Investigating the Anticancer Potential of Shikonin by Targeting PLK1 and Enhancing Therapeutic Efficacy Through Micellar Encapsulation in Oral Squamous Cell Carcinoma

Abstract

Natural bioactive alkaloid phytocompound shikonin is obtained from the Lithospermum erythrorhizon plant’s root and exhibits excellent pharmacological properties including anticancer activity. This study explores the mechanistic role of shikonin (Shk) in the proliferation and migration of oral squamous cell carcinoma (OSCC) cells. Shikonin suppresses the viability of SCC9 and H357 OSCC cells in a time and concentration-dependent manner. It promotes the generation of intracellular reactive oxygen species which then leads to the depletion of mitochondrial membrane potential (MMP). Further, this causes DNA damage and cell cycle arrest in the G2/M and S-G2/M phases in SCC9 and H357 cell lines respectively. Shk also induces apoptosis in OSCC cells via enhancing the expression of Bax and Caspase 3. It also suppresses colony formation and tumorigenicity in a dose-dependent manner. The molecular mechanism behind the anticancer activity of shikonin was analyzed using bioinformatics studies. It was found that out of all upregulated genes in oral cancer, polo-like kinase 1/PLK1 is the most significant one which is the key target of shikonin. The molecular docking and molecular dynamic simulation results showed that shikonin makes stable binding with PLK1 and inhibits its mode of action. It has been shown that after Shk treatment the mRNA expression level of PLK1 was decreased compared to the control. Knockdown of PLK1 reduces the proliferation and viability of OSCC cells. It also increases the apoptosis rate and DNA damage after siPLK1, similar to Shk treatment. Instead of having these excellent anticancer activities the clinical use of shikonin is still limited because of its poor bioavailability, solubility, and stability. To overcome this problem, polymeric micelles are used as a drug delivery vehicle. These micelles are smaller in size which helps micelles for easy penetration in cancer cells with increased permeability and retention effect. Here mPEG-SA micelles are used for shikonin encapsulation. The formation of blank and Shk-loaded micelles is characterized by 1HNMR, FTIR, CMC, drug-loading, and encapsulation efficiency, DLS, DSC, TEM, and drug release time. The shikonin-loaded micelles show better and prolonged toxicity compared to only shikonin treatment leading to cell death of OSCC cells. Hence it can be concluded that shikonin-loaded mPEG-SA micelles can be used as a therapeutic agent for effective delivery of shikonin in OSCC cells which can give prolonged and better anticancer effects.