The Novel Herbal Cocktail AGA Alleviates Oral Cancer through Inducing Apoptosis, Inhibited Migration and Promotion of Cell Cycle Arrest at SubG1 Phase

Abstract

Traditional Chinese medicines Antler's extract (A) and Ganoderma lucidum (G) and Antrodia Camphorata (A) have been known to individually contain a plethora of bioactive factors including triterpenoids, polysaccharides etc., exerting various curative impacts such as anti-inflammatory, anti-oxidative, anti-atherosclerotic and anti-viral activities. However, their combinatorial therapeutic efficacy for oral cancer has not been investigated. Hence, we synthesized a robust cocktail called AGA and investigated its anti-oral cancer potential in vitro and in vivo. An MTT assay revealed the IC₅₀ of AGA to be about 15 mg at 72 h. Therefore, 10 mg and 20 mg doses were selected to study the effect of AGA. The AGA significantly inhibited proliferation of oral cancer cells (HSC3, SAS, and OECM-1) in a dose- and time-dependent manner. AGA retarded cell cycle regulators (CDK4, CDK6, cyclin A, B1, D1 and E2) and apoptosis inhibitory protein Bcl-2, but enhanced pro-apoptotic protein Bax and a higher percentage of cells in Sub-G1 phase. Mechanistically, AGA suppressed all EMT markers; consequently, it decreased the migration ability of cancer cells. AGA significantly reduced xenograft tumor growth in nude mice with no adverse events in liver and renal toxicity. Conclusively, AGA strongly inhibited oral cancer through inducing apoptosis and inhibiting the migration and promotion of cell cycle arrest at subG1 phase, which may be mediated primarily via cocktail-contained triterpenoids and polysaccharides.

Keywords: Antler’s extract; Antrodia Camphorata; Ganoderma lucidum; anti-oxidation; cell cycle; oral cancer.

Figure 1

The cytotoxic effect of Antler’s extract, Ganoderma lucidum and Antrodia Camphorata (AGA) extract (0–20 mg/mL) on human oral squamous cell carcinoma cells (HSC3), human tongue squamous carcinoma (SAS) and oral epidermoid carcinoma cell, Meng-1 (OECM-1). (A) AGA dose optimization for cell viability revealing IC₅₀ value of about 15 mg. Thereafter, the dose of AGA was selected as 10 and 20 mg/mL and their effect on the (B) Colony-forming ability of oral cancer cells was determined for 72 h, which was later relatively quantified. (C) Relative expressions of Ki67 and proliferating Cell Nuclear Antigen (PCNA) were detected by Western blot (D). Data are represented in triplicates as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to control.

Figure 2

Impact of AGA on expression cell cycle-related factors in ) oral cancer cells (HSC3, SAS and OECM-1). (A) qPCR-dependent mRNA levels of CDK4 and CDK6. (B) Cyclins A, B1, D1 and E2 were determined by Western blot. (C) mRNA levels of apoptosis biomarkers, Bcl-2 and Bax. Data are represented in triplicates as mean ± SD. * p < 0.05 and ** p < 0.01 compared to control.

Figure 3

Effects of AGA on the phases of cell cycle distribution and apoptosis in oral cancer cells (HSC3, SAS and OECM-1). (A) Representative flow cytometric histograms cell cycle phases (Sub G1, G0–G1, S and G2-M) and their relative percentage population (B). Histogram revealing the live, early, late and necrotic phase of apoptosis of oral cancer cells after their treatment with AGA extract (C) and their relatively quantified population (D). Data are represented in triplicates as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to control.

Figure 4

Efficacy of AGA extract on cell migration in oral cancer cells. Cell motility of HSC3, SAS and OECM-1 upon AGA treatment was determined by wound-healing assay at 0, 10 and 20 mg/mL for 48 h and 72 h. Representative photomicrographs (magnification, 100 µm) of wound healing in (A) HSC3, (B) SAS and (C) OECM-1 cells with their relatively quantified wound area (%). Transwell assay-dependent analysis of invasive ability of (D) HSC3, (E) SAS and (F) OECM-1 cells (magnification, ×100), and their relative qualification. Data are represented in triplicates as mean ± SD. ** p < 0.01 and *** p < 0.001 compared to control (0 mg).

Figure 5

Influence of AGA on epithelial mesenchymal transition (EMT) markers. (A) Schematic representation of EMT showing increased motility of oral cancer cells and enabling them to develop into an invasive phenotype, (B) N-cadherin, β-catenin and vimentin, and its regulatory factors, i.e., (C) EpCAM and (D) survivin-1 in HSC3, SAS and OECM-1 oral cancer cells. * p < 0.05 and ** p < 0.01 compared to control (0 mg).

Figure 6

Impact of AGA on tumorigenesis and organ toxicity. (A) Representative photomicrographs of tumors and its quantified volume derived from mice with oral cancer. (B) AGA-associated hepatic and renal toxicity was assessed through hematoxylin and eosin-stained sections of liver and kidney. Magnification, 20X (scale: 100µm). (C) Blood serum levels of glutamate oxaloacetate (GOT) and glutamate pyruvate transaminase (GPT) represents liver function, whereas blood urea nitrogen (BUN) and creatinine indicate renal function. Data are represented in triplicates as mean ± SD, * <0.05 (Oral cancer (Control) = 5, AGA-Oral cancer = 5).

Figure 7

Schematic of possible mechanistic insight of therapeutic action by AGA in oral cancer.