lipofectamine and Prostatic-Neoplasms

This paper presents two water-soluble fullerene nanomaterials (HexakisaminoC

Topics: Cell Line, Tumor; Drug Delivery Systems; Drug Screening Assays, Antitumor; Fullerenes; Humans; Lipids; Male; Nanostructures; Prostatic Neoplasms; RNA, Small Interfering; Solubility

Herpes simplex virus (HSV)-thymidine kinase (TK)/ganciclovir (GCV) system is one of the most widely used and efficient suicide gene therapy for prostate cancer, but the lack of favorable gene vector and target limits its application. In this study, we established a novel system using nonviral gene vector G5-PAMAM-D to express HSV-TK and connexin43 (Cx43) gene driven by prostate-specific membrane antigen (PSMA) promoter, and evaluated the anti-tumor effect of this system. G5-PAMAM-D delivered PSMAe/p-TK-Cx43 showed expression of TK and Cx43 only in LNCaP cells, but not in PC-3 and other cells. The transfection efficiency of this system was comparable to lipofectamine 2000 by propidium iodide staining assay. With gemcitabine, folate-G5-PAMAM-D delivered PSMAe/p-TK-Cx43 (folate-G5-PAMAM-D/PSMAe/p-TK-Cx43) significantly decreased prostate cancer LNCaP cell proliferation and promoted apoptosis in vitro. With gemcitabine, the systemic deliver of folate-G5-PAMAM-D/PSMAe/p-TK-Cx43 significantly inhibited tumor growth in the LNCaP xenograft animal model. Our study demonstrates that this double-targeted and double-enhanced system is effective in inducing cell growth inhibition and apoptosis in vitro and suppressing tumor growth in vivo. In conclusion, Cx43 and gemcitabine combined with HSV-TK/GCV gene therapy using nonviral vector G5-PAMAM-D hold great potential as a novel approach for the gene therapy of prostate cancer.

Topics: Animals; Apoptosis; Bystander Effect; Connexin 43; Dendrimers; Deoxycytidine; Ganciclovir; Gemcitabine; Genes, Transgenic, Suicide; Genetic Therapy; Humans; Lipids; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Polyamines; Promoter Regions, Genetic; Prostatic Neoplasms; Simplexvirus; Thymidine Kinase; Transfection; Xenograft Model Antitumor Assays

KAI1/CD82, a tetraspanin membrane protein functions as a metastasis suppressor in many types of human cancers and has been shown to regulate cell adhesion properties. In the present study, we investigated the underlying mechanism of KAI1/CD82-mediated changes in cell adhesion to the extracellular matrix using human prostate cancer cells. We found that high KAI1/CD82 expression attenuated short-term cell adhesion to uncoated- or fibronectin-coated plates. Moreover, high KAI1/CD82 expression generated an extracellular environment unfavorable for cell adhesion as compared to low KAI1/CD82 expression, suggesting KAI1/CD82-dependent regulation of extracellular matrix (ECM) molecule(s) expression and/or secretion. Among ECM components examined, fibronectin exhibited decreased expression and secretion in high KAI1/CD82-expressing cells. Furthermore, high KAI1/CD82 expression interfered with the activation of β (1) integrin at the cell surface while total β (1) integrin levels remained unchanged, concomitant with reduced formation of focal adhesion complex and decreased bundling of actin filaments. Finally, high KAI1/CD82 expression significantly retarded cell motility in a scratch wound assay. Taken together, our results strongly suggest that KAI1/CD82 attenuates the activation of β (1) integrin, and thereby down-regulates outside-in signaling of β (1) integrin, leading to the reduction of focal adhesion formation and fibronectin expression/secretion, which subsequently interferes with cell adhesion properties and motility.

Topics: Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Extracellular Matrix; Fibronectins; Focal Adhesions; Gene Expression; Gene Silencing; Humans; Integrin beta1; Kangai-1 Protein; Lipids; Male; Microscopy, Confocal; Plasmids; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Up-Regulation

Nuclear factor-kappaB (NF-kB), a transcription factor, is abundantly expressed in prostate cancer and regulates many tumor-related genes. Given the important roles of these genes in tumor control, the present study was conducted to test the hypothesis that there was different expression of NF-kB in androgen- dependent or androgen-independent prostate cancer cells. In addition NF-kB decoy oligodeoxynucleotides (ODNs) were transfected into two prostate cancer cells to determine affects on growth and apoptosis.. First, NF-kB decoy ODNs were designed according to the NF-κB elements in the promoter region of c-myc gene. Then, NF-kB and control decoy ODNs were transfected with lipofectamine. Their influence on prostate cancer cell line proliferative activity was detected by MTT assay. Cell apoptosis was determined by flow cytometric(FCM) analysis and AO/EB study. Thirdly, nuclear extracts were prepared from PC-3M cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). Lastly, to confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kB decoy ODNs into PC-3M cells with lipofectamineTM2000. Expression levels of related endogenous genes were assessed by western blotting.. We found overexpression of NF-kB in the androgen-independent prostate cancer cell line PC-3M compared to the androgen-independent LNCaP. Treatment with NF-kB decoy ODNs resulted in strong suppression of proliferation, especially in the PC-3M case. Induction of apoptosis of PC-3M was observed in FCM and AO/EB studies. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that expression of some endogenous genes was reduced, while other genes transcripts were induced. EMSA demonstrated specific binding of the NF-kB decoy to NF-kB protein.. These findings indicate that NF-kB activation plays an important role in evolution of androgen-independent prostate cancer via manipulating expression of target genes. Inhibitors of NF-kB may thus offer promise as a therapeutic approach for the treatment of androgen-independent prostate cancer. NF-kB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.

Topics: Androgens; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cyclin D1; Electrophoretic Mobility Shift Assay; Forkhead Transcription Factors; Genes, myc; Humans; Inhibitor of Apoptosis Proteins; Lipids; Male; Nerve Tissue Proteins; NF-kappa B; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Prostatic Neoplasms

The goal of this study was to investigate the roles of PIM-1 in prostate cancer (CaP) cell proliferation and apoptosis, and to assess the potential of PIM-1 as a target for CaP therapy.. Using RNAi technology, we knocked down the expression of PIM-1 in PC-3 cell. After siRNA transfection, cell morphology, cell proliferation, cell cycle, and apoptosis rate were analyzed. PIM-1 siRNA with Lipofectamine were injected into xenograft models to evaluate its therapeutic effect.. PIM-1 siRNA significantly inhibited PIM-1 expression. In vitro, silencing of the PIM-1 gene resulted in irregular cell morphology, decreased cell proliferation, inhibition of cell-cycle progression, and induction of apoptosis. Compared with control groups, intratumoral injection of PIM-1 siRNA with Lipofectamine in nude mice dramatically suppressed PC-3 tumor progression.. PIM-1 could play important roles in the progression of CaP and may be an interesting target for CaP therapy.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Gene Silencing; Humans; Indicators and Reagents; Lipids; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection

To investigate whether microRNA-21 was involved in mediating the chemoresistance of prostate cancer cells to docetaxel.. A microarray technique was used to determine the miRNA profile in docetaxel-resistant PC3 cells. Real-time PCR was used to confirm the array results. miR-21 mimics and inhibitors were synthesized and introduced to cells using Lipofectamine 2000. Cell proliferation was examined with the CCK-8 assay. Luciferase reporter containing PDCD 3'UTR was constructed and the activity was detected by a dual luciferase assay. PDCD4 protein expression was evaluated using Western blot.. A docetaxel-resistant prostate cancer PC3 cell line (PC3R) was established . Using microarrays, miR-21 was found to be up-regulated in PC3R cells. Ectopic expression of miR-21 increased the resistance to docetaxel in PC3 wild type cells. In contrast, silencing of miR-21 in PC3R cells sensitized the cells to docetaxel. The IC(50) values for miR-21-silencing cells and control cells were 28.31 and 35.89 nmol/L, respectively. PDCD4, a direct target gene of miR-21, could mediate chemoresistance to docetaxel in PC3 cells.. Our findings suggest that miR-21 contributed to the resistance of PC3 cells to docetaxel, and that targeting miR-21 may offer a promising therapeutic approach in sensitizing prostate cancer to docetaxel treatment.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Docetaxel; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inhibitory Concentration 50; Lipids; Male; MicroRNAs; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; RNA-Binding Proteins; Taxoids

To investigate the apoptosis-promoting effect of PDCD5 on human prostate cancer cells PC-3M-1E8.. PCI-neo and PCI-neo-PDCD5 were transfected into PC-3M-1E8 cells by Lipofectamine 2000, the viability of the cells was analyzed by MTT assay 16 hours after removal of the serum, and the apoptosis was determined by in situ end-labeling and electron microscopy.. The viability and growing speed of the transfected cells were significantly decreased and their apoptotic indexes significantly increased as compared with the control group (P < 0.001).. PDCD5 may significantly inhibit the in vitro growth and promote the apoptosis of human prostate cancer cells PC-3M-1E8.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Humans; In Situ Nick-End Labeling; Lipids; Male; Neoplasm Proteins; Plasmids; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transfection

To investigate the inhibitory effect of RNA interference (RNAi) on the expression of survivin mRNA and its inducibility of the apoptosis of PC-3 cells.. siRNA expression vectors were designed and constructed to be directed at survivin and transfected into PC-3 cells by Lipofectamine in 4 groups: plasmid A, plasmid B, negative sequence and control E. RT-PCR, Western blot and flow cytometry were used to detect the expression of survivin mRNA and the apoptosis of PC-3 cells.. The expression rates of survivin protein were 18.94% +/- 0.63%, 16.35 +/- 0.23%, 46.41% +/- 0.76% and 46.20 +/- 1.47%, those of survivin mRNA were 27.94% +/- 1.43%, 24.51% +/- 1.37%, 49.46% +/- 0.71% and 48.49% +/- 1.32%, and the apoptosis rates of PC-3 cells were 12.80% +/- 1.33%, 16.48% +/- 1.00%, 3.03% +/- 0.62% and 2.96% +/- 0.41% respectively in different groups.. RNAi can effectively inhibit the expression of survivin mRNA and induce the apoptosis of PC-3 cells.

Topics: Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Lipids; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Plasmids; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Survivin; Transfection

To evaluate the effect of hypoxia-inducible factor-1 alpha (HIF-la) on angiogenesis in human prostate cancer cells.. Human prostate cancer cells of the line LNCaP were cultured and transfected by the recombinant plasmid pcDNA3. 1(-)/HIF-1alpha containing the gene HIF-1alpha with the Lipofectamine 2000 system. The positive clone cells were selected by G418 and confirmed by Western blotting and immunofluorescence (LNCaP/HIF-1alpha cells). The expressions of VEGF, iNOS and Ang-2 were detected by Western blotting.. The expression of HIF-1alpha in the LNCaP/HIF-1alpha cells was distinctly higher than that in the LN-CaP cells. Compared with the LNCaP cells, the expressions of VEGF and iNOS were up-regulated, whereas Ang-2 remained unchanged in the LNCaP/HIF-1alpha cells.. The over-expression of HIF-1alpha can induce an increase in angiogenesis proteins and improve the angiogenesis potency of prostate cancer.

Topics: Blotting, Western; Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lipids; Male; Nitric Oxide Synthase; Plasmids; Prostatic Neoplasms; Transfection; Vascular Endothelial Growth Factor A

Transfection efficiency of the novel reagent metafectene has not been compared with that of lipofectamine in the published English literature. We used these agents to transfect two prostate cancer cell lines, PC3 and G(s) alpha, with a deoxyribonucleic acid (DNA) expression vector that generates double-stranded ribonucleic acid (RNA) for RNA interference (RNAi). Cotransfection of the green fluorescent protein (GFP) reporter gene revealed that the mean (+/- standard deviation) transfection efficiencies with lipofectamine were 5.8+/-0.4% for PC3 cells and 3.6+/-1.5% for G(s) alpha cells. Mean transfection efficiency with metafectene declined to 0.1+/-0% for PC3 cells but improved to 54.6+/-5.5% for G(s) alpha cells. With G(s) alpha cells, metafectene transfection of GFP plasmid alone yielded 46.9% positive cells, and cotransfection with CD44v9 expression vector yielded 45.9% positive cells. The visual impact of the transfected RNAi construct was detectable at the protein level 4 to 6 d posttransfection and was more dramatic after using metafectene than after using lipofectamine. Thus, in vitro, metafectene transfection efficiency was sufficient to allow us to assess the functional significance of our RNAi construct, suggesting metafectene as an excellent candidate for RNAi-mediated anticancer gene therapy.

Topics: Biotechnology; Blotting, Western; Cell Line, Tumor; DNA; Drug Carriers; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; GTP-Binding Protein alpha Subunits; Humans; Lipids; Male; Models, Genetic; Plasmids; Prostatic Neoplasms; RNA Interference; RNA, Double-Stranded; Time Factors; Transfection