Chloroform Extract of Solanum lyratum Induced G0/G1 Arrest via p21/p16 and Induced Apoptosis via Reactive Oxygen Species, Caspases and Mitochondrial Pathways in Human Oral Cancer Cell Lines.

Abstract

Solanum lyratum (SLEC) Thunberg (Solanaceae) has been used as a traditional herbal medicine in China for centuries. Numerous studies have shown that SLEC Thunberg (Solanaceae) extract inhibited cancer cell growth in vitro. Herein, we investigated cell death-induced by EcoAc, water, chloroform, butanol extract of SLEC in human oral cancer cell lines (HSC-3, SAS, and CAL-27) in vitro. Different SLEC extract induced cytotoxic effects in human oral cancer cells were examined by contrast phase microscopy. We selected the chloroform extract of SLEC to examine the cytotoxic effects by using DAPI staining, comet assays, flow cytometric assay, Western blotting and examination of confocal laser microscopy. SLEC decreased the percentage of viable cells, induced G0/G1 arrest and apoptosis. These effects were concentration- and time-dependent manners. SLEC increased protein levels of p21, p16, CDK2, and cyclin D1 in HSC-3, SAS, and CAL-27 cells. Also, SLEC increased CDK6 in HSC-3 and CAL-27 cells, but inhibited CDK6 in SAS cells. Cyclin E in HSC-3 and SAS cells was increased by SLEC, but it was inhibited in CAL-27 cells. SLEC suppressed the anti-apoptotic proteins Bcl-2 and Bcl-xl, but increased the pro-apoptotic proteins Bax and Bad in HSC-3, SAS, and CAL-27 cells. SLEC promoted the production of reactive oxygen species (ROS) and Ca²⁺, decreased the mitochondrial membrane potential (Δψm) and stimulated NO production in HSC-3, SAS, and CAL-27 cells. Specific caspase inhibitors (caspase-8 inhibitor: Z-IETD-FMK; caspase-9 inhibitor: Z-LEHD-FMK and caspase-3 inhibitor: Z-DEVD-FMK) for caspase-8, -9, and -3 blocked SLE-activated caspase-8, -9, and -3 activities which were associated with an increase in the percentage of viable cells. Taken together, SLE induced G0/G1 arrest and apoptosis via extrinsic- and intrinsic-dependent pathways in HSC-3, SAS, and CAL-27 cells.