Evaluation of the cardioprotective effects of carvacrol and vitamin C against the cardiotoxic effect of doxorubicin

Doxorubicin (DOX) is an anthracycline derivative chemotherapeutic agent used for many years in the treatment of haematological malignancies and solid tumours. Cardiotoxic side effects limit its clinical use. Antioxidant effects of vitamin C and carvacrol have been shown in experimental studies. In our study, we aimed to investigate the individual and synergistic effects of vitamin C and carvacrol by modelling doxorubicin-induced cardiotoxicity, in which oxidative stress plays a role in the pathogenesis, on H9C2 cell line. The cell lines were divided into seven groups in order to evaluate the cardioprotective effects of carvacrol and vitamin C on both control and doxorubicin-exposed cells. The groups were as follows: control, carvacrol, vitamin C, DOX, DOX+carvacrol, DOX+vitamin C, DOX+carvacrol+vitamin C. Doxorubicin doses were determined as 1.25-2.5-5-10-20-40-50-50-60-75 µM. The viability level of each subgroup was measured separately at 24, 48 and 72 hours. Thus, the IC50 doses at which 50% of the cells remained viable were calculated as 14.71 µM at 24 h, 1.186 µM at 48 h and 0.272 µM at 72 h. Increasing DOX dose and time had a negative effect on cell viability (p<0.05). Cell viability was compared in the groups given varying doses of carvacrol and vitamin C compared to the control group. It was found that cell viability increased statistically at 24.hour in the vitamin C group and at both 24.and 72.hour in the carvacrol group compared to the control group (p<0,05). Cells treated with various doses of carvacrol and vitamin C together with DOX at IC50 dose were compared with the group receiving only DOX at IC50 dose. The percentage of viability of the DOX+ vitamin C group at 24th hour and the percentage of viability of the DOX+ carvacrol group at 48th and 72nd hours were statistically significantly higher than the DOX alone group (p<0.05). In order to evaluate the interaction between carvacrol and vitamin C, adding 20 mM and 80 mM carvacrol to doxorubicin, 100 µM vitamin C combination at IC50 dose and adding 20 mM, 40 mM and 80 mM carvacrol to doxorubicin, 500 µM vitamin C combination at IC50 dose decreased cell viability statistically significantly (p<0.05). In the wound healing test performed on the same groups, the wound surface area of the carvacrol group was significantly smaller than the control group (p=0,003). In the 24th hour analysis, there was a statistically significant difference between control and carvacrol, control and vitamin C, DOX and DOX+carvacrol+vitamin C groups (p<0.05). It was found that the addition of 40 mM carvacrol, 200 µM vitamin C and carvacrol + vitamin C to the doxorubicin treated group preserved the shape of cardiomyocytes with a decrease in BAX expression and vitamin C decreased apoptotic activity by decreasing caspase-3 expression. Vitamin C and carvacrol were found to decrease gene expressions of BAX, MMP-2, TGF-beta factors using real-time PCR method. Our results support that the separate use of carvacrol and vitamin C on doxorubicin-induced cardiotoxicity may be beneficial by showing cardioprotective effect. Key words: Doxorubicin, Cardiotoxicity, Carvacrol, Vitamin C, Cell culture