Design and synthesis of phenolic hydrazide hydrazones as potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors.

Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes responsible for catalyzing the formation and degradation of poly(ADP-ribose) (PAR) polymers, respectively. Activation of PARP has been shown to be involved in cell death induced by genotoxic stimuli. On the other hand, genetic disruption of PARG also leads to increased level of cell death by accumulation of PAR. Unlike PARP, where significant medicinal effort has been expended to identify potent inhibitors, PARG has been insufficiently investigated as a molecular therapeutic target. In this study, we report the design, synthesis, and biological evaluation of phenolic hydrazide hydrazones as potent PARG inhibitors. Compounds 3d, 3e, 5d, 5e, 8a, 8b and 8c showed their ability to inhibit the catalytic activity of PARG in vitro with IC50 values of 1.0, 2.1, 3.1, 3.2, 3.1, 2.8 and 1.6 µM, respectively.

Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer.

BACKGROUND: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear. METHODS AND RESULTS: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21CIP1/WAF1 in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the cellular growth and colony formation abilities, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene. CONCLUSION: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC.

Synergistic anti-tumor effects of a novel phosphatidyl inositol-3 kinase/mammalian target of rapamycin dual inhibitor BGT226 and gefitinib in non-small cell lung cancer cell lines.

Epidermal growth factor receptor (EGFR) and PI3K/mTOR pathway are drug targets for non-small cell lung cancer (NSCLC). Herein, we investigated anti-tumor effects of the combination of BGT226, a novel PI3K/mTOR dual inhibitor, and gefitinib on NSCLC cell lines which are high sensitive to gefitinib. The combination of BGT226 and gefitinib exhibited supra-additive growth inhibitory effects in PC-9 and HCC827 cells. Apoptotic induction and the inhibition of PI3K/mTOR signaling were enhanced by the combination. Significant tumor growth suppression was observed in xenograft model by the combination. These results suggest that the combination is effective in EGFR inhibitor-sensitive NSCLC therapy.

KRAS mutation confers resistance to antibody-dependent cellular cytotoxicity of cetuximab against human colorectal cancer cells.

Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab-mediated ADCC is not fully understood. Here, we investigated cetuximab-mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses FcγRIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin-dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin-independent ADCC was observed only in CRC cells with wild-type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas-Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin-independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth-inhibitory effect of cetuximab only in CRC cells with wild-type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab-mediated ADCC.

Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint.

Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma.

BACKGROUND: Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis. METHODS: To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined. RESULTS: LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells. CONCLUSIONS: In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.

Sunitinib inhibits lymphatic endothelial cell functions and lymph node metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3.

INTRODUCTION: Metastasis is a common event and the main cause of death in cancer patients. Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to be involved in the development of metastasis. Sunitinib is a multi-kinase inhibitor that blocks receptor tyrosine kinase activity, including that of vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is a clinically available angiogenesis inhibitor, its effects on lymphangiogenesis and lymph node metastasis remain unclear. The purpose of this study was to investigate the effects of sunitinib on vascular endothelial growth factor receptor 3 (VEGFR-3) and a related event, lymphangiogenesis. METHODS: The effects of sunitinib on the degree of phosphorylation of VEGFR-2/3 and other signaling molecules was examined in lymphatic endothelial cells (LECs) treated with the drug; VEGF-induced LEC growth, migration, and tube formation were also examined. For the in vivo study, luciferase-expressing breast cancer cells were transplanted into mammary fat pads of mice; the microvessel and lymphatic vessel density was then measured after treatment with sunitinib and anti-VEGFR-2 antibody. RESULTS: First, in human LECs, sunitinib blocked both VEGFR-2 and VEGFR-3 phosphorylation induced by VEGF-C or VEGF-D, and abrogated the activation of the downstream molecules extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. Furthermore, sunitinib attenuated the cell-proliferation activity induced by VEGF-C/D and prevented VEGF-C-induced migration and tube formation of the LECs; however, anti-VEGFR2 treatment shows only a partial effect on the growth and functions of the LECs. We used a breast cancer cell line expressing luciferase as a metastatic cancer model. Sunitinib treatment (40 mg/kg/day) inhibited the growth of the primary tumor transplanted in the mammary fat pad of the mice and significantly reduced the number of blood and lymphatic vessels in the tumor. Furthermore, the development of axillary lymph node metastasis, detected by bioluminescent imaging, was markedly suppressed. This effect of sunitinib was more potent than that of DC101, an anti-mouse VEGFR-2 antibody. CONCLUSIONS: The results suggest that sunitinib might be beneficial for the treatment of breast cancer by suppressing lymphangiogenesis and lymph node metastasis, through inhibition, particularly important, of VEGFR-3.

Efficacy of RAD001 (everolimus) against advanced gastric cancer with peritoneal dissemination.

Peritoneal dissemination occurs frequently in patients with unresectable advanced-stage gastric cancer. In this study, we tested the efficacy of the mTOR inhibitor RAD001 (everolimus) against advanced gastric cancer with peritoneal dissemination. Using the two cell lines, 58As1, a cell line exhibiting a high propensity for peritoneal metastasis, and its parental cell line, HSC-58, a human scirrhous gastric cancer cell line, we first examined the growth-inhibitory activity of everolimus in vitro. Methylene blue assay demonstrated a moderate inhibitory effect of the drug on both cell lines under normal culture conditions (maximal inhibitory effect: 50.5% at 1 µM, HSC-58, 65.3%, 58As1). However, under the hypoxic condition (1% O(2)), while the growth-inhibitory activity of everolimus was greatly reduced in the HSC-58 cell line, the degree of reduction of the inhibitory activity was much smaller in the 58As1 cell line. Western blotting revealed that the degree of phosphorylation of mTOR and its downstream signaling molecules, p70S6K and 4E-BP1, was decreased under hypoxic conditions in HSC-58. On the other hand, phospho-p70S6K and phospho-4E-BP1 remained active under hypoxic conditions in 58As1, the molecular activity was suppressed by everolimus. Cell-cycle analysis showed that hypoxia-induced G1 arrest was not manifested in the 58As1 cells, unlike in the HSC-58 cells. Separately, an in vivo orthotopic mouse model of 58As1 revealed that everolimus significantly reduced peritoneal dissemination as evaluated by the quantitative photon counting method. Taken together, our results suggest that everolimus may have favorable activity against gastric cancer, particularly in cases with peritoneal dissemination.

Synergistic interactions between the synthetic retinoid tamibarotene and glucocorticoids in human myeloma cells.

Tamibarotene (TM411) is a synthetic retinoic acid receptor-alpha/-beta selective retinoid that is chemically more stable than all-trans retinoic acid. This study was designed to evaluate the activity of TM411 in multiple myeloma (MM) and the effects of TM411 combined with a glucocorticoid (GC). In vitro, five human myeloma cells were treated with TM411 alone, GC alone, or TM411 + GC. Cell survival was analyzed by the tetrazolium dye assay and the Hoechst 33342/propidium iodide double-staining method. The effect of TM411 + GC was assessed by the isobologram method. In vivo, the growth-inhibitory effects of the drugs on RPMI-8226 cell xenografts established in SCID mice were examined. The effects of the agents on IL-6-mediated signaling pathways were also analyzed by Western blotting. TM411 was 2- to 10-fold more potent, in terms of its growth-inhibitory effect, than all-trans retinoic acid. The combination of TM411 and GC was found to show a markedly synergistic interaction. While increased expressions of the IL-6 receptor, phosphorylated MAPK, and Akt were observed after exposure to GC, TM411 attenuated this increase in the expressions, suggesting that such modification of the effect of GC by TM411 might be the possible mechanism underlying the synergistic interaction. Furthermore, TM411 + GC showed a supra-additive inhibitory effect in a xenograft model as compared with TM411 or GC alone. These results imply that the combination of TM411 + GC might be highly effective against MM, and suggest the need for clinical evaluation of TM411 + GC for the treatment of MM.