Sodium bicarbonate cotransporter NBCe1 affects the growth and motility of prostate cancer cell lines LNCaP and PC3
Authors: Lee Soojung , Li Jun Ming , Choi Inyeong 
Published: June 2021

Prostate cancer is the most common cancer in men. Prostate cancer develops mainly in older men: 6 cases in 10 are diagnosed in men aged 65 or older while it is rare before age 40, according to the report by the American Cancer Society. The average age at the time of diagnosis is ~66. Similar to other cancers, prostate cancer is developed from genetic and epigenetic changes in normal cells, and environmental and systemic stresses also make significant contributions to cancer aggravation. One of such stresses is acidic tumor microenvironments, which is caused by excessive glycolytic cancer cell metabolism, hypoxia, and inefficient blood perfusion. Acidic pH has pleiotropic effects on cancer cell proliferation, clonal evolution/selection and metastasis, and also modulates immune cell functions. The processes of the pH gradient in cancer cells have recently become targets for anti‐cancer therapies.In this study, we examined the effects of acidic pH environments on prostate cancer growth. We focuses on the Na/HCO3 cotransporter NBCe1 (SLC4A4) that moves Na+ and HCO3− into cells and buffers intracellular H+, resulting in extracellular acidification. To examine whether NBCe1 activity is stimulated in proliferating cancer cell lines, we incubated human prostate cancer cell lines LNCaP and PC‐3 cells and human breast cancer cell line MCF7 cells in hypoxic (1% O2, 5% CO2) conditions for 3 days and measured pHi using the fluorescent pH‐sensitive dye BCECF‐AM, and fluorescent images (excitation at 490 and 440 nm; emission at 535 nm) were captured at different time intervals. pH values were generated by the high K+‐nigericin calibration. The solutions contained 100 μM EIPA to inhibit endogenous NHE. After a rapid initial CO2‐induced acidification, the pHi was recovered. The recovery was significantly increased in hypoxic conditions. It is also inhibited by DIDS and S0859, which block most NBCs. In RT‐PCR with the primers that amplify NBCe1 transcripts, the amplification products with the expected size were detected in both prostate cancer cells and breast cancer cells. Next, we performed immunoblot to examine NBCe1 protein expression in cells. Determined by densitometric quantitation of immunoreactive bands and normalization of pixel intensities to β‐actin, the NBCe1 expression in LNCap and PC‐3 cells was increased in hypoxic conditions, whereas the expression in MCF7 remained unaffected. The NBCe1 upregulation in prostate cancer cells was small, but statistically significant (p < 0.05; paired, two‐tailed Student's test; n = 4). To test whether pH changes mediated by the Na/HCO3 transporter affects the growth of prostate cancer cells, we treated cells with the blocker N‐cyanosulphonamide S0859 (50 μM) in normoxic and hypoxic incubations and counted cells 5 days later using a propidium iodide staining protocol. S0859 reduced the number of cells that were proliferated under the hypoxic condition (p<0.05, n=6; unpaired two‐tailed Student t‐test). We also treated cells with higher amounts of the drug and observed more profound inhibition on cell growth (data not shown). These results demonstrate that NBCe1 is the key protein responsible for acidic microenvironments in human prostate cancer.Support or Funding InformationSupported by the Emory Winship Cancer Center Pilot GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.