Shikonin Stimulates Mitochondria-Mediated Apoptosis by Enhancing Intracellular Reactive Oxygen Species Production and
Stuti Biswal1, Munmun Panda1, Bijesh Kumar Biswal1
1Department of Life Science, Cancer Drug Resistance Laboratory, NIT Rourkela, Rourkela, Odisha, India.
Abstract:
Phytotherapy has rendered a new insight towards the treatment of various cancers, including oral cancer with fewer side effects, over the traditional chemotherapeutic drugs to overcome chemoresistance. Shikonin (Shk) is a natural biologically active alkaloid found in the Lithospermum erythrorhizon plant's root. It has potent cytotoxic activities against various cancers. Our study revealed the release time and anticancer potential of Shk on the SCC9 and H357 oral cancer cell lines. We investigated the antiproliferative, antimigratory, cell cycle arresting and apoptosis promoting activity of Shk in oral cancer cells by performing MTT and morphological assay, colony, and tumor sphere formation assay, AO/EtBr and DAPI staining, Annexin V-FITC/PI staining, assay for reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) measurement, comet assay, qRT-PCR, and western blot analysis. We also checked the interaction of DNA and Shk by docking and CD spectroscopy and EtBr displacement assay. As a result, we found that Shk reduced the viability, proliferation, and tumorigenicity of SCC9 and H357 cells in a time and concentration-dependent manner. We obtained half-maximal inhibitory concentration (IC50) at 0.5 µM for SCC9 and 1.25 µM for H357. It promotes apoptosis via overexpressing proapoptotic Bax and caspase 3 via enhancing ROS that leads to MMP depletion and DNA damage and arrests cells at the G2/M & G2/S phase. The antimigratory activity of Shk was performed by analyzing the expression of markers of epithelial-mesenchymal transition like E-cadherin, ZO-1, N-cadherin, and vimentin. These overall results recommended that Shk shows potent anticancer activity against oral cancer cell lines in both in vitro and ex vivo conditions. So, it could be an excellent agent for the treatment of oral cancer.
Insights
Shikonin, a natural compound from Lithospermum erythrorhizon, effectively reduces oral cancer cell viability and promotes apoptosis. This phytotherapy agent shows promise for treating oral cancer with fewer side effects.
Area of Science:
- Phytotherapy
- Natural Products Chemistry
- Cancer Biology
Background:
- Phytotherapy offers a promising alternative to traditional chemotherapy for oral cancer treatment, potentially overcoming chemoresistance with fewer side effects.
- Shikonin (Shk), a natural alkaloid from Lithospermum erythrorhizon root, exhibits potent cytotoxic effects against various cancer types.
- Oral cancer remains a significant health concern, necessitating the exploration of novel therapeutic agents.
Purpose of the Study:
- To investigate the anticancer potential of Shikonin (Shk) on SCC9 and H357 oral cancer cell lines.
- To determine the antiproliferative, antimigratory, cell cycle arresting, and apoptosis-promoting activities of Shk.
- To elucidate the mechanisms underlying Shk's anticancer effects, including DNA interaction and cellular pathway modulation.
Main Methods:
- Cell viability and proliferation assessed using MTT and colony formation assays.
- Apoptosis induction evaluated through AO/EtBr, DAPI, and Annexin V-FITC/PI staining.
- Cell cycle arrest, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyzed via various assays and molecular techniques (qRT-PCR, Western blot).
- DNA-Shk interaction studied using docking, CD spectroscopy, and EtBr displacement assays.
Main Results:
- Shikonin demonstrated time- and concentration-dependent reduction in oral cancer cell viability, proliferation, and tumorigenicity.
- Half-maximal inhibitory concentrations (IC50) were determined as 0.5 µM for SCC9 and 1.25 µM for H357 cells.
- Shk induced apoptosis by upregulating Bax and caspase 3, enhancing ROS production, leading to MMP depletion and DNA damage, and causing G2/M & G2/S phase arrest.
- Antimigratory effects were observed, with modulation of epithelial-mesenchymal transition markers.
Conclusions:
- Shikonin exhibits significant in vitro and ex vivo anticancer activity against oral cancer cell lines.
- The compound effectively inhibits proliferation, induces apoptosis, and reduces migration in oral cancer cells.
- Shikonin presents a potential therapeutic candidate for oral cancer treatment due to its potent anticancer properties and favorable side-effect profile.
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