Aspirin Selectively Targets Colorectal Cancer Stem Cells And Its Molecular Regulatory Mechanism

BackgroundColorectal cancer is a common malignancy. It was reported that the number of new cases and deaths of colorectal cancer figures among the first three of all malignancies throughout the United States in2012. Although the progress of basic research and early intervention has been made, its incidence and mortality rates continue to show a rising trend. Colorectal cancer increasingly becomes an important disease which seriously harms human health. It has been provn that cancer stem cells (CSC) exist in colorectal cancer and other malignancies. CSC is a subgroup with strong self-renewal capacity and pluripotent differentiation. Although accouting for a small fraction of tumor cells, it plays an important role in carcinogenesis, drug resistance, tumor relapse and metastasis. Hence, it is crucial to eradicate cancer colorectal cancer stem cells (CCSC) for the treatment of colorectal cancer.Aspirin, one of non-steroidal anti-inflammatory drugs, is a small molecule compound which has been the most and longest used in the world. In addition, it does not produce drug dependence. Epidemiological data and clinical trials indicate that Aspirin not only exerts antipyretic and anti-platelet aggregation, but also prevents several cancers including colorectal cancer, esophageal cancer and lung cancer. Increasing evidences also indicate that aspirin can significantly reduce the incidence and mortality of colorectal cancer, and improve the prognosis of patients. However, its mechanism is still unknown. Recently, a research reported that non-steroidal anti-inflammatory drugs (indomethacin) could reduce the number of CCSC in the colon cancer cell lines. However, whether Aspirin inhibits CCSC and what is the molecular mechanism involved has not been reported.Therefore, further investigation of Aspirin’s effect on CCSC has important clinical values. Because it is benefical to help us expand Aspirin’s applications, screen potential molecular targets for the treatment of colorectal cancer, and provide a theoretical basis for designing more efficient anticancer drugs.MethodsWe enriched CCSC from primary colorectal cancer cells and ATCC standards cell line by using serum-free culture technology, and identified its biological characteristics including the ability to form colonspheres, drug resistance, tumorigenicity, the proportion of cancer stem cells, and expression of stem genes. Different doses of Aspirin were added into the culture system, biological characteristics of CCSC were tested. MTS assay was used to analyze whether the inhibitory effect of Aspirin was selective. Immunofluorescence and flow cytometry analysis were also adopted to test the manner by which Aspirin selectively inhibited CCSC (apoptosis/autophagy). We investigated the apoptosis-related gene expression both in CCSC and non-CCSC through quantitative PCR, and screened apoptotic factors by which Aspirin induced the apoptosis of CCSC. On the contrary, biological characteristics of CCSC were further tested after blocking this apoptotic gene. Also, we discussed upstream regulatory mechanisms of the apoptotic gene from the perspective of epigenetics. Results1. CCSC enriched from serum-free culture technology has the typical characteristics of cancer stem cells, Aspirin can inhibit CSC’s featuresCCSC was successfully enriched from primary colorectal cancer cell and ATCC standards cell lines HT29by serum-free culture techniques. Compared with non-CCSC, the ability of colonspheres forming significantly strengthened in CCSC, expression of stem genes (Sox2, Oct4, Nanog, Notch1, Bmi-1and ALDH1) were obviously increased, stem cell marker ALDH1was distinctly increased, resistance to chemotherapy drugs oxaliplatin (OXA) greatly increased, tumorigenicity in vivo was significantly enhanced. Different concentrations of Aspirin (0,2.5,5.0,10.0mM) were added to CCSC, Aspirin could inhibit the numbers of colonspheres, reduce diameter of the colonspheres, lower expression of stem genes (Sox2, Oct4, Nanog, Notchl, Bmi-land ALDH1), abate expression of stem cell marker ALDH1, increase the sensitivity of OXA, and these effects were dose-dependent. Tumorigenicity in vivo experiments also showed that Aspirin could inhibit the growth of CCSC, reduce the volume of tumor. These results confirm that Aspirin can significantly inhibit CCSC.2. Aspirin selectively induced FasL mediated apoptosis of CCSCGiven that Aspirin can significantly inhibit the CCSC, whether there are differences between CCSC and non-CCSC when treated with Aspirin? Our study confirmed that Aspirin selectively inhibited the CCSC. Previous study reported that Aspirin inhibited colorectal cancer cells primarily by inducing apoptosis or autophagy. Our study showed that by Aspirin selectively induced apoptosis of CCSC rather than autophagy. Apoptosis includes exogenous and endogenous apoptosis pathway. Our study confirmed Aspirin up-regulated the expresson of FasL in CCSC, and induced extrinsic apoptotic pathway. But in the non-CCSC, the expression of FasL and Fas came down, so Aspirin selectively induced apoptosis of CCSC. When we used FasL antibody to block FasL, the survival rate of CCSC significantly increased and apoptosis decreased.3. Up-regulation of FasL by Aspirin is COX-2-independentThe classic mechanism of Aspirin is that it reduces the activity of COX-2which affects the synthesis of PGE2. Our study confirmed that Aspirin reduced COX-2enzyme activity in CCSC. In addition, knockdown of COX-2by specific siRNA, or a small molecule inhibitor NS-398did not induce apoptosis of CCSC and up-regulate the expression of FasL. Furthermore, Aspirin and PGE2co-treatment did not reduce apoptosis and down-regulate the expression of FasL in CCSC. These evidences suggest that Aspirin up-regulates FasL through non-COX-2pathway.4. Aspirin promotes the acetylation of histone H3at FasL promoterWe compared the expression of FasL after non-steroidal anti-inflammatory drugs treatment(including salicylic acid, Aspirin, indomethacin, ibuprofen, sulindac) in CCSC, and found that only Aspirin increased the expression of FasL. When compared chemical structure of these non-steroidal anti-inflammatory drugs, we found that only aspirin had acetyl moieties while other NSAIDs did not. It hints us that acetylation may play an important role in up-regulating FasL. The expression of FasL in CCSC treated with deacetylase inhibitor TSA was significantly upregulated. After analyzing the extent of histone acetylation, we found that Aspirin only elevated level of acetylated histone H3, which promoted FasL transcription.ConclusionWe demonstrated that Aspirin significantly and selectively inhibited CCSC and caused FasL-induced apoptosis of CCSC. Aspirin promoted the acetylation of histone H3at FasL promoter to regulate FasL transcription in a COX-2independent manner. The study not only expands the clinical application of Aspirin for targeting CCSC, but also provides a theoretical basis for screening potential molecular targets and designing more efficient anticancer drugs.