辛伐他汀通过调控PI3K/AKT通路介导的EMT参与放疗诱导的食管癌细胞耐受
|
摘要
目的明确辛伐他汀对食管癌细胞放疗耐受性、上皮间充质细胞转化(epithelialmesenchymal transition,EMT)发生的影响及其作用机制。方法食管癌细胞经辛伐他汀(5μmol/L)预处理8h后进行X线放射处理,克隆平板形成实验测定细胞生存分数;MTT检测放疗后细胞增殖能力;流式细胞术检测细胞凋亡;试剂盒检测caspase-3活性;Western blot检测对PI3K/AKT通路的影响。结果辛伐他汀预处理降低放疗细胞的克隆存活分数(P<0.05),放疗后细胞的增殖能力进一步降低(P<0.05),而细胞的凋亡率及caspase-3活性上调(P<0.05)。此外,辛伐他汀增加6Gy细胞处理组中上皮标记物E-cadherin的表达,同时抑制间充质标记物N-cadherin及Vimentin的表达,差异有统计学意义(P<0.05)。机制分析证实,放疗后细胞中p-AKT的表达水平明显增加,而辛伐他汀预处理可明显抑制p-AKT的表达。当用PI3K/AKT通路激动剂IGF-1预处理后,辛伐他汀介导的放疗细胞增敏效应及逆转放疗细胞EMT发生的能力明显减弱(P<0.05)。结论辛伐他汀可以阻断PI3K/AKT通路的活化,抑制放疗引发的食管癌细胞EMT发生,从而增加食管癌细胞的放疗敏感性,提示本研究将为食管癌放疗增敏的临床研究提供新的策略。
Objective To explore the role and mechanism of simvastatin in radioresistance and epithelialmesenchymal transition(EMT)of esophageal cancer.Methods Esophageal cancer cells were preconditioned with simvastatin(5μmol/L)for 8h,prior to exposure to x-ray irradiation.Clonogenic cell survival assay was performed to detect cell survival fraction.Cell proliferation was evaluated by MTT assay.Cell apoptosis was determined by flowcytometry.The activity of caspase-3was detected by the common commercial kit.Additionally,Western blot assay was performed to analyze the activation of the PI3K/AKT pathway.Results Pretreatment with simvastatin dramatically decreased the survival fraction(P <0.05)and proliferation(P <0.05)of the radiated cells,but aggravated cell apoptosis rate and caspase-3activity(P <0.05).Furthermore,simvastatin stimulation significantly increased the expression of epithelial marker E-cadherin in 6Gy-treated cells,but down-regulated the expression of mesenchymal marker N-cadherin and Vimentin(P <0.05).Mechanism analysis corroborated the activation of the PI3K/AKT pathway in cells after irradiation by elevating the expression of p-AKT,which was abrogated by simvastatin pre-treatment.More importantly,administration with IGF-1,an activator of PI3K/AKTpathway,attenuated the inhibitory effect of simvastatin on radioresistance and radiation-evoked EMT(P<0.05).ConclusionSimvastatin inhibits radiation-induced EMT of esophageal cancer cells by blocking the PI3K/AKT pathway,and ultimately elevates the radiosensitivity,indicating apromising therapeutic avenue for treatment of esophageal cancer radioresistance.
Objective To explore the role and mechanism of simvastatin in radioresistance and epithelialmesenchymal transition(EMT)of esophageal cancer.Methods Esophageal cancer cells were preconditioned with simvastatin(5μmol/L)for 8h,prior to exposure to x-ray irradiation.Clonogenic cell survival assay was performed to detect cell survival fraction.Cell proliferation was evaluated by MTT assay.Cell apoptosis was determined by flowcytometry.The activity of caspase-3was detected by the common commercial kit.Additionally,Western blot assay was performed to analyze the activation of the PI3K/AKT pathway.Results Pretreatment with simvastatin dramatically decreased the survival fraction(P <0.05)and proliferation(P <0.05)of the radiated cells,but aggravated cell apoptosis rate and caspase-3activity(P <0.05).Furthermore,simvastatin stimulation significantly increased the expression of epithelial marker E-cadherin in 6Gy-treated cells,but down-regulated the expression of mesenchymal marker N-cadherin and Vimentin(P <0.05).Mechanism analysis corroborated the activation of the PI3K/AKT pathway in cells after irradiation by elevating the expression of p-AKT,which was abrogated by simvastatin pre-treatment.More importantly,administration with IGF-1,an activator of PI3K/AKTpathway,attenuated the inhibitory effect of simvastatin on radioresistance and radiation-evoked EMT(P<0.05).ConclusionSimvastatin inhibits radiation-induced EMT of esophageal cancer cells by blocking the PI3K/AKT pathway,and ultimately elevates the radiosensitivity,indicating apromising therapeutic avenue for treatment of esophageal cancer radioresistance.
引文
[1]SIEGEL RL,MILLER KD,JEMAL A.Cancer statistics,2017[J].CA Cancer J Clin,2017,67(1):7-30.
[2]WANG ST,HO HJ,LIN JT,et al.Simvastatin-induced cell cycle arrest through inhibition ofSTAT 3/SKP2 axis and activation of AMPK to promote p27and p21accumulation in hepatocellularcarcinoma cells[J].Cell Death Dis,2017,8(2):e2626.
[3]WANG T,SEAH S,LOH X,et al.Simvastatin-induced breast cancer cell death and deactivation of PI3K/Akt and MAPK/ERK signalling are reversed by metabolic products of the mevalonate pathway[J].Oncotarget,2016,7(3):2532-2544.
[4]DE LLOBET LI,BARO M,MESIA R,et al.Simvastatin enhances the effects of radiotherapy and cetuximab on a cell line(FaDu)derived from a squamous cell carcinoma of head and neck[J].Transl Oncol,2014,7(4):513-522.
[5]THEYS J,JUTTEN B,HABETS R,et al.E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells[J].Radiother Oncol,2011,99(3):392-397.
[6]ZHANG H,LUO H,JIANG Z,et al.Fractionated irradiationinduced EMT-like phenotype conferred radioresistance in esophageal squamous cell carcinoma[J].J Radiat Res,2016,57(4):370-380.
[7]TORRE LA,BRAY F,SIEGEL RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[8]BURANRAT B,SENGGUNPRAI L,PRAWAN A,et al.Simvastatin and atorvastatin as inhibitors of proliferation and inducers of apoptosis in human cholangiocarcinoma cells[J].Life Sci,2016,153:41-49.
[9]LACERDA L,REDDY JP,LIU D,et al.Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with postmastectomy radiation[J].Stem Cells Transl Med,2014,3(7):849-856.
[10]金迎迎,卫阳,王力,等.辛伐他汀对食管癌细胞放疗敏感性的影响[J].西安交通大学学报(医学版),2016,37(4):565-568.
[11]LENG S,YANG M,ZHAO Y,et al.PTIP promotes recurrence and metastasis of hepatocellular carcinoma by regulating epithelial-mesenchymal transition[J].Oncotarget,2017,8(35):58184-58198.
[12]YU J,HAN Q,CUI Y.Decreased long non-coding RNA SPRY4-IT1contributes to ovarian cancer cell metastasis partly via affecting epithelial-mesenchymal transition[J].Tumour Biol,2017,39(7):1010428317709129.
[13]SKVORTSOVA I,SKVORTSOV S,RAJU U,et al.Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse[J].Radiother Oncol,2010,96(1):108-115.
[14]HE E,PAN F,LI G,et al.Fractionated ionizing radiation promotes epithelial-mesenchymal transition in human esophageal cancer cells through PTEN deficiency-mediated Akt activation[J].PLoS One,2015,10(5):e0126149.
[15]MENG X,DONG Y,YU X,et al.MREG suppresses thyroid cancer cell invasion and proliferation by inhibiting Akt-mTOR signaling[J].Biochem Biophys Res Commun,2017,491(1):72-78.
[16]XU Z,YAN Y,XIAO L,et al.Radiosensitizing effect of diosmetin on radioresistant lung cancer cells via Akt signaling pathway[J].PLoS One,2017,12(4):e0175977.
[2]WANG ST,HO HJ,LIN JT,et al.Simvastatin-induced cell cycle arrest through inhibition ofSTAT 3/SKP2 axis and activation of AMPK to promote p27and p21accumulation in hepatocellularcarcinoma cells[J].Cell Death Dis,2017,8(2):e2626.
[3]WANG T,SEAH S,LOH X,et al.Simvastatin-induced breast cancer cell death and deactivation of PI3K/Akt and MAPK/ERK signalling are reversed by metabolic products of the mevalonate pathway[J].Oncotarget,2016,7(3):2532-2544.
[4]DE LLOBET LI,BARO M,MESIA R,et al.Simvastatin enhances the effects of radiotherapy and cetuximab on a cell line(FaDu)derived from a squamous cell carcinoma of head and neck[J].Transl Oncol,2014,7(4):513-522.
[5]THEYS J,JUTTEN B,HABETS R,et al.E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells[J].Radiother Oncol,2011,99(3):392-397.
[6]ZHANG H,LUO H,JIANG Z,et al.Fractionated irradiationinduced EMT-like phenotype conferred radioresistance in esophageal squamous cell carcinoma[J].J Radiat Res,2016,57(4):370-380.
[7]TORRE LA,BRAY F,SIEGEL RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[8]BURANRAT B,SENGGUNPRAI L,PRAWAN A,et al.Simvastatin and atorvastatin as inhibitors of proliferation and inducers of apoptosis in human cholangiocarcinoma cells[J].Life Sci,2016,153:41-49.
[9]LACERDA L,REDDY JP,LIU D,et al.Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with postmastectomy radiation[J].Stem Cells Transl Med,2014,3(7):849-856.
[10]金迎迎,卫阳,王力,等.辛伐他汀对食管癌细胞放疗敏感性的影响[J].西安交通大学学报(医学版),2016,37(4):565-568.
[11]LENG S,YANG M,ZHAO Y,et al.PTIP promotes recurrence and metastasis of hepatocellular carcinoma by regulating epithelial-mesenchymal transition[J].Oncotarget,2017,8(35):58184-58198.
[12]YU J,HAN Q,CUI Y.Decreased long non-coding RNA SPRY4-IT1contributes to ovarian cancer cell metastasis partly via affecting epithelial-mesenchymal transition[J].Tumour Biol,2017,39(7):1010428317709129.
[13]SKVORTSOVA I,SKVORTSOV S,RAJU U,et al.Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse[J].Radiother Oncol,2010,96(1):108-115.
[14]HE E,PAN F,LI G,et al.Fractionated ionizing radiation promotes epithelial-mesenchymal transition in human esophageal cancer cells through PTEN deficiency-mediated Akt activation[J].PLoS One,2015,10(5):e0126149.
[15]MENG X,DONG Y,YU X,et al.MREG suppresses thyroid cancer cell invasion and proliferation by inhibiting Akt-mTOR signaling[J].Biochem Biophys Res Commun,2017,491(1):72-78.
[16]XU Z,YAN Y,XIAO L,et al.Radiosensitizing effect of diosmetin on radioresistant lung cancer cells via Akt signaling pathway[J].PLoS One,2017,12(4):e0175977.