Evaluation of the cardioprotective effects of all-trans retinoic acid and dexpanthenol on doxorubicin cardiotoxicity

Doxorubicin (DOX), an anthracycline derivative, is a drug used in the treatment of hematologic malignancies and solid neoplasms. Due to its long-term use, its well-defined side effects limit its application. One of these side effects is toxic myocarditis, which occurs through oxidative stress, mitochondrial dysfunction, apoptosis, pyroptosis, necroptosis, ferroptosis, and autophagy pathways. All-trans retinoic acid (ATRA) is known to regulate the antioxidant stress response, improve mitochondrial functions, and prevent cardiomyocyte apoptosis. On the other hand, dexpanthenol (DEX) is known for its regulatory effects on oxidative stress, endoplasmic reticulum stress, and inflammation. The fact that ATRA and DEX share multiple common pathways with DOX's cardiotoxic mechanisms is noteworthy. Therefore, we aimed to demonstrate the effects of ATRA and DEX individually and in combination in a DOX-induced toxic myocarditis model in H9c2(2-1) cells. For this purpose, seven different groups were designed using H9c2(2-1) cells: control, DOX, DEX, ATRA, DOX+DEX, DOX+ATRA, and DOX+DEX+ATRA. To determine the optimal drug doses in these groups, an XTT cell viability assay was employed. The IC50 (half-maximal inhibitory concentration) values for DOX at 24, 48, and 72 hours were calculated separately. For DEX and ATRA, the doses that most effectively increased cell viability were selected. Subsequently, the wound healing (Scratch Assay) experiment was applied to all groups at the determined doses to evaluate cell-cell interactions and cell migration. Caspase-3, Bax, Bcl-2, TGF-β, TIMP-1, and TIMP-2 primary antibodies were used for immunohistochemical evaluation to assess apoptosis pathways. Real-Time Polymerase Chain Reaction (RT-PCR) and TUNEL test was conducted to support the results. According to the experimental results, the DOX IC50 dose was determined to be 14.71 µM at 24 hours, 1.18 µM at 48 hours, and 0.27 µM at 72 hours. Experiments following the XTT assay were conducted using the 24-hour IC50 dose, while the selected doses for DEX and ATRA were 31.25 µM and 125 nM, respectively. In the cell viability experiments, the DEX group increased cell viability up to 167% within the 7.8 µM to 250 µM dose range, while the ATRA group increased cell viability up to 170% within the 3.9 nM to 1 µM dose range. However, ATRA exhibited toxic effects at concentrations above 2 µM. Upon combining DOX IC50 with increasing DEX doses, cell viability significantly increased at 15.6 µM, 31.25 µM, and 125 µM DEX doses compared to the DOX IC50 group alone. When DOX IC50 was combined with increasing doses of ATRA, cell viability remained significantly higher than the DOX IC50 group at ATRA doses ranging from 3.9 nM to 125 nM. No significant difference was observed between the DOX IC50+DEX and DOX IC50+ATRA groups. When DOX IC50 was combined with increasing doses of both ATRA and DEX, combinations containing low and moderate doses of DEX significantly increased cell viability. However, while these groups demonstrated higher cell viability than DOX+ATRA groups, they showed similar results to DOX+DEX groups, and no synergistic effect was observed. In the wound healing and migration experiments, none of the DOX IC50-treated groups reached the level of the control group, nor did the DEX and ATRA groups show significant results compared to the control group. Our immunohistochemistry results showed that increased TGF-β expression in DOX-treated groups was not reversed by DEX or ATRA, while isolated DEX application significantly reduced TGF-β expression compared to the control. Regarding Caspase-3 levels, both DEX and ATRA-treated groups showed significant reductions compared to the isolated DOX group; however, triple combination therapy showed no superiority over dual applications. Similarly, Bax expression was significantly suppressed by both DEX and ATRA in DOX-treated groups. DOX+DEX treatment resulted in Bax expression levels close to the control group, which was superior to the triple combination, with no evidence of a synergistic effect. Bcl-2 and TIMP-1 expression levels in DOX+DEX-treated groups were significantly lower than in all DOX-treated groups, while the DOX+ATRA group exhibited the highest TIMP-2 expression, even exceeding the triple combination group. In RT-PCR results, no significant differences were observed between groups, although the DOX+DEX+ATRA group notably showed significant reductions in Bcl-2, TIMP-1, and TIMP-2 expression levels. Although the TUNEL assay results generally supported the previous findings, a significant decrease in the apoptotic index was observed in the triple combination group compared to the DOX+DEX and DOX+ATRA groups. Our results demonstrate that dexpanthenol and all-trans retinoic acid exhibit protective effects against doxorubicin cardiotoxicity; however, sufficient evidence for synergistic effects was not obtained. During the experiments, some doses enhanced each other's effects, while others showed toxic effects. Therefore, additional studies are required to explore the cardioprotective and toxic doses of DEX and ATRA in various combinations. Keywords: H9c2(2-1), doxorubicin, dexpanthenol, all-trans retinoic acid, cardiotoxicity