Experimental Study On Protective Effect And Mechanism Of Hydrazinocurcumin On Hepatocarcinoma

Hepatocellular carcinoma (HCC) is the fifth commonest malignancyworldwide and is the third most common cause of cancer-related death.Incidence rate of HCC has been the most in China, where is the secondleading cause of cancer death now. Due to the onset occult disease, rapidprogress, and ineffective treatment,60%-70%of patients are usuallydiagnosed with advanced cancer and not available for the surgical treatment.They have to suffer from a palliative symptomatic treatment finally. Until now,the metastatic mechanisms and the regulated mechanisms of tumors are notknown clearly, so it is very important for understanding the mechanisms ofinvasion and metastasis and searching effective approaches to prevent therecurrence and metastasis of HCC is of great importance.Hydrazinocurcumin (HZC) is the analogue of curcumin (CUR).Compared with CUR, HZC has greatly improved water solubility and stability,and has high cell permeability, anti-tumor activity, improved bioavailabilitywith more favorable pharmacological activity. Persistent activation of signaltransduction pathway of JAKs/STATs is associated with the occurrence anddevelopment of tumors. STAT3is the important member of transductionpathway which connects extracellular signal to cell response. STAT3involvedin the regulation of tumor proliferation, survival, metastasis, angiogenesis andimmune response processes. Activation of the signal pathway of JAK2/STAT3,especially the activation of STAT3has an important influence on theoccurrence and development of tumors. In our study, the use of molecularbiology techniques and pathology on HZC which investigates theanti-hepatocarcinoma effect in vivo and vitro and its mechanism is applied tothe theoretical and experimental basis for the final clinical application. Part I Hydrazinocurcumin inhibits proliferation of humanhepatoma cell HepG2and induces cell apoptosisObjective: To investigate the effect of Curcumin analogue(hydrazinocurcumin, HZC) on proliferation and apoptosis of human hepatomacell HepG2and elucidate its mechanism.Methods: Different curcumin (CUR) and hydrazinocurcumin (HZC)concentrations (10,20,40μmol/L) were cultured for12h,24h and48h toHepG2cells proliferation was detected by MTT. For further study theinhibition of induction of apoptosis by CUR and HZC, we carried out differentconcentrations of CUR and HZC to culture the HepG2cells for48h in order todetermine the cell apoptosis rate by flow cytometry method (FCM).Expression of Bcl-2protein family in HepG2cells under differentconcentrations of CUR and HZC (10、20、40μmol/L) for24h, we compared theintensity of protein expression by Western blot.Results: MTT assay showed the IC50of CUR was25.43±2.86μmol/Lcompared with5.84±0.97μmol/L of HZC in HepG2cells. Both CUR and HZCcould inhibit the proliferation of HepG2cells, with a time and dose-dependentmanner (P<0.05). The inhibitory rate of HZC was higher than that of CUR at40μmol/L at different time (12、24and48h)(P<0.05). Compared with thecontrol group, both drugs had the ability to induce apoptosis in HepG2cells,and HZC was more potent than CUR. The level of apoptosis was higher in40μmol/L of CUR and HZC, especially the latter. It is shown that the expressionof Bcl-2and Bcl-xl is down-regulated while the expression of Bax isup-regulated after treated with CUR and HZC in HepG2cells, with adose-dependent manner（P＜0.05）. The stronger inhibitor of the Bcl-2andBcl-xl and promotion of Bax was HZC while the weaker one was CUR (P＜0.05).Conclusion: Both CUR and HZC could inhibit the proliferation andinduce apoptosis of HepG2cells, especially HZC. Impact of expression ofBcl-2protein family in HepG2cells by HZC was better than CUR. Part II Hydrazinocurcumin inhibits effect of signal transductionpathway of JAK2/STAT3on human hepatoma cellHepG2Objective: To investigate the effect of curcumin analogue(hydrazinocurcumin, HZC) on human hepatoma cell HepG2by inhibiting theJAK2/STAT3signal transduction pathway and elucidate its relevantmechanisms.Methods: The human hepatoma cell HepG2were divided into3groups,including DMSO control group, curcumin (CUR) treatment group andhydrazinocurcumin (HZC) treatment group. Treated with differentconcentrations of CUR and HZC (10、20、40μmol/L) at24h in HepG2cells, tostudy the effect on JAK2/STAT3signaling tranSDuction pathway, includingthe expression of P-JAK2, JAK2, P-STAT3, STAT3and its downstream targetswhich were detected by Western blot analysis.Results: The Western blot analysis demonstrated that HZC and CURdecreased the expressions of P-JAK2, P-STAT3in HepG2cells and regulatedits downstream targets which contributed to induction apoptosis. Comparedwith the same dose of CUR, HZC showed stronger effect on proteinexpression in P-JAK2、P-STAT3、Bcl-2，Bcl-xl，Bax，Cyt c，Caspase-3andCaspase-9（P<0.05or P<0.01）.Conclusion: Both CUR and HZC could significantly inhibit the theactiviation of JAK2and STAT3protein and affect expression of Bcl-2, Bcl-xl,Bax, Cyt c, Caspase-3and Caspase-9. CUR and HZC regulated thedownstream targets which contributed to suppression of cell proliferation, aswell as induction cell apoptosis through block of JAK2/STAT3signalingtransduction pathway, so did inhibition of proliferation of HepG2cells,induction of apoptosis and prevention the tumour progress. HZC was morefavorable pharmacological activity than CUR and might have translationalpotential as effective cancer therapeutics or preventive agent for human. Part III Preventive effect of hydrazinocurcumin oncarcinogenesis of diethylnitrosamine-inducedhepatocarcinoma in male SD ratsObjective: We studied the effect of CUR or HZC on hepatocarcinomainduced by diethylnitrosamine (DEN) in male SD rat, which could providetheoretical and experimental basis for clinical application.Methods:1Model preparation and drug intervention of hepatocarcinoma:120healthy male SD rats weighting100–120g were provided. Rats wererandomised divided into six groups accordance with the requirements afterone week of acclimatisation. Group1(control non-treated group) wasadministered intraperitoneally (80mg/kg) physiological saline twice a weekfor12weeks. CUR in group3(control CUR-treated group) was administeredintraperitoneally (80mg/kg) twice a week for12weeks. HZC in group5(control HZC-treated group) was administered intraperitoneally (80mg/kg)twice a week for12weeks. Rats in group2(DEN-bearing non-treated group)were given intraperitoneal injection of DEN (50mg/kg) twice a week for12weeks. Rats in group4(DEN-bearing CUR-treated group) were administeredwith both DEN as in group2and CUR as in group3. Rats in group6(DEN-bearing HZC-treated group) were administered with both DEN as ingroup2and HZC as in group5.2Determination of serum markers: The experiment was terminated at theend of24weeks and all the surviving living rats were sacrificed at the end ofthe experiment. Blood samples were taken before sacrifice. Determination ofserum alanine aminotransferase(ALT), aspartate aminotransferase(AST),alkaline phosphatase(ALP), γ-glutamyltransferase (GGT), total bilirubinlevel(TBL) were measured by automatic biochemical analyzer.3Tumor behavior observation: The surviving living rats wereanaesthetized and sacrificed at the end of the experiment. Liver was excisedimmediately and then counted tumors, observe tumor morphology. Meanwhilebody and liver weight were measured. 4HE staining and immunohistochemical analysis: Specimens were takenfrom the liver of rats. All the liver tissues were fixed routinely, and embeddedin paraffin. Comparison of pathological changes in liver tissues of each groupby haematoxylin and eosin (HE) staining. The expression of proliferating cellnuclear antigen (PCNA) in liver tissues of each group were detected byimmunohistochemical analysis.Results:1Incidence rate and Survival rate: There have47rats withhepatocarcinoma in all groups,30rats in group2(DEN-bearing non-treatedgroup),11in group4(DEN-bearing CUR-treated group),6in group6(DEN-bearing HZC-treated group),0in three control groups.12rats,5ratsand2rats were dead in group2, group4, group6, respectively. It showsSTATistical difference between group2and group4、group2and group6ininciDENce rate and survival rate (all. P<0.05). ALThough inciDENce rate andsurvival rate in group4and group6had no STATistical differences, the lattergroup was better.2Liver tumors: Nodule larger than6mm in diameter was considered ascancerous node. In rats of group2, the average count of nodule was17.17±1.08, while it was only13.36±2.17,11.83±3.90in the group4and group6,respectively. No hepatic nodule was observed in three control groups.although there was no statistical significance between group4and group6,they all declined count of nodules significantly compared with group2,especially group6.3Body weight and liver weight: The body weight in rats of group1werelarger than group2. Body weight and gain body weight in group2weresignificantly lower than group4and group6（all. p<0.01）. We measured theabsolute and relative liver weight which was significantly higher in group4orgroup6than in group2（all. p<0.01）. There were no differences in bodyweight, absolute and relative liver weight in three control groups (P>0.05).4Liver function index: The index of liver function in control groups werenormal. Rats from group2showed an significant increase in liver enzymesactivities in comparison to group1(P<0.01). The serum from rats in group2 showed a highly significant increase in levels of liver enzymes whencompared to those from group4or group6, especially the latter group(P<0.01).5Liver histopathology and immunohistochemical staining: Hepatictissues from the group1showed hepatic lobules with normal architecture;Hepatic tissues from group2and group5indicated moderate to severe venousand sinusoidal congestion; Hepatic tissues from rats in group2showed cancerfocus; Hepatic tissues from group4showed cancerous focus with focalnecrosis; Hepatic tissues from group6showed cancerous focus with patchynecrosis. Immunohistochemical staining in group1showed no PCNA stainingpattern. The positive tumor for PCNA was significantly lower in the group4and group6than in group2(all. P<0.05).Conclusion:1The course of hepatocarcinoma rat model induced by alow dose of DEN discontinuously is similar with the generating occurrenceand development of human primary hepatocarcinoma. So it is convenient forus to carry out the research and intervention test in mechanism ofhepatocarcinogenesis.2HZC had significantly preventive effect on carcinogenesis ofDEN-induced hepatocarcinoma in male SD rats and decreased the incidenceof hepatocarcinoma. Moreover administration of HZC had a protective effectagainst toxicity of the liver induced by DEN.3HZC could effectively reduce DEN-induced pathological changes inhepatocarcinoma of SD rats and decreased the expression of PCNA inhepatocarcinoma tissues, indicated a better prognosis.