This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, ...This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.展开更多
AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaP...AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test.RESULTS: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.展开更多
AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator prote...AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.展开更多
BACKGROUND Although treatment options for gastric cancer(GC)continue to advance,the overall prognosis for patients with GC remains poor.At present,the predictors of treatment efficacy remain controversial except for h...BACKGROUND Although treatment options for gastric cancer(GC)continue to advance,the overall prognosis for patients with GC remains poor.At present,the predictors of treatment efficacy remain controversial except for high microsatellite instability.AIM To develop methods to identify groups of patients with GC who would benefit the most from receiving the combination of a programmed cell death protein 1(PD-1)inhibitor and chemotherapy.METHODS We acquired data from 63 patients with human epidermal growth factor receptor 2(HER2)-negative GC with a histological diagnosis of GC at the Cancer Hospital,Chinese Academy of Medical Sciences between November 2020 and October 2022.All of the patients screened received a PD-1 inhibitor combined with chemotherapy as the first-line treatment.RESULTS As of July 1,2023,the objective response rate was 61.9%,and the disease control rate was 96.8%.The median progression-free survival(mPFS)for all patients was 6.3 months.The median overall survival was not achieved.Survival analysis showed that patients with a combined positive score(CPS)≥1 exhibited an extended trend in progression-free survival(PFS)when compared to patients with a CPS of 0 after receiving a PD-1 inhibitor combined with oxaliplatin and tegafur as the first-line treatment.PFS exhibited a trend for prolongation as the expression level of HER2 increased.Based on PFS,we divided patients into two groups:A treatment group with excellent efficacy and a treatment group with poor efficacy.The mPFS of the excellent efficacy group was 8 months,with a mPFS of 9.1 months after excluding a cohort of patients who received interrupted therapy due to surgery.The mPFS was 4.5 months in patients in the group with poor efficacy who did not receive surgery.Using good/poor efficacy as the endpoint of our study,univariate analysis revealed that both CPS score(P=0.004)and HER2 expression level(P=0.015)were both factors that exerted significant influence on the efficacy of treatment the combination of a PD-1 inhibitor and chemotherapy in patients with advanced GC(AGC).Finally,multivariate analysis confirmed that CPS score was a significant influencing factor.CONCLUSION CPS score and HER2 expression both impacted the efficacy of immunotherapy combined with chemotherapy in AGC patients who were non-positive for HER2.展开更多
BACKGROUND:KAI1/CD82 has been reported to attenuate the process of metastases in a variety of tumors;however,its mechanism of action in invasion has not been fully elucidated. The present study aimed to investigate th...BACKGROUND:KAI1/CD82 has been reported to attenuate the process of metastases in a variety of tumors;however,its mechanism of action in invasion has not been fully elucidated. The present study aimed to investigate the importance of KAI1 in invasion and its correlation with activation of sphingosine kinase(SPK)in human pancreatic cancer PANC1 and Miapaca-2 cell lines. METHODS:The expression of KAI1 in PANC1 and Miapaca-2 cells,which was mediated by recombinant adenovirus(Ad-KAI1), was assessed by a flow cytometer and Western blotting.After successful infection was established,in vitro growth curve and invasive ability in Boyden Chamber assay were studied.The presence of KAI1 correlating with c-Met and SPK was detected by co-immunoprecipitation and[γ-32P]ATP incorporation. RESULTS:KAI1 genes had no significant effects on the curve representing cell growth.After infection with the KAI1 gene,decreased invasive ability in the Boyden Chamber assay was observed in PANC1 and Miapaca-2 cells that were induced by hepatocyte growth factor.Over-expression of KAI1 in the cells led to the deactivation of SPK and a decreased level of intracellular sphingosine-1-phosphate.No correlation was observed between c-Met and KAI1 during co-immunoprecipitation. CONCLUSION:The results of this study for the first time demonstrated a regulatory role for KAI1 in SPK activation, which leads to decreased invasive ability in disease progression of human pancreatic cancer.展开更多
AIM： To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21^CIP1/WAF1. METHODS： The human gastric cancer...AIM： To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21^CIP1/WAF1. METHODS： The human gastric cancer cell lines were cultured, RNAs were purified, and RT-PCRs were carried out with specifically designed primer for each gene. Among them, the two cell lines SNU-5 and SNU-16 were cultured with activin A for 24, 48 and 72 h. The cell proliferation was measured by MTT assay. For SNU-16, changes in ActRIA, ActRIB, ActRIIA, ActRIIB, Smad2, Smad4, Smad7, and p21^CIP1/WAF1 mRNAs were detected with RT-PCR after the cells were cultured with activin A for 24, 48 and 72 h. RESULTS： The proliferation of SNU-16 cells was down regulated by activin A whereas other cells showed no change. Basal level of inhibin/activin subunits, activin receptors, Smads, and p21^CIP1/WAF1 except for activin βB mRNAs was observed to have differential expression patterns in the human gastric cancer cell lines, AGS, KATO III, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719. Interestingly, significantly higher expressions of ActR IIA and IIB mRNAs were observed in SNU-16 cells when compared to other cells. After activin treatment, ActR IA, IB, and IIA mRNA levels were decreased whereas ActR IIB mRNA level increased in SNU-16 cells. Smad4 mRNA increased for up to 48 h whereas Smad7 mRNA increased sharply at 24 h and returned to the initial level at 48 h in SNU-16 cells. In addition, expression of the p21^CIP1/WAF1 the mitotic inhibitor, peaked at 72 h after activin treatment in SNU-16 cells. CONCLUSION： Our results suggest that inhibition of cell growth by activin is regulated by the negative feedback effect of Smad7 on the activin signaling pathway, and is mediated through p21^CIP1/WAF1 activation in SNU-16 cells.展开更多
文摘This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.
基金Grant-in-aid No. 39970344 and No. 30470798the National Nature Science Foundation, China in 1999 and 2004
文摘AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test.RESULTS: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.
基金Supported by National Institute for Health Research Liverpool Pancreatic Biomedical Research Unit and the Pancreatic Cancer Research Fund (to Nedjadi T)
文摘AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.
基金Supported by Beijing CSCO Clinical Oncology Research Foundation,No.Y-HH202102-0314。
文摘BACKGROUND Although treatment options for gastric cancer(GC)continue to advance,the overall prognosis for patients with GC remains poor.At present,the predictors of treatment efficacy remain controversial except for high microsatellite instability.AIM To develop methods to identify groups of patients with GC who would benefit the most from receiving the combination of a programmed cell death protein 1(PD-1)inhibitor and chemotherapy.METHODS We acquired data from 63 patients with human epidermal growth factor receptor 2(HER2)-negative GC with a histological diagnosis of GC at the Cancer Hospital,Chinese Academy of Medical Sciences between November 2020 and October 2022.All of the patients screened received a PD-1 inhibitor combined with chemotherapy as the first-line treatment.RESULTS As of July 1,2023,the objective response rate was 61.9%,and the disease control rate was 96.8%.The median progression-free survival(mPFS)for all patients was 6.3 months.The median overall survival was not achieved.Survival analysis showed that patients with a combined positive score(CPS)≥1 exhibited an extended trend in progression-free survival(PFS)when compared to patients with a CPS of 0 after receiving a PD-1 inhibitor combined with oxaliplatin and tegafur as the first-line treatment.PFS exhibited a trend for prolongation as the expression level of HER2 increased.Based on PFS,we divided patients into two groups:A treatment group with excellent efficacy and a treatment group with poor efficacy.The mPFS of the excellent efficacy group was 8 months,with a mPFS of 9.1 months after excluding a cohort of patients who received interrupted therapy due to surgery.The mPFS was 4.5 months in patients in the group with poor efficacy who did not receive surgery.Using good/poor efficacy as the endpoint of our study,univariate analysis revealed that both CPS score(P=0.004)and HER2 expression level(P=0.015)were both factors that exerted significant influence on the efficacy of treatment the combination of a PD-1 inhibitor and chemotherapy in patients with advanced GC(AGC).Finally,multivariate analysis confirmed that CPS score was a significant influencing factor.CONCLUSION CPS score and HER2 expression both impacted the efficacy of immunotherapy combined with chemotherapy in AGC patients who were non-positive for HER2.
基金supported by grants from the National Nature Science Foundation of China(39970334 and 30470798)
文摘BACKGROUND:KAI1/CD82 has been reported to attenuate the process of metastases in a variety of tumors;however,its mechanism of action in invasion has not been fully elucidated. The present study aimed to investigate the importance of KAI1 in invasion and its correlation with activation of sphingosine kinase(SPK)in human pancreatic cancer PANC1 and Miapaca-2 cell lines. METHODS:The expression of KAI1 in PANC1 and Miapaca-2 cells,which was mediated by recombinant adenovirus(Ad-KAI1), was assessed by a flow cytometer and Western blotting.After successful infection was established,in vitro growth curve and invasive ability in Boyden Chamber assay were studied.The presence of KAI1 correlating with c-Met and SPK was detected by co-immunoprecipitation and[γ-32P]ATP incorporation. RESULTS:KAI1 genes had no significant effects on the curve representing cell growth.After infection with the KAI1 gene,decreased invasive ability in the Boyden Chamber assay was observed in PANC1 and Miapaca-2 cells that were induced by hepatocyte growth factor.Over-expression of KAI1 in the cells led to the deactivation of SPK and a decreased level of intracellular sphingosine-1-phosphate.No correlation was observed between c-Met and KAI1 during co-immunoprecipitation. CONCLUSION:The results of this study for the first time demonstrated a regulatory role for KAI1 in SPK activation, which leads to decreased invasive ability in disease progression of human pancreatic cancer.
基金Supported by the Research Fund 2003 from the Catholic University of Korea
文摘AIM： To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21^CIP1/WAF1. METHODS： The human gastric cancer cell lines were cultured, RNAs were purified, and RT-PCRs were carried out with specifically designed primer for each gene. Among them, the two cell lines SNU-5 and SNU-16 were cultured with activin A for 24, 48 and 72 h. The cell proliferation was measured by MTT assay. For SNU-16, changes in ActRIA, ActRIB, ActRIIA, ActRIIB, Smad2, Smad4, Smad7, and p21^CIP1/WAF1 mRNAs were detected with RT-PCR after the cells were cultured with activin A for 24, 48 and 72 h. RESULTS： The proliferation of SNU-16 cells was down regulated by activin A whereas other cells showed no change. Basal level of inhibin/activin subunits, activin receptors, Smads, and p21^CIP1/WAF1 except for activin βB mRNAs was observed to have differential expression patterns in the human gastric cancer cell lines, AGS, KATO III, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719. Interestingly, significantly higher expressions of ActR IIA and IIB mRNAs were observed in SNU-16 cells when compared to other cells. After activin treatment, ActR IA, IB, and IIA mRNA levels were decreased whereas ActR IIB mRNA level increased in SNU-16 cells. Smad4 mRNA increased for up to 48 h whereas Smad7 mRNA increased sharply at 24 h and returned to the initial level at 48 h in SNU-16 cells. In addition, expression of the p21^CIP1/WAF1 the mitotic inhibitor, peaked at 72 h after activin treatment in SNU-16 cells. CONCLUSION： Our results suggest that inhibition of cell growth by activin is regulated by the negative feedback effect of Smad7 on the activin signaling pathway, and is mediated through p21^CIP1/WAF1 activation in SNU-16 cells.
