Adaptation Of Colon Cancer Cells To Acidic Microenvironment-Cytological Changes And The Associated Mechanisms

Cancer is a major killer all over the world. According to World Cancer Report 2014, published by WHO, "Cancers are among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer related deaths in 2012". Colorectum was the third most common sites of cancer diagnosed and cancer death in 2012.694,000 people died of colorectal cancer (CRC) in 2012. In China, CRC is the third leading cancer type for the estimated new cancer cases and the fifth leading cancer type for cancer death.The concept of tumor microenvironment is developed from Stephen Paget’s "seed and soil" theory, proposed in 1880s. In this theory cancer cells are "seeds", and the stromal tissue and extracellular matrix (ECM) are "soil", i. e., the tumor microenvironment. Many key features of tumor microenvironment have been identified, including hypoxia, nutrition deficiency, presence of mesenchymal and immune cells, and low extracellular pH.Tumor cells can exhibit a glycolytic penotype, even under aerobic condition. This phenomenon was also called "Warburg Effect". In the meanwhile, elevated production of lactic acid via glyco lysis cannot be removed due to local poor perfusion. A rapid rate of glycolysis combined with poor perfusion produces extracellular acidosis. The pH of human tumor extracellular microenvironment is usually measured between 5.5-7.0.To most normal cells, extracellular acidic microenvironment is harmful, even lethal, but cancer cells can survive. However, survival mechanisms of cancer cells under acidic conditions have not been well characterized. Acute acidosis could disrupt adherent junctions by modifying E-cadherin. Exposure of cancer cells to an acidic environment for 6 hours was found to significantly enhance lung metastases. Those results imply that acidic microenvironment may enhance metastatic potential of cancer cells. It was showed that acidosis could promote the invasiveness of breast cancer cells. It has also been found that cancer-generated lactic acid may have immunosuppressive effect. Additionally, base excision repair (BER) was reported to play critical roles in the survival of cancer cells in an acidic microenvironment.Living cells are always subjected to the effects of reactive oxidizing molecules, both exogenously and endogenously produced. Reactive oxygen species (ROS) are the major source of oxidative stress. ROS can be defined as a group of oxygen-containing highly reactive species. At low and moderate levels, ROS may contribute to tumorigenesis either as signaling molecules or as mutagens. At high levels, ROS may inflict cellular damage and cause cell death. Cancer cells usually experience high levels of ROS. Therefore, robust antioxidant capacity is required for the survival of cancer cells. It was reported that acute acidosis increased ROS.Autophagy is a multi-step process, whereby cells degrade proteins, lipids and organelles for recycle. The relationship between autophagy and ROS is complex. On one hand, ROS induce autophagy; on the other hand, autophagy influences ROS. It was reported that tumor acidic microenvironment promote autophagy, and that chronic autophagy occurs as a survival mechanism in MDA-MB-231 cells.While tumor acidic microenvironment is known to affect tumor survival, growth, invasion and metastasis, most studies so far addressed acute acidosis, hours to days, and little is known about how cancer cells become adapted to chronic acidic environment, which is the case in real life. In order to understand what happens to cancer cells in chronic acidic microenvironment, we investigated the influences of chronic acidic microenvironment on CRC cells.PART ONECharacteristics of colon cancer cells adapted to acidic microenvironmentWe first treated CRC cells (HCT15, HCT116, LoVo and SW480 cells) with acidic medium (pH6.5,37℃,5% CO2) for more than 3 months to obtain CRC cells that became adapted to acidic microenvironment (we designated them "extended/6.5" cells, and "ext" for short). We were able to obtain HCT15-ext, HCT116-ext and LoVo-ext cells. SW480 failed to survive the chronic acidic stimulation.We examined CRC cells cultured at acidic medium (pH6.5) with microscopy. HCT15 cells exposed to pH6.5 for 3 days appeared to have reduced viability and density than their parental cells. HCT116 cells showed a similar trend. Morphologically while the control cells had the appearance of cobblestone that is typical of epithelial cells in culture, the CRC-ext cells were more elongated and more scattered. The morphological changes of CRC cells were suggestive of epithelial mesenchymal transition (EMT). We examined the EMT markers in HCT15 and HCT15-ext cells with quantitative real-time PCR and Western Blotting. We found that N-cadherin (N-cad), Snail 1 and Vimentin, markers of mesenchymal cells, were upregulated in HCT15-ext cells. E-cadherin (E-cad) and FN1, markers of epithelial cells, were down-regulated in HCT15-ext cells when compared with HCT15 cells. These results suggest that CRC cells adapted to acidic microenvironment have undergone some degree of EMT.Then we measured proliferation of CRC cells adapted to acidic tumor microenvironment.5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay showed that HCT15 cells cultured at low pH (pH6.5) for 3 days were reduced in proliferation when compared to control cells, and the proliferation of HCT15-ext cells was further reduced. Similar results were obtained in HCT116 cells. Then we examined cell cycle distribution and apoptosis of CRC cells cultured under low pH with flow cytometry (FCM). Acute and chronic exposure to acidic medium resulted in a significantly higher percentage of cells in Gl phase in HCT15, HCT116 and LoVo cells, but not in SW480 cells. Interestingly, both HCT15-ext and HCT116-ext cells were found to have reduced levels of apoptosis than their control cells.Together, these results suggest that CRC cells adapted to acidic microenvironment are different from their parental cells in morphology and proliferation. They even have a reduced rate of spontaneous apoptosis.PART TWOGSH-dependent Antioxidant Defense Contributes to theAcclimation of Colon Cancer Cells to Acidic MicroenvironmentPrevious studies showed that the levels of ROS in cancer cells exposed to acute acidosis were enhanced. We measured ROS levels in CRC cells cultured in acidic medium with FCM. Acute acidosis enhanced the ROS levels in CRC cells, while chronic exposure to acidic pH resulted in a reduction in ROS, in all three cell lines tested (HCT15, HCT116 and LoVo cells), suggesting that CRC cells may generally have a reduced level of ROS when adapted to low pH.As a strong antioxidant, reduced glutathione (GSH) can effectively eliminate ROS. We examined the expression of GSH-related genes with quantitative real-time PCR, Western Blotting and FCM. Compared with HCT15 cells, the upregulation of GSR, GLS1, GLS2, XCT, CD44, GCLM, GCLC, GSS, GPX1, GPX3, GPX5 and GPX6 and the downregulation of GGT1 and GGT6 in HCT15-ext cells suggested that GSH level might be elevated in acidic pH-acclimated cells. Notably, GSH-related antioxidant genes (for example, CD44, GPXs) were upregulated in HCT15-ext cells. Similar results were obtained in HCT116 cells. Indeed, the ratio of GSH/GSSG and GSH level were observed to be significantly higher in HCT15-ext cells than that in HCT15 cells.To test whether an increased level of GSH is responsible for adaptation of CRC cells to acidic pH, we measured the cytotoxicity of diethyl maleate (DEM, a depleting agent of GSH), buthionine sulfoximine (BSO, a GCL inhibitor) or GSR siRNA to CRC-ext cells and their parental cells. As expected, DEM, BSO and GSR siRNA were more toxic to CRC-ext cells than to controls, suggesting that the survival of CRC-ext cells were more dependent on GSH than control cells. ROS levels of colon cancer cells treated with BSO or GSR siRNA were higher than parental cells. Further more, apoptotic levels of CRC-ext cells treated with GSR siRNA were much higher than CRC-ext cells, while it remained unchanged in treated parental CRC.Together, these results indicate that a low level of ROS, which is probably mediated by increased level of GSH, is critical for CRC cells to become adapted to acidic microenvironment.PART THREEAutophagy Promotes Survival of Colon Cancer Cells under Acidic MicroenvironmenHigh levels of ROS can induce autophagy, and autophagy can affect ROS. Moreover, it was reported that autophagy was very important for breast cancer cells adapted to acidic microenvironment. We confirmed the upregulaton of pro-autophagy genes with quantitative real-time PCR and Western Blotting. We observed a highter LC3B Ⅱ/LC3B Ⅰ ratio in CRC-ext cells than in their parental cells. Increased autophagy in HCT15-ext and HCT116-ext cells was confirmed with Transmission Electron Microscope (TEM). In order to test the importance of autophagy in CRC cells survival under acidic microenvironment, we measured the apoptotic levels of CRC cells treated with 3-methyladenine (3-MA, an autophagy inhibitor) or ATG5 siRNA, We found significantly increased apoptosis in CRC-ext cells, when autophagy was inhibited, but not in parental cells. These results suggest that chronic acidosis promotes autophagy, and CRC-ext cells are more reliant on autophagy than their parental cells.To test whether autophagy contributes to the reduction of ROS in CRC cells adapted to acidic microenvironment, we measured the levels of ROS in CRC cells treated with 3-MA or ATG5 siRNA. Contrary to our expectation that inhibition of autophagy may enhance the ROS levels,3-MA or ATG5 siRNA treatment resulted in a reduction of ROS levels in CRC cells and CRC-ext cells. These results indicate that while autophagy contributes to survival of CRC cells under acidic pH, it does not affect the levels of ROS.Taken together, these results indicate that autophagy is enhanced in CRC cells adapted to acidic pH. While it is critical for cell survival in acidic condition, it does not contribute to the reduced ROS.