Calcium-sensing receptor antagonist NPS-2143 suppresses proliferation and invasion of gastric cancer cells.

NPS-2143 is a calcium-sensing receptor (CaSR) antagonist that has been demonstrated to possess anticancer activity. To date, the effects of NPS-2143 on gastric cancer (GC) cell growth, motility, and apoptosis have not been investigated. In the present study, we firstly investigated the expression of CaSR in GC tissues using immunohistochemistry and western blotting. Then Cell Counting Kit-8 and colony formation assays were conducted to explore the effect of the NPS-2143 on the proliferation of GC cell line AGS. Transwell invasion and migration assays were performed to test the effect of NPS-2143 on AGS cell motility. We determined the percentage of apoptotic cells by flow cytometry and explored the changes of apoptosis-related protein by western blotting. Furthermore, we constructed a CaSR knockdown AGS cell line to determine whether NPS-2143 acted via inhibition of CaSR. We found that the protein expression level of CaSR was higher in GC tissues compared with the paired adjacent normal tissues. In addition, NPS-2143 treatment caused an inhibitory effect on the proliferation, invasion, and migration of AGS cells and a promoting effect on AGS apoptosis. The expression of Bcl-2 was decreased while the levels of Bax and active caspase 3 were enhanced in AGS cells after NPS-2143 treatment. Mechanistically, NPS-2143 lead to a significant decrease in the expression of phosphorylated (p)-AKT, phosphorylated mechanistic target of rapamycin (p-mTOR), p70, and cyclin D1. Knockdown of CaSR also suppressed cell proliferation, invasion, and migration and promoted cell apoptosis. No significant difference was observed between CaSR-silenced AGS cells with and without NPS-2143 treatment. These results confirmed that NPS-2143 has an inhibitory influence on AGS cell growth via inhibiting CaSR, which then suppresses the PI3K/Akt signaling pathway.

Schisandrin A reverses doxorubicin-resistant human breast cancer cell line by the inhibition of P65 and Stat3 phosphorylation.

BACKGROUND: Multidrug resistance (MDR) in breast cancer therapy occurs frequently. Thus, anti-MDR agents from natural products or synthetic compounds were tested extensively. We have also explored the reverse effect and mechanism of Schisandrin A (Sch A), a natural product, on MCF-7 breast cancer doxorubicin (DOX)-resistant subline MCF-7/DOX. METHODS: MTT assay was performed to measure the viability of MCF-7 cells to assess the reverse effect of Sch A. Western blot analysis was used to study the protein levels. Laser scanning confocal microscopy was performed to detect the intercellular DOX and Rhodamine 123 accumulation. The qRT-PCR was used to analysis the target gene expression. Dual-luciferase reporter assay was performed to test the transcriptional activity of P-glycoprotein (P-gp). RESULTS: Sch A, at the concentration of 20 µM, showed selective reverse effect (better than the positive control, verapamil at 5 µM) on MCF-7/DOX cell line but not on BEL-7402/DOX, Hep G2/DOX, and K-562/DOX cells. In addition, Sch A enhanced DOX-induced cleavage of Caspase-9 and PARP levels by increasing intracellular DOX accumulation and inhibiting P-gp function. Furthermore, Sch A selectively suppressed P-gp at gene and protein levels in MCF-7/DOX cells which express high level of MDR1 but not MRP1, MRP3, or BCRP. Besides, Sch A showed inhibitory effect on P-gp transcriptional activity. Sch A significantly reduced p-IκB-α (Ser32) and p-Stat3 (Tyr705) levels which mediate P-gp expression. In addition, Stat3 knockdown enhanced the reverse effect of siP65. The combined effect of siStat3 and siP65 was better than Sch A single treatment in MCF-7/DOX cells. CONCLUSION: Sch A specifically reverses P-gp-mediated DOX resistance in MCF-7/DOX cells by blocking P-gp, NF-κB, and Stat3 signaling. Inhibition of P65 and Stat3 shows potent anti-MDR effect on MCF-7/DOX cells.