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Volume 17,Issue 4,2010 Table of Contents
2010, 17(4):363-367.
DOI: 10.3872/j.issn.1007-385X.2010.4.001
Abstract:
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite great advances made in diagnosis and treatment of HCC, its prognosis is poor and the mortality rate remains high. Differentiation therapy introduces an attractive concept that may shed new light on HCC treatment. Differentiation therapy has been successfully used in clinical treatment of hematological tumors; and classic example is all-trans retinoic acid in the treatment of acute promyelocytic leukemia, but the clinical use of differentiation therapy in the treatment of malignant solid tumor has been limited. Up to now there have been few HCC-specific differentiation-inducing agents, and their clinical application is limited. Agents that targeting signal transduction pathway molecules or transcriptional factors associated with the differentiation of cancer stem cells may induce the differentiation of HCC. Overexpression of hepatocyte nuclear factor 4α, which is a transcriptional factor closely related to the differentiation of hepatocytes, can effectively induce the differentiation of hepatoma cells, especially liver cancer stem cells. There are lots of aberrant epigenetic changes in the development and progression of tumors, such as altered methylation and acetylation as well as dysregulated expression of microRNA, and some are correlated to the differentiation of tumors. Agents targeting these epigenetic alterations such as inhibitor of histone deacetylase can induce the differentiation of tumors in vitro and in vivo. In all, the cancer stem cell theory and the development of epigenetic may cast new lights on differentiation therapy of HCC.
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite great advances made in diagnosis and treatment of HCC, its prognosis is poor and the mortality rate remains high. Differentiation therapy introduces an attractive concept that may shed new light on HCC treatment. Differentiation therapy has been successfully used in clinical treatment of hematological tumors; and classic example is all-trans retinoic acid in the treatment of acute promyelocytic leukemia, but the clinical use of differentiation therapy in the treatment of malignant solid tumor has been limited. Up to now there have been few HCC-specific differentiation-inducing agents, and their clinical application is limited. Agents that targeting signal transduction pathway molecules or transcriptional factors associated with the differentiation of cancer stem cells may induce the differentiation of HCC. Overexpression of hepatocyte nuclear factor 4α, which is a transcriptional factor closely related to the differentiation of hepatocytes, can effectively induce the differentiation of hepatoma cells, especially liver cancer stem cells. There are lots of aberrant epigenetic changes in the development and progression of tumors, such as altered methylation and acetylation as well as dysregulated expression of microRNA, and some are correlated to the differentiation of tumors. Agents targeting these epigenetic alterations such as inhibitor of histone deacetylase can induce the differentiation of tumors in vitro and in vivo. In all, the cancer stem cell theory and the development of epigenetic may cast new lights on differentiation therapy of HCC.
2010, 17(4):368-373.
DOI: 10.3872/j.issn.1007-385X.2010.4.002
Abstract:
Objective:To compare the serum protein mass spectra between trastuzumab resistant and non-resistant breast cancer patients by SELDI-TOF-MS (surface enhanced laser desorption/ionization-time of flight-mass spectrometry), so as to screen for biomarkers of trastuzumab resistance. Methods: Thirty-five breast cancer patients undergoing trastuzumab therapy in Fujian Tumor Hospital from Jan. 2008 to Oct. 2009 were included in this study. They included 11 trastuzumab resistant patients and 24 non-resistant patients according to clinical trastuzumab resistance standard. Serum protein mass spectrum difference between trastuzumab resistance and non-resistant patients was detected by SELDI-TOF-MS, and a protein peak ratio of 1.5 was used as SELDI-TOF-MS trastuzumab resistance standard. We analyzed the sensibility, specificity, positive and negative predictive values of the SELDI-TOF-MS trastuzumab resistance standard. Results: Trastuzumab resistance assessed by clinical trastuzumab resistance standard was not correlated with ages, clinical stages, lymph node metastases and double-negative expression of ER/PR in breast cancer patients. The expression of serum protein peaks 7 971 Da, 9 284 Da in trastuzumab resistant patients were significantly decreased (P<0.05) by comparing the serum proteomic mass spectra of trastuzumab resistant and non-resistant patients. We then used SELDI-TOF-MS trastuzumab resistant definition to judge trastuzumab resistant and non-resistant patients; the sensibility was 81.82% (9/11), specificity was 83.33% (20/24), positive predictive value was 69.23% (9/13), and negative predictive value was 9091% (20/22) when peak 7 971 Da was used; and the sensibility was 72.73% (8/11), specificity was 79.17% (19/24), positive predictive value was 61.54% (8/13) and negative predictive value was 86.36% (19/22) when peak 9 284 Da was used. Conclusions: Examination of serum protein peaks 7 971 Da and 9 284 Da expression by SELDI-TOF-MS technology in breast cancer patients may be used for predicting trastuzumab resistance.
Objective:To compare the serum protein mass spectra between trastuzumab resistant and non-resistant breast cancer patients by SELDI-TOF-MS (surface enhanced laser desorption/ionization-time of flight-mass spectrometry), so as to screen for biomarkers of trastuzumab resistance. Methods: Thirty-five breast cancer patients undergoing trastuzumab therapy in Fujian Tumor Hospital from Jan. 2008 to Oct. 2009 were included in this study. They included 11 trastuzumab resistant patients and 24 non-resistant patients according to clinical trastuzumab resistance standard. Serum protein mass spectrum difference between trastuzumab resistance and non-resistant patients was detected by SELDI-TOF-MS, and a protein peak ratio of 1.5 was used as SELDI-TOF-MS trastuzumab resistance standard. We analyzed the sensibility, specificity, positive and negative predictive values of the SELDI-TOF-MS trastuzumab resistance standard. Results: Trastuzumab resistance assessed by clinical trastuzumab resistance standard was not correlated with ages, clinical stages, lymph node metastases and double-negative expression of ER/PR in breast cancer patients. The expression of serum protein peaks 7 971 Da, 9 284 Da in trastuzumab resistant patients were significantly decreased (P<0.05) by comparing the serum proteomic mass spectra of trastuzumab resistant and non-resistant patients. We then used SELDI-TOF-MS trastuzumab resistant definition to judge trastuzumab resistant and non-resistant patients; the sensibility was 81.82% (9/11), specificity was 83.33% (20/24), positive predictive value was 69.23% (9/13), and negative predictive value was 9091% (20/22) when peak 7 971 Da was used; and the sensibility was 72.73% (8/11), specificity was 79.17% (19/24), positive predictive value was 61.54% (8/13) and negative predictive value was 86.36% (19/22) when peak 9 284 Da was used. Conclusions: Examination of serum protein peaks 7 971 Da and 9 284 Da expression by SELDI-TOF-MS technology in breast cancer patients may be used for predicting trastuzumab resistance.
2010, 17(4):374-380.
DOI: 10.3872/j.issn.1007-385X.2010.4.003
Abstract:
Objective:To study the effects of TGF-β1 on the growth of cancer stem cells and non-lung cancer stem cells of lung cancer, so as to lay a foundation for studying the molecular regulatory mechanism for proliferation and differentiation of lung cancer stem cells. Methods: Cancer cells were isolated and cultured from human large cell lung cancer specimens using a serum-free medium to establish large cell lung cancer cell line. Biologic characters of lung cancer stem cells were studied by cell sphere formation test and X-ray radiotherapy sensitive assay. The effects of TGF-β1 on the growth of cancer stem cells and non-cancer stem cells of lung cancer were analyzed by flow cytometry and cell growth inhibition test. Results: A large cell lung cancer cell line was established by primary culturing lung cancer tissues. TGF-β1 could inhibit lung cancer cell proliferation and induce them to the shape of mesenchymal cells. When CD44 and CD90 antibodies were co-stained and analyzed by flow cytometry, the lung cancer cells could be divided into 4 populations, i.e. CD44+CD90+, CD44+CD90-, CD44++CD90+ and CD44++CD90- cells. CD44++CD90+cells had the smallest population in the cell line, accounting for 0.4%; they had the strongest cell sphere formation ability and the strongest resistant ability to X-ray radiation among all the 4 populations. TGF-β1 had the weakest suppression effect against CD44++CD90+ cells and the strongest suppression effect against CD44+CD90+ cells. TGF-β1 could gradually increase the ratio of CD44++CD90+ cells when added to the culture medium. After depletion of TGF-β1 from the medium, the ratio of CD44++CD90+ cells gradually decreased again. Conclusion:CD44++CD90+ cells may be the stem cells of large cell lung cancer, and TGF-β1 can enrich lung cancer stem cells in vitro by inhibiting the non-stem cells of lung cancer.
Objective:To study the effects of TGF-β1 on the growth of cancer stem cells and non-lung cancer stem cells of lung cancer, so as to lay a foundation for studying the molecular regulatory mechanism for proliferation and differentiation of lung cancer stem cells. Methods: Cancer cells were isolated and cultured from human large cell lung cancer specimens using a serum-free medium to establish large cell lung cancer cell line. Biologic characters of lung cancer stem cells were studied by cell sphere formation test and X-ray radiotherapy sensitive assay. The effects of TGF-β1 on the growth of cancer stem cells and non-cancer stem cells of lung cancer were analyzed by flow cytometry and cell growth inhibition test. Results: A large cell lung cancer cell line was established by primary culturing lung cancer tissues. TGF-β1 could inhibit lung cancer cell proliferation and induce them to the shape of mesenchymal cells. When CD44 and CD90 antibodies were co-stained and analyzed by flow cytometry, the lung cancer cells could be divided into 4 populations, i.e. CD44+CD90+, CD44+CD90-, CD44++CD90+ and CD44++CD90- cells. CD44++CD90+cells had the smallest population in the cell line, accounting for 0.4%; they had the strongest cell sphere formation ability and the strongest resistant ability to X-ray radiation among all the 4 populations. TGF-β1 had the weakest suppression effect against CD44++CD90+ cells and the strongest suppression effect against CD44+CD90+ cells. TGF-β1 could gradually increase the ratio of CD44++CD90+ cells when added to the culture medium. After depletion of TGF-β1 from the medium, the ratio of CD44++CD90+ cells gradually decreased again. Conclusion:CD44++CD90+ cells may be the stem cells of large cell lung cancer, and TGF-β1 can enrich lung cancer stem cells in vitro by inhibiting the non-stem cells of lung cancer.
4 Enrichment and identification of cancer stem cell-like cells in mouse breast cancer cell line 4T1
WANG Xin-rong
,
SHAN Bao-en
,
AI Jun
,
LIU Li-hua
,
LIU Yue-cai
,
ZHANG Chao
,
ZHANG Hai-pu
,
LIU Deng-xiang
2010, 17(4):381-385.
DOI: 10.3872/j.issn.1007-385X.2010.4.004
Abstract:
Objective:To culture mouse breast cancer 4T1 cells in serum-free medium(SFM), and to screen for and identify the cancer stem-like cells in 4T1 cells. Methods: Breast cancer stem-like cells were enriched from 4T1 cells cultured in SFM containing EGF, bFEGF, and B27, etc. The suspension spheres were seeded in serum-supplement medium (SSM) and cell differentiation was observed. The proportion of cancer stem-like cells in 4T1 cells was determined through cell surface markers CD44+CD24-/low and Hoechst 33342 staining. Tumorigenic abilities of 4T1 cells in different culture conditions were detected by mouse tumorgenesis experiment. Results: 4T1 cells could survive, proliferate and form breast cancer suspension spheres in SFM for prolonged time period. 4T1 spheres seeded into SSM could differentiate and adhere to the culture plates. There were 6.4%-68.9% CD44+CD24-/low cells and 7.3%-61.2% side population (SP) cells in 4T1 spheres, which were significantly higher than those in 4T1 cells cultured in SSM (P<0.05). The ratios of CD44_CD24-/low and SP cells were gradually increased with the passage of 4T1 spheres in SFM. 4T1 spheres with enriched cancer stem cell were more tumorigenic than 4T1 cells cultured in the SSM. Conclusion: 4T1 cells can grow and form spheres in serum-free suspension medium containing growth factor, and they contain breast cancer stem-like cells, which can be enriched when cultured in SFM.
Objective:To culture mouse breast cancer 4T1 cells in serum-free medium(SFM), and to screen for and identify the cancer stem-like cells in 4T1 cells. Methods: Breast cancer stem-like cells were enriched from 4T1 cells cultured in SFM containing EGF, bFEGF, and B27, etc. The suspension spheres were seeded in serum-supplement medium (SSM) and cell differentiation was observed. The proportion of cancer stem-like cells in 4T1 cells was determined through cell surface markers CD44+CD24-/low and Hoechst 33342 staining. Tumorigenic abilities of 4T1 cells in different culture conditions were detected by mouse tumorgenesis experiment. Results: 4T1 cells could survive, proliferate and form breast cancer suspension spheres in SFM for prolonged time period. 4T1 spheres seeded into SSM could differentiate and adhere to the culture plates. There were 6.4%-68.9% CD44+CD24-/low cells and 7.3%-61.2% side population (SP) cells in 4T1 spheres, which were significantly higher than those in 4T1 cells cultured in SSM (P<0.05). The ratios of CD44_CD24-/low and SP cells were gradually increased with the passage of 4T1 spheres in SFM. 4T1 spheres with enriched cancer stem cell were more tumorigenic than 4T1 cells cultured in the SSM. Conclusion: 4T1 cells can grow and form spheres in serum-free suspension medium containing growth factor, and they contain breast cancer stem-like cells, which can be enriched when cultured in SFM.
2010, 17(4):386-391.
DOI: 10.3872/j.issn.1007-385X.2010.4.005
Abstract:
Objective: To construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors, and prepare purified MDA-7/IL-24-HT7 fusion protein, so as to study its cellular localization and apoptosis-inducing effect on tumor cells. Methods: MDA-7/IL-24 gene was amplified by PCR and cloned into vectors containing HaloTag (HT7) to construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors. MDA-7/IL-24-HT7 fusion protein was induced by IPTG and further puified. Cellular localization of MDA-7/IL-24-HT7 fusion protein in tumor cells was monitored by fluorescence-marked HT7 ligands. The effects of MDA-7/IL-24-HT7 fusion protein on growth and apoptosis of tumor cells were detected by MTT and Annexin V-PI staining assays. Results: MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors were successfully constructed. The MDA-7/IL-24-HT7 fusion protein was mainly expressed as inclusion bodies in E.coli BL21, and localized in the endoplasmic reticulum of tumor cells. MDA-7/IL-24-HT7 fusion protein inhibited growth of tumor cells. Apoptosis rates of colon cancer HCT116 cells and hepatic carcinoma SMMC7721 cells were (34.7±1.3)% and (22.1±0.9)%, respectively, after treatment with 1mg/ml MDA-7/IL-24-HT7 for 96 h, which were significantly higher than those of untreated tumor cells (P<0.01). Conclusion: MDA-7/IL-24-HT7 fusion protein containing HaloTag (HT7) can inhibit growth and induce apoptosis of tumor cells.
Objective: To construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors, and prepare purified MDA-7/IL-24-HT7 fusion protein, so as to study its cellular localization and apoptosis-inducing effect on tumor cells. Methods: MDA-7/IL-24 gene was amplified by PCR and cloned into vectors containing HaloTag (HT7) to construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors. MDA-7/IL-24-HT7 fusion protein was induced by IPTG and further puified. Cellular localization of MDA-7/IL-24-HT7 fusion protein in tumor cells was monitored by fluorescence-marked HT7 ligands. The effects of MDA-7/IL-24-HT7 fusion protein on growth and apoptosis of tumor cells were detected by MTT and Annexin V-PI staining assays. Results: MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors were successfully constructed. The MDA-7/IL-24-HT7 fusion protein was mainly expressed as inclusion bodies in E.coli BL21, and localized in the endoplasmic reticulum of tumor cells. MDA-7/IL-24-HT7 fusion protein inhibited growth of tumor cells. Apoptosis rates of colon cancer HCT116 cells and hepatic carcinoma SMMC7721 cells were (34.7±1.3)% and (22.1±0.9)%, respectively, after treatment with 1mg/ml MDA-7/IL-24-HT7 for 96 h, which were significantly higher than those of untreated tumor cells (P<0.01). Conclusion: MDA-7/IL-24-HT7 fusion protein containing HaloTag (HT7) can inhibit growth and induce apoptosis of tumor cells.
2010, 17(4):392-397.
DOI: 10.3872/j.issn.1007-385X.2010.4.006
Abstract:
Objective:To investigate the sensitivity of esophageal squamous cell carcinoma EC9706 cells to rapamycin after silencing mTOR expression by small interfering RNA targeting mTOR (mTOR-siRNA). Methods: EC9706 cells were transfected with mTOR-siRNA and the interference effect was investigated by RT-PCR. EC9706 cells were treated with rapamycin before and after mTOR-siRNA transfection, and the expressions of mTOR and its downstream p70S6K were detected by Western blotting analysis. Cell cycle, apoptosis and proliferation of EC9706 cells were determined by flow cytometry and CCK-8 kit, respectively. Results: mTOR-siRNA down-regulated the expression of mTOR mRNA in EC9706 cells(P<0.05 or P<0.01). Rapamycin inhibited mTOR and phosphorylated p70S6K (p-p70S6K) expressions and increased p70S6K expression in EC9706 cells(all P<0.05), and these effects of rapamycin were further enhanced by mTOR-siRNA transfection (P<0.05). Rapamycin also induced apoptosis, inhibited proliferation and arrested cell cycle in G1 phase of EC9706 cells (all P<0.01), and transfection with mTOR-siRNA significantly promoted these effects of rapamycin in EC9706 cells (P<0.05). Conclusion: mTOR-siRNA can specifically down-regulate mTOR expression in esophageal squamous cell carcinoma EC9706 cells, and increase the sensitivity of EC9706 cells to rapamycin.
Objective:To investigate the sensitivity of esophageal squamous cell carcinoma EC9706 cells to rapamycin after silencing mTOR expression by small interfering RNA targeting mTOR (mTOR-siRNA). Methods: EC9706 cells were transfected with mTOR-siRNA and the interference effect was investigated by RT-PCR. EC9706 cells were treated with rapamycin before and after mTOR-siRNA transfection, and the expressions of mTOR and its downstream p70S6K were detected by Western blotting analysis. Cell cycle, apoptosis and proliferation of EC9706 cells were determined by flow cytometry and CCK-8 kit, respectively. Results: mTOR-siRNA down-regulated the expression of mTOR mRNA in EC9706 cells(P<0.05 or P<0.01). Rapamycin inhibited mTOR and phosphorylated p70S6K (p-p70S6K) expressions and increased p70S6K expression in EC9706 cells(all P<0.05), and these effects of rapamycin were further enhanced by mTOR-siRNA transfection (P<0.05). Rapamycin also induced apoptosis, inhibited proliferation and arrested cell cycle in G1 phase of EC9706 cells (all P<0.01), and transfection with mTOR-siRNA significantly promoted these effects of rapamycin in EC9706 cells (P<0.05). Conclusion: mTOR-siRNA can specifically down-regulate mTOR expression in esophageal squamous cell carcinoma EC9706 cells, and increase the sensitivity of EC9706 cells to rapamycin.
2010, 17(4):398-402.
DOI: 10.3872/j.issn.1007-385X.2010.4.007
Abstract:
Objective:To prepare CD80-streptavidin (CD80-SA) fusion protein and immobilize it on the surface of nasopharyngeal carcinoma CNE2 cells (CD80-SA-CNE2 cells), so as to investigate the effect of CD80-SA-CNE2 cells on cytotoxicity of T cells. Methods: pET21a-CD80-SA-6His expression plasmid was transformed into E. coli BL21 (DE3). The CD80-SA fusion protein was induced by IPTG, purified by Ni-NTA affinity chromatography and refolded by dialysis. The immobilization rate of CD80-SA on CNE2 cell surface was analyzed by flow cytometry, and the effect of CD80-SA-CNE2 cells on cytotoxicity of T cells was detected by LDH assay. Results: CD80-SA fusion protein was successfully prepared and purified. CD80 was lowly expressed on CNE2 cells (\[2.233±0.176\]%). CD80-SA fusion protein was effectively immobilized on the surface of biotinylated-CNE2 cells, with the immobilization rate being 73%. Moreover, CD80-SA-CNE2 cells effectively induced cytotoxicity of T cells; the cytotoxic rates of T cells were (37±3.12)%, (51±263)% and (58±2.47)% at the E∶T ratios of 1∶1, 1∶20 and 1∶40, respectively, which were significantly higher than those of control CNE2 cells (all P<0.01). Conclusion: CD80-SA fusion protein can be effectively immobilized on the surface of biotinylated-CNE2 cells, enhancing the cyctotoxicty of T cells against CNE2 cells.
Objective:To prepare CD80-streptavidin (CD80-SA) fusion protein and immobilize it on the surface of nasopharyngeal carcinoma CNE2 cells (CD80-SA-CNE2 cells), so as to investigate the effect of CD80-SA-CNE2 cells on cytotoxicity of T cells. Methods: pET21a-CD80-SA-6His expression plasmid was transformed into E. coli BL21 (DE3). The CD80-SA fusion protein was induced by IPTG, purified by Ni-NTA affinity chromatography and refolded by dialysis. The immobilization rate of CD80-SA on CNE2 cell surface was analyzed by flow cytometry, and the effect of CD80-SA-CNE2 cells on cytotoxicity of T cells was detected by LDH assay. Results: CD80-SA fusion protein was successfully prepared and purified. CD80 was lowly expressed on CNE2 cells (\[2.233±0.176\]%). CD80-SA fusion protein was effectively immobilized on the surface of biotinylated-CNE2 cells, with the immobilization rate being 73%. Moreover, CD80-SA-CNE2 cells effectively induced cytotoxicity of T cells; the cytotoxic rates of T cells were (37±3.12)%, (51±263)% and (58±2.47)% at the E∶T ratios of 1∶1, 1∶20 and 1∶40, respectively, which were significantly higher than those of control CNE2 cells (all P<0.01). Conclusion: CD80-SA fusion protein can be effectively immobilized on the surface of biotinylated-CNE2 cells, enhancing the cyctotoxicty of T cells against CNE2 cells.
2010, 17(4):403-407.
DOI: 10.3872/j.issn.1007-385X.2010.4.008
Abstract:
Objective:To study the effect of PIN1 （protein interacting with N1MA1） gene on proliferation, cell cycle and tumorigenicity of lung cancer A549 cells by silencing PIN1 gene using RNA interference technique. Methods: The recombinant plasmid expressing short hairpin RNA (shRNA) targeting PIN1 gene was constructed and named pGPU6-GFP-Neo-PIN1. A549 cells were transfected with pGPU6-GFP-Neo-PIN1 and the negative control plasmid (pGPU6-GFP-Neo) by lipofectamine 2 000. Stable cell lines expressing PIN1 shRNA were obtained after G418 screening. Real-time PCR and Western blotting analysis were performed to determine the expressions of PIN1 at mRNA and protein levels, respectively. Proliferation and cell cycle distribution of A549 cells were detected by MTT assay and flow cytometry assay. Meanwhile, the growth of subcutaneously implanted tumors was observed in nude mice after inoculated with A549 cells with PIN1 stably silenced and the control A549 cells. Results: pGPU6-GFP-Neo-PIN1 plasmid vector was successfully constructed and transfected into A549 cells. PIN1 mRNA expression in A549 cells stably transfected with pGPU6-GFP-Neo-PIN1 decreased by 89.3% compared with that in pGPU6-GFP-Neo-transfected A549 cells, and PIN1 protein was also inhibited by pGPU6-GFP-Neo-PIN1 transfection. Proliferation of PIN1 -silenced A549 cells was significantly suppressed (P<0.01), and their cell cycle was arrested in G1 phase. The tumorigenicity of A549 cells in nude mice was inhibited when PIN1 was silenced (P<0.01). Conclusion: pGPU6-GFP-Neo-PIN1 plasmid stably transfecting into lung cancer A549 cells can effectively silence PIN1 gene expression, inhibit cell proliferation, influence cell cycle and inhibit tumorigenicity of A549 cells.
Objective:To study the effect of PIN1 （protein interacting with N1MA1） gene on proliferation, cell cycle and tumorigenicity of lung cancer A549 cells by silencing PIN1 gene using RNA interference technique. Methods: The recombinant plasmid expressing short hairpin RNA (shRNA) targeting PIN1 gene was constructed and named pGPU6-GFP-Neo-PIN1. A549 cells were transfected with pGPU6-GFP-Neo-PIN1 and the negative control plasmid (pGPU6-GFP-Neo) by lipofectamine 2 000. Stable cell lines expressing PIN1 shRNA were obtained after G418 screening. Real-time PCR and Western blotting analysis were performed to determine the expressions of PIN1 at mRNA and protein levels, respectively. Proliferation and cell cycle distribution of A549 cells were detected by MTT assay and flow cytometry assay. Meanwhile, the growth of subcutaneously implanted tumors was observed in nude mice after inoculated with A549 cells with PIN1 stably silenced and the control A549 cells. Results: pGPU6-GFP-Neo-PIN1 plasmid vector was successfully constructed and transfected into A549 cells. PIN1 mRNA expression in A549 cells stably transfected with pGPU6-GFP-Neo-PIN1 decreased by 89.3% compared with that in pGPU6-GFP-Neo-transfected A549 cells, and PIN1 protein was also inhibited by pGPU6-GFP-Neo-PIN1 transfection. Proliferation of PIN1 -silenced A549 cells was significantly suppressed (P<0.01), and their cell cycle was arrested in G1 phase. The tumorigenicity of A549 cells in nude mice was inhibited when PIN1 was silenced (P<0.01). Conclusion: pGPU6-GFP-Neo-PIN1 plasmid stably transfecting into lung cancer A549 cells can effectively silence PIN1 gene expression, inhibit cell proliferation, influence cell cycle and inhibit tumorigenicity of A549 cells.
2010, 17(4):408-413.
DOI: 10.3872/j.issn.1007-385X.2010.4.009
Abstract:
Objective: To construct short-hairpin RNA (shRNA) eukaryotic expression vector targeting CXC chemokine receptor 4 (CXCR4), and to observe its impact on the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells. Methods: The fragments of CXCR4 shRNA were synthesized and cloned into pGCsi-U6-Neo-GFP vector. The recombinant plasmids were transfected into 293T cells and the most effective interfering vector was selected. MDA-MB-231 cells were transfected by liposome assay. The effects of silencing CXCR4 expression by shRNA on the growth, adhesion and migration of MDA-MB-231 cells were determined by CCK8, cell-matrix adhesion and wound healing assays, respectively. Results: The shRNA eukaryotic expression vectors targeting CXCR4 (CXCR4-shRNA) were successfully constructed and transfected into 293T cells. RT-PCR and Western blotting results showed that the maximum inhibitory rate of CXCR4 expression was 81.3%. CXCR4-shRNA transfection significantly inhibited the proliferation of MDA-MB-231 cells (P<0.05) and the adhesion between MDA-MB-231 cells and extracellular matrix (P<0.05). Wound healing experiment showed that the migration distance of MDA-MB-231 cells in CXCR4-shRNA transfection group was significantly lower than those in the control plasmid and the blank control group (P<0.01). Conclusion: CXCR4-shRNA interfering vector can specifically inhibit CXCR4 expression, proliferation, adhesion and migration of MDA-MB-231 cells.
Objective: To construct short-hairpin RNA (shRNA) eukaryotic expression vector targeting CXC chemokine receptor 4 (CXCR4), and to observe its impact on the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells. Methods: The fragments of CXCR4 shRNA were synthesized and cloned into pGCsi-U6-Neo-GFP vector. The recombinant plasmids were transfected into 293T cells and the most effective interfering vector was selected. MDA-MB-231 cells were transfected by liposome assay. The effects of silencing CXCR4 expression by shRNA on the growth, adhesion and migration of MDA-MB-231 cells were determined by CCK8, cell-matrix adhesion and wound healing assays, respectively. Results: The shRNA eukaryotic expression vectors targeting CXCR4 (CXCR4-shRNA) were successfully constructed and transfected into 293T cells. RT-PCR and Western blotting results showed that the maximum inhibitory rate of CXCR4 expression was 81.3%. CXCR4-shRNA transfection significantly inhibited the proliferation of MDA-MB-231 cells (P<0.05) and the adhesion between MDA-MB-231 cells and extracellular matrix (P<0.05). Wound healing experiment showed that the migration distance of MDA-MB-231 cells in CXCR4-shRNA transfection group was significantly lower than those in the control plasmid and the blank control group (P<0.01). Conclusion: CXCR4-shRNA interfering vector can specifically inhibit CXCR4 expression, proliferation, adhesion and migration of MDA-MB-231 cells.
2010, 17(4):414-418.
DOI: 10.3872/j.issn.1007-385X.2010.4.010
Abstract:
Objective: To study the effect of small interfering RNA (siRNA) targeting Rac1b on the expression of angiogenesis-related molecules in gastric cancer AGS cells. Methods: siRNA targeting Rac1b ( Rac1b siRNA) was synthesized and transfected into AGS cells by lipofectamineTM reagent. The effect of Rac1b siRNA on mRNA and protein expressions of Rac1b in AGS cells was examined by RT-PCR and Western blotting analysis. ELISA and Western blotting analysis were used to assess VEGF production in the supernatants of transfected AGS cells and the expressions of angiogenesis-related molecules such as P53, VHL and HIF-1α in AGS cells under hypoxia situation. Results: DNA sequencing analysis confirmed that the sequence of Rac1b siRNA was correct. Transfection of Rac1b siRNA in AGS cells resulted in a specific decrease in Rac1b mRNA and protein expressions, while failed to knock down the expression of its homologous molecular Rac1. The secretion of VEGF in the supernatant of AGS cells transfected with Rac1b siRNA was significantly inhibited, and this inhibition effect was more obvious under hypoxia condition. Meanwhile, Rac1b siRNA down-regulated HIF-1α protein expression and up-regulated the expressions of angiogenesis-related molecules such as P53 and VHL. Conclusion: Rac1b siRNA can inhibit the mRNA and protein expressions of Rac1b in gastric cancer AGS cells; it can also inhibit VEGF production through regulating the expression of angiogenesis-related molecules such as P53, VHL and HIF-1α under hypoxia condition.
Objective: To study the effect of small interfering RNA (siRNA) targeting Rac1b on the expression of angiogenesis-related molecules in gastric cancer AGS cells. Methods: siRNA targeting Rac1b ( Rac1b siRNA) was synthesized and transfected into AGS cells by lipofectamineTM reagent. The effect of Rac1b siRNA on mRNA and protein expressions of Rac1b in AGS cells was examined by RT-PCR and Western blotting analysis. ELISA and Western blotting analysis were used to assess VEGF production in the supernatants of transfected AGS cells and the expressions of angiogenesis-related molecules such as P53, VHL and HIF-1α in AGS cells under hypoxia situation. Results: DNA sequencing analysis confirmed that the sequence of Rac1b siRNA was correct. Transfection of Rac1b siRNA in AGS cells resulted in a specific decrease in Rac1b mRNA and protein expressions, while failed to knock down the expression of its homologous molecular Rac1. The secretion of VEGF in the supernatant of AGS cells transfected with Rac1b siRNA was significantly inhibited, and this inhibition effect was more obvious under hypoxia condition. Meanwhile, Rac1b siRNA down-regulated HIF-1α protein expression and up-regulated the expressions of angiogenesis-related molecules such as P53 and VHL. Conclusion: Rac1b siRNA can inhibit the mRNA and protein expressions of Rac1b in gastric cancer AGS cells; it can also inhibit VEGF production through regulating the expression of angiogenesis-related molecules such as P53, VHL and HIF-1α under hypoxia condition.
2010, 17(4):419-423.
DOI: 10.3872/j.issn.1007-385X.2010.4.011
Abstract:
Objective: To evaluate the clinical efficacy of DC-activated and cytokine-induced killer cells (DCIK) combined with chemotherapy in the treatment of advanced lung cancer patients. Methods: Twenty-one patients, who were diagnosed as having advanced lung cancer in General Hospital of Chinese Armed Police Forces from Sept. 2005 to Oct. 2007, were treated by DCIK combined with systemic chemotherapy (combination therapy group); 20 advanced lung cancer patients treated with chemotherapy alone served as controls. Peripheral blood mononuclear cells (PBMC) were isolated from patients of combination therapy group before chemotherapy, and PBMC were induced to DCIK in vitro. DCIK were administered to patients in the combination group after 2 periods of systemic chemotherapy. The patients in chemotherapy group were treated with 2 periods of systemic chemotherapy alone. Short-term effect, quality of life, immunological indices and survival rates were observed. Results: The short-term effects were not significantly different between the 2 groups, with the clinical efficacy being 42.9% and 40.0%, disease control rates being 66.7% and 60.0% (P>0.05). KPS score was increased in the combination therapy group (P<0.05) after treatment and showed no improvement in the chemotherapy group (P>0.05). The numbers of CD3+CD18+, CD3+CD56+cells in the peripheral blood of combination therapy group were significantly increased (P<0.01) after treatment, while those in the chemotherapy group had no significant change (P>0.05). The 1-year, and 2-year survival rates of the combined therapy group were higher that those of the chemotherapy group (57.1% vs 50.0%, P<0.05; 28.6% vs 15.0％, P<0.01). Conclusion: DCIK combined with chemotherapy shows a better clinical efficacy in treatment of advanced lung cancer, with improved quality of life, immune function and survival rate.
Objective: To evaluate the clinical efficacy of DC-activated and cytokine-induced killer cells (DCIK) combined with chemotherapy in the treatment of advanced lung cancer patients. Methods: Twenty-one patients, who were diagnosed as having advanced lung cancer in General Hospital of Chinese Armed Police Forces from Sept. 2005 to Oct. 2007, were treated by DCIK combined with systemic chemotherapy (combination therapy group); 20 advanced lung cancer patients treated with chemotherapy alone served as controls. Peripheral blood mononuclear cells (PBMC) were isolated from patients of combination therapy group before chemotherapy, and PBMC were induced to DCIK in vitro. DCIK were administered to patients in the combination group after 2 periods of systemic chemotherapy. The patients in chemotherapy group were treated with 2 periods of systemic chemotherapy alone. Short-term effect, quality of life, immunological indices and survival rates were observed. Results: The short-term effects were not significantly different between the 2 groups, with the clinical efficacy being 42.9% and 40.0%, disease control rates being 66.7% and 60.0% (P>0.05). KPS score was increased in the combination therapy group (P<0.05) after treatment and showed no improvement in the chemotherapy group (P>0.05). The numbers of CD3+CD18+, CD3+CD56+cells in the peripheral blood of combination therapy group were significantly increased (P<0.01) after treatment, while those in the chemotherapy group had no significant change (P>0.05). The 1-year, and 2-year survival rates of the combined therapy group were higher that those of the chemotherapy group (57.1% vs 50.0%, P<0.05; 28.6% vs 15.0％, P<0.01). Conclusion: DCIK combined with chemotherapy shows a better clinical efficacy in treatment of advanced lung cancer, with improved quality of life, immune function and survival rate.
2010, 17(4):424-428.
DOI: 10.3872/j.issn.1007-385X.2010.4.012
Abstract:
Objective: To investigate the clinical quality control of PHA-CD3AK cells induced by PHA, anti-CD3 monoclonal antibody and rhIL-2, and their therapeutic effects against malignant tumors. Methods: Fifty-three patients with advanced malignant tumor were from Friendship Hospital of Shaanxi Province. Peripheral blood mononuclear cells (PBMC) were obtained and induced to differentiate into autologous PHA-CD3AK cells by PHA, anti-CD3mAb and rhIL-2. Quality control indices including quantity, viability, cytotoxicity, endotoxin contaminant and infection source of PHA-CD3AK cells were examined, and the immunophenotypes were studied by flow cytometry. The qualified autologous PHA-CD3AK cells were gathered and infused back to the tumor patients intravenously, once every 2 days; each episode included 6 times, with a total of 2 courses. Therapeutic effects and adverse reactions were observed, and the effective and clinical beneficial rates were calculated. Results: The prepared PHA-CD3AK cells met the quality standard of the expected immune activated cells. The ratios of CD3+T, CD4+T, CD8+T and CD16+ CD56+ cells in the peripheral blood were (49.36±9.21)%, (34.85±4.35)%, (29.20±5.12)% and (21.15±6.50)% respectively after treatment with autologous PHA-CD3AK cells, which were significantly higher than those before treatment (all P<0.05). The general symptoms of most patients were obviously improved (43/53), with 6 cases reaching CR, 14 cases reaching PR, 10 cases reaching MR, 14 cases reaching SD, 9 cases reaching progression; the total effective rate was 56.6%, and the clinical beneficial rate was 83.0%. There were no abnormal changes of the related chemical indices or toxicity reaction. Conclusion: Our quality control method for prepared PHA-CD3AK cells is feasible, and they have definite therapeutic effects against malignant tumor and can efficiently improve the immune function of tumor patients.
Objective: To investigate the clinical quality control of PHA-CD3AK cells induced by PHA, anti-CD3 monoclonal antibody and rhIL-2, and their therapeutic effects against malignant tumors. Methods: Fifty-three patients with advanced malignant tumor were from Friendship Hospital of Shaanxi Province. Peripheral blood mononuclear cells (PBMC) were obtained and induced to differentiate into autologous PHA-CD3AK cells by PHA, anti-CD3mAb and rhIL-2. Quality control indices including quantity, viability, cytotoxicity, endotoxin contaminant and infection source of PHA-CD3AK cells were examined, and the immunophenotypes were studied by flow cytometry. The qualified autologous PHA-CD3AK cells were gathered and infused back to the tumor patients intravenously, once every 2 days; each episode included 6 times, with a total of 2 courses. Therapeutic effects and adverse reactions were observed, and the effective and clinical beneficial rates were calculated. Results: The prepared PHA-CD3AK cells met the quality standard of the expected immune activated cells. The ratios of CD3+T, CD4+T, CD8+T and CD16+ CD56+ cells in the peripheral blood were (49.36±9.21)%, (34.85±4.35)%, (29.20±5.12)% and (21.15±6.50)% respectively after treatment with autologous PHA-CD3AK cells, which were significantly higher than those before treatment (all P<0.05). The general symptoms of most patients were obviously improved (43/53), with 6 cases reaching CR, 14 cases reaching PR, 10 cases reaching MR, 14 cases reaching SD, 9 cases reaching progression; the total effective rate was 56.6%, and the clinical beneficial rate was 83.0%. There were no abnormal changes of the related chemical indices or toxicity reaction. Conclusion: Our quality control method for prepared PHA-CD3AK cells is feasible, and they have definite therapeutic effects against malignant tumor and can efficiently improve the immune function of tumor patients.
LIU Yuan-yuan
,
ZHANG Lian-sheng
,
CHAI Ye
,
ZENG Peng-yun
,
YUE Ling-ling
,
WU Chong-yang
,
LI Li-juan
2010, 17(4):429-433.
DOI: 10.3872/j.issn.1007-385X.2010.4.013
Abstract:
Objective: To investigate the influence of PA-MSHA (Pseudomonas aeruginosa with mannose sensitive hemagglutination pili) vaccine on the inhibitory effect of dendritic cells derived from acute myeloid leukemia (AML-DCs) on regulatory T cells (Treg). Methods: AML-DCs were induced with rhGM-CSF and IL-4 and were divided into three groups: control group, PA-MSHA, and TNF-a groups. After 24 h, the morphological features of AML-DCs in different groups were observed; the phenotypes were detected by flow cytometry; and the effect of AML-DCs on T cell proliferation was measured by mixed lymphocyte reaction and MTT assay. CD4+T cells were collected by magnetic bead assay from healthy peripheral blood cells and were incubated with different AML-DCs to induce differentiation of Treg. Then IL-10 and TGF-β were detected in different Treg supernatants by ELISA; CD4 and CD25 expressions on different Treg were determined by flow cytometry; and Foxp3 mRNA expression was examined by RT-PCR. Results: AML-DCs in PA-MSHA and TNF-α groups showed typical dendritic morphology, increased expressions of CDla, CD80, CD83, CD86 and HLA-DR (P<005), and enhanced abilities to induce proliferation of T cells compared with those in the control group (P<0.05). In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3 mRNA in PA-MSHA and TNF-α groups were all significantly lower than those in the control group (P<0.05), and these indices had no differences between PA-MSHA and TNF-α groups. Conclusion: PA-MSHA vaccine can promote the maturation of AML-DC, inhibit the differentiation of Treg from CD4+T cells, and enhance the inhibitory effect of AML-DC on Treg of AML patients.
Objective: To investigate the influence of PA-MSHA (Pseudomonas aeruginosa with mannose sensitive hemagglutination pili) vaccine on the inhibitory effect of dendritic cells derived from acute myeloid leukemia (AML-DCs) on regulatory T cells (Treg). Methods: AML-DCs were induced with rhGM-CSF and IL-4 and were divided into three groups: control group, PA-MSHA, and TNF-a groups. After 24 h, the morphological features of AML-DCs in different groups were observed; the phenotypes were detected by flow cytometry; and the effect of AML-DCs on T cell proliferation was measured by mixed lymphocyte reaction and MTT assay. CD4+T cells were collected by magnetic bead assay from healthy peripheral blood cells and were incubated with different AML-DCs to induce differentiation of Treg. Then IL-10 and TGF-β were detected in different Treg supernatants by ELISA; CD4 and CD25 expressions on different Treg were determined by flow cytometry; and Foxp3 mRNA expression was examined by RT-PCR. Results: AML-DCs in PA-MSHA and TNF-α groups showed typical dendritic morphology, increased expressions of CDla, CD80, CD83, CD86 and HLA-DR (P<005), and enhanced abilities to induce proliferation of T cells compared with those in the control group (P<0.05). In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3 mRNA in PA-MSHA and TNF-α groups were all significantly lower than those in the control group (P<0.05), and these indices had no differences between PA-MSHA and TNF-α groups. Conclusion: PA-MSHA vaccine can promote the maturation of AML-DC, inhibit the differentiation of Treg from CD4+T cells, and enhance the inhibitory effect of AML-DC on Treg of AML patients.
WANG Yu-hong
,
ZHANG Lian-sheng
,
CHAI Ye
,
ZENG Peng-yun
,
SONG Fei-xue
,
YUE Ling-ling
,
LI Li-juan
,
WU Chong-yang
,
YI Liang-cai
,
LIU Ying
2010, 17(4):434-438.
DOI: 10.3872/j.issn.1007-385X.2010.4.014
Abstract:
Objective: To study the effects of lentinan (LNT) on maturation and function of dendritic cells (DCs) of acute myeloid leukemia (AML) patients, so as to explore new ways for leukemia immunotherapy. Methods: Bone marrow mononuclear cells (BMCs) were isolated from AML complete remission (AML-CR) patients, and were induced to differentiate into DCs by GM-CSF and IL-4 for 7 d. DCs were then divided into 3 groups: LPS positive control group, LNT group, and control group. After 48 h, the morphology of DCs was observed by Wright-Giemsa staining in different groups; CD80, CD83, CD86, CD1α, and HLA-DR expressions on DCs were examined by flow cytometry assay; and IL-12 production was determined by ELISA. DCs of AML patients were isolated from human peripheral blood mononuclear cells (PBMC) by magnetic cell sorting (MACS) after LNT therapy, and the concentration of IL-12 in DC supernatants was determined by ELISA. Results: In vitro, LNT-treated DCs showed a typical DC morphology; it concentration-dependently increased the expressions of CD80, CD83, CD86, CD1a and HLA-DR (P＜0.05) and level of IL-12 compared with the control group (P＜0.05). In vivo, IL-12 in the supernatant of DCs of AML patients after LNT therapy was significantly higher than that of untreated patients (P＜0.05). Conclusion: LNT can promote maturation and function of DCs from AML patients in vivo and in vitro, exrting its anti-tumor effect.
Objective: To study the effects of lentinan (LNT) on maturation and function of dendritic cells (DCs) of acute myeloid leukemia (AML) patients, so as to explore new ways for leukemia immunotherapy. Methods: Bone marrow mononuclear cells (BMCs) were isolated from AML complete remission (AML-CR) patients, and were induced to differentiate into DCs by GM-CSF and IL-4 for 7 d. DCs were then divided into 3 groups: LPS positive control group, LNT group, and control group. After 48 h, the morphology of DCs was observed by Wright-Giemsa staining in different groups; CD80, CD83, CD86, CD1α, and HLA-DR expressions on DCs were examined by flow cytometry assay; and IL-12 production was determined by ELISA. DCs of AML patients were isolated from human peripheral blood mononuclear cells (PBMC) by magnetic cell sorting (MACS) after LNT therapy, and the concentration of IL-12 in DC supernatants was determined by ELISA. Results: In vitro, LNT-treated DCs showed a typical DC morphology; it concentration-dependently increased the expressions of CD80, CD83, CD86, CD1a and HLA-DR (P＜0.05) and level of IL-12 compared with the control group (P＜0.05). In vivo, IL-12 in the supernatant of DCs of AML patients after LNT therapy was significantly higher than that of untreated patients (P＜0.05). Conclusion: LNT can promote maturation and function of DCs from AML patients in vivo and in vitro, exrting its anti-tumor effect.
2010, 17(4):439-443.
DOI: 10.3872/j.issn.1007-385X.2010.4.015
Abstract:
Objective: To study the expression of osteopontin (OPN) in breast carcinoma of different molecular subtypes and its clinical significance. Methods： A total of 99 breast cancer samples were collected from patients who were treated during Jan. 2000 to Dec. 2003 in Changhai Hospital. Expression of ER, PR and HER-2 was detected by inmmnohistochemical staining, and the samples were categorized as follows：luminal A, luminal B, HER-2 over-expression subtype, and basal-like subtype. The expression of OPN in all molecular subtypes was detected by immunohistochemical-SP method, then the correlation of OPN expression with patients’ clinical and pathological features was analyzed. Results: According to the expression of ER, PR and HER-2 in 99 breast cancer patients, there were 45 luminal A, 27 luminal B, 14 HER-2 over-expression and 13 basal-like subtypes. The positive rates of OPN in luminal A, luminal B, HER-2 over-expression and basal-like subtypes were 22.2%, 29.6%, 71.4% and 61.5%, respectively. The positive rates of OPN in HER-2 over-expression and basal-like subtypes were significantly higher than those in luminal A and luminal B subtypes (P<0.01). The survival rate of patients with low OPN expression was significantly higher than that of patients with high OPN expression (P<0.01). Conclusion: OPN is highly expressed in basal-like and HER-2 over-expression subtypes of breast cancer, which indicates that OPN may be a useful indicator for prognosis of breast carcinoma.
Objective: To study the expression of osteopontin (OPN) in breast carcinoma of different molecular subtypes and its clinical significance. Methods： A total of 99 breast cancer samples were collected from patients who were treated during Jan. 2000 to Dec. 2003 in Changhai Hospital. Expression of ER, PR and HER-2 was detected by inmmnohistochemical staining, and the samples were categorized as follows：luminal A, luminal B, HER-2 over-expression subtype, and basal-like subtype. The expression of OPN in all molecular subtypes was detected by immunohistochemical-SP method, then the correlation of OPN expression with patients’ clinical and pathological features was analyzed. Results: According to the expression of ER, PR and HER-2 in 99 breast cancer patients, there were 45 luminal A, 27 luminal B, 14 HER-2 over-expression and 13 basal-like subtypes. The positive rates of OPN in luminal A, luminal B, HER-2 over-expression and basal-like subtypes were 22.2%, 29.6%, 71.4% and 61.5%, respectively. The positive rates of OPN in HER-2 over-expression and basal-like subtypes were significantly higher than those in luminal A and luminal B subtypes (P<0.01). The survival rate of patients with low OPN expression was significantly higher than that of patients with high OPN expression (P<0.01). Conclusion: OPN is highly expressed in basal-like and HER-2 over-expression subtypes of breast cancer, which indicates that OPN may be a useful indicator for prognosis of breast carcinoma.
2010, 17(4):444-449.
DOI: 10.3872/j.issn.1007-385X.2010.4.016
Abstract:
Objective: To examine RASSF1A promoter methylation and Cyclin D1 and P53 expressions in colorectal cancer tissues, and to analyze their relationship with the clinicopathologic characteristics of colorectal cancer. Methods: Thirty-seven colorectal cancer and 14 peri-cancer tissue samples were obtained from Changhai Hospital during Aug. 2008 to Aug. 2009. RASSF1A promoter methylation in colorectal cancer tissues was detected by methylation-specific PCR (MSP); expressions of Cyclin D1 and P53 in colorectal cancer tissues were examined by immunohistochemistry assay. The relationship among RASSF1A promoter methylation, Cyclin D1 and P53 expressions and their relationship with clinicpathologic characteristics of colorectal cancer was analyzed. Results: Methylation of RASSF1A promoter was found 23(6216%) of the 37 colorectal cancer tissues and 12 (85.71%)of the 14 peri-cancer tissues; 14 (37.84%) of the 37 colorectal cancer tissues had Cyclin D1 expression and 15 (40.54%) had P53 expression. Cyclin D1 and P53 expressions was negative in 14 peri-cancer tissues. RASSF1A promoter methylation rate in rectum cancer was higher than that in the colon cancer (P<0.05). Cyclin D1 expression was negatively correlated with patient age (P<0.05), and P53 expression was positively correlated with lymph node metastasis (P<0.05); RASSF1A promoter methylation had no relationship with Cyclin D1 or P53 expressions (P>0.05). Conclusion: Combined detection of RASSF1A promoter methylation, Cyclin D1 and P53 expressions may lay a foundation for studying development and progression of colorectal cancer.
Objective: To examine RASSF1A promoter methylation and Cyclin D1 and P53 expressions in colorectal cancer tissues, and to analyze their relationship with the clinicopathologic characteristics of colorectal cancer. Methods: Thirty-seven colorectal cancer and 14 peri-cancer tissue samples were obtained from Changhai Hospital during Aug. 2008 to Aug. 2009. RASSF1A promoter methylation in colorectal cancer tissues was detected by methylation-specific PCR (MSP); expressions of Cyclin D1 and P53 in colorectal cancer tissues were examined by immunohistochemistry assay. The relationship among RASSF1A promoter methylation, Cyclin D1 and P53 expressions and their relationship with clinicpathologic characteristics of colorectal cancer was analyzed. Results: Methylation of RASSF1A promoter was found 23(6216%) of the 37 colorectal cancer tissues and 12 (85.71%)of the 14 peri-cancer tissues; 14 (37.84%) of the 37 colorectal cancer tissues had Cyclin D1 expression and 15 (40.54%) had P53 expression. Cyclin D1 and P53 expressions was negative in 14 peri-cancer tissues. RASSF1A promoter methylation rate in rectum cancer was higher than that in the colon cancer (P<0.05). Cyclin D1 expression was negatively correlated with patient age (P<0.05), and P53 expression was positively correlated with lymph node metastasis (P<0.05); RASSF1A promoter methylation had no relationship with Cyclin D1 or P53 expressions (P>0.05). Conclusion: Combined detection of RASSF1A promoter methylation, Cyclin D1 and P53 expressions may lay a foundation for studying development and progression of colorectal cancer.
2010, 17(4):450-454.
DOI: 10.3872/j.issn.1007-385X.2010.4.017
Abstract:
Objective: To investigate the infiltration and distribution of immunocytes in primary hepatocellular carcinoma (HCC) tissues. Methods: Thirty HCC tissues were obtained from Tumor Hospital of Fujian Province. The expressions of CD3+, CD4+, CD8+, FoxP3+, CD20+, CD56+, and CD68+ cells in liver tumor tissues, liver peri-tumor tissues and non-tumor liver tissues were detected by immunohistochemistry S-P assay. Results: Immunohistochemistry results showed that the numbers of CD3+, CD4+, FoxP3+ cells were highest in liver peri-tumor tissues, less in liver tumor tissues, and least in the corresponding non-tumor liver tissues (P<0.05); the numbers of CD8+, CD56+, CD68+ cells were highest in the liver peri-tumor tissues, less in non-tumor liver tissues, and least in liver tumor tissues (P<0.05); and the CD20+ cell numbers had no significant difference between liver tumor tissues, liver peri-tumor tissues, and non-tumor liver tissues (P>005). CD3+, CD4+, CD8+, CD56+, and CD68+ cells were mainly distributed in the mesenchyma of the liver tumor tissue, and FoxP3+ cells were scattered among the whole tumor tissue. Conclusion: Cytotoxic immunocyte is decreased and the inhibitory immunocyte is increased in the microenvironment of hepatocellular carcinoma, which results in an immunosuppression status in tumor tissues.
Objective: To investigate the infiltration and distribution of immunocytes in primary hepatocellular carcinoma (HCC) tissues. Methods: Thirty HCC tissues were obtained from Tumor Hospital of Fujian Province. The expressions of CD3+, CD4+, CD8+, FoxP3+, CD20+, CD56+, and CD68+ cells in liver tumor tissues, liver peri-tumor tissues and non-tumor liver tissues were detected by immunohistochemistry S-P assay. Results: Immunohistochemistry results showed that the numbers of CD3+, CD4+, FoxP3+ cells were highest in liver peri-tumor tissues, less in liver tumor tissues, and least in the corresponding non-tumor liver tissues (P<0.05); the numbers of CD8+, CD56+, CD68+ cells were highest in the liver peri-tumor tissues, less in non-tumor liver tissues, and least in liver tumor tissues (P<0.05); and the CD20+ cell numbers had no significant difference between liver tumor tissues, liver peri-tumor tissues, and non-tumor liver tissues (P>005). CD3+, CD4+, CD8+, CD56+, and CD68+ cells were mainly distributed in the mesenchyma of the liver tumor tissue, and FoxP3+ cells were scattered among the whole tumor tissue. Conclusion: Cytotoxic immunocyte is decreased and the inhibitory immunocyte is increased in the microenvironment of hepatocellular carcinoma, which results in an immunosuppression status in tumor tissues.
2010, 17(4):455-457.
DOI: 10.3872/j.issn.1007-385X.2010.4.018
Abstract:
目的： 探讨间皮素（mesothelin,MSLN）siRNA慢病毒对人卵巢癌裸鼠腹腔移植瘤的治疗作用。 方法： 利用人卵巢癌SKOV3细胞建立卵巢癌裸鼠腹腔移植瘤模型。实验分为3组：治疗组、阴性对照组和空白对照组。治疗组应用携间皮素siRNA慢病毒液(LV-MSLN-shRNA)对裸鼠模型进行治疗，阴性对照组采用空载体(LV-MSLN-neg)慢病毒液进行治疗，空白对照组注射PBS治疗。从裸鼠生长一般情况、成瘤率、体质量差、瘤体质量和肿瘤转移部位数等方面观察治疗效果， Western blotting法检测移植瘤组织中间皮素蛋白的表达情况。 结果： 用间皮素siRNA慢病毒液对荷瘤裸鼠进行注射的治疗组裸鼠的成瘤率、体质量差、瘤体质量及肿瘤形成部位数均明显低于空白对照组和空载体LV-MSLN-neg治疗组（P<0.01）。注射慢病毒液后瘤体组织中间皮素蛋白表达明显降低（P<0.05）。 结论： 间皮素siRNA慢病毒液对卵巢癌腹腔移植瘤的生长有显著抑制作用。
目的： 探讨间皮素（mesothelin,MSLN）siRNA慢病毒对人卵巢癌裸鼠腹腔移植瘤的治疗作用。 方法： 利用人卵巢癌SKOV3细胞建立卵巢癌裸鼠腹腔移植瘤模型。实验分为3组：治疗组、阴性对照组和空白对照组。治疗组应用携间皮素siRNA慢病毒液(LV-MSLN-shRNA)对裸鼠模型进行治疗，阴性对照组采用空载体(LV-MSLN-neg)慢病毒液进行治疗，空白对照组注射PBS治疗。从裸鼠生长一般情况、成瘤率、体质量差、瘤体质量和肿瘤转移部位数等方面观察治疗效果， Western blotting法检测移植瘤组织中间皮素蛋白的表达情况。 结果： 用间皮素siRNA慢病毒液对荷瘤裸鼠进行注射的治疗组裸鼠的成瘤率、体质量差、瘤体质量及肿瘤形成部位数均明显低于空白对照组和空载体LV-MSLN-neg治疗组（P<0.01）。注射慢病毒液后瘤体组织中间皮素蛋白表达明显降低（P<0.05）。 结论： 间皮素siRNA慢病毒液对卵巢癌腹腔移植瘤的生长有显著抑制作用。
2010, 17(4):458-461.
DOI: 10.3872/j.issn.1007-385X.2010.4.019
Abstract:
目的： 比较针对锌指蛋白A 20 基因不同位点的siRNA对正常人DC成熟度的影响。 方法： 根据A 20 基因序列特点，针对 A20 C端的锌指结构-5区（zinc finger-5,Zif-5）、Zif-2和N端的序列分别设计合成了Si1、Si2和Si3三条siRNA，以脂质体法转染正常人DC，RT-PCR和Western blotting检测DC中 A20 mRNA和蛋白表达水平，流式细胞术检测DC的成熟度。 结果： 成功合成了3条siRNA。RT-PCR和Western blotting检测显示，DC中 A20 在 TNF-α刺激后24 h表达最高;Si1在DC中干扰效果最好,Si2其次。 A20 下调表达促进了DC的成熟。 结论： 针对 A20 锌指结构区域的siRNA能有效下调A20的表达，从而促进DC成熟。
目的： 比较针对锌指蛋白A 20 基因不同位点的siRNA对正常人DC成熟度的影响。 方法： 根据A 20 基因序列特点，针对 A20 C端的锌指结构-5区（zinc finger-5,Zif-5）、Zif-2和N端的序列分别设计合成了Si1、Si2和Si3三条siRNA，以脂质体法转染正常人DC，RT-PCR和Western blotting检测DC中 A20 mRNA和蛋白表达水平，流式细胞术检测DC的成熟度。 结果： 成功合成了3条siRNA。RT-PCR和Western blotting检测显示，DC中 A20 在 TNF-α刺激后24 h表达最高;Si1在DC中干扰效果最好,Si2其次。 A20 下调表达促进了DC的成熟。 结论： 针对 A20 锌指结构区域的siRNA能有效下调A20的表达，从而促进DC成熟。
2010, 17(4):468-472.
DOI: 10.3872/j.issn.1007-385X.2010.4.021
Abstract:
Tumor angiogenesis plays an important role in tumor development and progression, and it is a complex process regulated by many factors. Vascular endothelial growth factor (VEGF) plays a key role in the regulation of tumor angiogenesis, and it is regulated by cell genetic characteristics and many factors in tumor microenvironment at transcriptional, translation and post-translation levels, of which the regulation via transcription factor is the most important one. In this paper, we review the five most important transcription factors（Sp1，HIF-1，AP-1，Stat3 and NF-κB）and their relationship with expression of VEGF and tumors. In fact, downstream targets of these transcription factors, in addition to VEGF, also include many other angiogenesis promoting factors such as platelet-derived growth factor (PDGF) and fibroblast growth factors (FGF). Therefore, biotherapy strategy targeting these transcription factors may provide effective approaches for tumor anti-angiogenesis therapy.
Tumor angiogenesis plays an important role in tumor development and progression, and it is a complex process regulated by many factors. Vascular endothelial growth factor (VEGF) plays a key role in the regulation of tumor angiogenesis, and it is regulated by cell genetic characteristics and many factors in tumor microenvironment at transcriptional, translation and post-translation levels, of which the regulation via transcription factor is the most important one. In this paper, we review the five most important transcription factors（Sp1，HIF-1，AP-1，Stat3 and NF-κB）and their relationship with expression of VEGF and tumors. In fact, downstream targets of these transcription factors, in addition to VEGF, also include many other angiogenesis promoting factors such as platelet-derived growth factor (PDGF) and fibroblast growth factors (FGF). Therefore, biotherapy strategy targeting these transcription factors may provide effective approaches for tumor anti-angiogenesis therapy.
2010, 17(4):473-477.
DOI: 10.3872/j.issn.1007-385X.2010.4.022
Abstract:
Eukaryotic translation initiation factor 4E (eIF4E) is one of the factors closely associated with the malignant tumors. It can specifically bind to the cap structure of mRNA 5′end and plays an important role in regulating the initial stage of protein synthesis. The activity of eIF4E is regulated by the phosphorylation, the phosphorylation of translation inhibitory protein and nuclear factors. EIF4E is highly expressed in many human malignancies and is related to development, invasion and metastasis of tumors. Previous researches about eIF4E have indicated it as a cancer therapeutic target; these researches included those using small interfering RNAs to suppress the expression of eIF4E, drugs to block AKT/mTOR or Raf/MEK/ERK signaling pathways, small molecule inhibitor of 4EG1 to inhibit cap-dependent translation; they inhibit tumor growth through decreasing the levels of eIF4E phosphorylation. EIF4E is expected to become a new target for malignant tumor biotherapy.
Eukaryotic translation initiation factor 4E (eIF4E) is one of the factors closely associated with the malignant tumors. It can specifically bind to the cap structure of mRNA 5′end and plays an important role in regulating the initial stage of protein synthesis. The activity of eIF4E is regulated by the phosphorylation, the phosphorylation of translation inhibitory protein and nuclear factors. EIF4E is highly expressed in many human malignancies and is related to development, invasion and metastasis of tumors. Previous researches about eIF4E have indicated it as a cancer therapeutic target; these researches included those using small interfering RNAs to suppress the expression of eIF4E, drugs to block AKT/mTOR or Raf/MEK/ERK signaling pathways, small molecule inhibitor of 4EG1 to inhibit cap-dependent translation; they inhibit tumor growth through decreasing the levels of eIF4E phosphorylation. EIF4E is expected to become a new target for malignant tumor biotherapy.
2010, 17(4):478-483.
DOI: 10.3872/j.issn.1007-385X.2010.4.023
Abstract:
Radiotherapy combined with chemotherapy is the standard therapy strategy for locally advanced unresectable non-small cell lung cancer (NSCLC); however, this treatment is intolerable for some NSCLC patients and its therapy outcome seems to have reached a “platform”. Epidermal growth factor receptor (EGFR) inhibitors may enhance radiosensitivity of NSCLC cells by increasing the cells in G2/M phase, reducing the cells in S phase, inhibiting phosphorylation of multi proteins in EGFR signal transduction pathway, and inhibiting the proliferation of NSCLC cells. The initial clinical results of radiotherapy or chemoradiotherapy in combination with EGFR-TKI show certain efficacy, but the long-term outcome is uncertain. Gefitinib combined with chemoradiotherapy resulted in a longer median survival and higher 2-year survival rate than the single therapy group; further randomized phase Ⅲ trials are needed to validate the efficacy of the combination treatment. Above all, radiotherapy or chemoradiotherapy combined with EGFR inhibitors has great potential for patients with locally advanced unresectable non-small cell lung cancer and is worthy of further study.
Radiotherapy combined with chemotherapy is the standard therapy strategy for locally advanced unresectable non-small cell lung cancer (NSCLC); however, this treatment is intolerable for some NSCLC patients and its therapy outcome seems to have reached a “platform”. Epidermal growth factor receptor (EGFR) inhibitors may enhance radiosensitivity of NSCLC cells by increasing the cells in G2/M phase, reducing the cells in S phase, inhibiting phosphorylation of multi proteins in EGFR signal transduction pathway, and inhibiting the proliferation of NSCLC cells. The initial clinical results of radiotherapy or chemoradiotherapy in combination with EGFR-TKI show certain efficacy, but the long-term outcome is uncertain. Gefitinib combined with chemoradiotherapy resulted in a longer median survival and higher 2-year survival rate than the single therapy group; further randomized phase Ⅲ trials are needed to validate the efficacy of the combination treatment. Above all, radiotherapy or chemoradiotherapy combined with EGFR inhibitors has great potential for patients with locally advanced unresectable non-small cell lung cancer and is worthy of further study.
2010, 17(4):484-485.
DOI: 10.3872/j.issn.1007-385X.2010.4.024
Abstract:
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- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
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- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.