Showed Cell Growth Inhibition Research Articles

Design, Synthesis, and Antitumor Potential of New Thiazole--contained 5-Fluoro-2-Oxindole Derivatives as Sunitinib Analogues.

Indole is considered the most promising scaffold for anticancer drug design due to its high bioavailability, unique chemical properties, and broad spectrum of pharmacological action. Twelve novel thiazole-containing the 5-fluoro-1,3-dihydro-2H-indol-2-one derivatives as sunitinib analogs were designed and synthesized, and their anticancer activity was evaluated against the NCI-60 cancer cell lines. The thiazole-contained 5-fluoro-1,3-dihydro-2H-indol-2-one derivatives were synthesized using Knoevenagel condensation of 1,3-thiazole-5-carboxylic acid 1. Their anticancer activities were evaluated by NCI-60 one-dose screen assay. The molecular docking studies were performed using AutoDock tools and the AutoDock Vina programs. The ADMETlab 2.0 web server predicted the physicochemical properties of compounds. Among the synthesized new 5-fluoro-2-oxindole derivatives, compound 3g demonstrated high antitumor activity (GI>70%) against eight types of cancer: leukemia, breast cancer, ovarian cancer, lung cancer, melanoma, CNS cancer, renal cancer, and colon cancer. The most activity was observed against breast cancer (T-47D, GI=96.17%), lung cancer (HOP-92, GI=95.95%), ovarian cancer (NCI/ADR-RES, GI=95.13%), and CNS cancer (SNB-75, GI=89.91%). The molecular docking results of compound 3g demonstrated the possibility of inhibiting VEGF2 receptors as his potential anticancer mechanism. The physicochemical properties predicted for compounds 3f and 3g showed positive results. Compound 3g demonstrated high in vitro NCI-60 anticancer activity against nine cancer types and showed cell growth inhibition against leukemia, CNS, and breast cancer at 6 - 31% higher than Sunitinib, and may represent the basis for further modification of the thiazole-containing analogs of the anticancer drug Sunitinib.

Targeted anti-tumor synergistic effects of Myc decoy oligodeoxynucleotides-loaded selenium nanostructure combined with chemoradiotherapy on LNCaP prostate cancer cells.

In the present study, we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer cells. Myc decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics assays. ODNs were loaded on the synthesized Se@BSA@Chi-MTX nanostructure. The physicochemical characteristics of nanostructures were determined by FTIR, DLS, UV-vis, TEM, EDX, in vitro release, and hemolysis tests. Subsequently, the cytotoxicity properties of them with and without X-irradiation were investigated by uptake, MTT, cell cycle, apoptosis, and scratch assays on the LNCaP cell line. The results of DLS and TEM showed negative charge (-9 mV) and nanometer size (40 nm) for Se@BSA@Chi-DEC-MTX NPs, respectively. The results of FTIR, UV-vis, and EDX showed the proper interaction of different parts and the correct synthesis of nanoparticles. The results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/mL. The ODNs release from the nanostructures showed a pH-dependent manner, and the release rate was 15% higher in acidic pH. The targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen (PSMA). The significant synergistic effects of nanostructure (containing MTX drug) treatment along with X-irradiation showed cell growth inhibition, apoptosis induction (~57%), cell cycle arrest (G2/M phase), and migration inhibition (up to 90%) compared to the control. The results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP cells. This nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.

Abstract A049: Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a complex disease with multiple sub-types, each characterized by unique clinical and molecular features, driving the need to develop targeted therapies which exploit specific vulnerabilities. Bromodomain-containing protein 9 (BRD9) is a component of the ncBAF chromatin remodeling complex, and has been recently indicated as a strong dependency in AML (Weisberg et al 2022). It has been shown that inhibitors of BRD9 induce growth inhibition and expression of apoptotic makers in AML cell lines (Hohmann et al 2016; Zhou et al 2021). It has also been reported that BRD9 is critical to AML cell survival through the maintenance of STAT5 signaling (Del Gaudio et al 2019). Herein, we profile the in vitro anti-proliferative effects of FHD-609, a potent and selective degrader of BRD9, in a panel of 40 AML cell lines representative of a broad range of AML sub-types. We observed that FHD-609 was effective at inhibiting the growth of a subset of AML cell lines, consistent with previous reports in the literature. We further investigated whether treatment of AML cell lines with FHD-609 would induce changes in cell cycle, and found an increase in G1 in the same subset of cell lines that showed cell growth inhibition. Furthermore, treatment with FHD-609 induced apoptosis in these cell lines. To identify the mechanisms of action of FHD-609 in AML, as well as potential predictive biomarkers for AML sensitivity to FHD-609, we performed a range of mechanistic studies including ATACseq, ChIPseq and RNAseq in AML cells treated with FHD-609. We observed significant changes in the chromatin landscape in sensitive AML cell lines, and negligible changes in insensitive cell lines. Further bioinformatics analysis identified a possible biomarker strategy that correlates with the sensitivity of AML cell lines to FHD-609 in vitro. To confirm whether this biomarker strategy would successfully predict response in vivo, we dosed AML CDX and PDX models with FHD-609 based on the biomarker selection strategy. We observed a strong anti-tumor effect in AML CDX models and significantly extended survival of the mice in the AML PDX models. In summary, we have shown that FHD-609 demonstrates strong anti-tumor efficacy in a subtype of AML and have identified a possible biomarker strategy to predict response. Citation Format: Claudia Dominici, David Mayhew, Ammar Adam, Flore Uzan, Victoria Garbitt-Amaral, Oliver Mikse, Brandon Antonakos, Hafiz Ahmad, Salonee Parikh, Mei Yun Lin, Gabriel Sandoval, David Lahr, Hsin-Jung Wu, Mengni Xu, Sean Brennan, Luis M. M. Soares, Jordana Muwanguzi, Huawei Chen, Zhaoxia Yang, Jason T Lowe, Matt Netherton, Laura Zawadzke, Johannes Voigt, Liyue Huang, Sabine Ruppel, Ho Man Chan, Ryan Kruger, David S Milan, Scott Innis, Qianhe Zhou, Steven F Bellon. Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A049.

Targeting the Oncofetal Transcription Factor Protein SALL4 in Cancer By a Non-IMiD Degrader

Sal-like protein 4 (SALL4) is a C2H2 zinc finger transcription factor (TF) with two naturally occurring isoforms; SALL4A and SALL4B. It is typically detected in fetal tissues and silenced in most normal adult tissues. It is therefore intriguing that SALL4 is aberrantly re-expressed in about one-third of almost all primary human malignancies, presumably by demethylation dependent mechanisms. Direct evidence of the causative role of SALL4 in cancer has been demonstrated in SALL4 transgenic mice, which developed myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and/or liver tumors. Loss-of-function studies by SALL4 knock-down using shRNA showed cell growth inhibition and death in leukemias and solid tumors in culture and in in vivo xenotransplants. TF's, such as SALL4, which are critical for cancer development and survival, have historically been viewed as “undruggable”. However, rather than blocking activity, an alternative approach to target TFs is to induce protein degradation. Immunomodulatory imide drugs (IMiDs), including thalidomide, lenalidomide and pomalidomide, are used in treating patients with multiple myeloma (MM), MDS with 5q deletion, mantle cell lymphoma (MCL), and other hematological malignancies. Recently, several groups have reported that IMiDs can degrade SALL4 in a proteasome-dependent manner. To that end, we sought to investigate whether IMiDS could be utilized to treat SALL4-positive/expressing cancers. Initially, our studies observed that IMiDs had no effect on SALL4-positive cancer cells. Additional investigations thereafter demonstrated that IMiDs could only degrade SALL4A. These findings suggested that SALL4B may not be affected by IMiDs, and may be essential for SALL4-mediated cancer cell survival. Further investigation revealed that SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. Moreover, through high-throughput screening, we identified a new non-IMiD SALL4 degrader that targets SALL4B via proteasomal degradation and which exhibited potent anti-cancer activity, inhibiting cancer cell proliferation in culture and in vivotumor growth by 70%. Our observation therefore suggest that protein degraders could possess isoform specific effects. Additionally, our results exemplify the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

Simultaneous XIAP and cIAP1/2 inhibition by a dimeric SMAC mimetic AZD5582 induces apoptosis in multiple myeloma

Overexpression of inhibitor of apoptosis (IAP) proteins is associated with poor prognosis. In multiple myeloma (MM), the IAP inhibitors (IAPi), LCL161, have been evaluated in preclinical and clinical settings but are not fully effective. Among IAPs, XIAP has the strongest anti-apoptotic function with direct binding activity to caspases and cIAP1 and cIAP2 are positive regulator of NF-κB signaling. Prior IAPi such as LCL161 has high affinity to cIAP1 and cIAP2 resulting in inferior inhibiting activity against XIAP. A novel dimeric IAPi, AZD5582 (C58H78N8O8), have high binding potency to XIAP with EC50 dose of 15 nM, enabling to simultaneous inhibit XIAP and cIAP1/2. AZD5582 monotherapy showed cell growth inhibition for all MM cell lines, MM1S, RPMI8226, U266 and KMS-5 and induced apoptosis. AZD5582 further showed anti-proliferation effect under the IL-6 additional condition and inhibited JAK-STAT signaling triggered by IL-6. AZD5582 combined with carfilzomib therapy showed a synergistic effect. Enhanced apoptosis was also observed in combination therapy. Synergistic effect was further observed with other conventional therapeutics. Simultaneous XIAP and cIAP1/2 inhibition by the dimeric IAPi AZD5582 is promising. This study provides a rationale of AZD5582 as a new treatment strategy in monotherapy and in combination therapy.

Glutamine and amino acid metabolism as a prognostic signature and therapeutic target in endometrial cancer.

Endometrial cancer (EC) is the most common female reproductive system cancer in developed countries with growing incidence and associated mortality, which may be due to the growing prevalence of obesity. Metabolism reprogramming including glucose, amino acid, and lipid remodeling is a hallmark of tumors. Glutamine metabolism has been reported to participate in tumor proliferation and development. This study aimed to develop a glutamine metabolism-related prognostic model for EC and explore potential targets for cancer treatment. Transcriptomic data and survival outcome of EC were retrieved from The Cancer Genome Atlas (TCGA). Differentially expressed genes related to glutamine metabolism were recognized and utilized to build a prognostic model by univariate and multivariate Cox regressions. The model was confirmed in the training, testing, and the entire cohort. A nomogram combing prognostic model and clinicopathologic features was established and tested. Moreover, we explored the effect of a key metabolic enzyme, PHGDH, on the biological behavior of EC cell lines and xenograft model. Five glutamine metabolism-related genes, including PHGDH, OTC, ASRGL1, ASNS, and NR1H4, were involved in prognostic model construction. Kaplan-Meier curve suggested that patients recognized as high risk underwent inferior outcomes. The receiver operating characteristic (ROC) curve showed the model was sufficient to predict survival. Enrichment analysis recognized DNA replication and repair dysfunction in high-risk patients whereas immune relevance analysis revealed low immune scores in the high-risk group. Finally, a nomogram integrating the prognostic model and clinical factors was created and verified. Further, knockdown of PHGDH showed cell growth inhibition, increasing apoptosis, and reduced migration. Promisingly, NCT-503, a PHGDH inhibitor, significantly repressed tumor growth in vivo (p = 0.0002). Our work established and validated a glutamine metabolism-related prognostic model that favorably evaluates the prognosis of EC patients. DNA replication and repair may be the crucial point that linked glutamine metabolism, amino acid metabolism, and EC progression. High-risk patients stratified by the model may not be sufficient for immune therapy. PHGDH might be a crucial target that links serine metabolism, glutamine metabolism as well as EC progression.

Treatment by PI3K/mTOR Inhibitor BEZ235 Combined with TLR-7/8 Agonist Interfere with Immune Evasion Mechanisms of WEHI-3 Mouse Leukemia Cells.

Several PI3K/Akt/mTOR pathway inhibitors and TLR agonists induce tumor cell death. However, the mechanisms of these therapeutic approaches in acute myeloid leukemia (AML) cells are still unknown. To investigate the effects of BEZ235, as a dual inhibitor of PI3K and mTOR pathways, and TLR7/8 agonist R848 on the expression and regulation of the immune inhibitory molecules in myeloid leukemia cells. WEHI-3 leukemia cells were incubated with dual PI3K and mTOR inhibitor BEZ235 and TLR7/8 agonist R848 for 48 hrs. Firstly, cell viability was assessed by MTT method. The semi-quantitative relative mRNA expression of Galectin-9 (Gal-9), PD-L1, PVR, and STAT3 was assessed according to HPRT as a housekeeping gene. Finally, the protein expression of phosphorylated STAT3 was evaluated by western blotting analysis. WEHI-3 cells showed growth inhibition following treatment with BEZ235 and R848 whose combination exerted more proliferation arrest. The mRNA expression of Gal-9, PD-L1 and PVR immune checkpoint molecules significantly reduced in treated cells with BEZ235 and R848. Combined treatment indicated more reduction compared with the single treatment. Finally, the expression and phosphorylation of STAT3 were down-regulated after a single or dual treatment with BEZ235 and R848. Our results conclude that treatment with the combination of BEZ235 and R848 interferes with immune evasion mechanisms through STAT3-signaling pathway in WEHI-3 leukemia cells.

Synthesis, X-ray Structure, Hirshfeld, DFT and Biological Studies on a Quinazolinone-Nitrate Complex.

The reaction of 4-hydroxyquinazoline (4HQZ) with aqueous solution of nitric acid afforded the corresponding quinazolinone-nitrate (4HQZN) complex in very good yield. The crystal structure of 4HQZN was determined and its structural and supramolecular structural aspects were analyzed. 4HQZN crystallized in the space group P21/c and monoclinic crystal system with one [4HQZ-H]+[NO3]− formula and Z = 4. Its supramolecular structure could be described as a 2D infinite layers in which the 4HQZN molecules are connected via N-H…O and C-H…O hydrogen bridges. Using DFT calculations, the relative stability of five suggested isomers of 4HQZN were predicted. It was found that the medium effects have strong impact not only on the isomers’ stability but also on the structure of the 4HQZN. It was found that the structure of 4HQZN in DMSO and methanol matched well with the reported X-ray structure which shed the light on the importance of the intermolecular interactions on the isomers’ stability. The structure of 4HQZN could be described as a proton transfer complex in which the nitrate anion acting as an e-donor whiles the protonated 4HQZ is an e-acceptor. In contrast, the structure of the isolated 4HQZN in gas phase and in cyclohexane could be described as a 4HQZ…HNO3 hydrogen bonded complex. Biological screening of the antioxidant, anticancer and antimicrobial activities of 4HQZ and 4HQZN was presented and compared. It was found that, 4HQZN has higher antioxidant activity (IC50 = 36.59 ± 1.23 µg/mL) than 4HQZ. Both of 4HQZ and 4HQZN showed cell growth inhibition against breast (MCF-7) and lung (A-549) carcinoma cell lines with different extents. The 4HQZ has better activity with IC50 of 178.08 ± 6.24 µg/mL and 119.84 ± 4.98 µg/mL, respectively. The corresponding values for 4HQZN are 249.87 ± 9.71 µg/mL and 237.02 ± 8.64 µg/mL, respectively. Also, the antibacterial and antifungal activities of 4HQZN are higher than 4HQZ against all studied microbes. The most promising result is for 4HQZN against A. fumigatus (MIC = 312.5 μg/mL).

Screening and molecular identification of marine sponges with cytotoxic activities, collected from Gulf of Mannar, Indian Ocean

The marine environment, a rich source of biological and chemical diversity, offers tremendous scope for discovering novel metabolites with pharmacological activities. Marine invertebrates, especially sponges, have gained more attention in drug discovery programmes since they exhibit unique metabolic and physiological capabilities and an extraordinarily symbiotic association with diverse bacterial communities. The present study focuses on screening the cytotoxic activity of the methanolic extracts of sponges against the Non-Small Cell Lung Cancer (NSCLC) cell line, NCI-H460. In the bioactivity screening, two sponges (IOS-11 and IOS-12) were found with potent cytotoxic activity. The sponge IOS-12 showed almost complete cell growth inhibition (99 %) at 50 µg/ml and 80 % inhibition at 5 µg/ml; whereas IOS-11 showed cell growth inhibition of 96 % and 77 % at 50 µg/ml and at 5 µg/ml, respectively. Amplification of the mitochondrial cytochrome C oxidase subunit I (COI) and subsequent nucleotide analysis enabled the identification of sponge samples, IOS-11 and IOS-12, as Rhabdastrella globostellata and Halichondria sp., respectively. The study concludes that the two marine sponges, Rhabdastrella globostellata and Halichondria sp., possess excellent cytotoxic activities and could be considered good candidates for the isolation of anticancer compounds with biomedical applications. The isolation, purification and structural elucidation of the lead molecules from these sponges are ongoing studies, which might yield potential new compounds with promising cytotoxic activities.

Exploration of Antioxidant and Anticancer Activity of Stephania japonica Leaves Extract

Aims: The demand for antioxidants from the natural source has drawn promising attention to outturn desired pharmacological effect by subsidizing the adverse effect for treating cancer. This study evaluated the antioxidant activity of Stephania japonica leaves extracts to explore the anticancer activity. 
 Methods: Antioxidant potential of crude extracts were evaluated using various methods which include total antioxidant activity, ferric reducing antioxidant assay, DPPH free radical, ABTS free radical, nitric oxide and superoxide anion radical scavenging assay. Anticancer activity was determined in vitro by MTT assay and in vivo on mice against Ehrlich ascites carcinoma (EAC) cell.
 Results: Phytoconstituents with free radical scavenging capacity were quantified in terms of inhibitory concentration (IC50) with the values of 17.00±3.22 µg/mL, 33.30 ± 5.45 µg/mL, 43.70±5.26 µg/mL and 52.30±1.07 µg/mL in DPPH, ABTS, superoxide anion radical and nitric oxide free radical scavenging assay, respectively as the highest quencher, acetone extract of S. japonica leaves (ASJL). ASJL and methanol extract (MSJL) showed low lethal dose (LD50) values of 21.76 and 26.63, respectively indicating higher toxicity. In vitro anti-proliferative activity (MTT assay), ASJL and MSJL were exhibited 15.44±2.96 to 80.94±2.87 and 11.76±3.74 to 74.25±1.49 percent of cell growth inhibition, respectively at the concentration of 10.28 µg/mL to 833.33 µg/mL. In in vivo test, ASJL and MSJL at the dose of 100 mg/kg/day and 200 mg/kg/day (i.p.) showed cell growth inhibition of 58.25±4.24% to 79.09±2.45% and 46.26±2.68% to 61.74±4.41%, respectively on EAC cell tumor-bearing mice. The life span of intraperitoneal induced EAC cell bearing mice was increased to 29.05% and 57.02% on the treatment of ASJL with 100 and 200 mg/kg/day, respectively.
 Conclusions: The free radical scavenger of S. japonica leaves extract was stimulated the host immunity and inhibited the EAC cell growth through initiating the apoptosis cell death program. Therefore, S. japonica leaves might be utilized as a potent anticancer natural source.

Suppressing the metastatic properties of the breast cancer cells using STAT3 decoy oligodeoxynucleotides: A promising approach for eradication of cancer cells by differentiation therapy.

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple-negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3-labeled ODNs is in the cytoplasm and the nucleus of MDA-MB-231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA-MB-231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach.

Tenovin-6 induces the SIRT-independent cell growth suppression and blocks autophagy flux in canine hemangiosarcoma cell lines

Canine hemangiosarcoma (HSA) is a commonly occurring aggressive tumor stemming from the vascular endothelial cells and is considered to be a good model for a similar disease in humans, called angiosarcoma. In this study, we reviewed drug libraries to identify new signal transduction inhibitors that can suppress the cell growth of canine HSA in vitro. We observed that tenovin-6, a sirtuin (SIRT) inhibitor, inhibited cell proliferation and induced cell death in three canine HSA cell lines (JuB4, Re12, and Ud6). These effects were induced through G1 cell cycle arrest and caspase-3 activation. Although tenovin-6 is known as an inhibitor of SIRT1 and SIRT2, knockout (KO) of genes encoding SIRT1 and/or SIRT2 had no apparent impact on cell proliferation in canine HSA. In addition, tenovin-6 showed cell growth inhibition in SIRT KO cells, as well as parental cells. These results indicated the cytotoxicity of tenovin-6 was a SIRT-independent event. Instead, we found that tenovin-6 inhibited autophagy flux in canine HSA cells, as evidenced by the suppression of lysosomal proteolysis. These results suggested that tenovin-6 induces cell growth suppression in canine HSA cells by impairing the lysosomal function. Therefore, tenovin-6 could be used in a new therapeutic strategy to treat canine HSA.

Two new dammarane triterpenes isolated from Cleome khorassanica Bunge & Bien with cytotoxicity against DU-145 and LNCaP prostate cancer cell lines

From the aerial parts of Cleome khorassanica Bunge & Bien, a new 3-oxo-4-oxa-A-homo-25,26,27-trinordammarano-24,20-lactone triterpene (1), a new natural product 20,25-dihydroxy-3-oxodammarane triterpene (2), together with known 5-hydroxy-3,6,7,8,3′,4′,5′-heptamethoxyflavone (3), have been isolated and characterized. The chemical structure of the new compounds was determined by 1D and 2D NMR and HR tandem MS measurements. All three compounds were subjected to biological tests for evaluation of their cytotoxicity against prostate (DU-145 and LNCaP) cancer cells. Compounds 1, 2, and 3 showed cell growth inhibition in a dose dependent manner against DU-145 and LNCaP cells.

MITF suppression improves the sensitivity of melanoma cells to a BRAF inhibitor

Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors.

Abstract 1550: Epigenetic downregulation and growth inhibition of IGFBP7 in gastric cancer

Abstract Insulin-like growth factor-binding protein 7 (IGFBP7) has been found to be a tumor suppressor in several human cancers, but the role of IGFBP7 in gastric cancer has not yet been fully investigated. In this study, we examined the epigenetic downregulation of IGFBP7 expression in gastric cancer. Analysis by qRT-PCR, western blot, and methylation specific-PCR revealed that DNA methylation was inversely correlated with IGFBP7 expression, and the expression of IGFBP7 was restored after treatment with 5-aza-2'-deoxycytidine. Immunohistochemistry showed that IGFBP7 expression was downregulated in 47.5% of advanced gastric cancer patients. Univariate and multivariate analysis showed that IGFBP7 expression and tumor stage were independent prognostic factors. In cell culture, IGFBP7 knockdown increased gastric cancer cell growth, invasion, and migration, whereas IGFBP7 overexpression in gastric cancer cells showed cell growth inhibition and apoptosis. Our data suggest that IGFBP7 functions as a tumor suppressor in gastric cancer via an epigenetic pathway. Citation Format: Jin Kim, Min A Kim, Sun-Ju Byeon, Woo Ho Kim. Epigenetic downregulation and growth inhibition of IGFBP7 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2017-1550

Growth Inhibition of Colon Cancer and Melanoma Cells by Versiol Derivatives from a Paraconiothyrium Species.

Bioassay-guided fractionation of a colon cancer growth inhibitory extract of the fungus Paraconiothyrium sp. led to the isolation of eight new versiol derivatives (1, 3-8, 10) along with two known compounds. The structures were elucidated by interpretation of combined MS and 2D NMR spectroscopic data. Compounds 8, 9, and 10 showed cell growth inhibition against COLO205 and KM12 cells, and both 8 and 9 displayed selectivity in their inhibition of melanoma cell lines in the NCI 60 one-dose test. In addition, compound 8 and the crude Paraconiothyrium sp. extract showed potent dose-dependent inhibitory effects in the five-dose NCI 60 cell line assay.

MITF suppression by CH5552074 inhibits cell growth in melanoma cells.

Although treatment of melanoma with BRAF inhibitors and immune checkpoint inhibitors achieves a high response rate, a subset of melanoma patients with intrinsic and acquired resistance are insensitive to these therapeutics, so to improve melanoma therapy other target molecules need to be found. Here, we screened our chemical library to identify an anti-melanoma agent and examined its action mechanisms to show cell growth inhibition activity. We screened a chemical library against multiple skin cancer cell lines and conducted ingenuity pathway analysis (IPA) to investigate the mechanisms of CH5552074 activity. Suppression of microphthalmia-associated transcription factor (MITF) expression levels was determined in melanoma cells treated with CH5552074. Cell growth inhibition activity of CH5552074 was evaluated in MITF-dependent melanoma cell lines. We identified an anti-melanoma compound, CH5552074, which showed remarkable cell growth inhibition activity in melanoma cell lines. The IPA results suggested that CH5552074-sensitive cell lines had activated MITF. In further in vitro studies in the melanoma cell lines, a knockdown of MITF with siRNA resulted in cell growth inhibition, which showed that CH5552074 inhibited cell growth by reducing the expression level of MITF protein. These results suggest that CH5552074 can inhibit cell growth in melanoma cells by reducing the protein level of MITF. MITF inhibition by CH5552074 would be an attractive option for melanoma treatment.

Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases

Walnut (Juglans regia L.) contains approximately 20-25% protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63±1.73% for breast cancer and 51±1.45% for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

Beta protein 1 homeoprotein induces cell growth and estrogen-independent tumorigenesis by binding to the estrogen receptor in breast cancer.

Expression of Beta Protein 1 (BP1), a homeotic transcription factor, increases during breast cancer progression and may be associated with tumor aggressiveness. In our present work, we investigate the influence of BP1 on breast tumor formation and size in vitro and in vivo. Cells overexpressing BP1 showed higher viability when grown in the absence of serum (p < 0.05), greater invasive potential (p < 0.05) and formed larger colonies (p < 0.004) compared with the controls. To determine the influence of BP1 overexpression on tumor characteristics, MCF-7 cells transfected with either empty vector (V1) or overexpressor plasmids (O2 and O4) were injected into the fat pads of athymic nude mice. Tumors grew larger in mice receiving O2 or O4 cells than in mice receiving V1 cells. Moreover, BP1 mRNA expression levels were positively correlated with tumor size in patients (p = 0.01). Interestingly, 20% of mice injected with O2 or O4 cells developed tumors in the absence of estrogen, while no mice receiving V1 cells developed tumors. Several mechanisms of estrogen independent tumor formation related to BP1 were established. These data are consistent with the fact that expression of breast cancer anti-estrogen resistance 1 (BCAR1) was increased in O2 compared to V1 cells (p < 0.01). Importantly, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Overall, BP1 overexpresssion in MCF-7 breast cancer cells leads to increased cell growth, estrogen-independent tumor formation, and increased proliferation. These findings suggest that BP1 may be an important biomarker and therapeutic target in ER positive breast cancer.

Abstract B188: Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model

Abstract Background: Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs. Several mechanisms of the acquired resistance have been described, including gatekeeper T790M mutation and MET amplification. Overexpression of AXL, a receptor type tyrosine kinase and/or its ligand, GAS-6 have been identified in a subset of NSCLC patients with acquired resistance, and inhibition of AXL has been shown to restore sensitivity to erlotinib in nonclinical resistant models, suggesting that AXL is also involved in EGFR-TKI resistance of NSCLC. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697, NCT02500927). We have previously reported that ASP8273 selectively inhibited kinase activities of EGFR activating and T790M mutations compared to wild type EGFR, and induced tumor regression in HCC827 and NCI-H1975 xenografted mice. In this study, we evaluated the antitumor activity of ASP8273 against AXL overexpressing PC-9 cells. Method: PC-9 vec. cells and PC-9 AXL cells were constructed by the infection of pMXs-puro vector and AXL coding vector, respectively into PC-9, a NSCLC cell line with EGFR exon 19 del activating mutation. Inhibitory effects of ASP8273 and other EGFR-TKIs, including erlotinib on EGFR and AXL signaling and cell proliferation were investigated in PC-9 vec. and PC-9 AXL cells. In vivo antitumor effect was also examined in subcutaneously implanted with PC-9 AXL. Result: ASP8273 at 1 μmol/L inhibited cell growth associated with the inhibition of EGFR, AXL, AKT, and ERK phosphorylation in PC-9 AXL. On the other hand, EGFR-TKIs at 1 μmol/L did not show cell growth inhibition in PC-9 AXL cells, whereas those showed cell growth inhibition in PC-9 vec. cells. EGFR-TKIs inhibited the phosphorylation of EGFR but not AXL, AKT, and ERK in PC-9 AXL cells. In an in vivo xenograft model using PC-9 AXL, once-daily oral administration of ASP8273 at 50 mg/kg induced statistically significant tumor growth inhibition although the other EGFR-TKIs did not. Conclusion: These results suggest that overexpression of AXL confers resistance to EGFR-TKIs in NSCLC cells and ASP8273 may show antitumor activity against EGFR-TKIs-resistant NSCLC patients with AXL expression. Citation Format: Naoki Kaneko, Hiroaki Tanaka, Satoshi Konagai, Hiroko Yamamoto, Hideki Sakagami, Tomohiro Eguchi, Takahiro Matsuya, Masamichi Mori, Hiroyuki Koshio, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B188.