Polydatin, a Glycoside of Resveratrol, Induces Apoptosis and Inhibits Metastasis Oral Squamous Cell Carcinoma Cells In Vitro

Abstract

Although various methods, such as surgery and chemotherapy, are applied to the treatment of OSCC, there are problems, such as functional and aesthetic limitations of the mouth and face, drug side effects, and lymph node metastasis. Many researchers are making efforts to develop new therapeutic agents from plant-derived substances to overcome the side effects that occur in oral cancer treatment. Polydatin is known as a natural precursor of resveratrol, and research on its efficacy is being actively conducted recently. Therefore, we investigated whether polydatin can induce apoptosis and whether it affects cell migration and invasion through the regulation of EMT-related factors in OSCC. Polydatin decreased the survival and proliferation rates of CAL27 and Ca9-22 cells, and induced the release of cytochrome c, a factor related to apoptosis, and fragmentation of procaspase-3 and PARP. Another form of cell death, autophagy, was observed in polydatin-treated cells. In addition, polydatin inhibits cell migration and invasion, and it has been shown to occur through increased expression of E-cadherin, an EMT related factor, and decreased expression of N-cadherin and Slug and Snail proteins and genes. These findings suggest that polydatin is a potential oral cancer treatment.

Keywords: apoptosis; autophagy; epithelial-mesenchymal transition; oral squamous cell carcinoma; polydatin.

Figure 1

Inhibitory effect of polydatin on OSCC cell viability and proliferation. Ca9-22 (A) and CAL27 (B) cells were treated with 0–2 mM polydatin for 24–72 h and then their viability was assessed. (C) Comparison of cell viability in human keratinocyte HaCaT and OSCC (Ca9-22 and CAL27) cells by polydatin treatment for 24 h. Ca9-22 (D) CAL27(E) cells were treated with 0–1 mM polydatin for 7 days. The Ca9-22 (D) and CAL27(E) cell colonies that formed were stained with 1% crystal violet and then photographed with a digital camera. The number of colonies that formed in each group was counted and graphed. Graphs showing the formation rate of each colony, with the control group representing a 100% formation rate. Results are expressed as mean ± SD. Data were derived from three independent experiments. (Ca9-22: * p < 0.05, ** p < 0.01, *** p < 0.001; CAL27: † p < 0.05, †† p < 0.01, ††† p < 0.001).

Figure 2

Polydatin-induced changes in nuclear morphology in OSCC cells. Ca9-22 and CAL27 cells were treated with 0, 1, and 1.5 mM polydatin. The nuclei morphology in both cell lines was observed through Hoechst staining. (A) The nuclear morphologies of Ca9-22 and CAL27 were observed and photographed under a fluorescence microscope. (B) The number of cells with a condensed nucleus is presented in a histogram. Results are expressed as mean ± SD. Data were derived from three independent experiments. (Ca9-22: ** p < 0.01, *** p < 0.001; CAL27: †† p < 0.01, ††† p < 0.001).

Figure 3

Expression of apoptosis-related proteins by polydatin in OSCC cells. After a 24-h treatment with 1 mM polydatin, the expression of (A) cytochrome c and (C) cleaved caspase-3 in Ca9-22 and CAL27 cells were confirmed through immunofluorescence staining as observed under a confocal microscope. After a 24-h treatment with 0–1 mM polydatin, the protein expression of (B) bcl-2, bax (D) caspase-3, cleaved caspase-3, and PARP was confirmed through Western blot analysis. β-actin was used as loading control.

Figure 4

Induction of autophagy by polydatin in OSCC cells. (A) MDC staining and (B) AO staining were performed to detect autophagosomes and AVOs. (C) Ca9-22 and CAL27 cells were treated with 0–1 mM polydatin, and the expression of the autophagy-related proteins Atg5 and LC3B was confirmed. β-actin was used as loading control.

Figure 5

Inhibition of cell migration and invasion by polydatin in OSCC cells. (A) Ca9-22 and CAL27 cells seeded in a six-well plate were scratched and then treated with 0, 0.25, and 0.5 mM polydatin; the cell migration distance was measured through Hoechst staining 24 h later. (B) The wound healing rates in the two cell lines were measured and graphed. Wound width was calculated and analyzed using the ImageJ software. (C) Ca9-22 and CAL27 cells were seeded in a transwell membrane and treated with 0.5 mM polydatin for 48 h; the invading cells were stained with H&E. (D) The number of invading cells was determined and graphed. Results are expressed as mean ± SD. Data were derived from three independent experiments. (Ca9-22: * p < 0.05; CAL27: † p < 0.05).

Figure 6

Polydatin-regulated expression of EMT-related proteins and genes in OSCC cells. (A) Ca9-22 and CAL27 cells were treated with 0.5 mM polydatin for 24 h, subjected to immunofluorescence assay, and imaged and analyzed by confocal microscopy. The nuclei, E-cadherin, and actin were stained with DAPI (blue), Alexa 488 (green), and rhodamine phalloidin (red), respectively. (B) Ca9-22 and CAL27 cells were treated with 0–1 mM polydatin, and their expression levels of autophagy-related proteins, namely, E-cadherin, N-cadherin, Snail, and Slug, were determined. β-actin protein was used as internal control. (C) Ca9-22 and (D) CAL27 cells were treated with 0, 0.5, and 1 mM polydatin for 24 h, and the gene expression levels of E-cadherin, N-cadherin, Snail, and Slug were determined through QPCR. Results are expressed as mean ± SD. Data were derived from three independent experiments. (Ca9-22: * p < 0.05, ** p < 0.01; CAL27: † p < 0.05, †† p < 0.01).