Weihua Sheng

Adenovirus-mediated ING4 expression suppresses lung carcinoma cell growth via induction of cell cycle alteration and apoptosis and inhibition of tumor invasion and angiogenesis

Previous studies demonstrated that ING4 as a novel member of ING (inhibitor of growth) family has potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in inhibition of human tumors has not been reported. To explore its therapeutic effect on human lung carcinoma, we constructed a recombinant adenoviral vector Ad-ING4 expressing the humanized ING4 gene derived from murine ING4 with two amino acid modifications at residue 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of A549 human lung carcinoma cells induced cell apoptosis, altered cell cycle with S phase reduction and G2/M phase arrest, suppressed cell invasiveness, and down-regulated IL-6, IL-8, MMP-2, and MMP-9 expression of transfected tumor cells. In athymic mice bearing A549 lung tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth and reduced the tumor microvessel formation. Therefore, Ad-ING4 may be useful in gene therapy of human lung carcinoma.

Adenovirus-Mediated ING4 Expression Suppresses Pancreatic Carcinoma Cell Growth via Induction of Cell-Cycle Alteration, Apoptosis, and Inhibition of Tumor Angiogenesis

Recent studies have demonstrated that ING4, as a novel member of the ING (inhibitor of growth) family, has a potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in the application of gene therapy for pancreatic carcinoma has not been reported. To explore its therapeutic effect on human pancreatic carcinoma, we constructed a recombinant adenoviral vector, Ad-ING4, expressing the green fluorescent protein (GFP) marker gene and the tumor-suppressor gene, humanized ING4 derived from murine ING4 with two amino-acid modifications at residues 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of PANC-1 human pancreatic carcinoma cells inhibited cell growth, altered the cell cycle with S-phase reduction and G2/M phase arrest, induced apoptosis, and downregulated interleukin (IL)-6 and IL-8 expression of transfected tumor cells. In athymic mice bearing the PANC-1 human pancreatic tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth, downregulated CD34 expression, and reduced the tumor microvessel formation. Therefore, this study will provide a framework for future clinical application of Ad-ING4 in human pancreatic carcinoma gene therapy.

Interleukin-17F Suppresses Hepatocarcinoma Cell Growth via Inhibition of Tumor Angiogenesis

ABSTRACT Previous studies have shown that interleukin-17F (IL-17F) can markedly inhibit the angiogenesis of endothelial cells, implying that it may play a role in antiangiogenic therapy for tumors. To explore its effect on antiangiogenic therapy for hepatocellular carcinoma (HCC), we constructed a recombinant retrovirus vector RV-IL-17F expressing IL-17F, transfected SMMC-7721 human hepatocarcinoma cells with RV-IL-17F, and investigated the effect of transgene IL-17F expression on human hepatocarcinoma cells in vitro and in vivo in animal model. We demonstrated that IL-17F expression exerted no direct effect on in vitro proliferation and cell cycle of SMMC-7721 hepatocarcinoma cells, while it downregulated IL-6, IL-8, and VEGF expression in SMMC-7721 cells at both protein and mRNA levels and IL-17F-expressing supernatant from SMMC-7721/RV-IL-17F directly inhibited ECV304 vascular endothelial cell growth. Moreover, SMMC-7721/RV-IL-17F exhibited a significant decrease in tumor size and microvessel density as compared to the SMMC-7721/RV control when transplanted in nude mice. This retarded tumor growth in vivo elicited by IL-17F was associated with direct suppression of vascular endothelial cells and reduced expression of proangiogenic factors IL-6, IL-8, and VEGF leading to the inhibition of tumor angiogenesis. Thus, our results indicate that IL-17F, a novel antiangiogenic factor, may be useful in antiangiogenic therapy for HCC.

Recombinant Human IL-24 Suppresses Lung Carcinoma Cell Growth via Induction of Cell Apoptosis and Inhibition of Tumor Angiogenesis

Previous studies have shown that interleukin-24 (IL-24; mda-7) as a novel tumor suppressor gene has tumor-suppressive activity against a broad spectrum of human cancers. However, the therapeutic effect of the recombinant human IL-24 (rhIL-24) protein purified from prokaryotic cells on human lung cancers has not been reported. In this study, we cloned the human gene coding for IL-24 from lipopolysaccharide-activated human peripheral blood mononuclear cells (PBMCs) by reverse-transcriptase polymerase chain reaction and constructed an expression vector pBV220-IL-24. We then transfected Escherichia coli DH5alpha with pBV220-IL-24. The soluble rhIL-24 was obtained from purified insoluble inclusion bodies of transfected cells by a denaturing and renaturing process. We demonstrated that the purified soluble rhIL-24 protein with 18.5 kappaDa was capable of (1) inducing in vitro apoptosis of A549 lung carcinoma cells; (2) activating PBMCs to secrete cytokines such as IL-6, tumor necrosis factor-alpha, and interferon-gamma; (3) inhibiting the formation of blood capillaries on chicken embryonic allantois and in vivo tumor angiogenesis; and (4) inhibiting A549 lung tumor cell growth in vitro and in vivo. Therefore, our results indicate its potent suppressive effect on human lung carcinoma cell line and warrant its further investigation for therapeutic application against human lung cancer.

Synergistic antitumor effect of adenovirus-mediated hING4 gene therapy and 125I radiation therapy on pancreatic cancer

Pancreatic cancer has a poor prognosis, even with surgery. ING4 is a member of the inhibitor of growth (ING) tumor suppressor family that has potent inhibitory effects on a variety of tumors; meanwhile, radiotherapy is a common adjunctive therapy for pancreatic cancer. Prior to this study, the effectiveness of a combination of ING4 gene-therapy and radiotherapy against pancreatic cancer had been unknown. In this study, we demonstrated that either ING4 or (125)I radiotherapy treatment could induce Panc-1 pancreatic cancer cell growth suppression and apoptosis in vitro. Furthermore, both treatments inhibited tumor growth and angiogenesis of Panc-1 pancreatic cancer subcutaneously xenografted in vivo. Moreover, the combination therapy had a synergistic effect.

Tumor-Suppressive Effect of Adenovirus-Mediated Inhibitor of Growth 4 Gene Transfer in Breast Carcinoma Cells In Vitro and In Vivo

The inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors. ING4 as a novel member of ING family has potential suppressive effect on different tumors via multiple pathways. However, the role of adenovirus-mediated ING4 (Ad-ING4) gene therapy for human breast carcinoma remains unknown. This study investigates the therapeutic effect of Ad-ING4 on human breast cancers in vitro and in vivo in an athymic nude mouse model, using two human breast carcinoma cell lines MDA-MB-231 (mutant p53) and MCF-7 (wild-type p53) and elucidated its underlying mechanism. It was found that Ad-ING4 treatment could induce in vitro significant growth suppression in both mutant p53 MDA-MB-231 and wild-type p53 MCF-7 breast carcinoma cells despite p53 status. This study further demonstrates that Ad-ING4 gene transfer resulted in G2/M phase arrest and apoptosis, upregulated P21, P27, and Bax, downregulated Bcl-2, IL-8, and Ang-1, promoted cytochrome c release from mitochondria into cytosol, and activated caspase-9, caspase-3, and PARP in mutant p53 MDA-MB-231 breast carcinoma cells. Moreover, intratumoral injections of Ad-ING4 in nude mice bearing mutant p53 MDA-MB-231 breast tumors remarkably inhibited the human breast xenografted tumor growth and reduced CD34 expression of tumor vessels and microvessel density. This retarded MDA-MB-231 breast carcinoma growth in vitro and in vivo elicited by Ad-ING4 closely correlated with the upregulation of cell cycle-related molecules P21 and P27, decrease in the ratio of anti- to proapoptotic molecules Bcl-2/Bax, release of cytochrome c from mitochondria into cytosol followed by caspase-9 and -3 activation leading to apoptosis via intrinsic apoptotic pathway, and the reduced expression of proangiogenic factors IL-8 and Ang-1 involved in the inhibition of tumor angiogenesis. Thus, the results indicate that Ad-ING4 is a potential candidate for breast cancer gene therapy.

The In Vitro and In Vivo Antitumor Activity of Adenovirus-Mediated Interleukin-24 Expression for Laryngocarcinoma

Interleukin-24 (IL-24)/melanoma differentiation associated gene-7 (mda-7) as a novel tumor-suppressor gene has potent antitumor activities in a broad spectrum of human cancers through the activation of various signaling pathways. However, the suppressive effect of adenovirus-mediated IL-24 (Ad-IL-24) expression on human laryngeal cancers is still elusive. In this study, we explored the therapeutic effect of Ad-IL-24 on human laryngeal cancers in vitro and in vivo in an athymic nude mouse model, using a Hep-2 human laryngocarcinoma cell line, and a WI-38 human diploid cell line served as a normal cell control. We demonstrated that Ad-IL-24 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27, and Bax, downregulated Bcl-2 expression, and activated caspase-3 in Hep-2 laryngeal tumor cells, while it exerted no direct effect on the in vitro proliferation of WI-38 normal diploid cells. Moreover, intratumoral injections of Ad-IL-24 in nude mice bearing Hep-2 tumors significantly suppressed the laryngeal xengrafted tumor growth and reduced microvessel density (MVD) and VEGF expression in tumors. This retarded tumor growth in vitro and in vivo elicited by Ad-IL-24 was closely associated with the upregulation of proliferation-related molecules P21 and P27, decrease in the ratio of anti- to proapoptotic molecules Bcl-2/Bax, followed by the activation of caspase-3, leading to apoptosis via intrinsic apoptotic pathways, and the reduced expression of proangiogenic factor VEGF involved in the inhibition of tumor angiogenesis. Thus, our results indicate that the potent, selective killing activity of Ad-IL-24 in laryngeal cancer cells, but not in normal cells, makes this vector a potential candidate for laryngeal cancer gene therapy.

Inhibition of pancreatic carcinoma growth by adenovirus-mediated human interleukin-24 expression in animal model.

Interleukin-24 (IL-24) has been shown to be a tumor-suppressor gene and the protein product found to be constitutively expressed by melanocytes, nerve cells, and some primary melanomas. The potential effect of adenovirus (AdV)-mediated IL-24 gene therapy was explored on human pancreatic carcinoma by using a pancreatic carcinoma cell line, patu8988. A recombinant adenovirus, AdVGFP/IL-24, expressing the marker, green fluorescent protein (GFP), and the tumor-suppressor gene, IL-24, was constructed. AdVGFP/IL-24 treatment of pancreatic carcinoma cells in vitro significantly induced pancreatic carcinoma cell cytotoxicity and apoptosis, compared with AdVGFP without IL-24 expression. In nude mice bearing patu8988 tumors, intratumoral injections of AdVGFP/IL-24 significantly inhibited pancreatic carcinoma growth. In addition, the molecular mechanism of tumor suppression was elucidated by downregulating the expression of vascular endothelial growth factor, CD34, and Bcl-2, as well as inhibiting tumor angiogenesis. Therefore, AdVGFP/IL-24 has the potential to serve as a novel tool for pancreatic carcinoma gene therapy.

Adenovirus-mediated ING4/PTEN double tumor suppressor gene co-transfer modified by RGD enhances antitumor activity in human nasopharyngeal carcinoma cells.

Inhibitor of growth-4 (ING4) is a member of the inhibitor of growth (ING) family and acts as a tumor suppressor protein. PTEN is a phosphatase and shows potent and extensive antitumor activity. In this study, we constructed an RGD-modified bicistronic ING4/PTEN adenovirus (Ad.RGD-ING4-PTEN) and comprehensively investigated its effects following modification of the CNE human nasopharyngeal carcinoma cell line both in vitro and in vivo. We demonstrated that Ad.RGD-ING4-PTEN enhanced growth inhibition and apoptosis. Furthermore, expression of P21, Bax and cleaved caspase-3 was upregulated, while that of Bcl-2 and survivin was downregulated in CNE cells and CNE xenografted tumors. Moreover, Ad.RGD-ING4-PTEN treatment additively downregulated CD34, VEGF and microvessel density in subcutaneously (s.c.) xenografted CNE cell tumors. The enhanced antitumor activity generated by Ad.RGD-ING4-PTEN was closely associated with activation of the intrinsic and extrinsic apoptotic pathways and additive inhibition of tumor angiogenesis both in vitro and in vivo. On the basis of this evidence, it is believed that cancer gene therapy combining two tumor suppressors such as ING4 and PTEN can be used to establish an effective and novel therapeutic strategy for nasopharyngeal carcinoma and other cancers.

Enhanced Capillary Formation by Transplanted Ad-VEGF165 Transgenic Epithelial Cells on Silk Fibroin Films

Vascular endothelial growth factors (VEGF) regulate multiple physiological and pathological processes including angiogenesis, i.e. growth of new blood vessels, a hallmark of wound healing. To improve VEGF blood life-time for enhanced angiogenesis in clinical applications, we investigate the possibility of cell transplant therapy using implanting silk protein film substrates loaded with VEGF-transfected THCE cells into a rabbit corneal model. Significantly enhanced corneal neovascularization is observed. THCE cells were cultivated on silk protein films (SF) and transfected with the Ad-VEGF165 for 48 hours. In vitro ELISA testing showed that Ad-VEGF165 transfected THCE cells expressed significantly higher levels of VEGF then self-secreted VEGF level in untransfected THCE cells. Interestingly, Ang1 and EGF levels were also higher in VEGF-transfected cells. After the transfection, the SF materials( 4 mm × 3mm ) were implanted into corneal stromal. Images of the corneal vasculature showed significant angiogenesis induced by VEGF-transfected THCE cells. One month after the implantation, corneal neovasculars extended to the margin of the pupil, with bifurcation and loops formation and the mean area of neovascularization was drastically increased. Immunostain- ing specimens all showed positive staining for CD34 on corneal stromal. It is concluded that cornea neovasculariza- tion was successfully induced by implanting SF/THCE-Ad- VEGF165 in rabbit corneal stroma. The regenerated silk fibroin films had good biological compatibility with THCE. Not only the expression level of the target VEGF gene was elevated, but also related genes such as Ang1 and EGF were also activated. These results indicate that regenerated silk fibroin films are a practical biomaterial platform that, once modified with transgenic seed cells, can provide multiple functions such as induced angiogenesis and enhanced tissue wound healing.