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The growth-inhibiting and apoptosis-inducing effects of WW domain-containing oxidoreductase (WWOX)gene on ovarian cancer cell line A2780 were investigated.The full length cDNA of human WWOX gene was amplified from normal human ovary tissues.The correct cDNA of full length WWOX was subcloned into eukaryocytic expression vector pCMV.After introduction of WWOX gene into cancer cells with liposome,the WWOX mRNA and protein level in the cancer cells were detected by reverse transcription polymerase chain reaction(RT-PCR)and immunoblotting.The growth activities of cancer cells were detected by Trypan blue staining.The clone formation assayin soft agar was employed to observe the proliferation of the cancer cells.Apoptosis was examined by DNA ladder and acridine orange-ethidium bromide fluorescent staining.The results showed that 72 h after WWOX gene transfection,the WWOX expression was increased significantly(P<0.01).The growth of ovarian cancer cells was decreased by 16.41% to 38.49%(P<0.01).The clone formation abilities were reduced(P<0.01).Some cancer cells presented the characteristic morphological changes of apoptosis with obvious ladder bands on electrophoresis.The apoptosis rate was(20.7±6.0)%(P<0.01).It was concluded that over-expression of WWOX gene could induce apoptosis and inhibit the growth of ovarian cancer cells,which might be potentially useful in the gene therapy of ovarian cancers.
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To investigate the efficacy and the clinical value of total laparoscopic radical hysterectomy (TLRH)for the treatment of uterine malignancies,we performed a retrospective review of 87patients with cervical cancer and 23 patients with endometrial carcinoma who underwent TLRH at Union hospital between June 2008 and September 2009.Data collected included operative time,estimatedblood loss,lymph node count,time for the recovery of normal temperature and time to resumption of normal bladder function,intraoperative and postoperative complications.The procedure was completed laparoscopically in 108 patients.Two patients were converted to laparotomy due to common iliac vein injury.The mean overall operative time was 200.6±38.6 min; the mean operative blood loss was 280.5±128.3 mL; the mean number of pelvic lymph nodes that were resected was26.0a:5.8.The time for recovery of normal temperature and the normal bladder function after the operation was 5.8±2.9 d and 15.2±4.3 d.There were 2(1.8%)common iliac vein injuries during the operationand 10(9.1%)bladder retentions post operation.It was concluded that TLRH is feasible,minimally invasive and provides promise for the treatment of uterine malignancies.
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Small cell carcinoma of cervix (SCCC) is a rare disease with highly aggressive behaviour and is pathologically hard to diagnose. In this study, the clinicopathological features, diagnosis,treatment and prognosis of the condition were examined. Clinical records and follow-up data of 7 cases of SCCC were retrospectively studied. Our results showed that five non-recurrent cases initially presented irregular vaginal bleeding or increased apocenosis of varying degrees. Pathological examination revealed that the stroma was diffusely infiltrated with small monomorphous cells ranging from round to oval shape. Three cases were immunohistochemically confirmed. One case was accompanied with squamous cell cancer. Of the 7 cases, one case was classified as stage Ⅰ bl, two stage Ⅰ b2,one stage Ⅱ a, one stage Ⅱ b, and one stage Ⅲb. On the basis of their stages of condition, one subject with stage Ⅲ b underwent chemotherapy, and one with stage Ib2 received extensive hysterectomy plus pelvic lymphadenectomy, while the other 5 cases were treated by extensive hysterectomy and pelvic lymphadenectomy in combination with pre- and/or post-operative adjuvant chemotherapy and radiotherapy. Of the 7 patients, 4 had relapse-free survival of 14, 14, 16 and 28 months respectively.It is concluded that SCCC is an aggressive tumor with propensity for early pelvis lymph node metastases. Early-stage patients should be treated by extensive hysterectomy and pelvic lymphadenectomy in combination with pre- and/or post-operative adjuvant chemotherapy and radiotherapy.
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In order to investigate the effects of vector-based hairpin small interference RNA (shRNA) on the reversal of multi-drug resistance (mdr) of A2780/Taxol cells, a novel vector pEGFP-H1/mdr1 containing mdr1-shRNA targeting at position 2943-2963 of mdr1 was designed and synthesized.Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/mdrl, and the expression of mdr1 mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration (1C50) of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdr1 mRNA was decreased to (52.1±1.0)% and (0.01+1.7)%, and that of P-gp decreased to (88.3±2.1)% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol.The tumor volume in pEGFP-H1/mdr1-transfected group was significantly reduced as compared with that in blank control group or pEGFP-H1-transfected group (807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm3, both P<0.01). These results suggested that transfection of pEGFP-H1/mdr1 could efficiently down-regulate the expression of mdr1 mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol cells to Taxol both in vitro and in vivo.
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The recombinant plasmids pGenesii-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells and cisplatin was added with different concentrations in order to study the inhibitory ef-fects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression of Bcl-2, Bax,caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations (3.0, 6.0 and 9.9 μg/mL) was added into the transfected Hela cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-1-BIRC71 and pGenesil-1-BIRC72 into Hela cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was sig-nificantly increased in transfection group as compared with control group (P<0.05). Cisplatin could in-crease the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expres-sion levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were in-creased in transfection group as compared with those in control group (P<0.05). It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in Hela cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the proc-ess of apoptosis induction.
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There has been emergence of evidence suggesting that specific variants of the vascular en-dothelia growth factor (VEGF) family, based on their ability to regulate angiogenesis, would be pivotal in the pathogenesis of endometriosis. This study was aimed at determining whether high levels of VEGF-A could be found in the serum and peritoneal fluid (PF) of patients with endometriosis. VEGF-A levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum and PF from 46 pa-tients with surgically confirmed endometriosis, and 40 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. The results showed the mean VEGF-A levels were significantly higher in the serum and PF of patients with endometriosis than in the controls. The VEGF-A levels in the serum and PF of patients with severe endometriosis (stages Ⅲ-Ⅳ) were significantly higher than in those with minimal endometriosis (P<0.001). It was concluded that endometriosis was associated with significant modulation in the levels of circulating VEGF-A.
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Voltage-gated sodium channels (VGSCs) are known to be involved in the initiation and progression of many malignancies,and the different subtypes of VGSCs play important roles in the metastasis cascade of many tumors.This study investigated the functional expression of Nav 1.5 and its effect on invasion behavior of human breast cancer cell line MDA-MB-231.The mRNA and pro-tein expression of Navl.5 was detected by real time PCR,Western Blot and immunofluorescence.The effects of Navl.5 on cell proliferation,migration and invasion were respectively assessed by MTT and Transwell.The effects of Nav1.5 on the secretion of matrix metalloproteases (MMPs) by MDA-MB-231 were analyzed by RT-PCR.The over-expressed Navl.5 was present on the membrane of MDA-MB-231 cells.The invasion ability in vitro and the MMP-9 mRNA expression were respec-tively decreased to (47.82±0.53)% and (43.97±0.64)% (P<0.05) respectively in MDA-MB-231 cells treated with VGSCs specific inhibitor tetrodotoxin (TTX) by blocking Navl.5 activity.It was con-eluded that Nav1.5 functional expression potentiated the invasive behavior of human breast cancer cell line MDA-MB-231 by increasing the secretion of MMP-9.
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In order to investigate the effect of chitosan/pshR.NA plasmid nanoparticles targeting MDRI genes on the resistance of A2780/TS cells to paclitaxel,chitosan/pshRNA plasmid nanoparticles were synthesized by means of a complex coacervation technique and transfected into A2780/TS cells.The cells transfected with MDR1-targeted chitosan/pshRNA plasmid nanoparticles were experimental cells and the cells transfected with chitosan/pGPU6/GFP/Neo no-load plasmid nanoparticles served as negative control cells.Morphological features of the nanoparticles were observed under transmission electron microscope (TEM).MDR1 mRNA expression was assessed by RT-PCR.Half-inhibitory concentration (IC50) of paclitaxel for A2780/TS cells was determined by MTT method.TEM showed that the nanoparticles were round-shaped,smooth in surface and the diameters varied from 80 to 120 nm.The MDR1 mRNA in the transfected cells was significantly decreased by 17.6%,27.8% and 52.6% on the post-transfection day 2,4 and 7 when compared with that in A2780/TS cells control (P<0.05).MTT assay revealed that the relative reversal efficiency was increased over time and was 29.6%,51.2% and 61.3% respectively in the transfected cells 2,4,7 days after transfection and IC50 (0.197±0.003,0.144±0.001,0.120±0.004) were decreased with difference being significant when compared with that in A2780/TS (0.269±0.003) cells control (P<0.05).It was concluded that chitosan/pshRNA plasmid nanoparticles targeting MDR1 can effectively reverse the paclitaxel resistance in A2780/TS cells in a time-dependent manner.
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he activity of MMP-2 and MMP-9 via ERK1/2 signaling pathway.
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adjunct to cytology screening, especially high-risk patients.
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This study investigated the effect of RhoC GTPase on the proliferation and metastasis of cervical cancer cells, SiHa cells, in vitro. RhoC siRNA was introduced into SiHa cells to silence the RhoC gene. The mRNA and protein expression of RhoC, before and after RhoC siRNA transfection,was examined by RT-PCR and Western blotting, respectively. The proliferation and apoptosis of SiHa cells were examined by MTT assay and flow cytometry (FACS), respectively. Adhesive rate was evaluated by Matrigel adhesive assay, and the invasive capability and migration capability were assessed by transwell invasive assay and migration assay, respectively. The results showed that after the RhoC siRNA transfection, the mRNA and protein expression of RhoC was down-regulated in SiHa cells. The down-regulation of RhoC GTPase did not affect the cell proliferation and apoptosis (P>0.05), but it did suppress SiHa cells' adhesion to matrigel (P<0.01), the invasive capability (P<0.01) and the migration capability (P<0.01). It was concluded that RhoC obviously promotes the adhesion, invasion and migration of SiHa cells in vitro, but not proliferation and apoptosis, suggesting that RhoC plays an important rote in the progression in cervical cancer.
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The activation of mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-IA cells respectively (P<0.01). There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells (P<0.05). mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.