Irreversible electroporation-mediated shRNA knockdown of the HPV18 E6 gene suppresses cervical cancer growth in vitro and in vivo.
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ABSTRACT:
SUBMITTER: Wang ZL
PROVIDER: S-EPMC5530149 | biostudies-other | 2017 Aug
REPOSITORIES: biostudies-other
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Project description:PurposeThe aim of this study was to develop a statistical model for cell death by irreversible electroporation (IRE) and to show that the statistic model is more accurate than the electric field threshold model in the literature using cervical cancer cells in vitro.MethodsHeLa cell line was cultured and treated with different IRE protocols in order to obtain data for modeling the statistical relationship between the cell death and pulse-setting parameters. In total, 340 in vitro experiments were performed with a commercial IRE pulse system, including a pulse generator and an electric cuvette. Trypan blue staining technique was used to evaluate cell death after 4 hours of incubation following IRE treatment. Peleg-Fermi model was used in the study to build the statistical relationship using the cell viability data obtained from the in vitro experiments. A finite element model of IRE for the electric field distribution was also built. Comparison of ablation zones between the statistical model and electric threshold model (drawn from the finite element model) was used to show the accuracy of the proposed statistical model in the description of the ablation zone and its applicability in different pulse-setting parameters.ResultsThe statistical models describing the relationships between HeLa cell death and pulse length and the number of pulses, respectively, were built. The values of the curve fitting parameters were obtained using the Peleg-Fermi model for the treatment of cervical cancer with IRE. The difference in the ablation zone between the statistical model and the electric threshold model was also illustrated to show the accuracy of the proposed statistical model in the representation of ablation zone in IRE.ConclusionsThis study concluded that: (1) the proposed statistical model accurately described the ablation zone of IRE with cervical cancer cells, and was more accurate compared with the electric field model; (2) the proposed statistical model was able to estimate the value of electric field threshold for the computer simulation of IRE in the treatment of cervical cancer; and (3) the proposed statistical model was able to express the change in ablation zone with the change in pulse-setting parameters.
| S-EPMC5919048 | biostudies-literature
Project description:PurposeGastric cancer has a high mortality rate worldwide. Although treatments, such as molecular-targeted therapy, have been introduced, the resulting long-term survival and prognosis remain unsatisfactory. Downregulation of the target genes using lentivirus-mediated short hairpin RNA (shRNA) can be an effective therapeutic strategy for patients with gastric cancer. Overexpressed vascular endothelial growth factor A (VEGF) in human gastric cancer cells can be an effective novel therapeutic target for human gastric cancer. Thus, this study aimed to evaluate the therapeutic effects of lentivirus-mediated knockdown of VEGF gene expression in human gastric cancer growth.Materials and methodsSpecific shRNA sequences targeting VEGF were designed to construct a lentiviral expression vector. After human gastric carcinoma cells (cell line NCI-N87) were infected with the lentiviral vector, the therapeutic effects of the lentivirus-mediated shRNA targeting VEGF were analyzed both in vitro and in vivo.ResultsStable suppression of VEGF gene expression in NCI-N87 cells using shRNA (ShVEGF) showed significant inhibition of cell proliferation, clonogenicity, and cell motility. ShVEGF also showed increased G0/G1 cell cycle arrest and apoptosis. In addition, in vivo results from nude mice xenografted ShVEGF showed significant inhibition of tumor growth. Assessing the therapeutic effects of intratumoral injection of lentivirus-targeting VEGF (Virus_VEGF) revealed that it significantly inhibited tumor growth compared to that in the Virus_Scramble or saline injection control groups.ConclusionThe constructed ShVEGF showed significant inhibition of NCI-N87 gastric cancer cell growth both in vitro and in vivo. These experimental results suggest a novel therapeutic strategy for patients with gastric cancer using lentivirus-mediated shRNA targeting VEGF.
| S-EPMC7025738 | biostudies-literature
Project description:BackgroundIrreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization.MethodsForty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 ?seconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days.ResultsAll animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p <0.01). Also, compared with pulse duration of 70 ?sec, pulses of 100 ?sec were associated with markedly reduced left ventricular function and markedly increased relative scar area ratio (28%±9% vs. 16%±3%, p = 0.02). Decreasing electroporation pulse frequency (1Hz vs. 2Hz, 2Hz vs. 4Hz) was associated with a significant increase in myocardial damage. Electroporation protocols with a greater number of pulses (20 vs. 10) correlated with more profound tissue damage (p<0.05). When compared with myocardial infarction damage, electroporation demonstrated a considerable likeness regarding the extent of the inflammatory process, but with relatively higher levels of extra-cellular preservation.ConclusionsIRE has a graded effect on the myocardium. The extent of ablation can be controlled by changing pulse length, frequency and number, as well as by changing electric field intensity.
| S-EPMC5125564 | biostudies-literature
Project description:BackgroundColorectal cancer is a one of the most common alimentary malignancies. Survivin has been proved by many studies to be an ideal target for cancer gene therapy because of its strong anti-apoptotic effect. The reduction of Survivin expression by means of chemically synthesized small interfering RNA or small hairpin RNA expressed from plasmid and resulted growth inhibition of cancer cells had been proved by many studies including ours, but the transfection efficiency was not encouraging. So for the first time we constructed the Survivin shRNA into an oncolytic adenovirus, tested its effects on colorectal cancer cell lines and nude mice xenograft model.MethodsIn this study, we constructed an oncolytic adenovirus with a Survivin targeted small hairpin RNA and a reporter gene (ZD55-Sur-EGFP). The expression of Survivin mRNA and protein were analyzed by RT-PCR and western blot. The cell growth and apoptosis were tested by in vitro cytopathic assay, MTT assay and flow cytometry respectively. The effect of the constructed virus on xenograft model was evaluated by tumor volume and western blot analysis.ResultsZD55-Sur-EGFP replicated in cancer cells specifically, reduced the expression of Survivin mRNA and protein expression effectively (P < 0.0001), induced cancer cell apoptosis and inhibited SW480 cell growth both in vitro and in vivo significantly.ConclusionWe conclude Survivin RNA interference combining with oncolytic adenovirus virotherapy to be a promising treatment for colorectal cancer.
| S-EPMC2703625 | biostudies-literature
Project description:Irreversible electroporation (IRE) is a predominantly nonthermal ablative technology that uses high-voltage, low-energy DC current pulses to induce cell death. Thermal ablative technologies such as radiofrequency ablation, microwave ablation, and cryoablation have several applications in oncology but have limitations that have been established. IRE has shown promise to overcome some of these limitations. This article reviews the basics of the technology, patient selection, clinical applications, practical pointers, and the published data.
| S-EPMC4640920 | biostudies-other
Project description:Renal cell carcinoma (RCC) accounts for approximately 2%-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance, and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-κB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-κB in RCC, and many have implicated NF-κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse RCC (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-κB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNA-NF-κB1 tumors. Thus, this study indicates that downregulation of NF-κB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-κB1 may be a potential therapeutic target for RCC.
| S-EPMC7844753 | biostudies-literature
Project description:Irreversible electroporation (IRE) is emerging as a powerful tool for tumor ablation that utilizes pulsed electric fields to destabilize the plasma membrane of cancer cells past the point of recovery. The ablated region is dictated primarily by the electric field distribution in the tissue, which forms the basis of current treatment planning algorithms. To generate data for refinement of these algorithms, there is a need to develop a physiologically accurate and reproducible platform on which to study IRE in vitro. Here, IRE was performed on a 3D in vitro tumor model consisting of cancer cells cultured within dense collagen I hydrogels, which have been shown to acquire phenotypes and respond to therapeutic stimuli in a manner analogous to that observed in in vivo pathological systems. Electrical and thermal fluctuations were monitored during treatment, and this information was incorporated into a numerical model for predicting the electric field distribution in the tumors. When correlated with Live/Dead staining of the tumors, an electric field threshold for cell death (500 V/cm) comparable to values reported in vivo was generated. In addition, submillimeter resolution was observed at the boundary between the treated and untreated regions, which is characteristic of in vivo IRE. Overall, these results illustrate the advantages of using 3D cancer cell culture models to improve IRE-treatment planning and facilitate widespread clinical use of the technology.
| S-EPMC3491727 | biostudies-literature
Project description:Y-box binding protein-1 (YB-1), a member of cold-shock protein superfamily, has been demonstrated to be associated with tumor malignancy, and is proposed as a prognostic marker in multiple carcinomas. However, the role of YB-1 in neuroblastoma has not been well studied. To investigate the functional role of YB-1 in neuroblastoma, we established a YB-1-silenced neuroblastoma cell strain by inhibiting YB-1 expression using a shRNA knockdown approach. YB-1-silenced neuroblastoma SH-SY5Y cells exhibited a pronounced reduction in cell proliferation and an increased rate of apoptosis in vitro and in vivo xenograft tumor model. At molecular level, YB-1 silencing resulted in downregulation of Cyclin A, Cyclin D1 and Bcl-2, as well as upregulated levels of Bax, cleaved caspase-3 and cleaved PARP-1. We further demonstrated that YB-1 transcriptionally regulated Cyclin D1 expression by chromatin-immunoprecipitation and luciferase reporter assays. In addition, xenograft tumors derived from neuroblastoma SH-SY5Y cell line were treated with YB-1 shRNA plasmids by intra-tumor injection, and YB-1 targeting effectively inhibited tumor growth and induced cell death. In summary, our findings suggest that YB-1 plays a critical role in neuroblastoma development, and it may serve as a potential target for neuroblastoma therapy.
| S-EPMC4438073 | biostudies-literature
Project description:Although it can be lethal in its advanced stage, prostate cancer can be effectively treated when it is localised. Traditionally, radical prostatectomy (RP) or radiotherapy (RT) were used to treat all men with localised prostate cancer; however, this has significant risks of post-treatment side effects. Focal therapy has emerged as a potential form of treatment that can achieve similar oncological outcomes to radical treatment while preserving functional outcomes and decreasing rates of adverse effects. Irreversible electroporation (IRE) is one such form of focal therapy which utilises pulsatile electrical currents to ablate tissue. This modality of treatment is still in an early research phase, with studies showing that IRE is a safe procedure that can offer good short-term oncological outcomes whilst carrying a lower risk of poor functional outcomes. We believe that based on these results, future well-designed clinical trials are warranted to truly assess its efficacy in treating men with localised prostate cancer.
| S-EPMC8230282 | biostudies-literature
Project description:As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insight into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have focused on establishing techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced in two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for most medusa-type cnidarians, or jellyfish, are quite limited, except for Clytia hemisphaerica, and reliable techniques are required to interrogate function of specific genes in different jellyfish species. Here, we present a method to knock down target genes by delivering small interfering RNA (siRNA) into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knock down gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNA electroporation, which circumvents the technical difficulty of microinjecting small eggs. Wnt3 knockdown in Cladonema causes gene expression changes in axial markers, suggesting a conserved Wnt/β-catenin-mediated pathway that controls axial polarity during embryogenesis. Our gene-targeting siRNA electroporation method is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.
| S-EPMC9525680 | biostudies-literature