| BackgroundsNowadays, colon cancer becomes one of the common malignant tumors and remains a serious threat to both males and females. In the global scope, the incidence and mortality of colorectal cancer (CRC) showed an upward trend year by year. The occurrence and development of colon cancer is a multistep process and multiple genes are involved in this pathological progress. The gene adenomatous polyposis coli (APC) mutation happened in 85% of the sporadic CRC. APC mutation in the early stage leads to colorectal adenomas and most of CRC are due to colorectal adenomas. The progress of carcinogenesis generally need more than ten years.Interferon-gamma (IFN-γ) is a potential endogenous anti-oncogenic protein and attracts widespread attention as a potential antitumor agent. IFN-γ are involved in many functions, including antitumor, antiviral and antimicrobial responses, regulation of cell cycle, cell differentiation and apoptosis, and modulation of immune responses. A recent study shows that genetic variations in IFN-γ and its receptor subunits (IFNyRl and IFNγR2) are closely associated with the risk of CRC and survival after diagnosis. However, the role of loss of IFN-γ or IFNyR function in the pathogenesis of colon cancer remains unclear. In Part 1 in this study, by using ApcMin/+ mouse model and APC mutant human colon cancer cells, we investigate the role and related molecular mechanism of IFN-y and IFNyRl in the progression of APC-mediated intestinal tumor.Autophagy is an adaptive response to the stress inside and outside of the cell. The cytoplasmic components and organelles, including damaged mitochondria, are degraded by lysosome in order to promote cell survival and restore cell homeostasis. Autophagy related gene-5 (ATG5), an essential ATG protein in autophagy, contributes to the early autophagosome formation and plays an important role in the progress of autophagy. The protein ATG5 is well expressed in normal colon cells, while it is lost in 23% of the patients with CRC. Despite the low incidence, loss of ATG5 might play an important role in the pathogenesis of CRC. Besides, in vitro studies show that knockdown of ATG5 by specific small interfering RNAs (siRNAs) enhances the antitumor efficacy of lots of chemotherapeutic agents. In Part 2, by using ApcMin/+mouse model and APC mutant human colon cancer cells, we investigate the role of loss of ATG5 in the pathogenesis of APC-mediated intestinal tumor and by in vivo studies, further validate the deficiency for ATG5 could sensitize the intestinal tumors to chemotherapies.Part 1 The role of deficiency of IFN-y in the progression of APC-mediated intestinal tumorChapter 1 The effects of deficiency of endogenous IFN-y on intestinal tumor growth in ApcMin/+miceObjective:Our goal in this part was to evaluate the role of deficiency of endogenous IFN-y in intestinal tumor growth in ApcMin/+mice.Methods:The double mutant ApcMin/+IFNy+/" mice are obtained by breeding male ApcMin/+mice with female IFNγ-/- and IFNy+/- mice and by genotyping. Through comparing the number and size of intestinal adenomas in ApcMin/+IFNy+/+ and ApcMin/+IFNγ+/-offsprings, we determined the effects of heterozygous deficiency of IFN-y on intestinal tumor growth in ApcMin/+ mice. Histopathology of intestinal adenomas was examined under a microscope to investigate the progression of intestinal tunors. The serum IFN-y levels in mice were detected by ELISA. Immunohistochemical assay was used to detect the levels and distributions of immune factors in tumor microenvironment. Further examination with western blotting analysis was employed to determine the effects of heterozygous deficiency of IFN-y on IFN-y signaling, Wnt/β-catenin and EGFR/Erkl/2 signaling pathways.Results:At age of 6 months, ApcMin/+IFNγ+/- mice developed a large number of adenomas in the small bowel and colon. Until they were sacrificed, about half of ApcMin/+IFNγ+/- mice died of intestinal obstruction with bloody stools and anal bleeding. Most of the remaining mice were in poor physical shape with dry hair and thin bodies. In contrast, most of the ApcMin/+IFNγ+/+ mice were healthy and active at the end of the experiment. Moreover, on sacrifice, none of the ApcMin/+IFNγ+/+ mice harbored carcinoma. In contrast, by histopathological assay, the polypoid and ulcerated intestinal tumors in 41.7% of the ApcMin/+IFNγ+/- mice showed the presence of adenocarcinomas. ELIS A showed that heterozygous deletion of IFN-y decreased the level of serum IFN-y in ApcMin/+ mice. Immunohistochemical assay showed that deficiency of IFN-y regulated the levels and distributions of CD4, CD8 and F4/80 in tumor microenvironment. Western blotting results revealed that heterozygous deficiency of IFN-γ inhibited the phosphorylation of signal transducer and activator of transcription (STAT1) and blocked IFN-y signal transduction. Heterozygous deficiency of IFN-y also promoted Wnt/β-catenin and EGFR/Erkl/2 signaling pathways in intestinal tumors. Hence, heterozygous deletion of IFN-y significantly promoted the growth of intestinal adenomas and induced adenocarcinoma, which was related to the regulation of EGFR/Erk1/2 and Wnt/β-catenin signaling pathways.Conclusion:Our results support the notion that IFN-y acts as a rate-limiting factor in the development of CRC, uncovering a novel role for them in cancer biology.Chapter 2 The effects of loss of IFNγR1 on the proliferation of HT-29 human colon cancer cellsObjective:To evaluate the effects of loss of IFNγR1 on the proliferation of HT-29 human colon cancer cells.Methods:IFNγR1 was knocked down in HT-29 colon cancer cells by using siRNA and lipofectamineTM 2000 transfection reagent. The level of IFNγR1 protein was detected by using western blotting analysis. The effects of IFNγR1 knockdown on cell growth were determined by using various methods, including MTT assay and colony formation assay. We verified the effects of IFNγR1 knockdown on Wnt/β-catenin and EGFR/Erkl/2 signal pathways in HT-29 cells by using western blotting. Moreover, from the opposite direction, we further verified the effects of IFN-y treatment on the proliferation of HT-29 and HCT-116 colon cancer cells by using MTT assay. The effects of IFN-y on Wnt/p-catenin signaling pathways were also investigated by using western blotting.Results:The siRNA transfection targeting IFNγRl almost knocked down more than 70% of protein expression of IFNγR1, compared with siRNA transfection for negative control (NC). SiRNA transfection for NC had no significant effect on IFNγR1 level, compared with untreated HT-29 cells. IFN-γ treatment significantly inhibited the proliferation of NC knockdown HT29 cells, but IFNyRl knockdown cells were protected from IFN-y-induced anti-proliferation. These results showed that IFN-y signaling was effectively blocked by the knockdown of IFNγR1. Colony formation assay showed that IFNγR1 knockdown significantly increased cell colony formation as compared with knockdown of NC. Western blotting showed that blocking IFNyRl could promote Wnt/β-catenin and EGFR/Erk1/2 signaling pathways. MTT assay revealed that IFN-y strongly inhibited the proliferation of HT-29 cells (carrying mutant APC) in a time dependent manner and however, HCT-116 cells (carrying WT APC) were not sensitive to IFN-y. Western blotting showed that IFN-y treatment induced STAT1 activation and inhibited Wnt/β-catenin signaling pathway in HT-29 cells, which might well explain the inhibition of HT-29 cell proliferation.Conclusion:IFN-y could significantly inhibit the proliferation of APC mutant colon cancer cell HT-29. Knockdown of IFNyRl could promote the colony formation of HT-29 cells, which is related to the regulation of Wnt/β-catenin and EGFR/Erkl/2 signaling pathways.Part 2 The role of deficiency of ATG5 in the progression of APC-mediated intestinal tumorChapter 1 The effects of deficiency of endogenous ATG5 on the growth of intestinal tumor in ApcMin/+ mice (In vivo)Objective:By using transgenic mice, we evaluate the effects of deficiency of endogenous ATG5 on the growth of intestinal tumor in ApcMin/+ mice.Methods:Considering homozygous knockout of ATG5 led to early postnatal lethal in mice, the compound mutant ApcMin/+ATG5+/- mice are obtained by breeding ApcMin/+ mice with ATG5+/- mice and by genotyping. Through comparing the number and size of intestinal adenomas in ApcMin/+ATG5+/+ and ApcMin/+ ATG5+/- offsprings, we determined the effects of heterozygous deficiency of ATG5 on intestinal tumor growth in ApcMin/+ mice. Histopathology of intestinal adenomas was examined under a microscope. We performed western blotting assay in intestinal tumors to investigate the effects of deficiency of ATG5 on autophagy, apoptosis and Wnt/β-catenin and EGFR/Erkl/2 signal pathways.Results:At 4.5 months of age, heterozygous deletion of ATG5 does not significantly impact on the number and size of adenomas of ApcMin/+mice. At the age of 6 months, compared with ApcMin/+ATG5+/+mice, the size of intestinal tumors were increased in mice bearing both APC and ATG5 mutations. The difference of the number of adenomas between ApcMin/+ATG5+/+ and ApcMin/+ATG5+/- mice also reached the statistical significance at the age of 6 months. ATG5 plays a significant role, but is insufficient to cause malignant progression of APC-mediated intestinal tumor. Western blotting showed that heterozygous deficiency of ATG5 had no significant effect on apoptosis and autophagy, but promoted Wnt/β-catenin and EGFR/Erkl/2 signaling pathways.Conclusion:Heterozygous deficiency of ATG5 could promote the growth of intestinal tumors in ApcMin/+ mice, but is insufficient to cause malignant progression of APC-mediated intestinal tumor. The effects are related to the regulation of Wnt/β-catenin and EGFR/Erkl/2 signaling pathways and have nothing to do with autophagy and apoptosis.Chapter 2 The effects of loss of ATG5 on the proliferation of HT-29 human colon cancer cells (In vitro)Objective:To investigate the effects of loss of ATG5 on the proliferation of HT-29 human colon cancer cells.Methods:ATG5 gene was knocked down in HT-29 colon cancer cells by using siRNA and lipofectamineTM 2000 transfection reagent. The level of ATG5 protein was detected by using western blotting analysis. The effects of ATG5 knockdown on cell growth were determined by using MTT and colony formation assay.Results:The siRNA transfection targeting ATG5 almost knocked down more than 90% of protein expression of ATG5, compared with siRNA transfection for NC. SiRNA transfection for NC had no significant effect on ATG5 level, compared with untreated HT-29 cells. MTT assay revealed that ATG5 knockdown evidently inhibited the proliferation of HT-29 colon cancer cells. Colony formation assay showed that ATG5 knockdown significantly reduced cell colony formation as compared with knockdown of NC.Conclusion:Knockdown of ATG5 gene could significantly inhibit the cell proliferation and colony formation of HT-29 human colon cancer cells. We speculated that the differences in gene deletion modes and species of experimental animals might be contributed to the discrepancy between in vitro and in vivo experimental results。Chapter 3 Deficiency of ATG5 enhances the antitumor efficacy of IFN-γ on the growth of intestinal tumor in ApcMin/+miceObjective:To investigate whether deficiency for ATG5 could sensitize tumor to chemotherapies, we compared the antitumor effects of IFN-γ between the ApcMin/+ ATG5+/+ and ApcMin/+ATG5+/- mice.Methods:The compound mutant ApcMin/+ATG5+/- mice are obtained by breeding ApcMin/+ mice with ATG5+/- mice and by genotyping. To investigate whether ATG5 deficiency could sensitize tumors to chemotherapies, we compared the anti-tumor effects of IFN-y between ApcMin/+ ATG5+/+ and ApcMin/+ ATG5+/- mice. We compared the number and size of intestinal adenomas in ApcMin/+ ATG5+/+ and ApcMin/+ ATG5+/-offsprings after IFN-γ treatment. Histopathology of intestinal adenomas was examined under a microscope. By body weight assay, food consumption assay and blood cell assay, we evaluated the toxic reaction of IFN-γ treatment. The protein levels were detected by western blotting assay.Results:IFN-γ was administered by abdomen injection using a 3-month schedule with two treatment months and one intermittent rest month. For early treatment, administration of IFN-y began at age 1.5 months and continued until 4.5 months of age. There were very few adenomas in control mice at age 1.5 months (when early treatment began). Late treatment did not begin until age 3 months, when adenomas already developed, and continued until 6 months of age. Early treatment of ApcMin/+ATG5+/-mice with IFN-y decreased the number, size and incidence rate of adenomas, compared with early treatment of ApcMin/+ ATG5+/- mice with saline. Moreover, early treatment of ApcMin/+ATG5+/- mice with IFN-y led to disappearance of macroscopic tumor nodules. Further histological analysis of intestinal adenomas demonstrated that following early treatment with IFN-γ, ApcMin/+ ATG5+/+ mice formed adenomas with severe dysplasia. However, after early treatment with IFN-y, most of the polypoid area in intestinal mucosa of ApcMin/+ATG5+/- mice showed hyperplastic morphology without obvious dysplasia. Besides, heterozygous deletion of ATG5 also enhanced the efficacy of late treatment with IFN-y. Therefore, deficiency of ATG5 enhances the efficacy of IFN-γ on the suppression of adenomas in ApcMin/+ mice. Treatment with IFN-γ did not cause any apparent decrease in body weight and food consumption and did not induce the decrease of the number of WBCs in the peripheral blood. Moreover, administration of IFN-y resulted in an improvement of bone marrow hematopoiesis as demonstrated by the increase of PLT numbers. Besides, heterozygous deletion of ATG5 enhances the effects of IFN-y-dependent suppression of EGFR/Erkl/2 and Wnt/β-catenin pathways.Conclusion:Heterozygous deletion of ATG5 could sensitize tumors to chemotherapeutic agents, which are consistent with the results from in vitro studies. Our results demonstrate that ATG5 plays important roles in intestinal tumor growth and the combination of IFN-y and ATG5 deficiency or ATG5-targeted inhibition is a promising strategy for prevention and treatment of CRC. |
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