Cytotoxic Synergy Research Articles

Cytotoxic Synergy Between Cytokines and NSAIDs Associated With Idiosyncratic Hepatotoxicity Is Driven by Mitogen-Activated Protein Kinases.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequent causes of idiosyncratic, drug-induced liver injury (IDILI). Mechanisms of IDILI are unknown, but immune responses are suspected to underlie them. In animal models of IDILI, the cytokines tumor necrosis factor-alpha (TNFα) and interferon-gamma (IFNγ) are essential to the pathogenesis. Some drugs associated with IDILI interact with cytokines to kill hepatocytes invitro, and mitogen-activated protein kinases (MAPKs) might play a role. We tested the hypothesis that caspases and MAPKs are involved in NSAID/cytokine-induced cytotoxicity. NSAIDs that are acetic acid (AA) derivatives and associated with IDILI synergized with TNFα in causing cytotoxicity in HepG2 cells, and IFNγ enhanced this interaction. NSAIDs that are propionic acid (PA) derivatives and cause IDILI that is of less clinical concern also synergized with TNFα, but IFNγ was without effect. Caspase inhibition prevented cytotoxicity from AA and PA derivative/cytokine treatment. Treatment with a representative AA or PA derivative induced activation of the MAPKs c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. Inhibition of either JNK or ERK reduced cytotoxicity from cytokine interactions with AA derivatives. In contrast, an ERK inhibitor potentiated cytotoxicity from cytokine interactions with PA derivatives. An AA derivative but not a PA derivative enhanced IFNγ-mediated activation of STAT-1, and this enhancement was ERK-dependent. These findings raise the possibility that some IDILI reactions result from drug/cytokine synergy involving caspases and MAPKs and suggest that, even for drugs within the same pharmacologic class, synergy with cytokines occurs by different kinase signaling mechanisms.

Ceramide‐enriched secreted microvesicles as a mediator of cytotoxic synergy between TNF‐α and IFN‐γ in oligodendrocyte cell death

A combination of TNFα and IFNγ induces cell death in several cell types including oligodendrocytes but the mechanism of their synergistic action is unclear. In this study, using a human oligodendroglioma cell line, HOG as a model, we show that secreted microvesicles (MVs) act as a paratropic mediator of synergistic cytotoxicity. Thus, TNFα and IFNγ are marginally cytotoxic individually but together induce marked cell death. MVs obtained from TNFα‐ but not IFNγ‐treated HOG cells, while mildly toxic to fresh cultures, induced extensive cell death in a fashion resembling the effect of cytokine combination when added to IFNγ‐primed target cultures. Since sphingolipids (SLs) including ceramide (Cer) form an integral part of secreted MVs, we determined the basic SL profiles of the secreted MVs as well as cytokine‐treated 'source’ cells by HPLC‐MS/MS. The treatment with the pro‐apoptotic cytokines time‐dependently increased Cer, dhCer & SM expression, in particular, of C24‐ and C24:1‐Cer species; C16‐, C24‐ and C24:1‐dhCer species; and C16‐, C24‐ and C24:1‐SM species in both intra‐ and extracellular compartments over a course of 48 hrs. That MV‐associated Cer contributes to the cytotoxic activity was supported by the observation that exogenous C6‐Cer replicated, albeit partially, the cytotoxic effect of Cer‐enriched MVs. We conclude that Cer‐laden MVs released from TNFα‐exposed cells collaterally 'spread’ the death signal to IFNγ‐primed target cells to elicit a robust synergistic cell death response.

The Synergistic Mechanism of Pemetrexed Followed by Kanglaite was due to KLT Subsequently Inhibiting the Pemetrexed-Activated MAPK Signaling Pathway.

Kanglaite (KLT) can enhance the cytotoxic effects of chemotherapy; however, the underlying mechanism remains unclear. We investigated the mechanism underlying the cytotoxic synergy between KLT and pemetrexed in NSCLC cell lines. A549 and H1975 cell lines were treated with pemetrexed and/or KLT in vitro. IC50 values, the combination index, cell cycle distribution, and signaling pathway analysis were assessed. Cytotoxic interactions between KLT and pemetrexed were dose-dependent in A549 and H1975 NSCLC cell lines. The administration of pemetrexed followed by KLT had a synergistic effect and an advantage over KLT followed by pemetrexed. Concomitant administration in both cell lines indicated that the cytotoxic interactions between KLT and pemetrexed were schedule-dependent. Cell cycle analysis showed that KLT arrested cells mainly in the G2/M phase, whereas pemetrexed arrested cells mainly in the S phase. Exposure to KLT first induced G2/M arrest and subsequently prevented the cytotoxicity of the S phase-specific drug pemetrexed. Signaling pathway analysis showed that exposure to pemetrexed resulted in increased phospho-p44/42MAPK levels which were inhibited by subsequent exposure to KLT. Thus pemetrexed followed by KLT inhibited the MAPK signaling pathway more obviously than KLT followed by pemetrexed. Pemetrexed followed by KLT had a synergistic effect and an advantage over other sequences in NSCLC cell lines. The synergistic mechanism was due to KLT subsequently inhibiting the pemetrexed-activated MAPK signaling pathway. These findings may provide molecular evidence to support clinical treatment strategies for patients with NSCLC.

Abstract 5516: Fenretinide and safingol synergistic cytotoxicity in neuroblastoma is, in part, mediated by increase of dihydroceramides

Abstract Introduction: N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) is a clinical-stage synthetic retinoid cytotoxic to many cancer cell lines in a dose- and time-dependent manner in vitro by mechanisms that include reactive oxygen species (ROS) generation and increase of D-erythro-dihydroceramides via de novo synthesis. The cytotoxicity of 4-HPR is synergistically increased by low, minimally cytotoxic concentrations (1 - 4 µM) of the stereochemical sphinganine analog, safingol (C18-L-threo-sphinganine). Currently reported activities of safingol include inhibition of sphingosine kinase 1 (SPHK1) and/or protein kinase C's (PKCs) at higher concentrations (20 - 50 µM) in vitro. However, previous work done in our lab has shown that parent safingol is substantially metabolized (90%) by +24 hrs, and as a result, activities dependent on the presence of parental safingol would not be expected to be maintained over time. Given these apparent discrepancies, we hypothesized that safingol does not synergize 4-HPR cytotoxicity through parental inhibition of SPHK1 or PKC's, but rather through a mechanism dependent on the concurrent increase of normal and variant, D-erythro- and L-threo-dihydroceramides. Methods: Human neuroblastoma (NB) cell lines CHLA-15, CHLA-90, and SK-N-RA, which display synergistic cytotoxicity to 4-HPR + safingol (H+S) were selected as a model system. In our washout experiments cells were treated with 4-HPR (0 - 6 µM) either concurrently with a 4-hr safingol (4 µM) exposure, or at later times after safingol exposure (+4_ +48 hours), to allow for safingol metabolism. Cytotoxicity was measured using a semi-automated, fluorescence-based assay system (DIMSCAN). Flow cytometry experiments to analyze ROS levels in response to H+S treatment have also been conducted. Subsequently, liquid chromatography tandem mass spectroscopy (LC/MS/MS) is being utilized to quantitate various ceramides, L-threo-, and D-erythro-dihydroceramides in order to determine any possible correlation between any particular species and cytotoxicity. Results and Conclusions: Comparable cytotoxicity (P<.05) was observed whether 4-HPR was administered concurrently with safingol or at various later time points. These data support that cytotoxic synergy was not dependent on the presence of parental safingol. Experiments quantitating ROS by flow cytometry do not show changes in the levels of these species in H vs. H+S groups, ruling out ROS generation as a mechanism of synergy between H+S. Preliminary LC/MS/MS experiments paired with DIMSCAN cytotoxicity assays indicate that certain sphingolipid species correlate with cytotoxicity in our NB models. Alternative hypotheses include that L-threo-dihydroceramides inhibit SPHK1 or PKC's activity. This information will help inform the clinical delivery schedules of H+S to decrease toxicities and maximize antitumor efficacy. Citation Format: Mohammad S. Abedi. Fenretinide and safingol synergistic cytotoxicity in neuroblastoma is, in part, mediated by increase of dihydroceramides. [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 5516. doi:10.1158/1538-7445.AM2014-5516

Abstract 4472: Targeted multifunctional lipid-PLGA hybrid nanosystems for metastatic breast cancer imaging and therapy

Abstract Introduction: Targeted theranostic nanoparticles based on near-infrared fluorescence is a novel concept in next-generation medicine that combines diagnostics and therapeutics simultaneously to radically change the way we diagnose, image and treat the breast cancer. Often these targeted therapies are efficacious and at the same time less toxic than traditional regimens. Our aim is to formulate a multifunctional theranostic agent capable of tumor targeting and tumor imaging along with anticancer therapy for metastatic breast cancer. Methods: We engineered lipo-polymeric composite multifunctional nanoparticles co-encapsulated with long lived dyes (like ADOTA) together with a combination of anticancer drugs, docetaxel and curcumin by nanoprecipitation. These anticancer agents have been found to show synergistic anti-angiogenic effects. Further, curcumin has the potential to downregulate P-glycoprotein expression which helps in reversing drug resistance seen with drugs like docetaxel, thereby potentiating the activity of the drug at lower concentrations. The formulated composite nanoparticles are conjugated to Annexin A2 (AnxA2) peptide (LCKLSL) to enable specific targeting to tumor site. LCKLSL hexapeptide in the amino-terminal domain of the AnxA2motif is essential for tissue plasminogen activator binding. Therefore AnxA2 peptide will serve as a useful therapeutic target against invasion and metastasis. Results: Multifunctional nanoparticles were successfully formulated and then characterized for drug loading efficiency, particle size, encapsulation efficiency, surface morphology, zeta potential and in vitro drug release profiles. The results show smooth and spherical nanoparticles when seen under TEM with drug encapsulation of 50% for curcumin and 19% for docetaxel. The particle size distribution showed uniform distribution with average size being110±10nm. Zeta potential varied between -35±5mV. Percent AnxA2 peptide attachment on the nanoparticles was determined by using Flow cytometry and found to be 74%. Intracellular uptake in breast cancer cell line (MDA MB231) was carried out and showed robust uptake. The nanoparticle formulations were further evaluated by cytotoxic studies and synergy between anticancer drugs was established. Mouse xenograft studies showed significant tumor regreesion for AnxA2 targeted curcumin and docetaxel loaded hybrid nanoparticles as compared to untreated mice. Conclusion: This study would help validate these targeted theranostic nanoparticles in metastatic breast cancer mouse model thus highlighting the immense clinical potential of targeted multifunctional theranostic nanoparticles for imaging and therapy of metastatic breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Amalendu P. Ranjan, Anindita Mukerjee, Jamboor K. Vishwanatha. Targeted multifunctional lipid-PLGA hybrid nanosystems for metastatic breast cancer imaging and therapy. [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 4472. doi:10.1158/1538-7445.AM2014-4472

Phase 1B Study of Oral Dual-Pi3K/Mtor Inhibitor Gdc-0980 in Combination with Carboplatin (Carbo)/Paclitaxel (Pac) ± Bevacizumab (Bev) and Cisplatin (Cis)/Pemetrexed (Pem) in Patients (Pts) with Advanced Solid Tumors and Nsclc

ABSTRACT Aim: The PI3K signaling pathway is altered in many tumor types, including NSCLC. GDC-0980 has single-agent activity as well as preclinical cytotoxic synergy with taxane and platinum agents and antiangiogenic synergy with anti-VEGF agents. Methods: This Phase 1b study evaluated the safety and tolerability of GDC-0980 in combination with platinum-based chemotherapy (CT) and Bev in pts with advanced solid tumors (AST) or first-line advanced NSCLC: Arm A – Carbo (AUC 6) + Pac (200 mg/m2); Arm B – Carbo/Pac + Bev (15 mg/kg); Arm C - Cis (75 mg/m2) + Pem (500 mg/m2). On a 21-day cycle, CT regimens were administered on Day 1, and GDC-0980 PO QD on Days 1-14. Pharmacokinetic (PK) analyses were performed. Archival tumor tissue and blood samples for pharmacogenetic and ctDNA analyses were collected. Results: Sixty-five pts (28 in Arm A [17 NSCLC]; 18 in Arm B [10 NSCLC]; 19 in Arm C [12 NSCLC]) were treated with GDC-0980 20 to 40 mg. For AST cohorts in Arms A and B, the MTD of GDC-0980 was 25 mg (DLTs were febrile neutropenia, rash, and fatigue). For NSCLC cohorts in Arms A and C, the maximum administered dose (MAD) was 30 mg GDC-0980. At this dose, Grade 3/4 adverse events (AEs) in >10% of all treated pts were neutropenia (59%), rash (24%), febrile neutropenia and anemia (12%) in Arm A and neutropenia (33%), anemia and fatigue (25%), hypertension, infection, and pneumonitis (17%) in Arm C. Other high-grade AEs of interest were less common: hyperglycemia in Arm A (6%) and Arm C (8%) and rash (8%) in Arm C. Among NSCLC pts treated with 30 mg GDC-0980, objective responses were observed in 9 of 14 (64%) pts in Arm A and 4 of 12 (33%) pts in Arm C. Other pts with follicular thyroid, ovarian, and urothelial cancers (Arm A) and angiosarcoma (Arm B) also had objective responses. There were no apparent PK interactions. Biomarker analysis is ongoing. Conclusions: The safety profile of GDC-0980 30 mg in combination with Carbo/Pac or Cis/Pem is consistent with that of the individual agents, and AEs were manageable and reversible. These GDC-0980 combinations have demonstrated preliminary activity in first-line NSCLC. Disclosure: O. Hamid: Consultant to Genenech; W. Lin: Employee of Genentech, Inc.; B. O'keeffe: Employee of Genentech, Inc.; M.R. Lackner: Employee of Genentech, Inc.; J. Spoerke: Employee of Genentech, Inc.; J. Ware: Employee of Genentech, Inc. All other authors have declared no conflicts of interest.

In vitro synergistic efficacy of conjugated linoleic acid, oleic acid, safflower oil and taxol cytotoxicity on PC3 cells

The aim of this study was to determine in vitro synergistic efficacy of conjugated linoleic acid (CLA), oleic acid (OLA), safflower oil and taxol (Tax) cytotoxicity on human prostate cancer (PC3) cell line. To determine synergistic efficacy of oil combinations, PC3 treated with different doses of compounds alone and combined with 10 μg/mL Tax. The MTT results indicated that OLA–Tax combinations exhibited cytotoxicity against PC3 at doses of 30 nM+10 μg-Tax, 15 nM+5 μg-Tax and 7.5 nM+2.5 μg-Tax. The treatment of OLA or Tax did not show significant inhibition on PC3, while OLA–Tax combinations showed effective cytotoxicity at treated doses. CLA–Tax combinations demonstrated the same effect on PC3 as combined form with 45.72% versus the alone form as 74.51% viability. Cytotoxic synergy between Tax, OLA and CLA shows enhanced cytotoxicity on PC3 which might be used in the therapy of prostate cancer.

UV-induced selective oxidation of Met5 to Met-sulfoxide leads to the formation of neurotoxic fibril-incompetent α-synuclein oligomers

Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the 1H-15N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.

A novel valproic acid prodrug as an anticancer agent that enhances doxorubicin anticancer activity and protects normal cells against its toxicity in vitro and in vivo

The poor survival of patients with malignant gliomas, underscores the need to develop effective treatment modalities for this devastating disease. Epigenetic agents used in combination with chemotherapy provide a promising approach to evoke synergistic cytotoxicity in glioblastomas. Previously we have described the cytotoxic synergy between a butyric acid prodrug and radiation in glioblastoma cell lines and the potentiation of radiation efficacy in glioma xenografts. Herein, we describe and compare the activities of AN446 (valproyl ester-valpramide of acyclovir) a novel histone deacetylase inhibitor (HDACI) to the previously described AN7 a HDACI prodrug of butyric acid. In various cancer cell lines, AN446 was a ∼2–5-fold more potent anticancer agent HDACI than AN7. While AN446 augmented the anticancer efficacy of doxorubicin (Dox) it also reduced the Dox toxicity in non-cancerous cells. The interaction between AN446 and Dox in U251 and in 4T1 cell lines was synergistic in inducing cytotoxicity. We examined the concomitant physical and molecular changes in the tumor and heart of glioblastoma xenografts treated with AN446, AN7, Dox and the combination of the prodrugs with Dox. A weekly dose of 4mg/kg Dox, caused toxicity in mice whereas AN446 (25mg/kg) or AN7 (50mg/kg) administered thrice weekly, did not. When Dox was administered with AN446 or AN7, the prodrugs ameliorated the decline in body weight, prolonged the time to failure and increased anticancer efficacy. Thus, the combination of Dox with AN446 or AN7 could add safety and efficacy to future treatment protocols for treating glioblastoma and other cancers.

A novel Cdk9 inhibitor preferentially targets tumor cells and synergizes with fludarabine.

Cdk9 is a key elongation factor for RNA transcription and functions by phosphorylating the C-terminal domain of RNA polymerase II. Here we present direct evidence that cdk9 is important for cancer cell survival and describe the characterization of the potent cdk9 inhibitor CDKI-73 in primary human leukemia cells. CDKI-73 induced caspase-dependent apoptosis that was preceded by dephosphorylation of cdk9 and serine 2 of RNA polymerase II. CDKI-73 was more potent than the pan-cdk inhibitor flavopiridol and showed >200-fold selectivity against primary leukemia cells when compared with normal CD34+ cells. Furthermore, CDKI-73 was equipotent in poor prognostic sub-groups of leukemia patients and showed cytotoxic synergy with the nucleoside analog fludarabine. The Mechanism of synergy was associated with CDKI-73-mediated transcriptional inhibition of MCL1 and XIAP that was maintained when used in combination with fludarabine. Our data present a strong rationale for the development of cdk9 inhibitors such as CDKI-73 as anticancer therapeutics.

Abstract P4-16-05: Hsp90 inhibition potentiates chemotherapy effectiveness in triple negative breast cancer: Role of glucocorticoid receptor signaling

Abstract Background: Few promising molecular targets exist for chemoresistant triple negative breast cancer (TNBC). Our laboratory has previously demonstrated that a subset of TNBCs express high glucocorticoid receptor (GR) levels. In breast epithelial cells, GR transcriptional activity directly upregulates the expression of several anti-apoptotic target genes including serum/glucocorticoid regulated kinase 1 (SGK1). High GR and/or SGK1 expression are both associated with decreased chemotherapy-induced cell death in TNBC models. A meta-analysis of patients with early-stage TNBC found that high primary tumor GR (NR3C1) expression significantly associates with earlier relapse compared to GR-non expressing TNBC. Inhibiting GR/SGK1 activity is therefore a testable strategy for improving TNBC responsiveness to chemotherapy. Both GR and SGK1 require Hsp90 for their activity; therefore, Hsp90 inhibition is predicted to disrupt both GR and SGK1 cell survival signaling in TNBC. Employing a second-generation Hsp90 inhibitor, ganetespib (Synta Pharmaceuticals), we determined the effectiveness of Hsp90 inhibition on GR/SGK1 activity and chemotherapy sensitivity using in vitro and in vivo TNBC models. Hypothesis: Hsp90 inhibition will decrease GR and SGK1 activity in TNBC in association with improved chemotherapy effectiveness. Results: Using TNBC cell lines treated with ganetespib at clinically relevant concentrations, we found that total GR and the relative proportion of phosphorylated Ser211 GR were significantly reduced beginning 2 hours following treatment. Reduced glucocorticoid-induced (SGK1) mRNA expression was also seen when cells were pre-treated with ganetespib. We next used fluorescent microscopy and live cell imaging to measure TNBC cell death over a 96 hour time course following chemotherapy +/- ganetespib therapy. Addition of ganetespib to paclitaxel treatment of MDA-MB-231 cells led to significant in vitro cytotoxic synergy; as hypothesized, this synergy was diminished in GR-depleted MDA-MB-231 cells. Cytotoxic synergy following ganetespib and chemotherapy treatment was also seen in vivo in MDA-MB-231 xenografts. Interestingly, tumor GR expression following Hsp90 inhibitor and paclitaxel treatment was significantly reduced compared to tumors from mice treated with paclitaxel alone. Using GR-depleted or control MDA-MB-231 xenografts, studies are currently underway to further characterize the role of GR/SGK1 activity as a target of Hsp90 inhibition to reverse TNBC chemoresistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-05.

Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.

Abstract Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR. Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies. Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P < 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P < 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm. Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC. Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [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 1788. doi:10.1158/1538-7445.AM2013-1788

Pazopanib Enhances Paclitaxel-Induced Mitotic Catastrophe in Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) has perhaps the worst prognosis of any cancer, with a median survival of only about 5 months regardless of stage. Pazopanib monotherapy has promising clinical activity in differentiated thyroid cancers (generally attributed to vascular endothelial growth factor receptor inhibition), yet has less effective single-agent activity in ATC. We now report that combining pazopanib with microtubule inhibitors such as paclitaxel produced heightened and synergistic antitumor effects in ATC cells and xenografts that were associated with potentiated mitotic catastrophe. We hypothesized that combined effects may reflect enhanced paclitaxel-induced cytotoxicity mediated by cell cycle regulatory kinase inhibition by pazopanib. Indeed, pazopanib potently inhibited aurora A, with pazopanib/paclitaxel synergy recapitulated by aurora A short hairpin RNA knockdown or by specific aurora A pharmacological inhibition. Pazopanib/paclitaxel synergy was reversed by aurora A knockdown. Moreover, aurora A (but not B or C) message and protein levels were significantly increased in patient ATCs, and durable benefit resulted from pilot clinical translation of pazopanib/paclitaxel therapy in a patient with metastatic ATC. Collectively, these results suggest that the pazopanib/paclitaxel combination is a promising candidate therapeutic approach in ATC and that aurora A may represent a potentially viable therapeutic molecular target in ATC.

Replicating Human Tumor Biology in Vitro

Genetic Engineering & Biotechnology NewsVol. 33, No. 1 TutorialReplicating Human Tumor Biology in Vitro3D Culture Systems Integrated for Robust Imaging and Functional AnalysisRobin Felder, Juliana Cano, Rachael Shevin, Brad Justice, and Raj SinghRobin FelderRobin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. (E-mail Address: rsingh@vivobiotech.com), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.Search for more papers by this author, Juliana CanoRobin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. (E-mail Address: rsingh@vivobiotech.com), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.Search for more papers by this author, Rachael ShevinRobin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. (E-mail Address: rsingh@vivobiotech.com), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.Search for more papers by this author, Brad JusticeRobin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. (E-mail Address: rsingh@vivobiotech.com), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.Search for more papers by this author, and Raj SinghRobin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. (E-mail Address: rsingh@vivobiotech.com), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.Search for more papers by this authorPublished Online:31 Dec 2012https://doi.org/10.1089/gen.33.01.11AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byCombinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions30 May 2018 | Scientific Reports, Vol. 8, No. 1Patient-Derived Xenografts as a Model System for Radiation ResearchSeminars in Radiation Oncology, Vol. 25, No. 4 Volume 33Issue 1Jan 2013 Information© 2013 by GEN PublishingTo cite this article:Robin Felder, Juliana Cano, Rachael Shevin, Brad Justice, and Raj Singh.Replicating Human Tumor Biology in Vitro.Genetic Engineering & Biotechnology News.Jan 2013.19-19.http://doi.org/10.1089/gen.33.01.11Published in Volume: 33 Issue 1: December 31, 2012PDF download

The Order of Exposure to Cytotoxic Chemotherapy and the Proteasome Inhibitor Bortezomib Dictates the Extent of Reversal of Chemotherapy Resistance in T-ALL

AbstractAbstract 1355 Introduction:While most children with T-cell acute lymphoblastic leukemia (T-ALL) are cured, the outcome for children who respond poorly to therapy or relapse is almost universally fatal. Our laboratory is interested in understanding the mechanisms contributing to therapy resistance and has developed a mutliparameter phosphoflow cytometry assay to study signal transduction events at a single cell level.Nuclear Factor kappa B (NF-κB) is a family of transcription factors that plays an important role in cancer development by preventing apoptosis and facilitating tumor cell growth. Consistent with this, constitutive NF-κB activity has been reported in T-ALL, and has been suggested as a predictor of relapse. We hypothesized that the proteasome inhibitor Bortezomib, which blocks NF-κB activation, induces apoptosis in T-ALL. We further hypothesized that combining Bortezomib with conventional chemotherapeutic agents sensitizes T-ALL cells to apoptosis and overcomes chemotherapy resistance by altering the balance of downstream pro- and anti- apoptotic mediators. Methods:A panel of T-ALL cell lines was cultured in the presence of vehicle alone, conventional cytotoxic drugs (Dexmethasone, Etoposide, Cyclophosphamide, Cytarabine), Bortezomib, or combinations of these. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3. Multiparameter phosphoflow cytometry was used to measure expression levels of downstream apoptosis mediators. An advantage of multiparameter phosphoflow cytometry is the ability to gate on caspase 3-negative (i.e., chemotherapy resistant) cells, allowing us to interrogate potential mechanisms of resistance by comparing expression profiles of cell survival proteins before and after treatment. Results:T-ALL cells were exposed to cytotoxic chemotherapy or vehicle for 24 to 48 hours and subjected to phosphoflow cytometry. Analysis of caspase-3 negative cells indicated that chemotherapy resistant cells rewired their signaling networks by upregulating their MAPK, NF-κB, and AKT/PI3K/S6 signaling networks in response to genotoxic stress. This correlated with upregulation of the pro-survival protein survivin, suggesting a potential mechanism for chemotherapy resistance. To test whether Bortezomib could reverse this resistance, cells were exposed to subtoxic concentrations of Bortezomib alone or in combination with cytotoxic drugs. Combination therapy produced only a modest synergistic effect. We then wondered whether the sequence of administration of cytotoxic drug and Bortezomib could potentiate this effect. T-ALL cell lines were therefore 1) pretreated with Bortezomib for 24 hours followed by cytotoxic drug for an additional 24 hours, 2) pretreated with cytotoxic drug for 24 hours followed by addition of Bortezomib for an additional 24 hours, or 3) treated with both agents simultaneously. Surprisingly, the most pronounced cytotoxic synergy was observed when T-ALL cells were pretreated with cytotoxic drug followed by Bortezomib. While simultaneous or pretreatment with Bortezomib had no effect on upregulation of surivin in response to cytoxic chemotherapy, addition of Bortezomib post genotoxic stress resulted in markedly decreased levels of survivin. Conclusions:We found that Bortezomib lowered the apoptotic threshold to conventional cytotoxic drugs and can reverse drug resistance in T-ALL cell lines. Interestingly, the order of exposure to cytotoxic chemotherapy and Bortezomib determines the extent of reversal of chemotherapy resistance. Our data is in agreement with studies in multiple myeloma and provides evidence supporting the use of Bortezomib in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease. Disclosures:No relevant conflicts of interest to declare.

Antitumor Activity of Triolimus: A Novel Multidrug-Loaded Micelle Containing Paclitaxel, Rapamycin, and 17-AAG

Triolimus is a first-in-class, multidrug-loaded micelle containing paclitaxel, rapamycin, and 17-AAG. In this study, we examine the antitumor mechanisms of action, efficacy, and toxicity of Triolimus in vitro and in vivo. In vitro cytotoxicity testing of Triolimus was conducted using two aggressive adenocarcinomas including the lung cancer cell line, A549, and breast cancer cell line, MDA-MB-231. The three-drug combination of paclitaxel, rapamycin, and 17-AAG displayed potent cytotoxic synergy in both A549 and MDA-MB-231 cell lines. Mechanistically, the drug combination inhibited both the Ras/Raf/mitogen-activated protein kinase and PI3K/Akt/mTOR pathways. Triolimus was advanced into tumor xenograft models for assessment of efficacy, toxicity, and mechanisms of action. In vivo, a three-infusion schedule of Triolimus inhibited A549 and MDA-MB-231 tumor growth far more potently than paclitaxel-containing micelles and effected tumor cures in MDA-MB-231 tumor-bearing animals. Tumor growth delays resulted from a doubling in tumor cell apoptosis and a 50% reduction in tumor cell proliferation compared with paclitaxel-containing micelles. Enhanced antitumor efficacy was achieved without clinically significant increases in acute toxicity. Thus, Triolimus displays potent synergistic activity in vitro and antitumor activity in vivo with comparable toxicity to paclitaxel. These observations provide strong support for further development of Triolimus and an important proof of concept for safe, effective nanoparticle-based delivery of three complementary anticancer agents.

Development of a combination drug-eluting bead

Drug-eluting beads (DEBs) are becoming a mainstay locoregional therapy for hepatic malignancies but are currently loaded with single drugs alone. Here, we wished to prepare DEB containing different drug combinations, to screen their efficacy using an in-vitro cell culture assay and to include any promising combinations that demonstrate additive efficacy in an in-vivo model of locoregional tumour treatment. A modified in-vitro assay was used based upon the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) with either HepG2 liver cancer or PSN1 pancreatic cancer cell lines. The comparative cytotoxicity of DEB combinations prepared containing doxorubicin, irinotecan, topotecan and rapamycin was evaluated. Those combinations that demonstrated an additive cytotoxicity effect were investigated in vivo using a nude mouse xenograft model of pancreatic cancer. Although many of the DEB combinations showed either no effect or a slight antagonistic effect, the combination of doxorubicin and rapamycin DEBs demonstrated synergistic activity. On the basis of these findings, a method was developed to prepare a doxorubicin/rapamycin dual-loaded DEB, which was shown to possess the same drug-loading capacities, drug elution properties and HepG2 cell cytotoxicity synergy as the single drug-loaded DEB combination. Evaluation of this dual-loaded combination DEB versus the respective single drug-loaded DEBs in a mouse xenograft model of pancreatic cancer showed an equivalent tumour volume reduction as the doxorubicin DEB, but with less toxicity than the rapamycin DEB. The doxorubicin/rapamycin combination DEB offers great potential for enhanced efficacy in the locoregional treatment of malignant tumours.

P1-12-07: Pharmacokinetics (PK) of Trastuzumab Emtansine and Paclitaxel or Docetaxel in Patients with HER2−Positive MBC Previously Treated with a Trastuzumab-Containing Regimen.

Abstract Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate composed of trastuzumab, DM1 (a potent cytotoxic tubulin polymerization inhibitor), and the stable MCC linker. Based on in vitro and preclinical data suggesting synergy in tumor cell cytotoxicity with T-DM1 + taxanes, these combinations are being tested in phase Ib studies. The assessment of risk for PK-based drug interaction (PK-DI) with these combinations is important. T-DM1 is expected to undergo proteolytic degradation with no significant involvement of cytochrome P450 isoenzymes (CYPs), and DM1 is metabolized mainly by CYP3A4 (and to a lesser extent by CYP3A5). Paclitaxel (P) and docetaxel (D) are metabolized primarily by CYP2C8 and CYP3A4, respectively. None of these drugs inhibits or induces CYPs at clinically relevant concentrations. Since DM1 and D are both metabolized by CYP3A4, PK-DI may occur. This is less likely with DM1 and P since they are primarily metabolized by different CYPs. Methods: The tolerability, PK, and dose-limiting toxicities of T-DM1 + P or D were investigated in 2 studies (TDM4652g and BP22572) of patients with HER2−positive MBC previously treated with a trastuzumab-containing regimen. For PK-DI analysis, both studies used a staggered dosing design in the first cycle, with same-day dosing in subsequent cycles. Study BP22572 was later amended to give both drugs on the same day in all cycles. Either P (65 mg/m2, n=11; 80 mg/m2, n=6) or D (60 mg/m2, n=12; 75 mg/m2, n=6) was combined with T-DM1 at various doses (2 mg/kg or 2.4 mg/kg q3w or 1.2 mg/kg qw for TDM4652g; 2.4 mg/kg or 3.6 mg/kg q3w for BP22572). PK of T-DM1, total trastuzumab (conjugated + unconjugated trastuzumab), DM1, P, and D were evaluated and compared with historical singleagent data and across cycles within the study, using population analysis and/or noncompartmental analysis (NCA), as data allowed. Preliminary safety and efficacy data for the historical T-DM1 singleagent studies were reported previously (Krop JCO 2010; Burris JCO 2010; Krop ESMO 2010). Results: The PK of P and D were similar from cycle 1 to cycle 2 for maximum concentration (Cmax), area under the concentration-time curve, halflife, clearance (CL) and steady-state volume of distribution, and were also similar to historical single-agent data. The Cmax of DM1 was <8 ng/mL in both studies, and the average Cmax values were similar to historical single-agent data. T-DM1 PK parameters for the combination studies are within the range of historical single-agent data (Table). Data from additional patients and doses in the combination studies will be presented. Conclusion: The risk of clinically relevant PK-DI between T-DM1 and P or D appears to be low. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-07.

Anticancer activity of an extract from needles and twigs of Taxus cuspidata and its synergistic effect as a cocktail with 5-fluorouracil

BackgroundBotanical medicines are increasingly combined with chemotherapeutics as anticancer drug cocktails. This study aimed to assess the chemotherapeutic potential of an extract of Taxus cuspidata (TC) needles and twigs produced by artificial cuttage and its co-effects as a cocktail with 5-fluorouracil (5-FU).MethodsComponents of TC extract were identified by HPLC fingerprinting. Cytotoxicity analysis was performed by MTT assay or ATP assay. Apoptosis studies were analyzed by H & E, PI, TUNEL staining, as well as Annexin V/PI assay. Cell cycle analysis was performed by flow cytometry. 5-FU concentrations in rat plasma were determined by HPLC and the pharmacokinetic parameters were estimated using 3p87 software. Synergistic efficacy was subjected to median effect analysis with the mutually nonexclusive model using Calcusyn1 software. The significance of differences between values was estimated by using a one-way ANOVA.ResultsTC extract reached inhibition rates of 70-90% in different human cancer cell lines (HL-60, BGC-823, KB, Bel-7402, and HeLa) but only 5-7% in normal mouse T/B lymphocytes, demonstrating the broad-spectrum anticancer activity and low toxicity to normal cells of TC extract in vitro. TC extract inhibited cancer cell growth by inducing apoptosis and G2/M cell cycle arrest. Most interestingly, TC extract and 5-FU, combined as a cocktail, synergistically inhibited the growth of cancer cells in vitro, with Combination Index values (CI) ranging from 0.90 to 0.26 at different effect levels from IC50 to IC90 in MCF-7 cells, CI ranging from 0.93 to 0.13 for IC40 to IC90 in PC-3M-1E8 cells, and CI < 1 in A549 cells. In addition, the cocktail had lower cytotoxicity in normal human cell (HEL) than 5-FU used alone. Furthermore, TC extract did not affect the pharmacokinetics of 5-FU in rats.ConclusionsThe combinational use of the TC extract with 5-FU displays strong cytotoxic synergy in cancer cells and low cytotoxicity in normal cells. These findings suggest that this cocktail may have a potential role in cancer treatment.

C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 μM) and DM102 (IC(50) 20 μM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a >3-fold increase in caspase 3/7 activation, PARP cleavage, and a >70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.