞剂对宫颈癌具有放疗增敏作用,为进一步研究如何提高宫颈癌的放射治疗效果提供了新思路。
Objective:The research on enhancing radiosensitization is one of the highlights of tumor therapy research. Phosphoinositide-3-kinase (PI3K)/AKT pathway appears to be principal in regulating resistance to radiotherapy and is deregulated in many human cancers, and PI3K inhibition can sensitize these cancer cells to radiation. However, no research on cervical cancer in vivo has been reported. The present study further investigated whether PI3K inhibition could sensitize cervical cancer to radiation in vivo.
Methods:HeLa cells with sustained PI3K activity and AKT phosphorylation were injected subcutaneously into BALB/c nude mice to establish tumors(1×106/200μl). LY294002 was selected as PI3K inhibitor due to its confirmed effects in former studies. The tumor bearing mice were randomly assigned to control (sham radiation+DMSO), radiation alone(radiation+DMSO), LY294002 alone(sham radiation+LY294002), or combined application of LY294002 and radiation group(radiation+LY294002). LY294002 and/or control solution (DMSO) was administered intraperitoneally. Part of the mice after treatments in each group sacrificed for paraffin sections and evaluation of the blocking efficiency of LY294002 on PI3K activity (phospho-Ser 473 AKT) by Western blotting. The survival efficiency of cells after treatments with LY294002 (0,100,150mg/kg) and/or radiation (0,2,4, 6Gy) in vivo was measured by clonogenic assays and the suppression of treatments on cervical cancer was evaluated. Besides, apoptosis happened after radiosensitizing effect was tested by Hoechst 33258 staining. Part of the mice after treatments were kept for tumor regrowth assay, and the effect of tumor regrowth delay was evaluated by a long-term observation of tumor volume and tumor growth curve.
Results:Mice bearing cervical cancer cell xenografts were established successfully. Western blotting showed remarkable reduction of AKT phosphorylation in all the three treatment groups and the most of which was in the combined application group, indicating the remarkably suppressed PI3K activity. Clonogenic assay showed that combined application of radiation and LY294002 lead to the most significant suppression of the regrowth of cervical cancer cells, and the effect was radiation dose-dependent and LY294002 dose-dependent. The synergism of radiation and LY294002 was detected at each level of them by Factorial analysis (P<0.05). The rate of cell apoptosis was higher in combined group (35.5±13.67)% than that in the control group (8.67±4.37)%, radiation (21.67±13.17)% and LY294002 group (15±9.90)% (P=0.003). Tumor regrowth assay showed the lowest increase of tumor volume in combined application group (P=0.003) and the time to tumor volume of 240 mm3 Was 10±6.2 days in control and 19±12.0 days in the LY294002 alone, while it increased to 22±5.0 days in radiation alone and 37±9.0 days in combined group. Besides, LY294002 (100mg/kg) alone decreased cell survival and produced xenograft regrowth delay.
Conclusions:Compared with radiation alone, the combined application of PI3K inhibitor LY294002 and radiation can significantly enhance dephosphorylation of AKT in cervical cancer, increase tumor cell apoptosis and suppress tumor regrowth, indicating that PI3K inhibition can significantly enhance radiation efficacy via dephosphorylation of AKT in cervical cancer. Besides, PI3K inhibition alone can also suppress tumor regrowth. The present study initially proved the significant radiosensitization of PI3K inhibition in cervical cancer in vivo with nude mice bearing HeLa cell xenograft. This may provide novel therapeutic opportunities to enhance the effect of radiotherapy against cervical cancer.
引文
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