Ribosomal protein S3 affects colon cancer Caco-2 cell growth by modulating the levels of p53 and lactate dehydrogenase

Abstract

Colorectal cancer is the second leading cause of death worldwide accounting for around nine percent of cancer incidence. Studies have shown that there is an increase in the levels of several ribosomal proteins in many types of cancer. In colon adenocarcinomas and adenomatous polyps, the RNA levels of ribosomal protein S3 (RPS3), a component of the 40S ribosomal subunit, are increased. Although RPS3 is overexpressed in colon cancer, its role in initiating and promoting tumor formation is poorly studied. In this study, we induce RPS3 Knock Down (KD) using four combinations of siRNAs in human epithelial colorectal adenocarcinoma cells Caco-2 and normal cell mucosa NCM-460 cells. The aim of our current research is to decipher the molecular mechanisms that are affected by RPS3 knockdown in both Caco2 and NCM-460 cells. We started by studying the proliferation rate and apoptosis rate of both Caco-2 and NCM-460 cells upon RPS3 knockdown using trypan blue, a dye that stains dead cells, and Annexin V apoptosis kit with flow cytometry as a detection tool, respectively. For proliferation, there was a decrease by around 40 % in Caco-2 cells but just by 2 % in NCM-460 at day 4 after the transfection with RPS3-siRNA compared to the control. On the other hand, flow cytometry analysis of apoptotic cells showed a five folds increase in cell death rate in Caco-2 cells treated by siRNAs compared to untreated cells. However, in NCM-460 cells there was no significant change in apoptosis level after RPS3 KD. Then using the wound healing assay, we demonstrated that there was a significant decrease in migration rate in Caco-2 cells after the KD of RPS3 compared to the control. RPS3 is known to interact with p53, a tumor suppressor gene involved in cell cycle arrest and induction of apoptosis. Using western blot analysis, we showed that upon RPS3 knockdown in Caco2 cells, there is a significant increase in p53 levels by 1.95 folds. No notable change in p53 level was shown in the case of NCM-460 cells. This suggests a possible involvement of RPS3 in controlling apoptosis rate in Caco-2 cells through modulation of p53 levels selectively in cancer cells. On the other hand, lactate dehydrogenase (LDH), an enzyme in glycolysis responsible for transforming pyruvic acid into lactic acid, has a high activity in most tumorigenic cells. The elevated level of glycolysis followed by the high rate of lactic acid fermentation even in the presence of high oxygen level is known as the Warburg effect. To understand this regulation, we measured the level of LDH enzyme upon RPS3 KD. Western blot analysis showed a decrease in LDH protein level by 2.82 folds after the knockdown in Caco2 cells suggesting that RPS3 is modulating LDH levels in these cancer cells. No change in LDH level was seen after RPS3 KD in NCM-460. P53 knock down had no effect on either RPS3 nor LDH level in both Caco-2 and NCM-460 cells suggesting that p53 is downstream of RPS3 in Caco-2 cells. Double KD of both RPS3 and p53 had no significant effect on the level of LDH in both Caco-2 and NCM-460. This indicated that maybe the decreased level of LDH observed in Caco-2 after RPS3 KD is due to the high p53 level induced by the KD of RPS3. Taken altogether, our results demonstrate that RPS3 could be a promising therapeutic agent in treating colorectal cancer since its KD had major effect on colon tissues cell but not on normal colon cells. The combination of RPS3 knockdown with other therapeutic options against colorectal cancer must be studied to detect to which extent targeting RPS3 would be beneficial in treating colorectal cancer.