Advent and maturation of regenerative medicine.

The field of regenerative medicine (RM), encompassing stem cell (SC) technologies, therapeutics, tissue engineering (TE), biomaterials, scaffolds and other enabling technologies provides a wide gamut of tools and tracks to combat, manage and hopefully cure serious human and animal injuries, dysfunctions and diseases. This review illustrates the trends that are becoming the major platforms in this field. The last 10 years in itself has seen major definitive observations, including multi-track directives of adult stem cell translational technologies, tissue and organ engineering protocols, iPS cell applications and understanding of the role of cancer stem cells to develop effective anti-cancer regimens. With the rapid advances of RM translational research, further advances are expected to be implemented for personalized repair and curative outcomes. RM future is bright although laden with challenges of global fragmentation which needs coherent consolidation, stringent cost and time effective regulation and long-term funding mechanisms, so clinical and diagnostic solutions are realized and recognized to combat unmet medical needs.

Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.

BACKGROUND: The present study examined whether transplantation of adherent bone marrow-derived stem cells, termed pMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractile function in the infarct zone and border zone (BZ) after coronary artery occlusion. METHODS AND RESULTS: Permanent left anterior descending artery occlusion in swine caused left ventricular remodeling with a decrease of ejection fraction from 55+/-5.6% to 30+/-5.4% (magnetic resonance imaging). Four weeks after left anterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a marked decrease in subendocardial phosphocreatine/ATP (31P magnetic resonance spectroscopy; 1.06+/-0.30 in infarcted hearts [n=9] versus 1.90+/-0.15 in normal hearts [n=8; P<0.01]). This abnormality was significantly improved by transplantation of allogeneic pMultistem cells (subendocardial phosphocreatine/ATP to 1.34+/-0.29; n=7; P<0.05). The BZ protein expression of creatine kinase-mt and creatine kinase-m isoforms was significantly reduced in infarcted hearts but recovered significantly in response to cell transplantation. MRI demonstrated that the infarct zone systolic thickening fraction improved significantly from systolic "bulging" in untreated animals with myocardial infarction to active thickening (19.7+/-9.8%, P<0.01), whereas the left ventricular ejection fraction improved to 42.0+/-6.5% (P<0.05 versus myocardial infarction). Only 0.35+/-0.05% donor cells could be detected 4 weeks after left anterior descending artery ligation, independent of cell transplantation with or without immunosuppression with cyclosporine A (with cyclosporine A, n=6; no cyclosporine A, n=7). The fraction of grafted cells that acquired an endothelial or cardiomyocyte phenotype was 3% and approximately 2%, respectively. Patchy spared myocytes in the infarct zone were found only in pMultistem transplanted hearts. Vascular density was significantly higher in both BZ and infarct zone of cell-treated hearts than in untreated myocardial infarction hearts (P<0.05). CONCLUSIONS: Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global left ventricular ejection fraction. These improvements may have resulted from paracrine effects that include increased vascular density in the BZ and spared myocytes in the infarct zone.

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10.

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G(2)/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27(Kip1). In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.

MDA-7/IL-24-based cancer gene therapy: translation from the laboratory to the clinic.

Despite recent advances in treatment strategies, the overall 5-year survival rate for patients with common epithelial cancers is poor largely because of the difficulty in treating metastatic cancers. Therefore, therapeutic agents are urgently needed that can effectively inhibit both primary epithelial tumors and their metastases. One such agent that has shown promise in preclinical studies is the tumor suppressor/cytokine, melanoma differentiation associated gene-7 also known as interleukin-24 (mda-7/IL-24). Preclinical studies from our and other laboratories have shown that overexpression of MDA-7/IL-24 causes a strong tumor- suppressive effect in many human cancer cells but spares normal cells. This gene therapy also enhances the tumor-suppressive activity of radiotherapy and chemotherapy. Secreted MDA-7 protein that is glycosylated also has been shown to have potent antiangiogenic activity both in vitro and in vivo. Studies examining the immune properties of mda-7 have shown that MDA-7/IL-24 unlike the related IL-10, functions as a Th1 cytokine. Recently, an MDA-7 protein-mediated "bystander effect" on tumor cells has been documented. Building on these findings we successfully completed a Phase I clinical trial of adenovirus-based mda-7 cancer therapy that confirmed the safety of this gene therapy. Phase II trials evaluating the efficacy of mda-7-based gene therapy are warranted. The outcome of such ongoing mda-7-based gene therapy trials will allow us to better understand this therapy's clinical utility.

Housekeeping genes in cancer: normalization of array data.

Biological maintenance of cells under variable conditions should affect gene expression of only certain genes while leaving the rest unchanged. The latter, termed "housekeeping genes," by definition must reflect no change in their expression levels during cell development, treatment, or disease state anomalies. However, deviations from this rule have been observed. Using DNA microarray technology, we report here variations in expression levels of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy model system. To highlight, differential expression was observed for ribosomal protein genes in the prostate cancer cells and beta-actin in treated colorectal cells. High-throughput differential gene expression analysis via microarray technology and quantitative PCR has become a common platform for classifying variations in similar types of cancers, response to chemotherapy, identifying disease markers, etc. Therefore, normalization of the system based on housekeeping genes, such as those reported here in cancer, must be approached with caution.

Activation of the Fas-FasL signaling pathway by MDA-7/IL-24 kills human ovarian cancer cells.

The tumor-suppressive activity of melanoma differentiation-associated gene-7 (mda-7), also known as interleukin 24 (IL-24), has been shown in a spectrum of human cancer cells in vitro and in vivo. However, mechanisms responsible for antitumor activity of mda-7 in human ovarian cancer cells have not been identified. We investigated the therapeutic activity and underlying mechanisms of adenovirus-mediated mda-7 gene (Ad-mda7) transfer in human ovarian cancer cells. Ad-mda7 treatment resulted in overexpression of MDA-7/IL-24 protein in both ovarian cancer and normal ovarian epithelial cells. However, Ad-mda7 significantly (P = 0.001) inhibited cell proliferation and induced apoptosis only in tumor cells and not in normal cells. Studies addressing the mechanism of action of Ad-mda7-induced tumor cell apoptosis revealed early activation of the transcription factors c-Jun and activating transcription factor 2, which in turn stimulated the transcription of an immediate downstream target, the death-inducer Fas ligand (FasL), and its cognate receptor Fas. Associated with the activation of Fas-FasL was the activation of nuclear factor kappaB and induction of Fas-associated factor 1, Fas-associated death domain, and caspase-8. Promoter-based reporter gene analyses showed that Ad-mda7 specifically activated the Fas promoter. Inhibition of Fas using small interfering RNA resulted in a significant decrease in Ad-mda7-mediated tumor cell death. Additionally, blocking of FasL with NOK-1 antibody abrogated Ad-mda7-mediated apoptosis. Collectively, these results show that Ad-mda7-mediated killing of human ovarian cancer cells involves activation of the Fas-FasL signaling pathway, a heretofore unrecognized mediator of MDA-7 apoptosis induction.

mda-7/IL24 kills pancreatic cancer cells by inhibition of the Wnt/PI3K signaling pathways: identification of IL-20 receptor-mediated bystander activity against pancreatic cancer.

The melanoma differentiation-associated gene (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose protein expression in normal cells is restricted to the immune system and to melanocytes. Recent studies have shown that mda-7 gene transfer inhibits cell growth and induces apoptosis in melanoma, lung cancer, breast cancer, and other tumor types through activation of various intracellular signaling pathways. In the current study, we demonstrate that Ad-mda7 transduction of human pancreatic cancer cells results in G2/M cell cycle arrest and cell killing. Cytotoxicity is mediated via apoptosis in a time- and dose-dependent manner. Tumor cell killing correlates with regulation of proteins involved in the Wnt and PI3K pathways: beta-catenin, APC, GSK-3, JNK, and PTEN. Additionally, we identify bystander cell killing activated by exposure of pancreatic tumor cells to secreted human MDA-7 protein. In pancreatic tumor cells, exogenous MDA-7 protein activates STAT3 and kills cells via engagement of IL-20 receptors. The specificity of bystander killing is demonstrated using neutralizing anti-MDA-7 antibodies and anti-receptor antibodies, which inhibit the apoptotic effects. In sum, we show that Ad-mda7 is able to induce growth inhibition and apoptosis in pancreatic cancer cells via inhibition of the Wnt/PI3K pathways and identify a novel bystander mechanism of MDA-7 killing in pancreatic cancer that functions via IL-20 receptors.

Sulindac enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human lung cancer.

Several studies have shown antitumor activities of the melanoma differentiation-associated gene 7 (mda-7) and the nonsteroidal anti-inflammatory drug sulindac when used as a monotherapies against a wide variety of human cancers. However, the combined effects of mda-7 and sulindac have not previously been tested. Therefore, we tested the antitumor activity of an adenoviral vector expressing mda-7 (Ad-mda7) in combination with sulindac against non-small cell lung cancer cells in vitro and in vivo. When treated with Ad-mda7 in combination with sulindac, human lung cancer cells (A549 and H1299) underwent growth suppression resulting in apoptosis. The growth inhibition induced by Ad-mda7 in combination with sulindac was significantly greater than that observed with Ad-mda7 or sulindac alone. Furthermore, the degree of growth inhibition induced using this combination was dose-dependent for sulindac. Treatment with Ad-mda7 in combination with sulindac had no growth inhibitory effects on human normal lung (CCD-16) fibroblasts. We then investigated the mechanism by which sulindac enhances Ad-mda7-mediated apoptosis. Sulindac increased expression of ectopic MDA-7 protein in tumor cells, thereby increasing the expression of downstream effectors RNA-dependent protein kinase, p38MAPK, caspase-9, and caspase-3 and enhancing apoptosis of non-small cell lung cancer cells. Pulse-chase experiments showed that the increased expression of MDA-7 protein in sulindac-treated cells was due to increased half-life of the MDA-7 protein. Finally, treatment of human lung tumor xenografts in nude mice with Ad-mda7 plus sulindac significantly suppressed growth (P = 0.001) compared with Ad-mda7 or sulindac alone. Our results show for the first time that combined treatment with Ad-mda7 plus sulindac enhances growth inhibition and apoptosis of human lung cancer cells. The increased antitumor activity observed with the combination treatment is a result of increased half-life of MDA-7 protein. Regulation of protein turnover is a heretofore-unrecognized mechanism of this nonsteroidal anti-inflammatory drug.

Selective induction of cell cycle arrest and apoptosis in human prostate cancer cells through adenoviral transfer of the melanoma differentiation-associated -7 (mda-7)/interleukin-24 (IL-24) gene.

We have previously reported that overexpression of the melanoma differentiation-associated gene -7 (mda-7) using a replication-defective adenovirus (Ad-mda7), results in tumor-specific growth suppression and induction of apoptosis in wide variety of cancer cells. In the present study, we investigated the antitumor activity of Ad-mda7 and the underlying mechanism in human prostate cancer cells and normal prostate epithelial cells. Overexpression of MDA-7 induced significant (P=.001) suppression of cell growth and apoptosis in prostate cancer cells (DU 145, LNCaP, and PC-3). In normal prostate epithelial cells (PrEC) some degree of growth inhibition but not apoptosis was observed. However, the inhibitory effects in normal cells were less compared to tumor cells. Growth inhibitory effects were mediated by the intracellular and not by extracellular MDA-7 protein. Molecular effectors that are involved in Ad-mda7-mediated tumor killing included activation of the caspase cascade, and the induction of G2 phase cell cycle arrest through the inhibition of Cdc25C pathway. These results demonstrate the mechanisms by which Ad-mda7 exerts its antitumor activity in human prostate cancer cells. The antitumor activity combined with previously reported antiangiogenic and proimmune properties of Ad-mda7 can serve as a potential therapeutic agent for treatment of primary and disseminated prostate cancer.

Intratumoral injection of INGN 241, a nonreplicating adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/IL24): biologic outcome in advanced cancer patients.

The mda-7 gene (approved gene symbol IL24) is a novel tumor suppressor gene with tumor-apoptotic and immune-activating properties. We completed a Phase I dose-escalation clinical trial, in which a nonreplicating adenoviral construct expressing the mda-7 transgene (INGN 241; Ad-mda7) was administered intratumorally to 22 patients with advanced cancer. Excised tumors were evaluated for vector-specific DNA and RNA, transgenic MDA-7 expression, and biological effects. Successful gene transfer as assessed by DNA- and RT-PCR was demonstrated in 100% of patients evaluated. DNA analyses demonstrated a dose-dependent penetration of INGN 241 (up to 4 x 10(8) copies/mug DNA at the 2 x 10(12) vp dose). A parallel distribution of vector DNA, vector RNA, MDA-7 protein expression, and apoptosis induction was observed in all tumors, with signals decreasing with distance away from the injection site. Additional evidence for bioactivity of INGN 241 was illustrated via regulation of the MDA-7 target genes beta-catenin, iNOS, and CD31. Transient increases (up to 20-fold) of serum IL-6, IL-10, and TNF-alpha were observed. Significantly higher elevations of IL-6 and TNF-alpha were observed in patients who responded clinically to INGN 241. Patients also showed marked increases of CD3+CD8+ T cells posttreatment, suggesting that INGN 241 increased systemic TH1 cytokine production and mobilized CD8+ T cells. Intratumoral delivery of INGN 241 induced apoptosis in a large volume of tumor and elicited tumor-regulatory and immune-activating events that are consistent with the preclinical features of MDA-7/IL-24.

Clinical and local biological effects of an intratumoral injection of mda-7 (IL24; INGN 241) in patients with advanced carcinoma: a phase I study.

The melanoma differentiation-associated gene-7 (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose expression induces selective apoptosis in tumor cells. To characterize the safety and biologic activity of mda-7 gene transfer, we conducted a phase I trial using intratumoral injections of an adenovirus containing the mda-7 construct (Ad-mda7; INGN 241; 2 x 10(10) to 2 x 10(12) vp) in 28 patients with resectable solid tumors. One hundred percent of injected lesions demonstrated INGN 241 vector transduction, transgenic mRNA, elevated MDA-7 protein, and apoptosis induction, with the highest levels near the injection site. Apoptosis of cells in injected tumors was consistently observed even in heavily pretreated patients. INGN 241 vector DNA and mRNA were detected more than 1 cm from the injection site, whereas MDA-7 protein and bioactivity were more widely distributed. Toxicity attributable to the injections was self-limiting and generally mild; however, one patient experienced a grade 3 SAE possibly related to the study drug. Evidence of clinical activity was found in 44% of lesions with the repeat injection schedule, including complete and partial responses in two melanoma patients. Thus intratumoral administration of INGN 241 is well tolerated, induces apoptosis in a large percentage of tumor cells, and demonstrates evidence of clinically significant activity.

Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism.

The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF.

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.

Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells-new perspectives for the generation of universal donor cells for tissue transplantation.

BACKGROUND: The seeding of small-calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source-however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti-MHC I intrabody might reduce graft failure. The intrabody consists of a single-chain variable fragment (sFv) of an anti-MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum. METHODS: Adenoviral gene transfer was used to express the intrabody in HUVEC. The MHC I surface expression was measured 48 h after transduction by flow cytometry. Functional effects of the intrabody expression were analyzed in a calcein release cytotoxicity assay. RESULTS: A transduction efficiency of more than 95% with EGFP-adenovirus indicates a sufficient gene transfer into HUVEC. Intrabody-adenovirus-transduced HUVEC show a massive reduction in MHC I surface expression creating almost a complete 'knockout' phenotype. Stimulation with inflammatory cytokines could not overcome this effect. The cell lysis of anti-MHC I intrabody-expressing HUVEC in a cytotoxicity assay is reduced when compared with the level of the MHC mismatched control. CONCLUSIONS: Our data indicate that HUVEC with reduced levels of MHC I might be used as universal donor cells for the seeding of vascular grafts.

MDA-7/IL-24 is a unique cytokine--tumor suppressor in the IL-10 family.

The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer--normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in different tumor types, suggesting that Ad-mda7 may regulate various signaling pathways. mda-7 encodes a secreted protein, MDA-7, which has now been designated as IL-24, and is a novel member of the IL-10 cytokine family. MDA-7/IL-24 protein is actively secreted from cells after mda-7 gene transfer. In human peripheral blood mononuclear cells (PBMC), STAT3 activation by MDA-7/IL-24 is followed by elaboration of secondary Th1 cytokines, demonstrating that MDA-7/IL-24 is a pro-Th1 cytokine. Furthermore, MDA-7/IL-24 is antagonized by the prototypic Th2 cytokine IL-10. MDA-7/IL-24 protein is endogenously expressed in cultured NK and B-cells and is also expressed in dendritic cells in tissues. MDA-7/IL-24 protein is expressed in nevi and melanoma primary tumors, to varying degrees, but is rarely expressed in malignant melanoma or other human tumors evaluated. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The "bystander" effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent antitumor utility in a clinical setting.

Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells.

We have previously observed the suppression of lung tumor growth in response to overexpression of melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24; approved gene symbol IL24) in vitro and in vivo. MDA-7/IL-24 exerts its tumor-suppressive effects by multiple mechanisms, including the activation of the caspase cascade and the inhibition of angiogenesis. In this study, we used an adenoviral vector (Ad-mda7) to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human non-small-cell lung carcinoma cells. Lung tumor cells (H1299 and A549) treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline (PBS) or Ad-Luc (vector control). MDA-7/IL-24 inhibited migration and invasion by down-regulating the production of phosphatidylinositol 3-kinase/protein kinase B, focal adhesion kinase, and matrix metalloproteinase-2 and -9 relative to PBS and Ad-Luc. Furthermore, tumor cells treated with Ad-mda7 ex vivo or with DOTAP:Chol-mda7 complex in vivo formed significantly fewer tumors in an experimental lung metastasis model. These results show that MDA-7/IL-24 inhibits invasion and migration by lung cancer cells by down-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.

The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells.

mda-7/IL-24 (HGMW-approved symbol IL24) is a tumor suppressor gene whose expression is lost during tumor progression. Gene transfer using adenoviral mda-7/IL-24 (Ad-mda7) exhibits minimal toxicity on normal cells while inducing potent apoptosis in a variety of cancer cell lines. Ad-mda7-transduced cells express high levels of MDA-7 protein intracellularly and also secrete a soluble form of MDA-7 protein. In this study, we sought to determine whether the intracellular or secreted MDA-7 protein was responsible for anti-tumor activity in H1299 lung tumor cells. Ad-mda7 transduction of lung tumor cells increased expression of stress-related proteins, including BiP, GADD34, PP2A, caspases 7 and 12, and XBP-1, consistent with activation of the UPR pathway, a key sensor of endoplasmic reticulum (ER)-mediated stress. Blocking secretion of MDA-7 did not inhibit apoptosis, demonstrating that intracellular MDA-7 was responsible for cytotoxicity. Consistent with this result, when applied directly to lung cancer cells, soluble MDA-7 protein exhibited minimal cytotoxic effect. We then generated mda-7 expression constructs using vectors that target the expressed protein to various subcellular compartments, including cytoplasm, nucleus, and ER. Only full-length and ER-targeted MDA-7 elicited cell death in tumor cells. Thus in lung cancer cells, Ad-mda7 activates the UPR stress pathway and induces apoptosis via intracellular MDA-7 expression in the secretory pathway.

Cytokine- and chemokine-based gene therapy for cancer.

Cytokines are protein/glycoprotein messengers of the immune system and have distinct autocrine and paracrine functions to modulate immunity. Recombinant cytokine proteins have been employed as biological drugs for cancer, viral and autoimmune targets. Unfortunately, systemic delivery of pharmacological doses of proteins often results in severe side effects and toxicities. As these therapeutic proteins tend to have very short half-lives and are complex to manufacture and deliver, many investigators are evaluating the genetic delivery of cytokine genes. Here, some of the promising cytokines currently under investigation for cancer therapies are examined, including interleukin (IL)-2, IL-4, IL-12, IL-24, interferon (IFN)-alpha, IFN beta, IFN gamma, granulocyte-monocyte colony-stimulating factor and tumor necrosis factor (TNF)-alpha. Chemokines are smaller chemotactic cytokines which induce migration of leukocytes, activate inflammatory responses, and are implicated in the regulation of tumor development and growth. Chemokines can modulate tumor growth via regulation of tumor-associated angiogenesis, by activation of host immunological responses or by direct inhibition of tumor cell proliferation. In this review, chemokines that have been proposed as antitumor drugs will be discussed, including Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG and SDF-1 alpha from the human CXC and C-C chemokine families.

Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells.

The tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene is a negative regulator of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt/PKB) signaling pathway. Overexpression of PTEN in cancer cells results in cell-cycle arrest and cell death through inhibition of PI3K. Caffeine, a xanthine analogue, is well known to enhance the cytocidal and growth-inhibitory effects of DNA-damaging agents such as radiation, UV light, and anticancer agents on tumor cells by abrogating DNA-damage checkpoints through inhibition of ataxia-telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR) kinase activity. In this study, we demonstrate that treatment with a combination of adenovirus-mediated transfer of PTEN (Ad-PTEN) and caffeine synergistically suppressed cell growth and induced apoptosis in colorectal cancer cells but not in normal colorectal fibroblast cells. This synergistic effect was induced through abrogation of G(2)/M arrest, downregulation of the Akt pathway, and modulation of the p44/42MAPK pathway. Thus, combined treatment with Ad-PTEN and caffeine is a potential therapy for colorectal cancer.