Inhibition Of Survival Signals Research Articles

Strategies for Improving Photodynamic Therapy Through Pharmacological Modulation of the Immediate Early Stress Response.

Photodynamic therapy (PDT) is a minimally to noninvasive treatment modality that has emerged as a promising alternative to conventional cancer treatments. PDT induces hyperoxidative stress and disrupts cellular homeostasis in photosensitized cancer cells, resulting in cell death and ultimately removal of the tumor. However, various survival pathways can be activated in sublethally afflicted cancer cells following PDT. The acute stress response is one of the known survival pathways in PDT, which is activated by reactive oxygen species and signals via ASK-1 (directly) or via TNFR (indirectly). The acute stress response can activate various other survival pathways that may entail antioxidant, pro-inflammatory, angiogenic, and proteotoxic stress responses that culminate in the cancer cell's ability to cope with redox stress and oxidative damage. This review provides an overview of the immediate early stress response in the context of PDT, mechanisms of activation by PDT, and molecular intervention strategies aimed at inhibiting survival signaling and improving PDT outcome.

DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease.

Huntington disease (HD) is a devastating neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Disrupted cortico-striatal transmission is an early event that contributes to neuronal spine and synapse dysfunction primarily in striatal medium spiny neurons, the most vulnerable cell type in the disease, but also in neurons of other brain regions including the cortex. Although striatal and cortical neurons eventually degenerate, these synaptic and circuit changes may underlie some of the earliest motor, cognitive, and psychiatric symptoms. Moreover, synaptic dysfunction and spine loss are hypothesized to be therapeutically reversible before neuronal death occurs, and restoration of normal synaptic function may delay neurodegeneration. One of the earliest synaptic alterations to occur in HD mouse models is enhanced striatal extrasynaptic NMDA receptor expression and activity. This activity is mediated primarily through GluN2B subunit-containing receptors and is associated with increased activation of cell death pathways, inhibition of survival signaling, and greater susceptibility to excitotoxicity. Death-associated protein kinase 1 (DAPK1) is a pro-apoptotic kinase highly expressed in neurons during development. In the adult brain, DAPK1 becomes re-activated and recruited to extrasynaptic NMDAR complexes during neuronal death, where it phosphorylates GluN2B at S1303, amplifying toxic receptor function. Approaches to reduce DAPK1 activity have demonstrated benefit in animal models of stroke, Alzheimer’s disease, Parkinson’s disease, and chronic stress, indicating that DAPK1 may be a novel target for neuroprotection. Here, we demonstrate that dysregulation of DAPK1 occurs early in the YAC128 HD mouse model, and contributes to elevated extrasynaptic GluN2B S1303 phosphorylation. Inhibition of DAPK1 normalizes extrasynaptic GluN2B phosphorylation and surface expression, and completely prevents YAC128 striatal spine loss in cortico-striatal co-culture, thus validating DAPK1 as a potential target for synaptic protection in HD and warranting further development of DAPK1-targeted therapies for neurodegeneration.

Potential Anticancer Effect of Calcium-mediated Src Degradation on Hormone-dependent Breast Cancer.

The antitumor effect of sustained calcium supply on Src degradation was investigated in the context of hormone-dependent breast cancer, followed by elucidation of the underlying mechanisms. Hormone-dependent T-47D breast cancer cells were used. Lactate calcium salt (LCS) was used as the source of sustained calcium supply, and the applicable concentration of LCS was determined by the colorimetric MTT assay. LCS-mediated deactivation of downstream signaling via Src degradation was identified by western blot and immunocytochemistry. Calcium-mediated degradation of Src decreased survival signaling via phosphoinositide 3-kinase and protein kinase B and resulted in significant inhibition of the clonogenic ability of hormone-dependent breast cancer cells. Tumor volume was significantly decreased in response to LCS injection in a heterotopic xenograft model, and immuno histochemistry revealed tumor necrosis. Sustained supply of calcium inhibited survival signaling via degradation of Src in hormone-dependent breast cancer cells.

3201 - Inhibition of Survival Signaling in B-Cell Lymphoma Using TLR9-Targeted Delivery of NF-Kb Decoy Oligodeoxynucleotides in Vitro and in Vivo

3201 - Inhibition of Survival Signaling in B-Cell Lymphoma Using TLR9-Targeted Delivery of NF-Kb Decoy Oligodeoxynucleotides in Vitro and in Vivo

The CXCR4 inhibitor BL-8040 induces the apoptosis of AML blasts by downregulating ERK, BCL-2, MCL-1 and cyclin-D1 via altered miR-15a/16-1 expression.

CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.

Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells.

Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5.

Abstract 3556: CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells

Abstract Background: CXCR4 is overexpressed in the majority of tumor cells and its degree of expression often correlates with disease severity. Binding of CXCL12 to the CXCR4 receptor activates signaling pathways which are crucial for the interaction of hematopoietic cells with the microenvironment and cell survival. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy in many types of cancer. CXCR4-antagonists, such as BL-8040, currently in phase II trials, were shown to selectively inhibit tumor cell growth and to induce apoptosis in vitro and in vivo. However, the molecular mechanism by which CXCR4 overexpression triggers tumor cell survival and its inhibition leads to cell death is not fully understood. Objective: To study the mechanism by which the CXCR4 pathway controls malignant cell survival and death through regulating miR-15a/16-1 expression. Method: We assessed the effect of CXCR4 overexpression, its activation and inhibition, on the expression of miR-15a/16-1 and their target genes, BCL-2, MCL-1 and cyclin D1, in a variety of tumor cells in vitro and in vivo. Results: We found that overexpression of CXCR4 in tumor cells or stimulation of cells with its ligand, CXCL12, lead to up-regulation of miR-15a/16-1, resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorgenesis and shifts their oncogenic dependency from the BCL-2 to the CXCR4/ERK signaling pathway. Antagonists of CXCR4 such as BL-8040 were shown to induce apoptotic cell death of malignant cells. BL-8040 was found to increase the expression of miR-15a/16-1 and reduce the expression of BCL2, MCL1 and cyclin D1. Importantly, CXCR4 inhibition using BL-8040 induced apoptosis in vitro and in vivo in AML and neuroblastoma tumors. This was mediated by inhibition of survival signals by ERK and down-regulation of BCL-2 expression. In support of these results overexpression of miR-15a/16-1 in AML and neuroblastoma cells was shown to induce their apoptosis. Conclusions: Our results demonstrate, for the first time to the best of our knowledge, that CXCR4 signaling regulates the expression of miR-15a/16-1 and their target genes. Our results suggest that overexpression of CXCR4 may override the survival dependency of tumor cells on BCL-2, MCL-1, and cyclin D1 leading to resistance of tumor cells to inhibition of these pathways. Furthermore, these results indicate that ligands of CXCR4 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 suppression of BCL2, MCL1 and cyclin D1 expression. Citation Format: Shiri Klein Silberman, Michal Abraham, Baruch Bulvik, Hanna Wald, Orly Eizenberg, Dvorah Olam, Lola Weiss, Katia Beider, Ori Wald, Shlomo Bulvik, Abraham Avigdor, Ohad Benjamini, Eithan Galun, Arnon Nagler, Yaron Pereg, Amnon Peled. CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3556.

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells.

Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment.

Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy.

Treatment of patients with B-NHL with rituximab and CHOP has resulted in significant clinical responses. However, a subset of patients develops resistance to further treatments. The mechanism of unresponsiveness in vivo is not known. We have reported the development of rituximab-resistant clones derived from B-NHL cell lines as models to investigate the mechanism of resistance. The resistant clones exhibit hyper-activated survival/anti-apoptotic pathways and no longer respond to a combination of rituximab and drugs. Recent studies reported the therapeutic efficacy in mice bearing B-cell lymphoma xenografts following treatment with the anti-CD20-hIFNα fusion protein. We hypothesized that the fusion protein may bypass rituximab resistance and inhibit survival signaling pathways. Treatment of the rituximab-resistant clones with anti-CD20-hIFNα, but not with rituximab, IFNα, or rituximab+IFNα resulted in significant inhibition of cell proliferation and induction of cell death. Treatment with anti-CD20-hIFNα sensitized the cells to apoptosis by CDDP, doxorubicin and Treanda. Treatment with anti-CD20-hIFNα inhibited the NF-κB and p38 MAPK activities and induced the activation of PKC-δ and Stat-1. These effects were corroborated by the use of the inhibitors SB203580 (p38 MAPK) and Rottlerin (PKC-δ). Treatment with SB203580 enhanced the sensitization of the resistant clone by anti-CD20-hIFNα to CDDP apoptosis. In contrast, treatment with Rotterin inhibited significantly the sensitization induced by anti-CD20-hIFNα. Overall, the findings demonstrate that treatment with anti-CD20-hIFNα reverses resistance of B-NHL. These findings suggest the potential application of anti-CD20-hIFNα in combination with drugs in patients unresponsive to rituximab-containing regimens.

Abstract IA10: Improving existing cancer therapy by targeting the tumor microenvironment

Abstract Many human cancers fail to respond to chemotherapy, and cancers that initially respond frequently acquire drug resistance and relapse. This process of tumor relapse is particularly confounding, as patients can be in remission for years following treatment prior to the reemergence of a cancer. Additionally, tumors that relapse have generally acquired resistance to the initial treatment. While conventional anti-cancer therapies have been in clinical use for decades, little is known about the mechanisms by which a tumor cell can survive treatment and persist in a patient for extended period of time. In fact, a major cause of cancer deaths is the inability to eradicate small sets of surviving tumor cells, termed “minimal residual disease” or “MRD”. Work from our laboratory has found that chemotherapy paradoxically elicits a pro-survival response in certain anatomical cites. Specifically, normal cells proximal to tumor cells secrete factors that counter the effects of the chemotherapy. This survival response likely serves to protect normal progenitor and stem cells to allow for tissue regeneration following damage, but it is coopted by tumor cells that find themselves in these specialized sites. Thus, effective cancer therapy may not only involve targeting cancer cells but also may require inhibiting survival signals emanating from surrounding cells. We have developed multiple models of MRD that have not only identified mechanisms of tumor cell persistence, but also strategies to sensitize chemorefractory tumors to front-line chemotherapy. Citation Format: Christian Pallasch, Luke Gilbert, Michael Hemann. Improving existing cancer therapy by targeting the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA10.

Abstract 4221: HSP90 as a therapeutic target in colorectal cancer

Abstract Purpose/Objectives: Activation of PI3K /Akt/ NF-κB and MAPK signaling pathway contributes to colorectal cancer cell growth, survival and resistance to therapy. HSP90 is required for PI3K/Akt and MAPK stability, folding and trafficking. Ganetespib is a potent functional inhibitor of HSP90 currently under evaluation in clinical trials. We tested the hypothesis that HSP90 functional inhibition by ganetespib can inhibit PI3K/Akt/ NF-κB and MAPK signaling pathways in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50nM for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of ganetespib on protein molecules involved in cell survival and proliferation. Electrophoretic mobility shift assay (EMSA) was performed in CRC cells to evaluate the effects of ganetespib on NF-κB activity. A subcutaneous xenograft mouse model was used to evaluate the in vivo effects of ganetespib. Results: Treatment of HCT-116 and HT-29 CRC cell lines with ganetespib inhibited survival signaling (PI3K/Akt) and proliferative signaling (MAPK) at the transcriptional and translational level. As validation, HSP90 knockdown similarly inhibited PI3K/Akt and MAPK protein and mRNA levels in both cell lines. Functional inhibition of HSP90 resulted in inhibition of PI3K/Akt and MAPK transcription. Functional inhibition or down regulation of HSP90 was associated with inhibition of NF-κB trafficking (downregulation of NF-κB in nucleus). Ganetespib sensitized colon cancer cell lines to the effects of oxaliplatin and 5FU. Similar effects were also observed in tumors from animals treated with ganetespib. Conclusion: In this study, we present in vitro and animal data supporting the targeting of HSP90 as a method to inhibit the PI3K/Akt/ NF-κB and MAPK signaling pathways in colorectal cancer. We demonstrate that inhibition of HSP90 downregulates PI3K/Akt/ NF-κB and MAPK leading to a decrease in colon cancer cell survival and proliferation, as well as sensitization to chemotherapy. Combining HSP90 inhibitors with chemotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Ganji Purnachandra Nagaraju, Field F. Willingham, Kevin E. Woods, Patrick Sullivan, Jerome C. Landry, Roberto Diaz, Bassel F. El-Rayes. HSP90 as a therapeutic target in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4221. doi:10.1158/1538-7445.AM2014-4221

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.

New Perspectives of Curcumin in Cancer Prevention

Numerous natural compounds have been extensively investigated for their potential for cancer prevention over the decades. Curcumin, from Curcuma longa, is a highly promising natural compound that can be potentially used for chemoprevention of multiple cancers. Curcumin modulates multiple molecular pathways involved in the lengthy carcinogenesis process to exert its chemopreventive effects through several mechanisms: promoting apoptosis, inhibiting survival signals, scavenging reactive oxidative species (ROS), and reducing the inflammatory cancer microenvironment. Curcumin fulfills the characteristics for an ideal chemopreventive agent with its low toxicity, affordability, and easy accessibility. Nonetheless, the clinical application of curcumin is currently compromised by its poor bioavailability. Here, we review the potential of curcumin in cancer prevention, its molecular targets, and mechanisms of action. Finally, we suggest specific recommendations to improve its efficacy and bioavailability for clinical applications.

Emodin sensitizes the gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine via downregulation of NF-κB and its regulated targets

The aim of this study was to evaluate whether emodin can overcome the chemoresistance of the gemcitabine-resistant cancer cell line (Bxpc-3/Gem) in vitro. The cell line Bxpc-3/Gem was derived from the human pancreatic cancer cell line Bxpc-3. We found that Bxpc-3/Gem cells were characterized by a series of morphological changes with a resistance index of 43.51 comparing with the parental cell line. Emodin reduced Bxpc-3/Gem cell proliferation in a dose-dependent manner. Emodin and gemcitabine combination treatments resulted in decreased cell proliferation and increased apoptosis in Bxpc-3/Gem cells. In addition, combination treatments resulted in downregulation of gene and protein expression of MDR-1 (P-gp), NF-κB, XIAP, survivin, as well as inhibition of NF-κB activity and P-gp function. These observations suggest that emodin may sensitize the pancreatic cancer gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine therapy via inhibition of survival signaling.

Science Signaling Podcast: 29 January 2013

Vemurafenib induces endoplasmic reticulum stress in melanoma cells.

Cannabisin B induces autophagic cell death by inhibiting the AKT/mTOR pathway and S phase cell cycle arrest in HepG2 cells

This study investigates the anticancer properties of cannabisin B, purified from hempseed hull, in HepG2 human hepatoblastoma cells. The results indicate that cannabisin B significantly inhibited cell proliferation by inducing autophagic cell death rather than typical apoptosis. Cell viability transiently increased upon the addition of a low concentration of cannabisin B but decreased upon the addition of high concentrations. Cannabisin B-induced changes in cell viability were completely inhibited by pre-treatment with 3-methyladenine (3-MA), indicating that the induction of autophagy by cannabisin B caused cell death. Additionally, cannabisin B induced S phase cell cycle arrest in a dose-dependent manner. Moreover, cannabisin B was found to inhibit survival signaling by blocking the activation of AKT and down-stream targets of the mammalian target of rapamycin (mTOR). These findings suggest that cannabisin B possesses considerable antiproliferative activity and that it may be utilised as a promising chemopreventive agent against hepatoblastoma disease.

Abstract 19722: Cardiac Deletion of miRNA-17-92 Cluster Augments Acute Myocardial Ischemia-Reperfusion Injury

Objective: MicroRNAs (miRNA) are small, endogenous, conserved, noncoding RNAs that down-regulate protein production by translational repression. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in stem cells and is up-regulated by ischemia, but little is known about the postnatal role of miR-17-92 in myocardial ischemia-reperfusion injury (I/R). Methods and Results: We established a transgenic mouse model with cardiac-specific deletion of miR-17-92 by crossing miR-17-92 flox/flox (F/F) mice with myh6-MerCreMer (MCM) via tamoxifen dependent Cre-loxP mediated DNA recombination. Two weeks after tamoxifen induced miRNA-17-92 inactivation, no significant differences in histology were observed between tamoxifen and corn oil treated animals despite downregulation of miR-17, -18a, -19a/b, -20a, and miR-92a in the heart. However, in response to 45min ischemia by LAD ligation followed by 24hrs reperfusion in vivo, cardiac miR-17-92 knockout resulted in increased I/R injury, and TTC and phthalocyanine blue staining revealed significantly larger infarct size as compared with control (63.6%±3.9%, n=10 vs. 41.0%±3.1%, n=7, P<0.05). Consistent with increased infarct size, cardiac miR-17-92 inactivation led to increased cardiomyocyte apoptosis after I/R injury, as identified by TUNEL staining. Furthermore, PTEN levels were increased while Phospho-Akt levels were significantly decreased in miR-17-92 inactivated hearts. Conclusions: We conclude that cardiac deletion of miR-17-92 augments the ischemia-reperfusion injury by inhibiting survival signaling. .

Icariside II potentiates paclitaxel-induced apoptosis in human melanoma A375 cells by inhibiting TLR4 signaling pathway

Combination therapy of paclitaxel (taxol) with natural anti-tumor agents that are capable of inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Our previous study showed that icariside II (IS), derived from Herba Epimedii, inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. In this report, the combination effects of paclitaxel and IS were investigated in human melanoma A375 cells. As compared to the treatment with paclitaxel alone, the co-administration of IS and paclitaxel resulted in an enhancement of apoptosis as revealed by WST-8 and PI assays. Meanwhile, Western blot analysis showed that the co-administration of IS and paclitaxel resulted in increases of cleaved caspase-3, one of the terminal pro-apoptotic proteins. In melanoma, IL-8 and VEGF are positively correlated with disease stage and a high probability of progression. We demonstrated that treatment of A375 cells with IS in combination with paclitaxel resulted in a significant decrease in the production of IL-8 and VEGF, compared with paclitaxel alone. Recent studies suggest that TLR4–MyD88–ERK signaling may be a novel target for reversing chemoresistance to paclitaxel. Our flow cytometry and Western blot data showed that paclitaxel activated TLR4–MyD88–ERK signaling and that IS treatment could effectively inhibit this paclitaxel-induced activation of TLR4–MyD88–ERK signaling. In conclusion, this study demonstrated for the first time that IS could potentiate paclitaxel-induced apoptosis in melanoma cells. These effects were mediated, at least in part, by inhibiting the activation of the TLR4 signal transduction pathways. These findings support further preclinical evaluation of IS as a new potential anti-tumor agent.

Abstract 4945: Targeting ErbB3-addicted cancers across the HER2 spectrum

Abstract ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in EGFR-, HER2-, and MET-addicted cancers, and ligand-driven activation of ErbB3 is a prominent method by which cancers become resistant to targeted inhibitors of ErbB signaling, as well as anti-hormonal and chemotherapeutic agents. With the goals of inhibiting survival signaling in cancer cells, delaying the onset of resistance to standard-of-care therapies, and re-sensitizing cancer cells to prior therapies, we developed an anti-ErbB3 monoclonal antibody, MM-121, that effectively blocks ligand-dependent activation of ErbB3 and induces internalization and degradation of the receptor. The activity of single-agent MM-121 was examined in xenograft models and a machine-learning algorithm was developed that accurately predicts which preclinical models of cancer will respond to MM-121 based on five protein-based biomarkers. By simulating ErbB3 inhibition using a computational model of ErbB signaling, we predicted that a bispecific molecule that docks onto HER2 and subsequently binds to ErbB3 would effectively block ErbB3-driven signaling in HER2-amplified tumors. We therefore developed a bispecific antibody comprising two human scFv antibodies linked by a modified form of human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with HER2 and ErbB3, effectively inhibiting ErbB3 signaling and demonstrating antitumor activity in preclinical models that are dependent on HER2 overexpression. Both MM-121 and MM-111 are now in early stages of clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4945. doi:1538-7445.AM2012-4945

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.