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Vol.9 No.3-2:Biomolecular evaluation of apoptosis, cell cycle, oxidative stress, and limiting enzymes of the glycolytic pathway in hepatocellular carcinoma cell line HepG2 treated with crude snake venom with or without sorafenib

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By: Maha Y. Abdel Al Shakour1, Emad M. Elzayat2*, Khalid M. Mahmoud3,

Mamdouh I. Nassar4, Abdel Hamid Z. Abdel-Hamid5

1PhD student at Zoology Department, Faculty of Science, Cairo University, Giza, Egypt

2* Biotechnology Department, Faculty of Science, Cairo University, Giza, Egypt

3Pharmacognosy Department, NRC, Giza, Egypt

4Entomology Department, Faculty of Science, Cairo University, Giza, Egypt

5Therapeutic Chemistry Department, NRC, Giza, Egypt

Abstract

Background: Natural venoms have biological activities including anti-inflammatory, antimicrobial, and anticancer effects.  Hepatocellular carcinoma (HCC) is still a worldwide problem and difficult to treat by chemotherapeutic agents especially sorafenib (SOR), as it evokes many harsh side effects and is disable to differentiate between normal and cancer cells. Objective: The present study aimed to test the hypothesis that combining crude venoms of the snake or the bee or the scorpion could synergistically enhance the antiproliferative effects of SOR in hepatocellular carcinoma cell line (HepG2). Experimental design: Separate crude venoms have been applied to HepG2 cells and normal human retinal cells(RBE1) for estimation of IC50. The most effective venom has been combined with sorafenib in five nonconstant ratios and the combination index (CI) was estimated to expose their synergistic or antagonistic action. The best combination was used for downstream analysis. Results: The crude snake venom exhibited the most cytotoxic effect and the least IC50. It has been combined with sorafenib, and the combination index (CI) was calculated. IC25 SV + IC10 SOR was the best combination with CI=0.209 indicating high synergistic cytotoxic activity against HepG2. The underlining molecular mechanisms of action, in terms of the expression level of apoptotic genes (p53, Bax, Caspase 3, and Bcl2), flow cytometric analysis of cell cycle, oxidative stress markers as well as the activity of some limiting enzymes in the glycolytic pathway (ALDOB, PK and LDH) have been investigated.

Conclusion: Our results suggest a novel synergistic, and anti-proliferative effect of snake venom with sorafenib on HepG2 cells.

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Vol.5 No.3 – 7 : Downregulation of miR-23a and miR-24 in human hepatocellular carcinoma cells by Sorafenib via transforming growth factor beta 1 in a SMAD dependent manner

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By: Eman G. Ayad1, Mohga S. Abdulla*1, Hayat M. Sharada1, Abdel Hady A. Abdel Wahab2, and Abeer M. Ashmawy2.

1Department of chemistry, Faculty of Science, Helwan University, Egypt.

2 Departments of Cancer Biology, National Cancer Institute, Cairo University, Egypt.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through post-transcriptional interactions with mRNA. MiRNAs have recently considered as key regulators of various cancers including liver cancer. Sorafenib is one of antitumor drug for treatment of advanced hepatocellular carcinoma. It acts as a multikinase inhibitor suppressing cell proliferation and angiogenesis. This study try to investigate a potential microRNA-based mechanism of action of the drug,by studying the effect of sorafenib on miR-23a and miR-24 levels in HCC cell lines HepG2 /Huh7 and revealing the possible drug mechanism against these oncogenic mi-RNAS,in this study cell viability of cultured HepG2 /Huh7 after treatment with sorafenib were evaluated using Sulphorhodamine-B (SRB) assay, cell cycle and apoptosis estimated by flowcytometry assay. Caspase-3 level was determined using ELISA assay. Moreover, MiR-23a and miR-24 expressions levels analyzed by qPCR. Finally, TGF-β levels and phosorylated smad2, 3 were examined after treatment with sorafenib using ELISA and western blotting. Our data confirmed the Sorafenib inhibition of cell growth in both cell lines which was accompanied by significantly increased in cell apoptosis and cell cycle arrest. Cells treated with sorafenib showed a significant decrease in miR-23a and miR-24 levels in both cell lines. Interestingly,   the change in these oncogenic miRNA was accompanying with a significant decrease of (TGF-β1) and phosorylated smad2, 3 proteins levels. Our study suggested that inhibition of tgf beta pathway in smad dependent manner could be the way characteristic of sorafenib to inhibit the oncogenic miR-23a and miR-24 levels in HCC.  

Downregulation of miR-23a and miR-24 in human hepatocellular carcinoma cells by Sorafenib via transforming growth factor beta 1 in a SMAD dependent manner

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