Molecular Mechnisms Of MMPs Expression Alterations And Tumor Metastasis After Microcystin-LR Exposure

Cyanobacteria blooms occur more and more frequently because of the severe lake eutrophication. The bloom-froming cyanobacteria can produce excess microcystins to damage the liver of mammals, birds and fishes, which have received more toxicological concerns. Epidemiological and experimental evidences suggest that liver cancer incidence is usually higher for local residents in the regions undergoing algae bloom pollution, which imply the potential carcinogentic risk induced by exposure to cyanobacteria bloom. Cellular migration and tumor metastasis mediated by matrix metalloproteinases (MMPs) expression is an important molecular mechanism inducing cancer recurrence. This thesis aimed to explore the effects of microcystins exposure on tumor metastasis and to undercover the implied molecular mechanisms so as to access the potential health rsik induced by cyanobacteria blooms. Experimental methods and results are summarized as follows:Hepatotoxicity and MMPs expression changes after exposure of mice to Taihu Lake water:Water was sampled from four Taihu Lake locations, Meiliang Bay2group (M2), Meiliang Bay1group (M1), Lake Center (H) and Xukou Bay (X), along a gradient of decreasing degree of eutrophication. The experimental design consists of five groups of male mice (Mus musculus, ICR):one control and four groups ingesting water from the four sampling sites for90days. Compared to control, M1and M2mice showed hepatic histopathological changes including swollen, vacuolar degeneration or inflammatory. Immunohistochemical staining demonstrated a higher expression of MMP-2proteins in M2group and a lower expression of MMP-9in Ml. Enzyme-linked immunosorbent assay indicated that MMP-2concentration was significantly increased from2.9to3.5ng/g liver weight in M2(p<0.05). Real time PCR revealed a down-regulation of MMP-9mRNA by2.2fold in M1and an up-regulation of MMP-2mRNA by1.73fold in H. Conclusion:Taihu Lake water contaminated with cyanobacteria blooms caused severe histopathological demage in mice livers and the water exposure exerted an significant influence on hepatic MMP-2/-9expression which is closely related to tumor metastasis, revealing the potential health risks induced by the exposure to Taihu Lake water.Hepatotoxicity and MMPs expression changes after exposure of the mice to microcystin-LR (MC-LR):Mice were orally administered with MC-LR in drinking water (0,1,40and80μg/l) for180d or270d. Histopathologic observation demonstrated the obvious hepatic demages in the mice exposed to40and80μg/l MC-LR. Low-concerntration MC-LR (1μg/l) also resulted in slight histopathological changes after270-d exposure. Immunohistochemical staining and enzyme-linked immunosorbent assay revealed that excess MMP-2/-9proteins were produced in livers of the mice exposed to MC-LR at the higher concentrations, and hepatic MMP-2level was elevated from2.1ng/g liver weight in control to3.5ng/g liver weight in80μg/l group after270-d exposure (p<0.05). Real time PCR showed that180-d exposure caused4.9-and6.9-fold up-regulation of MMP-2mRNA expression and2.8-and5.0-fold up-regulation of MMP-9in40and80μg/l group, respectively. Conclusion: Chronic exposure to low concertrations of MC-LR caused slight and potential hepatotoxicity and stimulated MMP-2/-9expressions at the levels of enzyme activity, protein level and mRNA expression.The effects of in vitro MC-LR exposure on tumor cells migration and MMP-2/-9expression: Breast cancer cells (MDA-MB-435s) were exposed to MC-LR to assess the effects on cancer cell migration and cellular MMP-2/-9expression. MTT and Flow cytometry showed no obvious changes in celluar survivability and cycle after the cells were exposed to MC-LR (<50nM). Wound healing and transwell assay demonstrated that MC-LR stress posed a time/dose-dependent stimulation effect on breast cancer cell migration and infiltration. Gelatin zymography and Western blot showed significant increase in extracellular MMP-2/-9and intracellular pro-MMP-2expression after MC-LR exposure. Real time PCR demonstrated that the transcription of cellular mmp-2was up-regulated by5.5fold in25nM MC-LR treatment group and9.0fold in100nM group (p<0.05). Conclusion:In vitro MC-LR exposure stimulated the MMP-2/-9overexpression in MDA-MB-435s cells and induced the tumor cells migration and infiltration increased the potencial risk of tumor metastasis.The molecular functional mechanism of MC-LR as tumor promoter:The transcription of84genes related to tumor metastasis was analyzed by Tumor metastasis PCR Array (Supperarray, USA) after the MDA-MB-435s cell line was exposed to25nM MC-LR for72h. Statistics analyses revealed the up-regulations of52genes and the down-regulations of32ons. Among the differenciatly expressed genes,12ones were up-regulated and4ones were down-regulated by more than1.5fold. Eight genes were found to be up-regulated by over2.0fold:Cdhl1, Igf1, Il8rb, mmp13, mmp2, mmp9, Pnn and Rb1. Gene function annotation revealed that each of these8genes encoded the protein factors which positively regulate tumor metastasis progress. Conclusion:MC-LR exposure caused signicant stimulation of cancer cell invasion and migration, which is possibly mediated by abnormal expression of some specific genes. In summary, this study revealed that microcystins exposure influenced expressions of crucial enzyme or cell factors, especially MMP-2/-9, and stimulated the tumor cells migration and invasion, increasing the potential risk of metastasis and recurrence of malignant tumors.