Induction of Apoptosis by Matrine Derivative ZS17 in Human Hepatocellular Carcinoma BEL-7402 and HepG2 Cells through ROS-JNK-P53 Signalling Pathway Activation

Round 1

Reviewer 1 Report

Xiang Wang et al. describe in their article the effect of a new component - the Matrine derivate ZS17 - on two hepatocellular cell lines in vitro and in vivo and observe the overall toxicity of the component in zebra fish. 

The component exhibits a strong anti-proliferative on the cell cultures, reduced the number of migrating cell when cancer cells were implanted into zebra fish and no obvious toxic side effects in zebra fish embryos was observed. They conclude that ZS17 might be a good candidate for the treatment of hepatocellular cancer patients. 

This article reflects an extensive investigation of several derivatives of Matrine, in particular of ZS17, which represents the most promising candiate. They investigate the dose and exposure time dependents of viability of cells from two cancer cell lines, BEL-7402 and HepG2 in comparison to normal human liver cells, the number of cell colonies in culture and the ability of closing a gap in culture. They observer that a large number of cells is arrested in S phase of the cell division cycle. The study the effect the drug treatment has on the generation of reactive oxygen species - a class of molecules that when too abundant can activate apoptosis - and the stability of the mitochondrial membrane. Mitochondria are the a major source of reactive oxygen molecules. On top of all this they study the fate of HepG2 cells when injected into living zebra fish as an in-vivo system. 

 

All of this points into the direction that ZS17 inhibits growth of hepatic cancer cells and has at the dose level required for its action no major toxic effects on zebra fish embryos. 

All together I think this article presents convincing evidence to support the authors claim and should be published as it is. 

 

Author Response

Dear Editors and Reviewers:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled " Induction of Apoptosis by Matrine Derivative ZS17 in Human Hepatocellular Carcinoma BEL-7402 and HepG2 Cells through ROS-JNK-P53 Signalling Pathway Activation" (ID ijms-2078511). we will continue to work hard.

Reviewer 2 Report

In this study, the authors synthesised twelve matrine derivatives and tested them on cancer cell lines viability. They identified a compound named ZS17 that inhibits the growth, migration, and invasion of hepatocellular carcinoma cells in vivo and in vitro by activating the ROS-JNK-P53 pathway. They concluded that ZS17 may represent a promising agent for the treatment of liver tumors in hepatocellular carcinoma patients.

 The authors present here a very comprehensive study using both in vitro (human cancer cell lines) and in vivo (zebrafish and mice) models. The results obtained are very convincing and attractive. The paper is well written and clear, and the conclusions are supported by the results. This is a significant study that greatly contributes to this field of research.

I have only minor remarks/questions.

 1/ Among the 12 molecules tested, the authors chose to keep ZS17 on the basis of its IC50 (μM) value for antiproliferative effect on the 4 cell lines tested. "ZS17 showed broad antitumor toxicity against four tumor cell types." This choice is not totally clear for me since ZS-07 and ZS-10 exhibited an antiproliferative effect with a lower IC 50 on the 4 lines. Can the authors justify this choice?

2/ The authors should enlighten the reader on the natures of the 4 lines used in experiment 1.

3/ Why the authors did not used HepG2 cell line in the first experiment? The authors should specify why they used these HepG2 cells in addition to the BEL-7402 line to test the properties of ZS17.

4/ The authors concluded that ZS17 exerted anti-tumour effect by activating the ROS-JNK-P53 signalling pathway and mitochondrial dysfunction. How do they explain, in this case, the activation of caspase 8 by ZS17?

 5/ Flow cytometry analysis did not show the presence of apoptotic cells (subG0) after ZS17 while apoptosis seen clearly induced by ZS17 treatment (Western blot, cell viability assay …). Could the authors explain this apparent discrepancy?

Author Response

Please see the attachment.

Author Response File: Author Response.docx