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,Praveen Kumar Shetty,Mahendra Gowdru Sreenivas,Madhyastha Radha,Shilpa Sharathraj Shetty,Revanasiddappa Bistuvalli Chandrashekharappa,Suchetha Kumari Nalilu,Satheesh Kumar Bhandary Bavabeedu,Madhyastha Harishkumar,Remya Varadarajan

doi:10.3390/molecules26247430

Sharmila Kameyanda Poonacha, Praveen Kumar Shetty + Show 8 more

Open Access

https://doi.org/10.3390/molecules26247430

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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.

Highlights

A long-established history of alternative medicine from plant origin has continuously been practiced in southeastern countries since ancient times
We investigated the molecular interaction of major phytochemicals in Oroxylum indicum (OI) stem bark extract identified in Liquid Chromatography–Mass Spectroscopy (LC–MS) [3] using molecular docking studies to evaluate their binding affinities with targeted apoptosis protein and poly-ADP-ribose polymerase (PARP)
In silico studies carried out using the compound Oroxylin-A-7-O-beta-D-glucuronide identified in the Oroxylum indicum bark extract docked effectively against PARP (Figure 1A,B) and caspase-3 proteins (Figure 1C,D), suggesting the possible induction of apoptosis in HSC-3 cancer cells

Summary

Introduction

A long-established history of alternative medicine from plant origin has continuously been practiced in southeastern countries since ancient times. Commonly known as the trumpet tree (L.) Benth ex Kurz., is a medicinal plant that is found in India, Thailand, Vietnam, Malaysia, Indonesia, the Philippines, China, and Japan. It may reach a height of 8–15 m and has a thick bark [1]. As far as the anticancer property of OI bark extract is concerned, not much research has been done and, as such, the mechanism of its cytotoxic activity is yet to be established. OroxylinA-7-O-β-D-glucuronide is a flavonoid derivative and has a principal effect as radical scavenging properties with potent anticancer aspects. OG was observed in mitochondria, indicating that OG may have an additional target in hepatic cancer cells

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