# Evaluating the Antitumor Effect of Root Extract of Salvadora persica in Hepatocarcinoma Through the Induction of Apoptosis via an Intrinsic Pathway: An In Vitro Study

## TL;DR

This study shows that an extract from the Salvadora persica plant can kill liver cancer cells by inducing apoptosis, while being safe for normal liver cells.

## Contribution

The study demonstrates the antitumor effect of Salvadora persica root extract on hepatocarcinoma via intrinsic apoptosis induction.

## Key findings

- Salvadora persica extract showed dose- and time-dependent cytotoxicity in HepG2 liver cancer cells.
- The extract induced apoptosis in HepG2 cells through the intrinsic pathway, increasing proapoptotic proteins and mRNA markers.
- The extract was non-toxic to normal THLE-2 hepatic cells, indicating selective antitumor activity.

## Abstract

Background: Hepatocarcinoma (HCC) is the primary form of liver cancer, which is a highly prevalent cancer associated with an increased rate of mortality worldwide. The plant Salvadora persica (miswak) has been reported for its cytotoxic effect in various cancer cell lines. The purpose of the current research study was to assess the cytotoxicity and apoptosis-inducing capability of Salvadora persica aqueous extract from the root part (miswak sticks) on human HCC HepG2 cells and to evaluate its safety in human normal hepatic THLE-2 cells in a dose-dependent manner.

Materials and methods: The preparation of aqueous extract from the root part of Salvadora persica was carried out using a maceration extraction process. The effect on cytotoxicity and cellular proliferation in HepG2 cells and THLE-2 cells at 10, 50, 100, 150, and 200 µg/mL concentrations after 24, 48, and 72 hours was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In further experiments, IC50 (24 hours) was 65.8 µg/mL, and increasing concentrations were employed: 115 µg/mL (twofold) and 165 µg/mL (threefold). The apoptosis at the quantitative level was evaluated using a flow cytometry assay by the Annexin V-FITC/PI apoptosis detection kit in HepG2 cells and THLE-2 cells. The expression levels of the proapoptotic proteins Annexin V and p53 in the HepG2 cell line were assessed using an immunocytochemistry assay. The mRNA levels of apoptosis markers, caspase 9, caspase 3, cytochrome c, BAX, and p53, and the antiapoptotic marker, Bcl-2, in the HepG2 cell line were determined using the RT-qPCR (real-time, quantitative-polymerase chain reaction) assay.

Results: Salvadora persica aqueous extract demonstrated time-dependent and dose-dependent growth inhibitory effect and cytotoxicity in the HepG2 cell line in the MTT test. The IC50 values were 65.8 µg/mL at 24 hours, 51.4 µg/mL at 48 hours, and 30.3 µg/mL at 72 hours. In contrast, a non-toxic effect was observed in the THLE-2 cell line. The flow cytometry results indicated a significantly enhanced apoptotic effect, characterized by increased early apoptosis, late apoptosis, and cell death in HepG2 cells. In contrast, the normal THLE-2 cell line showed insignificant apoptotic cell numbers. In the immunostaining assay, a pronounced elevation in the expression levels of proapoptotic proteins, including p53 and Annexin V, was observed. The RT-qPCR assay revealed an increase in the fold change of caspase 9, caspase 3, cytochrome c, BAX, and p53 mRNA levels, along with a decrease in Bcl-2 mRNA levels. Salvadora persica aqueous extract induced apoptosis via the intrinsic pathway in the HepG2 cell line. Salvadora persica aqueous extract exhibited the most significant apoptotic effect in HepG2 cells at high concentrations.

Conclusions: In the present study, Salvadora persica aqueous extract demonstrated dose-dependent antitumor potential in the HCC cell line HepG2 through the induction of intrinsic apoptosis and showed non-toxic activity in the normal hepatic THLE-2 cell line, emphasizing its therapeutic efficacy in HCC treatment.