Our study demonstrated that thymol has an anticancer effect on the HepG2 cell line, a cell model of hepatocellular cancer [21]. We found that thymol decreased oxidative stress in these cells by decreasing TOS levels and increasing TAS levels. Furthermore, we observed significant increases in apoptosis as well as alterations in expression of certain genes related to apoptosis after treatment with thymol. Finally, our results showed considerable genotoxicity effects caused using this compound on HepG2 cells.
The use of essential oils with a certain concentration of thymol has been widely studied, and the results have shown that these thymol-containing essential oils can be used for their cytotoxic action against HepG2 cells [22, 23]. In particular, it was found that the thymol-rich essential oil extracted from leaves of L. gracilis had both in vitro and in vivo anticancer action, with tumor growth inhibition rates ranging from 38.5–41.9% [24]. Moreover, another study suggested that combining thymol-artemicin could inhibit the gene expression of CDK2 and P38 which may make this compound an effective new anticancer agent as well [25]. Thus overall, there is strong evidence to suggest potential therapeutic applications using essential oils containing a certain concentration of thymol for cancer treatment purposes. Thymol is widely regarded as the most important component of these extracts, with many references being made to its effectiveness. Despite this, it is difficult to definitively say which components are responsible for the efficacy of these extracts due to their complex composition and multiple ingredients.
As therefore, we performed our research using pure thymol, and the results clearly demonstrated that thymol causes cytotoxicity in HepG2 cells even at a low dose of 11 micromoles. This is in contrast to the semi-lethal dose range of 90-4000 µM reported in previous studies [14, 19, 26]. Our research showed that the 11 µM concentration destroyed half of the HepG2 cells, demonstrating its potent anticarcinogenic activity. It is possible that this discrepancy between our results and those from other studies could be attributed to differences in metabolic processes within individual cell lines or measurement techniques employed for assessing cytotoxicity.
The results of this study indicate that the calculated IC50 values for HepG2 cells are lower than those of healthy HUVEC cells. This effect needs to be further investigated with other cell lines in order to confirm its validity. Elshafie et al.'s research showed similar findings, as their reported IC50 values for HepG2 were lower than those for healthy kidney cells [27]. These results suggest that there may be a difference between cancerous and non-cancerous cell lines when it comes to drug sensitivity, which could prove useful in developing targeted therapies.
Thymol is a compound that has been studied for its potential medicinal properties, and the evidence suggests that it displays pro-oxidant activities at high concentrations and antioxidant activities at low quantities [28]. In our study, thymol decreased the oxidant status, increased the antioxidant status, and thus lowered oxidative stress in HepG2 cell line. This result was further corroborated by other studies which showed that thymol lowers oxidative stress while strengthening antioxidant defense systems as well as suppressing cytokines responsible for inflammation [7, 23, 29]. These results suggest that thymol does not seem to be able to destroy cancer cells through an oxidative mechanism alone.
Thymol has been widely studied for its anti-tumor effects, with numerous studies demonstrating its ability to decrease cell proliferation and trigger apoptosis in a variety of cancers [14, 30]. Our own research confirms these findings. We have shown that thymol significantly induced apoptosis in HepG2 cells compared to the control group (1%) and H2O2 group (52%) as seen in Fig. 3. These results demonstrate the potential of thymol as an effective treatment option for various types of cancer.
In this study, we investigated the effects of thymol on bax and bcl-2 proteins in HepG2 cells. As is well known, these two counteracting members of the bcl-2 family are key factors in apoptotic cell death [31]. The researchers have shown that it is not only the levels of individual proteins but also their ratio (bax/bcl-2) which determines susceptibility to apoptosis [32]. bax is a pro-apoptotic protein that responds to apoptotic signals by activating the intrinsic pathway of apoptosis. It works in tandem with bcl-2, an anti-apoptotic protein, which promotes cellular survival and inhibits the functions of other pro-apoptotic proteins. Together, these two proteins work to regulate cell death and survival pathways within cells in order to maintain homeostasis.
In our work, gene expressions of bax and bcl-2 did not alter following the thymol treatment. However, although there was no statistically significant difference, the bax/bc-l2 ratio tended to increase after thymol treatment. All these results suggest that there is a predisposition to apoptosis in HepG2 cells with the effect of thymol in our study. Our current apoptosis results also support this proposition. Indeed, our results demonstrate that thymol treatment significantly increases the rate of apoptosis in comparison to untreated cells, indicating its potential as a highly effective agent for inducing cell death in this cell line. The results of our study support the findings of other studies which show that an increase in the bax/bcl-2 ratio is a reliable indicator for apoptosis [33, 34]. This has been demonstrated by previous research and is further confirmed through our own analysis. The results of this study suggest that thymol may be an effective agent for treating hepatocellular cancer and further research is needed to confirm its efficacy.
Caspase 3 (casp3) is an effector caspase which proteolytically degrades a number of intracellular proteins and carries out apoptosis. Our study found that there was no significant change in the gene expression of casp3 after thymol treatment. This indicates that the apoptotic effect of thymol on HepG2 cells does not seem to be mediated by casp3. But our findings suggest this is not the case with regards to thymol's effects on HepG2 cells. Therefore, it can be concluded that other pathways may be involved in mediating these effects instead.
Unrepaired or incorrectly repaired DNA damage can have serious consequences for a cell, as it can trigger apoptosis, senescence and even carcinogenesis [35, 36]. There are two main pathways that are activated when DNA is damaged: extrinsic death receptors and intrinsic mitochondrial apoptosis [37]. These pathways play an important role in the body's ability to identify damaged cells and initiate the necessary repair processes. Failure to do so could result in potentially dangerous cellular changes. The comet assay is a powerful tool for measuring DNA damage and was used in this experiment to investigate whether thymol has a cancer cell-destroying effect due to DNA damage. The results of the comet assay showed that thymol treatment did indeed induce significant amounts of DNA damage in the cells, suggesting that it may be an effective agent for destroying cancer cells through inducing genotoxicity.
Our findings are consistent with those of another study, which found that C. aromatic leaves ethanolic extract is highly enriched with the presence of phytochemicals like thymol and caused statistically significant DNA damage in HepG2 cells in a dose-dependent manner [38]. These results suggest that thymol may have genotoxic effects against HepG2 cell.