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Autophagy is a conserved catabolic process that degrades cytoplasmic proteins and organelles for recycling. The role of autophagy in tumorigenesis is controversial because autophagy can be either protective or damaging to tumor cells, and its effects may change during tumor progression. A number of cancer cell lines have been exposed to chloroquine, an anti-malarial drug, with the aim of inhibiting cell growth and inducing cell death. In addition, chloroquine inhibits a late phase of autophagy. This study was conducted to investigate the anti-cancer effect of autophagy inhibition, using chloroquine together with 5-fluorouracil (5-FU) in a colon cancer cell line. Human colon cancer DLD-1 cells were treated with 5-FU (10 μΜ) or chloroquine (100 μΜ), or a combination of both. Autophagy was evaluated by western blot analysis of microtubule-associated protein light chain3 (LC3). Proliferative activity, alterations of the cell cycle, and apoptosis were measured by MTT assays, flow cytometry, and western blotting. LC3-II protein increased after treatment with 5-FU, and chloroquine potentiated the cytotoxicity of 5-FU. MTT assays showed that 5-FU inhibited proliferation of the DLD-1 cells and that chloroquine enhanced this inhibitory effect of 5-FU. The combination of 5-FU and chloroquine induced G1 arrest, up-regulation of p27 and p53, and down-regulation of CDK2 and cyclin D1. These results suggest that chloroquine may potentiate the anti-cancer effect of 5-FU via cell cycle inhibition. Chloroquine potentiates the anti-cancer effect of 5-FU in colon cancer cells. Supplementation of conventional chemotherapy with chloroquine may provide a new cancer therapy modality.
Autophagy, from the Greek, means ‘self-eating’, and is a conserved eukaryotic catabolic process involving the degradation and recycling of cytoplasmic proteins and organelles in response to stress or starvation. At the outset of autophagy, double-membrane structures called autophagosomes form in the cytosol and sequester cytoplasmic material. These autophagosomes fuse with lysosomes to form autolysosomes, and the sequestered material is degraded by lysosomal enzymes. Autophagy plays important roles in survival, development, and homeostasis, as well as in pathologic conditions such as neurodegenerative disease, infection, immune responses, and cancer [1-3]. However, the role of autophagy in tumorigenesis remains controversial, because it can both protect and destroy tumor cells, and its role may change during tumor progression [3, 4].
There are several autophagy inhibitors that can be used to study the role of autophagy. Chloroquine is one of the autophage inhibitors. It is lysosomotropic and inhibits the fusion of autophagosomes and lysosomes. Chloroquine is widely used as an anti-malarial, and also as an anti-inflammatory in the treatment of rheumatoid arthritis and lupus erythematosus. Recently, attention has focused on its potential as an anti-cancer agent and chemotherapy sensitizer [5, 6]. Although the precise basis of the anti-cancer effects of chloroquine is still under investigation, inhibition of autophagy most probably plays a part in anti-tumoral effect.
Colon cancer cell lines are resistant to nutritional deprivation due to activation of autophagy . A previous report showed that activation of autophagy contributes to the survival of colon cancer cells, and that inhibition of autophagy augments 5-fluorouracil (5-FU)-induced apoptosis of colon cancer cells . In addition, it was recently reported that chloroquine enhanced the effects of 5-FU chemotherapy in a human colon cancer cell line .
The aim of this study was to investigate the anti-cancer effect of combination therapy with chloroquine and 5-FU in a colon cancer cell line.
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In this study, we found that chloroquine enhanced the anti-proliferative effect of 5-FU in DLD-1 colon cancer cells, and the combination of 5-FU and chloroquine induced G1 arrest, up-regulation of p27 and p53, and down-regulation of CDK2 and cyclin D1. However, chloroquine did not induce the apoptosis. These results suggest that the cell growth inhibitory effect of chloroquine may be associated with G1 arrest of cell cycle rather than apoptosis.
Before performing this study, we had hypothesized that pre-treatment of the colon cancer cells with chloroquine to inhibit the autophagy would enhance the anti-cancer activity of 5-FU by activating the apoptosis. A recent report showed that inhibition of autophagy using 3-methyladenine augmented 5-FU induced apoptosis in human colon cancer cell lines . In addition, this synergistic effect was also seen in a DLD-1 xenograft tumor model. These results showed that 5-FU induced the autophagy activation and the autophagy process occurred within several hours after treatment of drug. Since chloroquine inhibits the fusion of the autophagosome and lysosome, and then, induces accumulation of autophagosome, the LC3-II expression level is increased after chloroquine treatment. In addition, LC3 (cytoplasmic vacuoles by confocal microscope) was detected in chloroquine or 5-FU/chloroquine treated DLD-1 cells.
There are several reports of the effects of chloroquine on the cell cycle. A recent study revealed that chloroquine protected against breast cancer in a p53-dependent manner . In that study, chloroquine reduced the incidence of N-methyl-N-nitrosourea-induced mammary tumors and the growth rate of tumors in animal models. Chloroquine induced up-regulation of tumor suppressor p53 and the p53 downstream target gene p21, leading to G1 cell cycle arrest. In addition, p27 was reported to be up-regulated after chloroquine treatment, and p27 also played a role in cell cycle arrest in that study. On the contrary, in our study, neither chloroquine nor 5-FU alone induced G1 arrest, but the combination did, and the same was true for up-regulation of p27. The differences between our observations and those results may be resulted from the relatively short exposure time of chloroquine in our experiments or different cancer cell line. Our results suggest that chloroquine may enhance the anti-cancer effect of 5-FU in colon cancer cells by promoting p27-induced cell cycle arrest.
DLD-1 was known to be a mutated p53 transcription factor. Therefore, it might not be reasonable that synergistic anti-tumor effect of 5-FU and chloroquine was induced via up-regulation of p53 in our experiment. However, many evidences have been accumulated that small molecules could reactivate the p53 mutant, and 5-FU is one of the small molecules. Aizu et al. demonstrated that AJ02-MM cells were not mutated p53 gene, but lacked in p53 DNA-binding activity. However, treatment of AJ02-MM cells with 5-FU resulted in the reversal of p53 DNA-binding activity . Endo et al. also reported that up-regulation of p53 protein could be seen in NUCG-1, mutated p53 gastric cell line, after 5-FU treatment for 24 h . In our experiments, PUMA was up-regulated, and mdm2 was down-regulated after treatment of 5-FU and chloroquine, indicating that 5-FU might reactivate the mutated p53 in DLD-1 cells.
The mammalian class I phosphatidylinositol-3-kinase (PI3K)-target of rapamycin (mTOR) pathway plays a key role in regulation of cell growth, protein translation, metabolism, and autophagy, and alterations of the mTOR pathway are common in many types of cancer [3, 17, 18]. The class I PI3K generates PIP3, which binds to AKT and PDK1. AKT activates kinase-mTOR signaling, which suppresses autophagy. The tumor suppressor PTEN inhibits class I PI3K and allows the initiation of autophagy. Recently, p53 activation was shown to inhibit mTOR activity and affect its downstream targets, such as autophagy, by activating AMP kinase followed by the tuberous sclerosis (TSC) 1/TSC2 complex . Other investigators have reported that 5-FU treatment increased p53 expression in colon cancer cells at the translational level . We also found that 5-FU treatment caused up-regulation of p53 and p-PTEN, followed by reductions in p-PDK1 and p-mTOR, and the eventual initiation of autophagy. These results are consistent with previous studies , and suggest that 5-FU may suppress the mTOR pathway by up-regulating p53 and PTEN. Chloroquine is known to block the fusion of the autophagosome and lysosome. Therefore, chloroquine pre-treatment appeared to have the opposite effects to 5-FU on PTEN, PDK1, and mTOR expression in this study.
The present study had several limitations. We used only one colon cancer cell line, and did not perform any in vivo expression experiments. In addition, the expression of cell cycle proteins was assessed by western blotting only. More precise results could be obtained by immunoprecipitation and kinase assays. Nevertheless, we have shown that chloroquine potentiates the anti-cancer effects of 5-FU in DLD-1 colon cancer cells by affecting the cell cycle. Our results indicate that chloroquine may be an effective chemotherapy sensitizer, and supplementation of conventional chemotherapy with chloroquine may provide a novel therapeutic modality in colon cancer.