Greater Inhibition Of Proliferation Research Articles

Biological Activity of Extracts from Differently Produced Blueberry Fruits in Inhibiting Proliferation and Inducing Apoptosis of HT-29 Cells.

For several decades, people have been searching for natural substances of plant origin that, when introduced into the diet, could strengthen immunity, have anticancer properties, and support conventional therapy. The development of agriculture with the implementation of various plant cultivation systems, apart from the economic aspect, results in the search for such cultivation conditions that would contribute to obtaining the most beneficial product for health. Therefore, the aim of our research is as follows: (a) to compare the antiproliferative activity and the ability to induce apoptosis of HT-29 cells by extracts from blueberry fruits deriving from different types of cultivation systems (conventional, organic, and biodynamic); (b) to examine whether the interaction of extracts with anticancer drugs used in the treatment of colorectal cancer is influenced by the type of cultivation, and (c) to investigate whether extracts obtained from fruits from subsequent years of cultivation retain the same biological activity. The results of our study are promising but inconclusive. A statistically significant difference occurred in only one of the two years of the study. The greatest inhibition of proliferation is observed for biodynamic cultivation compared to organic cultivation, while the highest levels of apoptosis and necrosis of HT-29 cells are induced by blueberry fruit extracts obtained from organic cultivation. The complementary effect of the extracts on the inhibition of HT-29 cell proliferation by anticancer drugs (5-FU and Erbitux) is not demonstrated. The induction of apoptosis by 5-FU is not enhanced by blueberry extracts, in contrast to necrosis. The level of apoptosis and necrosis induced by Erbitux is potentiated, but no dependence on crop type is shown. Blueberry fruit extracts from two consecutive years of cultivation did not maintain the same activity. A plausible reason for the variability in the composition and biological activity of fruit extracts obtained from two years of cultivation is the varying environmental conditions.

Abstract 4074: Reversing acquired PARPi resistance of TNBC through combined inhibition of cMet and EGFR

Abstract Front line therapeutic failure happened to half of triple-negative breast cancer (TNBC) patients represent the effective treatment strategies for these patients are urgently needed. Poly (ADP-ribose) polymerase (PARP) is currently the most promising drug target for BRCA-mutated TNBC, and PARP inhibitors (PARPi), olaparib and talazoparib, were recently approved for the treatment of metastatic breast cancer (including TNBC) in patients with germline BRCA1/2 mutations. Despite impressive response rates of ~60%, the prolongation in median progression-free survival with a PARPi is modest, suggesting the emergence of resistance. Several studies have reported that receptor tyrosine kinases (RTKs), such as c-MET (also known as hepatocyte growth factor receptor), are involved in resistance to various anti-neoplastic agents, including PARPi. However, the mechanism by which c-MET contributes to acquired resistance to PARPi in TNBC is not fully understood. In this study, we analyzed acquired PARPi resistant TNBC cells and found c-Met is hyperactivated in these cells with PARPi treatment. We further treated these cells with talazoparib and crizotinib (a multi-kinase inhibitor that inhibits c-MET) and found synergistic inhibition effects of cell proliferation. Unexpectedly, limited effects of talazoparib sensitivity showed while depleting c-MET in PARPi-resistant cells. Interestingly, we found epidermal growth factor receptor (EGFR) is also hyperactivated and interaction of EGFR/c-Met in these cells. Notably, combination of c-MET and EGFR inhibitors increased sensitivity to talazoparib in TNBC cells with acquired resistance to PARPi. Combining EGFR and PARP inhibitors also resulted in greater inhibition of proliferation in c-MET-depleted TNBC cells. Collectively, our findings suggested a potential combination therapy of inhibiting c-MET and EGFR to overcome acquired PARPi resistance in TNBC. Citation Format: Yu Yi Chu, Clinton Yam, Mei-Kuang Chen, Li-Chuan Chan, Lei Nie, Mien-Chie Hung. Reversing acquired PARPi resistance of TNBC through combined inhibition of cMet and EGFR [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 4074.

Abstract B60: Optimization of biologic scheduling of gemcitabine and abraxane improves treatment response compared to the standard concurrent regimen in preclinical models of pancreatic cancer

Abstract Introduction: Gemcitabine (gem) and Abraxane (albumin-bound paclitaxel, abx) combination chemotherapy delivered on the same day is now one of the preferred chemotherapy regimens for metastatic and locally advanced pancreatic ductal adenocarcinoma (PDAC) based on results of the phase III MPACT clinical trial that showed improved overall survival of gem+abx compared to gem alone in the metastatic/advanced setting. Our previous published works revealed that caveolin-1 (Cav-1), the principal structural component of caveolae and mediator of endocytosis, is highly expressed in PDAC cells and associated with enhanced tumor progression and resistance to gem in PDAC preclinical models. We also found that loss of Cav-1 through genetic knockdown inhibited both PDAC cell uptake of albumin and response to albumin-based chemotherapies, i.e., abx. In addition, gem treatment resulted in a time-dependent increase in Cav-1 protein expression. Taken together, we hypothesized pretreatment with gem would increase the efficacy of the gem/abx combination via increased Cav-1 dependent albumin-bound chemotherapy uptake. Results: ASPC1 and HPAFII PDAC cell lines were exposed to gem (25-100nM) for 24-48 hours followed by a 1-hour albumin pulse and displayed a time-dependent increase in intracellular albumin via Western blotting. We next tested PDAC cell proliferation and cell survival via alamarBlue and colony formation assays with various different gem/abx scheduling combinations. HPAFII and ASPC1 cells were treated for 24 hours with the following schedules: (1) vehicle, (2) gem alone, (3) abx alone, (4) gem+abx on same day (d1gem+abx), (5) gem on day 1 and abx day 2 (d1gem_d2abx), or (6) gem on day 1, no drug on day 2 and abx on day 3 (d1gem_d3abx). Both alternative schedules of d1gem_d2abx and d1gem_d3abx had greater inhibition of proliferation and colony formation compared to the current standard of care d1gem+abx, and of all the combinations, d1gem_d3abx was the most efficacious (p<0.01). Genetic knockdown of Cav-1 through short-hairpin loop RNA (shRNA) in Mia-Paca2 cells resulted in reversal of the observed altered scheduling effect on cell proliferation and survival. In vivo, we compared tumor growth rates with altering gem-abx scheduling following subcutaneous injection into athymic nude mice of ASPC1 or HPAFII cells. Similar to our in vitro studies, mice were treated with either the control-vehicle or three cycles delivered over 9 days of the following gem-abx combinations: 1) d1gem+abx, 2) d1gem_d2abx, or 3) d1gem_d3abx. The d1gem_d3abx scheduling had the most significant delay in tumor growth and tumor doubling time compared to the other gem-abx schedules (p<0.001). Conclusion: We found 24- to 48-hour gem treatment increased albumin uptake, leading to maximal treatment efficacy with an optimized schedule of gem delivered on day 1 and abx on day 3 compared to the standard gem+abx administered on the same day. These findings support further testing of an altered and biologically optimized scheduling of gemcitabine and Abraxane. Citation Format: Adam R. Wolfe, Ryan Robb, Ahmad Hegazi, Duan-Liang Shyu, Laith Abushahin, Terence M. Williams. Optimization of biologic scheduling of gemcitabine and abraxane improves treatment response compared to the standard concurrent regimen in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B60.

MP45-05 UROTHELIUM-SPECIFIC AND TEMPORALLY CONTROLLED BI-ALLELIC INACTIVATION OF BOTH PTEN AND P53 TRIGGERS BASAL-SUBTYPE MUSCLE-INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: PTEN is a tumor suppressor whose inhibitory activity towards phosphoinositide 3 kinase replies on membrane association, a process that is tightly regulated by electrostatic charges of PTEN tail domain. Precisely how and to what extent this domain affects PTEN’s activity remains unclear and is the subject of the present study. METHODS: Urothelial carcinoma cell lines without PTEN (UMUC3 and J82), or that expressing a mutated PTEN (T24), were transiently transfected with vector only, vector containing a full-length PTEN or vector containing a tailless PTEN, and their effects on cell proliferation, AKT activity, cell cycle and apoptosis were assessed under different culture conditions. To circumvent the extreme deleterious effects of the tailless PTEN, stable transfectants of UMUC3 were created that only expressed the tailless PTEN (or the full-length PTEN as a control) upon doxycycline exposure (e.g., stable transfection of CMV/rtTA and TRE-tailless PTEN (or TRE/full-length PTEN). The effects of their inducible expression were assessed in vitro and in xenograft mouse models. RESULTS: Introduction of full-length PTEN and tailless PTEN into PTEN-deficient UMUC3 and J82 cells inhibited proliferation at comparable levels in serum free and low serum (2%) media. However, in high serum (10%) medium, tailless PTEN was three times more inhibitory than full-length PTEN. This was due primarily to the phosphorylation of the serine/threonine clusters of the full-length PTEN’s tail domain, an effect absent in the tailless PTEN. Similar results were observed with transiently transfected T24 cells which expressed an endogenous mutant PTEN. UMUC3, J82 and T24 transfected with tailless PTEN could not sustain long-term culture because of its extreme deleterious effects, prompting us to establish doxycycline-inducible expression cell lines. UMUC3 inducibly expressing tailless PTEN exhibited significantly greater inhibition of proliferation, AKT phosphorylation, G1/S transition and significantly more apoptosis than UMUC3 inducibly expressing full-length PTEN. Finally, xenografted UMUC3 cells inducibly expressing full-length PTEN established tumors, albeit 30% smaller than the vector only controls. In contrast, those inducibly expressing tailless PTEN were devoid of any tumor in nude mice. CONCLUSIONS: Our in vitro and in vivo data are the first demonstration of the high potency of tailless PTEN as a tumor suppressor. They suggest that delivering tailless PTEN via viral vectors or other vehicles can be an effective approach to tackle advanced urothelial carcinomas with wild-type, mutated or deleted PTEN.

Abstract 2430: Treatment sequence and molecular biomarker in EGFR mutant lung cancer cells

Abstract Introduction Lung cancer is the leading cause of cancer related death in the US. A subset of patients with lung cancer has a mutation in the Epidermal Growth Factor Receptor (EGFR) which makes them extremely responsive to EGFR tyrosine kinase inhibitor (TKI) erlotinib. The emergence of EGFR TKI resistance is new challenge in the management of these patients. We conducted a study to determine the best treatment sequence after resistance to EGFR TKI, testing the use of chemotherapy Pemetrexed (P), Afatinib (A), Rociletinib (R) and the combination of these agents in lung cancer cell lines with EGFR mutation that were pretreated with erlotinib. Material and methods Lung cancer cell lines with EGFR mutation CRL-2868 (E746 - A750 deletion) and CRL-2871 (L747 - E749 deletion, A750P) mutation were treated with 5 nM erlotinib for 24h and 72 h. The erlotinib resistance cells were then treated with 10nM of A or 10nM of P, or 21.5 nM of R, or the combination AP or the combination of RP. As control, we used lung cancer HTB-177 which does not have EGFR mutation. MTT proliferation assay was performed after 24, 48, 72 and 144h after the treatment. microRNA profiling was done by real time PCR in all cells after erlotinib treatment to find potential biomarkers as hallmark of erlotinib resistance cells. Results Rociletinib induced the maximum inhibition of cell proliferation at 144h in both cell lines with EGFR mutation. The RP also induced decreased cell proliferation in both cell lines with EGFR mutation. RP combination was more effective then R alone in CRL 2871. The use of A alone, P alone or AP combination was not different in CRL2871 but P had antagonistic effect when combined with A or R in CRL 2868. microRNA 10b, 27a and 27b were upregulated in the erlotinib resistant cells. Conclusion. EGFR resistance arises after treatment with erlotinib in lung cancer cell lines with EGFR mutation. Treatment with R and the combination of RP induced greater inhibition of proliferation compared to A, P alone. The combination of AP was antagonistic. This may have implications in the management of patients resistant to EGFR TKI. microRNA analysis showed up-regulation of several microRNA that should be tested in clinical setting. Citation Format: Hannah Motes, Emma Borrego-Diaz Reyes, Jared Kevern, Chao H. Huang, Peter J. Van Veldhuizen. Treatment sequence and molecular biomarker in EGFR mutant lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2015-2430

IMiDs® Immunomodulatory Agents Regulate Interferon-Stimulated Genes through Cereblon-Mediated Aiolos Destruction in Multiple Myeloma (MM) Cells: Identification of a Novel Mechanism of Action and Pathway for Resistance

Numerous cellular effects have been attributed to IMiDs lenalidomide (LEN) and pomalidomide (POM) over the years. However, the zinc finger transcription factors Aiolos (IKZF3) and Ikaros (IKZF1) were identified only recently as substrates targeted for destruction in the presence of IMiDs compounds. LEN and POM bind directly to cereblon (CRBN), a substrate receptor of the cullin ring E3 ligase 4 (CRL4). Binding of these compounds to CRBN triggers the activation of CRL4CRBN, ubiquitylation and destruction of Aiolos and Ikaros.We systematically compared growth, gene expression, and signal transduction responses elicited by doxycycline-inducible, stable Aiolos knockdown or POM treatment in MM cell lines, and their POM-resistant counterparts. Upon exposure to POM, U266 MM cells undergo rapid degradation of Aiolos and Ikaros (90 min) and subsequent downregulation of IRF4 and Myc at 72 hr. At 72-96 hr, a decrease in proliferation and an increase in apoptosis are also observed. U266 MM cells selected in vitro for resistance to POM have lost CRBN expression and consequently no longer downregulate Aiolos and Ikaros after POM administration. Knockdown of Aiolos in these POM-resistant U266 MM cells is sufficient to inhibit their proliferative capacity by 50%, while Aiolos knockdown in POM-sensitive cells caused a greater inhibition of proliferation (90%). This suggests that MM cells with acquired resistance are still dependent on Aiolos for growth, but that a second mechanism may contribute to the antitumorigenic effect of Aiolos downregulation in POM-sensitive U266 cells.We have found that treatment with LEN or POM induces expression of antiviral response genes in MM cells. The induction of interferon (IFN)-stimulated genes (ISGs) such as DDX58, IFIT1, IFIT3, XAF1, ISG15, IFI44, and IFI27 are seen by qPCR in 8 hr of compound treatment (1.8- to 5-fold increase in transcript level) and this effect is further enhanced at 24 and 72 hr. Of note, this response is not accompanied by an increase in β IFN production. The IMiD compound-induced upregulation of the antiviral response correlates with CRL4-CRBN-mediated destruction of the lymphoid restricted transcription factor, Aiolos. In agreement with this, Aiolos knockdown by shRNA is sufficient to trigger a similar effect. These data suggest that Aiolos functions as a transcriptional repressor of ISGs, regulating the antiviral response. Consequently, Aiolos chromatin immunoprecipitation and sequencing (ChIP-Seq) experiments were performed, demonstrating that Aiolos binds near the transcription start site of numerous ISGs, including DDX58, IFIT1, ISG15, XAF1, IFI44, and IFI35. In addition, our data suggest that Aiolos co-binds with STAT and IRF family transcription factors and thereby co-regulates expression of these genes. STAT1 is part of the ISGF3 complex that drives ISG transcription upon viral infection. POM-resistant MM cells lacking CRBN expression do not have STAT1 activity and do not upregulate ISGs upon Aiolos knockdown, even though Myc and IRF4 are still being downregulated. In order to elucidate the relevance of the ISG expression in patients receiving IMiD treatment, we compared the gene expression profile of 12 patients after relapse or disease progression. Paired pre- and posttreatment samples from bone marrow-isolated CD-138 cells were evaluated with RNAseq and gene set enrichment analysis. We found an overall decrease in expression of ISGs, with significant negative enrichment of genes involved in IFN α, β, and γ signaling in relapsed patients. These data from clinical samples confirmed the importance and relevance of the ISGs in the response to IMiDs.In conclusion, our results indicate that Aiolos is a substrate of consequence in IMiD-sensitive MM cells, based on at least 2 pathways: driving the Myc-IRF4 feedback loop and repressing the antiviral pathway. Both in vitro and in vivo patient data suggest that one mechanism of IMiD resistance may be the abrogation of the STAT1 pathway resulting in subsequent blunting of the ISG induction. Finally, while upregulation of ISGs by IMiD treatment may serve as a relevant diagnostic marker of patient responsiveness to these drugs, these data highlight how response and resistance of the IMiD drugs are regulated by the interplay between complex pathway networks, suggesting that the measurement of only one component will not necessarily define the clinical course and outcomes for an individual patient. DisclosuresHavens:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Kang:Celgene Corp: Employment, Equity Ownership. Ortiz:Celgene Corp: Employment, Equity Ownership. Fontanillo:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corporation: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Bahlis:Celgene Corp: Honoraria, Research Funding. Thakurta:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Human Breast Tumor Cells Are More Resistant to Cardiac Glycoside Toxicity Than Non-Tumorigenic Breast Cells

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.

Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

BackgroundDocetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family. MethodsProstate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin. ResultsIn vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels. Transfection siRNA against survivin enhanced the cytotoxicity of docetaxel in PC3 cells. ConclusionThe rapamycin-dependent enhancement of the cytotoxic effects of docetaxel was associated with the downregulation of survivin expression. Our results suggest that the combination of docetaxel and rapamycin is a candidate for the improved treatment of advanced prostate cancer.

P1-12-03: Combined Targeting of the PI3K Pathway and HER2 Overcomes Acquired and De Novo Trastuzumab Resistance.

Abstract Background: Although trastuzumab and lapatinib provide clinical benefit for women with HER2−positive breast cancer, both de novo and acquired resistance to these agents exist. There is increasing evidence to suggest that aberrant activity of the PI3K/AKT/mTOR signaling pathway is one of the key mechanisms responsible for resistance. Thus, pharmacologically targeting the PI3K pathway is a rational approach for overcoming resistance to HER2−targeted therapy. However, specifically targeting individual elements of the PI3K pathway may result in feedback activation or in activation of compensatory pathways, whereas targeting the pathway at multiple points may be more effective in permanently shutting down proliferation and inducing cell death. Materials and Methods: In this study we evaluated the activity of three small molecule tyrosine kinase inhibitors that target specific critical portions of the PI3K pathway; BKM120 (pan-PI3K), RAD001 (mTORC1 specific) and BEZ235 (dual PI3K & mTOR) both individually and in combination with trastuzumab in a panel of HER2−amplified breast cancer cell lines that have previously been characterized for trastuzumab response/resistance. Results: In the trastuzumab sensitive BT-474 and SKBR3 cell lines, 72 hours of trastuzumab treatment efficiently inhibited cell proliferation and deactivated AKT (S473 & Th308), PS6K and ERK signaling. In contrast, in the trastuzumab-resistant MDA453 and SUM225 cell lines, as well as the BT-474 cells conditioned to acquire trastuzumab resistance (BT-TR), trastuzumab treatment did not inhibit pAKT, pS6K and pERK levels. RAD001 had significant antiproliferative activity in both trastuzumab sensitive and resistant cell lines and resulted in rapid down regulation of pPS6K (mTOR) activity (15 min). However, this deactivation was followed by a reactivation of AKT and ERK signaling (24 to 72 hours). BKM120 also demonstrated anti-proliferative activity independent of trastuzumab sensitivity. Specifically targeting PI3K with BKM120 directly decreased pAKT/pS6K/pERK signaling and feedback activation was less pronounced than that observed with RAD001. Of the three molecules tested, BEZ235 was the most efficacious in the cell line panel (all IC50gs < 20 nM). BEZ235 rapidly (15 min) decreased pAKT/pS6K/pERK signaling in both trastuzumab sensitive and resistant cells lines and this inhibition was maintained at 72 hours. Thus, BEZ235 is effective in shutting down PI3K feedback activation and efficiently shuts down PI3K signaling in trastuzumab resistant HER2−amplified breast cancer cell lines. The combined treatment of trastuzumab and each of the PI3K/mTOR targeting agents resulted in greater inhibition of proliferation and induction of cell death in the HER2−amplified cell lines than each agent used alone. No increase in activity was observed in the HER2−normal control cell lines, MCF-7 and KPL-1. Discussion: Together, these data indicate that targeting the PI3K/AKT/mTOR pathway either alone or in combination with trastuzumab is effective in overcoming trastuzumab resistance. These findings are currently being validated in in vivo models of trastuzumab resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-03.

RNAi Screening of the Human Kinome Identifies Inhibition of WEE1 Kinase As Potent and Universal Sensitizing Target to Cytarabine in Leukemias

Abstract 229 Introduction:Optimal rational combination targets with Cytarabine (AraC) have not been identified. After the development of a high-throughput, small-interfering RNA (siRNA) platform for suspension cells, we performed the first RNAi sensitizer screen of the human kinome with AraC in myeloid cell lines. Herein we present the final data of this first siRNA-sensitizer screen in leukemias in combination with AraC. Our results include full validation of the selected targets as well as ex vivo data assessing the first in class clinically tested WEE1 inhibitor MK1775 with AraC providing a pre-clinical rationale for using the combination of AraC with WEE1 inhibitors in leukemias. RNAi Screens and siRNA Validation:In siRNA sensitizer screens, we silenced 572 kinases of the human kinome in combination with AraC. Few kinases strongly sensitized to AraC and the targets CHEK1 and the WEE1 related kinase PKMYT1 emerged as the most potent genes whose silencing sensitized leukemia cells to AraC. In secondary siRNA validation experiments, silencing of WEE1 kinase was found to be more potent than silencing of CHEK1, PKMYT1 or other kinases (i.e. ATR, BRCA, AURKB) in combination with AraC treatment. Importantly, the sensitizing activity observed with WEE1 inhibition was universal across all AML cell lines examined, whereas it was not for CHEK1 inhibition. siRNA assays were robust, exhibiting little (<10%) non-specific toxicity and siRNA silencing was specific for individual genes (i.e. WEE1 or CHEK1) as demonstrated by selective reduction of mRNA and protein level for the respective genes; confirming the validity of the siRNA assay. Pharmacological WEE1 Inhibitor Experiments In vitro and Ex vivo:In vitro treatment of eight AML, ALL and CML cell lines with commercial and the first clinically developed WEE1 kinase inhibitor MK1775, demonstrated strong sensitization to AraC up to 97-fold in individual cell lines. Ex vivo, MK1775 substantially reduced viability in 13 of 14 AML, MDS and CML patient samples compared to AraC alone. The optimal dose to achieve maximum sensitization was observed for AraC between ∼10–120 nM and for MK1775 between 200–400 nM. Synergy at these optimal concentrations was confirmed in all ex vivo samples tested for which sufficient data for analysis with CalcuSyn Software was available (n=7): with Combination Indices (CI) ranging from 0.21–0.45. At higher AraC and MK1775 concentrations antagonism was observed both ex vivo and in vitro. Thus, we have also established optimal suggested concentration dose ranges for the currently ongoing design of a clinical trial.Clinically relevant, WEE1 is expressed in primary AML, ALL and advanced CML specimens with a trend towards higher expression in relapsed samples. However, there was no direct correlation with WEE1 mRNA expression or TP53 status. Our initial in vitro experiments further suggest that inhibition of WEE1 with MK1775 in combination with AraC at optimal concentrations was associated with higher degree of caspase 3 cleavage, DNA damage induction as measured by gamma-H2AX, and greatest inhibition of proliferation. Thereby establishing a context of optimal drug concentration ranges with molecular correlates and anti-leukemic activity directly informing design of the clinical trial and correlative biomarkers. In conclusion,data from this first siRNA sensitizer screen of the kinome in leukemias identifies inhibition of WEE1 kinase as the most potent sensitizing target to AraC. Inhibiting WEE1 with pharmacological inhibitors in combination with AraC is a rational treatment strategy for myeloid and lymphoid leukemias that warrants clinical investigation. Furthermore, our RNAi loss-of-function genomics approach has high potential to rapidly identify and validate crucial genes whose inhibition modulate the response to cytotoxic agents. Establishment of the siRNA sensitizer platform allows accelerating the development of new rational combinations for leukemia therapy. Disclosures:Off Label Use: AraC in combination with the experimental Wee1 inhibitor MK1775. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding. Demuth:Merck and CO: Employment.

734 EAST MEETS WEST: THE MUSHROOM EXTRACT PSK ENHANCES DOCETAXEL THERAPY FOR CASTRATE RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011734 EAST MEETS WEST: THE MUSHROOM EXTRACT PSK ENHANCES DOCETAXEL THERAPY FOR CASTRATE RESISTANT PROSTATE CANCER Joel Slaton, Michael Verneris, Haling Lu, and Cynthia Wenner Joel SlatonJoel Slaton Minneapolis, MN More articles by this author , Michael VernerisMichael Verneris Minneapolis, MN More articles by this author , Haling LuHaling Lu Seattle, WA More articles by this author , and Cynthia WennerCynthia Wenner Kenmore, WA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1703AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Polysaccharide Krestin (PSK), an extract of the mushroom Trametes versicolor and a known immunomodulator, has proven to enhance survival in randomized clinical trials in Asia.when combined with chemotherapy in patients with breast, colon and stomach cancer. The role of PSK in prostate cancer has not previously been investigated due to the lack of effective chemotherapy. However, with the demonstration of improved survival in patients with castrate resistant prostate cancer (CPRC) with the use of docetaxel, there is now an opportunity to investigate the effect of adding PSK to docetaxel in CRPC. METHODS C57BL/6 mice bearing established orthotopic CRPC PC3-LN4 tumors (100 mg) were randomized to four groups for treatment with a) saline b) docetaxel 5mg/kg ip every other day, c) PSk 300 mg/kg po and d) docetaxel plus PSK Mice were treated were 12 days and then sacrificed the following day 14. Mice were weighed daily. At time of sacrifice, blood and organs were collected for evidence of toxicity. Tumors and lymph nodes were weighed and preserved in either nitrogen or formalin and analyzed for immunomodulatory changes. Spleens were assessed for NK cell activity. RESULTS A combination of docetaxel and PSK resulted in the greatest tumor inhibition (399+62 mg) compared to saline ((826 +82 mg), PSK (780+75 mg), and docetaxel (528+ 62 mg) alone. Combination therapy resulted in the highest level of apoptosis (determined by TUNEL) and greatest inhibition of proliferation (determined by Ki67). Simultaneously, combination therapy resulted in a 25% higher total white count and a 10% higher animal weight. Treatment with PSK, a known inhibitor of toll-like receptor 4 (TLR4) resulted in a complete inhibition of expression of interferon gamma within the tumors and upregulation of T-regulatory cell. NK cell activity and CD4 count was higher in the mice treated with combination therapy compared to controls and docetaxel treated group. CONCLUSIONS Combination therapy with docetaxel and the mushroom extract PSK has superior antitumor and immunoprotective activity compared to docetaxel alone. This data served as the basis for a recently NIH-funded Phase I clinical trial combining PSK and docetaxel in CPRC patients which will soon be open for accrual. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e295 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joel Slaton Minneapolis, MN More articles by this author Michael Verneris Minneapolis, MN More articles by this author Haling Lu Seattle, WA More articles by this author Cynthia Wenner Kenmore, WA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lactoferrin inhibits the growth of nasal polyp fibroblasts

The aim of this study was to evaluate the effects of lactoferrin (LF) on the growth of fibroblasts derived from nasal polyps. We showed that the proliferation of fibroblasts was inhibited in a dose-dependent manner by both native and recombinant LF. The greatest inhibition of proliferation was caused by human milk-derived, iron-saturated LF. The inhibition of fibroblast proliferation was not species specific because bovine LF also was active. The interaction between LFs and a putative cell receptor did not depend on the sugar composition of the glycan moiety of the LF molecule because lactoferrins of different origins were active and the addition of monosaccharides to the cultures did not block proliferation. However, the treatment of fibroblasts with sodium chlorate (an inhibitor of glycosaminoglycan sulfation) or the addition of heparin abolished the inhibitory effect of LF, suggesting that LF binds heparan sulfate-containing proteoglycans. The significance of LF in nasal excretions in controlling polyp formation is discussed.

Abstract 4070: Mutant tristetraprolin enhances growth factor downregulation and inhibition of cell growth in malignant glioma

Abstract Background: Tristetraprolin (TTP) negatively regulates short-lived mRNAs by binding to AU-rich elements in the 3′ untranslated region (3′UTR). A number of mRNA targets, including vascular endothelial growth factor (VEGF), and interleukin (IL)-8, play an important role in tumor progression. We have shown previously that TTP downregulates IL-8 and VEGF in malignant glioma (MG) cells via RNA destabilization. In primary glioblastoma tumors, TTP is heavily phosphorylated which may render the protein less active. Hypothesis: We postulate that the destabilizing effect of TTP on target growth factor mRNAs is held in check by hyperphosphorylation which leads to altered subcellular localization and protein stability. Methods: Wild-type TTP and two mutant forms in which select serines were exchanged with alanines were cloned with a Flag epitope into pTRE2 and pEGFP vectors. U251 Tet On MG cells were transfected with pTRE2–TTP plasmids to produce stable clones. TTP expression was induced using doxycycline. Cellular location was assessed by fluorescence microscopy and Western blotting. Protein stability was measured by assessment of TTP levels following cycloheximide treatment. VEGF and IL-8 mRNA and protein levels were measured by qRT-PCR and ELISA. For assessment of RNA half-lives, cells were treated with actinomycin D. Cell growth, proliferation and cell viability were assessed using biochemical assays, soft agar, and Trypan Blue method. Results: Both IL-8 and VEGF mRNA levels were significantly lowered in the mutant TTP clones compared to wild-type. Half-lives of these mRNAs were also shortened, and ELISA analysis showed a correspondingly greater decrease in protein expression. There was a marked impact on cellular phenotype with greater inhibition of proliferation and loss of cell viability. A Soft agar colony formation was decreased by five-fold in mutant clones versus wild-type and a greater than ten-fold decrease versus the parent cell line. With TNF-α, there was a significant shift of mutant TTP to the nuclear compartment compared to WT TTP. Treatment with cycloheximide revealed that the WT TTP was less stable compared to dephosphorylated mutant. Conclusion: Removal of key serine residues from TTP enhances its function as an RNA destabilizer of VEGF and IL-8 mRNs and as a negative regulator of tumor cell growth in MG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4070.

The Green Tea Polyphenol EGCG Potentiates the Antiproliferative Activity of c-Met and Epidermal Growth Factor Receptor Inhibitors in Non–small Cell Lung Cancer Cells

Activation of the c-Met and epidermal growth factor receptors (EGFR) promotes the growth and survival of non-small cell lung cancer (NSCLC). Specific receptor antagonists have shown efficacy in the clinic, but tumors often become resistant to these therapies. We investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to inhibit cell proliferation, and c-Met receptor and EGFR kinase activation in several NSCLC cell lines. NSCLC cell lines with variable sensitivity to the EGFR antagonist erlotinib were studied. Cell growth was evaluated using proliferation and colony formation assays. Kinase activation was assessed via Western blot analysis. Experiments were conducted with EGCG, the EGFR antagonist erlotinib, and the c-Met inhibitor SU11274. The antagonists were also tested in a xenograft model using SCID mice. EGCG inhibited cell proliferation in erlotinib-sensitive and -resistant cell lines, including those with c-Met overexpression, and acquired resistance to erlotinib. The combination of erlotinib and EGCG resulted in greater inhibition of cell proliferation and colony formation than either agent alone. EGCG also completely inhibited ligand-induced c-Met phosphorylation and partially inhibited EGFR phosphorylation. The triple combination of EGCG/erlotinib/SU11274 resulted in a greater inhibition of proliferation than EGCG with erlotinib. Finally, the combination of EGCG and erlotinib significantly slowed the growth rate of H460 xenografts. EGCG is a potent inhibitor of cell proliferation, independent of EGFR inhibition, in several NSCLC cell lines, including those resistant to both EGFR kinase inhibitors and those overexpressing c-Met. Therefore, EGCG might be a useful agent to study as an adjunct to other anticancer agents.

The Effect of Doxorubicin on MEK-ERK Signaling Predicts Its Efficacy in HCC

Hepatocellular cancer (HCC) is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) has been widely used against unresectable HCC with variable response rates. We hypothesized that DOX combined with mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) targeted therapy may provide enhanced anti-cancer effects. Human HCC cell lines (HepG2, Hep3B) were treated with DOX and MEK enzyme inhibitors, U0126 or PD184161, alone or in combination. Growth, apoptosis, and ERK expression/MEK activity were respectively determined by proliferation assay, DNA fragmentation enzyme-linked immunoassay or fluorochrome inhibitor of caspases, and Western blot. DOX (0.01-1 microM) decreased cell proliferation in Hep3B cells (IC(50) approximately 0.12 microM) at 48 to 72 h; DOX was less effective in HepG2 cells (IC(50) approximately 0.25 microM). At early time points (30 min) after DOX treatment of Hep3B cells, MEK activity was unchanged at low doses and decreased at higher doses; after 24 h, phospho-ERK levels increased at higher doses. Contrarily, in HepG2 cells, DOX caused a sustained, dose-dependent increase in phospho-ERK levels at early and late time points. The MEK inhibitor U0126 decreased phospho-ERK in both HCC lines. In contrast to DOX, HepG2 cells were more sensitive than Hep3B cells to U0126. The combination of DOX with U0126 (or PD184161) resulted in greater inhibition of proliferation in HepG2 but not in Hep3B cells. This effect may be mediated in part by enhanced apoptosis. The effect of DOX on early and late induction of MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.

Inhibition of Vitamin D 3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells

Induction of growth arrest and differentiation by 1α,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH) 2D 3. This appears to be due to increased 1,25-(OH) 2D 3-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2 H-naphthalen-1-one ( 4) was found to be a potent inhibitor of Vitamin D 3 metabolising enzymes (IC 50 3.5 μM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC 50 20 μM). The combination of the inhibitor and 1,25-(OH) 2D 3 caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH) 2D 3 plus inhibitor of Vitamin D 3 metabolising enzymes co-ordinately upregulated CYP24, p21 waf1/cip1 and GADD45α.

Proteasome Inhibitor Combination with Cytarabine or Arsenic Trioxide Can Potentiate Apoptosis in AML Cells.

Proteasome inhibitors block degradation of the NF-κB regulator, IκB, resulting in inhibition of NF-κB nuclear localization. Proteasome inhibitors have been tested in the setting of refractory acute leukemia, with proteasome inhibition demonstrated within leukemic blasts. Arsenic trioxide (AsO3) has also been shown to inhibit NF-κB in leukemia. Since NF-κB is constitutively activated in primitive AML cells and serves as a regulator of many genes which encode proliferative and survival responses, we have begun to explore the effects of AsO3 and bortezomib (PS-341, VelcadeTM ), on AML cell lines and primary cells. The farnesyl transferase inhibitors (FTIs) also inhibit AML proliferation, and their effects in combination with bortezomib have also been explored. Because FTIs may inhibit signal transduction pathways independent of those affected by NF-κB inhibition, and because FTIs may indirectly inhibit NF-κB function via Akt inhibition, the effects of combining these agents with other NF-κB agents on AML cells in vitro have also been explored. Bortezomib, at concentrations of 4nM or greater, inhibited NF-κB in AML cell lines and primary cells as did AsO3 at concentrations of 1 nM or greater. NF-kappa B was measured by ELISA for p65 NF-κB activity. The nonpeptidomimetic FTI, R115777 (J&J), did not inhibit NF-κB at concentrations up to 100 nM, concentrations which effectively inhibit farnesylation of target proteins, whereas the FTI, L-744832 (Merck), was able to inhibit NF-κB expression at 1 μM from 24 to 72 hours of exposure. In the HL60 cell line, inhibited by FTI and bortezomib independently, the combination did not appear to have additive or synergistic effects. Furthermore, the effects of combined exposure to FTI and bortezomib on expression of activated caspase 3 or activated PARP cleavage were no greater than with bortezomib alone. Likewise, combination effects on expression of phosphorylated AKT or ERK were not observed. In contrast, the combination of bortezomib and AsO3 resulted in decreased phospho-ERK expression and increased expression of cleaved PARP, suggesting increased apoptosis. When cytarabine, 100 nM was combined with bortezomib at 1 to 4 nM, no effect on timing of administration was noted, and apoptosis was increased with the combination as evidenced by an increase in cleaved PARP expression. Greater inhibition of proliferation was seen with this combination than with individual agents as demonstrated in MTT assays with combination index calculations suggesting synergism. With this combination, co-culture with an endothelial monolayer did not prevent the increased apoptosis noted with combined cytarabine and bortezomib. These studies suggest that future studies combining proteasome inhibition with standard chemotherapeutic agents or with other inhibitors of NF-κB like Arsenic trioxide may have greater antileukemic activity by inducing apoptosis in AML cells in vivo as well as in vitro, without obvious limitations of other targeted agents and drugs that are extruded by multridrug resistance transporters.

Doxorubicin’s effect on MEK activity predicts its chemotherapeutic response in hepatocellular carcinoma

Abstract Introduction: HCC is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) was widely used against unresectable HCC with variable response rates. Methods: We hypothesized that doxorubicin combined with MEK-ERK targeted therapy may provide enhanced anti-tumor effects. Three HCC lines (HepG2, Hep3B, and PLC) were treated with DOX (100–1000nM) and a MEK enzyme inhibitor, U0126 (1–5mcM), alone or in combination. Growth, apoptosis, and ERK expression/ MEK activity were respectively determined by MTS assay and trypan blue excluded counts, DNA fragmentation ELISA and FLICA, and immunoblot. Results: DOX treatment in Hep3B and PLCs effectively decreased MEK activity as evidenced by decreased phospho-ERK (1–48hrs). Contrarily, in HepG2s, DOX increased phospho-ERK. ERK expression remained unchanged with DOX. DOX dose-dependently decreased cell proliferation in Hep3Bs (IC50 = 100nM) and PLCs (IC50 = 300nM) at 48–72 hrs. Up to 5X higher doses resulted in equivalent decreases in HepG2s. U0126 decreased phospho-ERK in all three HCC lines. In contrast to DOX, the HepG2s were most sensitive to U0126. The combination of DOX with U0126 in HepG2s resulted in 34% greater inhibition of proliferation and a synergistic increase in apoptosis. DOX upregulated the expression of Bcl-xL in HepG2s, an effect blocked with U0126. Conversely, combination treatment in Hep3B and PLC cells resulted in protection against cell death. Conclusions: DOX's effect on MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.

Activin A regulates growth and acute phase proteins in the human liver cell line, HepG2

Activin, and its binding protein, follistatin, are up-regulated by mediators of inflammation, and recent studies have demonstrated that activin A can block the activity of the key inflammatory cytokine, interleukin-6 (IL-6). These findings thereby implicate activin and follistatin in the control of the inflammatory cascade. In this study, interactions between interleukin-1 β (IL-1 β), IL-6 and activin were examined the human liver cell line, HepG2, for their effect on cell proliferation and the production of the acute phase proteins, haptoglobin and α1-acid glycoprotein ( α1-AGP). IL-1 β and activin A, but not IL-6, inhibited the proliferation of HepG2 cells. Activin A together with IL-1 β caused a greater inhibition of proliferation than either factor alone, and the inhibitory effects of activin A were blocked by the addition of follistatin to the cultures. Activin A alone inhibited the production of haptoglobin but did not affect α1-AGP concentrations. However, activin A suppressed the stimulatory effects of IL-6 on the production of both haptoglobin and α1-AGP. Production of follistatin by HepG2 cells was stimulated by activin A, but was inhibited by both IL-1 β and IL-6, indicating a complex regulatory loop is operable to modulate the effects of activin A during inflammation. Taken together, these data suggest that activin A interacts with IL-1 β and IL-6 to regulate and coordinate the production of acute phase proteins during an inflammatory episode.

Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities.