| Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. In addition to surgical procedures, adjuvant chemotherapy is often administered to prolong survival time, especially in advanced stages of the disease. Over the past 40 years, 5-Fluorouracil (5-FU) has been the most commonly used chemotherapeutic agent for CRC and is still the mainstay therapy for patients with advanced colorectal cancer. However, less than 25% of patients with advanced CRC were observed to have major responses after 5-FU-based chemotherapy, and, in many cases, patients responding initially to 5-FU ultimately became resistant. Consequently, resistance to this drug is a major obstacle in CRC chemotherapy. Previous studies have demonstrated that apoptosis is one of the primary cytotoxic mechanisms of chemotherapeutic agents, including 5-FU, and inhibition of the apoptotic pathway is one of the factors that may be responsible for drug resistance.Apoptosis can be induced via two different pathways. The extrinsic pathway is triggered by ligation of death receptors, such as Fas. On the other hand, the intrinsic pathway is initiated by the release of cytochrome c into the cytosol from the mitochondria. This molecule interacts with APAF-1, pro-caspase-9, and deoxyadenosine triphosphate to form the apoptosome complex,(1) resulting first in activation of the initiator caspase and then of the effector caspases. Recent experimental evidence supports the hypothesis that 5-FU induces cytotoxicity through the Fas/FasL pathway in colon cancer cells. Further, the inhibitor of apoptosis protein (IAP) family was thought to prevent apoptosis through direct caspase and pro-caspase inhibition in human colon cancer cells.(2) In addition, Bcl-2 proteins have also recently arisen as apoptosis regulators in 5-FU treatment of human colon cancer cell lines. Bcl-2 family proteins regulate mitochondrial outer membrane permeability, thereby determining cellular commitment to apoptosis. Significantly,5-FU for the majority of anticancer drugs is the notable exception to identify the apoptotic machinery and understand its regulation initiated by caspase-9.miRNAs negatively regulate the expression of target genes by interacting with complementary sites in the 3'untranslated region (3'UTR). Recently, an increasing amount of evidence suggests that aberrant expression of miRNA in cancer cells could modulate the functional expression of critical genes involved in cell survival in response to chemotherapeutic stress; additionally, patterns of miRNA have been shown to accurately define specific cancer types. For example, Lorena Rossi et al. found that miR-23a was upregulatedin 5-FU-treated C22.20 and HC.21 colon cancer cells. Moreover, miRNA expression has been repeatedly observed in serum and plasma and the use of miRNAs as novel, minimally invasive biomarkers has demonstrated reasonable sensitivity for CRC detection. In a study by Chen et al., the serum of CRC patients had a significantly different miRNA profile compared to the serum of healthy subjects. Moreover, CRC patients and lung cancer patients had increased serum miR-23a. Differentially expressed miR-92 in the plasma of patients with CRC has also been reported. Further, Pearson correlation further indicated that miRNAs are becoming increasingly recognized as regulatory molecules in human colorectal cancer chemotherapy. The mechanisms of chemotherapeutic resistance modulated through miRNA regulation of apoptotic processes in malignant human colorectal cancer are poorly understood. The discovery of miRNAs based on expression profiling have proven there were significant changes of miRNA expression levels in CRC tissue in comparison to colorectal epithelium, and these miRNAs enabled prognostic stratification of CRC patients and prediction of their responses to selected chemotherapeutic regimens. To explore the role of miRNA in cell survival and chemotherapeutic response, we questioned whether current treatments targeted appropriate molecular mechanisms in the intrinsic pathway of apoptosis.In this study, we evaluated that microRNA-23a decreased the chemosensitivity of 5-fluorouracil (5-FU) in colon cancer cells through Mitochondria-mediated apoptotic signaling pathway. Quantitative real-time PCR was used to evaluate the differential expression of microRNA-23a in 5-FU-based chemotherapy or not colon cancer cells and colonrectal cancer tissue samples. Cell proliferation, drug cytotoxicity and apoptosis assay were used to find the function of microRNA-23a on colon cancer cells in vitro. Luciferase 3'UTRs reporter assay and western blot analysis for APAF-1 protein expression level were used to determine whether microRNA-23a could target APAF-1 gene in the cytochrome c/APAF-1/caspase-9 pathway. HCT116 and HT29 tumor xenografts model were used to determine whether down-regulation of microRNA-23a could enhance 5-FU sensitivity in vivo, as assessed by monitoring the bioluminescence and tumor volume. Suppressing the function of APAF-1 with small interfering RNA (siRNA) and preventing the cytochrome c, APAF-1, pro-caspase-9, and deoxyadenosine triphosphate (dATP) to form the apoptosome complex and blunted caspas-9 activation after 5-FU treated, can inverse the effect of microRNA-23a inhibitors combined with 5-FU increased the activation of caspas 9. We then reported that chemotherapy with 5-FU in colon cancer cells induced microRNA-23a expression increased, the expression of protein for target gene APAF-1 decreased and the activity of caspas-9 decreased. In patients who received 5-FU therapy, high miR-23a expression indicated a poor response to therapy. Subsequent inhibition of target gene APAF-l's protein expression with small interfering RNA blunted caspas-9 activation. Up-regulation of cytochrome c/APAF-1/caspase-9 signaling with microRNA-23a inhibitors enhanced chemosensitivity in vitro and in vivo.Our studies identified that the expression of miR-23a was consistently altered and dramatically upregulated in 5-FU-treated human HCT116 and HT29 colon carcinoma cells. This result was consistent with the report that showed upregulation of miR-23a in human colon cancer cells following exposure to 5-FU in vitro and in vivo. Moreover, we provide evidence that miR-23a was differentially expressed and the expression level was significantly increased in 5-FU-based chemotherapy-treated colorectal cancers tissues when compared to untreated samples. Overexpression of miR-23a has previously been reported in other solid tumors, but this is the first report correlating its affect on the mitochondria-mediated apoptotic pathway in colon cancer. For example, current understanding suggests that miR-23a is an up-stream regulator of TOP2B and confers chemoresistance to cisplatin in tongue squamous cell carcinoma lines. Further, some reports found that miR-23a expression is altered in other types of cancer, including acute leukemia and bladder cancer. The data from these studies suggest that miR-23a controls a wide array of biological processes, including apoptosis, a process often dysregulated in cancer. Moreover, our results support a functional role for miR-23a-mediated chemotherapy-induced apoptosis in both HCT116 and HT29 cells. We postulated that aberrant expression of miR-23a in cancer cells could modulate the functional expression of critical genes involved in tumor cell apoptosis in response to chemotherapeutic stress.Fortunately, we identified an apoptosis-related target gene, APAF-1, for miR-23a by luciferase 3'UTRs reporter and Western blot analysis. Moreover, we also reported that APAF-1 is an important molecule involved in cytochrome c/APAF-1/caspase-9 signaling in 5-FU-treated tumor cells. Recent findings indicate that the expression of APAF-1 and caspase-9 were significant prognostic indicators in stageâ…¡/â…¢colon carcinoma. Furthermore, analysis of apoptosis biomarkers suggested that APAF-1 as potential prognostic markers. It may help to guide chemotherapy for colorectal cancer patients. We found that there is a pathway mediated by miR-23a that inhibits the apoptosis of 5-FU-treated human colon cancer cells. A specific complex, which contains several key molecules, controls the apoptosis of human colon cancer cells. Cytochrome c and APAF-1 are critical members of this network. Release of cytochrome c into cytosol from the mitochondria can be significantly induced by 5-FU, in the cytosol it binds to APAF-1. The cytochrome c/APAF-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases. In addition, caspase-9 is regulated by APAF-1 through miR-23a. The novel 5-FU/Cytochrome c/miR-23a/APAF-1/caspase-9 pathway is an important complement to the network. The distinct role of high levels of miR-23a involved in apoptosis elucidates the 5-FU chemotherapeutic-apoptosis cascade and provides new insight on colorectal cancer treatment.The present work indicates that 5-FU-induced inhibition of miR-23a expression could promote tumor cells apoptosis through up-regulation of this anti-apoptotic mitochondrial pathway in vitro and in vivo. We report that colon cancer cell lines and xenografts could be rendered more sensitive to 5-FU chemotherapy by miR-23a inhibitors. The modulation of aberrantly expressed miR-23a may be a useful strategy to limit tumor cell proliferation or to improve responses to cytotoxic therapies. Over expression of miR-23a has been consistently observed in cancers, including colorectal carcinoma, P-493 B lymphoma cells, PC3 prostate cancer cells, tongue squamous cell carcinoma lines, hepatocellular carcinoma cells, gastric adenocarcinoma cells, acute leukemia, and bladder cancer cells. Further work based on miR-23a over-expression in cancers needs to be performed to evaluate their efficacy in other tumor models and to develop clinically relevant therapeutic strategies. Knowledge of specific processes that are regulated by miR-23a and identification of apoptotic mechanisms involved in the mitochondrial pathway will yield useful information and novel insight into the mechanisms of chemoresistance of colorectal cancer and other types of cancer.
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