Combination of Epigenetics Regulators with Chemotherapeutic Agents: A Novel Approach for Treatment of Hepatocellular Carcinoma

Abstract

Method: We tested our hypothesis using in silico, in-vitro and in-vivo models. For the in silico analysis, molecular docking was performed for the initial screening of the selected ligands on DNMT1 protein (PDB ID: 4WXX) using AutoDockTools-1.5.6. software. Ligand preparation and energy minimization step was performed using Chemdraw Professional 15.0 and Chem3D softwares, respectively. In-vitro experiments were performed on human hepatocellular carcinoma HepG2 cells. MTT assay was performed to evaluate the effects of gemcitabine alone or in combination with Olsalazine pretreatment on the viability of selected cell line. For animal experiments, thirty animals were allocated to five different groups (n=5 per group): Normal Control (NC), Diseased control (DC) treated with N-Nitrosodiethylamine (NDEA), Disease control treated with standard drug (DC-SD) gemcitabine (50mg/kg) once a week at a dose of intraperitoneally, Disease control treated with standard drug (Gemcitabine) and a low pretreatment dose (50mg/kg) of Olsalazine (DC-SD-LD) and Disease control treated with standard drug (Gemcitabine) and a higher pretreatment dose (100mg/kg)of Olsalazine (DC-SD-HD). For induction of Hepatocellular carcinoma, Carbon tetrachloride (CCL4) at a dose of 50 μl/kg body weights in liquid paraffin was administered orally for 4 consecutive days followed by NDEA administration at a cumulative dose of 350mg/kg twice a week intraperitoneally for six weeks. NDEA was administered for two weeks at 25mg/kg followed by increasing doses at 50mg/kg and 100mg/kg, respectively for next two weeks. Following induction, treatment with Gemcitabine alone or in combination with Olsalazine pretreatment was continued for 3 weeks. Bodyweight, food and water intake were measured for 3 weeks. At the end of third week, blood samples were collected and serum biochemistry and ELISA were performed. Isolated livers at the end of third week of treatment were weighed and subjected to histopathological analysis and immunohistochemistry (IHC). Oxidative stress parameters were performed on liver tissue homogenates. Results: Among the selected ligands Olsalazine, showed the highest binding affinity (-5.54) among all selected ligands. Therefore, Olsalazine was selected for further in-vitro and in-vivo evaluation. Cytotoxicity of gemcitabine against the HepG2 cells was improved upon pretreatment with Olsalazine (at 10um concentration). In in-vivo study, reduced tumor lesions were seen in animals pretreated with Olsalazine (20mg/kg and 40mg/kg) followed by treatment with gemcitabine (50mg/kg). Pretreatment with Olsalazine improved the treatment response to gemcitabine in comparison to gemcitabine alone treated group. Cancer progression was reduced, which was evident from improved body weight, food and water intake. Serum biochemistry showed reduced levels of ALP, LDH, CRP, SGPT, SGOT and total bilirubin levels in Olsalazine pretreated groups in comparison to diseased animals. Reduced levels of IL-6 and TNF-alpha were seen in Olsalazine pretreated animals, with a significant increase in P53 levels, suggesting improved treatment response. Furthermore, treatment showed reduced levels of LPO and increased activity of GSH and SOD in liver tissues. Results from histopathological examination showed increased number of double nucleus, congestion in central and portal veins, decreased sinusoidal space in disease control group. In contrast, the sinusoidal space was increased with decreased number of double nucleus in treated group compared to the diseased control group. Immunohistochemistry results showed increase in apoptosis evident with increased levels of p53 in treated group as compared to the disease control group. Conclusion: Our findings from the current study indicate that combining the chemotherapeutic drug gemcitabine with the epigenetic regulator Olsalazine enhances the antitumor response and also increases the sensitivity of gemcitabine for antitumor responses.