Gene Therapy For Treatment Of Cancer Research Articles

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography

BackgroundOral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods.MethodsThe present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC).ResultsTwo types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin.ConclusionsThe increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction, while the increased trends of cellular proliferation and survival in the protein profile of SCC-2 were implicative of its rapid growing tumor mass and early lymph node metastasis. These analyses of the essential oncogenic protein expression profiles in OSCC provide important information for genetic counseling or customized gene therapy in cancer treatment. Therefore, protein expression profile analysis through IP-HPLC is helpful not only for the molecular genetic diagnosis of cancer but also in identifying target molecules for customized gene therapy in near future.

Epigenetic Modification of the CCL5/CCR1/ERK Axis Enhances Glioma Targeting in Dedifferentiation-Reprogrammed BMSCs

SummaryThe success of stem cell-mediated gene therapy in cancer treatment largely depends on the specific homing ability of stem cells. We have previously demonstrated that after in vitro induction of neuronal differentiation and dedifferentiation, bone marrow stromal cells (BMSCs) revert to a primitive stem cell population (De-neu-BMSCs) distinct from naive BMSCs. We report here that De-neu-BMSCs express significantly higher levels of chemokines, and display enhanced homing abilities to glioma, the effect of which is mediated by the activated CCL5/CCR1/ERK axis. Intriguingly, we find that the activated chemokine axis in De-neu-BMSCs is epigenetically regulated by histone modifications. On the therapeutic front, we show that De-neu-BMSCs elicit stronger homing and glioma-killing effects together with cytosine deaminase/5-fluorocytosine compared with unmanipulated BMSCs in vivo. Altogether, the current study provides an insight into chemokine regulation in BMSCs, which may have more profound effects on BMSC function and their application in regenerative medicine and cancer targeting.

Nanoparticles for cancer gene therapy: Recent advances, challenges, and strategies

Compared to conventional treatments, gene therapy offers a variety of advantages for cancer treatment including high potency and specificity, low off-target toxicity, and delivery of multiple genes that concurrently target cancer tumorigenesis, recurrence, and drug resistance. In the past decades, gene therapy has undergone remarkable progress, and is now poised to become a first line therapy for cancer. Among various gene delivery systems, nanoparticles have attracted much attention because of their desirable characteristics including low toxicity profiles, well-controlled and high gene delivery efficiency, and multi-functionalities. This review provides an overview on gene therapeutics and gene delivery technologies, and highlight recent advances, challenges and insights into the design and the utility of nanoparticles in gene therapy for cancer treatment.

A novel cationic lipid with intrinsic antitumor activity to facilitate gene therapy of TRAIL DNA.

Metformin (dimethylbiguanide) has been found to be effective for the treatment of a wide range of cancer. Herein, a novel lipid (1,2-di-(9Z-octadecenoyl)-3-biguanide-propane (DOBP)) was elaborately designed by utilizing biguanide as the cationic head group. This novel cationic lipid was intended to act as a gene carrier with intrinsic antitumor activity. When compared with 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), a commercially available cationic lipid with a similar structure, the blank liposomes consisting of DOBP showed much more potent antitumor effects than DOTAP in human lung tumor xenografts, following an antitumor mechanism similar to metformin. Given its cationic head group, biguanide, DOBP could encapsulate TNF-related apoptosis-inducing ligand (TRAIL) plasmids into Lipid-Protamine-DNA (LPD) nanoparticles (NPs) for systemic gene delivery. DOBP-LPD-TRAIL NPs demonstrated distinct superiority in delaying tumor progression over DOTAP-LPD-TRAIL NPs, due to the intrinsic antitumor activity combined with TRAIL-induced apoptosis in the tumor. These results indicate that DOBP could be used as a versatile and promising cationic lipid for improving the therapeutic index of gene therapy in cancer treatment.

Gene Electrotransfer: A Mechanistic Perspective.

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

Herpes simplex virus type 1 VP22-mediated intercellular delivery of PTEN increases the antitumor activity of PTEN in esophageal squamous cell carcinoma cells in vitro and in vivo

In the past decade, studies have revealed that the phosphatase and tensin homolog (PTEN) protein, a tumor suppressor, comprises a potential biological marker and therapeutic target for esophageal squamous cell carcinoma (ESCC). As such, the delivery of the PTEN gene represents a powerful strategy for ESCC therapy. The tegument protein VP22 of herpes simplex virus type 1 (HSV-1) has been reported to act as a transporter of heterologous proteins across the host cell membrane, thereby enhancing the biological functions of these proteins. In the present study, the intercellular delivery and antitumor activity of the fusion protein PTEN-VP22 were examined in the esophageal squamous cell carcinoma cell line Eca109 both in vitro and in vivo. VP22-mediated PTEN intercellular delivery was confirmed in the Eca109 cells by western blot analysis and by quantitation of immunofluorescence. VP22 alone did not exert antiproliferative effects or induce cell cycle arrest, induction of apoptosis, blockage of the Akt and focal adhesion kinase (FAK) pathways, tumor growth inhibition, or antiangiogenic effects in Eca109 cells. However, compared with PTEN alone, PTEN-VP22 exerted significantly higher antiproliferative effects and induced cell cycle arrest at G1 stage, apoptosis and antiangiogenic effects in Eca109 cells. Together, our findings demonstrate that VP22 alone does not exert antitumor activity directly; however, this protein mediates the intercellular delivery of PTEN and thereby increases its intracellular concentration to achieve a therapeutic steady state, leading to an overall increase in the antitumor activity of PTEN. This study provides further experimental data to confirm the potential of VP22-based intercellular delivery strategies for enhancing the efficacy of gene therapy for cancer treatment.

Fluorinated dendrimer for TRAIL gene therapy in cancer treatment.

The delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene into cancer cells is a promising strategy for cancer treatment. However, the low transfection efficacy and/or the toxicity of vectors severely hamper the translation of TRAIL gene therapy into the clinics. In this article, we employed our recently developed fluorinated dendrimer as a vector to deliver plasmid encoding TRAIL (pTRAIL) into cancer cells for cancer treatment, which holds the advantages of both excellent transfection efficacy and low toxicity. Fluorinated poly(amidoamine) dendrimer (G4-F735) represented much higher TRAIL gene transfection efficacy than a series of transfection reagents including poly(ethylene imine) (PEI), SuperFect and Lipofectamine 2000, leading to a much higher cell apoptosis efficacy. The G4-F735/pTRAIL complex, compared with PEI/pTRAIL, could more efficiently destroy three-dimensional multicellular spheroids consisting of MDA-MB-231 cells, and suppress the tumor growth in vivo. Furthermore, G4-F735 showed minimal toxicity in vitro and undetectable systemic toxicity in vivo. From this study, the fluorinated dendrimer offers a promising prospect for TRAIL gene therapy.

Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy

Event Abstract Back to Event Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy Kishor Sarkar1, Harini V. Krishnan1, G Vinoth Kumar2, K Suresh Babu2, Vinayak Sant1 and Shilpa Sant1, 3, 4 1 University of Pittsburgh, Department of Pharmaceutical Sciences, United States 2 Pondicherry University, Centre for Nano Science and Technology, India 3 University of Pittsburgh, Department of Bioengineering, United States 4 University of Pittsburgh, McGowan Institute for Regenerative Medicine, United States Introduction: Gene therapy has become alternative therapeutic approach to treat genetic diseases such as cancer[1]. Nonviral vectors have gained tremendous attraction over the viral vector due to its low toxicity, non-immunogenicity and low production cost[2]-[4]. Recently, rare earth metal oxide especially cerium oxide nanoparticle (CNP) has widely been used in biomedical fields such as drug delivery, bio-imaging, nanomedicine and tissue engineering application because of its inherent antioxidant activity, free radical scavenging activity and radioprotection to normal cell[5],[6]. It is also reported that CNP increased the ROS (reactive oxygen species) in cancer cell[7]. Therefore, we hypothesized that therapeutic gene (like siRNA, p53 etc.) delivery by means of CNP may give the dual therapeutic effect in conjugation with increased ROS level in cancer cell for the treatment of cancer. Here we report the functionalization of CNP by low molecular weight polyethyleneimine (LPEI, 2 kDa) followed by conjugation of folic acid (FA) for targeted gene delivery. Methods: LPEI was conjugated with CNP (LPEI-conj-CNP) through carboxylation followed by amidation reaction. Then, FA was reacted with LPEI-conj-CNP by carbodiimide to get FA-LPEI-conj- CNP. CNP/pDNA (enhanced green fluorescent protein, EGFP encoding pDNA) complexes were prepared at various weight ratios and characterized by agarose gel electrophoresis assay and dynamic light scattering (DLS). In vitro transfection efficiency, toxicity, ROS and underlying mechanistic pathway were also evaluated in HeLa cell. Results: Poor water dispersibility of CNP at physiological pH is a major drawback for biological field especially gene/ drug delivery application. CNP forms stable dispersion in acidic pH only. Despite the positive zeta potential of CNP, it did not bind any DNA at any weight ratio due to poor dispersibility. But, the DNA complexation capability of CNP significantly increased after conjugation of LPEI and LPEI-conj-CNP complexed all pDNA at a very low weight ratio of 0.5:1. Due to presence of lots of primary amine groups in LPEI, the water dispersibility of CNP increased drastically and resulted better DNA complexation through the cationic primary amine groups. The transfection efficiency of CNP increased after conjugation of LPEI but the efficiency increased significantly after FA conjugation with LPEI-conj-CNP. FA-LPEI-conj-CNP/pDNA complex at weight ratio of 15:1 showed highest transfection efficiency which also surpassed the efficiency offered by PEI (25 kDa) (Figure 1). It was also observed that FA-LPEI-conj-CNP internalized into cell via folate receptor as well as caveolae mediated pathway and resulted enhanced transfection efficiency. While LPEI-conj-CNP followed clathrin mediated pathway followed by lysosomal compartment entrapment and as a result showed low transfection efficiency. It was found that green fluorescence (fluorescent dichlorofluorescein, DCF obtained through oxidation of nonfluorescent dichlorofluorescein diacetate, DCFA by ROS) increased drastically by FA-LPEI-conj-CNP compared to all CNPs and H2O2 treated cell indicating the increase in ROS (Figure 2). Conclusion: The transfection efficiency as well as ROS of CNP increased after functionalization of CNP especially after FA conjugation. Therefore, FA-LPEI-conj-CNP may be useful candidate in nonviral gene therapy for cancer treatment by using its dual property including gene delivery and ROS enhancement. This work was financially supported by Start-up funds from University of Pittsburgh School of Pharmacy

Synergistic effects of co-administration of suicide gene expressing mesenchymal stem cells and prodrug-encapsulated liposome on aggressive lung melanoma metastases in mice

The success of conventional suicide gene therapy for cancer treatment is still limited because of lack of efficient delivery methods, as well as poor penetration into tumor tissues. Mesenchymal stem cells (MSCs) have recently emerged as potential vehicles in improving delivery issues. However, these stem cells are usually genetically modified using viral gene vectors for suicide gene overexpression to induce sufficient therapeutic efficacy. This approach may result in safety risks for clinical translation. Therefore, we designed a novel strategy that uses non-viral gene vector in modifying MSCs with suicide genes to reduce risks. In addition, these cells were co-administrated with prodrug-encapsulated liposomes for synergistic anti-tumor effects. Results demonstrate that this strategy is effective for gene and prodrug delivery, which co-target tumor tissues, to achieve a significant decrease in tumor colonization and a subsequent increase in survival in a murine melanoma lung metastasis model. Moreover, for the first time, we demonstrated the permeability of MSCs within tumor nests by using an in vitro 3D tumor spheroid model. Thus, the present study provides a new strategy to improve the delivery problem in conventional suicide gene therapy and enhance the therapeutic efficacy. Furthermore, this study also presents new findings to improve our understanding of MSCs in tumor-targeted gene delivery.

Hydrophobic polyalanine modified hyperbranched polyethylenimine as high efficient pDNA and siRNA carrier.

Successful gene therapy for cancer treatment and various human genetic diseases relies on high efficient gene carriers, and many carriers can efficiently deliver plasmid DNA to cells, while most of them are unefficient in siRNA delivery. In this study, a series of amphiphilic copolymers are synthesized for pDNA and siRNA delivery by grafting hydrophobic polyalanine to polyethylenimine (named PPAs). There are 12.5, 7.2, 12.3 and 11.3 times of gene expression more than PEI in HeLa, 293T, A549 and CHO cell lines for the optimal PPAs, and the highest luciferase knockdown efficiency in Huh7 and CT26 cells are investigated to be 90.4% and 89.0%, respectively. This meant the PPAs have the potential as efficient transfection reagents for pDNA and siRNA delivery in future gene therapy.

Abstract 3920: In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology.

Abstract Photoacoustic imaging is a powerful tool for assessing tumor vasculature and oxygen saturation as well as detecting contrast agents for molecular imaging. Another exciting possibility is to visualize gene expression by using a reporter which produces a photoacoustic contrast agent. Tyrosinase is such a reporter in that its expression results in the production of melanin, which gives a strong photoacoustic signal. Here we describe the use of a commercially available photoacoustic (PA) imaging system (Vevo LAZR, VisualSonics, Toronto) to image inducible gene expression in subcutaneous xenograft tumors using tyrosinase as a reporter gene. The photoacoustic imaging system generated light from a tunable laser (680 - 970 nm) which was delivered through fiber optic bundles integrated into a linear array transducer (LZ-250, fc = 21 MHz), mounted to a linear stepper motor for 3D imaging. Animals (n=3) having MCF-7 xenograft tumors on either flank transfected with (+TYR) or without (-TYR) doxycycline-regulated tyrosinase were imaged before and one week after the induction of tyrosinase expression. 3D images of the tumors were acquired using multiple wavelengths (680, 750, 800, 850, 900 and 950nm) and 2D images were acquired across the entire wavelength range of the laser to generate absorption curves for blood and melanin. To confirm the presence of melanin and distinguish it from the endogenous blood signal, one animal was exsanguinated and the tumor was imaged again. Approximately 1mm-thick slices of the tumors were taken and photographed for visual confirmation of the presence of melanin. Comparison of pre- and post-doxycycline images of +TYR tumors clearly showed enhanced contrast in the tumor which closely matched the absorption spectrum of melanin. This signal persisted upon exsanguination. -TYR tumors showed signal which corresponded with the spectra of oxy and deoxy hemoglobin and changed little over the course of the experiment. Visual inspection of the excised sliced tumors revealed pigmentation in the +TYR tumors which was absent in the -TYR tumors. Here we have shown the ability of the Vevo LAZR photoacoustic imaging system to visualize inducible reporter gene expression in vivo. This has implications for assessing genetically controlled cellular processes and their response to anti-cancer drugs but also for monitoring gene therapy for cancer treatment non-invasively. Citation Format: Andrew Heinmiller, Minalini Lakshman, Dave Bates, Andrew Needles, Catherine Theodoropoulos, Robert J. Paproski, Roger J. Zemp. In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3920. doi:10.1158/1538-7445.AM2013-3920

Enhanced siRNA Delivery Using a Combination of an Arginine-Grafted Bioreducible Polymer, Ultrasound, and Microbubbles in Cancer Cells

RNAi-based gene therapy for cancer treatment has not shown significant clinical impact due to poor siRNA delivery to the target site. Here, we design a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), microbubbles (MB), and ultrasound (US), for targeting vascular endothelial growth factor (VEGF) in a human ovarian cancer cell line. Newly designed MBs with a perfluorocrownether gas core show higher stability compared to controls. Further, MBs in combination with polyplexes show a significant higher loading capacity compared to naked siRNA. Lastly, only siRNA-ABP-MB (SAM) complexes in combination with US show significant VEGF knock down in A2780 human ovarian cancer cell line compared to naked siRNA when incubated for a short time after sonication treatment.

Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD)

The application of gene therapy in cancer treatment is limited by non-specific targeting. In the present study, we constructed a recombinant plasmid, containing a carcinoembryonic antigen (CEA) promoter and double suicide genes thymidine kinase (TK) and cytosine deaminase (CD), henceforth referred to as pCEA-TK/CD. Our results showed that the CEA promoter can specifically drive target gene expression in CEA-positive lung cancer cells. In the presence of prodrugs 5-flucytosine and ganciclovir, pCEA-TK/CD transfection decreased inhibitory concentration 50 and increased apoptosis and cyclomorphosis. Our result suggests that gene therapy using pCEA-TK/CD may be a promising new approach for treating lung cancer.

Par-4 Sensitizes Human Colon Cancer Cells to Chemoradiotherapy

Despite aggressive treatment and recent advances, cancers of the colon and rectum remain a leading cause of death worldwide. In the case of rectal cancer, chemo-radiotherapy (CRT) is given prior to surgery to provide a greater chance of sphincter sparing surgery and lower the incidence of local recurrence. However, toxicity of CRT remains a problem. The tumor suppressor, Par-4, serves to sensitize tumor cells to chemotherapy and radiation therapy. This study tested the effects of increased Par-4 expression in HT29 human colon cancer cells. The results show the success of Par-4 in sensitizing the tumor cells to both radiation and chemo therapies. In addition, the presence of increased Par-4 caused a positive response to lower doses of both therapies. This supports the idea that Par-4 may be a viable target for gene therapy in cancer treatment, particularly in the case of accessible tumors such as rectal cancers, serving to reduce the required doses of CRT, and consequent side effects, for a successful treatment of rectal cancer.

Abstract LB-370: A placental growth factor variant unable to recognize VEGFR-1 inhibits VEGF-dependent tumor angiogenesis via heterodimerization

Abstract Angiogenesis is one of the crucial events for cancer development and growth. Two members of vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if co-expressed in the same cell, are both required for pathological angiogenesis. We generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala. PlGF1-DE is unable to bind and activate VEGFR-1 but can heterodimerize with VEGF (J. Biol. Chem., 279: 43929-43939, 2004). Here we demonstrate that overexpression of PlGF1-DE variant in tumor cells by adenoviral delivery or stable transfection significantly reduced the production of VEGF homodimer via heterodimerization, leading to a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel size, vessel stabilization by smooth muscle cells and monocyte-macrophage infiltration. PlGF1-DE/VEGF heterodimer, differently from wild type heterodimer able to induce VEGFR-1 dimerization as well as VEGFR-1/R-2 heterodimerization and activation, failed to induce receptors phosphorylation demonstrating that co-expression of PlGF1-DE variant in VEGF-producing cells effectively sequestered active VEGF, biasing toward the generation of a non-functional heterodimer. Conversely, overexpression of PlGF1wt, while reducing VEGF homodimer production comparably to PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, did not inhibit tumor growth and vessel density compared to controls, but induced an increase of vessel size, vessel stabilization and monocyte-macrophage infiltration. The ability of PlGF and VEGF-A to heterodimerize represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a ‘dominant negative’ of VEGF molecule and is a new candidate for anti-angiogenic gene therapy in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-370.

Structural importance of residue 334 in the stability of cytochromes P450 2B6 and 2B11

Human P450 2B6 and canine P450 2B11 play an important role in xenobiotic metabolism, bioremediation research, and activation of anti‐cancer prodrugs. Thorough biochemical and biophysical characterization of the enzymes has been impeded by relatively low stability compared to the extensively studied rat 2B1 and rabbit 2B4. Comparison of the amino acid sequences of N‐terminal truncated and C‐terminal His‐tagged constructs revealed seven sites that are conserved between the more stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. Site‐directed mutagenesis at these positions showed that P334→ Ser substitution increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure‐perturbation spectroscopy. Compressibility of the heme pocket in the four P450 2B enzymes and their P334S (2B6 and 2B11) or S334P (2B1 and 2B4) mutants was assessed from the pressure induced displacement of the Soret band. The variants of all four enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterparts. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Mutation of the non‐active site residue P334→ Ser could find applications in drug metabolism and gene therapy for cancer treatment.

A Placental Growth Factor Variant Unable to Recognize Vascular Endothelial Growth Factor (VEGF) Receptor-1 Inhibits VEGF-Dependent Tumor Angiogenesis via Heterodimerization

Angiogenesis is one of the crucial events for cancer development and growth. Two members of the vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if coexpressed in the same cell, are both required for pathologic angiogenesis. We have generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala, which is unable to bind and activate VEGF receptor-1 but is still able to heterodimerize with VEGF. Here, we show that overexpression in tumor cells by adenoviral delivery or stable transfection of PlGF1-DE variant significantly reduces the production of VEGF homodimer via heterodimerization, determining a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel lumen and stabilization, and monocyte-macrophage infiltration. Conversely, the overexpression of PlGF1wt, also reducing the VEGF homodimer production comparably with PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, does not inhibit tumor growth and vessel density compared with controls but induces increase of vessel lumen, vessel stabilization, and monocyte-macrophage infiltration. The property of PlGF and VEGF-A to generate heterodimer represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a "dominant negative" of VEGF and is a new candidate for antiangiogenic gene therapy in cancer treatment.

Targeting the Urokinase Plasminogen Activator Receptor with Synthetic Self-Assembly Nanoparticles

Targeting specific receptors is attracting growing interest in the fields of drug delivery and gene therapy for cancer treatment. The urokinase plasminogen activator receptor (uPAR) is overexpressed on many tumors,particularly that of prostate and breast cancers. The aim of this study is to design, prepare, and characterize a synthetic self-assembled nanoparticle that presents targeting ligands at a certain conformation and molar ratio onthe surface of the particles. Here, we describe the synthesis of a novel uPAR targeting ligand consisting of an 11-amino-acid sequence named U11 peptide modified with an alkyl chain to form an U11 peptide-lipid amphiphile.This peptide-lipid is inserted into the outer layer of a parent stealth liposome by post-modification to derive a U11 peptide-targeted nanoparticle. We demonstrate that the peptide moieties become separated into more singular conformations as they are inserted into a liposome membrane, rendering them to be sufficiently biologically active to observe specific receptor-mediated endocytosis (RME) and delivery of plasmid DNA to uPAR positive cells (DU145 cells). The U11 peptide targeted nanoparticle transfection of DU145 cells is essentially 10-fold higher compared to transfection achieved by nanoparticles having a scrambled peptide sequence on their surface.U11 peptide targeted nanoparticles also proved to be uPAR-specific, as they did not improve transfection levels on the uPAR-negative cell line, HEK293.

A dual role of p21waf1/cip1 gene in apoptosis of HEp-2 treated with cisplatin or methotrexate

We investigated the effects of p21(waf1/cip1) gene overexpression in human laryngeal squamous carcinoma cells HEp-2 lacking p53 protein expression on apoptosis induction upon the treatment with two commonly used chemotherapeutic agents, cisplatin and methotrexate. For that purpose, we employed cDNA arrays and qPCR to monitor gene expression upon treatment with AdCMV-p21 alone or in combination with the chemotherapeutic compounds. We found that p21(waf1/cip1) gene overexpression provoked apoptosis of HEp-2 through the induction of the TNFRSF9 gene and activation of caspase 7. In addition, we have proved that p21(waf1/cip1) can assume a dual role in apoptosis in the same cell system depending on the chemotherapeutic agent: its overexpression enhances apoptosis in cisplatin-treated cells and attenuates apoptotic signals in methotrexate-treated cells. The observed dual role of p21(waf1/cip1) was in direct correlation with the modulation of caspases 3 and 7 activation and changes in the expression of GADD45a gene. The results presented herein encourage future use of targeted p21(waf1/cip1) gene therapy in cancer treatment in a well-defined therapeutic and genetic context.

Microdevice-based delivery of gene products using sonoporation

This paper presents a proof-of-concept miniature device for delivery of antisense oligonucleotides (ASO). A piezoelectric, lead zirconate titanate (PZT) plate (0.5 cm(2) x 0.75 mm) is used to transfect cells using cavitation-induced sonoporation. Both human umbilical vein endothelial cells (HUVEC) and human prostate cancer cells (PC3) are investigated in vitro. Preliminary results show that after sonication, the transfection rate for HUVEC increases by 96% compared to controls (p < 0.01). For PC3, the transfection rate increases by 31% compared to controls (p < 0.02). This research can potentially be applied in realizing a microelectromechanical system (MEMS)-based device for gene therapy in cancer treatment.