[Expression of prostate stem cell antigen and Claudin-4 in human pancreatic carcinoma].

OBJECTIVE: To investigate the expressions of prostate stem cell antigen (PSCA) and Claudin-4 in human pancreatic carcinoma and to discuss its role in the ontogenesis of pancreatic cancer. METHODS: Pancreatic carcinoma tissue microarray was constructed, containing 100 cores of 10 normal adult pancreas tissues, 12 chronic pancreatitis tissues, and 78 pancreatic carcinomas. The expressions of PSCA and Claudin-4 were detected using immunohistochemical method and the relationship between PSCA and Claudin-4 and the pancreatic carcinoma was analyzed. RESULTS: The positive expression rates of PSCA and Claudin-4 protein in pancreatic carcinoma were 79. % and 88. % respectively. PSCA and Claudin-4 staining were more intense in malignant cells than in chronic pancreatic tissues and normal adult pancreas tissues. No evidence was found for an association between expressions of PSCA and Claudin-4 and other variables, including gender, age at surgery, and tumor grade. CONCLUSIONS: The expressions of PSCA and Claudin-4 are related to the pancreatic carcinomas. PSCA and Claudin-4 play a role in the development of pancreatic cancer, but PSCA and Claudin-4 are not correlated with the clinical pathology of tumor.

[Biological significance of expression of calcyclin in human pancreatic carcinoma: a tissue microarray-based study].

OBJECTIVE: To investigate the expression of calcyclin in pancreatic carcinoma and its relation to the patients' prognosis. METHODS: Human pancreatic carcinoma tissue microarray was constructed, which contained 63 cores of 3 normal adult pancreas tissues, 6 chronic pancreatitis tissues, 51 pancreatic carcinoma tissues and 3 islet cell carcinoma tissues. Immunohistochemistry was performed to detect the expression of calcyclin in these tissues, and the relationship between calcyclin and the clinicopatholoical features of pancreatic carcinoma was analyzed. RESULTS: The positivity rate of calcyclin in the pancreatic carcinoma tissue was 76.5% (39/51), and calcyclin staining was more intense in the malignant cells than in the benign cells (P=0.007), suggesting a correlation between calcyclin expression and pancreatic carcinoma. No evidence was found for an association of calcyclin expression with the variables including the patients' gender, age at surgery, and tumor grade. Weak staining for calcyclin was noted in chronic pancreatitis tissues. CONCLUSION: Calcyclin expression is related to the pancreatic carcinomas and up-regulation of calcyclin expression is possibly an early event in pancreatic and pragression of development cancer.

[Effects of siRNA targeted to survivin in suppressing proliferation and inducing apoptosis in breast cancer MCF-7 cells].

OBJECTIVE: Blocking the expression of survivin with RNA interference techniques, the effects of suppressing proliferation and inducing apoptosis of breast cancer MCF-7 cells were investigated. METHODS: A siRNA eukaryotic expression vector against survivin was constructed and transfected into breast cancer MCF-7 cells with lipofectamine 2000. The changes of survivin expression were detected by semi-quantitive RT-PCR and immunohistochemistry. The effect of suppressing proliferation of MCF-7 cell was detected by MTT assay. The effect of inducing MCF-7 cell apoptosis was detected by TUNEL assay. RESULTS: The sequence-specific siRNA can efficiently block the expression of survivin both at mRNA and protein levels. The expression inhibition rate was 64.9% at mRNA level detected by semi-quantitive RT-PCR and 79.7% at protein level detected by immunohistochemistry. Blocking the expression of survivin can suppress proliferation of MCF-7 cells significantly. At 24 and 48 h after the cells were reseeded, the proliferation inhibition rates were 31.6% and 33.0%, respectively. At 24 h after transfection, apoptosis was induced in 12.9% of the cells as detected by TUNEL assay. CONCLUSION: Blocking the expression of survivin with RNA interference technology can significantly suppress proliferation of MCF-7 cells and induce apoptosis to a certain degree. RNAi targeted to survivin has a potential value in gene therapy of breast cancer.

Knockdown of survivin expression by small interfering RNA suppresses proliferation of two human cancer cell lines.

OBJECTIVE: To construct an expression vector of small interfering RNA (siRNA) against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. METHODS: Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine 2000. The expression of survivin was detected by semi-quantitive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. RESULTS: The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level. In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79.72% at protein level. The proliferation of PC-2 and MCF-7 cells was also suppressed, and 24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28.00% and 33.38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. CONCLUSIONS: The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.

Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity.

AIM: To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS: A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with Lipofectamine 2000. The down regulation of survivin expression was detected by semi-quantitative RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS: The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semi-quantitative RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION: The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce apoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene therapy of pancreatic cancer.

[siRNA against survivin coupling with epirubicin enhances to induce breast cancer cell MCF-7 to apoptosis].

OBJECTIVE: To use the sequence-specific siRNA knocking down the expressions of Survivin gene and inducing breast cancer MCF-7 cell line to apoptosis, and to couple the siRNA with Survivin for investigating the effects of MCF-7 cell induced to apoptosis and the chemotherapy sensitivity of breast cancer cell treated to epirubicin. METHODS: The molecular cloning technique was applied to construct the eukaryotic expression vector of siRNA against Survivin, and lipofectamine 2000 was used to transfect MCF-7 cell. Survivin expressions were detected by semi-quantitive RT-PCR and immunohistochemical SABC methods. The effects of inducing MCF-7 cell apoptosis and enhanced chemotherapy sensitivity to epirubicin were assessed by TUNEL method. RESULTS: The sequence-specific siRNA can, effectively and specifically, knock the expressions of Survivin gene down at both mRNA and protein levels, in which the expression inhibition rates were 64.91 and 79.72% respectively. After 48 h, 8.75% cells transfected with siRNA expression vector were induced to apoptosis; Coupling siRNA against Survivin with epirubicin can induce the cell apoptosis rate up to 24.21%. CONCLUSIONS: In the study, the siRNA against Survivin can, effectively and specifically, decrease the expressions of Survivin gene in MCF-7 cell; blocking the expressions of Survivin can, in certain degree, induce MCF-7 cell to apoptosis and enhance cell chemotherapy sensitivity to epirubicin significantly; Survivin RNAi has a great potential value in the gene therapy of breast cancer.

[siRNA targeted against survivin induces apoptosis of pancreatic cancer cells].

OBJECTIVE: To investigate the effect of a sequence-specific small interfering RNA (siRNA) in suppressing survivin expression and cell proliferation and inducing apoptosis of PC-2 cells. METHODS: The plasmid expression vector of siRNA targeted against survivin was constructed and transfected into PC-2 cells with Lipofectamine 2000. The changes of survivin expression were detected by semi-quantitative RT-PCR and immunohistochemical SP methods. The effect of siRNA in suppressing the proliferation of PC-2 cells was detected by MTT assay, and its role in inducing PC-2 cell apoptosis evaluated by flow cytometry. RESULTS: The sequence-specific siRNA effectively suppressed survivin expression at both mRNA and protein levels with inhibition rate of 81.25% at mRNA level and 74.24% at protein level. Survivin expression suppression significantly inhibited the proliferation of PC-2 cells, and at 24 and 48 h after cell seeding, the proliferation inhibition rate was 28.00% and 33.38% respectively; 24, 48 h after the transfection, apoptosis occurred in 8.46% and 7.53% of the cells, respectively. CONCLUSIONS: The plasmid expression vector for the siRNA against survivin constructed in the study can effectively and specifically suppress survivin expression in PC-2 cells, and blocking survivin expression suppresses PC-2 cell proliferation and induces cell apoptosis. siRNA targeted against survivin has a potential value in gene therapy for pancreatic cancer.

[Effects of Ras antisense oligoribonucleotide on multidrug resistance of pancreatic carcinoma Pc-2 cells].

OBJECTIVE: To investigate the role of Ras antisense oligoribonucleotide (ASODN) in multidrug resistance (MDR) of pancreatic carcinoma Pc-2 cells. METHODS: Ras and P-gp expression was suppressed by Ras ASODN. Sensitivity of Pc-2 cells to chemotherapy was determined by the MTT assay. MDR-1 mRNA level was detected by fluorogenic probe quantitative reverse transcription polymerase chain (RT-PCR) method. Flow cytometry (FCM) was used to detect the accumulative concentration of adriamycin (ADR) in the cells. RESULTS: Ras ASODN significantly inhibited the Ras and P-gp expression (P < 0.05), increased the sensitivity of Pc-2 cells to chemotherapeutic agents (P < 0.05), decreased MDR-1 gene level in Pc-2 cells (P < 0.05), and increased the intracellular intake of ADR in Pc-2 cells (P < 0.05). CONCLUSION: Ras ASODN may enhance the sensitivity of multidrug-resistant pancreatic cancer Pc-2 cells to chemotherapeutic agents by regulating MDR-1 gene level.

[Construction of a DNA vaccine against extracellular domain 1-3 of Flk1 and its inhibitory effect on growth of liver cancer cell line H22].

BACKGROUND & OBJECTIVE: Angiogenesis plays an important role in the growth,invasion,and metastasis of most solid tumors. Vascular endothelial growth factor (VEGF) and its receptor flk-1 play a key role in tumor angiogenesis. Blocking VEGF-flk1 pathway may inhibit tumor growth. This study was to construct a DNA vaccine against extracellular domain 1-3 of flk1,and test its inhibitory effect on growth of liver cancer cell line H22. METHODS: Extracellular domain 1-3 of flk1 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR),and inserted into plasmid pcDNA3.1(+) to construct vaccine pcDNA3.1(+)-flk1-domain 1-3. The vaccine was transfected into COS7 cells,and protein expression of flk1-domain 1-3 was detected by Western blot. Standard 4-h (51)Cr releasing test was used to detect specific cytotoxic T lymphocyte (CTL) activity in vaccine-inoculated mice. To test vaccine's preventive effect,mice were divided into V,P, and S groups,treated with vaccine,pcDNA3.1(+), and saline, respectively. H22 cells were inoculated into mice 10 days later. The tumor size, tumor weight, mice survival time, tumor latent period, and microvessel density were recorded and analyzed. RESULTS: Extracellular domain 1-3 of flk1 was cloned,and vaccine against it was constructed,both have been proved by DNA sequencing and comparing with data in GenBank. A protein of 44 kDa,which is consisted with flk1-domain 1-3 protein,expressed in COS7 cells inoculated with the DNA vaccine,specific CTL activity in these cells raised. After inoculated with H22 cells,tumor latent time of V group was (5.2+/-0.9) d,of P group was (4.0+/-0.7) d,of S group was (3.8+/-0.6) d; survival time of V group was (24.5+/-3.2) d,of P group was (14.7+/-2.6) d,of S group was (14.3+/-2.0) d; microvessel density of V group was 10.1+/-1.7,of P group was 27.3+/-3.3,of S group was 25.3+/-4.6; tumor weight of V group was (1.4+/-0.1) g,of P group was (1.8+/-0.2) g,of S group was (1.8+/-0.2)g. In comparison among groups,all data in V group were significantly different from P group and S group (P< 0.05),no significant difference existed between P group and S group (P >0.05). CONCLUSION: The DNA vaccine against flk-1 may stimulate potent specific CTL activity, and inhibit growth of H22 cells by its anti-endothelial cell property.

[Effects of hypertonic saline on erythrocyte adherence function and bacterial infection of hemorrhagic shock rabbits].

AIM: To explore the effect of hypertonic saline on the erythrocyte adherence function and bacterial infection of hemorrhagic shock rabbits. METHODS: 60 Japanese rabbits were randomly divided into 6 groups, 10 for each group. Artery catheterization and heparin were given to the rabbits in group 1 (sham shock group). Hemorrhagic shock model was set up by bleeding resulting from carotid artery catheter in group 2 (normal saline group )and group 3 (hypertonic saline group). 30 minutes after shock, the rabbits in group 1 and group 2 were treated with normal saline and balanced salt solution containing 1 x 10(9)/kg E.coli, respectively. And the rabbits in group 3 were treated with 75 g/L NaCl solution and balanced salt solution containing 1 x 10(9)/kg E.coli. Then the survival rates of the rabbits in group 1-3 were observed. Rabbits in group 4-6 were same treatment as received, group 1-3, respectively, except that there was no E.coli in balanced salt solution. The erythrocyte immune adherence function of rabbits in group 4-6 were detected 5 hours after shock by RBC-C3bR and RBC-IC rosette forming assays. RESULTS: The survival rate of rabbits in hypertonic saline group was significantly higher than that in normal saline group. The RBC-C3bR rosette forming rate of the normal saline treated rabbits were pronouncedly decreased, while RBC-IC rosette forming rate was notably elevated, as compared with those of either sham shock group or hypertonic saline group(P<0.01). Hypertonic saline markedly increased RBC C3bR rosette forming rate. CONCLUSION: The above findings suggest that hypertonic salt solution can remarkably improve the depressed erythrocyte immune adherence function and enhance the rabbit's resistance to E.coli challenge after hemorrhagic shock.

A DNA vaccine against extracellular domains 1-3 of flk-1 and its immune preventive and therapeutic effects against H22 tumor cell in vivo.

AIM: To construct a DNA vaccine against extracellular domains 1-3 of fetal liver kinase-1 (flk-1), and to investigate its preventive and therapeutic effect against H22 cell in vivo. METHODS: Flk-1 DNA vaccine was produced by cloning extracellular domains 1-3 of flk-1 and by inserting the cloned gene into pcDNA3.1 (+). Fifteen mice were divided into 3 groups and inoculated by vaccine, plasmid and saline respectively to detect specific T lymphocyte response. Thirty Mice were equally divided into preventive group and therapeutic group. Preventive group was further divided into V, P, and S subgroups, namely immunized by vaccine, pcDNA3.1 (+) and saline, respectively, and attacked by H22 cell. Therapeutical group was divided into 3 subgroups of V, P and S, and attacked by H22, then treated with vaccine, pcDNA3.1 (+) and saline, respectively. The tumor size, tumor weight, mice survival time and tumor latency period were compared within these groups. Furthermore, intratumoral microvessel density (MVD) was assessed by immunohistochemistry. RESULTS: DNA vaccine pcDNA3.1 (+) flk-1-domains 1-3 was successfully constructed and could raise specific CTL activity. In the preventive group and therapeutic group, tumor latency period and survival time were significantly longer in vaccine subgroup than that in P and S subgroups (P<0.05); the tumor size, weight and MVD were significantly less in vaccine subgroup than that in P and S subgroups (P<0.05). The survival time of therapeutic vaccine subgroup was significantly shorter than that of preventive vaccine subgroup (P<0.05); the tumor size, and MVD of therapeutic vaccine subgroup were significantly greater than that of preventive vaccine subgroup (P<0.05). CONCLUSION: DNA vaccine against flk-1 domains 1-3 can stimulate potent specific CTL activity; and has distinctive prophylactic effect on tumor H22; and also can inhibit the tumor growth in vivo. This vaccine may be used as an adjuvant therapy because it is less effective on detectable tumor.

Clinical significance of expression of p21 and p53 proteins and proliferating cell nuclear antigen in pancreatic cancer.

OBJECTIVE: To study the clinical significance and effect of p21, p53 protein as well as proliferating cell nuclear antigen (PCNA) on the occurrence and development of pancreatic carcinoma. METHOD: p21, p53 protein and PCNA expressions were detected in specimens from 30 patients with pancreatic carcinoma and 3 samples of normal pancreatic tissue by immunohistochemistry. The data were analyzed together with clinical findings. RESULTS: The positive expression rates of p21 and p53 proteins were 75.0% and 57.3% respectively in pancreatic carcinoma, which were significantly different from those in the normal tissue (P<0.05). p21 and p53 proteins were positively correlated (P<0.05). The positive expression of PCNA was 43.33%+/-17.99%, that was significantly higher than that in the normal pancreatic tissue (P<0.05). The expression of PCNA was correlated with the histological grade (P<0.05). The positive expression rate was consistent with the exacerbation of cancer. The expression was also correlated significantly with prognosis and p53 expression (P<0.05). CONCLUSIONS: The occurrence and development of pancreatic cancer are the result of associated function for many oncogenes and antioncogenes. PCNA may be helpful to identify malignant degree and prognosis of pancreatic cancer.