In vitro analyzes of synergistic anticancer effects of sorafenib and lipoic acid combination on hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC), the most common type of liver cancer, represents the third leading cause of cancer death worldwide. Its complex pathogenesis and molecular heterogeneity complicate the treatment of liver cancer cases. Therefore, the need for the development of targeted therapies is increasing. It is anticipated that the use of Alpha Lipoic Acid (ALA) together with various antineoplastic drugs will be promising for cancer control and treatment due to its ability to interact with cells at molecular resolution and its protective effects. This study aimed to determine the synergistic, anti-proliferative and anti-cancer effects of different combinations of ALA on HepG2 cell line by cell viability test (MTT) and expression levels of some genes (Tnf-α, Bax, Caspase-3 and Bcl2) and to evaluate the protein levels of the same genes by ELISA method, as well as to detect possible DNA damage by Comet test. According to the findings obtained from MTT test, the group that was administered only Sorafenib showed approximately 42% cell viability after 24 hours of application, indicating that the compound alone showed significant cytotoxic effect. Similarly, the group that was administered only 5 µM ALA showed approximately 56% cell viability, and it was observed that ALA alone reduced cell viability. As a result of the application of ALA + Sorafenib combination, 85% cell viability was detected. In the 48 hour application, cell viability was approximately 45% in the Sorafenib group, and 47% in the 5 µM ALA group. In the ALA + Sorafenib combination group, cell viability was 48%. In the comet test results, DNA damage was determined as 20.33±2.52 for 24 hours at the Sorafenib 20 µM dose and 25.33±4.16 for 48 hours. The lowest DNA damage in the data was determined to be the ALA dose with 4.67±2.08 at 24 hours. An increase in DNA damage was observed in 48 hour ALA application, and this damage was determined as 15.67±1.15. DNA damage caused by Sorafenib decreased in both 24 and 48 hour applications compared to the combination application. The effects of Sorafenib and ALA combinations for 24 and 48 hours in HepG2 liver cancer cell line were evaluated by examining the expression levels of Bax, Bcl-2, Caspase-3 and Tnf-α genes involved in apoptotic and inflammatory processes. According to the qRT-PCR data obtained, significant changes were observed in gene expressions in both periods. Again, the levels of key gene products related to apoptosis and inflammation, such as Bax, Bcl-2, Caspase-3 and TNF-α, were determined in both 24 hour and 48 hour applications, and it was found that gene expression levels and protein products were generally consistent. The findings from all these studies suggest that combination therapy produces more pronounced effects compared to single agents in most cases. While increases in the expression of pro-apoptotic genes, especially Bax and Caspase-3, reveal the effects of the applied agents on cell death mechanisms, changes in anti-apoptotic genes, such as Bcl-2, are critical to understanding how this process is regulated. Keywords: Alpha-Lipoic Acid; Hepatocellular Carcinoma; HepG2 Cell Line; Sorafenib; Cytotoxicity, Genotoxicity, BAX, BCL-2, CASPASE-3, TNF-α