A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality.

Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.

Bioreducible polymer-mediated delivery of oncolytic adenovirus can attenuate antiviral immune response and concurrently enhance the induction of antitumor immune response to effectively prevent metastasis.

Oncolytic virotherapy is a highly promising and novel treatment modality for cancer. Several clinical trials with oncolytic viruses have illustrated that the potent antitumor efficacy of these viruses may rely on the efficient induction of antitumor immune response. In contrast, antiviral immune response is attributed to adverse side defects and diminishing therapeutic efficacy. In the present report, we generated a nanohybrid complex incorporating immune stimulatory oncolytic adenovirus (oAd) co-expressing decorin (DCN) and interleukin (IL)-12 with a bioreducible nanomaterial composed of PEI-Arg-mPEG-S-S-mPEG-Arg-PEI blocks (PAPS), ultimately aiming to modulate both antitumor and antiviral immune responses to be favorable toward oncolytic virotherapy. The transduction efficacy of the PAPS-incorporated nanohybrid vector (Ad/PAPS) was significantly higher than that of a complex using our previously reported polymer PPSA (Ad/PPSA) regardless of the cellular coxsackievirus and adenovirus receptor expression level of cancer cells. oAd complexed with PAPS (oAd/PAPS) also elicited a more potent cancer cell killing effect, antitumor efficacy, and metastasis inhibition than naked oAd or oAd complexed with PPSA (oAd/PPSA) through a higher level of therapeutic transgenes (DCN and IL-12), viral replication, and more efficient infiltration of T cells into tumor tissues. Notably, oAd/PAPS induced the highest level of antitumor immune response while the antiviral immune response was mediated at a significantly lower level than those of naked oAd. Adaptive immune response against the virus was also significantly attenuated in the oAd/PAPS group. oAd/PAPS treatment also led to the highest level of antitumor central memory T cells and the lowest level of immunosuppressive regulatory T cells in the spleen. Collectively, our findings illustrate that oAd/PAPS can simultaneously regulate both antitumor and antiviral immune responses to be more favorable to oncolytic virotherapy, leading to improved gene expression, viral replication, and growth inhibition of both primary and metastatic tumors.

Erratum: Oh, E., et al. Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect). Cancers 2019, 11, 966.

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Regulatory T Cells Induce Metastasis by Increasing Tgf-ß and Enhancing the Epithelial­Mesenchymal Transition.

Malignant melanoma is the most aggressive form of skin cancer; a substantial percentage of patients present with distant metastases. However, the mechanism of metastasis is not well understood. Here, we demonstrate that the administration of exogenous regulatory T cells (Tregs) into melanoma tumor-bearing mice results in a significant increase in lung metastasis. An increase in the invasive and metastatic phenotype of melanoma was mediated by cell-to-cell contact between melanoma cells and Tregs, which elevated the expression level of transforming growth factor-ß (TGF-ß) and the subsequent induction of the epithelial-to-mesenchymal transition (EMT).B16-BL6 melanoma tumors co-cultured with Tregs showed a larger population of migrating cells compared to B16-BL6 tumors cultured without Tregs. Additionally, the injection of exogenous Tregs into B16-BL6 melanoma tumors led to the recruitment and infiltration of endogenous Tregs into tumor tissues, thus increasing the overall Treg percentage in the tumor infiltrating lymphocyte population. Collectively, our findings propose novel mechanisms in which exogenous Treg-dependent upregulation of TGF-ß and mesenchymal markers is important for augmenting the migration capacity and invasiveness of melanoma, thereby contributing to the metastasis.

A Helical Polypeptide-Based Potassium Ionophore Induces Endoplasmic Reticulum Stress-Mediated Apoptosis by Perturbing Ion Homeostasis.

Perturbation of potassium homeostasis can affect various cell functions and lead to the onset of programmed cell death. Although ionophores have been intensively used as an ion homeostasis disturber, the mechanisms of cell death are unclear and the bioapplicability is limited. In this study, helical polypeptide-based potassium ionophores are developed to induce endoplasmic reticulum (ER) stress-mediated apoptosis. The polypeptide-based potassium ionophores disturb ion homeostasis and then induce prolonged ER stress in the cells. The ER stress results in oxidative environments that accelerate the activation of mitochondria-dependent apoptosis. Moreover, ER stress-mediated apoptosis is triggered in a tumor-bearing mouse model that suppresses tumor proliferation. This study provides the first evidence showing that helical polypeptide-based potassium ionophores trigger ER stress-mediated apoptosis by perturbation of potassium homeostasis.

Cryopreserved Human Natural Killer Cells Exhibit Potent Antitumor Efficacy against Orthotopic Pancreatic Cancer through Efficient Tumor-Homing and Cytolytic Ability (Running Title: Cryopreserved NK Cells Exhibit Antitumor Effect).

Pancreatic cancer is known to be highly aggressive, and desmoplasia-induced accumulation of extracellular matrix (ECM), which is a hallmark of many pancreatic cancers, severely restricts the therapeutic efficacy of both immunotherapeutics and conventional chemotherapeutics due to the ECM functioning as a major physical barrier against permeation and penetration. In the case of cell-based immunotherapeutics, there are several other bottlenecks preventing translation into clinical use due to their biological nature; for example, poor availability of cell therapeutic in a readily usable form due to difficulties in production, handling, shipping, and storage. To address these challenges, we have isolated allogeneic natural killer (NK) cells from healthy donors and expanded them in vitro to generate cryopreserved stocks. These cryopreserved NK cells were thawed to evaluate their therapeutic efficacy against desmoplastic pancreatic tumors, ultimately aiming to develop a readily accessible and mass-producible off-the-shelf cell-based immunotherapeutic. The cultured NK cells post-thawing retained highly pure populations of activated NK cells that expressed various activating receptors and a chemokine receptor. Furthermore, systemic administration of NK cells induced greater in vivo tumor growth suppression when compared with gemcitabine, which is the standard chemotherapeutic used for pancreatic cancer treatment. The potent antitumor effect of NK cells was mediated by efficient tumor-homing ability and infiltration into desmoplastic tumor tissues. Moreover, the infiltration of NK cells led to strong induction of apoptosis, elevated expression of the antitumor cytokine interferon (IFN)-γ, and inhibited expression of the immunosuppressive transforming growth factor (TGF)-ß in tumor tissues. Expanded and cryopreserved NK cells are strong candidates for future cell-mediated systemic immunotherapy against pancreatic cancer.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/ß-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.

Systemic administration of human mesenchymal stromal cells infected with polymer-coated oncolytic adenovirus induces efficient pancreatic tumor homing and infiltration.

Oncolytic adenovirus (oAd)-mediated gene therapy is a promising approach for cancer treatment because of its cancer cell-restricted replication and therapeutic gene expression. However, systemic administration of oAd is severely restricted by their immunogenic nature and poor tumor homing ability, thus oAd cannot be utilized to treat disseminated metastases. In this study, human bone marrow-derived mesenchymal stromal cell (hMSCs) was used as a viral replication-permissive carrier for oAd with an aim to improve the systemic delivery of the virus to tumor tissues. To overcome the poor delivery of oAd into hMSCs, a relaxin (RLX)-expressing oncolytic Ad (oAd/RLX), which degrades dense tumor extracellular matrix of highly desmoplastic pancreatic cancer, was complexed with biodegradable polymer (poly (ethyleneimine)-conjugated poly(CBA-DAH); PCDP), generating oAd/RLX-PCDP complex. oAd/RLX-PCDP complex enhanced the internalization of oAd into hMSC, leading to superior viral production and release from hMSCs, along with high RLX expression. Furthermore, systemic administration of oAd/RLX-PCDP-treated hMSCs elicited more potent antitumor effect compared to naked oAd/RLX or oAd/RLX-treated hMSC in pancreatic tumor model. This potent antitumor effect of systemically administered oAd/RLX-PCDP-treated hMSCs was achieved by superior viral replication in tumor tissues than any other treatment group. In conclusion, these results demonstrate that hMSCs are effective carriers for the systemic delivery of oAd to tumor sites and treatment of pancreatic cancer.

Identification and Functional Characterization of Anti-metastasis and Anti-angiogenic Activities of Triethylene Glycol Derivatives.

We had previously reported anticancer activity in the water extract (WEX) of Ashwagandha leaves, and identified Triethylene glycol (TEG) as an active tumor suppressor component. In this study, we investigated anti-migratory and anti-angiogenesis activities of WEX and TEG. We conducted in vitro and in vivo experiments using TEG, and its two derivatives, Triethyleneglycol dimethacrylate (TD-10), and Tetraethyleneglycol dimethacrylate (TD-11). The data revealed strong anticancer and anti-metastasis potentials in the derivatives. Non-toxic, anti-migratory doses of the derivatives showed inhibition of canonical Wnt/ß-catenin axis and consequent downregulation of EMT-signaling proteins (Vimentin, MMPs and VEGF). These results endorse that the TD-10 and TD-11 have potential to safely put a check on the aggressiveness of the metastatic cells and therefore represent promising candidates for the treatment of metastatic cancers.

A hypoxia- and telomerase-responsive oncolytic adenovirus expressing secretable trimeric TRAIL triggers tumour-specific apoptosis and promotes viral dispersion in TRAIL-resistant glioblastoma.

Glioblastoma is a highly aggressive and malignant type of cancer that is apoptosis resistant and difficult to cure by conventional cancer therapies. In this regard, an oncolytic adenovirus that selectively targets the tumour tissue and induces tumour cell lysis is a promising treatment option. We designed and constructed a hypoxia-responsive and cancer-specific modified human telomerase reverse transcriptase (H5CmTERT) promoter to drive replication of an oncolytic adenovirus (H5CmTERT-Ad). To enhance the anti-tumour efficacy of H5CmTERT-Ad against malignant glioblastoma, we also generated an H5CmTERT-Ad expressing secretable trimeric tumour necrosis factor-related apoptosis-inducing ligand (H5CmTERT-Ad/TRAIL). H5CmTERT promoter-regulated oncolytic adenoviruses showed cancer-specific and superior cell-killing effect in contrast to a cognate control oncolytic adenovirus replicating under the control of the endogenous adenovirus promoter. The cancer cell-killing effects of H5CmTERT-Ad and H5CmTERT-Ad/TRAIL were markedly higher during hypoxia than normoxia owing to hypoxia responsiveness of the promoter. H5CmTERT-Ad/TRAIL showed more potent anti-tumour efficacy than H5CmTERT-Ad did in a xenograft model of TRAIL-resistant subcutaneous and orthotopic glioblastoma through superior induction of apoptosis and more extensive virus distribution in the tumour tissue. Altogether, our findings show that H5CmTERT-Ad/TRAIL can promote dispersion of an oncolytic adenovirus through robust induction of apoptosis in a highly TRAIL-resistant glioblastoma.

A hydrogel matrix prolongs persistence and promotes specific localization of an oncolytic adenovirus in a tumor by restricting nonspecific shedding and an antiviral immune response.

Currently, intratumoral injection of an oncolytic adenovirus (Ad) is the conventional administration route in clinical trials. Nonetheless, the locally administered Ad disseminates to the surrounding nontarget tissues and has short biological activity due to immunogenicity of Ad, thus necessitating multiple injections to achieve a sufficient therapeutic index. In the present study, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-expressing oncolytic Ad (oAd-TRAIL) was encapsulated in a gelatin hydrogel (oAd-TRAIL/gel) to enhance and prolong antitumor efficacy of the virus after a single intratumoral injection. oAd-TRAIL/gel showed greater antitumor efficacy than naked oAd-TRAIL did due to enhanced and prolonged intratumoral accumulation of Ad up to a 20-day period, showing potent induction of apoptosis and inhibition of tumor cell proliferation. Furthermore, the gel system effectively prevented shedding of oncolytic Ad from the injection site to hepatic and other healthy tissues. oAd-TRAIL/gel treatment resulted in a markedly weaker antiviral immune response against Ad relative to naked oAd-TRAIL, further contributing to prolonged persistence of the oncolytic Ad in tumor tissue. Moreover, the hydrogel matrix preserved oAd-TRAIL's ability to induce an antitumor immune response, resulting in higher intratumoral infiltration by CD4+/CD8+ T cells. Taken together, these findings show that single intratumoral administration of the Ad/hydrogel modality may prolong and potentiate the therapeutic efficacy of Ad, modulate the immune reaction in favor of the virotherapy, and enhance intratumoral localization of the virus, ultimately overcoming limitations of oncolytic virotherapy revealed in recent clinical trials.

Optimized biodegradable polymeric reservoir-mediated local and sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF for cancer immunotherapy.

Administration of dendritic cells (DCs) combined with oncolytic adenovirus (Ad) expressing antitumor cytokines induces a potent antitumor effect and antitumor immunity by ameliorating the immunosuppressive tumor microenvironment. However, this combination therapy has significant limitations due to rapid dissemination and inactivation of the therapeutics at the tumor site, necessitating multiple injections of both therapeutics. To overcome these limitations, we have utilized gelatin-based hydrogel to co-deliver oncolytic Ad co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (oAd) and DCs for sustained release of both therapeutics. The injectable and biodegradable hydrogels were prepared by mixing the polymer solutions containing horseradish peroxidase and hydrogen peroxide. Gel matrix enabled sustained release of both oAd and DCs while preserving their biological activity over a considerable time period, leading to efficient retention of both therapeutics in tumor tissue. Further, tumors treated with oAd- and DC-loaded gel (oAd+DC/gel) showed a significantly greater expression level of IL-12, GM-CSF, and interferon-γ (IFN-γ) than either single treatment (oAd or DC) or oAd in combination with DC (oAd+DC), resulting in efficient activation of both endogenous and exogenous DCs, migration of DCs to draining lymph nodes, and tumor infiltration of CD4+ and CD8+ T cells. Moreover, oAd+DC/gel resulted in a significantly higher number of tumor-specific IFN-γ-secreting immune cells compared with oAd+DC. Lastly, oAd+DC/gel significantly attenuated tumor-mediated thymic atrophy, which is associated with immunosuppression in the tumor microenvironment, compared with oAd+DC. Taken together, these results demonstrate that gelatin gel-mediated co-delivery of oncolytic Ad and DCs might be a promising strategy to efficiently retain both therapeutics in tumor tissue and induce a potent antitumor immune response for an extended time period via a single administration.

Oncolytic adenovirus coexpressing interleukin-12 and decorin overcomes Treg-mediated immunosuppression inducing potent antitumor effects in a weakly immunogenic tumor model.

Interleukin (IL)-12 is a potent antitumor cytokine. However, immunosuppressive tumor microenvironments containing transforming growth factor-ß (TGF-ß) attenuate cytokine-mediated antitumor immune responses. To enhance the efficacy of IL-12-mediated cancer immunotherapy, decorin (DCN) was explored as an adjuvant for overcoming TGF-ß-mediated immunosuppression. We designed and generated a novel oncolytic adenovirus (Ad) coexpressing IL-12 and DCN (RdB/IL12/DCN). RdB/IL12/DCN-treated tumors showed significantly greater levels of interferon (IFN)-γ, tumor necrosis factor-α, monocyte chemoattractant protein-1, and IFN-γ-secreting immune cells than tumors treated with cognate control oncolytic Ad expressing a single therapeutic gene (RdB/DCN or RdB/IL12). Moreover, RdB/IL12/DCN attenuated intratumoral TGF-ß expression, which positively correlated with reduction of Treg cells in draining lymph nodes and tumor tissues. Furthermore, tumor tissue treated with RdB/IL12/DCN showed increases infiltration of CD8+ T cells and proficient viral spreading within tumor tissues. These results demonstrated that an oncolytic Ad co-expressing IL-12 and DCN induces a potent antitumor immune response via restoration of antitumor immune function in a weakly immunogenic murine 4T1 orthotopic breast cancer model. These findings provide new insights into the therapeutic mechanisms of IL-12 plus DCN, making it a promising cancer immunotherapeutic agent for overcoming tumor-induced immunosuppression.

Combined therapy with oncolytic adenoviruses encoding TRAIL and IL-12 genes markedly suppressed human hepatocellular carcinoma both in vitro and in an orthotopic transplanted mouse model.

BACKGROUND: Gene-based virotherapy mediated by oncolytic viruses is currently experiencing a renaissance in cancer therapy. However, relatively little attention has been given to the potentiality of dual gene virotherapy strategy as a novel therapeutic approach to mediate triplex anticancer combination effects, particularly if the two suitable genes are well chosen. Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interleukin-12 (IL-12) have been emerged as promising pharmacological candidates in cancer therapy; however, the combined efficacy of TRAIL and IL-12 genes for treatment of human hepatocellular carcinoma (HCC) remains to be determined. METHODS: Herein, we investigated the therapeutic efficacy of concurrent therapy with two armed oncolytic adenoviruses encoding human TRAIL gene (Ad-ΔB/TRAIL) and IL-12 gene (Ad-ΔB/IL-12), respectively, on preclinical models of human HCC, and also elucidated the possible underlying mechanisms. The effects of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy were assessed both in vitro on Hep3B and HuH7 human HCC cell lines and in vivo on HCC-orthotopic model established in the livers of athymic nude mice by intrahepatic implantation of human Hep3B cells. RESULTS: Compared to therapy with non-armed control Ad-ΔB, combined therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 elicited profound anti-HCC killing effects on Hep3B and HuH7 cells and on the transplanted Hep3B-orthotopic model. Efficient viral replication and TRAIL and IL-12 expression were also confirmed in HCC cells and the harvested tumor tissues treated with this combination therapy. Mechanistically, co-therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 exhibited an enhanced effect on apoptosis promotion, activation of caspase-3 and-8, generation of anti-tumor immune response evidenced by upregulation of interferon gamma (IFN-γ) production and infiltration of natural killer-and antigen presenting cells, and remarkable repression of intratumor vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) expression and tumor microvessel density. CONCLUSIONS: Overall, our data showed a favorable therapeutic effect of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy against human HCC, and may therefore constitute a promising and effective therapeutic strategy for treating human HCC. However, further studies are warranted for its reliable clinical translation.

Potent antitumor effect of neurotensin receptor-targeted oncolytic adenovirus co-expressing decorin and Wnt antagonist in an orthotopic pancreatic tumor model.

Pancreatic cancer is highly aggressive, malignant, and notoriously difficult to cure using conventional cancer therapies. These conventional therapies have significant limitations due to excessive extracellular matrix (ECM) of pancreatic cancer and poor cancer specificity. The excess ECM prevents infiltration of drugs into the inner layer of the solid tumor. Therefore, novel treatment modalities that can specifically target the tumor and degrade the ECM are required for effective therapy. In the present study, we used ECM-degrading and Wnt signal-disrupting oncolytic adenovirus (oAd/DCN/LRP) to achieve a desirable therapeutic outcome against pancreatic cancer. In addition, to overcome the limitations in systemic delivery of oncolytic Ad (oAd) and to specifically target pancreatic cancer, neurotensin peptide (NT)-conjugated polyethylene glycol (PEG) was chemically crosslinked to the surface of Ad, generating a systemically injectable hybrid system, oAd/DCN/LRP-PEG-NT. We tested the targeting and therapeutic efficacy of oAd/DCN/LRP-PEG-NT toward neurotensin receptor 1 (NTR)-overexpressing pancreatic cancer cells, both in vitro and in vivo. The oAd/DCN/LRP-PEG-NT elicited increased NTR-selective cancer cell killing and transduction efficiency when compared with a cognate control lacking NT (oAd/DCN/LRP-PEG). Furthermore, systemic administration of oAd/DCN/LRP-PEG-NT significantly decreased induction of innate and adaptive immune responses against Ad, and blood retention time was markedly prolonged by PEGylation. Moreover, NTR-targeting oAd elicited greater in vivo tumor growth suppression when compared with naked oAd and 9.5 × 10(6)-fold increased tumor-to-liver ratio. This significantly enhanced antitumor effect of oAd/DCN/LRP-PEG-NT was mediated by active viral replication and viral spreading, which was facilitated by ECM degradation and inhibition of Wnt signaling-related factors (Wnt, ß-catenin, and/or vimentin) in the tumor tissues. Taken together, these results demonstrate that oAd/DCN/LRP-PEG-NT has strong therapeutic potential for systemic treatment of NTR-overexpressing pancreatic cancer due to its NTR-targeting ability, enhanced therapeutic efficacy, and safety.

Potent and long-term antiangiogenic efficacy mediated by FP3-expressing oncolytic adenovirus.

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells.

c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death.

Withanone-rich combination of Ashwagandha withanolides restricts metastasis and angiogenesis through hnRNP-K.

Ashwagandha is an important herb used in the Indian system of traditional home medicine, Ayurveda. Alcoholic extract (i-Extract) from its leaves and its component, withanone, were previously shown to possess anticancer activity. In the present study, we developed a combination of withanone and withaferin A, major withanolides in the i-Extract, that retained the selective cancer cell killing activity and found that it also has significant antimigratory, -invasive, and -angiogenic activities, in both in vitro and in vivo assays. Using bioinformatics and biochemical approaches, we demonstrate that these phytochemicals caused downregulation of migration-promoting proteins hnRNP-K, VEGF, and metalloproteases and hence are candidate natural drugs for metastatic cancer therapy.

Oncolytic adenovirus expressing IL-23 and p35 elicits IFN-γ- and TNF-α-co-producing T cell-mediated antitumor immunity.

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4(+) and CD8(+) T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.