Summary： This study aimed to examine the effect of the 24 N-terminal amino acids （N24） ofp55PIK, a regulatory subunit of phosphatidylinositol 3-kinase （PI3K）, on the endotoxin lipopolysaccharide （LPS）-stimulate...Summary： This study aimed to examine the effect of the 24 N-terminal amino acids （N24） ofp55PIK, a regulatory subunit of phosphatidylinositol 3-kinase （PI3K）, on the endotoxin lipopolysaccharide （LPS）-stimulated release of the cytokines （CKs） by HaCaT cells. The fusion protein, trans-acting activator of transcription （TAT）-N24 （an experimental peptide, EP） containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E.coli. HaCaT cells （a human keratinocyte cell line） was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha （TNF-ct）, interleukin-6 （IL-6） and interleukin-8 （IL-8） released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay （ELISA） and real-time polymerase chain reaction （PCR）. Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells （NF-r,B p65） in HaCaT cells. The expression of the NF-kB inhibitor alpha （Iv, B-a） protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The resillts showed that EP treatment increased TNF-a secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-a, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-a, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-kB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-kB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-kB p65 protein expression was inhibited after the addition of EP. Western blotting showed that Ir, B-a expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. Ir, B-a expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater Ir, B-a expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-a, IL-6, and IL-8, which involves the inhibition of the hydrolysis of Ir, B-a and thereby blockage of the nuclear transloca- tion of NF-kB p65.展开更多
Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative ...Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative study between normal and carcinoma tissues and in different grades of tumor cells differentiation tissue in colorectal cancer patients. Ex-pression of NF-κΒ were assessed by immunohistochemical method using rabbit polyclonal antibodies against human p65 NF-κΒ proteins. Results: There was none or very little expression of NF-κB in non-neoplastic colon epithelial cells, while the expression of it's protein was significantly increased (P < 0.01) in adjacent cancerous cells. Moreover, there was a significant increase in the mean expression of NF-κB-p65 between poorly differentiated malignant epithelial cell and well-differentiated cells (P < 0.05). Conclusion: NF-κB-p65 may play an essential role in colorectal carcinogenesis and may be valuable for diagnosis, evaluating malignancy extent and prognosis.展开更多
基金supported by a grant from the National Natural Science Foundation of China(No.81072431)
文摘Summary： This study aimed to examine the effect of the 24 N-terminal amino acids （N24） ofp55PIK, a regulatory subunit of phosphatidylinositol 3-kinase （PI3K）, on the endotoxin lipopolysaccharide （LPS）-stimulated release of the cytokines （CKs） by HaCaT cells. The fusion protein, trans-acting activator of transcription （TAT）-N24 （an experimental peptide, EP） containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E.coli. HaCaT cells （a human keratinocyte cell line） was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha （TNF-ct）, interleukin-6 （IL-6） and interleukin-8 （IL-8） released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay （ELISA） and real-time polymerase chain reaction （PCR）. Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells （NF-r,B p65） in HaCaT cells. The expression of the NF-kB inhibitor alpha （Iv, B-a） protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The resillts showed that EP treatment increased TNF-a secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-a, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-a, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-kB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-kB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-kB p65 protein expression was inhibited after the addition of EP. Western blotting showed that Ir, B-a expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. Ir, B-a expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater Ir, B-a expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-a, IL-6, and IL-8, which involves the inhibition of the hydrolysis of Ir, B-a and thereby blockage of the nuclear transloca- tion of NF-kB p65.
基金Supported by a grant from the National Science Foundation of China (No. 30973496)
文摘Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative study between normal and carcinoma tissues and in different grades of tumor cells differentiation tissue in colorectal cancer patients. Ex-pression of NF-κΒ were assessed by immunohistochemical method using rabbit polyclonal antibodies against human p65 NF-κΒ proteins. Results: There was none or very little expression of NF-κB in non-neoplastic colon epithelial cells, while the expression of it's protein was significantly increased (P < 0.01) in adjacent cancerous cells. Moreover, there was a significant increase in the mean expression of NF-κB-p65 between poorly differentiated malignant epithelial cell and well-differentiated cells (P < 0.05). Conclusion: NF-κB-p65 may play an essential role in colorectal carcinogenesis and may be valuable for diagnosis, evaluating malignancy extent and prognosis.
