| Objective Microcystin-LR(MCLR)is monocyclic heptapeptide hepatotoxin,and long-term low-dose MCLR exposure is associated with high incidence of digestive tract tumors such as liver cancer.However,the mechanisms of its carcinogenic action remain obscure.The catalytic subunit of glutamylcysteine ligase(GCLC)primarily regulates de novo synthesis of glutathione and is central to the antioxidant capacity of the cell,but a large amount of data suggest that the GCLC expression is associated with the occurrence and development of cancer.Our previous study found that MCLR induced the malignant transformation of WRL68 cells,accompanied by the continuous decrease of GCLC protein expression level.It is speculated that GCLC down-regulation may be involved in MCLR-induced hepatocyte malignant transformation.In this study,human liver cell line WRL68 with or without GCLC stable overexpression was exposed to low-dose MCLR to inducemalignant transformation.The effects of GCLC overexpression on oxidative stress level and malignant phenotype of MCLR-induced malignant transformed cells were observed to investigate the effect of GCLC in MCLR-induced malignant transformation.Methods1.Inducing malignant transformation.Human liver cell line WRL68 and GCLC stable overexpressed WRL68 cell line were divided into un-transfected cells,un-transfected cells treated with MCLR,empty vector-transfected cells,empty vector-transfected cells treated with MCLR,GCLC-transfected cells,GCLC-transfected cells treated with MCLR.Un-transfected group treated with MCLR,empty vector-transfected group treated with MCLR and GCLC-transfected group treated with MCLR were continuously treated and maintained in the medium containing MCLR(10 nmol/L)for 72 h per passage.The process was continued for 25 passages(about 11 weeks).Un-transfected group,empty vector-transfected group and GCLC-transfected group were mock-treated with phosphate-buffered saline(PBS).Colony formation assay was used to assess the ability of cells to anchor-independently grow.Transwell migration assay was used to detect the migration ability of cells in vitro.Transwell invasion assay was used to detect the invasion ability of cells in vitro.2.Detection of cellular GCLC protein expression,oxidative stress level and PP2 A activity.GCLC protein expression was detected by Western blot.Oxidative stress levels were detected by Nanjing Jiancheng commercial kit: theactivities of glutamate-cysteine ligase(GCL),glutathione S-transferase(GST),catalase(CAT),and superoxide dismutase(SOD),the contents of glutathione(GSH),8-hydroxy-2’-deoxyguanosine(8-OHdG),malondialdehyde(MDA)by the kits.Protein phosphatase 2A(PP2A)activity was measured UV-visible spectrophotometrically by measuring the content of phosphorus at 630 nm.Results1.Effects of long-term low-dose MCLR exposure on the growth,migration and invasion of WRL68 cells and the role of GCLC overexpression(1)Western Blot results showed that,the expressions of GCLC protein were decreased in un-transfected cells treated with MCLR compared with un-transfected cells(P<0.05).The GCLC protein expression in GCLC-transfected group was up-regulated to 3.2 folds higher than that in vector-transfected group(P<0.05).The GCLC protein expression of vector-transfected cells treated with MCLR was decreased by 25.01% compared with vector-transfected cells(P<0.05).The expressions of GCLC protein in GCLC-transfected cells treated with MCLR was higher than that in vector-transfected cells treated with MCLR(P<0.05).(2)The results of colony formation of cells showed that,the colony formation rate of un-transfected cells with MCLR exposure was significantly higher than that of un-transfected cells(P<0.05).The colony formation rate in vector-transfected cells with MCLR exposure was significantly higher than that in vector-transfected cells(P<0.05).The colony formation rate inGCLC-transfected cells with MCLR exposure was less than that in vector-transfected cells with MCLR exposure(P<0.05).(3)The results of transwell migration of cells showed that,the number of cells penetrating the chamber in un-transfected cells with MCLR exposure were significantly higher than that in un-transfected cells(P<0.05).The number of cells penetrating the chamber in GCLC-transfected cells was significantly lower than that in vector-transfected cells(P<0.05).The number of cells penetrating the chamber in vector-transfected cells with MCLR exposure was significantly higher than that in vector-transfected cells(P<0.05),while the number of cells penetrating the chamber in GCLC-transfected cells with MCLR exposure was significantly less than that in vector-transfected cells with MCLR exposure(P<0.05).(4)The results of transwell invasion of cells showed that,the number of cells penetrating the Matrigel membrane in un-transfected cells with MCLR exposure were significantly higher than that in un-transfected cells(P<0.05).The number of cells penetrating the Matrigel membrane in GCLC-transfected cells was significantly lower than that in vector-transfected cells(P<0.05).The number of cells penetrating the Matrigel membrane in vector-transfected cells with MCLR exposure was significantly higher than that in vector-transfected cells(P<0.05),while the number of cells penetrating the Matrigel membrane in GCLC-transfected cells with MCLR exposure was significantly less than that in vector-transfected cells with MCLR exposure(P<0.05).2.Effects of long-term low-dose MCLR exposure on oxidative stress level and PP2 A activity in WRL68 cells and,role of GCLC overexpression(1)The results of GCL activity and GSH content of cells showed that,the GCL activity and GSH content of the un-transfected cells treated with MCLR were all decreased,which were decreased to 44.16% and 64.56%,respectively,compared to un-transfected cells(P<0.05).The activity of GCL and the content of GSH in GCLC-transfected cells were 4.32 U/mgprot and 7.41 μmol/gprot,respectively;and had significantly increase compared with vector-transfected cells(P<0.05).GCL activity and GSH content in vector control group treated with MCLR were decreased compared with vector-transfected group(P<0.05),while the GSH level and GCL activity in GCLC-transfected cells with MCLR exposure were significantly higher than that of vector-transfected cells with MCLR exposure(P<0.05).(2)The results of GST activity of cells showed that,the GST activity of the un-transfected cells treated with MCLR was lower than that of the un-transfected cells(P<0.05).The GST activity in GCLC-transfected cells were higher than that in vector-transfected cells,GST activity in GCLC-transfected group was up-regulated to 1.2 folds higher than that in vector-transfected group(P<0.05).The activity of GST in vector-transfected group treated with MCLR were decreased compared with vector-transfected group(P<0.05),while GST activity in GCLC-transfected cells with MCLR exposure was significantly higher than that in vector-transfected cells with MCLR exposure(P<0.05).(3)The results of contents of 8-OHdG and MDA in cells showed that,the levels of 8-OHdG and MDA in un-transfected cells treated with MCLR were higher than that in the un-transfected cells(P<0.05).The contents of 8-OHdG and MDA in GCLC-transfected cells were lower than that in vector-transfected cells(P<0.05).The contents of 8-OHdG and MDA in vector-transfected group treated with MCLR were decreased compared with vector-transfected group(P<0.05),while the 8-OHdG and MDA levels of GCLC-transfected cells with MCLR exposure were significantly lower than that of vector-transfected cells with MCLR exposure(P<0.05).(4)The results of CAT and SOD activities in cells showed that,the activities of CAT and SOD in un-transfected cells treated with MCLR were lower than that in un-transfected cells(P<0.05).There was no significant difference in activities of CAT and SOD between GCLC-transfected cells and vector-transfected cells(P>0.05).The activities of CAT and SOD in vector-transfected cells treated with MCLR were decreased compared with vector-transfected cells(P<0.05),while CAT and SOD activities of GCLC-transfected cells with MCLR exposure were all significantly higher than that of vector-transfected cells with MCLR exposure(P<0.05).(5)The results of PP2 A activity in cells showed that,the activity of PP2 A in un-transfected cells treated with MCLR decreased to 68.01% compared to un-transfected cells(P<0.05).There was no significant difference in PP2 A activities between GCLC-transfected group and vector-transfected group(P>0.05).PP2 A activity of GCLC-transfected group treated with MCLR by19.09% compared with GCLC-transfected group(P<0.05),whereas PP2 A activity of vector-transfected group treated with MCLR was decreased by29.86% compared with vector-transfected group(P<0.05),while PP2 A activity of GCLC-transfected cells with MCLR exposure was significantly higher than that of vector-transfected cells with MCLR exposure(P<0.05).Conclusions1.Chronic low-dose exposure to MCLR inhibited the activity of PP2 A,reduced GCLC protein expression and GSH level,increased oxidative DNA damage,eventually induced malignant transformation of cells,migration and invasion in vitro.2.Overexpression of GCLC can enhance GCL activity and GSH level,inhibit DNA oxidative damage,and eventually reduce the degree of malignant transformation and the abilities of migration and invasion induced by MCLR;and also attenuate the inhibition of PP2 A activity;suggesting that GCLC plays an important role in MCLR-induced malignant transformation of human liver cells. |
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