| Glyoxalase 1 (Glo1), mainly distributed in the cytosol of all mammalian cells, comprised the glyoxalase system with glyoxalase 2 (Glo2) and a catalytic amount of reduced glutathione (GSH). The system is responsible for methylglyoxal (MG) detoxification in animals. MG is a byproduct of glycolysis, and quite cytotoxic. The healthy people maintain the equilibrium of MG concentration by glyoxalase system. Many publications had been reported that Glo1 was up-regulated in malignancies, and involved in the multidrug resistance of tumors. In contrast, reports about Glo1’s role in HCC development are quite limited. So we want to do some exploration on Glo1’s role in HCC development by our study.We detected the Glo1 expression in 73 paired tumor and nontumorous of HCC tissues at mRNA levels by RT-PCR. The results displayed that Glo1 expression was significantly up-regulated in 43 tumor tissues, compared to the nontumorous tissues, accounting for 58.9% of the total tissues. Only in 5 cases, Glo1 showed down-regulation. The up-regulation indicates Glo1 may play quite important role in HCC progression.We also studied which factors promoted Glo1’s up-regulation. Increased glycolytic capacity is always a characteristic for tumors. However, we cannot detect the glycolytic capacity in HCC tissues directly at present. We choose to detect the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a very important enzyme in glycolysis, in HCC tissues, and discovered that GAPDH also displayed an up-regulation trend in HCC tissues. What is more, the expression of Glo1 was quite correlated with that of GAPDH in these tissues. Meanwhile, we observed that MG can up-regulate the expression of Glol in HCC cell lines. The results indicated that tumor tissues with high glycolysis activities need to up-regulate Glo1’s expression to detoxify the cytotoxic byproducts like MG, to keep the progression of tumor. Besides, oxidative stress is also existed in tumor microenviroment, and we discovered that H2O2 can also up-regulate Glo1 expression.To study the role of Glol in HCC cell proliferation, we knocked down the expression of Glo1 by RNA interference in Hep3B, SK-HEP1 and SMMC-7721 cell lines. We found that the cell proliferation was significantly inhibited upon Glol being silenced. This suggested that Glol was essential for HCC cell proliferation. However, the cell growth rate was not affected when Glo1 was overexpressed in these cells.Why the Glo1 knockdown could lead to cell proliferation inhibition? We speculated that Glo1’s cytotoxic substrate, MG, may accumulate in this process. To verify the hypothesis, we set up the HPLC-UV method to detect MG levels in these cells, and discovered that MG levels were up-regulated when Glo1 was silenced. The result was confirmed in Hep3B, SK-HEP1 and SMMC-7721 cells. The cytotoxic effect of MG was also verified in HCC cell lines by culturing cells in media spiked with indicated concentrations of MG.To figure out whether the expression level of Glo1 in HCC cell lines could also affect the cell’s sensitivity to anticancer drugs. We overexpressed Glol in Hep3B cell with relative low background of its expression. Through screening 9 generally used anticancer drugs, we found that the cells turned less sensitive to most of the drugs. Correspondingly, we silenced the expression of Glol in SK cells, and discovered that the cells became more sensitive to most of the drugs. The results suggested expression level of Glol can also affect HCC cells’ sensitivity to anticancer drugs.Finally, we analysed an SNP site (rs2736654) in Glol coding region by DNA sequencing. The base variant for this site is A to C, which are correspondent to the amino acid variant:Glu to Ala. The variants may differ in enzyme activity. By sequencing this site in 205 healthy controls and 332 HCC patients, we discovered that frequency of C is higher in patients and show significance.In summary, we discovered that Glol, as the main detoxification enzyme for MG, was significantly up-regulated in HCC tissues. As soon as Glo1 was silenced, MG levels in cells were increased, which resulted in cell proliferation inhibition. Besides, Glo1 expression level significantly affected cells sensitivity to anticancer drugs. Our study made an initial exploration of Glo1’s function in HCC development, and suggested that Glo1 could be designated as a potential therapeutic target for HCC in the future.HCC is one of the most common malignancies worldwide. The asymptomatic nature of HCC and lack of early-detection markers always result in the diagnosis of the disease at an advanced stage enforcing the patients to rely on chemotherapy. Anthracyclines are among the most efficient anticancer drugs. However their clinical uses are limited because of tumor resistance and severe cardiotoxicity. Carbonyl reduction is one of the generally accepted mechanisms responsible for anthracyclines’ resistance and cardiotoxcity, and mainly publications are focusing on carbonyl reductase 1(CBR1). By reducing the carbonyl group on C-13 positions of daunorubicin and doxorubicin to their corresponding alcohol metabolite, CBR1 participates in the development of drug resistance and cardiotoxicity of anthracyclines. So developing inhibitors targeting CBR1 to against anthracyclines’resistance and cardiotoxicity has drawn much attention.The previous research work of our lab had found a new effective inhibitor for CBR1, named epigallocatechin-3-gallate (EGCG). By combining EGCG and daunorubicin to treat on HCC cell lines, we discovered that EGCG can significantly enhance the efficacy of daunorubicin, which depends on the CBR1 expression. And similar phenomenon was also observed in vivo. What is more, EGCG reduced the cardiotoxicity of daunorubicin to mice notably. This research provided us a new combination strategy for HCC chemotherapy. However, new questions should be raised, such as whehter CBR1 can metabolise other analogues of anthracyclines except daunorubicin; whether other members of carbonyl reductase family can reduce anthracyclines; whether EGCG can inhibit the activity of other members in carbonyl reductase family except for inhibiting CBR1. And this work will make further researches on such inquiring.we expressed and purified the recombinant CBR1 with carbonyl reductase activity and detected the CBR1 reducing ability on four anthracyclines, including daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), and idarubicin (IDA). And we discovered that CBR1 showed more metabolism activity on IDA, except for DNR, while DOX and EPI could only be weakly reduced by CBR1 relatively. So we decided to choose IDA for the following research. We combined EGCG and IDA to treat the HCC cell lines, HepG2 and SMMC-7721, both of which show relative high CBR1 expression. The result showed that EGCG can also enhance the efficacy of IDA. However, similar effect can not be observed in Hep3B, which show much low CBR1 expression. These results indicated that EGCG enhancing ability may still depend on CBR1 expression. To make deeper confirming, we overexpressed CBR1 in Hep3B cells, interfered the CBR1 expression in HepG2 cells, and confirmed that the antitumor activity of IDA enhanced by EGCG is mainly dependent on CBR1.Among the four members of human carbonyl reductase family, carbonyl reductase 2 (CBR2) is expressed extraordinarily high in liver tissue. This suggests that CBR2 might play very important role in liver metabolism and detoxicification. So we choose CBR2 to explore its further role in anthracyclines metabolism. We expressed and purified recombinant CBR2 with enzyme activity, and also studied its metabolite ability on the above four anthracyclines. The results displayed that CBR2 show strongest activity on IDA, and relative low activity on the other three.We discovered that EGCG also can inhibit the enzyme activity of CBR2. So we’d like to explore whether CBR2 played any role in enhancing the antitumor activity of IDA by EGCG next. We detected CBR2 expression level in several common HCC cell lines, and found that CBR2 expressed especially high in HepG2. Then we interfered the CBR2 expression in HepG2, and the cells turned more sensitive to IDA. Meanwhile, EGCG’s enhancing for IDA was partly impaired. Besides, EGCG’s enhancing ability disappeared totally when both CBR1 and CBR2 were down-regulated in HepG2 cells. Correspondingly, we overexpressed CBR2 in Hep3B, and the cells became less sensitive to IDA. EGCG also show enhancing ability on IDA’s efficacy in these cells. All these results indicated us that EGCG partly targeted CBR2 in the enhancing effect.At last, we detected the relative CBR1 and CBR2 mRNA levels in 68 paired tumor and nontumorous tissues of HCC, and discovered that both genes displayed down-regulated expression in tumor tissues, compared to the nontumorous tissues, which were in consistent with previous reports. Besides, the expression level of CBR1 and CBR2 are quite correlate (R=0.6117, P<10-7). And we want to know whether the down-regulation means there is faint value for EGCG and anthracyclines cotreatment. That is not the fact. Many publications had been reported about CBRl’s activity up-regulation by anthracyclines’stimulation. In our study, we used the all the four anthracyclines to treat with Hep3B and HepG2 cells, and discovered that both CBR1 and CBR2 protein level was up-regulated by all the anthracyclines. These suggested that it is quite necessary to combining EGCG when treated tumors with anthracyclines.In summary, our study indicates that EGCG can enhancing the antitumor activity of IDA. This is a new combination strategy for chemotherapy, which depends on the expression of both CBR1 and CBR2. These results revealed a further mechanism elucidation and theoretical support for such combination strategy. |
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