.hcr2/cbr2 Relations And Egcg Targeting Of Cbr1 Treatment Of Liver Cancer And Liver Cancer

Hepatocellular cancer (HCC) is the fifth most common malignancy worldwide and third leading cause of cancer death. China alone accounts for 55%liver cancer death worldwide. HCC is highly resistant to conventional systemic therapies, and prognosis for advanced HCC patients remains poor. To date, it is important for us to make any significant progress in understanding tumor associated genes, so it is with the cancer treatment and prevention. HCR2 was cloned by homogenous screening strategy in our lab, and previous work found it was obviously expressed lower in the HCC tissues than in the normal liver tissues. In this work, we further detected 59 pairs of hepatocellular carcinoma (HCC) samples and found it exhibited remarkable down-regulation at both mRNA and protein levels.Further experiments demonstrated that hypoxia and hepatitis B virus X protein (HBx) were both the elements contributing to down-regulating HCR2 by stabilizing HIFlαin hepatocellular carcinoma cell lines, In order to explore the role of HIFla on HCR2 expression, we tansfected HIF1αinto Hep3B and HepG2 cell lines and the results show that HCR2 was down-regulated at both mRNA and protein levels. With detailed analysis of promoter of HCR2, two ARNT binding sites in the promoter were predicted. A 2602-bp HCR2 promoter construct containing two ARNT binding sites was generated from human genomic DNA. Promoter luciferase assay indicated that ARNT can increase transcription activity of HCR2 and the(-358bp) - (-353bp)and(-1538bp) - (-1533bp) binding sites on the HCR2 promoter were essential for transcriptional activation of HCR2 by mutation assays. We also demonstrated that ARNT directly bound to the promoter of HCR2 and the binding was specific revealed by chromatin immunoprecipitation assay. Further experiments show that ARNT can up-regulate the expression of HCR2 and the effect was inhibited by HIF1α. Furthermore, HCR2 can also be down-regulated by Reactive oxygen species (ROS) through inhibiting ARNT expression. In conclusion, our work demonstrates HCR2 was down-regulated by hypoxia and hepatitis B virus X protein (HBx), as well as ROS in hepatocarcinogenesis.In order to study the function of HCR2 in HCC, we carried out various experiments including cell growth and colony formation by over expressing HCR2 or knockdown HCR2 in Hep3B and SMMC7721 cell lines. All the results indicate that HCR2 inhibits proliferation of hepatocellular carcinoma cell lines. To further determine the role of HCR2 in progression of HCC, we generated HCR2 stable expression cell lines and did an in vivo animal experiment. In nude mice model, stable cell lines Hep3B expressing HCR2 grew significantly slower than the control cell lines. The inhibition role of HCR2 expression in tumor growth was correlation with the carbonyl reductase activity of HCR2 and the ROS level.Here we also explore the function of HCR2 on intracellular ROS level and its relevant effect. The MTS experiments showed that HCR2 can reduce the damnification caused by ROS. These findings establish a new mechanism for the negative regulation of intracellular ROS level by HCR2 and shed new light on the regulation of cellular redox homeostasis.Therefore, it strongly suggests that HCR2 is probably a novel and functional factor during HCC development and progress. Thus HCR2, a down-regulating gene in HCC, is a potential target for diagnosis, therapy and new drug development of HCC. Human carbonyl reductase 1 (CBR1) converts the antitumor drug and anthracycline daunoru-bicin (DNR) into the alcohol metabolite daunorubicinol (DNROL) with significantly reduced antitumor activity and cardiotoxicity, and this limits the clinical use of DNR. (-)-epigallocatechin gallate(EGCG) is the natural active compound of tea without outstanding anticancer effect, but it can be use as an assistant drug because if its low toxicity. We found that EGCG directly interacts with CBR1 and acts as a noncompetitive inhibitor, and the inhibition was strong in neutral or alkali conditions. EGCG specifically enhanced the antitumor activity of DNR against hepatocellular carcinoma cells which expressing high levels of CBR1, including anti-proliferation and pro-apoptosis effect of DNR. To verify the inhibiting effect of EGCG in cells, we separate and mensurate the levels of metabolite DNROL in cell extract which was decreasing with gradient concentrations of EGCG.The result show that EGCG enhance the activity of DNR by inhibiting the metabolism of DNR. In order to examine the ability of EGCG targeting CBR1 and on DNR anti-cancer effect in vivo, we established human carcinoma xenograft model with SMMC7721 and Hep3B cell lines in nude mice. For SMMC7721 xenografts, EGCG can enhance the anti-tumor effect of DNR, and there is no obviously difference in Hep3B xenografts. We test the body weight, liver function and cardiotoxicity with drug injection. The results show that EGCG as a safe drug can decrease the cardiotoxicity of the anticancer drug DNR and reverse the weight loss caused by DNR.These results strongly suggest that EGCG can inhibit CBR1 activity and enhance the effectiveness and decrease the cardiotoxicity of the anticancer drug DNR. These findings also indicate that a combination of EGCG and DNR might broaden the clinical use of DNR, and represent a novel approach for hepatocellular carcinoma therapy or chemoprevention.