Synergistic Inhibition Of Cell Proliferation Research Articles

Abstract 4231: Enhanced antitumor efficacy of T-DM1 in combination with pertuzumab in HER2-positive breast cancer models

Abstract Background: The HER2 oncogene is amplified in approximately 20-25% of human breast cancers and is a well established therapeutic target. HER2-targeted therapy by trastuzumab (T) has become increasingly important for treating HER2+ breast cancers, and following the EMILIA trial, T-DM1 is expected to serve as a suitable alternative to trastuzumab. Pertuzumab (P), a HER2 targeted monoclonal antibody that prevents HER2 dimerization, showed progression-free survival when used with trastuzumab and docetaxel on HER2+ metastatic breast cancer (CLEOPATRA trial). In this preclinical study, we investigated the effect of combining T-DM1 and P on xenografted HER2+ breast tumors. Methodology: Growth inhibition, apoptosis, and antitumor efficacy were examined using the 3D-ON-TOP assay, annexinV staining and mouse xenografts respectively, in BT474 (T- sensitive), BT474HR (T-resistant) and HCC1954 (PIK3CA mutated) breast cancer cell lines. Results: Treatment of HER2-overexpressing tumor cells (T-sensitive, T-resistant and PIK3CA mutant) in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation (3D-ON-TOP and 2D clonogenic assays) and induction of apoptotic cell death (annexinV positive cells). The combination (T-DM1+P) also inhibited p-AKT (Ser473 and Thr308) in HER2-overexpressing cells. T-sensitive and T-resistant xenografts regressed completely after 6 weeks of therapy with T-DM1 plus P whereas either single agent inhibited tumor growth partially in both groups. Mice with HCC1954 (HER2+/PIK3CA mutated) xenograft treated with the same combination exhibited significant tumor growth inhibition when compared with vehicle treated mice after 6 weeks of treatment. Conclusions: Our in vitro and in vivo data clearly demonstrated that dual-targeting HER2 with a combination of T-DM1 plus P was highly efficacious both in HER2+/T-resistant and HER2+/PIK3CA mutated xenograft models. Citation Format: Yuliang Sun, Nandini Dey, Jennifer Carlson, Melissa Brammer, Pradip De, Brian Leyland-Jones. Enhanced antitumor efficacy of T-DM1 in combination with pertuzumab in HER2-positive breast cancer models. [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 4231. doi:10.1158/1538-7445.AM2014-4231

The Akt inhibitor MK-2206 enhances the cytotoxicity of paclitaxel (Taxol) and cisplatin in ovarian cancer cells.

Abnormalities in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway are commonly observed in human cancers and contribute to chemotherapy resistance. Combination therapy, involving the use of molecular targeted agents and traditional cytotoxic drugs, may represent a promising strategy to lower resistance and enhance cytotoxicity. Here, we demonstrate the efficacy of an Akt inhibitor, MK-2206, in increasing the cytotoxic effect of either paclitaxel (Taxol) or cisplatin against the ovarian cancer cell lines SKOV3 (with constitutively active Akt) and ES2 (with inactive Akt). Sequential treatment of Taxol or cisplatin, followed by MK-2206, induced a synergistic inhibition of cell proliferation and effectively promoted cell death, either by inhibiting the phosphorylation of Akt and its downstream effectors 4E-BP1 and p70S6K in SKOV3 cells or by restoring p53 levels, which were downregulated after Taxol or cisplatin treatment, in ES2 cells. Combination treatment also downregulated the pro-survival protein Bcl-2 in both SKOV3 and ES2 cells, which may have contributed to cell death. In addition, we discovered that Taxol/MK-2206 or cisplatin/MK-2206 combination treatment resulted in significant enhancement of intracellular reactive oxygen species (ROS)induced by MK-2206, in both SKOV3 and ES2 cells; however, MK-2206-induced growth inhibition was reversed by a ROS scavenger only in ES2 cells. MK-2206 also suppressed DNA repair, particularly in SKOV3 cells. Taken together, our results demonstrate that the Akt inhibitor MK-2206 enhances the efficacy of cytotoxic agents in both Akt-active and Akt-inactive ovarian cancer cells but through different mechanisms.

Gamma-tocotrienol and hydroxy-chavicol synergistically inhibits growth and induces apoptosis of human glioma cells.

BackgroundGamma-tocotrienol (GTT), an isomer of vitamin E and hydroxy-chavicol (HC), a major bioactive compound in Piper betle, has been reported to possess anti-carcinogenic properties by modulating different cellular signaling events. One possible strategy to overcome multi-drug resistance and high toxic doses of treatment is by applying combinational therapy especially using natural bioactives in cancer treatment.MethodsIn this study, we investigated the interaction of GTT and HC and its mode of cell death on glioma cell lines. GTT or HC alone and in combination were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) by [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)- 2H- tetrazolium, inner salt] MTS assay. The interactions of each combination were evaluated by using the combination index (CI) obtained from an isobologram.ResultsIndividually, GTT or HC displayed mild growth inhibitory effects against glioma cancer cell lines at concentration values ranging from 42–100 μg/ml and 75–119 μg/ml respectively. However, the combination of sub-lethal doses of GTT + HC dramatically enhanced the inhibition of glioma cancer cell proliferation and exhibited a strong synergistic effect on 1321N1 with CI of 0.55, and CI = 0.54 for SW1783. While in LN18 cells, moderate synergistic interaction of GTT + HC was observed with CI value of 0.73. Exposure of grade II, III and IV cells to combined treatments for 24 hours led to increased apoptosis as determined by annexin-V FITC/PI staining and caspase-3 apoptosis assay, showing caspase-3 activation of 27%, 7.1% and 79% respectively.ConclusionIn conclusion, combined treatments with sub-effective doses of GTT and HC resulted in synergistic inhibition of cell proliferation through the induction of apoptosis of human glioma cells in vitro.

The Phosphoinositide 3-Kinase α Selective Inhibitor BYL719 Enhances the Effect of the Protein Kinase C Inhibitor AEB071 in GNAQ/GNA11-Mutant Uveal Melanoma Cells

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

Targeting HER2 with multiple HER2-directed therapies represents a promising area of treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody-drug conjugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta). Drug combination studies with T-DM1 and pertuzumab were performed on cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3+3 phase Ib/II study design. Treatment of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3 ligand, heregulin (NRG-1β), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Results from mouse xenograft models showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity. Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture and mouse xenograft models resulted in enhanced antitumor activity. In patients, this combination showed an encouraging safety and tolerability profile with preliminary evidence of efficacy.

Epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A inhibits the growth of cholangiocarcinoma cells

Enhancer of zeste homolog 2 (EZH2) is involved in malignant transformation and the biological aggressiveness of several human malignancies. Growing evidence indicates that EZH2 may be an appropriate therapeutic target for malignancies, including cholangiocarcinoma. Recently, an S-adenosyl-L-homocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep) was shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep and the combination of gemcitabine and DZNep in cholangiocarcinoma cells. The effects of DZNep and its combination with gemcitabine were assessed in the cholangiocarcinoma cell lines RBE and TFK-1. DZNep depleted the cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. DZNep treatment resulted in the inhibition of proliferation in the cholangiocarcinoma cell lines, and the combination of DZNep and gemcitabine showed synergistic inhibition of cell proliferation. DZNep induced apoptosis and G1 phase cell cycle arrest in cholangiocarcinoma cells, and the combination of DZNep and gemcitabine enhanced the induced apoptosis and G1 arrest when compared with gemcitabine alone. Inhibition of cell proliferation by DZNep was partially associated with upregulation of p16INK4a and p17KIP1. The present study shows that DZNep inhibits cell proliferation by inducing G1 arrest and apoptosis. These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach for the treatment of cholangiocarcinoma.

The effect of resveratrol in combination with irradiation and chemotherapy

Merkel cell carcinoma (MCC) is a rare, but highly malignant tumor of the skin. In case of systemic disease, possible therapeutic options include irradiation or chemotherapy. The aim of this study was to evaluate whether the flavonoid resveratrol enhances the effect of radiotherapy or chemotherapy in MCC cell lines. The two MCC cell lines MCC13 and MCC26 were treated with increasing doses of resveratrol. Combination experiments were conducted with cisplatin and etoposide. Colony forming assays were performed after sequential irradiation with 1, 2, 3, 4, 6, and 8 Gy and apoptosis was assessed with flow cytometry. Expression of cancer drug targets was analyzed by real-time PCR array. Resveratrol is cytotoxic in MCC cell lines. Cell growth is inhibited by induction of apoptosis. The combination with cisplatin and etoposide resulted in a partially synergistic inhibition of cell proliferation. Resveratrol and irradiation led to a synergistic reduction in colony formation compared to irradiation alone. Evaluation of gene expression did not show significant difference between the cell lines. Due to its radiosensitizing effect, resveratrol seems to be a promising agent in combination with radiation therapy. The amount of chemosensitizing depends on the cell lines tested.

Synergistic Targeting of PI3K/AKT Pathway and Androgen Receptor Axis Significantly Delays Castration-Resistant Prostate Cancer ProgressionIn Vivo

The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC.

Targeting focal adhesion kinase in ER+/HER2+ breast cancer improves trastuzumab response

The HER2 transmembrane receptor is a well-characterised predictive marker for trastuzumab benefit and may be associated with decreased benefit from endocrine therapy use. Despite the clinical effectiveness of anti-HER2 agents in such cases, resistance represents a significant limiting factor. Focal adhesion kinase (FAK) plays an important role in HER2 signalling, mediating downstream Akt activation in addition to HER2 cross talk with other growth factor receptors. In this study, we investigated the therapeutic potential of FAK in oestrogen receptor-positive (ER+)/HER2+ breast cancer using the novel FAK-specific inhibitor PF4554878 ('PF878'). The activation of the FAK/HER2 signalling pathway was assessed in ER+/HER2- (MCF7 and T47D) and ER+/HER2+ (BT-474 and MDAMB361) breast cancer cells in the presence or absence of PF878 and PF878±trastuzumab. The effects of PF878 on cell growth as a monotherapy and in combination with trastuzumab were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Coulter counting with isobologram analysis to determine synergy/additive effects. FAK activation (at Y861 but not at Y397) was highest in ER+/HER2+ cells, which also demonstrated the greatest sensitivity to PF878. As a monotherapy, PF878 prevented heregulin-induced MDA361 cell migration, but had no significant effect on cell growth. The treatment of ER+/HER2+ cells with PF878 and trastuzumab in combination resulted in the synergistic inhibition of cell proliferation. Underlying this was an abrogation of Akt activity and increased poly(ADP-ribose) polymerase cleavage, effects that were greatest in trastuzumab-refractory MDA361 cells. Collectively, these data support a role for FAK in ER+/HER2+ breast cancer, where its targeting has the potential to improve trastuzumab response. This is particularly important in the context of ER+/HER2+, trastuzumab-refractory disease, where FAK inhibition may present an important strategy to restore trastuzumab sensitivity.

Abstract 3281: Combination therapy to target KIT mutant cells.

Abstract Purpose: Gastrointestinal stromal tumors (GIST), the most common abdominal sarcoma, arise from the interstitial cells of Cajal in the wall of the gut. Eighty percent of GISTs harbor activating mutations in the receptor tyrosine kinase KIT. Despite progress in medical treatment of GIST, advanced disease remains incurable. In addition to GIST, activating KIT mutations are found in cases of mast cell neoplasms, AML, melanoma and seminoma. Therefore, there is a compelling need to identify new targets whose inhibition is synergistic when combined with biochemical inhibition of KIT. Based on shared oncogenic kinase mechanisms with CML, we hypothesized that combined inhibition of KIT and a novel Wnt/Ca++/Calcineurin/NFAT pathway would synergistically inhibit the proliferation of KIT mutant cells. Experimental methods: We sequentially combined four different calcineurin inhibitors with five different KIT inhibitors and measured cell proliferation and caspase 3/7activity in nine unique KIT mutant cell lines. Using the Chou and Talalay method we calculated combination indices to quantify drug-drug interactions. Changes in NFAT phosphorylation and localization in KIT mutant cells were assessed by immunoblotting. Finally, we measured NFAT transcriptional activity using an NFAT-responsive reporter cell line. Results: In contrast to other cellular models, NFAT appeared to be constitutively active and localized in the nucleus in many KIT mutant cell lines. We observed synergy when we combined a calcineurin inhibitor with a KIT inhibitor in all KIT mutant cell lines where NFAT was constitutively active. Combination index (CI) values for the different KIT mutant cell lines ranged from 0.3-0.5. To investigate whether NFAT was required for this synergy, we combined NFAT specific inhibitors with KIT inhibitors and found synergistic inhibition of cell proliferation. We created a KIT-mutant reporter cell line where NFAT binding drives the expression of luciferase. As expected, NFAT transcriptional activity decreased when our promoter-reporter cell line was treated with calcineurin inhibitors and increased with TPA and ionomycin treatment. Unexpectedly, we also found that KIT inhibitors decreased NFAT transcriptional activity in this cell model. Conclusions: These data indicate that combination therapy using a calcineurin phosphatase inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant cells. Additionally, the constitutive activation of NFAT in these cell lines indicate aberrant signaling in the Wnt/Ca2+/calcineurin/NFAT pathway, which has not been previously reported in KIT mutant cell lines. The observed synergy between NFAT inhibitors and KIT inhibitors suggests this effect is mediated by NFAT rather than other calcineurin targets. Finally, the fact that KIT inhibitors modulated NFAT transcriptional activity suggests crosstalk between KIT and NFAT signaling pathways. Citation Format: Alison C. Macleod, Michael C. Heinrich, Diana Griffith, Ajia Town. Combination therapy to target KIT mutant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3281. doi:10.1158/1538-7445.AM2013-3281

Synergistic Effects of Exemestane and Aspirin on MCF-7 Human Breast Cancer Cells

The purpose of this study is to investigate the combined effects of exemestane and aspirin on MCF-7 human breast cancer cells. Antiproliferative effects of exemestane and aspirin, alone and in combination, on growth of MCF-7 human breast cancer cells were assessed using the MTT assay. Synergistic interaction between the two drugs was evaluated in vitro using the combination index (CI) method. The cell cycle distribution was analyzed by flow cytometry and Western blotting was used to investigate the expression of cyclooxygenase-1, cyclooxygenase-2 and Bcl-2. MTT assays indicated that combination treatment obviously decreased the viability of MCF-7 human breast cancer cells compared to individual drug treatment (CI<1). In addition, the combination of exemestane and aspirin exhibited a synergistic inhibition of cell proliferation, significantly arrested the cell cycle in the G0/G1 phase and produced a stronger inhibitory effect on COX-1 and Bcl-2 expression than control or individual drug treatment. These results indicate that the combination of exemestane and aspirin might become a useful method to the treatment of hormone- dependent breast cancer. The combination of the two inhibitors significantly increased the response as compared to single agent treatment, suggesting that combination treatment could become a highly effective approach for breast cancer.

Abstract LB-388: Combination treatment of a dual IGF-1R/IR kinase inhibitor, OSI-906, with EGFR inhibitor erlotinib in models of non-small cell lung cancer with an EGFR activating mutation

Abstract Erlotinib is a tyrosine kinase inhibitor of EGFR approved for use in non-small cell lung cancer (NSCLC) and pancreatic cancer in combination with Gemcitabine. Within the context of NSCLC it has been observed that patients harboring an activating mutation within the kinase domain of EGFR demonstrate a high response to treatment. However, their disease often progresses within 1 year. OSI-906 targets IGF-1R and IR receptor tyrosine kinases and has shown combination efficacy with erlotinib in preclinical models of NSCLC with wild type EGFR. Here we report preclinical data suggesting that the combination of erlotinib and OSI-906 is more efficacious in NSCLC models expressing an EGFR mutation than either single agent. A panel of NSCLC cells lines with confirmed mutations in the EGFR kinase domain was screened for erlotinib and OSI-906 single agent drug sensitivities. All mutant EGFR models exhibited sensitivity to EGFR inhibition by erlotinib (IC50 7–33 nM) while none showed sensitivity to OSI-906 (IC50 7gt;10 uM). In vitro, the combination of erlotinib and OSI-906 resulted in synergistic inhibition of cell proliferation and induction of apoptosis in PC3 (JCP-1) cells. Mechanistically we found the combination had enhanced inhibition of signaling through the PI3K/AKT pathway compared to treatment with either single agent. We also observed that while OSI-906 showed no induction of cell death, erlotinib treatment caused almost complete cell death. However after 9 days of erlotinib treatment small colonies of viable cells still remained. Removal of drug allowed the cells to grow and expand. When cells were treated with both erlotinib and OSI-906, complete cell death occurred without any evidence of cell regrowth following removal of both drugs. In order to evaluate this combination effect in vivo the EGFR mutant human tumor xenograft models NCI-H1650 and PC-14 were employed. Both lines are highly sensitive to erlotinib single agent treatment but show no anti-tumor activity with OSI-906 single agent treatment. The combination of 100 mg/kg erlotinib and 10 mg/kg OSI-906 demonstrated enhanced tumor growth delay when compared to either single agent dosed at MTD. However, as a first step towards understanding preclinical mechanism of this interaction we investigated the combination of 25 mg/kg of erlotinib with 30 mg/kg of OSI-906 and identified potential in vivo synergy. These studies with 25 mg/kg of erlotinib and 30 mg/kg of OSI-906 showed initial tumor regressions, enhanced tumor inhibition and substantially prolonged tumor growth delay when compared to either single agent. Drug-drug interaction PK studies suggest that OSI-906 is not acting to enhance erlotinib exposure. This data provides preclinical proof-of-concept for the use of OSI-906 in combination with erlotinib to obtain greater anti-tumor activity in the mutant EGFR setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-388. doi:10.1158/1538-7445.AM2011-LB-388

Abstract A50: Targeting 3-phosphoinositide-dependent kinase-1 and mammalian target of rapamycin synergistically inhibits cell proliferation of breast cancer cells

Abstract Aberration of receptor tyrosine kinases (RTKs), PTEN (phosphatase and tensin homolog deleted on chromosome 10), and PIK3CA (encodes the p110 subunit of phosphatidylinositol 3-kinase [PI3K]) frequently contribute to tumor progression through their ability to regulate the intracellular level of phosphatidylinositol-3,4,5- triphosphate (PIP3). PIP3 subsequently recruits 3-phosphoinositide- dependent kinase-1 (PDK-1), a serine/threonine kinase, to the plasma membrane and initiates PDK-1 kinase activity to phosphorylate AKT within the activation loop of the catalytic domain at the residue threonine-308 (T308). The mammalian target of rapamycin (mTOR) plays a critical role in the PI3K/AKT pathway. The mTOR kinase is present as two protein complexes, TORC1 and TORC2. The TORC1 complex is activated by PI3K/AKT and phosphorylates p70S6K and 4E-BP1 to modulate protein translation whereas the TORC2 complex phosphorylates AKT in the regulatory domain at residue serine 473 (S473). Phosphorylation of AKT at both T308 and S473 fully activates AKT function through downstream signaling to mediate cell proliferation, survival, migration, angiogenesis and other pathways which are critical for tumor progression. We have developed a potent and selective PDK1 inhibitor (PF-05177624) that is sufficient to inhibit the phosphorylation of AKT atT308 and block cell proliferation of cancer cells in vitro. In this study, we combined this PDK1 inhibitor (PF-05177624) and a selective mTOR kinase inhibitor (WYE-354) to perform the combination studies in breast cancer cell lines. Our data demonstrate that targeting both PDK1 and mTOR simultaneously produces a synergistic inhibitory effect in AKT phosphorylation. Moreover, this results in a synergistic inhibition of cell proliferation in breast cancer cells. Our data suggest that a combination strategy of targeting both PDK-1 and mTOR may have potential improved clinical efficacy in the development of cancer drugs for breast cancer patients. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A50.

Abstract 5607: Dual targeting of HER2: Enhanced antitumor efficacy of trastuzumab-DM1 combined with pertuzumab

Abstract The EGFR family of transmembrane receptors consists of HER1 (ErbB1/EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These receptors are often dysregulated in human solid tumors. For example, amplification and overexpression of HER2 occur in approximately 20 percent of human breast cancers and are predictive of poor clinical outcome. A principal means by which these receptors exert their growth-stimulatory effect is through homo- and hetero-dimerization which can occur in either a ligand-dependent or -independent manner and subsequently promotes receptor tyrosine kinase activity, leading to autophosphorylation and activation of downstream signal transduction pathways. The humanized HER2 antibody, trastuzumab (Herceptin®), is approved for use in adjuvant and metastatic HER2-positive breast cancer and has shown significant clinical benefit. Trastuzumab-DM1 (T-DM1, TMAb-mcc-DM1, trastuzumab emtansine) is an antibody-cytotoxic drug conjugate composed of the maytansine derivative DM1 directly coupled, through a thioether SMCC linker, to trastuzumab. We previously reported the potent in vitro and in vivo efficacy of T-DM1 in trastuzumab-sensitive and -refractory breast tumor models. Both trastuzumab and T-DM1 bind domain IV of the HER2 extracellular domain (ECD) and inhibit HER2 function in a ligand-independent manner. T-DM1 provides additional efficacy through selective delivery of the cytotoxic agent DM1 to HER2-overexpressing cancer cells. Pertuzumab is a humanized HER2 antibody that binds domain II of the HER2 ECD and thereby inhibits ligand-induced activation of HER2 by blocking dimerization of HER2 with other HER family members. Pertuzumab in combination with trastuzumab has shown impressive preclinical as well as clinical activity in HER2-positive breast cancer. The purpose of our studies was to evaluate the in vitro and in vivo efficacy of T-DM1 combined with pertuzumab. Our in vitro results show that combination treatment resulted in synergistic inhibition of cell proliferation in human breast and lung cancer cells that over-express HER2. Drug combination effects were analyzed by the Chou and Talalay method for determining combination index values. Similarly, we observed enhanced in vivo efficacy when administering T-DM1 in combination with pertuzumab, as compared with either agent alone, in breast and lung cancer xenograft models. Treatment of cells with T-DM1 combined with pertuzumab also resulted in a synergistic increase in apoptosis as measured by increased activity of the apoptotic enzymes, caspases 3 and 7, and increased levels of the apoptotic population in cell cycle experiments. Together, these studies support the hypothesis that T-DM1 in combination with pertuzumab for HER2-amplified cancer may offer an additional therapeutic approach for patients whose disease progresses on trastuzumab and lapatinib-based therapy. 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 5607.

Abstract 610: Metformin augments rituximab and chemotherapeutic agents-induced apoptosis via mTOR inhibition in rituximab-resistant B lymphoma cell lines

Abstract With elusive mechanisms, a subgroup of NHL patients who treated with rituximab in combination with cytotoxic chemotherapeutic agents becomes unresponsive. Patients who relapsed lymphoma are known to have poorer outcome when treated with rituximab compared with treated without rituximab. Since activation of mTOR pathway has been recently suggested for progression of NHL, especially in salvage setting, we'd like to examine the effect of metformin which inhibits mTOR pathway in rituximab-resistant NHL. Three lymphoma cell lines (Raji, Raji-2R, Raji-4RH) were used, which were kindly given by Dr Czuczman MS from Rosewell Park Cancer Institute (Ref. Czuczmana MS et al. Clin Cancer Res 2008;14:1561, 2008;14:1550). These cells were treated with cisplatin, adriamycin, vincristine and rituximab in the absence or presence of metformin for 24, 48, 72 hrs. Cell growth and proliferation was evaluated by MTT assay and cell cycle progression was determined FACS analysis. To characterize molecular mechanisms involved in metformin-induced inhibition of cell proliferation, the level of BAX, BAK, Bcl-2, Bcl-XL, Mcl-1, AMPK, pAMPK, pACC, mTOR, p70S6K and S6K was determined by western blot analysis. Compared with rituximab-sensitive cell line (RSCL) which is parental lymphoma cell, Raji, rituximab-resistant cell lines (RRCLs), that are Raji-2R and Raji-4RH, have shown decreased cytotoxicity in response to chemotherapeutic agents including cisplatin, adriamycin and vincristine as well as rituximab. However, cytotoxic effect of these chemotherapeutic agents and rituximab was improved in combination of metformin in both RSCL and RRCLs. To us surprise, a better synergistic inhibition of cell proliferation was found in RRCLs as a result of combination of metformin and chemotherapeutic agents or rituximab, suggesting that intracellular signaling pathway induced by metformin might not be overlapped to that by chemotherapeutic agent and rituximab. Notably, it has known that in RRCLs, the level of proapototic proteins, BAX, BAK, is relatively low compared to in RSCL decreased, but the expression of anti-apoptotic protein and AMPK activity is increased and dysregulation of mTOR pathway is found. Our data showed that metformin restored dysregulation of mTOR pathway in RRCLs, since activation of p70S6K and S6K posphorylation was decreased. At the same time, expression of BAX and BAK was increased and that of anti-apoptotic proteins was decreased. Metformin enhances rituximab-induced and chemotherapy-induced apoptosis via mTOR inhibition in rituximab-resistant NHL. 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 610.

Synergistic activity of letrozole and sorafenib on breast cancer cells

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Human melatonin MT 1 receptor induction by valproic acid and its effects in combination with melatonin on MCF-7 breast cancer cell proliferation

We have reported that valproic acid upregulates melatonin MT 1 receptor expression in rat C6 glioma cells. In addition to its anticonvulsant and mood stabilizing properties, valproic acid can also inhibit the growth of cancer cells. Since the melatonin MT 1 receptor has been implicated in the oncostatic action of melatonin on human MCF-7 breast cancer cells, the effect of valproic acid on its expression was examined in this cell line. Treatment of MCF-7 cells with valproic acid (0.5 or 1 mM) for 24 or 72 h caused a significant increase in melatonin MT 1 receptor mRNA or protein expression, as shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis and western blotting, respectively. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays revealed a concentration-dependent inhibition of MCF-7 cell proliferation by valproic acid (0.5, 1.0 or 5 mM), but melatonin (1 or 10 nM) was ineffective alone or in combination with valproic acid, in the first (MCF-7A) subline examined. However, in subsequent experiments using a different (MCF-7B) subline, which expressed higher levels of MT 1 receptor mRNA and showed modest sensitivity to melatonin, a combination of this hormone with valproic acid produced a significant synergistic inhibition of cell proliferation. These findings indicate that clinically relevant concentrations of valproic acid upregulate melatonin MT 1 receptor expression in human breast cancer cells. Moreover, the enhanced antiproliferative effect observed with a combination of valproic acid and melatonin suggests that a similar therapeutic approach may be beneficial in breast cancer.

The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root ( Panax quinquefolius L.)

American ginseng root ( Panax quinquefolius) has a number of purported therapeutic effects, including inhibition of cancer cell proliferation. The ability of environmentally relevant heavy metals to alter ginseng effects on cancer cell growth was the subject of this study. A water extract of American ginseng root was applied alone or in combination with physiologically relevant doses of either lead (Pb) or arsenite to MCF-7 breast cancer cells in vitro and effects on cell proliferation were determined. Ginseng alone produced a significant dose-dependent inhibition of MCF-7 cell proliferation starting at 0.5 mg ml − 1 . Treatment of MCF-7 cells with 2.5 μM arsenite significantly decreased MCF-7 cell proliferation ( p < 0.01). When cells were treated with arsenite (1.25 or 2.5 μM) in combination with ginseng extract (0.5 mg ml − 1 ), there was an apparent synergistic inhibition of cell proliferation. Treatment of MCF-7 breast cancer cells with 50 μM Pb significantly decreased cell proliferation relative to control ( p < 0.01), and concomitant ginseng and Pb treatment did not lead to a further decrease. These results suggest that contaminant heavy metals, some of which have been detected in ginseng root extracts or commercial ginseng preparations, may alter the biological activity of ginseng.

A study of cytotoxic synergy of UCN-01 and flavopiridol in syngeneic pair of cell lines

Flavopiridol and UCN-01 are two novel protein kinase inhibitors with diverse cellular effects that may complement each other with regards to induction of apoptosis. HeLa cells engineered to overexpress human survivin (HeLa-S) were at least approximately 4.8-fold resistant to UCN-01 relative to proliferation observed in control HeLa cells (HeLa-V). Flavopiridol cytotoxicity as measured using the MTT assay was unaffected in HeLa-S cells when compared with HeLa-V cells. Similarly, simultaneous treatment of HeLa-V cells with flavopiridol and UCN-01 for 72 hours did not result in synergistic inhibition of proliferation; however, in HeLa-S cells, this combination resulted in synergistic inhibition of cell proliferation. Flavopiridol and UCN-01 augmented apoptosis in HeLa-S cells (as compared with HeLa-V cells) as measured by caspase-3 cellular activity assay, DNA fragmentation and PARP cleavage by western blot. In HeLa-V and -S cells, combination treatment resulted in caspase-8 cleavage. Caspase-9 was expressed in HeLa-V cells; however, there was a marked reduction of caspase-9 content in HeLa-S cells only. Combination treatment resulted in a significant reduction in survivin abundance in HeLa-S and SKBR3-UR cells, but not in their respective parental lines. The synergy of Flavopiridol and UCN-01 are selectively toxic to survivin-overexpressing cell lines and the mechanism of toxicity involves caspase-dependent cell death.

Synergistic Anti-Oxidative Effects of Lycopene with Other Bioactive Compounds

Lycopene belongs to the carotenoid family, which has more than 700 members. In human serum, only 14 caroteinoids have been identified, and, among these, lycopene was the most abundant. Lycopene is regarded as one of the most efficient singlet oxygen quencher and peroxyl radical scavenger of all the carotenoids and may represent an important defense mechanism in the human body. Singlet oxygen and free radicals can cause biological damage to important macromolecules and membrane constituents, and the presence of natural antioxidants may help to control these degradative reactions.The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis which makes the antioxidants that protect LDL potential antiatherogenic agents. There is growing evidence that certain combinations of natural antioxidants have synergistic antioxidant activity, that is, the actual inhibitory activity of the mixture is greater than the additive activities of the individual antioxidants. Lycopene on its own can retard the oxidation of LDL, but in combination with vitamin E, a synergistic effect is observed. There is clear evidence that lycopene in association with α-tocopherol synergistically inhibits the proliferation of two prostate carcinoma cell lines. Polyphenols are very potent antioxidants found in tomatoes and many other fruits, vegetables, and herbs. The polyphenols glabridin, rosmarinic acid, and carnosic acid, when used in combination with lycopene, inhibited the oxidation of LDL to a greater extent than the additive values by 32%, 32%, and 15%, respectively. Extracts and powders containing mixtures of antioxidants also showed synergism. Tomato oleoresin produced a 5-fold increase in antioxidant activity against LDL oxidation when compared to pure lycopene at the same concentration. A 33% increase in LDL inhibition was observed when lycopene was used with garlic powder. Of the six major carotenoids found in human blood and tissue, lycopene plus lutein produced the greatest synergistic effect against oxidation of multilamellar liposomes. It also has been shown that low concentrations of 1,25-dihydroxylvitamin D3 and lycopene synergistically inhibit cell proliferation and differentiation in HL-60 leukemic cells. A number of hypotheses have been put forward to explain the synergistic effects between lycopene and other natural antioxidants. It has been suggested that the synergistic effects may be related to their physicochemical properties and or their location within the biomembrane or LDL particle; that one antioxidant is able to regenerate the second antioxidant; or that vitamin E and lycopene affect different signal transduction pathways leading to the synergistic inhibition of cell proliferation.