Background and aims: Endoplasmic reticulum (ER) stress is a growing concern for drug-induced toxicity which causes several side effects. Ritonavir, a potent HIV protease inhibitor, induces both ER and mitochondrial stress; however, the missing link between ER stress and mitochondrial damage has been unknown. In the present study, we have studied the sequential events that occur during ritonavir-induced cell cytotoxicity and elucidate the link between ER stress and mitochondrial damage.
Methods: Cytotoxicity of ritonavir was calculated on different cells; Huh-7.5, 293T, HeLa, and Hepa RG cells using the MTT assay and also by measuring total protein content. Cellular stress response was evaluated by RT-PCR for stress marker genes. Entry of drug into the mitochondrial compartment was evaluated by HPLC. Mitochondria-mediated apoptosis was analyzed by western blotting.
Results: Ritonavir treatment initially triggered ER stress during the early hours of treatment. Consequently, the BAX was activated which permeabilized the mitochondrial outer membrane. Simultaneously, upon entry of the drug into the mitochondrial compartment, change in mitochondrial membrane potential was observed which led to the release of cytochrome c in the cytoplasm. Release of cytochrome c activated mitochondria-mediated apoptosis by the activation of caspase-9/7 and parp-1.
Conclusion: The cytotoxic effects of ritonavir involved the interplay of ER stress and mitochondria-mediated apoptosis. This unusual mechanism of drug-induced toxicity expands our knowledge in understanding side effects caused by ritonavir.
Keywords: Apoptosis; BIP; ER stress; HIV; Mitochondrial dysfunctions; Ritonavir.