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Volume 15,Issue 4,2008 Table of Contents
2008, 15(4):301-304.
DOI: 10.3872/j.issn.1007-385X.2008.4.001
Abstract:
Aptamers are small singlestranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent nonconventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.
Aptamers are small singlestranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent nonconventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.
2008, 15(4):305-310.
DOI: 10.3872/j.issn.1007-385X.2008.4.002
Abstract:
Although great achievement has been made on the diagnosis and treatment of malignant tumors, metastasis of tumor remains to be the major reason for treatment failure and death of their victims. The development of systematic biology and functional genomics give us a deeper understanding of the nature of metastasis at the molecular level. Based on the consensus reached on the metastasis mechanisms of cancer, including the genetic heterogeneity of cancer, premetastatic niche, epithelialmesenchymal transition, anoikis resistance, angiogenesis and lymphangiogenesis, etc, the targeted therapy at molecular level should be emphasized, the treatment should target the tumor stem cells, and RNAi technique and other techniques should be used in clinical antimetastatic therapy.
Although great achievement has been made on the diagnosis and treatment of malignant tumors, metastasis of tumor remains to be the major reason for treatment failure and death of their victims. The development of systematic biology and functional genomics give us a deeper understanding of the nature of metastasis at the molecular level. Based on the consensus reached on the metastasis mechanisms of cancer, including the genetic heterogeneity of cancer, premetastatic niche, epithelialmesenchymal transition, anoikis resistance, angiogenesis and lymphangiogenesis, etc, the targeted therapy at molecular level should be emphasized, the treatment should target the tumor stem cells, and RNAi technique and other techniques should be used in clinical antimetastatic therapy.
2008, 15(4):311-315.
DOI: 10.3872/j.issn.1007-385X.2008.4.003
Abstract:
Abstract Objective: To investigate the inducing effects of sunitinib malate on expression of NKG2D ligands in nasopharyngeal carcinoma cell ABCG2high CNE2/DDP. Methods:ABCG2highCNE2/DDP cells and AlloNK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of isolated cells and the expression of NKG2Dligands on target cells before and after incubation with sunitinib malate. Then the cytotoxic sensitivity of treated and untreated ABCG2high CNE2/DDP cells to AlloNK cells were measured by LDH releasing assay. Results: The positive rate of ABCG2 in ABCG2highCNE2/DDP cells was (91.40±2.32)%. More than 90% of isolated AlloNK cells were proven to be CD3-CD16+CD56+ cells. The expression of MICA, MICB,ULBP1, ULBP2 and ULBP3 on ABCG2high CNE2/DDP cells incubated with sunitinib malate increased from (2.92±0.33)%, (427±0.33)%, (5.80±0.62)%, (11.10±3.15)%, and (7.75±1.14)% to (89.12±4.56)%, (66.10±222)%, (67.56±4.19)%, (69.37±8.83)%, and (63.28±3.31)%, respectively. At the E∶T ratios of 10∶1 and 20∶1, the cytotoxic sensitivities of ABCG2high CNE2/DDP cells to AlloNK cells increased from (15.32±13.86)% and (27.26±6.81)% to (41.12±4.12)% and (57.25±2.37)%, respectively, after treatment with sunitinib malate, with significantly difference found in the cytotoxic sensitivities of target cells in each group before and after sunitinib malate treatment (F=1558, P＝0.000). Conclusion: Sunitinib malate can upregulate expression of NKG2Dligands (MICA/B, ULBP13) in ABCG2high nasopharyngeal carcinoma cells, which results in higher cytotoxic sensitivity to AlloNK cells.
Abstract Objective: To investigate the inducing effects of sunitinib malate on expression of NKG2D ligands in nasopharyngeal carcinoma cell ABCG2high CNE2/DDP. Methods:ABCG2highCNE2/DDP cells and AlloNK cells were isolated by magnetic activated cell sorting (MACS). Flow cytometry was used to evaluate the purity of isolated cells and the expression of NKG2Dligands on target cells before and after incubation with sunitinib malate. Then the cytotoxic sensitivity of treated and untreated ABCG2high CNE2/DDP cells to AlloNK cells were measured by LDH releasing assay. Results: The positive rate of ABCG2 in ABCG2highCNE2/DDP cells was (91.40±2.32)%. More than 90% of isolated AlloNK cells were proven to be CD3-CD16+CD56+ cells. The expression of MICA, MICB,ULBP1, ULBP2 and ULBP3 on ABCG2high CNE2/DDP cells incubated with sunitinib malate increased from (2.92±0.33)%, (427±0.33)%, (5.80±0.62)%, (11.10±3.15)%, and (7.75±1.14)% to (89.12±4.56)%, (66.10±222)%, (67.56±4.19)%, (69.37±8.83)%, and (63.28±3.31)%, respectively. At the E∶T ratios of 10∶1 and 20∶1, the cytotoxic sensitivities of ABCG2high CNE2/DDP cells to AlloNK cells increased from (15.32±13.86)% and (27.26±6.81)% to (41.12±4.12)% and (57.25±2.37)%, respectively, after treatment with sunitinib malate, with significantly difference found in the cytotoxic sensitivities of target cells in each group before and after sunitinib malate treatment (F=1558, P＝0.000). Conclusion: Sunitinib malate can upregulate expression of NKG2Dligands (MICA/B, ULBP13) in ABCG2high nasopharyngeal carcinoma cells, which results in higher cytotoxic sensitivity to AlloNK cells.
2008, 15(4):316-320.
DOI: 10.3872/j.issn.1007-385X.2008.4.004
Abstract:
Abstract Objective: To study the inhibitory effects of AdODCAdoMetDCas, a recombinant adenovirus with biantisense RNA of ornithine decarboxylase (ODC）and sadenosylmethionine decarboxylase（AdoMetDC）, on cell proliferation and invasion of esophageal cancer cells. Methods: The AdODCAdoMetDCas was used to infect esophageal cancer Eca109 cells. Western blotting was used to examine the protein expression of ODC and AdoMetDC in Eca109 cells. The content of polyamine in Eca109 cells was determined by HPLC. The inhibition on the proliferation of Eca109 cells was investigated by viable cell counting. Matrigel invasion assay was used to study the invasion ability of Eca109 cells. Furthermore, the antitumor effect of AdODCAdoMetDCas was evaluated in a nude mouse xenograft model. Results: Western blotting assay showed that AdODCAdoMetDCas inhibited the expression of ODC and AdoMetDC; HPLC indicated that the contents of putrescine, spermidine and spermine were markedly decreased in Eca109 cells after infection with AdODCAdoMetDCas（P<0.01）. Viable cell counting showed that AdODCAdoMetDCas significantly inhibited the proliferation of Eca109 cells（P<0.01）. Matrigel invasion assay showed that AdODCAdoMetDCas significantly decreased the invasion ability of Eca109 cells in vitro(P<0.01). Meanwhile, experiments with nude mouse xenograft model demonstrated that AdODCAdoMetDCas had significant antitumor ability in vivo （P<0.05, P<0.01）. Conclusion: AdODCAdoMetDCas can significantly inhibit the proliferation and invasion of esophageal cancer, which makes it a potential therapy for the treatment of esophageal cancer.
Abstract Objective: To study the inhibitory effects of AdODCAdoMetDCas, a recombinant adenovirus with biantisense RNA of ornithine decarboxylase (ODC）and sadenosylmethionine decarboxylase（AdoMetDC）, on cell proliferation and invasion of esophageal cancer cells. Methods: The AdODCAdoMetDCas was used to infect esophageal cancer Eca109 cells. Western blotting was used to examine the protein expression of ODC and AdoMetDC in Eca109 cells. The content of polyamine in Eca109 cells was determined by HPLC. The inhibition on the proliferation of Eca109 cells was investigated by viable cell counting. Matrigel invasion assay was used to study the invasion ability of Eca109 cells. Furthermore, the antitumor effect of AdODCAdoMetDCas was evaluated in a nude mouse xenograft model. Results: Western blotting assay showed that AdODCAdoMetDCas inhibited the expression of ODC and AdoMetDC; HPLC indicated that the contents of putrescine, spermidine and spermine were markedly decreased in Eca109 cells after infection with AdODCAdoMetDCas（P<0.01）. Viable cell counting showed that AdODCAdoMetDCas significantly inhibited the proliferation of Eca109 cells（P<0.01）. Matrigel invasion assay showed that AdODCAdoMetDCas significantly decreased the invasion ability of Eca109 cells in vitro(P<0.01). Meanwhile, experiments with nude mouse xenograft model demonstrated that AdODCAdoMetDCas had significant antitumor ability in vivo （P<0.05, P<0.01）. Conclusion: AdODCAdoMetDCas can significantly inhibit the proliferation and invasion of esophageal cancer, which makes it a potential therapy for the treatment of esophageal cancer.
2008, 15(4):321-325.
DOI: 10.3872/j.issn.1007-385X.2008.4.005
Abstract:
Abstract Objective: To screen for functional monoclonal antibody which can inhibit the adhesion between colon cancer cells and human liver sinusoidal endothelial cells (LSECs), and to identify the specific antigen of the screened antibody. Methods: BALB/c mice were immunized with LSECs, the spleen cells of the immunized mice were fused with SP2/0 cells and cultured on methyl cellulose to establish antiLSECs monoclonal antibody library. Functional monoclonal antibodies which inhibited the adherence between colon cancer cells (Ls174 T cells) and LSECs were screened and identified by immunofluorescence, ELISA and adhesive assay. The membrane proteins were extracted from LSECs and the specific antigen recognized by monoclonal antibody was detected by Western blotting assay. Results:Totally 1 440 monoclonal antibody clones were obtained by the fusing spleen cells of immunized mice with SP2/0 cells, 199 of the monoclonal antibodies recognized LSECs; and 20 clones, including 15 IgM type, 3 IgG type, and 2 with the heavy strand undetectable, significantly suppressed the adherence between colon cancer cells and LSECs. One of these clones with the strongest antiadhesive ability (inhibitory rate 51%) significantly inhibited the adhesion between colon cancer cells and HLSECs at 15-200 μg/ml in a dosedependent manner and reacted with antigens of 46 kD. Conclusion: We have successfully established an antiHLSEC monoclonal antibody library and obtained 20 functional monoclonal antibodies which can suppress the adherence of colon cancer cells with LSECs. One adhesive molecule which might mediate colon cancer liver metastasis has been preliminarily identified. These antibodies may help to understand the mechanism of organspecific metastasis of colon cancer cells and shed light on antimetastasis therapy of colon cancer.
Abstract Objective: To screen for functional monoclonal antibody which can inhibit the adhesion between colon cancer cells and human liver sinusoidal endothelial cells (LSECs), and to identify the specific antigen of the screened antibody. Methods: BALB/c mice were immunized with LSECs, the spleen cells of the immunized mice were fused with SP2/0 cells and cultured on methyl cellulose to establish antiLSECs monoclonal antibody library. Functional monoclonal antibodies which inhibited the adherence between colon cancer cells (Ls174 T cells) and LSECs were screened and identified by immunofluorescence, ELISA and adhesive assay. The membrane proteins were extracted from LSECs and the specific antigen recognized by monoclonal antibody was detected by Western blotting assay. Results:Totally 1 440 monoclonal antibody clones were obtained by the fusing spleen cells of immunized mice with SP2/0 cells, 199 of the monoclonal antibodies recognized LSECs; and 20 clones, including 15 IgM type, 3 IgG type, and 2 with the heavy strand undetectable, significantly suppressed the adherence between colon cancer cells and LSECs. One of these clones with the strongest antiadhesive ability (inhibitory rate 51%) significantly inhibited the adhesion between colon cancer cells and HLSECs at 15-200 μg/ml in a dosedependent manner and reacted with antigens of 46 kD. Conclusion: We have successfully established an antiHLSEC monoclonal antibody library and obtained 20 functional monoclonal antibodies which can suppress the adherence of colon cancer cells with LSECs. One adhesive molecule which might mediate colon cancer liver metastasis has been preliminarily identified. These antibodies may help to understand the mechanism of organspecific metastasis of colon cancer cells and shed light on antimetastasis therapy of colon cancer.
2008, 15(4):326-330.
DOI: 10.3872/j.issn.1007-385X.2008.4.006
Abstract:
Abstract Objective: To study the inhibitory effect of demethylating agent 5Aza2′deoxycytidine（5AzaCdR）and trichostatin A (TSA) against human endometrial cancer cell line Ishikawa in vitro. Methods: Ishikawa cells were treated with 5AzaCdR, TSA or a combination of both and the growth curves of cells in each group were obtained by trypanblue exclusion assay and cell counting. The cell apoptosis was examined by Annexin V, cell cycle by flow cytometry, and cleavage of poly ADPribose polymerase (PARP) and activation of caspase8 by Western blotting. The effect of the 2 inhibitors on the survival rates of transplanted tumors, tumor sizes and weights were studied in mouse transplantation model of Ishikawa cells. Results: 5AzaCdR and TSA inhibited the growth of Ishikawa cells in a dose and timedependent manner. 5AzaCdR combined with TSA resulted in greater growth inhibition than they were used alone（P<0.05）. 5AzaCdR and TSA both induced apoptosis of Ishikawa cells, and a combination of the two caused higher apoptosis rate（P<0.05）. 5AzaCdR combined with TSA also induced the G1 phase arrest of Ishikawa cells. Caspase8 activation and cleavage of PARP were confirmed by Western blotting. 5AzaCdR and TSA inhibited the growth of transplanted tumors and reduced the volume and weight of tumors. Conclusion: 5AzaCdR and TSA can inhibit the growth of Ishikawa cells and induce their apoptosis, and combination of both has more potent effect, which might be related to the activation of caspase8 and the cleavage of PARP.
Abstract Objective: To study the inhibitory effect of demethylating agent 5Aza2′deoxycytidine（5AzaCdR）and trichostatin A (TSA) against human endometrial cancer cell line Ishikawa in vitro. Methods: Ishikawa cells were treated with 5AzaCdR, TSA or a combination of both and the growth curves of cells in each group were obtained by trypanblue exclusion assay and cell counting. The cell apoptosis was examined by Annexin V, cell cycle by flow cytometry, and cleavage of poly ADPribose polymerase (PARP) and activation of caspase8 by Western blotting. The effect of the 2 inhibitors on the survival rates of transplanted tumors, tumor sizes and weights were studied in mouse transplantation model of Ishikawa cells. Results: 5AzaCdR and TSA inhibited the growth of Ishikawa cells in a dose and timedependent manner. 5AzaCdR combined with TSA resulted in greater growth inhibition than they were used alone（P<0.05）. 5AzaCdR and TSA both induced apoptosis of Ishikawa cells, and a combination of the two caused higher apoptosis rate（P<0.05）. 5AzaCdR combined with TSA also induced the G1 phase arrest of Ishikawa cells. Caspase8 activation and cleavage of PARP were confirmed by Western blotting. 5AzaCdR and TSA inhibited the growth of transplanted tumors and reduced the volume and weight of tumors. Conclusion: 5AzaCdR and TSA can inhibit the growth of Ishikawa cells and induce their apoptosis, and combination of both has more potent effect, which might be related to the activation of caspase8 and the cleavage of PARP.
2008, 15(4):331-335.
DOI: 10.3872/j.issn.1007-385X.2008.4.007
Abstract:
Abstract Objective:To investigate the antitumor immune responses against renal cell carcinoma induced by dendritic cells (DCs) transfected with survivin gene mediated by recombinant adenovirus.Methods: The DCs derived from human peripheral blood were infected with recombined adenovirus vector carrying the survivin gene. The expression of survivin protein in the infected DCs was examined by Western blotting assay; the surface expression of CD83, MHCⅡ, CD80 and CD86 by flow cytometry (FCM), Interleukin12 (IL12) in the supernatants of DCs and IFNγ released by the cytotoxic T lymphocytes (CTLs) by ELISA, the ability of DCs in proliferating allolymphocytes by mixed lymphocyte reaction (MLR), and specific killing activity of CTLs by MTT assay. Results: The DCs presented mature DCs phenotype after transfection with Adsvv. The expression of survivin protein in transfected DCs was confirmed by Western blotting analysis. The IL12 level in the supernatant of DCs transfected with Adsvv was significantly higher than that transfected with empty vector (AdCMV, P<0.01); and both of them were significantly higher than that in the control group(P<001). During MLR assay the DCs infected and not infected with adenovirus both stimulated allogeneic lymphocyte proliferation, with DCs infected with Adsvv having the strongest stimulating activity (P<0.01). After MLR the DCs infected and not infected with adenovirus both stimulated IFNγ secretion by T lymphocytes, with DCs infected with Adsvv having the strongest secreting activity (P<0.01). The CTL activity of DCs was significantly higher against renal cancer cell line 7860 positive for survivin than against hepatic cancer cell line L02 negative for survivin(P<0.01). DCs in the AdCMV group and empty control group had not cytotoxic effect on 7860 cells. Conclusion: Infection with the recombinant adenovirus encoding survivin can greatly enhance the antigen presenting ability of DCs and subsequently induce survivinspecific CTL activity and antitumor effect.
Abstract Objective:To investigate the antitumor immune responses against renal cell carcinoma induced by dendritic cells (DCs) transfected with survivin gene mediated by recombinant adenovirus.Methods: The DCs derived from human peripheral blood were infected with recombined adenovirus vector carrying the survivin gene. The expression of survivin protein in the infected DCs was examined by Western blotting assay; the surface expression of CD83, MHCⅡ, CD80 and CD86 by flow cytometry (FCM), Interleukin12 (IL12) in the supernatants of DCs and IFNγ released by the cytotoxic T lymphocytes (CTLs) by ELISA, the ability of DCs in proliferating allolymphocytes by mixed lymphocyte reaction (MLR), and specific killing activity of CTLs by MTT assay. Results: The DCs presented mature DCs phenotype after transfection with Adsvv. The expression of survivin protein in transfected DCs was confirmed by Western blotting analysis. The IL12 level in the supernatant of DCs transfected with Adsvv was significantly higher than that transfected with empty vector (AdCMV, P<0.01); and both of them were significantly higher than that in the control group(P<001). During MLR assay the DCs infected and not infected with adenovirus both stimulated allogeneic lymphocyte proliferation, with DCs infected with Adsvv having the strongest stimulating activity (P<0.01). After MLR the DCs infected and not infected with adenovirus both stimulated IFNγ secretion by T lymphocytes, with DCs infected with Adsvv having the strongest secreting activity (P<0.01). The CTL activity of DCs was significantly higher against renal cancer cell line 7860 positive for survivin than against hepatic cancer cell line L02 negative for survivin(P<0.01). DCs in the AdCMV group and empty control group had not cytotoxic effect on 7860 cells. Conclusion: Infection with the recombinant adenovirus encoding survivin can greatly enhance the antigen presenting ability of DCs and subsequently induce survivinspecific CTL activity and antitumor effect.
8 Preparation of an oral DNA vaccine based on VEGFR2 and investigation of its immune effects in mice
2008, 15(4):336-341.
DOI: 10.3872/j.issn.1007-385X.2008.4.008
Abstract:
Abstract Objective: Vascular endothelial growth factor receptor2 plays an important role in tumor growth and metastasis. This study is to prepare an oral VEGFR2 DNA vaccine and to evaluate its immune effects in mice. Methods: The recombinant plasmid pcDNA3.1VR2 targeting the extracellular domains of VEGFR2 was constructed by gene engineering technique. After confirmated by sequencing the pcDNA3.1VR2 vector was transfected into COS7 cells via lipid; the protein expression of VEGFR was identified by Western blotting. Then pcDNA3.1VR2 was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against the extracellular domains of VEGFR2. C57BL/6 mice were immunized with the prepared oral vaccine; the serum level of VEGFR2Ag specific antibody was detected by ELISA. The cytotoxic T lymphocyte (CTL) was detected by modified MTT assay. Results：We successfully prepared VEGFR2 DNA vaccine. ELISA results showed that the VEGFR2specific IgG level in oral vaccine group (1.07±0018) was significantly higher than those of the empty vector group (0.14±0.038) and saline group (0.14±0.033)（F=853.17，P=0.000）. The titer of serum antibody increased with the immunization period and the immunization times. As shown by MTT assay, the prepared oral vaccine had specific and efficient cytotoxicity against murine endothelium cells （Ms1） in a dosedependent manner in vitro. The CTL activity in the oral vaccine group (89.38±1.51) was significantly higher than those of the empty vector group (12.99±4.31) and saline group (15.17±2.54) （F=315.42，P=0.000）．Conclusion：We have successfully prepared an oral VEGFR-2 DNA vaccine which can stimulate specific cellular and humoral immune responses in mice, paving a way for further anticancer research.
Abstract Objective: Vascular endothelial growth factor receptor2 plays an important role in tumor growth and metastasis. This study is to prepare an oral VEGFR2 DNA vaccine and to evaluate its immune effects in mice. Methods: The recombinant plasmid pcDNA3.1VR2 targeting the extracellular domains of VEGFR2 was constructed by gene engineering technique. After confirmated by sequencing the pcDNA3.1VR2 vector was transfected into COS7 cells via lipid; the protein expression of VEGFR was identified by Western blotting. Then pcDNA3.1VR2 was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against the extracellular domains of VEGFR2. C57BL/6 mice were immunized with the prepared oral vaccine; the serum level of VEGFR2Ag specific antibody was detected by ELISA. The cytotoxic T lymphocyte (CTL) was detected by modified MTT assay. Results：We successfully prepared VEGFR2 DNA vaccine. ELISA results showed that the VEGFR2specific IgG level in oral vaccine group (1.07±0018) was significantly higher than those of the empty vector group (0.14±0.038) and saline group (0.14±0.033)（F=853.17，P=0.000）. The titer of serum antibody increased with the immunization period and the immunization times. As shown by MTT assay, the prepared oral vaccine had specific and efficient cytotoxicity against murine endothelium cells （Ms1） in a dosedependent manner in vitro. The CTL activity in the oral vaccine group (89.38±1.51) was significantly higher than those of the empty vector group (12.99±4.31) and saline group (15.17±2.54) （F=315.42，P=0.000）．Conclusion：We have successfully prepared an oral VEGFR-2 DNA vaccine which can stimulate specific cellular and humoral immune responses in mice, paving a way for further anticancer research.
2008, 15(4):342-346.
DOI: 10.3872/j.issn.1007-385X.2008.4.009
Abstract:
Abstract Objective: To construct the recombinant DNA vaccine by fusing cEGFR with IgG Fc, and to observe the inhibitory effect of the constructed DNA vaccine against the transplanted melanoma in mice. Methods: The extracellular domain of xenogeneic chicken epidermal growth factor receptor (cEGFR) was fused with rabbit IgG Fc; and the product was insert into PVAX1 eukaryotic expression vector to construct the recombinant pVAX1/cEGFRrFc DNA vaccine. Expression of the fusion protein in melanoma B16 cells was detected by immunofluorescent assay after transfection. In vivo expression of the fusion protein in immunized C57BL/6J mice was detected by Western blotting assay. The DNA vaccines were used to immunize C57 mice 3 times before transplantation of B16 melanoma cells . The weights of tumors were determined after 14 d to calculate the antitumor rate and the survival rate of the mice was observed. The serum level of the specific antibody against human EGFR was determined by ELISA. Subgroups of spleen T lymphocytes were examined by FACS. Results: The eukaryotic expression vector pVAX1/ cEGFRrFc was successfully constructed. Effective expression of fusion protein was detectable in B16 cells after transfection with recombinant plasmid and stable expression was detected in mice after vaccine immunization. The fused DNA vaccine inhibited the growth of transplanted melanoma, with the inhibitory rate being 54% (P<0.01). The vaccine also prolonged the survival period of the mice transplanted with B16 cancer cells (the survival rate was 40% 30 d after inoculation). The vaccine also induced production of specific antibody against human EGFR (1∶1 000) and T cell immunoresponse (The CD8+ subgroups of spleen T lymphocyte were significantly increased \[P<0.01\]). Conclusions: The DNA vaccine containing cEGFR and IgG Fc is an effective antitumor vaccine against EGFRpositive tumor.
Abstract Objective: To construct the recombinant DNA vaccine by fusing cEGFR with IgG Fc, and to observe the inhibitory effect of the constructed DNA vaccine against the transplanted melanoma in mice. Methods: The extracellular domain of xenogeneic chicken epidermal growth factor receptor (cEGFR) was fused with rabbit IgG Fc; and the product was insert into PVAX1 eukaryotic expression vector to construct the recombinant pVAX1/cEGFRrFc DNA vaccine. Expression of the fusion protein in melanoma B16 cells was detected by immunofluorescent assay after transfection. In vivo expression of the fusion protein in immunized C57BL/6J mice was detected by Western blotting assay. The DNA vaccines were used to immunize C57 mice 3 times before transplantation of B16 melanoma cells . The weights of tumors were determined after 14 d to calculate the antitumor rate and the survival rate of the mice was observed. The serum level of the specific antibody against human EGFR was determined by ELISA. Subgroups of spleen T lymphocytes were examined by FACS. Results: The eukaryotic expression vector pVAX1/ cEGFRrFc was successfully constructed. Effective expression of fusion protein was detectable in B16 cells after transfection with recombinant plasmid and stable expression was detected in mice after vaccine immunization. The fused DNA vaccine inhibited the growth of transplanted melanoma, with the inhibitory rate being 54% (P<0.01). The vaccine also prolonged the survival period of the mice transplanted with B16 cancer cells (the survival rate was 40% 30 d after inoculation). The vaccine also induced production of specific antibody against human EGFR (1∶1 000) and T cell immunoresponse (The CD8+ subgroups of spleen T lymphocyte were significantly increased \[P<0.01\]). Conclusions: The DNA vaccine containing cEGFR and IgG Fc is an effective antitumor vaccine against EGFRpositive tumor.
2008, 15(4):347-350.
DOI: 10.3872/j.issn.1007-385X.2008.4.010
Abstract:
Abstract Objective: To establish a glioma cell line U251 stably expressing programmed cell death 4(PDCD4) gene, and to observe the influence of exogenous PDCD4gene on the proliferation and cell cycle of U251 cells.Methods: Recombinant eukaryotic expression vector pEGFPPDCD4 was transfected into human glioma cell line U251 by Lipofectamine 2000, and the U251 cells stably expressing PDCD4 were established by G418 selection. Reverse transcription polymerase chain reaction (RTPCR) and Western blotting were employed to detect the expression ofPDCD4 mRNA and protein. Furthermore, cell proliferation and colony forming ability were determined by cell counting and colony formation assay; the cell cycle was detected by FACS. Results: High expression of PDCD4 mRNA and protein was observed in U251 cells transfected with pEGFPPDCD4, whereas no PDCD4 mRNA and protein expression was detected in the nontransfected and vectortransfected cells. Further, cells transfected with pEGFPPDCD4 grew more slowly and had lower colony formation rate than cells of the other two control groups(P<0.01). Moreover, transfection of PDCD4 significantly reduced the number of cells at the G2/M phase and increased the cells at the S phase(P<0.05). Conclusion: Tumor suppressor gene PDCD4 can effectively inhibit cell proliferation and colony formation by altering their cell cycle.
Abstract Objective: To establish a glioma cell line U251 stably expressing programmed cell death 4(PDCD4) gene, and to observe the influence of exogenous PDCD4gene on the proliferation and cell cycle of U251 cells.Methods: Recombinant eukaryotic expression vector pEGFPPDCD4 was transfected into human glioma cell line U251 by Lipofectamine 2000, and the U251 cells stably expressing PDCD4 were established by G418 selection. Reverse transcription polymerase chain reaction (RTPCR) and Western blotting were employed to detect the expression ofPDCD4 mRNA and protein. Furthermore, cell proliferation and colony forming ability were determined by cell counting and colony formation assay; the cell cycle was detected by FACS. Results: High expression of PDCD4 mRNA and protein was observed in U251 cells transfected with pEGFPPDCD4, whereas no PDCD4 mRNA and protein expression was detected in the nontransfected and vectortransfected cells. Further, cells transfected with pEGFPPDCD4 grew more slowly and had lower colony formation rate than cells of the other two control groups(P<0.01). Moreover, transfection of PDCD4 significantly reduced the number of cells at the G2/M phase and increased the cells at the S phase(P<0.05). Conclusion: Tumor suppressor gene PDCD4 can effectively inhibit cell proliferation and colony formation by altering their cell cycle.
11 Tumor suppressor gene OVCA1 inhibits migration and invasion of ovarian cancer cells A2780 in vitro
2008, 15(4):351-355.
DOI: 10.3872/j.issn.1007-385X.2008.4.011
Abstract:
Abstract Objective:To investigate the inhibitory effects of tumor suppressor gene OVCA1, also named diphthamide synthesis protein 2like(DPH2L), on migration and invasion of ovarian cancer cell A2780 in vitro. Methods: Ovarian cancer cell line A2780 was transfected with GFPtaggedOVCA1 vector or empty vector via Lipofetamine 2000. Cells stably expressing GFPtaggedOVCA1 (A2780OVCA1) or GFP (A2780GFP) were screened by G418. Stably transfected monoclones were obtained by limiting dilution. GFP protein in the cells was examined by fluorescence microscopy and OVCA1 fragment was examined by RTPCR. Wound healing assay, transwell migration assay and matrigel invasion assay were employed to study the effects of OVCA1 on A2780 cell migration and invasion, while taking A2780GFP and A2780 cells as controls. Results:The cell line stably expressing GFPtagged 〖STBX〗OVCA1〖STBZ〗 (A2780OVCA1) was successfully established. The migration speed of cells in A2780OVCA1 group was significantly lower than those of the other 2 groups (P<0.05). Cells passing the pores of transwell filter was less in A2780OVCA1 cells than in the cells of the 2 control groups (P<0.05). Cells passing matrigelcoated filter was significantly decreased in A2780OVCA1 group than in the 2 control groups (P<0.05). Conclusion: OVCA1 can effectively inhibit A2780 cell migration and invasion in vitro.
Abstract Objective:To investigate the inhibitory effects of tumor suppressor gene OVCA1, also named diphthamide synthesis protein 2like(DPH2L), on migration and invasion of ovarian cancer cell A2780 in vitro. Methods: Ovarian cancer cell line A2780 was transfected with GFPtaggedOVCA1 vector or empty vector via Lipofetamine 2000. Cells stably expressing GFPtaggedOVCA1 (A2780OVCA1) or GFP (A2780GFP) were screened by G418. Stably transfected monoclones were obtained by limiting dilution. GFP protein in the cells was examined by fluorescence microscopy and OVCA1 fragment was examined by RTPCR. Wound healing assay, transwell migration assay and matrigel invasion assay were employed to study the effects of OVCA1 on A2780 cell migration and invasion, while taking A2780GFP and A2780 cells as controls. Results:The cell line stably expressing GFPtagged 〖STBX〗OVCA1〖STBZ〗 (A2780OVCA1) was successfully established. The migration speed of cells in A2780OVCA1 group was significantly lower than those of the other 2 groups (P<0.05). Cells passing the pores of transwell filter was less in A2780OVCA1 cells than in the cells of the 2 control groups (P<0.05). Cells passing matrigelcoated filter was significantly decreased in A2780OVCA1 group than in the 2 control groups (P<0.05). Conclusion: OVCA1 can effectively inhibit A2780 cell migration and invasion in vitro.
2008, 15(4):356-360.
DOI: 10.3872/j.issn.1007-385X.2008.4.012
Abstract:
Abstract Objective: To determine the promoting effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NFκB, on the sensitivity of human ovarian cancer cell line SKOV3 to chemoagent cisplatin and the possible mechanisms. Methods:SKOV3 cells were treated with various concentrations of cisplatin and PDTC combination for different periods. Then the growth inhibition of SKOV3 cells were observed by MTT assay, expression of IκBα and P65 protein by Western blotting assay, and apoptosis of cells by flow cytometry. Results: Separate application of both cisplatin（0.1-10 μg/ml）and PDTC（10-40 μmol/L L）significantly inhibited the growth of SKOV3 cells and caused cell apoptosis(P<0.05 orP<0.01). Even at lower concentration, cisplatin（0.01 μg/ml）combined with PDTC （2.5, 5 μmol/L）resulted more significant growth inhibition and apoptosis of SKOV3 cells than cisplatin alone (both P<0.05). Cisplatin alone reduced cytoplasmic IκBα protein in the cytoplasm and increased nuclear P65 protein compared with control group(both P<0.05), which could be reversed by addition of PDTC. Conclusion: Low dose of PDTC can enhance the chemosensitivity of ovarian cancer cells to cisplatin, which might be related to PDTCinduced IκBα protein and the subsequent inhibition of nuclear translocation of P65 protein.
Abstract Objective: To determine the promoting effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NFκB, on the sensitivity of human ovarian cancer cell line SKOV3 to chemoagent cisplatin and the possible mechanisms. Methods:SKOV3 cells were treated with various concentrations of cisplatin and PDTC combination for different periods. Then the growth inhibition of SKOV3 cells were observed by MTT assay, expression of IκBα and P65 protein by Western blotting assay, and apoptosis of cells by flow cytometry. Results: Separate application of both cisplatin（0.1-10 μg/ml）and PDTC（10-40 μmol/L L）significantly inhibited the growth of SKOV3 cells and caused cell apoptosis(P<0.05 orP<0.01). Even at lower concentration, cisplatin（0.01 μg/ml）combined with PDTC （2.5, 5 μmol/L）resulted more significant growth inhibition and apoptosis of SKOV3 cells than cisplatin alone (both P<0.05). Cisplatin alone reduced cytoplasmic IκBα protein in the cytoplasm and increased nuclear P65 protein compared with control group(both P<0.05), which could be reversed by addition of PDTC. Conclusion: Low dose of PDTC can enhance the chemosensitivity of ovarian cancer cells to cisplatin, which might be related to PDTCinduced IκBα protein and the subsequent inhibition of nuclear translocation of P65 protein.
2008, 15(4):361-364.
DOI: 10.3872/j.issn.1007-385X.2008.4.013
Abstract:
Abstract Objective: To investigate the inhibitory effect of recombinant mutant human tumor necrosis factor (rmhTNF-α) combined with cisplatin (DDP) against transplanted Lewis lung carcinoma (LLC) in mice, and to explore the related mechanism. Methods: LLC cells were subcutaneously inoculated into C57BL/6 mice at the right axilla and the mice were randomly divided into 4 groups: sodium chloride (control group),DDP group, rmhTNFα group, rmhTNFα plus DDP group. On the 7th day after inoculation the diameter of the tumor reached 0.6 cm; the corresponding agents were intratumorally injected for 3 d. All mice were sacrificed 1 d later and the tumors were obtained, weighed, and the tumor inhibitory rate was calculated. Flow cytometric analysis was used to examine cells apoptosis and cell cycle. Expression of survivin mRNA was examined by RTPCR.Results: (1)The tumor inhibitory rates of the DDP plus rmhTNFα group (3661%) was significantly higher than those of the DDP group (17.12%) and rmhTNF-α group (15.83%, P＜0.05）.The q value of Jin′s formula was 1.2, indicating a good synergistic effect between rmhTNFα and DDP. (2) The cell apoptotic rate of the rmhTNFα plus DDP group (\[28.2±1.8\]%) was significantly higher than those of the DDP group (\[21.6±1.0\]%) and rmhTNFα group (\[19.3±2.0\]%, P＜0.05）; the cells were arrested G2 phase in the DDP plus rmhTNFα group. (3) RTPCR showed that the expression of survivin gene was significantly inhibited in all the 3 groups, with the inhibition in the DDP plus rmhTNFα group more potent than that in the other 2 groups (P＜0.01). Conclusion: The combination of rmhTNFα and DDP has synergic effect in treatment of transplanted Lewis lung cancer, which might be related to the inhibition of survivin gene expression and induction of tumor cell apoptosis.
Abstract Objective: To investigate the inhibitory effect of recombinant mutant human tumor necrosis factor (rmhTNF-α) combined with cisplatin (DDP) against transplanted Lewis lung carcinoma (LLC) in mice, and to explore the related mechanism. Methods: LLC cells were subcutaneously inoculated into C57BL/6 mice at the right axilla and the mice were randomly divided into 4 groups: sodium chloride (control group),DDP group, rmhTNFα group, rmhTNFα plus DDP group. On the 7th day after inoculation the diameter of the tumor reached 0.6 cm; the corresponding agents were intratumorally injected for 3 d. All mice were sacrificed 1 d later and the tumors were obtained, weighed, and the tumor inhibitory rate was calculated. Flow cytometric analysis was used to examine cells apoptosis and cell cycle. Expression of survivin mRNA was examined by RTPCR.Results: (1)The tumor inhibitory rates of the DDP plus rmhTNFα group (3661%) was significantly higher than those of the DDP group (17.12%) and rmhTNF-α group (15.83%, P＜0.05）.The q value of Jin′s formula was 1.2, indicating a good synergistic effect between rmhTNFα and DDP. (2) The cell apoptotic rate of the rmhTNFα plus DDP group (\[28.2±1.8\]%) was significantly higher than those of the DDP group (\[21.6±1.0\]%) and rmhTNFα group (\[19.3±2.0\]%, P＜0.05）; the cells were arrested G2 phase in the DDP plus rmhTNFα group. (3) RTPCR showed that the expression of survivin gene was significantly inhibited in all the 3 groups, with the inhibition in the DDP plus rmhTNFα group more potent than that in the other 2 groups (P＜0.01). Conclusion: The combination of rmhTNFα and DDP has synergic effect in treatment of transplanted Lewis lung cancer, which might be related to the inhibition of survivin gene expression and induction of tumor cell apoptosis.
2008, 15(4):365-368.
DOI: 10.3872/j.issn.1007-385X.2008.4.014
Abstract:
Abstract Objective:To investigate the inhibitory effect of Resveratrol (Res) on human gastric cancer cell line SGC7901 and the related mechanism.Methods： Human gastric cancer SGC7901 cells were treated with different concentrations of Res（10, 20, 40 μg/ml）; cells treated with DMSO and culture medium served as controls. The growth inhibition rate of SGC7901 cells was examined by MTT assay; the morphological changes of cells were observed under phasecontrast microscope; caspase3 activity was assessed by colorimetry; and the cell cycle was detected by flow cytometry(FCM). Results: Res inhibited the growth of SGC7901 cells in a time and dosedependent manner, with maximal inhibitory rate being (53.39±5.32)%. After Res treatment the amount of suspended cell increased and the cells shrank, became round and smashed, with particlelike substance found in the cells. The most obvious changes were found 48 h after treatment with 40 μg/ml Res. Besides, Res upregulated the Caspase3 activity in SGC7901 cells in a time and concentrationdependent manner. Flow cytometry revealed that Res induced the S phase arrest of SGC7901 cells. Conclusion：Res can obviously inhibit the growth of human gastric cancer SGC7901 cells, possibly through activating Caspase3,inducing cell apoptosis and influencing cell cycle.
Abstract Objective:To investigate the inhibitory effect of Resveratrol (Res) on human gastric cancer cell line SGC7901 and the related mechanism.Methods： Human gastric cancer SGC7901 cells were treated with different concentrations of Res（10, 20, 40 μg/ml）; cells treated with DMSO and culture medium served as controls. The growth inhibition rate of SGC7901 cells was examined by MTT assay; the morphological changes of cells were observed under phasecontrast microscope; caspase3 activity was assessed by colorimetry; and the cell cycle was detected by flow cytometry(FCM). Results: Res inhibited the growth of SGC7901 cells in a time and dosedependent manner, with maximal inhibitory rate being (53.39±5.32)%. After Res treatment the amount of suspended cell increased and the cells shrank, became round and smashed, with particlelike substance found in the cells. The most obvious changes were found 48 h after treatment with 40 μg/ml Res. Besides, Res upregulated the Caspase3 activity in SGC7901 cells in a time and concentrationdependent manner. Flow cytometry revealed that Res induced the S phase arrest of SGC7901 cells. Conclusion：Res can obviously inhibit the growth of human gastric cancer SGC7901 cells, possibly through activating Caspase3,inducing cell apoptosis and influencing cell cycle.
2008, 15(4):369-373.
DOI: 10.3872/j.issn.1007-385X.2008.4.015
Abstract:
Abstract Objective: To screen for primary drug resistancerelated genes of human malignant glioma using cDNA microarray, so as to provide evidence for drugsensitive predicting gene for chemodrugs for malignant gliomas of different patients. Method: Six fresh glioma specimens were obtained immediately after surgical resection and were primary cultured. MTT method was used to determine the inhibitory effect of Teniposide (VM26) against gliomas. When 4.5 μg/ml（peak blood drug concentration, 1PPC）was used, the inhibitory rate >30% was considered sensitive and the rate ≤30% was considered resistant; and the 6 specimens were divided into 2 group according to the above standard. cDNA microassay combined with clustering analysis was used to screen for resistancerelated genes. Semiquantitative RTPCR was used for verification of HDAC1 gene expression. Results: Three of the 6 glioma specimens belonged to the drug resistance group and the other 3 to the drug sensitive group. cDNA microarray analysis combined with cluster analysis screened out 21 genes, with 6 upregulated and 15 downregulated. High expression of gene HDAC1 was noticed in all the 6 specimens by semiquantitative RTPCR, and the trend was similar to that by microassay. Conclusion: The primary drug resistance of glioma may be associated with the 21 genes screened by cDNA microarray; the detailed mechanisms for drug controlling still need to discussed in the future.
Abstract Objective: To screen for primary drug resistancerelated genes of human malignant glioma using cDNA microarray, so as to provide evidence for drugsensitive predicting gene for chemodrugs for malignant gliomas of different patients. Method: Six fresh glioma specimens were obtained immediately after surgical resection and were primary cultured. MTT method was used to determine the inhibitory effect of Teniposide (VM26) against gliomas. When 4.5 μg/ml（peak blood drug concentration, 1PPC）was used, the inhibitory rate >30% was considered sensitive and the rate ≤30% was considered resistant; and the 6 specimens were divided into 2 group according to the above standard. cDNA microassay combined with clustering analysis was used to screen for resistancerelated genes. Semiquantitative RTPCR was used for verification of HDAC1 gene expression. Results: Three of the 6 glioma specimens belonged to the drug resistance group and the other 3 to the drug sensitive group. cDNA microarray analysis combined with cluster analysis screened out 21 genes, with 6 upregulated and 15 downregulated. High expression of gene HDAC1 was noticed in all the 6 specimens by semiquantitative RTPCR, and the trend was similar to that by microassay. Conclusion: The primary drug resistance of glioma may be associated with the 21 genes screened by cDNA microarray; the detailed mechanisms for drug controlling still need to discussed in the future.
2008, 15(4):374-378.
DOI: 10.3872/j.issn.1007-385X.2008.4.016
Abstract:
Abstract Objective: To investigate the expression of minichromosome maintenance proteins 4(MCM4) and cell division cycle 6 (CDC6) in uterine cervical carcinomas and its relationship with human papilloma virus (HPV)16/18 infection. Methods：The expression of MCM4 and CDC6 was examined in 50 squamous cell carcinoma specimens, 20 cervical intraepithelial neoplasia (CIN)ⅡⅢ specimens, 20 CINⅠ specimens, and 20 normal cervical tissues by immunohistochemical method. The infections of HPV type 16, 18 DNA were determined by PCR. Results：(1) The expression of MCM4 and CDC6 in uterine cervical carcinoma tissues was significantly higher than that in the CIN specimens and normal cervical tissues (Both P<0.05). Expression of MCM4 and CDC6 was correlated with tumor grades and lymph node metastasis (all P<0.05), but not with age and clinical stages (all P>0.05). (2) The positive rates of (HPV)16/18 were significant different between cervical carcinomas, CIN and normal tissues (P<0.05), and was not associated with age, tumor grades, clinical stages or lymphnode metastasis (all P>0.05). (3) MCM4 expression were positively correlated with the expression of CDC6 in uterine cervical carcimonas（r=0.390, P<0.05）. The positive rate of HPV16/18 was positively correlated with the expression of MCM4 and CDC6 （r= 0.634, P<0.05; r=0.386 P<0.05）.Conclusion: The results suggest that changes in MCM4 and CDC6 expression in CIN and cervical carcinoma specimens are associated with HPV16/18 infection, which is related to the progression of CIN and carcinogenesis of cervical carcinomas.
Abstract Objective: To investigate the expression of minichromosome maintenance proteins 4(MCM4) and cell division cycle 6 (CDC6) in uterine cervical carcinomas and its relationship with human papilloma virus (HPV)16/18 infection. Methods：The expression of MCM4 and CDC6 was examined in 50 squamous cell carcinoma specimens, 20 cervical intraepithelial neoplasia (CIN)ⅡⅢ specimens, 20 CINⅠ specimens, and 20 normal cervical tissues by immunohistochemical method. The infections of HPV type 16, 18 DNA were determined by PCR. Results：(1) The expression of MCM4 and CDC6 in uterine cervical carcinoma tissues was significantly higher than that in the CIN specimens and normal cervical tissues (Both P<0.05). Expression of MCM4 and CDC6 was correlated with tumor grades and lymph node metastasis (all P<0.05), but not with age and clinical stages (all P>0.05). (2) The positive rates of (HPV)16/18 were significant different between cervical carcinomas, CIN and normal tissues (P<0.05), and was not associated with age, tumor grades, clinical stages or lymphnode metastasis (all P>0.05). (3) MCM4 expression were positively correlated with the expression of CDC6 in uterine cervical carcimonas（r=0.390, P<0.05）. The positive rate of HPV16/18 was positively correlated with the expression of MCM4 and CDC6 （r= 0.634, P<0.05; r=0.386 P<0.05）.Conclusion: The results suggest that changes in MCM4 and CDC6 expression in CIN and cervical carcinoma specimens are associated with HPV16/18 infection, which is related to the progression of CIN and carcinogenesis of cervical carcinomas.
2008, 15(4):379-387.
DOI: 10.3872/j.issn.1007-385X.2008.4.017
Abstract:
Abstract Objective: To examine the expression of VEGF gene in the endometrial carcinoma tissues, paratumor tissues, normal endometria and peripheral blood, and analyze the role of VEGF in tumor growth and tumor metastasis. Methods: Realtime fluorescence quantitative PCR was used to detect the expression of VEGF gene in 51 endometrial carcinoma samples and the corresponding paratumor tissues, 40 normal endometria samples and their corresponding peripheral blood samples. The relation between the VEGF expression and clinical pathological parameters was analyzed.Results: The expression of VEGF gene was higher in the endometrial carcinoma tissues than in the corresponding paratumour tissues and normal endometrial tissues (P<0. 05) . VEGF expression in endometrial carcinoma was significantly correlated with the clinical stage, histological grade, lymph node metastasis and depth of myometrial invasion (all P<0.05), but not with the presence of menopause or the pathological types of the tumor (P>0.05). The expression of VEGF in peripheral blood was higher in patients with endometrial carcinoma than that in the normal controls; and the expression was significantly correlated with the clinical stage, histological grades, pathological types and the presence of lymph node metastasis (P<0.05), but not with the depth of myometrial invasion and the presence of menopause( P>0.05). Conclusion: Realtime fluorescent quantitative PCR can sensitively, specifically detect the expression of VEGF in the endometrial carcinoma tissues and peripheral blood samples. VEGF might play an important role in the development, invasion, and metastasis of endometrial carcinoma.
Abstract Objective: To examine the expression of VEGF gene in the endometrial carcinoma tissues, paratumor tissues, normal endometria and peripheral blood, and analyze the role of VEGF in tumor growth and tumor metastasis. Methods: Realtime fluorescence quantitative PCR was used to detect the expression of VEGF gene in 51 endometrial carcinoma samples and the corresponding paratumor tissues, 40 normal endometria samples and their corresponding peripheral blood samples. The relation between the VEGF expression and clinical pathological parameters was analyzed.Results: The expression of VEGF gene was higher in the endometrial carcinoma tissues than in the corresponding paratumour tissues and normal endometrial tissues (P<0. 05) . VEGF expression in endometrial carcinoma was significantly correlated with the clinical stage, histological grade, lymph node metastasis and depth of myometrial invasion (all P<0.05), but not with the presence of menopause or the pathological types of the tumor (P>0.05). The expression of VEGF in peripheral blood was higher in patients with endometrial carcinoma than that in the normal controls; and the expression was significantly correlated with the clinical stage, histological grades, pathological types and the presence of lymph node metastasis (P<0.05), but not with the depth of myometrial invasion and the presence of menopause( P>0.05). Conclusion: Realtime fluorescent quantitative PCR can sensitively, specifically detect the expression of VEGF in the endometrial carcinoma tissues and peripheral blood samples. VEGF might play an important role in the development, invasion, and metastasis of endometrial carcinoma.
2008, 15(4):384-387.
DOI: 10.3872/j.issn.1007-385X.2008.4.018
Abstract:
Abstract Objective: To investigate the expression of extracellular matrix protein 1 (ECM1) in breast cancer tissues and cell lines and to assess its significance. Methods: Immunohistochemical and immunocytochemical EnVision methods were used to detect expression of ECM1 in the breast tissues（normal tissues and cancer tissues） and in breast cancer cell lines of different malignancies ( MCF7，SkBr3，and MDA435). The ECM1 positive rates were compared between normal tissues and cancer tissues and between cancer tissues with and without lymph node metastasis. Western blotting was used to identify the subtypes of ECM1. Results: The positive rate of ECM1 was 8.7%(2/23) in the normal breast tissues, 60.4%(29/48) in breast cancer tissues, 38.5% (10/26) in breast cancer tissues without lymph node metastasis, and 864% (19/22) in those with lymph node metastasis. Statistical analysis indicated that the ECM1 expression was higher in the breast cancer tissues than in the normal tissues (P<0.01), and the expression in the metastatic breast cancer was higher than that in the primary cancer tissues (P<0.01). Cell lines with low malignancy, such as MCF7 and SkBr3 cells, were negative of ECM1; cell lines with high malignancy, such as MDA435 cells, were strongly positive of ECM1. Western blotting showed that the highly expressed ECM1 protein was mainly ECM1a subtype, with a relative molecular mass of 68 000. Conclusion: ECM1, mainly ECM1a subtype, is overexpressed in breast cancer tissues and cell lines of high malignancy, which is associated with the metastasis of breast cancer.
Abstract Objective: To investigate the expression of extracellular matrix protein 1 (ECM1) in breast cancer tissues and cell lines and to assess its significance. Methods: Immunohistochemical and immunocytochemical EnVision methods were used to detect expression of ECM1 in the breast tissues（normal tissues and cancer tissues） and in breast cancer cell lines of different malignancies ( MCF7，SkBr3，and MDA435). The ECM1 positive rates were compared between normal tissues and cancer tissues and between cancer tissues with and without lymph node metastasis. Western blotting was used to identify the subtypes of ECM1. Results: The positive rate of ECM1 was 8.7%(2/23) in the normal breast tissues, 60.4%(29/48) in breast cancer tissues, 38.5% (10/26) in breast cancer tissues without lymph node metastasis, and 864% (19/22) in those with lymph node metastasis. Statistical analysis indicated that the ECM1 expression was higher in the breast cancer tissues than in the normal tissues (P<0.01), and the expression in the metastatic breast cancer was higher than that in the primary cancer tissues (P<0.01). Cell lines with low malignancy, such as MCF7 and SkBr3 cells, were negative of ECM1; cell lines with high malignancy, such as MDA435 cells, were strongly positive of ECM1. Western blotting showed that the highly expressed ECM1 protein was mainly ECM1a subtype, with a relative molecular mass of 68 000. Conclusion: ECM1, mainly ECM1a subtype, is overexpressed in breast cancer tissues and cell lines of high malignancy, which is associated with the metastasis of breast cancer.
2008, 15(4):388-392.
DOI: 10.3872/j.issn.1007-385X.2008.4.019
Abstract:
Abstract Objective: To explore the expression of the epidermal growth factor receptor (EGFR) mRNA and vascular endothelial growth factor (VEGF) mRNA in ovarian carcinoma and its significance. Methods: Reverse transcription polymerase chain reaction (RTPCR) was employed to determine the expression of EGFR mRNA and VEGF mRNA in 58 specimens, including 12 normal, 11 benign and 35 malignant ovarian carcinomas, which were obtained during operation from patients treated in the Affiliated Hospital of Qingdao University Medical College. The relationship between EGFR and VEGF mRNA expression and the clinicopathological features was analyzed. Results: The positive rates of EGFR and VEGF mRNA were significantly higher in the malignant ovarian specimens than in the normal and benign ones （P＜0.05). EGFR mRNA expression was correlated with the clinical stages of ovarian carcinomas, with the expression at stage ⅢⅣ significantly higher than that of stageⅠⅡ（P<0.05）. The expression of VEGF mRNA was correlated with the clinical stages and lymph node metastasis of ovarian carcinomas. VEGF mRNA expression in carcinomas of stage ⅢⅣ or those with lymph node metastasis was significantly higher than that at stageⅠⅡ and those without lymph node metastasis（all P<005）. Besides, the expression of EGFR mRNA was correlated with the expression of VEGF mRNA（r=0.438，P<0.05）.Conclusion: The expression of EGFR and VEGF is positively correlated with the carcinogenesis and metastasis of ovarian carcinoma, and there might be a synergistic effect between the 2.
Abstract Objective: To explore the expression of the epidermal growth factor receptor (EGFR) mRNA and vascular endothelial growth factor (VEGF) mRNA in ovarian carcinoma and its significance. Methods: Reverse transcription polymerase chain reaction (RTPCR) was employed to determine the expression of EGFR mRNA and VEGF mRNA in 58 specimens, including 12 normal, 11 benign and 35 malignant ovarian carcinomas, which were obtained during operation from patients treated in the Affiliated Hospital of Qingdao University Medical College. The relationship between EGFR and VEGF mRNA expression and the clinicopathological features was analyzed. Results: The positive rates of EGFR and VEGF mRNA were significantly higher in the malignant ovarian specimens than in the normal and benign ones （P＜0.05). EGFR mRNA expression was correlated with the clinical stages of ovarian carcinomas, with the expression at stage ⅢⅣ significantly higher than that of stageⅠⅡ（P<0.05）. The expression of VEGF mRNA was correlated with the clinical stages and lymph node metastasis of ovarian carcinomas. VEGF mRNA expression in carcinomas of stage ⅢⅣ or those with lymph node metastasis was significantly higher than that at stageⅠⅡ and those without lymph node metastasis（all P<005）. Besides, the expression of EGFR mRNA was correlated with the expression of VEGF mRNA（r=0.438，P<0.05）.Conclusion: The expression of EGFR and VEGF is positively correlated with the carcinogenesis and metastasis of ovarian carcinoma, and there might be a synergistic effect between the 2.
2008, 15(4):393-395.
DOI: 10.3872/j.issn.1007-385X.2008.4.020
Abstract:
摘 要 目的: 探讨鸦胆子油乳对宫颈癌Hela细胞的抑制作用及其可能的机制。方法: 采用倒置显微镜及巴氏染色法，观察药物质量浓度分别为5、10 μg/ml作用下细胞形态的变化；用MTT法检测5个不同药物质量浓度(40、20、10、5、2.5 μg/ml)作用下药物对细胞的抑制率；用流式细胞仪检测药物质量浓度分别为10、20 μg/ml的实验组细胞周期的变化。结果: 鸦胆子油乳作用后，颈癌Hela细胞形态上都发生了改变，凋亡小体出现；鸦胆子油乳显著抑制了宫颈癌Hela细胞的增殖，作用48 h后各组中抑制率都已超过60%，72 h后抑制率都超过80%；鸦胆子油乳阻滞S期细胞进入G2~M期，并且有凋亡峰，10、20 μg/ml药物作用后细胞凋亡率分别为59.9%、69.8%。结论: 鸦胆子油乳可有效抑制宫颈癌Hela细胞的增殖且呈时间依赖性，其机制可能与诱导细胞凋亡和阻滞细胞于S期有关。
摘 要 目的: 探讨鸦胆子油乳对宫颈癌Hela细胞的抑制作用及其可能的机制。方法: 采用倒置显微镜及巴氏染色法，观察药物质量浓度分别为5、10 μg/ml作用下细胞形态的变化；用MTT法检测5个不同药物质量浓度(40、20、10、5、2.5 μg/ml)作用下药物对细胞的抑制率；用流式细胞仪检测药物质量浓度分别为10、20 μg/ml的实验组细胞周期的变化。结果: 鸦胆子油乳作用后，颈癌Hela细胞形态上都发生了改变，凋亡小体出现；鸦胆子油乳显著抑制了宫颈癌Hela细胞的增殖，作用48 h后各组中抑制率都已超过60%，72 h后抑制率都超过80%；鸦胆子油乳阻滞S期细胞进入G2~M期，并且有凋亡峰，10、20 μg/ml药物作用后细胞凋亡率分别为59.9%、69.8%。结论: 鸦胆子油乳可有效抑制宫颈癌Hela细胞的增殖且呈时间依赖性，其机制可能与诱导细胞凋亡和阻滞细胞于S期有关。
2008, 15(4):396-400.
DOI: 10.3872/j.issn.1007-385X.2008.4.021
Abstract:
摘 要 真核细胞主要通过高度结构化的5′端非翻译区(5′ untranslated region, 5′UTR)实现mRNA翻译起始的调控，其主要作用方式有3种，即通过本身高度复杂的二级结构在空间上阻碍翻译起始、通过其包含的上游AUG密码子(uAUG)和上游开放阅读框(uORF)元件来抑制翻译起始，以及通过其包含的内部核糖体进入位点(IRES)元件的“非帽依赖”(cap-indepen-dent)起始途径来抑制翻译起始。肿瘤细胞通常会过表达真核起始因子4E(eukaryotic initiation factor 4E, eIF4E)、 eIF4A、eIF4G等，这些因子可以通过解开5′UTR的复杂结构特异性地解除5′UTR的翻译抑制作用。目前应用5′UTR进行肿瘤靶向性基因治疗的思路是把肿瘤杀伤基因置于5′UTR调控之下，利用肿瘤细胞过表达翻译起始因子来发挥5′UTR在肿瘤细胞中的翻译竞争优势，实现治疗基因在肿瘤细胞中的特异性表达，从而达到靶向杀伤肿瘤的目的。
摘 要 真核细胞主要通过高度结构化的5′端非翻译区(5′ untranslated region, 5′UTR)实现mRNA翻译起始的调控，其主要作用方式有3种，即通过本身高度复杂的二级结构在空间上阻碍翻译起始、通过其包含的上游AUG密码子(uAUG)和上游开放阅读框(uORF)元件来抑制翻译起始，以及通过其包含的内部核糖体进入位点(IRES)元件的“非帽依赖”(cap-indepen-dent)起始途径来抑制翻译起始。肿瘤细胞通常会过表达真核起始因子4E(eukaryotic initiation factor 4E, eIF4E)、 eIF4A、eIF4G等，这些因子可以通过解开5′UTR的复杂结构特异性地解除5′UTR的翻译抑制作用。目前应用5′UTR进行肿瘤靶向性基因治疗的思路是把肿瘤杀伤基因置于5′UTR调控之下，利用肿瘤细胞过表达翻译起始因子来发挥5′UTR在肿瘤细胞中的翻译竞争优势，实现治疗基因在肿瘤细胞中的特异性表达，从而达到靶向杀伤肿瘤的目的。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.