Effect of miR-5581-5p/TRIM22 on acute promyelocytic leukemia cell differentiation / 国际肿瘤学杂志

Objective:To investigate the function of miR-5581-5p and its interaction with tripartite motif 22 (TRIM22) during the terminal differentiation of human acute promyelocytic leukemia (APL) cells into granulocytes.Methods:APL cells (NB4) were differentiated into granulocytes by all-trans retinoic acid (ATRA), using dimethylsulfoxide (DMSO) as the control. The expression of TRIM22 was detected by real-time fluorescent quantitative PCR (qRT-PCR) and Western blotting, and the expression of miR-5581-5p was detected by qRT-PCR during cell differentiation. miRNA expression was regulated by cell transfection with miR-5581-5p mimic and inhibitor, and negative control was set, and qRT-PCR was used to verify the regulatory effect. Luciferase binding assay was performed to detect the presence of targeted binding. Western blotting was used to detect the expression of TRIM22 after miRNA differential expression. Flow cytometry was used to detect the effects of the regulation of miR-5581-5p on the differentiation of NB4 cells induced by ATRA.Results:After ATRA induced NB4 cells to differentiate into granulocytes, the gene expression level of TRIM22 was significantly higher than that of the control group (24.56±2.80 vs. 1.02±0.13; t=8.392, P=0.001). The level of protein expression was also significantly higher than that of the control group (0.80±0.01 vs. 0.17±0.01; t=44.900, P<0.001). The expression level of miR-5581-5p in NB4 cells differentiation group was significantly lower than that in the control group (0.14±0.02 vs. 1.01±0.08; t=10.840, P<0.001). The results of the dual luciferase reporter gene showed that the luciferase activity of the co-transfected miR-5581-5p mimic and TRIM22 WT group was significantly lower than that of the co-transfected miR-5581-5p mimic and TRIM22 MUT group (0.73±0.02 vs. 0.98±0.03; t=7.534, P=0.002). Western blotting showed that after transfection with miR-5581-5p inhibitor, the expression of TRIM22 was significantly higher than that of the negative control (0.44±0.01 vs. 0.21±0.01; t=18.290, P<0.001). While after transfection with miR-5581-5p mimic, the expression of TRIM22 decreased significantly compared with the negative control (0.62±0.01 vs. 0.80±0.02; t=6.402, P=0.003). CD11b expression of miR-5581-5p mimic group after ATRA treatment was significantly lower than that of the control group (45.80±1.80 vs. 56.61±1.88; t=4.159, P=0.014). The expression of CD11b in miR-5581-5p inhibitor group was significantly higher than that in the control group (66.48±2.54 vs. 52.60±1.70; t=4.539, P=0.011). Conclusion:miRNA-5581-5p can bind to TRIM22 3′UTR and negatively regulate TRIM22 expression. The decrease of miR-5581-5p can increase the expression of TRIM22, then promote the differentiation of ATRA-induced NB4 cells into granulocytes.

Function and mechanism of TRIM22 targeting eIF4E in the process of NB4 cells differentiation / 国际肿瘤学杂志

Objective To investigate the function of tripartite motif protein 22 (TRIM22) and the interaction with eukaryotic translation initiation factor-4E (eIF4E) in the differentiation of NB4 cells, one kind of acute promyelocytic leukemia cells, which elucidates the mechanism of TRIM22 targeting to regulate eIF4E.Methods The model of NB4 cells inducing differentiation was established in vitro.The expression changes of gene and protein of TRIM22 and eIF4E were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.In addition, the effect on cell function and protein expression level of eIF4E after adopting electroporation technology to depress or over-express TRIM22 was detected by CCK-8 and flow cytometry.Finally, the interaction of TRIM22 and eIF4E was verified by using co-immunoprecipitation (CO-IP).Results The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79 (F=280.700, P=0.000), and the protein relative expression level was gradually increasing from 0.22±0.03 to 0.51±0.05 (F=51.430, P=0.000) after the all-trans-retinoic acid (ATRA) induction for NB4 cell.However, the mRNA relative expression level of eIF4E was gradually decreasing from 1.01±0.09 to 0.47±0.06 (F=20.520, P=0.000), with the same trend, the protein relative expression level was gradually decreasing from 0.97±0.02 to 0.64±0.09 (F=14.700, P=0.001).The expression level of PE-CD11b in the TRIM22 over-expression group with ATRA detected by flow cytometry [(78.80±2.00)%] was higher than that in the transfection group of empty vetor with ATRA [(58.70±2.70)%] (t=9.535, P=0.000) and the cotransfection group with ATRA [(61.60±3.80)%] (t=8.187, P=0.000).Meanwhile, the protein level of eIF4E changed reversely after over-expressing the gene level of TRIM22 (t=4.985, P=0.007).The CO-IP experiment was used to verify the interaction of TRIM22 and eIF4E.ConclusionTRIM22 is able to promote the cell differentiation during the process of NB4 cells differentiation.Furthermore, eIF4E is a target of TRIM22 for binding with, which plays an important role in depressing the expression of eIF4E.

Helicobacter pylori and gastric stump cancer / 国际肿瘤学杂志

The gastric stump cancer is closely associated with Helicobacter pylori.Helicobacter pylori can promote the proliferation of gastric remnant mucosa epithelial cells,the production of nitroso compounds in gastric juice and abnormal expressions of some genes in human body,and finally to promote the occurrence of gastric stump cancer.The eradication of Helicobacter pylori infection is expected to reduce the incidence of gastric stump cancer.

Risk factors analysis of initial surgery and postoperative complications of Crohn's disease / 中华消化外科杂志

Objective To explore the risk factors of initial surgery and postoperative complications of Crohn's disease (CD).Methods The retrospective case-control study was conducted.The clinical data of 227 patients with CD who were admitted to the Cangzhou Central Hospital from April 2011 to July 2015 were collected.Treatment principles included reducing the clinical symptoms,promoting healing of intestinal mucosa under endoscopy,delaying CD progression and preventing intestinal exhaustion and related complications.The medication was performed in the early period.The resection of partial intestines was applied to patients if there was poor effect of medication or combined with intestinal obstruction,intestinal fistula,digestive tract perforation,abdominal abscess and complex anal fistula.Observation indicators:(1) treatment situation,(2) follow-up situation,(3) related factors analysis affecting initial surgery of patients with CD,(4) related factors analysis affecting postoperative complications of patients after initial surgery for CD.Follow-up using regular telephone interview and outpatient examination was performed up to May 2016.Follow-up included the wound infection,abdominal abscess,intestinal obstruction,anastomotic fistula and pulmonary infection.Measurement data with normal distribution were represented as-x ± s and comparison between groups was analyzed using the t test.Count data were represented as the proportion and analyzed by the chi-square test.The univariate analysis was done using the chi-square test and Kruskal-Wallis test,and multivariate analysis was done using the Logistic regression model.Results (1) Treatment situation:of 227 patients,68 underwent initial surgery and 159 didn't undergo surgery.The duration from diagnosis to initial surgery in 68 patients was (4.7 ± 2.5) months.Of 68 patients with surgery,28 received the emergency surgery and 40 received the selective surgery.Operation time and volume of intraoperative blood loss were (175 ±44) minutes and (285 ± 110) mL,respectively.The side-to-side anastomosis was conducted in 47 patients and non-side-to-side anastomosis in 21 patients.Other 159 patients without surgery received the medication of mesalazine,hydrocortisone,methotrexate and infliximab.(2) Follow-up situation:68 patients with initial surgery were followed up for 5-61 months,and 22 had postoperative complications.Of 9 patients with anastomotic fistula,6 had enterocutaneous fistula (5 patients with enterocutaneous fistula were improved by selective surgery,and the other patient was progress to acute diffuse peritonitis and then was improved by peritoneal lavage,adequate drainage and nutritional support therapy after emergency surgery).Three patients with anastomotic abscess were improved by adequate drainage.Six patients with secondary intestinal obstruction were improved by conservative treatment.Three patients with abdominal abscess were improved after antiinflammatory treatment and adequate drainage.Two patients with wound infection were improved by regular dressing change.Two patients with pulmonary infection were improved by anti-inflammatory and phlegm conservative treatment.(3) The related factors analysis affecting initial surgery of patients with CD.The results of univariate analysis showed that age of diagnosis,smoking history and behavior of disease were the related factors affecting initial surgery of patients with CD (Z =-2.120,x2 =5.082,50.512,P＜ 0.05).The results of multivariate analysis showed that A3 of age of diagnosis,B2 and B3 of pattern of disease were the independent risk factors affecting initial surgery of patients with CD [OR =15.624,10.535,28.509,95％ confidence interval (CI):4.856-29.375,3.609-17.637,8.526-79.228,P ＜ 0.05].(4) The related factors analysis affecting postoperative complications of patients after initial surgery for CD.The results of univariate analysis showed that preoperative levels of albumin (Alb) and hemoglobin (Hb),emergency surgery,operation time and anastomotic method were the related factors affecting postoperative complications of patients after initial surgery for CD (x2 =10.757,7.639,6.773,4.309,16.346,P ＜ 0.05).The results of multivariate analysis showed that preoperative Alb≤28 g/L,Hb≤ 100 g/L,emergency surgery and non-side-to-side anastomosis were the independent risk factors affecting postoperative complications of patients after initial surgery for CD (OR =9.592,8.849,6.538,12.645,95％CI:2.209-25.235,2.034-24.773,1.846-15.893,3.935-38.873,P ＜ 0.05).Conclusions The age of diagnosis ＞ 40 years,B2 and B3 of CD are high risk group of initial surgery.The poor preoperative nutritional status,emergency surgery and non-side-to-side anastomosis are independent risk factors affecting postoperative complications of patients after initial surgery for CD.

Sorafenib inhibits tumor growth through modulating vasculogenic mimicry in a hepatocellular carci-noma xenograft model / 国际肿瘤学杂志

Objective To investigate the effects and the mechanism of sorafenib on hepatocellular car-cinoma growth and vasculogenic mimicry (VM)in mice.Methods A subcutaneous implantation mouse model of human hepatocellular carcinoma (HCC)HepG2 cells were established.Mice inoculated with HepG2 cells were randomly divided into the treatment group (sorafenib 30 mg·kg -1 ·d -1 )and the control group by using of paired comparison method.Growth of established tumor xenografts was monitored at least twice weekly by vernier caliper measurements.VMwas assessed by immunohistochemical assay and periodic acid schiff reaction (PAS)histochemical double-staining.The expressions of HIF-1 α,VEGFA,VEGFR-1 and MMP-2 in tumor tissues were also assessed by immunohistochemical assay,Western blotting and real-time quantitative PCR. Results The tumor volume in the sorafenib group was obviously decreased compared with the control group (809.69 mm3 ±208.71 mm3 vs 1 678.00 mm3 ±31 3.29 mm3 ),with a statistically significant difference (t =6.1 03,P =0.030).Haematoxylin and eosin (HE)staining showed that tumor tissues treated with sorafenib were characterized by obvious necrosis,but there were not the same cases in the control group.Sorafenib group significantly reduced the number of tumor functional vessel in HepG2 xenografts compared with the control group,as assessed by tumor vasculature uptake of DiOC7 (4.77 ±0.1 5 vs 8.44 ±0.68,t =9.1 92,P =0.01 3).The number of VMwas significantly decreased by sorafenib (1 .04 ±0.46 vs 2.66 ±0.42,t =4.51 0, P =0.041 ).Relative to controls,CD31 -positive vessels decreased after treatments (3.42 ±0.1 0 vs 1 .26 ± 0.1 4),with a statistically significant difference (t =21 .580,P =0.002).Compared with the control group, the protein levels of HIF-1 α(0.65 ±0.03 vs 1 .00 ±0.00),VEGFA (0.51 ±0.02 vs 1 .00 ±0.00), VEGFR-1 (0.45 ±0.04 vs 1 .00 ±0.00)and MMP-2 (0.69 ±0.02 vs 1 .00 ±0.00)were significantly decreased in the sorafenib group (t =1 9.650,P =0.003;t =40.493,P =0.000;t =23.429,P =0.002;t =26.071 ,P =0.002).Compared with the control group,the mRNA levels of HIF-1 α(0.78 ±0.05 vs 1 .00 ±0.00),VEGFA (0.52 ±0.05 vs 1 .00 ±0.00),VEGFR-1 (0.45 ±0.02 vs 1 .00 ±0.00)and MMP-2 (0.71 ±0.02 vs 1 .00 ±0.00)were also significantly decreased in sorafenib group (t =6.840,P =0.021 ;t =27.71 0,P =0.001 ;t =62.740,P =0.000;t =23.850,P =0.002).Conclusion Sorafenib can inhibit the tumor growth and VMchannels formation,which may be related with the HIF-1 αand VEGFA /VEGFR-1 signa-ling pathway.

Experimental study of correlation between T-cell paradigm and bone loss induced by estrogen deficiency / 中国免疫学杂志

Objective:To demonstrate the relationship between the Th 1/Th2/Th17/Treg paradigm and the bone loss induced by estrogen deficiency and looking for potential target for clinical treatment.Methods:30 BALB/c mice were divided randomly into the normal control group , the sham operation group , and the ovariectomy group.The serum estradiol ( E2 ) was assessed by ELISA.Bone mineral density (BMD) of thigh bone was measured with dual energy X ray absorptiometry.Meanwhile,the T-cell subsets (Th1:CD4+TNFα+, Th2: CD4+IL-4+, Th17: CD4+IL-17 A+, Treg: CD4+CD25+Foxp3+) in spleen lymphocytes were analyzed by flow cytometry.Results:Compared with the normal group and the sham operation group , both E2 and BMD in the ovariectomy group decreased significantly ( P<0.05 ).The percentage of Th 1 and Th17 subset increased while the percentage of Th 2 and Treg decreased significantly in ovariectomy mice compared with sham operation mice.Correlation analysis showed that BMD was positively related to E 2 level and the percentage of Th 2 and Treg subset;however ,BMD was negatively related to the percentage of Th 1 and Th17 subset ( P<0.05 ).Conclusion: Conclusion: T-cell paradigm was involved in the bone loss induced by estrogen deficiency.Modifying T-cell paradigm may become a potential target for reducing bone loss induced by estrogen deficiency .

Roles and mechanisms of Fas/FasL in the apoptosis of HL-60 cells induced by euphorbiasteroid / 国际肿瘤学杂志

Objective To investigate the effect of euphorbiasteroid on inducing the apoptosis of HL-60 cells and demonstrate whether the Fas/FasL signaling pathway is involved in the induction of apoptosis. Methods HL-60 cells were treated with dose of 2.5,10,40 μg/ml of euphorbiasteroid in vitro for 24 h respectively.After that,cell counting Kit-8 was used to detect cell proliferation.The morphology of HL-60 cells were observed under light and fluorescent microscopy.The early cell apoptosis was detected by using flow cytometry with Annexin Ⅴ-FITC /PI double staining.The expressions of Fas,FasL,caspase-8 and caspase-3 mRNA were analyzed by the method of RT-PCR.The activities of caspase-8 and caspase-3 were examined by chromatometry.Results Compared with 1640 control group,HL-60 cell proliferation was inhibited significant-ly by euphorbiasteroid.The inhibition rates were (34.9 ±3.7)%,(54.6 ±5.2)% and (61.3 ±4.3)%respectively.Moreover,HL-60 cells exhibited typical morphological features.Early cell apoptosis rates of HL-60 cells were (23.4 ±3.1)%,(35.7 ±4.3)% and (53.2 ±3.9)% respectively.Furthermore,the expressions of Fas,FasL,caspase-3 and caspase-8 mRNA were up-regulated significantly after euphorbiasteroid administration in a dose-dependent manner (P<0.01 ).After treated with euphorbiasteroid,the activities of caspase-8 and caspase-3 were significantly enhanced (P<0.01 ).Conclusion The up-regulation effect of euphorbiasteroid on Fas/FasL signaling pathway might contribute to the apoptosis of HL-60 cells.

Induction of apoptosis by osthole in HL-60 cells and the molecular mechanism research / 国际肿瘤学杂志

Objective To detect the effect of osthole on proliferation and apoptosis of HL-60 cells and its molecular mechanism.Methods HL-60 cells proliferation was measured through the CCK8 assay method.The cell morphological changes were observed by Hoechst33342 staining after 8 h of drug effect.Induction of apoptosis was determined by flow cytometry and fluorescent microscopy.Expressions of Bcl-2 and Bax mRNA were evaluated by RT-PCR,and the expressions of cleaved caspase-3,caspase-8,caspase-9,Fas and FasL were evaluated by using western bolt assay.Results Osthole could inhibit the proliferation of HL-60 cells,the maximum inhibiting rate was (90.7 ±4.5)％,F =138.46,P =0.000; the apoptosis rate was 33.6％,F =27.75,P =0.006.The changes of apoptosis of cells and nucleus were shown in cell morphological observation.Osthole affected the decrease of the mRNA levels of Bcl-2 and the increase of the Bax mRNA levels via a dosedependent manner(F =210.12,P =0.000).Western blotting demonstrated that osthole could lead to the increase of the expression levels of cleaved caspase-3,caspase-8,caspase-9,Fas and FasL in the HL-60 cell line via a time-dependent manner.Conclusion Data suggests that osthole inhibits proliferation and induces apoptosis of HL-60 cells through mitochondria-dependent pathway and death-receptor pathway.

Study on molecular regulation mechanism of VEGF in HL-60 cells after induced differentiation / 国际肿瘤学杂志

Objective To study the molecular regulation mechanism of VEGF in the model of ATRA induced differentiation in HL-60 cells,and to provide new targets for leukemia anti-angiogenic therapy.Methods The morphology was observed by Wright-Gimesa staining; HL-60 cells differentiation was detected by NBT reduction experiment.VEGF,STAT3,c-myc mRNAs were measured by reverse transcription-PCR;VEGF,STAT3 and c-myc proteins were determined by Western blot.Results The proliferation of HL-60 cells was inhibited obviously by ATRA(1 μmol/L) with the induction of differentiation,NBT positive rate was 82.59％ (t =-24.157,P ＜ 0.01) ; VEGF mRNA (t =7.339,P ＜ 0.05),STAT3 mRNA (t =3.667,P ＜0.05) and c-myc mRNA (t =6.858,P ＜ 0.05) were all down-regulated.VEGF protein (t =3.386,P ＜0.05),STAT3 protein(t =4.074,P ＜ 0.05) and c-myc protein (t =3.333,P ＜ 0.05) were all down-regulated.Conclusion VEGF expression level is reduced with the procession of differentiation of HL-60 cells,which may be largely correlated with the down regulation of STAT3 and c-myc.

Effects of honokiol on the proliferation and apoptosis of human acute leukemia U937 cells / 国际肿瘤学杂志

ObjectiveTo detect the mechanism of the growth inhibition and apoptosis of human acute leukemia cell line U937 cells induced by honokiol.MethedsThe proliferation inhibition was detected by MTT method.Cell apoptosis was tested by Hoechst 33342 staining and flow cytometry with Annexin V/PI staining.RT-PCR was used to detect the mRNA expression of the apoptosis gene Bcl-2,Bax,Caspase 3,Caspase 8 and Caspase 9.ResultsThe inhibition effect of honokiol(5 μg/ml,48 h) on U937 cells proliferation could he observed,and the inhibition rate of 10 μg/ml honokiol on cell proliferation reached above 50％ (48 h).U937 cells proliferation could be completely inhibited for 120 h. U937 cells apoptosis rate reached 26.8％ (P ＜0.01)after being treated with 10 μg/ml honokiol.After being treated with 10 μg/ml honokiol for 48 h,the Bcl-2 gene expression in U937 cells was reduced (control group:0.33 ± 0.02,experimental group:0.14 ±0.01,P ＜ 0.01 ),and the Bax gene expression was elevated ( control group:0.1 ± 0.01,experimental group:0.87 ± 0.08,P ＜ 0.01 ).The gene expressions of Caspase 3 ( control group:0.48 ± 0.01,experimental group:0.87±0.06,P ＜0.01),Caspase 8(control group:0.23±0.02,experimental group:0.41 ±0.07,P ＜ 0.01 ) and Caspase 9 ( control group:0.44 ± 0.05,experimental group:0.76 ± 0.06,P ＜ 0.01 ) were all increased.The activity of Caspase-3 was 0.325 ±0.089,which was significantly higher than that of the control group ( P ＜0.01 ).ConclusionHonokiol can significantly inhibit the proliferation and induce cell apoptosis of human acute leukemia cell line U937 cells.The mechanism is related to the up-regulation of Bax and down-regulation of Bcl-2,and the endogenous and exogenous pathways are both inolved in the apoptosis process.

Growth inhibition and apoptosis induced by oridonin combined with valproic acid in HL-60 cells / 国际肿瘤学杂志

Objective To investigate the apoptosis-inducing effect of oridonin combined with valproic acid( VPA)on leukemic cell line HL-60,and study the feasibility of oridonin combined with VPA to be used in clinical practice. Methods Oridonin of 6-12 μmol/L combined with VPA of 0. 5-1 mmol/L were added in exponential growth HL-60 cells respectively. Cell count assays were used to measure the growth inhibitory effect of oridonin combined with VPA or alone. Flow cytometry was used to evaluate apoptosis with Annexin V and propidium iodide (PI) double staining. Results Combined use of oridonin and VPA could synergistically inactivate HL-60 cells,and inhibit the cell proliferation and induce apoptosis in a dose-and time-dependent manner (P < 0.05 ). Conclusion Oridonin has a synergistic effect combined with VPA. Oridonin has a promising prospect in clinical use of leukemia.

Experimental study on inducing differentiation of K562 cells into dendritic cells by A23187 / 国际肿瘤学杂志

Objective To explore the method for differentiation induction of leukemia cells into dendritic cells(DC) by A23187 in vitro. Methods Chronic myeloid leukemia K562 cells were cultured with A23187 or cytokine to induce differentiation and form DC. The morphologic features of cells were observed under inverted microscope, the changes of DC surface marks were determined by flow cytometry and RT-PCR, the ability to stimulate lymphocyte proliferation was tested by MTT colorimetry. Results Under the condition of the does (385 ng/ml) of A23187 for four days, some of K562 cells were found in typical dendritic appearance.The expression of DC markers CD1a,CD83 ,HLA-DR,CD86 and CD80 was 6.65 ±2.70,7.37 ±2.40,6.24 ±4.29, 21.60 ± 3.84, 18.52 ± 4.48 repectively, and increased obviously compared with the negative control group(2.80 ±0.52,1.85 ±0.56,2.25 ±0.47,6.69 ±0.83,9.96 ±3.53). The differences had statistical significance (P < 0.05). K562 cells derived from DC acquired the ability to stimulate lymphocyte proliferation.Conclusion A23187 can induce the leukemia cells differenntiation into activated DC-like cells rapidly.

Effect of polypeptide extract from scorpion venom (PESV) on immune escape of Lewis lung carcinomas / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of polypeptide extract from scorpion venom (PESV) on immune escape of Lewis lung carcinomas (LLC) and its mechanism.</p><p><b>METHOD</b>Forty C57BL/6J mice were inoculated with LLC cells suspension (1 x 10(7) cells/ mL) in right armpit subcutaneously. The tumor-bearing mice were randomly divided into two groups: the control group and the PESV group. PESV was intragastrically subjected to the mice of the experimental group for 18 days. The tumor volume and tumor inhibitory rate were determined. The expression levels of VEGF,TGF-beta1 and IL-10 in tumor microenvironment were determined by immunohisto-chemistry-staining and ELISA. Surface co-stimulatory molecules CD80 and CD86 of tumor infiltrating dendritic cells (DC) were determined by immunohistochemistry-staining and flow cytometry.</p><p><b>RESULT</b>The growth inhibitory rate of PESV was 56. 60%. The expression levels of VEGF,TGF-beta1 and IL-10 were decreased in tumor and serum, while the expression of co-stimulatory molecules CD80 and CD86 on DC were increased in tumor. Compared with the control group, the differences were all significant (P < 6.05).</p><p><b>CONCLUSION</b>PESV was effective in recovering immuno-surveillance and intervening immune escape of lung cancer through multi-pathway. And its effects might be attained by decreasing the level of VEGF, TGF-beta1 and IL-10 in tumor microenvironment and increasing the expression of co-stimulatory molecules CD80 and CD86 on DC.</p>

Sencitivity enhancement of U251 cells to TRAIL by cisplatin / 国际肿瘤学杂志

Objective To evaluate the positive effects of cisplatin on sensitivity of human glioma U251 to tumor necrosis factor-related apoptosis inducing ligand and to investigate the potential mechanism. Methods The expression of green fluorescent protein (GFP) in U251 which was transfected with pAdxsi-GFP-TRAIL was observed by inverted fluorescent microscope ×400) and to ascertain the MOI. The proliferation inhibition was studied by MTT method. Morphological change was detected through inverted florescent microscope and the Hoechst33342 staining assay was used to verify whether cell apoptosis could be induced or not. The cell apoptosis was also analyzed by flow cvtometry with propidium iodide staining. Semi-quantitative RT-PCR was introduced to detect the mRNA expression of apoptosis related gene.Results The expression of TRAIL mRN A was significantly upregulated after transfection. Compared with treatment group of cisplatin and TRAIL alone, the proliferation of U2S1 was significantly inhibited in the cisplatin sensitizing TRAIL group (P < 0.05 ). Nuclear shrinkage and pyknosis fragmentation were observed by Hoechst 33342 staining assay; Apoptotic peak was detected from the results of flow cvtometry and there were significant differences between the sensitizing group and the other two groups ( P < 0.05 ) ; Moreover, the relatively high expression of TRAIL, DR5, caspaseS and down - regulated survivin genes were also observed. There was no significant changes in DR4 expression. Conclusion Cisplatin could extremely enhance the sensitivity of U251 cells to TRAIL And the potential mechanism may related to the increase of TRAIL, DRS, caspaseS genes while the reduction of surivivin gene.

Effect of TGF-β1/SMAD signaling pathway on K562 cells growth inhibition caused by HMBA / 国际肿瘤学杂志

Objective To investigate the effect of TGF-β1/SMAD signaling pathway on K562 cells growth inhibition caused by HMBA. Methods After establishing the in vitro differentiation model with HMBA on K562 cells, the MTT assay was used to detect the proliferation of K562 cells, the cell cycle profile was detected by flow cytometry, and the mRNA expression of TGF-β1, SMAD3, SMAD4 and EVI1 was measured by RT-PCR assay. Results HMBA could inhibit the proliferation and promote the differentiation of K562 cells obviously, which was time and concentration-dependent, and the 72 h corresponding IC50, was about 2 mmol/L. Within 72 h, flow cytometry assay indicated that the ration of G0-G1 phase cells was up-regulated, and the results of RT-PCR showed that relative mRNA expression of TGF-β1, SMAD3 and SMAD4 at mRNA level was increased gradually while that of EVI1 was decreased gradually. Conclusion HMBA can inhibit K562 cells proliferation through TGF-β1/SMAD signaling pathway.

Anticancer effect of polysaccharide isolated from Polyporus sp. M05 / 国际肿瘤学杂志

Objective To study effect and mechanism of fungus polysaccharide PSM-a of Polyporus sp.M05 on S180 bearing mice. Methods MTT method was used to detect the inhibiting role of PSM-a on S180 cells proliferation in vitro. S180 mice model was established,and was administered by gavage. Tumor volume was detected, and the ratio of tumor to mile weight and inhibiting tumor rate. The activity of NK and LAK cells on the target cells was analyzed by MTT colorimetric assay ;HE stain was used to detect the necrosis of tumor cells. Results PSM-a could inhibit S180 cells grouth in vitro. PSM-a could decrease the tumor weight and increase the ratio of tumor volume and mice weight; Tumor inhibiting rate reached 80% and above when treated with 250 μg/nml PSM-a. PSM-a could increase the activity of NK and LAK cells, and necrosis happened. Conclusion PSM-a could significantly inhibit the growth of S180 cells, and the mechanism bnay be related with the increased killing activity of immunne cells to tumor cells.

Molecular mechanism of different sensitivities of PML-RARα to apoptosis induced by apoptosis inducing agents / 国际肿瘤学杂志

Objective To study the molecular mechanism of different sensitivities to apoptosis induced by low concentration of As2O3 in PML-RARα negative HL-60 cells and PML-RARα positive NB4 cells. Meth-ods NB4 and HL-60 cells were cultured with As2O3 for 1 to 4 days; cell proliferation were detected by MTT method; the apoptosis was detected by flow cytometry,Bcl-2,Bax and Fas mRNA were determined by RT-PCR. Results The proliferation of NB4 cells was inhibited obviously by As2O3(1.0 μmol/L)with the induction of apoptosis( P <0.05) ,which was accompanied by the down-regulation of Bcl-2 mRNA expression( P <0.05)and the ratio of Bcl-2/Bax(P <0.05), but there was no obvious variation of Bax and Fas expression( P >0.05). Inhibition of proliferation and apoptosis were not obvious in PML-RARα negative HL-60 cells induced by low concentration As2O3 ( P >0.05), and there was no obvious variation of Bcl-2, Bax, Fas mRNA expres-sion or Bcl/Bax ratio( P >0.05). Conclusion The ratio of Bcl-2/Bax is contributed to the different sensitiv-ities of PML-RARα negative HL-60 cells and positive NB4 cells induced by low concentration of As2O3.

Mechanism of VEGF expression and secretion in HL-60 cells induced by all-trans refinoic acid / 国际肿瘤学杂志

Objective To explore molecular mechanism of expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein in HL-60 cells induced by all-trans refinoic acid (ATRA). Methods MTr method was used to detect the proliferation of HL-60 cells induced by ATRA,cell cycle and CD11b expression in HL-60 cells were detected by flow cytometry. Expression of VEGF, c-myc, by-poxia-inducible factor(HIF)-lα, matrix metalloproteinase (MMP)-9 and MMP-2 mRNA were detected by semi-quantitative RT-PCR. VEGF protein in HL-60 cells culture supernatant was measured by ELISA before and after being induced by ATRA. Results After treatment with ATRA,the proliferation of HL-60 cells was obviously inhibited, CD11b expression increased, trend of granulocyte directional differentiation emerged, and differentiation degree was increasd(P <0. 05) ;expression level of c-my and VEGF mRNA was down-regulated (P < 0. 05), but expression level of HIF-1α mRNA was up-regulated (P < 0. 05). VEGF protein level in HL-60 cells culture supernatant was decreased by blocking the expression of MMP-9 or MMP-2(P <0. 05).Conclusion VEGF expression has positive correlation with c-myc expression,but has negative correlation with HIF-1α expression. MMP-9 and MMP-2 may be the main factors regulating VEGF secretion in HL-60 cells.

Reversal of multidrug resistance of K562/ADM cells in vitro by the monomer of traditional Chinese medicine extract and investigation of its molecular mechanism / 国际肿瘤学杂志

Objective To investigate the reversal effect of the monomer of traditional Chinese medicine on muhidrug resistance(MDR) and its possible mechanism in K562/ADM cell line in vitro. Methods With different concentrations of baicalin, geniposide administered to K562/ADM cells, the proliferation of K562/ ADM cells was detected by the MTY assay. Expression of mdr-1 mRNA, Topo Ⅱ mRNA was measured by semi-quantitive RT-PCR. Results Thatbaicalin and geniposide could increase the sensitivity of K562/ADM cells to adriamycin, multiples of reversion were 1.95 times and 1.46 times. The proliferation of K562/ADM cells was in-hibited obviously by baicalin and geniposide, the level of mdr-1 mRNA expression was down-regulated and the Topo Ⅱ mRNA was up-regulated(P<0.01 ). Conclusion Baicalin and geniposide may reverse the multi-drag-resistance of K562/ADM cells, which was related to the down-regulation of mdr-1 expression and up-reg-ulation of Topo Ⅱ beta expression.

Changes and function of Bax and Fas in HL-60 cell apoptosis induced by two fungus polysaccharides / 国际肿瘤学杂志

Objective To detect the proliferation inhibition and apoptosis of HL-60 cell induced by APS-1 and APS-2 isolated from Polyporus sp. M05 and to investigate its mechanism. Methods The proliferation inhibition was detected by living cells count method,and chosed proper concentration.Flow cytometry with propidium iodide staining was used to detect cell apoptosis. Semi-quantitative RT-PCR was used to detect the expression of apoptosis related gene. Results APS-1 and APS-2 could significantly inhibit the proliferation of HL-60 cells on a time and dose dependent manner. Apoptosis ratio increased to 47. 9% and 26. 8% after HL-60 cells were exposed to APS-1 and APS-2 respectively for 48 h,and the differences had statistical significance (P <0.01). After being induced by APS-1,mRNA of Bax,Fas,Caspase-3 was upregulated. And after being induced by APS-2,mRNA of Bax,Caspase-3 was upregulated,while Fas mRNA did not change. Conclusion APS-1 and APS-2 can inhibit the proliferation and induce apoptosis of HL-60 cells. Mechanism of HL-60 cell apoptosis induced by APS-1 is related to both mitochondrial pathway and Fas signaling pathway,while apoptosis induced by APS-2 is only related to mitochondrial pathway.