Construction of siRNA-survivin and GRIM-19 coexpression plasmid and its growth inhibitory effect on prostatic cancer DU145 cells

AIM: To construct the recombinant plasmid that expresses siRNA-survivin and GRIM-19 simultaneously, and to identify the validity of the recombinant plasmid and observe its effect on expression of survivin and GRIM-19 and proliferation ability of prostate cancer DU145 cells. METHODS: The recombinant plasmid coexpressing siRNA-survivin and GRIM-19 was constructed using gene cloning technique. The prostatic cancer DU145 cells were transfected with the coexpression plasmid and control plasmids. Survivin and GRIM-19 mRNA expression was detected by semi-quantitative RT-PCR. The proliferation ability affected by coexpression plasmid was measured by MTT assay. RESULTS: The coexpression plasmid pGRIM-19-si-survivin was successfully constructed according to DNA recombinant technique and identified through restriction enzyme digestion and plasmid sequencing. Compared with the mock, survivin mRNA expression levels were 0.55?0.05,0.62?0.08 and 0.35?0.05 in psi-survivin, pGRIM-19 and pGRIM-19-si-survivin groups, respectively. Compared with psi-survivin and pGRIM-19, pGRIM-19-si-survivin inhibited survivin mRNA expression markedly (P<0.05), while the expression levels of GRIM-19 mRNA were 1.93?0.14, 2.57?0.20 and 4.12?0.21 in psi-survivin, pGRIM-19 and pGRIM-19-si-survivin groups, respectively (P<0.01). Compared with pGRIM-19 group, pGRIM-19-si-survivin enhanced GRIM-19 mRNA expression more obviously (P<0.05). After transfection for 48 h, the proliferation rates were 58.0%?7.2%, 62.1%?6.1% and 50.2%?4.8% in the 3 experiment groups compared with the mock (P<0.05). After transfection for 72 h, the proliferation rate were 43.4%?4.3%, 51.3%?6.7% and 26.8%?7.1% in experiment groups compared with the mock (P<0.05). Compared with psi-survivin and pGRIM-19, pGRIM-19-si-survivin significantly inhibited the cell growth (P<0.05). CONCLUSION: Transfection of coexpression plasmid pGRIM-19-si-survivin dramatically changes the mRNA expression of survivin and GRIM-19 and inhibits the cell growth.     

Inhibitory effect of survivin-siRNA on prostatic subcutaneous xenografts in nude mice

AIM: To investigate the inhibitory effect of survivin-siRNA recombinant plasmid on prostate cancer xenografts. METHODS: Prostatic cancer DU145 cells were cultured and subcutaneously injected into nude mice. When the tumor grew to 8 mm in diameter, it was aseptically removed and divided into about 2 mm blocks through surgery and subcutaneously implanted into another nude mice. After the prostatic cancer xenograft model was reconstructed, the mice were treated with survivin-siRNA plasmid and control scrambled siRNA plasmid using electric transfection method. The tumor growth curve was plotted and the inhibitory rate was calculated. HE staining, immunohistochemical staining and TUNEL assay were applied to observe the effect of survivin-siRNA on the xenografts. RESULTS: The prostatic cancer xenograft model was successfully constructed in vivo. Compared with mock and scrambled siRNA groups, transfection of survivin-siRNA recombinant plasmid obviously inhibited the tumor growth with the inhibitory rates of 61.81% and 62.87%, respectively. Compared with both controls, survivin-siRNA depressed the protein expression of survivin and promoted the cell apoptosis. CONCLUSION: Survivin-siRNA recombinant plasmid significantly inhibits the growth of prostatic tumor xenografts by inhibiting the protein expression of endogenous survivin and promoting cell apoptosis.     

Effect of HGF gene transfection on human lymphoma xenografts in nude mice

AIM：To explore the promotion effect of hepatocyte growth factor (HGF) gene transfection on human lymphoma xenografts in nude mice. METHODS：The model of human lymphoma xenograft in nude mice was established by transplantation of Raji cells, which were transfected with recombinant plasmid pVITRO2-HGF harboring the HGF gene. The body weight of the nude mice and the tumor size were dynamically monitored and the tumor tissues were obtained after 8 weeks. Additionally, the methods of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry were used to detect the apoptotic index (AI) and microvessel density (MVD). RESULTS：The success rate of the human lymphoma xenografts in nude mice was 96.7%. The tumor volume in HGF transfection group was significantly greater than that in HGF transfection+VP-16 group and control groups (non-transfection group and empty vector group). The tumor volume in HGF transfection+VP-16 group was also bigger than that in control groups. No difference of the tumor volume between non-transfection group and empty vector group was observed. AI in HGF transfection group was substantially lower than that in control groups. AI in HGF transfection+VP-16 group showed a little higher than that in HGF transfection group, yet was still lower than that in control groups. MVD in HGF transfection group was extraordinary higher than that in control groups, but decreased after VP-16 induction (P<0.01), which was still higher than that in control groups. CONCLUSION：HGF gene transfection significantly promotes the growth of human lymphoma xenografts in nude mice and substantially inhibits the apoptosis presumably owing to promoting tumor angiogenesis and inhibiting tumor cell apoptosis.     

Inhibitory effect of survivin antisense oligodeoxyribonucleotide combined with DDP in nude mice bearing human osteosarcoma xenograft

AIM:To investigate the feasibility and its mechanisms of improving therapeutic effect by antisense gene therapy combined with chemotherapy in osteosarcoma. METHODS:The human osteosarcoma implanted tumor model in the nude mice was established. By intratumoral injection and abdominal cavity administration, the tumor bearing mice were treated with survivin ASODN in combination with diamminedichloroplatinum (DDP) for a week. Comparison with each single-agent therapy and control group was performed in aspects such as tumor growth condition, pathological changes of tumor tissues;survivin protein expression in tumor tissues by immunohistochemistry, survivin mRNA expression levels by RT-PCR method and tumor apoptosis by Tdt-mediated dUTP nick end labeling (TUNEL). RESULTS:All nude mice survived the therapy. As compared with the control group, the antisense gene therapy group presented synchronous decrease in survivin mRNA and protein expression;all therapy group displayed tumor growth inhibition and cell apoptosis with different extent;while in contrast to single-agent therapy group, the combined therapy group showed stronger inhibition of tumor growth and abundant tumor cell apoptosis with the highest apoptotic rate. CONCLUSION:Synergistic effect was achieved by combination of DDP with ASODN that may overcome drug resistant of DDP and the combined strategy may shed new light on the cancer therapy.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Effects of baicalin on CA46 cell xenografts in nude mice

AIM: To study the effects of baicalin on CA46 cell xenografts in nude mice. METHODS: The nude mice with CA46 cell xenografts were treated with drugs via intraperitoneal injection daily, and were divided into 5 groups: negative control group, 15 mg/kg baicalin group, 30 mg/kg baicalin group, 60 mg/kg baicalin group and 4 mg/kg etoposide (VP-16) positive control group. After 12-day treatment, the weight of CA46 cell xenografts stripped from some nude mice in the 5 groups was used to evaluate the effect of baicalin on xenograft growth in the nude mice. The apoptosis, necrosis and pathological changes of the xenograft cells were examined under light microscope and transmission electronic microscope respectively. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway-related proteins extracted from xenografts were determined by Western blotting. The other nude mice with CA46 cell xenografts in the 5 groups continued to be treated with the drugs until death in order to evaluate the effect of balcalin on survival time of the nude mice with CA46 cell xenografts. RESULTS: Baicalin remarkably inhibited the growth of CA46 cell xenografts, induced apoptosis and necrosis of xenograft cells, and reduced the protein expression of phospho-Akt (p-Akt), nuclear factor-kappa B (NF-κB), mammalian target of rapamycin (mTOR) and phospho-mTOR (p-mTOR) in the xenografts after 12-day treatment. Furthermore, baicalin prolonged the survival time of the nude mice with CA46 cell xenografts in a dose-dependent manner. CONCLUSION: Baicalin inhibits the growth and induces apoptosis of CA46 cell xenografts in the nude mice, and prolongs the survival time of the nude mice with CA46 cell xenografts through the mechanism of down-regulating PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathways.     

The inhibitory effects of endostatin gene transfer on the growth of breast cancer cells in vivo and in vitro

AIM: To investigate the therapeutic action of secreted endostatin (ES) on breast cancer cells. METHODS: Retroviral-mediated endostatin gene was transferred to breast cancer cell line MDA-MB-231. The ES biological properties and function were evaluated by polymerase chain reaction (PCR), MTT and a murine xenograft model. RESULTS: After retroviral transduction, endostatin genetically modified breast tumor cells were confirmed by PCR, and the integration and durative expression of endostatin gene was successfully committed. Compared with controls, endostatin secreted by genetically modified cells markedly inhibited endothelial cell proliferation (P<0.05) while the influences on the growth of MDA-MB-231 cell line in vitro were not found (P>0.05). The results of the transplanted subcutaneous tumor model in nude mice suggested that the subcutaneous growth of MDA-MB-231 was significantly inhibited by the expression of endostatin gene (P<0.05). In experimental groups, the tumor microvascular density (MVD) and VEGF expression were decreased. CONCLUSION: Retroviral- mediated overexpression of endostatin inhibits the proliferation of vascular endothelial cells and angiogenesis that associated with tumor growth in vivo via the paracrine pathway, which has a potential effect in the angiostatic gene therapy for breast cancer.     

The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Apoptosis of the prostate cancer cell line PC-3M induced by E2F decoy DNA

AIM：To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS：E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry (FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS：The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION：These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.     

Chutan-Jiedu decoction reverses resistance of lung cancer to gefitinib by processing EMT

AIM: To investigate the mechanism of Chutan-Jiedu decoction (CJD) reversing the resistance of lung cancer to gefitinib via epithelial-mesenchymal transition (EMT) pathway.METHODS: BALB/c nude mice (n=60) were selected to establish lung cancer xenograft model with human lung adenocarcinoma drug-resistant cell line H1975, which were randomly divided into 6 groups (10 mice per group):model group, gefitinib (0.04 g/kg) group, low-dose (13.52 g/kg) CJD group, middle-dose (27.04 g/kg) CJD group, high-dose (54.08 g/kg) CJD group, and combined medication group (27.04 g/kg CJD+0.04 g/kg gefitinib). The mice in each group were treated for 2 weeks before the tumor size and tumor weight were detected for the calculation of the tumor inhibitory rate. The mRNA and protein expression levels of E-cadherin, Snail and vimentin were determined by immunohistochemistry, Western blot and real-time PCR.RESULTS: Compared with model group and gefitinib group, the tumor size and the tumor weight in middle-dose CJD group, high-dose CJD group and combined medication group were decreased significantly (P<0.05). The results of immunohistochemistry, Western blot and real-time PCR showed that the expression of E-cadherin at mRNA and protein levels was increased significantly, while the expression of Snail and vimentin at mRNA and protein levels was decreased significantly (P<0.05).CONCLUSION: The growth of lung adenocarcinoma H1975 xenografts in nude mice is inhibited by CJD. In addition, the resistance of lung cancer to gefitinib is reversed. The mechanism may be related to the regulation of EMT-related protein expression.     

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

AIM：To investigate the role of human wings apart-like (hWAPL) protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA (shRNA) duplexes. METHODS：The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell proliferation was detected by MTT assay, and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining. Western blotting was used to analyze the expression of cleaved caspase-3, p21 and p27. The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model. RESULTS：Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA. The shRNA-mediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells. Additionally, the expression levels of cleaved caspase-3, p21 and p27 were up-regulated in hWAPL knockdown cells. Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells. CONCLUSION：hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo, and might serve as a therapeutic target in cancer treatment.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Effects of XPD on expression of Rb and MAD2 in human hepatoma HepG2 cells

AIM：To investigate the effects of xeroderma pigmentosum group D (XPD) protein on the growth of human hepatoma HepG2 cells and the expression of retinoblastoma (Rb) and mitotic arrest deficient 2 (MAD2) proteins. METHODS：The recombinant plasmid pEGFP-N2-XPD and empty plasmid pEGFP-N2 were transfected into HepG2 cells by LipofectamineTM 2000. The cells were divided into 4 groups including blank control group, liposome group, pEGFP-N2 group (N2 group) and pEGFP-N2-XPD group (XPD group). The expression of XPD, Rb and MAD2 at mRNA and protein levels was detected by RT-PCR and Western blotting. The cell growth was measured by MTT assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS：Overexpression of XPD up-regulated the expression of Rb, and down-regulated the expression of MAD2 at mRNA and protein levels. XPD inhibited the proliferation of HepG2 cells and exacerbated the apoptosis. XPD prevented the hepatoma cells from G1 stage to S stage. CONCLUSION：XPD suppresses the growth of hepatoma cells, up-regulates the expression of Rb, and down-regulates the expression of MAD2.     

Effect of metronomic cyclophosphamide combined with recombinant human endostatin on a nude mouse xenograft model of non-small-cell lung cancer

AIM：To investigate the effects of combination of metronomic (MET) cyclophosphamide (CPA) with recombinant human endostatin (Endostar) as maintenance therapy on treating non-small-cell lung cancer (NSCLC). METHODS：The lung adenocarcinoma model of BALB/c nude mice was established by subcutaneous inoculation of A549 cells. Following 4 circles of CPA chemotherapy at maximum tolerated dose (MTD), the tumor-bearing mice were randomly divided into 4 groups：the mice in control group were treated with saline, the mice in MET CPA group were treated with CPA, the mice in Endo group were treated with Endostar, and the mice in MET CPA+Endo group were treated with CPA+Endostar. The volume of xenograft tumors and the survival rate of the mice were recorded. The circulating endothelial cells (CECs) and viable CECs in peripheral blood of tumor-bearing mice were detected by flow cytometry. The microvessel density (MVD) and pericyte coverage were observed by confocal microscopy. RESULTS： At the 6th week of maintenance therapy, the tumor volume in both MET CPA group and Endo group was significantly smaller than that in control group, and the highest inhibitory effect was observed in MET CPA+Endo group. The survival time of the mice in both MET CPA group and Endo group was significantly longer than that in control group, and the mice in MET CPA+Endo group showed the longest survival time. Compared with control group, MET CPA or Endostar significantly reduced the total number and viable CECs in peripheral blood of tumor-bearing mice and the MVD in the xenograft tumors. Endostar also considerably reduced pericyte coverage in xenograft tumors. MET CPA combined with Endostar even more greatly inhibited the angiogenesis-related indicators mentioned above. CONCLUSION：Combination of MET CPA with Endostar shows effective anti-tumor activity and leads to improved survival in a xenograft model of lung adenocarcinoma, which might be partly attributed to the enhanced anti-angiogenic effect of the combination therapy.     

Cholesterol depletion up-regulates expression of prohibitin in human prostate carcinoma PC-3 cells

AIM: To study how cholesterol delpetion affects prohibitin expression and the development of prostate cancer. METHODS: Human prostate carcinoma PC-3 cells were cultured in normal medium (NM) and cholesterol delpetion medium (CDM) for 48 h.The mRNA expression of prohibitin was detected by real-time PCR. Prohibitin promoter was cloned and inserted into PGL3-Basic to reconstruct plasmid. The plasmid was transiently transfected into PC-3 cells. The cells were cultured in the medium with different concentrations of cholesterol for 48 h and luciferase expression was detected.RESULTS: The results of real-time PCR showed that the mRNA level of prohibitin increased about 19 times in PC-3 cells in the presence of CDM. After transfected with prohibitin promoter plasmid for 48 h, PC-3 cells cultured in CDM had higher luciferase expression than the cells cultured in NM or in CDM with cholesterol. CONCLUSION: Cholesterol depletion up-regulates prohibitin expression in PC-3 cells, which may be one of the self-prophylactic and regulatory mechanisms to protect PC-3 cells from apoptosis caused by cholesterol insufficiency.