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@#Objective To precisely knock out MCT1in humancolorectalcancercellline RKO by using CRISPR/Cas9 gene editingtechnique,and to detect its biological function. Methods CRISPRv2-MCT1-KO # 1 and CRISPRv2-MCT1-KO # 2 plasmids weretransfected into RKO cells. The monoclonal cells were selected by the medium containing puromycin after 48 h. The expression of MCT1 was detected by Western blotting and DNA sequencing. The change of expression and distribution of MCT1 protein were detected by immunofluorescence.Intracellular lactic acid level was detected by lactic acid detection kits. Colonyformation assayand CCK-8 assay were performed to detect cell proliferation ability. The potential signaling pathways of MCT1 in colorectal cancer were explored by GSEA software. Results CRISPRv2-MCT1-KO plasmid was well constructed. Western blottingand DNAsequencingresults showedthat MCT1 was successfully knocked outinthe humancolorectal cancer RKOcells. Compared withthecontrol group, MCT1 protein was notobserved onthecell membraneof MCT1-nockoutcells,andtheintracellularlactatelevel was significantlyreduced(P <0.05). The proliferation ability of RKO cells was significantly decreased after MCT1 knockout(P <0.05). GSEA analysis showedthat MCT1 may promotethe occurrence and development ofcolorectal cancerthrough oxidative phosphorylation and MYC signaling pathway. Conclusion The stable MCT1-knockout human colorectal cancer RKO cell line was successfully constructed by applying CRISPR/Cas9 technology, which might become an ffectivetoolforstudyingtherole of MCT1 inthe occurrence and development ofcolorectal cancer.
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Phosphatidylinositol 3-kinase (PI3K) signaling plays an important role in the regulation of cellular lipid metabolism and non-alcoholic fatty liver disease (NAFLD). However, little is known about the role of the regulatory subunits of PI3K in lipid metabolism and NAFLD. In this study, we characterized the functional role of PIK3R3 in fasting-induced hepatic lipid metabolism. In this study, we showed that the overexpression of PIK3R3 promoted hepatic fatty acid oxidation via PIK3R3-induced expression of PPARα, thus improving the fatty liver phenotype in high-fat diet (HFD)-induced mice. By contrast, hepatic PIK3R3 knockout in normal mice led to increased hepatic TG levels. Our study also showed that PIK3R3-induced expression of PPARα was dependent on HNF4α. The novel PIK3R3-HNF4α-PPARα signaling axis plays a significant role in hepatic lipid metabolism. As the activation of PIK3R3 decreased hepatosteatosis, PIK3R3 can be considered a promising novel target for developing NAFLD and metabolic syndrome therapies.
Subject(s)
Animals , Mice , Diet, High-Fat , Fatty Liver , Lipid Metabolism , Non-alcoholic Fatty Liver Disease , Phenotype , Phosphatidylinositol 3-KinaseABSTRACT
Objective To investigate the effect of enteral nutrition tolerance assessment form on the early enteral nutrition tolerance in patients with gastric cancer after operation. Methods According to the admission sequence, 108 patients with gastric cancer were divided into the control group and the experiment group with 54 cases in each group. Enteral nutrition was used in the control group, while enteral nutrition tolerance assessment form was used to evaulate and care patients in experiment group. Finally, the two groups were compared in the anal exhaust time and the rate of complications. Result Anal exhaust in the experiment group was significantly earlier than that in the control group and the rate of complications was significantly smaller than that of the control group (P < 0.05). Conclusions The enteral nutrition tolerance assessment form for systemic evaluation and intervention is effective in improvement of the patients′tolerance to enteral nutrition in early postoperative enteral nutrition support to patients with gastric cancer. It can promote the recovery of patients.
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Objective To explore the function of p53 on regulating the expression of miR-148b in lung cancer cell line PC-9 and its corresponding molecular mechanism and the impact on cell proliferation. Methods Transient transfection of p53 eukaryotic expressing plasmids into lung cancer cell line PC-9 was performed to establish a cell model over-expressing p53. RT-PCR was used to explicit the impact of p53 on the expression of miR-148b. A reporter vector containing miR-148b promoter was used to investigate the function of p53 on regulating the transcription of miR-148b. Low-expressing miR-148b by transfecting its specific inhibitors , a CCK-8 assay was performed to explore the influence of miR-148b on the lung cancer cell proliferation inhibited by p53. Results Over-expression of p53 promoted miR-148b expression in lung cancer cell line PC-9. P53 could increase the luciferase activity driven by miR-148b promoters. Knockdown of miR-148b attenuated the impact of p53 on inhibiting the proliferation of PC-9 cells. Conclusion P53 inhibits the proliferation of lung cancer cell line PC-9 partially depending on miR-148b.
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Objective To investigate the effects of huaier granules on invasion and metastasis of colorectal cancer SW480 cells in vitro, and explore the basic mechanism. Methods The appropriate concentration and duration of huaier granules promoting SW480 cell apoptosis were determined by SubG1 method. Wound healing assay and transwell assay were used to observe the effect of huaier granules on SW480 cell invasion and metastasis. The changes of E-cadherin, twist, snail and vimentin at protein and mRNA levels were examined by Western blotting and Real-Time PCR. Results After treatment with huaier granules at 3. 0 g·L-1 for 36 h, apoptosis of SW480 cells was most significant, and wound healing assay revealed that relative mobility was (31. 36±2. 39)%, compared with (61. 11±1. 09)% in control group (P<0. 01). Number of invaded cells per field of view was (129±12) in treatment group and (354±20) in control group (P<0. 01). After treatment with huaier granules at 3. 0 g·L-1 for 36 h, protein and mRNA levels of E-cadherin were increased, while those of twist, snail and vimentin were decreased. Conclusion Huaier granules can inhibit invasion and metastasis of colorectal cancer SW480 cells in vitro through effectively depressing epithelial-mesenchymal transition.
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Transforming growth factor-β (TGF-β) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF-β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF-β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF-β induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF-β-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I-ε (CKI-ε). CKI inhibition by small molecule D4476 could abrogate TGF-β-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI-ε and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF-β-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.
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Humans , Apoptosis , Genetics , Apoptosis Regulatory Proteins , Bcl-2-Like Protein 11 , Carcinoma, Hepatocellular , Genetics , Pathology , Cell Line, Tumor , Forkhead Box Protein O3 , Forkhead Transcription Factors , Genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms , Genetics , Pathology , Membrane Proteins , Proto-Oncogene Proteins , Threonine , Genetics , Transforming Growth Factor beta , GeneticsABSTRACT
This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohistochemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xenografts were established by subcutaneous injection of PC9 cell suspension (about 5×10(7)/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.
Subject(s)
Humans , Carcinogenesis , Metabolism , Pathology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Cell Line, Tumor , Lung Neoplasms , Metabolism , Pathology , Oligopeptides , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Signal Transduction , PhysiologyABSTRACT
This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohistochemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xenografts were established by subcutaneous injection of PC9 cell suspension (about 5×10(7)/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.
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This study examined the synergetic effect of class IA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-V FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G(0)-G(1) phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wild-type group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.
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This study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.
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DNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.
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Objective:To investigate the effect of retinoid-interferon-induced mortality (GRIM-19) gene on the apoptosis of colon cancer. Methods: A GRIM-19 eukaryotic expression vector (pCMV-Flag-GRIM-19) was constructed and transfected into SW480 cells. Expressions of GRIM-19 and apoptosis-related proteins were detected by Western blotting analysis. Apoptosis of SW480 cells was measured by Annexin-V/PI assay and mitochondrial membrane potential JC-1 staining. Results: The GRIM-19 eukaryotic expression vector pCMV-Flag-GRIM-19 was successfully constructed. Expression of GRIM-19 in SW480 cells was up-regulated and that of apoptosis-related protein Bcl-xl was down-regulated after transfection with pCMV-Flag-GRIM-19. Apoptosis rate was (7.7±1.39)% in SW480 cells transfected with pCMV-Flag empty vector and (15.0 ± 2.52)% in pCMV-Flag-GRIM-19 transfected cells (P<0.05). Mitochondrial membrane potential was decreased in (7.5±2.09)% of pCMV-Flag transfected cells and (17.5±3.07)% of pCMV-Flag-GRIM-19 transfected cells (P<0.05). Conclusion: In vitro GRIM-19 transfection can effectively induce apoptosis of colon cancer SW480 cells.
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In this study, CD133+ subpopulations were isolated from 41 primary colorectal cancer tissues, the proliferation and cell cycle distribution of the cells were examined without in vitro expansion, and then compared to those of cell lines. The detection of CD133 in colorectal cancer tissues, isolation of CD133+ and CD133- epithelial subpopulations, Ki-67/DNA multiparameter assay and cell volume analysis were flow cytometrically conducted. The results showed that Ki-67 expression was correlated with CD133 level in primary cancer tissues, while cell cycle G2/M phase distribution or clinicopathological characteristics was not. In addition, the CD133+ cells showed larger cell volume and higher Ki-67 expression as compared with CD133- cells. But there was no statistically significant difference in G(2)/M phase distribution between the two subpopulations. Our results demonstrated that the CD133+ subpopulation in colorectal cancer tissue contained more actively cycling and proliferating cells, which was not correlated to clinicopathological factors but might contribute to tumor progression and poor clinical outcome.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , AC133 Antigen , Antigens, CD , Metabolism , Cell Cycle , Physiology , Cell Proliferation , Colorectal Neoplasms , Pathology , Glycoproteins , Metabolism , Ki-67 Antigen , Metabolism , Neoplastic Stem Cells , Metabolism , Pathology , Peptides , Metabolism , PrognosisABSTRACT
In this study, CD133+ subpopulations were isolated from 41 primary colorectal cancer tissues, the proliferation and cell cycle distribution of the cells were examined without in vitro expansion, and then compared to those of cell lines. The detection of CD133 in colorectal cancer tissues, isolation of CD133+ and CD133- epithelial subpopulations, Ki-67/DNA multiparameter assay and cell volume analysis were flow cytometrically conducted. The results showed that Ki-67 expression was correlated with CD133 level in primary cancer tissues, while cell cycle G2/M phase distribution or clinicopathological characteristics was not. In addition, the CD133+ cells showed larger cell volume and higher Ki-67 expression as compared with CD133- cells. But there was no statistically significant difference in G(2)/M phase distribution between the two subpopulations. Our results demonstrated that the CD133+ subpopulation in colorectal cancer tissue contained more actively cycling and proliferating cells, which was not correlated to clinicopathological factors but might contribute to tumor progression and poor clinical outcome.
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Little is reported about the role of PTEN gene in the progression and prognosis of GISTs. This study examined the clinical implications of the tumor suppressor gene PTEN as a prognostic factor in the GISTs. Immunohistological staining and immunoblotting were employed to examine the PTEN protein expression, and its association with clinical measures. Clinicopathological features were reviewed by a retrospective examination of medical records. Reduced PTEN expression was significantly associated with tumor diameter, mitotic figure count, metastasis and pathological stage of tumor (P0.05). The 3-year survival rate of the patients with reduced PTEN expression was significantly lower than those with high PTEN expression (P<0.01). These results suggest that the expression of PTEN gene was significantly linked with the progression and metastasis of GISTs and it is an independent prognostic factor.
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Objective To study the mechanism of unscheduled Cyclin B1 expression at G_1 phase which is usually at G_2/M phase.Methods Human peripheral blood lymphocytes(PBL) from healthy volunteers were firstly activated by PHA and then went into cell cycle.The cells were collected at 0,36,48 and 60 h after activation and divided into two parts:one for Cyclins/DNA muhiparameter assay,and another for Post-sorting Western blot.Results After activation by PHA,Cyclin B1 and CDK1 of lymphocytes were expressed at G_1 phase.Conclusion Unscheduled Cyclin B1/CDKl probably contributes to lymphocytes in vivo into cell cycle.
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<p><b>OBJECTIVE</b>To study the effects of sodium butyrate (NaBu) on cell cycle checkpoint and the apoptosis sensitivity in U937 cells.</p><p><b>METHODS</b>Two mutant U937 cell lines, U937-ASPI3K (ATM negative) and U937-pZeosv2(+) (ATM wild-type), were used as the cell model system. Immunoprecipitation and kinase assay were used to examine the p38 MAPK and ERK1 kinase activities. Western blot was used to analyze the phosphorylation of Bad protein.</p><p><b>RESULTS</b>U937-pZeosv2(+) pretreated with NaBu exhibited enhanced apoptotic response in a NaBu dose dependent fashion upon (137)Cs irradiation, which could be abolished by olomoucine (OLM), a p38 MAPK specific inhibitor. On the other hand, Cyclin dependent kinase 2 (CDK2) specific inhibitor CDK2-I and p34cdc2/cyclinB inhibitor alsterpaullone (ALP) failed to block the effects of NaBu. Similar results were also observed in U937-ASPI3K. The effect of irradiation on p38 MAPK and ERK1 was strikingly potentiated by NaBu. Furthermore, inactivation of irradiated Bad protein via phosphorylation on serine 136 was also enhanced.</p><p><b>CONCLUSION</b>NaBu is able to enhance the apoptotic response in U937 cells, which is mediated by p38 MAPK activation but not ATM status.</p>
Subject(s)
Humans , Apoptosis , Butyrates , Pharmacology , Carrier Proteins , Metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , U937 Cells , bcl-Associated Death Protein , p38 Mitogen-Activated Protein KinasesABSTRACT
To study the therapeutic effects of herpes simplex virus thymidine kinase (HSV-TK) gene transferred by the EBV-based expression vector (pDR2) on experimental hepatocellular carcinoma, pDR2-TK gene was delivered into human hepatocellular carcinoma cell line SMMC-7721 by using liposome-mediated transfection technique，and then gene expression was detected by RT-PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model，the antitumor effects of pDR2-TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2-TK /GCV had cytotoxic effect and about 70 % SMMC-7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2-TK gene was significantly smaller than that in control group (P<0.01). On the 10th day the tumor in 3 mice (60 %） disappeared completely after GCV treatment. It is concluded that the pDR2-TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
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To study the therapeutic effects of herpes simplex virus thymidine kinase (HSV-TK) gene transferred by the EBV-based expression vector (pDR2) on experimental hepatocellular carcinoma, pDR2-TK gene was delivered into human hepatocellular carcinoma cell line SMMC-7721 by using liposome-mediated transfection technique，and then gene expression was detected by RT-PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model，the antitumor effects of pDR2-TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2-TK /GCV had cytotoxic effect and about 70 % SMMC-7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2-TK gene was significantly smaller than that in control group (P<0.01). On the 10th day the tumor in 3 mice (60 %） disappeared completely after GCV treatment. It is concluded that the pDR2-TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.