P-79: Doxorubicin Induced Apoptosis in H9c2 Cells Through Intrinsic Pathway

Hosseinzadeh L *, Karimi G , Behravan J , Mosaffa F ,


Objective: Doxorubicin (DOX) has been one of the most prescribed chemotherapeutic drugs for the treatment of a variety malignancies. Unfortunatly, use of doxorubicin is associated with cardiomyocytes apoptosis. The exact mechanism of doxorubicin –induced apoptosis has not fully understood yet. Some evidences demonstrated that doxorubicin induced apoptosis of isolated cardiomyocytes proceeds through activation of Nuclear factor-kappaB. In the current study we evaluated effects of doxorubicin on some important genes involved in apoptosis and also Nf-kB activity in H9c2 cardiomyoblast cells. Materials and Methods: Cell viability was determined by MTT assay. Apoptosis was assessed one step Quantitative Real Time RT-PCR was used to evaluate the expression of Bcl-2, Bax, cIAP1, caspase-8 and caspase-9. The DNA-binding capacity of NF-κB (P65 subunit) in nuclear extracts was examined by ELISA method. Results: Doxorubicin induced cytotoxicity in H9c2 cells in a dose dependent manner. Real time RT-PCR analysis shows a significantly reduction (42.7 % of control level) of the expression level of Bcl-2 after 22 hours treatment with DOX. Moreover, induction of apoptosis was accompanied by increase in pro-apoptotic Bax level. The Bax/Bcl-2 ratio increased 2.83±0.164 – fold upon treatment with doxorubicin. In contrast to remarkable down- regulation of Bcl-2, we did not show a significant change in the cIAP1 mRNA expression. To determine which apoptotic pathway is activated by doxorubicin, we examined the mRNA expression of caspase-8 and caspase-9. Addition of DOX to H9c2 cells increased significantly mRNA expression of caspase-9 (1.227 ± 0.05- fold of control). However, treatment of H9c2 cells with DOX had no effect on the expression of caspase-8. Also, we observed that NF-κB activity in H9c2 cells was sharply increased (6.86-fold) by incubation in the presence of 3µM doxorubicin after 22 hours. Conclusion: Previous studies have shown that depending on the cell models, apoptosis from DOX can follow different pathways. Our findings demonstrated that apoptosis induced by doxorubicin occurred through intrinsic pathway rather than by extrinsic pathway. Also, DOX sharply increased NF-kB activation. It was indicated that NF- kappaB induces multiple factors to regulate apoptosis at many steps along the cell death cascade including cIAP1 and 2, caspase-8 in various cellular model system. Based on these results, it is possible that the increase in nuclear translocation of the transcription factor, NF- kB (p65 subunit), may contribute in inhibition down regulation of anti-apoptotic cIAP 1, caspase-8 and consequently, inhibition of extrinsic pathway of apoptosis in H9c2 cells.