The reducing effect of TNF-α on carbonic anhydrase III gene expression in colon carcinoma and osteosarcoma cells

The appropriate acid-base balance in organisms is maintained by Carbonic Anhydrase proteins (CAs), which have hydratase activity and regulate intracellular pH. CAs are required for both physiological and pathophysiological processes such as cancer development, and there are differences in their expression profiles in different cancer types. Some members of the CA family like CAIX and CAXII have been suggested as potential cancer biomarkers in various studies. CAIX has been proposed as a possible biomarker for hypoxic colorectal carcinoma. Expression of CAIII, another member of the CA family, was also found to be reduced by TGF-beta via the MAPK and PI3K signaling pathways in human colon cancer cells. Additionally, not much is known regarding the interaction between TNF-alpha, inflammation-related cytokine, and CAIII in colorectal carcinoma (CRC). This study investigates the effect of TNF-alpha on CAIII expression in different cancer cells, namely HT-29 and Saos-2. CAIII mRNA expression was analyzed by qRT-PCR at both late (24, 48, and 72 h) and early time points (1, 3, and 6 h). Western blot analysis was also used to confirm the reducing effect of TNF-alpha at the CAIII protein level. To analyze the transcriptional regulation of CAIII by TNF-alpha, CAIII promoter constructs were transiently transfected to HT-29 and Saos-2 cells, and transcriptional activities of truncated promoter constructs were analyzed with luciferase/SEAP activities. 500U/mL TNF-alpha led to a drastic decrease at 24 and 48 h time points at CAIII mRNA level. Western blot analysis showed that this decreasing effect of 500 U/mL TNF-alpha at 24 h, 48 h, and 72 h resulted in a reduction of the CAIII protein level by 0.5 times. P1 (- 939/+ 86), P2 (- 699/+ 86), P3 (- 236/+ 86), and P4 (- 108/+ 86) CAIII promoter constructs were transiently transfected to HT-29 cells, P4 (- 108/+ 86) promoter basal activity is greater than the other promoter constructs. Transcriptional activity of all CAIII promoter constructs was reduced by TNF-alpha. As a result of pathway inhibition analysis, we deduce that TNF- alpha decreases CAIII mRNA expression in a colon cancer cell via the PI3K pathway. In addition, the similar reducing effect of TNF-alpha on CAIII in Saos-2 cells, which is a model of osteosarcoma, was also obtained at mRNA and transcriptional levels.