Significant Dose-dependent Growth Inhibition Research Articles

Abstract A074: Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway

Abstract Background: The Wnt/β-catenin pathway controls the growth of pancreatic carcinoma by regulating processes such as cell cycle progression, apoptosis, and the tumor immune microenvironment. Treating pancreatic cancer with Wnt pathway inhibitors was found to be toxic in clinical studies. Namodenoson, an A3 adenosine receptor (A3AR) agonist, is under clinical development as a treatment of advanced liver cancer, where namodenoson was shown to have an excellent safety profile in phase II studies and is a phase III study is ongoing. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. The objectives of the currents study were to examine the anti-growth effect of namodenoson on pancreatic carcinoma cells both in vitro and in vivo and to investigate the molecular mechanisms involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson, or in the presence of a combination of namodenoson (0.1 nM) and gemcitabine (0.2 µM). Cell growth was monitored via 3[H]-thymidine proliferation, MTT, and PrestoBlue assays. Western blot analyses were conducted to identify the regulatory cell growth proteins impacted by the treatment. A xenograft model with human BxPC-3 cells in nude mice was established to investigate the in-vivo effect of namodenoson. Results: A significant dose-dependent growth inhibition of BxPC-3 cells was observed with namodenoson in the3[H]-thymidine proliferation assay (1 nM: 67.4% ± 1.7%, p < 0.001; 0.1 nM: 53.7% ± 6.3%, p< 0.05; 0.01 nM: 27.9 % ± 2.3%, p< 0.005). The combination of namodenoson and gemcitabine showed an additive inhibitory effect in the MTT assay (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p<0.001 for all). Western blot analyses demonstrated that namodenoson treatment was associated with de-regulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, β-catenin and up-regulation of the BAD and BAX apoptotic proteins. In the xenograft model, daily oral treatment with 10 µg/kg of namodenoson led to a significant progressive inhibition of tumor growth. Conclusions: Nanomolar concentrations of namodenoson inhibited the growth of pancreatic carcinoma in cell culture and in a xenograft model. This effect involved A3AR activation and de-regulation of the Wnt/β-catenin pathway. Our results support a potential clinical utility for namodenoson in pancreatic cancer, a disease where an unmet clinical need exists, and novel therapeutic opportunities are required. Citation Format: Pnina Fishman, Avital Bareket-Samish, Inbal Izhak. Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A074.

Abstract C145: ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer

Abstract Mesenchymal epithelial transition (MET) proto-oncogene receptor tyrosine kinase (RTK) is a cell surface receptor selective for hepatocyte growth factor (HGF), and is involved in embryogenesis regulation, wound healing, organ regeneration, angiogenesis, and immunomodulation. Aberrant MET oncogenic alterations include: MET exon 14 skipping (MET∆ex14) mutations; activating mutations in the kinase domain; MET gene amplification; MET fusions; and MET protein overexpression. These oncogenic alterations occur in a wide range of human solid cancers. Small-molecule type I c-Met inhibitors bind to the active (Asp-Phe-Gly “DFG-in” state) of c-Met within the adenosine triphosphate (ATP) binding pocket via the Y1230 residue, resulting in down-regulation of multiple pro-oncogenic downstream factors. Presently, type I c-Met inhibitors such as Capmatinib are used as monotherapies in patients with locally advanced or metastatic NSCLC with MET∆ex14 mutations. However, development of post-treatment resistance to type I c-Met inhibitors occurs clinically, including through acquired mutations in codons D1228 and Y1230. No drugs have been approved globally for MET alteration indications other than MET∆ex14 mutations. Next generation MET inhibitors are thus needed to treat patients harboring various MET oncogenic alterations beyond MET∆ex14, including post-treatment acquired mutations. ANS014004 is a newly developed small-molecule type II inhibitor of c-Met, binding to the inactive state of c-Met (“DFG-out”) in the ATP pocket. ANS014004 showed greater kinase inhibition activity against c-Met mutant kinases Y1230H and D1228A in vitro (IC50’s = 2.596 and 2.971 nM, respectively), when compared with Capmatinib, Cabozantinib, and Merestinib (IC50 values = >5000, ~10, and ~5 nM respectively). Further, ANS014004 showed a stronger anti-proliferative effect in MET mutant Ba/F3 cells TPR-Met D1228A and TPR-Met-Y1230H in vitro (IC50’s = 49.34 nM and 30.32 nM, respectively), when compared with Capmatinib (IC50 values>10000nM) and Merestinib (IC50 values: 37~100 nM). ANS014004 also showed potent antitumor effects in vitro in the human cancer tumor cell lines harboring various pathogenetic MET alterations: Hs-746T (gastric); MKN-45 (gastric); EBC-1 (NSCLC); NCI-H1993 (NSCLC); with IC50 ranging from 6.38~62.32 nM. In several subcutaneous xenograft tumor model studies using these same cell lines implanted in BALB/c nude mice, orally administered ANS014004 produced significant dose-dependent tumor growth inhibition, and the corresponding exposures of ANS014004 in plasma, tumor and brain samples increased with increasing dose. ANS0104001 was also observed to induce tumor regression in a MET-amplified intracranial xenograft model. Overall, the non-clinical studies of ANS014004 demonstrated it is a potent, orally-bioavailable type II c-Met inhibitor with activity against various pathogenetic MET alterations and with favorable absorption, distribution, pharmacokinetic, efficacy, and tolerability profiles in vivo. The molecule will enter a Phase 1 clinical study in the US soon. Citation Format: Gong Li, Hepeng Shi, Peilong Zhang, Xiangqiu Li, Wenli Lan, Linna Li, David Chung, Kevin Schaab. ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer [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 C145.

Effects of 24-Epibrassinolide, Bikinin, and Brassinazole on Barley Growth under Salinity Stress Are Genotype- and Dose-Dependent

Brassinosteroids (BRs) are involved in the regulation of many plant developmental processes and stress responses. In the presented study, we found a link between plant growth under salinity stress and sensitivity to 24-epibrassinolide (24-EBL, the most active phytohormone belonging to BRs), brassinazole (Brz) and bikinin (inhibitors of BR biosynthesis and signaling pathways, respectively). Plant sensitivity to treatment with active substances and salinity stress was genotype-dependent. Cv. Haruna Nijo was more responsive during the lamina joint inclination test, and improved shoot and root growth at lower concentrations of 24-EBL and bikinin under salinity stress, while cv. Golden Promise responded only to treatments of higher concentration. The use of Brz resulted in significant dose-dependent growth inhibition, greater for cv. Haruna Nijo. The results indicated that BR biosynthesis and/or signaling pathways take part in acclimation mechanisms, however, the regulation is complex and depends on internal (genotypic and tissue/organ sensitivity) and external factors (stress). Our results also confirmed that the lamina joint inclination test is a useful tool to define plant sensitivity to BRs, and to BR-dependent salinity stress. The test can be applied to manipulate the growth and stress responses of crops in agricultural practice or to select plants that are sensitive/tolerant to salinity stress in the plant breeding projects.

N-myristoyltransferase proteins in breast cancer: prognostic relevance and validation as a new drug target

PurposeN-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood.MethodsWe generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706).ResultsNMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo.ConclusionsThese results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients.

Abstract 5607: Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade

Abstract We previously reported that DRP-104, a novel broad acting glutamine antagonist, has significant therapeutic potential in cancer via directly targeting tumor metabolism and inducing a potent antitumor immune response. Here we sought to further elucidate the immunomodulatory effect of DRP-104 on tumor growth inhibition as a single agent and in combination with PD-1/PD-L1 checkpoint inhibitors in the MC38 and CT26 colon cancer syngeneic models. We utilized multiple technologies to assess mechanistic effects on immune cells in vivoincluding flowcytometry, multiplex immunoassay, gene expression profiling and GeoMx™ digital spatial profiling. DRP-104 mediated tumor growth inhibition was associated with increased tumor-infiltrating leukocytes (TIL) including T, NKT, and NK cells, M1-polarized tumor associated macrophages, and decreased immune-suppressive cells such as MDSCs. Nanostring® IO360 analysis revealed broad immunological modulation such as increase in cytotoxicity/antigen presentation score, increase in tumor metabolic stress/apoptotic score and decrease in cell proliferation score. GeoMx™ profiling also showed increased tumoral T cells and granzyme B expression. Luminex analysis of tumor lysates revealed DRP-104 treatment decreased pro-tumorigenic proteins, such as VEGF and IL-8. DRP-104 showed significant dose dependent tumor growth inhibition and regressions as a single agent in mouse syngeneic tumor models, including those resistant to immune checkpoint inhibitors. Lastly, combination of DRP-104 with anti-PD-1 Ab or anti-PD-L1 Ab further reduced tumor growth resulting in significant improvement in survival with long-term durable cures. In summary, DRP-104 treatment resulted in broad remodeling of the tumor microenvironment including enhanced infiltration and function of multiple immune cells distinct from activities obtained by a checkpoint inhibitor treatment. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti-tumor efficacy including long-term durable cures even in checkpoint inhibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in combination with PD-1/PD-L-1 checkpoint inhibitors. Citation Format: Yumi Yokoyama, Robert Wild. Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5607.

Abstract 1782: N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target

Abstract N-myristoyltransferases (NMTs) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. This activity regulates numerous membrane-bound signal transduction pathways important in cancer biology, and the pan-NMT inhibitor PCLX-001 is in clinical development as a cancer therapy. The physiologic distribution and relative contributions of the two human NMTs, NMT1 and NMT2, remain poorly understood as previous studies used polyclonal antibodies with potential cross-reactivity. We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to the human isotypes of NMT1 and NMT2. These mAbs were used to perform an immunohistochemical (IHC) analysis of the abundance and distribution of NMT1 and NMT2 in normal human breast epithelial samples and a large (n=703) cohort of primary breast adenocarcinomas from the BCIRG001 study. While NMT1 protein was readily identifiable in most normal and transformed breast epithelial tissue, NMT1 abundance was associated with higher overall histologic grade, higher Ki67, and lower hormone expression. While NMT2 protein was readily detected in normal breast epithelial tissue, NMT2 protein was undetectable in the majority of malignant breast cancers, but detectable NMT2 protein correlated with significantly poorer overall survival outcomes (hazard ratio for death 1.36; p < 0.029) and significantly worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. NMT status was unrelated to HER2 status. NMT1 and NMT2 protein abundances were positively correlated with each other. Treatment of cultured breast cancer cells with the pan-NMT inhibitor PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts induced significant dose-dependent tumor growth inhibition in vivo. These results support the further evaluation of NMT immunohistochemistry for selection of patients for NMT inhibitor therapy, and clinical trials of NMT inhibition in breast cancer patients. Citation Format: John R. Mackey, Justine Lai, Utkarsh Chauhan, Wei-Feng Dong, Darryl Glubrect, Sunita Ghosh, Gilbert Bigras, Raymond Lai, Luc G. Berthiaume. N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1782.

Abstract 3080: Chronic exposure to arsenic induces malignant transformation in human kidney epithelial cells

Abstract Arsenic contamination is a significant environmental and public health issue worldwide including the United States. Accumulating evidence suggests that kidney is one of the target organ for arsenic-induced carcinogenesis and toxicological effects. However, the mechanism of arsenic-induced renal carcinogenesis is not well understood. Therefore, the objective of this study was to evaluate the carcinogenicity of chronic exposure to low dose arsenic on kidney epithelial cells, and identify the molecular mechanism underlying this process. HK-2 kidney epithelial cells derived from normal human kidney were treated with arsenic for acute, long-term and chronic durations and cellular response to arsenic exposure at these time points was determined by evaluating the changes in growth, morphology, and expression of genes. The results revealed a significant dose-dependent growth inhibition after acute exposure, whereas increased growth after long-term and chronic exposure to arsenic of HK-2 cells as compared to untreated control cells. The anchorage-independent growth assay for colony formation further confirmed malignant transformation of arsenic exposed cells. Additionally, the morphological changes of EMT and stemness were also observed in long-term arsenic exposed cells. These changes observed above were further confirmed by analysis of gene and protein expression. Our data suggest that cellular response of EMT and stemness act a driving factor and mechanistic basis for increased survival and malignant transformation in arsenic exposed cells. In summary, results of this study suggest that acute exposure to arsenic can cause loss of kidney epithelial cells with potential significance to acute kidney injury, whereas the chronic exposure to arsenic can increase the risk of kidney cancer through enhancing cell proliferation. Citation Format: Yu-Wei Chang, Kamaleshwar Singh. Chronic exposure to arsenic induces malignant transformation in human kidney epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3080.

Ginkgetin induces G2-phase arrest in HCT116 colon cancer cells through the modulation of b‑Myb and miRNA34a expression.

Ginkgetin has been reported to display antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in CRC cells remains to be identified. In this study, ginkgetin-treated HCT116 CRC cells exhibited significant dose-dependent growth inhibition with a GI50 value of 4.0µM for 48-h treatment, together with apoptosis, via G2-phase cell cycle arrest. When HCT116 cells were treated with 10µM ginkgetin for 48h, the percentage of cells in G2/Mphase increased by 2.2-fold (43.25%) versus the untreated control(19.69%). Ginkgetin regulated the expression of genes that are critically involved in G2phase arrest cells, such as b‑Myb, CDC2 and cyclinB1. Furthermore, we found that the suppression of b‑Myb expression by ginkgetin was rescued ~5.1-fold by treatment with a miR-34a inhibitor (500nM) and b‑Myb was downregulated by >80% by 100nM miR‑34a mimic. These data suggest that the miRNA34a/b‑Myb/cyclinB1 cascade plays a critical role in ginkgetin-induced G2 cell cycle arrest, as well as in the inhibition of HCT116 cell proliferation. Moreover, the administration of ginkgetin (10mg/kg) reduced tumor volumes by 36.5% and tumor weight by 37.6% in the mice xenografted with HCT116 cells relative to their vehicle-treated counterparts. Therefore, ginkgetin is the first compound shown to regulate b‑Myb by modulating miR-34a, and we suggest the use of ginkgetin as an inducer of G2arrest for the treatment of CRC.

Abstract 2175: Vietnamese medicinal plant compounds show potent anti-pancreatic cancer activity in vitro

Abstract BACKGROUND: Pancreatic cancer is a devastating disease with a dismal survival rate of less than 7%. This is due to its late diagnosis and aggressive, metastatic disease. Currently treatment typically consists of surgery and/or treatment with gemcitabine; gemcitabine/nab-paclitaxel or FOLFIRINOX - all chemotherapeutic agents that have natural product-derived components that only prolong survival by 5-6 months at best. As traditional Vietnamese medicinal plants have been used to treat a number of diseases for several thousands of years, there is great potential to investigate their importance as sources of novel therapeutic agents for pancreatic cancer. METHODS: Pancreatic cancer cell lines (BxPC3, MiaPaCa2 and CFPAC1) and the normal pancreatic ductal epithelial (HPDE) cell line were treated with pure compounds isolated from the Vietnamese plants Croton tonkinensis and Salacia cochinchinensis and the effects on cell viability (CCK-8 assay) and apoptosis (caspase-3/-7 activation assay) were assessed to determine their anti-cancer capacity. RESULTS: Treatment of pancreatic cancer cell lines with Pristimerin (from Salacia cochinchinensis) and CT1 (from Croton tonkinensis) resulted in significant dose-dependent growth inhibition. Pancreatic cancer cell lines showed significantly greater sensitivity to Pristimerin treatment after 24 and 72hrs, with a 1.7 – 2.3 fold lower IC50 value in MiaPaCa2 pancreatic cancer cells compared to normal HPDE cells, respectively (24hrs: 48 vs. 111nM; 72hrs: 63.85 vs. 111nM). Significant cytotoxicity was also seen in other pancreatic cancer cell lines (BxPC3 103 - 210nM; CFPAC1 77nM). Treatment with CT1 also resulted in significant dose-dependent cytotoxicity with an IC50 value that was 1.5 – 1.8 fold lower in MiaPaCa2 cells compared to normal HPDE cells, respectively (24hrs: 116 vs. 177nM; 72hrs: 46.5 vs. 84.5nM). Treatment of BxPC3 and CFPAC1 cells with CT1 also led to significant cytotoxicity with IC50 values of 300nM and 170nM, respectively. Moreover, treatment with Pristimerin and CT1 at concentrations of 625nM and 1μM induced caspase-dependent apoptosis of MiaPaCa2 and HPDE cells after 22hrs of treatment. CONCLUSIONS: Pure compounds from Vietnamese medicinal plants show significant pancreatic cancer growth inhibition and induce apoptosis in vitro at concentrations that have minimal effect on normal pancreatic cells and therefore show promise as novel anti-pancreatic cancer therapies. Note: This abstract was not presented at the meeting. Citation Format: Danielle Bond, Phuong Thien Thuong, Do Thi Ha, Nguyen Minh Khoi, Judith Weidenhofer, Christopher J. Scarlett. Vietnamese medicinal plant compounds show potent anti-pancreatic cancer activity in vitro [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 2175. doi:10.1158/1538-7445.AM2017-2175

Abstract 4038: Exploring optimal targeted combination therapies with neratinib for HER2+ breast cancer

Abstract Activation by amplification or overexpression of the proto-oncogene HER2 (also known as ERBB2) is associated with the development and progression of breast cancer. Neratinib is a novel, irreversible, pan-HER tyrosine kinase inhibitor which selectively inhibits EGFR, HER2 and HER4. In this preclinical study, we explored the efficacy of neratinib in combination with other clinically relevant targeted agents for optimal treatment of HER2-positive breast cancer in in vitro and in vivo models. Western blot (WB) analysis of a panel of breast cancer cell lines showed that BT-474, SKBR-3, and HCC-1954 cells displayed elevated HER2 expression levels (here denoted as HER2+), while MDA-MB-361, MDA-MB-453, and CAMA-1 breast cancer cells expressed relatively lower HER2 expression levels. Interestingly, neratinib inhibition positively correlated with HER2 expression levels as assessed by cell survival assays. The IC50 of neratinib in the HER2+ cell lines was in the 3.6 - 113nM range. Consistent with the in vitro data, treatment of a HER2+ breast cancer PDX model with neratinib single agent (p.o. 5 days/week) led to a significant dose-dependent tumor growth inhibition with tumor regressions observed at a low dose of 10 mg/kg (p<0.0001). The activity of neratinib in combination with other targeted agents was then evaluated in vitro in the HER2+ breast tumor cell lines using a Chou-Talalay method. Neratinib showed synergistic anti-proliferative activity in combination with the PI3Kα inhibitor BYL719 in all the HER2+ cell lines tested. By contrast, synergy with mTOR inhibitors (everolimus and TAK228) and with the MEK inhibitor trametinib was only observed in BT-474 and HCC-1954 cells. HCC-1954 cells had the best response to neratinib combinations among all HER2+ cell lines tested, with combination index values <0.5. Immunoblot analysis demonstrated that neratinib treatment displayed complete inhibition of HER2 phosphorylation in these cells. Substantial inhibition of downstream signaling pathways following single agent neratinib treatment was evident by reduced phosphorylation levels of AKT, S6K1, S6, 4EBP1, ERK1/2 and MEK1/2. Consistently with the cell viability data, the combination of neratinib with PI3K/Akt/mTOR and MAPK inhibitors showed a synergy on signaling blockage in these two downstream pathways respectively. Taken together this data shows that neratinib has efficacy as single agent in preclinical models of HER2+ breast tumors and its anti-proliferative activity in vitro was enhanced when combined with PI3K/Akt/mTOR and MAPK pathway inhibitors. Further studies of neratinib-based combination treatments are underway in HER2+ breast cancer xenograft and PDX models to further corroborate these findings. Citation Format: Ming Zhao, Stephen Scott, Kurt Evans, Erkan Yuca, Rashmi Murthy, Francesca Avogadri-Connors, Richard Cutler, Alshad S. Lalani, Sarina Piha-Paul, Funda Meric-Bernstam. Exploring optimal targeted combination therapies with neratinib for HER2+ breast 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 4038. doi:10.1158/1538-7445.AM2017-4038

In Vitro Activity of Lactobacillus fermentum LF5 Against Different Candida Species and Gardnerella vaginalis

This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and Gardnerella vaginalis to weigh its potential effectiveness even in mixed vaginal infections. A wide female population is suffering from various vulvovaginal infections. These diseases are often associated with a decrease in the concentration of Lactobacilli in the vagina. Mixed vaginal infections represent >20% of women with vulvovaginal infection. LF5 strain was cocultured in De Man, Rogosa and Sharpe with Candida according to a 1:100 ratio in favor of the yeast. Each culture was sampled after 24 hours of incubation for the selective enumeration of the yeasts performed on yeast extract glucose chloramphenicol agar medium.The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants of L. fermentum LF5 ranging from 5% to 20% was quantified by means of optical density at 600 nm (OD600). L. fermentum LF5 demonstrated the ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms.Furthermore, L. fermentum LF5 showed a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth. The biotherapeutic LF5 could be the only documented strain effective in mixed forms. For this purpose, a human clinical trial is in progress.

Chemical Composition, Anticancer, Anti-neuroinflammatory, and Antioxidant Activities of the Essential Oil of Patrinia scabiosaefolia.

To study the chemical composition, anticancer, anti-neuroinflflammatory, and antioxidant activities of the essential oil of Patrinia scabiosaefolia (EO-PS). Patrinia scabiosaefolia was analyzed by gas chromatography-mass spectrometry. Eight human carcinoma cell lines, including SGC-7901, AGS, HepG2, HT-29, HCT-8, 5-FU/HCT-8, HeLa, and MDA-MB-231, were assessed by methylthiazolyldiphenyltetrazolium bromide (MTT) assay. Anti-neuroinflflammatory activity was assessed by production of interleukin (IL)-1β and IL-6 induced by lipopolysaccharide in BV-2 cells (microglia from mice). The antioxidant activity was evaluated with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. Forty-four components, representing 83.919% of the total oil, were identifified in the EO-PS. The major constituents were caryophyllene oxide (12.802%), caryophyllene (6.909%), α-caryophyllene (2.927%), β-damascenone (3.435%), calarene (5.621%), and phenol (3.044%). The MTT assay showed that the EO-PS exhibited significant dose-dependent growth inhibition in the 50-200 μg/mL dilution range. The EO-PS exhibited a dose-dependent scavenging activity against the DPPH radical, with an half of maximal inhibitory concentration 1.455 mg/mL. The EO-PS possesses a wide range of antitumor, anti-neuroinflflammatory and antioxidant activities, suggesting that it may be a good candidate for further investigations of new bioactive substances.

Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy

The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40%. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated β-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.

Abstract A39: 3,4',5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase

Abstract 3,4',5-trismethoxybenzophenone (TMBP) is an analogue of the natural dietary polyphenolic compound resveratrol (3,5,4'-trihydro-trans-stilbene, abbreviated as RVT). Natural RVT belongs to the phytoalexin class of phytochemicals that are antibiotics produced by plants in response to stimuli such as injury, ultraviolet irradiation, stress, and fungal attack. RVT and its analogues have also been shown to prevent hepatic steatosis. In this study we focused on TMBP which is a methylated derivative of RVT. Studies have shown that TMBP exhibits added biological effects and a better pharmacokinetic profile compared to RVT due to the presence of methoxy groups. The human hepatoma cell line (FCA4 cells) that harbors a subgenomic selectable HCV replicon was used for this study. The Huh7 cell line lacking the replicon served as the corresponding control. MTS assay was used to determine the cytotoxicity of TMBP at various concentrations. Wound healing assay was performed to determine the effect of the analogue on cell migration. Flow cytometry and magnetic levitation were used to measure the effect of compound on cell cycle and 3D spheroid formation. Our data showed that treating liver cancer cells with TMBP resulted in a significant dose and time dependent growth inhibition combined with G2/M-phase cell cycle arrest at 5 μM with (IC50) at 7.5 μM. TMBP at 5μM inhibited the formation of 3D spheroids, and also repressed the migration of cancer cells. Current therapies available for treating hepatocellular carcinoma (HCC) have a high incidence of recurrence and postoperative death in patients. Exploring different strategies with high pharmaceutical function and low toxicity is of great urgency for treating HCC. Our studies show that we have identified a promising analogue for treating HCC. Citation Format: Christopher J. Patton, Pritika Khadka, Hari Kotturi. 3,4',5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase. [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 A39.

P7170: A Novel Molecule with Unique Profile of mTORC1/C2 and Activin Receptor-like Kinase 1 Inhibition Leading to Antitumor and Antiangiogenic Activity.

The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFβ receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.

Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts.

Tagging of RNases, such as the ribotoxin α-sarcin, with the variable domains of antibodies directed to surface antigens that are selectively expressed on tumor cells endows cellular specificity to their cytotoxic action. A recombinant single-chain immunotoxin based on the ribotoxin α-sarcin (IMTXA33αS), produced in the generally regarded as safe (GRAS) yeast Pichia pastoris, has been recently described as a promising candidate for the treatment of colorectal cancer cells expressing the glycoprotein A33 (GPA33) antigen, due to its high specific and effective cytotoxic effect on in vitro assays against targeted cells. Here we report the in vivo antitumor effectiveness of this immunotoxin on nude mice bearing GPA33-positive human colon cancer xenografts. Two sets of independent assays were performed, including three experimental groups: control (PBS) and treatment with two different doses of immunotoxin (50 or 100 μg/ injection) (n = 8). Intraperitoneal administration of IMTXA33αS resulted in significant dose-dependent tumor growth inhibition. In addition, the remaining tumors excised from immunotoxin-treated mice showed absence of the GPA33 antigen and a clear inhibition of angiogenesis and proliferative capacity. No signs of immunotoxin-induced pathological changes were observed from specimens tissues. Overall these results show efficient and selective cytotoxic action on tumor xenografts, combined with the lack of severe side effects, suggesting that IMTXA33αS is a potential therapeutic agent against colorectal cancer.

Abstract 4562: Ginkgetin inhibits the growth of cancer cells via the cell cycle arrest at G2-phase

Abstract Ginkgetin, isolated from herbal medicines, has been reported to play a role in antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in cancer cells remains to be identified. In the present study, ginkgetin treatment of colon cancer cells resulted in significant dose-dependent growth inhibition together with apoptosis, G2-phase cell cycle arrest at a 5 μM (IC50) dose in HCT116colon cancer cells and ginkgetin also inhibits tumor growth in a mouse xenograft model of HCT116 cells. We found that ginkgetin regulated the expression of genes which are critically involved in cell cycle, cell signaling transduction, and cell proliferation using DNA microarray analysis. Furthermore, we provided evidence that ginkgetin inhibited BUB3, cyclin B, CDC2, and b-Myb expression. Especially ginkgetin induced G2-phase arrest by down-regulates expression of b-Myb Our data have suggested that ginkgetin exerts its anticancer effects through induction of cell cycle arrest at G2 phase as well as inhibition cell proliferation. Note: This abstract was not presented at the meeting. Citation Format: Byoung-Mog Kwon, Yu-Jin Lee. Ginkgetin inhibits the growth of cancer cells via the cell cycle arrest at G2-phase. [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 4562. doi:10.1158/1538-7445.AM2014-4562

Anti-neoplastic properties of hydralazine in prostate cancer.

Prostate cancer (PCa) is a major cause of cancer-related morbidity and mortality worldwide. Although early disease is often efficiently managed therapeutically, available options for advanced disease are mostly ineffective. Aberrant DNA methylation associated with gene-silencing of cancer-related genes is a common feature of PCa. Therefore, DNA methylation inhibitors might constitute an attractive alternative therapy. Herein, we evaluated the anti-cancer properties of hydralazine, a non-nucleoside DNA methyltransferases (DNMT) inhibitor, in PCa cell lines. In vitro assays showed that hydralazine exposure led to a significant dose and time dependent growth inhibition, increased apoptotic rate and decreased invasiveness. Furthermore, it also induced cell cycle arrest and DNA damage. These phenotypic effects were particularly prominent in DU145 cells. Following hydralazine exposure, decreased levels of DNMT1, DNMT3a and DNMT3b mRNA and DNMT1 protein were depicted. Moreover, a significant decrease in GSTP1, BCL2 and CCND2 promoter methylation levels, with concomitant transcript re-expression, was also observed. Interestingly, hydralazine restored androgen receptor expression, with upregulation of its target p21 in DU145 cell line. Protein array analysis suggested that blockage of EGF receptor signaling pathway is likely to be the main mechanism of hydralazine action in DU145 cells. Our data demonstrate that hydralazine attenuated the malignant phenotype of PCa cells, and might constitute a useful therapeutic tool.

MP39-14 METFORMIN IS A NEW THERAPEUTIC STRATEGY FOR TARGETING CD44 VARIANT IN BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Metformin is an oral biguanide drug widely used to treat type 2 diabetes mellitus and has recently been shown to have an antitumor effect through selectively targeting chemotherapy-resistant cancer stem cells, characterized by CD44+CD24phenotype. We focused particularly on variant isoforms of CD44 (CD44v) which have been recently reported as new markers for targeting cancer stem cells and evaluated whether metformin could have a therapeutic role for treating bladder cancer cells with a positive CD44v phenotype. METHODS: The efficacy of metformin alone and in combination with cisplatin was examined in T24 and HT1376 bladder cancer cell lines. WST assay was used to evaluate the cytotoxicity. The expressions of CD44v were evaluated by Western blotting analysis. The percentage of CD44v-positive cells in T24 cells treated with CDDP, metformin, or both were assessed by flow cytometry. A xenograft model was used to study the effects of metformin alone and in combination with cisplatin on bladder cancer tumor growth. Mice were divided into 4 groups; (1) no treatment, (2) metformin 200 ml/ml daily p.o., (3) cisplatin 2.5 mg/kg intraperitoneally for 1 day, or (4) combined metformin (200 ml/ml) and cisplatin (2.5 mg/kg). RESULTS: CD44v was positive in T24 cells, but not in HT1376 cells and significant dose-dependent growth inhibition by metformin was observed in T24 cells but not in HT1376 cells. Cell growth inhibition relative to vehicle control was significantly higher in the combination therapy (52.1%) compared to 1mM metformin alone (77.5%) or 1mM cisplatin alone (90.2%) in T24 cells. Flow cytometry showed that the percentage of CD44v-positive cells was 29.4%, 21.7%, 45.6%, and 42.6% in vehicle control, 1mM metformin, 1mM cisplatin, and the combination, respectively. The increased CD44v-positive cell population by cisplatin was reduced by metformin. In the T24 bladder cancer xenograft model, tumor volume at day 30 for the combination therapy was 467 75 mm3, which was significantly smaller than the vehicle control (1139 136 mm3), cisplatin monotherapy (1072 160 mm3), and metformin monotherapy (857 188 mm3) (p<0.001, p1⁄40.002 and p1⁄40.006, respectively). CONCLUSIONS: Metformin targeting CD44v-positive cells showed significant antitumor effects when given in combination with cisplatin for bladder cancer. Combined metformin and cisplatin therapy might be a novel therapeutic modality for treating bladder cancer.

Anti-Tumoral Effect of the Non-Nucleoside DNMT Inhibitor RG108 in Human Prostate Cancer Cells

Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA reexpression was only attained for GSTP1 and APC. RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.