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Objective:To investigate the effect of a short hairpin RNA (shRNA) targeting epidermal growth factor receptor (EGFR) combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16, and to explore underlying mechanisms.Methods:Cultured Colo-16 cells were divided into 5 groups: normal cell group receiving conventional culture and treatment with phosphate-buffered saline (PBS) , negative control group transfected with a shRNA-NC-expressing plasmid and treated with PBS, sirolimus group receiving conventional culture and sirolimus treatment, EGFR shRNA group transfected with an EGFR shRNA-expressing plasmid and treated with PBS, and combined group transfected with an EGFR shRNA-expressing plasmid and treated with sirolimus. Methyl thiazol tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity in the above groups from 24 to 96 hours, and flow cytometry to detect cell apoptosis after 48-hour treatment. Semiquantitative RT-PCR was conducted to determine the mRNA expression of Bcl-2 and Bax, and Western blot analysis to determine the expression of apoptosis-related proteins cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, cell proliferation-related proteins phosphorylated mammalian target of rapamycin (p-mTOR) , phosphorylated protein kinase B (p-AKT) , phosphorylated 70-kDa ribosomal protein S6 kinase (p-P70S6k) , and cyclin D1. Comparisons among groups were carried out by using one-way analysis of variance, and multiple comparisons between 2 groups by using Student-Newman-Keuls q test. Results:MTT assay showed that the proliferative activity of Colo-16 cells was significantly lower in the sirolimus group, EGFR shRNA group and combined group during 24 - 96 hours than in the normal cell group (all P < 0.05) , and higher in the combined group than in the sirolimus group and EGFR shRNA group at 24-96 hours (all P < 0.001) , and there was no significant difference in the cellular proliferative activity at any time points between the normal cell group and negative control group (all P > 0.05) . Flow cytometry showed that the apoptosis rate was significantly higher in the sirolimus group, EGFR shRNA group and combined group (9.52% ± 0.25%, 12.65% ± 0.23%, 19.81% ± 0.31%, respectively) than in the normal cell group (3.33% ± 0.18%, q = 60.07, 78.08, 122.81, respectively, all P < 0.001) and negative control group (3.42% ± 0.19%, q = 59.90, 77.91, 122.64, respectively, all P < 0.001) , and was highest in the combined group. As RT-PCR and Western blot analysis revealed, the sirolimus group, EGFR shRNA group and combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2, but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax compared with the normal cell group (all P < 0.05) . Compared with the sirolimus group and EGFR shRNA group, the combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2 (all P < 0.05) , but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax (all P < 0.01) . Conclusion:EGFR shRNA and sirolimus exerted a synergistic effect in inhibiting the proliferation and promoting the apoptosis of Colo-16 cells, which may be related to the inhibition of the phosphoinositide 3-kinase (PI3K) /AKT/mTOR pathway.
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Objective To assess the mortality,complications and major morbidity of pneumonectomy for non -small cell lung cancer(NSCLC)and to establish the importance of various prognostic factors.Methods The data of 64 consecutive patients who underwent pneumonectomy for NSCLC were retrospectively reviewed.Results The 30 -day mortality rate was 7.8%(5 /64).Complications developed in 29.7%(1 9 /64)and overall 5 -year survival was 1 9.0%.Pathological stage Ⅲ(P =0.030)and right pneumonectomy(P =0.01 0)were independent risk factors of an adverse outcome.Survival was not significantly influenced by histological types (P =0.1 25)or curability (P =0.587).Conclusion Pneumonectomy is associated with acceptable overall morbidity and mortality.However,the patients with pathological stage Ⅲ or right pneumonectomy require special consideration.Pneumonectomy should be performed only in selected patients.
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Objective To evaluate effects of cucurbitacin Ⅰ on in vitro proliferation of HaCaT cells and the expression of keratin 17 (K17),signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in cultured HaCaT cells.Methods In vitro cultured HaCaT cells were divided into 6 groups to be treated with cucurbitacin Ⅰ at different concentrations of 0.0125,0.025,0.05 and 0.1 μmol/L (0.0125,0.025,0.05 and 0.1 μmol/L cucurbitacin Ⅰ groups),DMEM containing the same volume of DMSO as 0.1 pmol/L cucurbitacin Ⅰ (DMSO group),DMEM (negative control group) and 10 nmol/L calcipotriol (positive control group),respectively.Cell counting kit-8 (CCK8) assay was performed to assess in vitro cellular proliferative activity after 12-,24-,36-hour treatment,reverse transcription (RT)-PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment,and Western blot analysis to determine the protein expression of K17,STAT3,phosphorylated-STAT3 (p-STAT3) and VEGF after 24-hour treatment.Statistical analysis was carried out by one-way analysis of variance (ANOVA),repeated measures ANOVA,Student-Newman-Keuls (SNK)-q test and Pearson correlation analysis.Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin Ⅰ at the concentration of 0.0125 μmol/L.When the concentration of cucurbitacin Ⅰ increased up to 0.1 μmol/L,the cell proliferation rates were inhibited by 43.00％ ± 2.11％ and 48.98％ ± 2.27％ after 24-and 36-hour treatment respectively.Compared with the negative control group,cucurbitacin Ⅰ at different concentrations all could inhibit in vitro proliferation of HaCaT cells (all P ＜ 0.05),and the inhibitory effects increased gradually with the increase of cucurbitacin Ⅰ concentration and treatment duration.After 24-hour treatment,the mRNA expression of K17 and VEGF and the protein expression of K17,VEGF and P-STAT3 were significantly decreased along with the increase of concentration of cucurbitacin Ⅰ (all P ＜ 0.05).Conclusion Cucurbitacin Ⅰ can inhibit in vitro proliferation of HaCaT cells,and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17,VEGF and P-STAT3.
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Objective To study the curative effect of minimally invasive surgery in the treatment of rectal cancer.Methods 100 patients with rectal cancer were researched.They were randomly divided into observation group and control group,50 cases in each group.The control group received traditional abdominal radical operation,the observation group was treated with minimally invasive surgery.The operation effect was compared between the two groups.Results The operation time,length of stay and feeding time after operation in the observation group were shorter than those in the control group[(78.45±12.34)min vs.(98.24±25.31)min,(3.12±0.43)d vs.(7.53±1.12)d,(1.23±0.30)d vs.(3.56±0.57)d](t=4.967,25.992,25.578,all P0.05).Conclusion The effect of minimally invasive surgery for patients with rectal cancer is significant,it is worthy of promoting.
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Objective To analyze the changes of serum fatty acid synthase (FAS),carcinoembryonic antigen (CEA) and carbohydrate antigen 72-4 (CA72-4) in patients with gastric cancer and its clinical diagnostic value.Methods Forty five patients with gastric cancer and 45 patients with benign gastric cancer treated in our hospital from January 2016 to December 2016 were enrolled in this study.Forty five healthy subjects were enrolled in this study.The levels of FAS,CEA,and CA72-4 in three groups were analyzed.Results The levels of FAS,CEA,and CA72-4 in patients with gasttric cancer [(12.73 ± 5.48) mg/L,(31.36 ± 14.51) ng/ml,and (39.32 ± 18.76) U/ml] were significantly higher than those in benign gastric cancer group [(2.26 ± 1.15) ng/L,(3.24 ± 1.47) ng/ml,and (3.75 ± 1.69) U/ml],and normal control group [(1.83 ± 0.92) mg/L,(2.71 ± 1.54) ng/ml,and (3.13 ± 1.82) U/ml] (P ＜ 0.05).FAS,CEA,and CA72-4 levels in patients with lymph node metastasis of gastric cancer [(13.58 ± 6.09) mg/L,(6.25 ± 11.54) ng/ml,and (41.31 ± 13.67) U/ml] were significantly higher than those without lymph node metastasis [(9.21 ± 5.42) mg/L,(28.38 ± 9.72) ng/ml,and (26.75 ± 11.86) U/ml] (P ＜ 0.05).The sensitivity of FAS,CEA,and CA72-4 in patients with gastric cancer was significantly lower than that in combined detection,the specificity of FAS,CEA,and CA72-4 in patients with gastric cancer was significantly higher than that in combination test (P ＜ 0.05).The sensitivity of FAS,CEA,and CA72-4 in patients with lymph node metastasis was significantly lower than that in combination test,and the specificity of FAS,CEA and CA72-4 was significantly higher than that of combined detection (P ＜ 0.05).Conclusions FAS,CEA,and CA72-4 can be used as indicators of gastric cancer and diagnosis of lymph node metastasis.Combined detection of three indexes can improve the diagnostic sensitivity and have good clinical significance.
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Objective To analyze the changes of serum fatty acid synthase (FAS),carcinoembryonic antigen (CEA) and carbohydrate antigen 72-4 (CA72-4) in patients with gastric cancer and its clinical diagnostic value.Methods Forty five patients with gastric cancer and 45 patients with benign gastric cancer treated in our hospital from January 2016 to December 2016 were enrolled in this study.Forty five healthy subjects were enrolled in this study.The levels of FAS,CEA,and CA72-4 in three groups were analyzed.Results The levels of FAS,CEA,and CA72-4 in patients with gasttric cancer [(12.73 ± 5.48) mg/L,(31.36 ± 14.51) ng/ml,and (39.32 ± 18.76) U/ml] were significantly higher than those in benign gastric cancer group [(2.26 ± 1.15) ng/L,(3.24 ± 1.47) ng/ml,and (3.75 ± 1.69) U/ml],and normal control group [(1.83 ± 0.92) mg/L,(2.71 ± 1.54) ng/ml,and (3.13 ± 1.82) U/ml] (P ＜ 0.05).FAS,CEA,and CA72-4 levels in patients with lymph node metastasis of gastric cancer [(13.58 ± 6.09) mg/L,(6.25 ± 11.54) ng/ml,and (41.31 ± 13.67) U/ml] were significantly higher than those without lymph node metastasis [(9.21 ± 5.42) mg/L,(28.38 ± 9.72) ng/ml,and (26.75 ± 11.86) U/ml] (P ＜ 0.05).The sensitivity of FAS,CEA,and CA72-4 in patients with gastric cancer was significantly lower than that in combined detection,the specificity of FAS,CEA,and CA72-4 in patients with gastric cancer was significantly higher than that in combination test (P ＜ 0.05).The sensitivity of FAS,CEA,and CA72-4 in patients with lymph node metastasis was significantly lower than that in combination test,and the specificity of FAS,CEA and CA72-4 was significantly higher than that of combined detection (P ＜ 0.05).Conclusions FAS,CEA,and CA72-4 can be used as indicators of gastric cancer and diagnosis of lymph node metastasis.Combined detection of three indexes can improve the diagnostic sensitivity and have good clinical significance.
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Objective To evaluate effects of acitretin on HaCaT cells cultured in hypoxic condition,and to preliminarily explore the possible therapeutic mechanisms of acitretin in psoriasis.Methods HaCaT cells were divided into several groups to be cultured in hypoxic condition with the presence of acitretin at concentrations of 10-5,10-6,10-7 and 10 8 mol/L respectively,with cells treated with dimethyl sulfoxide (DMSO) as DMSO control group and those receiving no treatment as blank control group.Cellular proliferative activity was evaluated by CCK-8 assay after 12-,24-and 36-hour hypoxic culture in vitro.The mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were determined by reverse transcription (RT)-PCR and Western-blot analysis,respectively,after 24-hour hypoxic culture.Results After 24-hour hypoxic culture,the cellular proliferation rate was inhibited by 13.31％ ± 1.15％,21.86％ ± 5.31％,32.05％ ± 2.99％ and 37.28％ ± 3.21％ in the 10 8-,10-7-,10-6-and 10-5-mol/L acitretin groups respectively.With the increase of culture duration and acitretin concentrations,the degree of inhibition on cellular proliferation increased gradually.Compared with the blank control group,the 10-5-mol/L acitretin group showed significantly decreased protein expression of HIF-1α (0.319 ± 0.180 vs.1.196 ± 0.088,P ＜0.05),as well as decreased mRNA and protein expressions of VEGF (mRNA:0.442 ± 0.090 vs.1.108 ± 0.073;protein:0.216 ± 0.066 vs.1.174 ± 0.186;both P ＜ 0.05).However,no significant difference was found in the mRNA expression of HIF-lα between the 10-5-mol/L acitretin group and blank control group.Conclusion Acitretin can suppress the in vitro proliferation of HaCaT cells cultured in hypoxic condition,and down-regulate the expressions of HIF-1α and VEGF proteins as well as VEGF mRNA.
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The current treatments of metastatic malignant melanoma include chemotherapy,targeted therapy,immune therapy and radiation therapy,but the treatment outcome is far from optimism.In order to im-prove the treatment efficiency,it is urgent to improve early diagnosis,and develop more effective treatment drugs and delivery systems.The application of nanotechnology in the diagnosis and therapy of melanoma can re-duce the resistance to the drugs,increase efficacy and reduce side effects.
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Objective To investigate the effects of rottlerin on in vitro proliferation of and expressions of interleukin (IL)-17C,CCL20 chemokine,and nuclear factor (NF)-κB in cultured human HaCaT keratinocytes.Methods Some HaCaT cells were divided into several test groups treated with rottlerin at concentrations of 0.5,1.0,2.0 and 4.0 μmol/L,a solvent group treated with RPMI 1640 culture solution containing the same volume of dimethyl sulfoxide (DMSO) as that of 4.0 μmol/L rottlerin,and a control group treated with RPMI 1640 culture solution.Cell counting kit-8 (CCK8) assay was conducted to estimate the proliferative activity of HaCaT cells after 24-,48-and 72-hour culture,RT-PCR to determine the mRNA expressions of IL-17C and CCL20 after 48-hour culture,and Western blot to measure the protein expressions of IL-17C,CCL20 and NF-κB after 48-hour culture.Statistical analysis was carried out by using repeated-measures analysis of variance,one-way analysis of variance and Pearson correlation analysis with the SPSS16.0 software.Results Rottlerin showed an inhibitory effect on the proliferation of HaCaT cells,and the inhibitory effect increased over time (F =126.936,P ＜ 0.05) and with the increase of rottlerin concentrations (F =838.308,P ＜ 0.05),with a significant interaction effect between rottlerin concentrations and treatment duration (F =15.961,P ＜ 0.05).After 48-hour treatment,a significant decrease was observed in the mRNA and protein expressions of IL-17C (F =206.041,233.887,respectively,both P ＜ 0.05) and CCL20 (F =143.883,162.431,respectively,both P ＜ 0.05),as well as in the protein expression of NF-κB (F =577.915,P ＜ 0.05) in the test groups with the increase in rottlerin concentrations.Conclusions Rottlerin can inhibit the proliferation of HaCaT cells in vitro,and decrease the mRNA and protein expressions of IL-17C and CCL20 likely by downregulating the protein expression of NF-κB.
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Objective To investigate the effects of recombinant human pigment epithelium derived factor (rhPEDF)on in vitro proliferation of and expressions of interleukin 6(IL-6), IL-8 and vascular endothelial growth factor (VEGF)in cultured human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with rhPEDF at various concentrations(25, 50, 100 μg/L)for different durations, and some treated with RPMI 1640 medium only served as the control group. Cell counting kit-8(CCK8)assay was performed to evaluate cell proliferation after 24-, 48- and 72-hour treatment, reverse transcription (RT)-PCR to measure the mRNA expressions of IL-6, IL-8 and VEGF in HaCaT cells after 24-hour treatment, and Western blot to detect the protein expressions of IL-6, IL-8 and VEGF in HaCaT cells after 48-hour treatment. Statistical analysis was carried out by two- and one-way analysis of variance, Student-Newman-Keuls(SNK)-q test and Pearson correlation analysis. Results After treatment with rhPEDF of 25-100 μg/L for 24 - 72 hours, the proliferation of HaCaT cells was significantly inhibited to different extents compared with the control group(all P 0.05). Conclusions rhPEDF can inhibit the proliferation of HaCaT cells, and down-regulate the mRNA and protein expressions of IL-6, IL-8 and VEGF.
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Objective To investigate the in vitro effects of acitretin on the apoptosis and expressions of insulin-like growth factor binding protein 7 (IGFBP7) and vascular endothelial growth factor (VEGF) in HaCaT cells.Methods Cultured HaCaT cells were treated with various concentrations (10-5,10-64,10-7,10-8 mol/L) of acitretin for various durations,with those cultured in acitretin-free medium serving as the control group.Then,CCK-8 assay was performed to evaluate the proliferation of cells after 24-,48-and 72-hour treatment,flow cytometry to detect the apoptosis of HaCaT cells,and Western blot and reverse transcription-PCR to quantify the protein and mRNA expressions of IGFBP7 and VEGF in HaCaT cells,respectively,after 48-hour treatment.Statistical analysis was carried out by one-way analysis of variance and Pearson correlation analysis.Results The proliferation of HaCaT cells was inhibited by the treatment with acitretin,and the inhibitory effect increased with the elevation of concentration and prolongation of treatment duration of acitretin.A significant decrease was observed in the proliferative activity of HaCaT cells treated with acitretin of 10-8 mol/L for 48 hours,and when the concentration of acitretin was 10-5 mol/L,the proliferation of HaCaT cells was inhibited by 39.94％ ± 2.27％ and 49.77％ ± 1.87％ at 48 and 72 hours respectively,compared with the control cells.The HaCaT cells treated with acitretin of 10-5 mol/L for 48 hours showed a significant elevation in apoptosis rate (7.617％ ± 0.767％ vs.1.803％ ± 0.313％,P ＜ 0.05),IGFBP7 protein and mRNA expressions (0.939 ± 0.040 vs.0.436 ± 0.013,0.872 ± 0.079 vs.0.190 ± 0.056,both P ＜ 0.05),but a significant reduction in VEGF protein and mRNA expressions (0.213 ± 0.032 vs.0.798 ± 0.036,0.274 ± 0.041 vs.0.933 ± 0.054,both P ＜ 0.05) in comparison to the control cells.Conclusions Acitretin can induce the apoptosis of HaCaT cells,and up-regulate IGFBP7 but down-regulate VEGF expressions in HaCaT cells at protein and mRNA levels.
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ObjectiveTo investigate the inhibitory effect of dacarbazine and an oncolytic adenovirus carrying interleukin-24 (IL-24) on transplanted melanoma in nude mice.MethodsNude mice were inoculated with human A375 melanoma cells to establish a model of malignant melanoma.Then,the mice were divided into 4 groups to be treated with an oncolytic adenovirus carrying interleukin-24 (ZD55-IL-24),dacarbazine,the combination of ZD55-IL-24 and dacarbazine,and phosphate buffer(PBS),respectively,for 3 days.Seven days after the end of the treatment,some mice were sacrificed followed by the determination of IL-24 and E1A protein levels in tumor tissue by Western blot.The tumor volume was measured on a daily basis for 30 days.ResultsIL-24 and E1A were highly expressed in melanoma cell-bearing nude mice treated with ZD55-IL-24 and dacarbazine.At 30 days after the inoculation,the average volume of transplanted melanoma was (2346.5 ± 576.0) mm3 in the combination group,significantly different from that in the ZD55-IL-24 group((4141.6 ± 1348.2) mm3,P ＜ 0.05),dacarbazine group((5230.1 ± 922.8) mm3,P ＜ 0.05),and the control group ((7135.1 ± 1002.3) mm3,P ＜ 0.05).ConclusionThe ZD55-IL-24 in combination with dacarbazine exhibits a remarkably inhibitory effect on the proliferation of melanoma transplanted into nude mice.
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Objective To estimate the effects ot rosiglitazone on cultured HaCaT human keratinocytes and their possible mechanism.Methods HaCaT cells were cultured and treated with different concentrations ( 10,20,40,80 μ mol/L) of rosiglitazone or solvent for 24,48,72 and 96 hours,respectively.Cell proliferation was detected with methyl thiazolyl tetrazolium (MTT) assay.Western blot was performed to measure the protein expression of β-catenin and cyclin D1.Results Compared with the solvent-treated cells,the proliferation of HaCaT cells was significantly inhibited by 18.9％,23.7％,35.1％ and 44.6％ (all P＜ 0.05) after treatment with rosiglitazone of 10,20,40 and 80 μmol/L,respectively,for 48 hours.The expressions of β-catenin and cyclin D 1 were significantly lower in rosiglitazone-treated HaCaT cells than in solvent-treated cells (all P ＜ 0.05).Conclusion Rosiglitazone could inhibit the proliferation of HaCaT cells,likely by downregulating the expressions of β-catenin and cyclin D 1.
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Objective To investigate the effects of a short hairpin RNA targeting epidermal growth factor recereceptor (EGFR-shRNA) on Colo-16 cell apoptosis and sensitivity to rapamycin. Methods The expression vector of EGFR-specific shRNA was constructed. Colo-16 cells were classified into 4 groups, normal control group remaining untreated, liposome group transfected with lipofectamine 2000, negative control group transfected with shRNA-NC/Iipofectamine 2000 and positive interference group transfected with the expression vector of shRNA-EGFR/Lipofectamine 2000. After additional culture, immunocytochemistry and Western blot were conducted to detect the protein expression of EGFR, and flow cytometry to measure the apoptosis in Colo-16cells. MTT assay was performed to measure the sensitivity of Colo-16 cells to rapamycin. Results Compared with the normal control group, the expression of EGFR was down-regulated by 43.3% in positive interference group (F= 44.6, P< 0.05), and the sensitivity to rapamycin was increased by 2.44 folds. The apoptosis rate in positive interference group was (12.65±0.091)%, significantly different from that in the normal control group (F = 2042.9, P < 0.05). Conclusion The plasmid expression vector containing shRNA targeting EGFR can effectively suppress the expression of EGFR by Colo-16 cells, enhance the sensitivity of Colo-16 cells to rapamycin and induce the apoptosis in Colo-16 cells.
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Objective To study the effects of oncolytic adenoviruses ZD55 harboring IL-24 gene (ZD55-IL-24) on the apoptosis of human melanoma cell line A375. Methods The oncolytie adenoviruses ZD55-IL-24 were verified by PCR. Then, the viruses were propagated, purified, and titrated by HEK293 cell plaque assay. A375 cells were cultured, divided into three groups transfected with ZD55-1L-24, ZD55 fused with enhanced green fluorescent protein (ZD55-EGFP), and replication-deficient adenovirus ZD55 carrying IL-24 gene (AD-IL-24), respectively. The multiplicity of infection was 0.1, 1, 10 and 100, respectively.Subsequently, the eytotoxity of these viruses and proliferation of A375 cells were determined by crystal violet staining and methyl thiazolyl tetrazolium (MTT) assay, respectively. The expressions of EIA and IL-24 protein were detected by Western blot in A375 cells. Results PCR verified that the adenoviruses ZD55-IL-24 contained IL-24 gene without wild adenovirns contamination. Crystal violet staining revealed that ZD55-IL-24 had an obvious eytotoxic effect on A375 cells, and MTT assay indicated that ZD55-IL-24 inhibited the proliferation of A375 cells in a time-and concentration-dependent manner. As shown by Western blot analysis, ZD55-1L-24 expressed IL-24 and E1A protein in A375 cells with a high efficiency. Conclusions The oncolytic adenoviruses ZD55-IL-24 can efficiently express IL-24 gene, inhibit the proliferation of, and induce the apoptosis in A375 cells.
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Objective To evaluate the antitumor effect of oncolytic adenovirus expressing human IL-18 gene on malignant melanoma implanted in nude mice.Methods BALB/c nude mice were subcutaneously inoculated with A375 cells to establish a model of malignant melanoma.When the volume of implanted tumor reached 100-150 mm3,murine models were randomly divided into three groups to receive a 3-day intratumoral injection of IL-18 gene-expressing human oncolytic adenovirus named ZD55-IL-18,IL-18 gene-expressing adenovirus named Ad-IL-18,phosphate buffer saline(PBS),respectively.The tumor size was measured at an interval of 4 days for 9 weeks.Hematoxylin and eosin (HE)staining was performed to observe the morphological changes of tumor cells.The protein expression of IL-18 and E1A.microvessel density in tumor tissue,and apoptosis of tumor xenografts were detected by immuno fluorescence assay,immunohistochemistry and in situ end labeling technique (TUNEL).respectively.Results The treatment with ZD55-IL-18 significantly inhibited the growth of tumor.Forty-four days after the treatment,the mean tumor volume was 1039.378±29.67 mm3 in ZD55-IL-18-treated mice.significantly smaller than that in Ad-IL-18 treated mice(2900.46±62.65 mm3)and PBS-treated mice(3980.24±63.78 mm3).HE staining showed that the nuclei of tumor cells were heavily stained with few nucleoli in ZD55-IL-18-treated mice.Increased positivity rate of IL-18 was noticed in ZD55-IL-18-treated mice vs.AD55-IL-18-treated mice(83.4%±3.2%vs 24.4%±2.1%.P＜0.01).Moreover,immunofluorescence assay revealed the presence of E1A protein in tumor tissue.A decrease was found in the microvessel density in ZD55-IL-18-treated mice compared with the PBS-treated mice(P＜0.01).The apoptosis rate in tumor cells from high to low was 86.28%±3.25%in ZD55-IL-18-treated mice,43.67%±3.46%in Ad-IL-18-treated mice,and 10.73%±2.48%in PBS-treated mice;there was a significant difference between the three groups(all P＜0.05).Conclusion The oncolytic adenovirus expressing human IL-18 gene,ZD55-IL-18,has a significant inhibitory effect on the growth and metastasis of malignant melanoma implanted in nude mice.
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Objective To investigate the effects of COX-2 antisense oligonucleotide (AsODN) on the proliferation and expression of COX-2 in human skin squamous cell carcinoma cell line Coio-16. Methods The COX-2 AsODN was synthesized artificially, and various concentrations (50, 100, 200, 400 nmol/L) of the AsODN were transfected into Colo-16 cells with lipofectin followed by additional culture for different durations. The transfection results were observed with fluorescence microscopy. Subsequently, MTT assay,Western blotting and reverse transcription PCR were used to detect the cell proliferation, protein and mRNA expression of COX-2 in Coio-16 cells, respectively. Restults Compared with untreated cells, the proliferation of Colo-16 cells was inhibited significantly at 24, 48, 72 and 96 hours after transfection with different concentrations of COX-2 AsODN (all P < 0.05), and the COX-2 AsODN of 400 nmol/L exerted the highest inhibition rate of 60.3% at 48 hour. The average gray scale was 0.763±0.070, 0.600±0.065, 0.430±0.074 and 0.251±0.045 for COX-2 protein, 0.778±0.025, 0.602±0.041, 0.417±0.031 and 0.297±0.051 for COX-2 mRNA in Colo-16 cells transfected with COX-2 AsODN of 50, 100, 200, and 400 nmol/L respectively,significantly lower than that in untreated Colo-16 cells (all P < 0.05); there was a significant difference in the expression of COX-2 protein and mRNA among the cells transfected with the four concentrations of COX-2 AsODN and untreated cells (F = 83.54, 132.48, respectively, both P < 0.05). Conehtsions COX-2 AsODN can inhibit the proliferation, as well as the expression of COX-2 protein and mRNA in Colo-16 cells, which suggests that COX-2 AsODN has a potential therapeutic effect on skin squamous cell carcinoma.
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Objective To investigate the in vitro effect of sirolimus on the apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells. Methods Cultured Colo-16 cells were treated with different concentrations (50, 100, 150, 200 nmol/L) of sirolimus for various durations ( 12, 24, 48, 72 hours). Subse-quently, cell proliferation was detected by MTT assay, and cell apoptosis by Annexin V-FITC and PI double staining. Morphological changes of the cells were observed with Hoechst 33258 fluorescent staining. Total RNA was extracted from Colo-16 cells treated with sirolimus for 48 hours, and subjected to reverse tran-scription (RT)-PCR for the detection of mRNA expression of B cell lymphoma/leukmia-2 (Bcl-2) and Bcl-2-associated X Protein (Bax). Results Sirolimus inhibited the proliferation of Colo-16 cells in a time-and dose-dependent fashion. The early apoptosis rate was 7.26%±0.26%, 8.34% ±0.19%, 9.86%±0.14%, 11.92% ±0.15% in Colo-16 cells treated with sirolimus of 50, 100, 150, and 200 nmol/L, respectively, signifi-candy higher than that in untreated cells (1.53%±0.09%, P < 0.05); a positive correlation was observed between the apoptosis rate and concentrations of sirolimus (r = 0.955, P = 0.000). Typical morphological changes of apoptosis, such as chromatin condensation and margination as well as nuclear fragmentation were observed by fluorescence staining. After treatment with sirolimus for 48 hours, a significant decrease was observed in the mRNA expression of Bcl-2, while an increase in that of Bax was noticed. Conclusion Sirolimus could induce Colo-16 cells apoptosis in vitro, which may be associated with the modulation of expression of apoptosis-regnlating genes, such as Bcl-2 and Bax.
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Objective To investigate the prevalence of mite sensitivity in patients with urticaria or other skin rashes, and to observe the clinical efficacy of a specific immunotherapy(SIT) by the Injectio dermatophagoidei farinae for the patients.Methods In 7-year period(1998-2005), skin prick test(SPT) with a dust mite(Df) allergen was carried out to detect the prevalence of mite sensitivity in OPD patients suffering from skin rashes.Among the patients sensitive to mite with SPT ≥++ response, 3 groups were established.In group A, routine SIT with Injectio dermatophagoidei farinae was conducted.In 9-week increasing dose phase, three stepwise increasing volumes(0.3ml, 0.6 ml and 1.0 ml) each case was injected subcutaneously with mite concentration of 1 ∶ 100 000(w/v) , 1 ∶ 10 000(w/v) or 1 ∶ 5 000(w/v) respectively once a week, followed by a maintenance dose phase for an injection with 1 ∶ 5 000(w/v) 1.0 ml/wk for 6 weeks.Group B received rush SIT with mite injections.A total of 15 injections in a course of therapy with same concentration and volume was given as those for the routine ones except shortened intervals, namely, 9 initial injections completed in 3 days by three injections of each concentration per day with two 30 min intervals, maintenancedoses were then provided in 6 days with 1 ∶ 5 000(w/v) 1.0 ml/d.Thereafter, both groups A and B were maintained for one year with a dose of 1 ∶ 5 000(w/v) 1.0 ml every 2 wk.Group C received antihistamine treatment as control, the patients received daily oral Ebastine 10 mg in the morning and Cetirizine dihydrochloride 10 mg in the evening for one week course and pro re nata later.Levels of serum tIgE and serum mite sIgE were detected by ELISA in 20 urticaria cases before and after one year mite SIT.Results Altogether, 2 685 cases with skin rashes were detected by Df allergen SPT.The prevalence of urticaria cases sensitive to mite was 70.3%(1 754/2 496), which was higher than that of eczema 63.5%(54/85) and anaphylactoid purpura 60.6%(63/104)(P