AIM:To investigate whether the expression of kallikrein 12(KLK12) is related to the development of gastric cancer(GC) and to determine the role of KLK12 in gastric cancer cells growth,invasion and migration.METHODS:Be...AIM:To investigate whether the expression of kallikrein 12(KLK12) is related to the development of gastric cancer(GC) and to determine the role of KLK12 in gastric cancer cells growth,invasion and migration.METHODS:Between September 2007 and March 2008,133 patients with histologically confirmed GC were recruited for the study.Expression of KLK12 was detected in samples from GC patients by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry.The relationship between KLK12 protein expression and clinicopathological features of GC was analyzed.The difference in 5-year survival rates between the high KLK12 protein expression group and the low KLK12 expression group was compared.Additionally,the expression of KLK12 was examined in various human GC cell lines,including MKN-28,SGC-7901 and MKN-45.Small interfering RNA(siRNA) was used to inhibit KLK12 expression in MKN-45 cells.Cell clones stably transfected with KLK12 siRNA were tested for KLK12 expression by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting.Furthermore,a series of functional assays were performed in this study to assess the biological features of transfected cells.Cell proliferation was assessed using the methylthiazolyltetrazoliumassay.Finally,cell migration and invasion were assessed using transwell chamber assays.RESULTS:Of the 133 GC patients included in the study,126(94.7%) showed a higher expression level of KLK12 mRNA when compared to noncancerous tissue specimens.Expression of KLK12 mRNA was significantly higher in GC tissues than in normal tissue(P < 0.001).KLK12 protein expression was detected in 96 of 133(72.2%) GC samples with moderate or strong staining primarily in the cytoplasm.In contrast,negative immunostaining for KLK12 protein was observed in the corresponding normal gastric mucosal tissue.Overexpression of KLK12 protein was significantly associated with lymph node metastasis(P = 0.001),histological type(P < 0.001) and tumor-node-metastasis stage(P = 0.005),while no significant correlation was observed between expression of KLK12 protein and sex,age,depth of invasion,tumor size or lymphatic invasion.Furthermore,patients with high KLK12 expression had a significantly poorer 5-year survival rate than those with low KLK12 expression(P = 0.002).Expression of KLK12 mRNA was significantly higher in MKN-45 GC cells compared to normal mucosal cells or two other GC cell lines(P < 0.01).Expression of KLK12 in MKN-45 cells was downregulated after transfection with siRNA.Knockdown of KLK12 markedly decreased the proliferation of MKN-45 cells when compared with parent or mock-transfected cells(P = 0.001),especially from the 3rd to the 5th day of the assay.In migration assays,fewer KLK12 siRNA cells migrated through the chambers(22.00 ± 1.81) when compared to the parent(46.47 ± 2.42) or mock-transfected cells(45.40 ± 1.99);these differences were statistically significant(P < 0.001).However,in the invasion assay,the number of KLK12 siRNA cells that invaded the chambers was 18.40 ± 1.12,closely similar to both the parent(18.67 ± 0.98) and mock-transfected cells(18.53 ± 0.92).There was no significantly difference between the three groups in the invasion assay(P = 0.054).CONCLUSION:The KLK12 gene is markedly overexpressed in GC tissue,and its expression status may be a powerful prognostic indicator for patients with GC.KLK12 might serve as a novel diagnosis and prognosis biomarker in GC.展开更多
AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT...AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by M-I-I- assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 （COX-2） and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction （RT- PCR） was used for mRNA analysis. RESULTS： From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol （0.024-1.504 mmol/L） reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION： One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is upregulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.展开更多
AIM： To study the alterations in p53 gene among Indian gastric cancer patients and to correlate them with the various clinicopathological parameters. METHODS： A total of 103 gastric cancer patients were included in ...AIM： To study the alterations in p53 gene among Indian gastric cancer patients and to correlate them with the various clinicopathological parameters. METHODS： A total of 103 gastric cancer patients were included in this study. The p53 alterations were studied by both immunohistochemical method as well as polymerase chain reaction （PCR）-single strand conformation polymorphism （SSCP） analysis. We only studied four （exon 5, 6, 7, and 8） of the 11 ,p53 exons. The alterations in p53 were also correlated with respect to various clinicopathological parameters. RESULTS： Among 103 cases, p53 over-expression and alteration were detected in 37 （35.92%） and 19 （18.44%） cases, respectively. Most of the ,p53 alterations were found at exon 5 （31.54%）, followed by exon 6 （26.31%）, exon 7 （21.04%） and exon 8 （21.04%）. A significant correlation of p53 overexpression was found with p53 alteration （P = 0.000）. Concordance between ,p53 alteration （as detected by SSCP） and over-expression [as detected by immunohistochemistry （IHC）] was found in 75% cases. We found that IHC-positive/SSCP-negative cases accounted for 21% of cases and IHC-negative/SSCP- positive cases accounted for remaining 4% cases. CONCLUSION： Our results show that p53 gene mutations are significantly correlated with p53 protein over-expression, with 75% concordance in over-expression and alteration in the p53 gene, but 25% disconcordance also cautions against the assumption that p53 over-expression is always associated with a gene mutation. There may be other mechanisms responsible for stabilization and accumulation of p53 protein with no evidence of gene mutation that reflect an accumulation of a non-mutated protein, or a false negative SSCP result.展开更多
基金Supported by Scientific Research Fund from Shanghai Science and Technology Committee,No.08411964200
文摘AIM:To investigate whether the expression of kallikrein 12(KLK12) is related to the development of gastric cancer(GC) and to determine the role of KLK12 in gastric cancer cells growth,invasion and migration.METHODS:Between September 2007 and March 2008,133 patients with histologically confirmed GC were recruited for the study.Expression of KLK12 was detected in samples from GC patients by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry.The relationship between KLK12 protein expression and clinicopathological features of GC was analyzed.The difference in 5-year survival rates between the high KLK12 protein expression group and the low KLK12 expression group was compared.Additionally,the expression of KLK12 was examined in various human GC cell lines,including MKN-28,SGC-7901 and MKN-45.Small interfering RNA(siRNA) was used to inhibit KLK12 expression in MKN-45 cells.Cell clones stably transfected with KLK12 siRNA were tested for KLK12 expression by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting.Furthermore,a series of functional assays were performed in this study to assess the biological features of transfected cells.Cell proliferation was assessed using the methylthiazolyltetrazoliumassay.Finally,cell migration and invasion were assessed using transwell chamber assays.RESULTS:Of the 133 GC patients included in the study,126(94.7%) showed a higher expression level of KLK12 mRNA when compared to noncancerous tissue specimens.Expression of KLK12 mRNA was significantly higher in GC tissues than in normal tissue(P < 0.001).KLK12 protein expression was detected in 96 of 133(72.2%) GC samples with moderate or strong staining primarily in the cytoplasm.In contrast,negative immunostaining for KLK12 protein was observed in the corresponding normal gastric mucosal tissue.Overexpression of KLK12 protein was significantly associated with lymph node metastasis(P = 0.001),histological type(P < 0.001) and tumor-node-metastasis stage(P = 0.005),while no significant correlation was observed between expression of KLK12 protein and sex,age,depth of invasion,tumor size or lymphatic invasion.Furthermore,patients with high KLK12 expression had a significantly poorer 5-year survival rate than those with low KLK12 expression(P = 0.002).Expression of KLK12 mRNA was significantly higher in MKN-45 GC cells compared to normal mucosal cells or two other GC cell lines(P < 0.01).Expression of KLK12 in MKN-45 cells was downregulated after transfection with siRNA.Knockdown of KLK12 markedly decreased the proliferation of MKN-45 cells when compared with parent or mock-transfected cells(P = 0.001),especially from the 3rd to the 5th day of the assay.In migration assays,fewer KLK12 siRNA cells migrated through the chambers(22.00 ± 1.81) when compared to the parent(46.47 ± 2.42) or mock-transfected cells(45.40 ± 1.99);these differences were statistically significant(P < 0.001).However,in the invasion assay,the number of KLK12 siRNA cells that invaded the chambers was 18.40 ± 1.12,closely similar to both the parent(18.67 ± 0.98) and mock-transfected cells(18.53 ± 0.92).There was no significantly difference between the three groups in the invasion assay(P = 0.054).CONCLUSION:The KLK12 gene is markedly overexpressed in GC tissue,and its expression status may be a powerful prognostic indicator for patients with GC.KLK12 might serve as a novel diagnosis and prognosis biomarker in GC.
文摘AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by M-I-I- assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 （COX-2） and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction （RT- PCR） was used for mRNA analysis. RESULTS： From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol （0.024-1.504 mmol/L） reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION： One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is upregulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.
文摘AIM： To study the alterations in p53 gene among Indian gastric cancer patients and to correlate them with the various clinicopathological parameters. METHODS： A total of 103 gastric cancer patients were included in this study. The p53 alterations were studied by both immunohistochemical method as well as polymerase chain reaction （PCR）-single strand conformation polymorphism （SSCP） analysis. We only studied four （exon 5, 6, 7, and 8） of the 11 ,p53 exons. The alterations in p53 were also correlated with respect to various clinicopathological parameters. RESULTS： Among 103 cases, p53 over-expression and alteration were detected in 37 （35.92%） and 19 （18.44%） cases, respectively. Most of the ,p53 alterations were found at exon 5 （31.54%）, followed by exon 6 （26.31%）, exon 7 （21.04%） and exon 8 （21.04%）. A significant correlation of p53 overexpression was found with p53 alteration （P = 0.000）. Concordance between ,p53 alteration （as detected by SSCP） and over-expression [as detected by immunohistochemistry （IHC）] was found in 75% cases. We found that IHC-positive/SSCP-negative cases accounted for 21% of cases and IHC-negative/SSCP- positive cases accounted for remaining 4% cases. CONCLUSION： Our results show that p53 gene mutations are significantly correlated with p53 protein over-expression, with 75% concordance in over-expression and alteration in the p53 gene, but 25% disconcordance also cautions against the assumption that p53 over-expression is always associated with a gene mutation. There may be other mechanisms responsible for stabilization and accumulation of p53 protein with no evidence of gene mutation that reflect an accumulation of a non-mutated protein, or a false negative SSCP result.
