Insight into OroxylinA-7-O-β-d-Glucuronide-Enriched Oroxylum indicum Bark Extract in Oral Cancer HSC-3 Cell Apoptotic Mechanism: Role of Mitochondrial Microenvironment
Sharmila Kameyanda Poonacha, Harishkumar Madhyastha, Radha Madhyastha, Remya Varadarajan, Suchetha Kumari Nalilu, Shilpa S. Shetty, Praveen Kumar Shetty, Revanasiddappa Bistuvalli Chandrashekharappa, Mahendra Gowdru Sreenivas, Satheesh Kumar Bhandary Bavabeedu
Abstract
Oroxylum indicum, of the Bignoniaceae family, has various ethnomedical uses such as an astringent, anti-inflammatory, anti-bronchitis, anti-helminthic and anti-microbial, including anticancer properties. The druggability of OI stem bark extract was determined by its molecular docking interactions with PARP and Caspase-3, two proteins involved in cell survival and death. Note that 50 µg/mL of Oroxylum indicum extract (OIE) showed a significant (p < 0.05%) toxicity to HSC-3 cells. MTT aided cell viability and proliferation assay demonstrated that 50 µg/mL of OIE displayed significant (p < 0.5%) reduction in cell number at 4 h of incubation time. Cell elongation and spindle formation was noticed when HSC-3 cells were treated with 50 µg/mL of OIE. OIE initiated DNA breakage and apoptosis in HSC-3 cells, as evident from DNA ladder assay and calcein/EB staining. Apoptosis potential of OIE is confirmed by flow cytometer and triple-staining (live cell/apoptosis/necrosis) assay. Caspase-3/7 fluorescence quenching (LANCE) assay demonstrated that 50 µg/mL of OIE significantly enhanced the RFU of caspases-3/7, indicating that the apoptosis potential of OIE is probably through the activation of caspases. Immuno-cytochemistry of HSC-3 cells treated with 50 µg/mL of OIE showed a significant reduction in mitochondrial bodies as well as a reduction in RFU in 60 min of incubation time. Immunoblotting studies clearly showed that treatment of HSC-3 cells with OI extract caused caspase-3 activation and PARP deactivation, resulting in apoptotic cell death. Overall, our data indicate that OIE is an effective apoptotic agent for human squamous carcinoma cells and it could be a future cancer chemotherapeutic target.