Published online Mar 27, 2023. doi: 10.4254/wjh.v15.i3.393
Peer-review started: October 27, 2022
First decision: December 31, 2022
Revised: January 17, 2023
Accepted: March 3, 2023
Article in press: March 3, 2023
Published online: March 27, 2023
Conventional chemotherapy and radiotherapies based on x-ray and gamma-ray radiations are the most widespread techniques in the world for the treatment of malignant diseases due to their ability to penetrate tissues and thus allow them to reach deep sites. The only limitation of these treatments is the lack of selectivity between the tumour and the healthy surrounding tissues. Interestingly, previous studies have shown that silver nanoparticles (AgNPs) have the ability to selectively induce cytotoxic effects on cancer cells, as compared to normal cells. Therefore, the present study aims to evaluate the cytotoxic effects of AgNPs synthesised by C. roseus aqueous extract against liver carcinoma cells HepG2 and normal liver cells THLE-3, by assessing the proliferative activity followed by the mRNA transcriptome profiling analysis.
Due to the limitations of the conventional treatment like non-specificity and less effectiveness, novel strategies are in demand to solve these issues. Amongst all, the use of plant-synthesised silver nanoparticles has gained attention as they are known for non-toxic properties, are cost-effective, are easily assessable and environmentally friendly. The unique properties of nano-sized nanoparticles have been reported can penetrate cancer cells effectively. In this study, the anticancer activity was evaluated at both cellular and molecular levels to gain insight into its mechanisms.
To evaluate the proliferative activity of the human hepatocellular carcinoma cells HepG2 in response to the Catharanthus roseus-silver nanoparticles (C. roseus-AgNPs), in comparison to the normal liver cells THLE-3 cells.
To evaluate the proliferative activity, the hepatocellular carcinoma cells HepG2 and normal human liver cells THLE3 were treated with standardised Catharanthus roseus-silver nanoparticles (C. roseusAgNPs) in a double dilution manner and analysed using MTT assay. To elucidate the gene expression study, the RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, DEGs analysis, GO analysis, and pathway analysis.
The proliferative activity revealed selective effects, indicating that the Catharanthus roseus-silver nanoparticles were cytotoxic on hepatocellular carcinoma cells HepG2 cells but not on the normal liver cells THLE3 cells. The transcriptome analysis has resulted in the regulation of 296 protein-coding genes, of which 182 genes were upregulated while 114 genes were downregulated. The most intriguing finding is the expression of tumour suppressor gene GADD45A, responsible for the regulation of DNA repair, cell cycle control and genotoxic stress. The expression of this gene is regulated by p53. The upregulated GADD45A was supported by the downregulated MDM2, which is the negative regulator for p53. Our findings revealed the activation of several signalling pathways including the mitogen-activated protein kinase signalling pathway, TNF signalling pathway and TGF-β signalling pathway. These pathways are the main regulator in fundamental intracellular activities such as apoptosis, cell cycle and cellular growth. The upregulation of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25 and TSG101 indicated that the C. roseus-AgNPs were taken up by HepG2 cells via both clathrin-dependent and clathrin-independent.
The selective proliferative activity between cancerous and normal liver cells indicates a promising potential of Catharanthus roseus-silver nanoparticles (C. roseus-AgNPs) as an effective anticancer agent. The understanding of the molecular signalling pathways induced by the genes associated with oxidative stress, apoptosis and cell cycle arrest provides the novelty towards the development and establishment of C. roseus-AgNPs as an anticancer drug for hepatocellular carcinoma. Moreover, we propose that the uptake was via both clathrin-dependent and clathrin-independent endocytosis. These findings would explain the cytotoxicity mechanisms of the C. roseus-AgNPs at cellular and molecular level towards hepatocellular carcinoma cells HepG2.
While the endocytic pathways emphasise the action of the selectively permeable plasma membrane on the nanomaterials, cytotoxicity of silver nanoparticles (AgNPs) generally involves the cells’ downstream activity, including reactive oxygen species (ROS)- dependent pathway, cell cycle arrest and genotoxicity. Moreover, the small-sized AgNPs can easily penetrate the cells and bind to macromolecules including proteins and DNA, either directly or indirectly although the exact mechanism for this interaction has not been clarified. The physicochemical characteristics that make AgNPs so useful can be the main reason they might be dangerous to cells, and at a higher level to human health. Therefore, to avoid these problems, the AgNPs must be engineered from either biocompatible, nontoxic, biodegradable material or materials have with minimal toxic effects.