Low Concentrations Of Paclitaxel Research Articles

Surface Modified Carvacrol-rich Satureja khuzestanica Essential Oil Nanoemulsion: A Novel Paclitaxel Formulation Induced Apoptosis on Paclitaxel-Resistant Breast Cancer Cells

Background: The ability of cancer cells to develop multidrug resistance (MDR) is a major challenge in modern chemotherapy. The current generation of commercially available paclitaxel formulations have not been designed to treat resistant tumours. In this study, a nanoemulsion-based delivery system was developed to enhance the efficacy of paclitaxel against resistant breast cancer cells. Methods: The nanoemulsion was formulated using carvacrol-rich Satureja khuzestanica essential oil. Modification of nanoemulsion was performed by incorporating tocopheryl polyethylene glycol 1000 succinate (TPGS) which could inhibit drug resistance in cancer cells. Fabrication of paclitaxel nanoemulsion was performed by high speed homogenization. The cytotoxicity of prepared formulation against resistant breast cancer cells was investigated by MTT assay. Flow cytometry technique was used for cell cycle arrest analysis and examination of the apoptosis induction ability of prepared nanoemulsion. Results: The nanoemulsion had a relatively small mean droplet diameter (93.6 ± 4.2 nm) and good long-term stability. The ability of paclitaxel to inhibit P-gp function in paclitaxel-resistant breast cancer cells (MCF-7/PTX) was synergistically enhanced by administering it within the nanoemulsion. The cytotoxicity of the prepared nanoemulsion on the HUVEC normal cells was much lower than that of MCF-7/PTX cells. Cell cycle analysis utilizing flow cytometry showed that the paclitaxel-loaded nanoemulsion promoted G2-M arrest. Flow cytometry also demonstrated that this nanoemulsion induced apoptosis in MCF-7/PTX cells. Interestingly, apoptosis increased from 20.0% for the free paclitaxel treated group to 85.2 % for the paclitaxel-loaded nanoemulsion treated group. Conclusion: This novel paclitaxel nanoemulsion efficiently suppressed the drug resistance of breast cancer cells and induced effective apoptosis in very low concentrations of paclitaxel.

Microtubule Dynamics Deregulation Induces Apoptosis in Human Urothelial Bladder Cancer Cells via a p53-Independent Pathway.

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

The Glutaminase Inhibitor Compound 968 Exhibits Potent In vitro and In vivo Anti-tumor Effects in Endometrial Cancer.

Glutamine is one of the primary nutrients utilized by cancer cells for energy production and biosynthesis. Hence, interfering with glutamine metabolism may impose anti-tumor effects. In this study, we assessed the anti-tumorigenic effects of glutaminase-1 enzyme (GLS1) inhibition in endometrial cancer in vitro and in vivo. The human endometrial cancer cell lines Ishikawa and HEC-1B were used. The effects of compound 968 on cell proliferation, cell cycle, apoptosis, cellular stress, and AKT/mTOR pathway inhibition were assessed. The synergistic effects of compound 968 and paclitaxel were also analyzed. The in vivo effect of compound 968 was evaluated using tumor xenografts. We found that the GLS1-targeting compound 968 was able to reduce cancer cell proliferation in a dose- and time-dependent manner. Compound 968 combined with a low concentration of paclitaxel showed stronger inhibitory effects. Further analyses indicated that compound 968 induced cell cycle arrest at the G1 phase, as well as increased the production of cellular reactive oxygen species (ROS) and promoted cellular stress and cancer cell apoptosis. Additionally, the treatment of endometrial cancer with compound 968 downregulated the expression of GLS1 and cyclin D1 and upregulated the expression of P21 and E-cadherin. Moreover, the treatment of endometrial cancer cells with compound 968 significantly reduced the levels of phospho-S6 ribosomal protein and phospho-AKT (Ser473), indicative of AKT/mTOR/S6 signaling pathway inhibition. In xenograft mouse models of endometrial cancer, compound 968 significantly suppressed tumor growth. In addition, western blotting analysis indicated that GLS1 expression was upregulated in human endometrial cancer tissues. Compound 968 may be a promising approach for the management of human endometrial cancer.

The MEK 1/2 inhibitor PD98059 exhibits synergistic anti-endometrial cancer activity with paclitaxel in vitro and enhanced tissue distribution in vivo when formulated into PAMAM-coated PLGA-PEG nanoparticles.

Endometrial cancer is the most common gynecological cancer that affects the female reproductive organs. The standard therapy for EC for the past two decades has been chemotherapy and/or radiotherapy. PD98059 is a reversible MEK inhibitor that was found in these studies to increase the cytotoxicity of paclitaxel (PTX) against human endometrial cancer cells (Hec50co) in a synergistic and dose-dependent manner. Additionally, while PD98059 arrested Hec50co cells at the G0/G1 phase, and PTX increased accumulation of cells at the G2/M phase, the combination treatment increased accumulation at both the G0/G1 and G2/M phases at low PTX concentrations. We recently developed poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) modified with polyethylene glycol (PEG) and coated with polyamidoamine (PAMAM) (referred to here as PGM NPs) which have favorable biodistribution profiles in mice, compared to PD98059 solution. Here, in order to enhance tissue distribution of PD98059, PD98059-loaded PGM NPs were prepared and characterized. The average size, zeta potential, and % encapsulation efficiency (%EE) of these NPs was approximately 184nm, + 18mV, and 23%, respectively. The PD98059-loaded PGM NPs released ~ 25% of the total load within 3days in vitro. In vivo murine studies revealed that the pharmacokinetics and biodistribution profile of intravenous (IV) injected PD98059 was improved when delivered as PD98059-loaded PGM NPs as opposed to soluble PD98059. Further investigation of the in vivo efficacy and safety of this formulation is expected to emphasize the potential of its clinical application in combination with commercial PTX formulations against different cancers.

Constitutive BAK/MCL1 complexes predict paclitaxel and S63845 sensitivity of ovarian cancer

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.

Plastin 3 down-regulation augments the sensitivity of MDA-MB-231 cells to paclitaxel via the p38 MAPK signalling pathway

Plastin 3 (PLS3) overexpression may serve as a marker for predicting chemotherapeutic outcomes in drug-resistant cancer cells, but the mechanism is unclear. Herein, we show that the down-regulation of PLS3 by PLS3 gene silencing augments the sensitivity of MDA-MB-231 triple-negative breast cancer cells to paclitaxel. Interestingly, a low concentration of paclitaxel was able to induce strong apoptosis in the PLS3-silenced cells. Further study revealed that p38 MAPK signalling was responsible for the increased sensitivity to paclitaxel in these cells, as the p38 MAPK inhibitor SB203580 impaired the changes mediated by PLS3 down-regulation in response to paclitaxel. Therefore, our study identifies PLS3 as a potential target for enhancing the p38 MAPK-mediated apoptosis induced by paclitaxel. Unlike paclitaxel, Abraxane was unable to induce strong apoptosis in the PLS3-silenced cells. As PLS3 was found to be involved in the process of endocytosis in breast cancer cells, the reliance of cellular Abraxane uptake on this process may render it not as efficient as paclitaxel in PLS3-depleted tumour cells. The finding that PLS3 could be a critical regulator of paclitaxel sensitivity may have important implications for breast cancer chemotherapy.

Inhibition of BUB1 Kinase by BAY 1816032 Sensitizes Tumor Cells toward Taxanes, ATR, and PARP Inhibitors In Vitro and In Vivo.

The catalytic function of BUB1 is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors and plays only a minor role in spindle assembly checkpoint activation. Here, we present the identification and preclinical pharmacologic profile of the first BUB1 kinase inhibitor with good bioavailability. The Bayer compound library was screened for BUB1 kinase inhibitors and medicinal chemistry efforts to improve target affinity and physicochemical and pharmacokinetic parameters resulting in the identification of BAY 1816032 were performed. BAY 1816032 was characterized for kinase selectivity, inhibition of BUB1 signaling, and inhibition of tumor cell proliferation alone and in combination with taxanes, ATR, and PARP inhibitors. Effects on tumor growth in vivo were evaluated using human triple-negative breast xenograft models. The highly selective compound BAY 1816032 showed long target residence time and induced chromosome mis-segregation upon combination with low concentrations of paclitaxel. It was synergistic or additive in combination with paclitaxel or docetaxel, as well as with ATR or PARP inhibitors in cellular assays. Tumor xenograft studies demonstrated a strong and statistically significant reduction of tumor size and excellent tolerability upon combination of BAY 1816032 with paclitaxel or olaparib as compared with the respective monotherapies. Our findings suggest clinical proof-of-concept studies evaluating BAY 1816032 in combination with taxanes or PARP inhibitors to enhance their efficacy and potentially overcome resistance.

Abstract 287: BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity

Abstract The spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance. Citation Format: Gerhard Siemeister, Anne Mengel, Wilhelm Bone, Jens Schröder, Sabine Zitzmann-Kolbe, Hans Briem, Amaury E. Fernández-Montalván, Simon Holton, Ursula Mönning, Oliver von Ahsen, Sandra Johanssen, Arwed Cleve, Marion Hitchcock, Kirstin Meyer, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2017-287

Abstract NTOC-105: RUXOLITINIB SENSITIZES OVARIAN CANCER CELLS TO LOW–DOSE PACLITAXEL THERAPY

Abstract In ovarian cancer chemotherapy initially reduces tumor burden; however, >70% of patients experience tumor recurrence, creating a need for additional therapies. We evaluated the ability of Ruxolitinib, a FDA approved JAK 1/2 kinase inhibitor, as a potential adjunctive therapy for paclitaxel treatment of ovarian cancer. Using murine ID8 and human TOV-112D ovarian cancer cells we tested the ability of Ruxolitinib alone and in combination with low-dose paclitaxel to limit cell proliferation and colony formation in vitro. Additionally, using flow cytometry we assessed the capacity of paclitaxel to induce the expression of cell surface markers associated with stemness and the cellular stress response. We next determined if Ruxolitinib could reduce paclitaxel-induced expression of stemness and stress markers. Finally daily administration of Ruxolitinib, both alone and in combination with a single low-dose administration of paclitaxel, was tested for the capacity to reduce tumor growth and extend survival using the syngeneic ID8 luciferase-expressing model of ovarian cancer in C57Bl/6 mice. Our in vitro data demonstrate that Ruxolitinib can sensitize both murine ID8 and human TOV-112D cells to low concentrations of paclitaxel, as evidenced by a significant reduction of cell proliferation and colony formation. We find that pre-incubation with Ruxolitinib is sufficient to sensitize cells to subsequent incubation with paclitaxel. Paclitaxel-induced expression of stemness and stress markers (SCA-1, GRP78 and CD133) is reduced by co-incubation with Ruxolitinib. In vivo, Ruxolitinib plus administration of a single low-dose paclitaxel treatment is sufficient to limit tumor growth and significantly extend median survival from in mice bearing ID8 ovarian cancer tumors (Control 59days vs Ruxolitinib-paclitaxel 77 days). Together, these data indicate that Ruxolitinib can increase the susceptibility of both murine and human ovarian cancer cells to reduced concentrations paclitaxel chemotherapy in vitro and that combination therapy with Ruxolitinib and a single low-dose paclitaxel administration extends survival in mice. Our data provide important pre-clinical data to support the use of Ruxolitinib to enhance the efficacy of paclitaxel therapy for patients with ovarian cancer. Citation Format: Patrick Reeves PhD, Mojgan Abbaslou MD, Mark C. Poznansky MD PHD. RUXOLITINIB SENSITIZES OVARIAN CANCER CELLS TO LOW–DOSE PACLITAXEL THERAPY [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-105.

Abstract 4822: Combination efficacy of the selective HDAC inhibitor ACY-241 and paclitaxel in solid tumor models

Abstract ACY 241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor in clinical development in combination with pomalidomide and dexamethasone in multiple myeloma. Like the structurally related drug ricolinostat (ACY-1215), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. One of the major effects of HDAC6 inhibition is hyperacetylation of á-tubulin, which is suggestive of potential combination activity with taxanes in cancer treatment. Paclitaxel is a chemotherapeutic agent approved for use in the treatment of multiple solid tumor types, including breast cancer, ovarian cancer, non-small cell lung cancer and Kaposi's sarcoma (Weaver, 2014). Recent studies indicate that clinically relevant low concentrations of paclitaxel caused multipolar spindle formation resulting in aberrant mitosis and cell death, which further contributes to the anti-cancer efficacy of paclitaxel (Zasadil et al., 2014). In cell lines, combination treatment with ACY-241 and paclitaxel resulted in enhanced inhibition of proliferation and increased cell death. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft models of pancreatic and ovarian cancer. Cell-cycle analysis indicated that ACY-241 treatment in combination with paclitaxel caused synergistic inhibition of tumor cell proliferation through enhanced reduction of DNA replication in S-phase and increased induction of cell death and aneuploidy. ACY-241 treatment in combination with paclitaxel did not increase mitotic arrest but did increase the frequency of multipolar spindle formation during mitosis, leading to aberrant mitoses, abnormal nuclei, and cell death in agreement with a previous report (Zasadil et al). At the molecular level, the enhanced anti-cancer efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with further increased hyperacetylation of á-tubulin, suggesting these agents synergistically impact the regulation of tubulin biology. Interestingly,gamma-tubulin was not always detectable at each pole of these multipolar spindles, suggesting that multipolar spindle formation may be independent of centrosome amplification and/or splitting. Additional studies investigating the association between localization of spindle proteins and multipolar spindle formation are in progress. The enhanced efficacy of paclitaxel plus ACY-241 observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors. Citation Format: Pengyu Huang, Ingrid Almeciga-Pinto, Min Yang, Simon S. Jones, Steven N. Quayle. Combination efficacy of the selective HDAC inhibitor ACY-241 and paclitaxel in solid tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4822.

Low doses of paclitaxel enhance liver metastasis of breast cancer cells in the mouse model.

Paclitaxel is the most commonly used chemotherapeutic agent in breast cancer treatment. In addition to its well-known cytotoxic effects, recent studies have shown that paclitaxel has tumor-supportive activities. Importantly, paclitaxel levels are not maintained at the effective concentration through one treatment cycle; rather, the concentration decreases during the cycle as a result of drug metabolism. Therefore, a comprehensive understanding of paclitaxel's effects requires insight into the dose-specific activities of paclitaxel and their influence on cancer cells and the host microenvironment. Here we report that a low dose of paclitaxel enhances metastasis of breast cancer cells to the liver in mouse models. We used microarray analysis to investigate gene expression patterns in invasive breast cancer cells treated with low or clinically relevant high doses of paclitaxel. We also investigated the effects of low doses of paclitaxel on cell migration, invasion and metastasis in vitro and in vivo. The results showed that low doses of paclitaxel promoted inflammation and initiated the epithelial-mesenchymal transition, which enhanced tumor cell migration and invasion in vitro. These effects could be reversed by inhibiting NF-κB. Furthermore, low doses of paclitaxel promoted liver metastasis in mouse xenografts, which correlated with changes in estrogen metabolism in the host liver. Collectively, these findings reveal the paradoxical and dose-dependent effects of paclitaxel on breast cancer cell activity, and suggest that increased consideration be given to potential adverse effects associated with low concentrations of paclitaxel during treatment. Gene expression microarray data are available in the GEO database under accession number GSE82048.

BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers.

IntroductionIntrinsic or acquired chemoresistance is a major problem in oncology. Although highly responsive to chemotherapies such as paclitaxel, most triple negative breast cancer (TNBC) patients develop chemoresistance. Here we investigate the role of BRCA1-IRIS as a novel treatment target for TNBCs and their paclitaxel-resistant recurrences.MethodsWe analyzed the response of BRCA1-IRIS overexpressing normal mammary cells or established TNBC cells silenced from BRCA1-IRIS to paclitaxel in vitro and in vivo. We analyzed BRCA1-IRIS downstream signaling pathways in relation to paclitaxel treatment. We also analyzed a large cohort of breast tumor samples for BRCA1-IRIS, Forkhead box class O3a (FOXO3a) and survivin expression. Finally, we analyzed the effect of BRCA1-IRIS silencing or inactivation on TNBCs formation, maintenance and response to paclitaxel in an orthotopic model.ResultsWe show that low concentrations of paclitaxel triggers BRCA1-IRIS expression in vitro and in vivo, and that BRCA1-IRIS activates two autocrine signaling loops (epidermal growth factor (EGF)/EGF receptor 1 (EGFR)-EGF receptor 2 (ErbB2) and neurogulin 1 (NRG1)/ErbB2-EGF receptor 3 (ErbB3), which enhances protein kinase B (AKT) and thus survivin expression/activation through promoting FOXO3a degradation. This signaling pathway is intact in TNBCs endogenously overexpressing BRCA1-IRIS. These events trigger the intrinsic and acquired paclitaxel resistance phenotype known for BRCA1-IRIS-overexpressing TNBCs. Inactivating BRCA1-IRIS signaling using a novel inhibitory mimetic peptide inactivates these autocrine loops, AKT and survivin activity/expression, in part by restoring FOXO3a expression, and sensitizes TNBC cells to low paclitaxel concentrations in vitro and in vivo. Finally, we show BRCA1-IRIS and survivin overexpression is correlated with lack of FOXO3a expression in a large cohort of primary tumor samples, and that BRCA1-IRIS overexpression-induced signature is associated with decreased disease free survival in heavily treated estrogen receptor alpha-negative patients.ConclusionsIn addition to driving TNBC tumor formation, BRCA1-IRIS overexpression drives their intrinsic and acquired paclitaxel resistance, partly by activating autocrine signaling loops EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. These loops activate AKT, causing FOXO3a degradation and survivin overexpression. Taken together, this underscores the need for BRCA1-IRIS-specific therapy and strongly suggests that BRCA1-IRIS and/or signaling loops activated by it could be rational therapeutic targets for advanced TNBCs.

Abstract 4540: SAC abrogation by MPS1 kinase inhibition: preclinical proof of concept of a novel approach to tumor treatment

Abstract Cell cycle deregulation represents one of the classical hallmarks of cancer and consequently cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids). Targeted disruption of the cell cycle checkpoint offers a novel approach to cancer treatment: driving tumor cells into cell division despite DNA damage or unattached/misattached chromosomes resulting in a lethal degree of DNA damage or aneuploidy. MPS1, a mitotic kinase that is overexpressed in several human cancers, has been shown to function as the key kinase which activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the identification and functional characterization of novel inhibitors of MPS1 from two structurally unrelated chemical classes. In biochemical assays, these molecules inhibited the kinase activity of MPS1 with IC50 values in the single digit nanomolar range and have shown an exquisite selectivity against a panel of kinases. In cellular assays the compounds were able to abrogate nocodazole-induced SAC, to reduce time in mitosis, to induce multinuclearity and apoptotic cell death, and to inhibit tumor cell proliferation with IC50 values in the low nanomolar range. In combination experiments MPS1 inhibitors showed cooperativity with low concentrations of paclitaxel. MPS1 inhibitors showed limited efficacy in monotherapy (T/C ca. 0.6) in tumor xenograft studies. However, when combined with paclitaxel dosed at the maximal tolerated dose, low doses of MPS1 inhibitor strongly improved efficacy over paclitaxel or MPS inhibitor monotreatment in intrinsically paclitaxel-insensitive xenograft models. The combination treatment was well tolerated. These results validate the concept of SAC abrogation preclinically and pave the way to a clinical proof of concept. Citation Format: Dominik Mumberg, Gerhard Siemeister, Antje M. Wengner, Marcus Koppitz, Volker Schulze, Benjamin Bader, Stefan Prechtl, Bertolt Kreft, Karl Ziegelbauer. SAC abrogation by MPS1 kinase inhibition: preclinical proof of concept of a novel approach to tumor treatment. [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 4540. doi:10.1158/1538-7445.AM2014-4540

Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture

Peripheral neuropathy (PN) is a debilitating and dose-limiting side effect of treatment with the chemotherapeutic agent, paclitaxel. Understanding the effects of paclitaxel on sensory neuronal function and the signaling pathways which mediate these paclitaxel-induced changes in function are critical for the development of therapies to prevent or alleviate the PN. The effects of long-term administration of paclitaxel on the function of sensory neurons grown in culture, using the release of the neuropeptide calcitonin gene-related peptide (CGRP) as an endpoint of sensory neuronal function, were examined. Dorsal root ganglion cultures were treated with low (10nM) and high (300nM) concentrations of paclitaxel for 1, 3, or 5days. Following paclitaxel treatment, the release of CGRP was determined using capsaicin, a TRPV1 agonist; allyl isothiocyanate (AITC), a TRPA1 agonist; or high extracellular potassium. The effects of paclitaxel on the release of CGRP were stimulant-, concentration-, and time-dependent. When neurons were stimulated with capsaicin or AITC, a low concentration of paclitaxel (10nM) augmented transmitter release, whereas a high concentration (300nM) reduced transmitter release in a time-dependent manner; however, when high extracellular potassium was used as the evoking stimulus, all concentrations of paclitaxel augmented CGRP release from sensory neurons. These results suggest that paclitaxel alters the function of sensory neurons in vitro, and suggest that the mechanisms by which paclitaxel alters neuronal function may include functional changes in TRP channel activity. The described in vitro model will facilitate future studies to identify the signaling pathways by which paclitaxel alters neuronal sensitivity.

Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells

Drugs that target microtubules are potent inhibitors of angiogenesis, but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; however, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish, rather than elongated, cells, rapid but nondirectional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells, whereas more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay.

Tumor RNA-transfected dendritic cells and combined therapy with low dose 5-FU induce regression of murine colon cancer

Background We have recently observed that treatment of colon tumor cells with low concentration of paclitaxel increased the expression of several genes associated with antigen-presenting machinery. Since 5-fluoruracil (5-FU) is the main antineoplastic agent for colon cancer, in this study we aimed to evaluate: a) whether transfection of dendritic cells (DC) with drug-treated tumor cells RNA, enhances the effectiveness of DC-based vaccine; b) if the modulatory effects of vaccine can be observed in vivo, and c) if the combination of DC with low dose chemotherapy schedule improves the antitumor responsiveness.

Low-dose SN-38 with paclitaxel induces lethality in human uterine cervical adenocarcinoma cells by increasing caspase activity

Combination of anticancer drugs may provide a rational molecular basis for novel chemotherapeutic strategies. Paclitaxel and SN-38 (an active metabolite of CPT-11) are effective for many kinds of cancer. Therefore, we investigated the possibility that combination of these drugs could be effective against cervical adenocarcinoma cells. In this study, we examined cell growth inhibition after 96h using the MTT assay and examined the release of fragmented DNA into the cytoplasm during apoptotic cell death by PI staining. Single and combined use of paclitaxel and SN-38 produced significant cytolethality against the cervical adenocarcinoma cell line CAC-1. Addition of a low concentration of SN-38 reduced the IC50 value of paclitaxel compared to that without SN-38, although the low concentration of paclitaxel did not enhance the cytotoxicity of SN-38. FACS scan analysis suggested that these drugs induced apoptosis and cell cycle arrest, and that caspase-3 and -7 were activated in the process. MTT assay and the IC50 demonstrated that paclitaxel had strong cytotoxicity against CAC-1 as well as other cancer cells. In this study, though only a single cell line was used for the experiment and the data are limited, our results suggest that paclitaxel together with low-dose CPT-11 is a promising basis for a new combination cancer chemotherapy.

Abstract B14: Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA.

Abstract The central role of dendritic cells (DCs) in antigen presentation and induction of antitumor immunity supported the development of antitumor vaccines based on these cells. In previous studies, we have observed that treatment with ultra-low doses of paclitaxel (PAC) modulated human DCs to express the phenotype of mature and activated cells, and stimulated their antigen presentation activity. At the same time, tumor cells treated with low concentrations of drugs became more immunogenic and more susceptible to the lytic activity of cytotoxic T lymphocytes in vitro. Therefore, we aimed to verify if the transfection of DCs with drug-treated tumor cells RNA enhances the effectiveness of DC-based vaccines and if the in vitro effects are reproducible in vivo. Murine colon tumor cells (MC-38) were subcutaneously inoculated in C57Bl/6 mice. At the 10th and 17th days after inoculation, they received a DC vaccine transfected with RNA of MC-38 cells pre-treated with minimum effective concentration of PAC. These DCs expressed 12% higher levels of CD40 and were able to delay the tumor growth when compared with non-treated animals. Although the differences were not significant, our results suggest that the treatment of tumor cells with low concentration of PAC can enhance the immunogenicity of tumor cells, which is transfered for DCs by RNA transfection, and delay tumor growth. Financial support: FAPESP 2009/18331-8; FAPESP 2010/06013-9. Citation Format: Marcela Rodrigues Camargo, Cecília Pessoa Rodrigues, Carolina Mendonça Gorgulho, Fabiana Albani Zambuzi, Victória Elizabeth Galvão, Juliana Cristina Longo Frederico, Marcimara Penitenti, Ramon Kaneno. Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B14.

Abstract 1924: The aurora kinase inhibitor CYC3 synergizes with low concentrations of paclitaxel in pancreatic cancer cells in vitro

Abstract CYC3, an Aurora Kinase A specific inhibitor, suppresses the pancreatic cancer cell growth (72h GI50 2.4μM in MiaPaCa-2 cells and 1.95μM in Panc-1 cells), arrest the cells at M phase and induces apoptosis. In order to evaluate CYC3's clinical potential, we employed mathematical models and simulations to look for possible synergistic combinations of CYC3 with paclitaxel in MiaPaCa-2 and Panc-1 cells. We have identified low concentrations of paclitaxel (3nM) and CYC3 is synergistic in inhibiting pancreatic cell growth, achieving similar effects as high concentrations of paclitaxel (30nM). Liquid Chromatography-Mass Spectrometry (LC-MS) analysis shows that CYC3 does not alter the cellular uptake of paclitaxel, supporting a synergistic mechanism at molecular level. In addition, the combination of CYC3 with low concentration of paclitaxel (3nM) displays less myelotoxicity compared to high concentrations of paclitaxel (30nM) in colony formation assay using Colony Forming Unit of Granulocyte and Macrophage (CFU-GM) (60-70% vs 100% inhibition), suggesting a potentially safer but equally efficient application of paclitaxel in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1924. doi:1538-7445.AM2012-1924

Abstract 3064: An alternative mechanism of action for paclitaxel in breast cancer

Abstract Paclitaxel is a microtubule-stabilizing drug that binds to the β-tubulin subunit and accumulates in mitotic cells. As a chemotherapeutic agent, paclitaxel is used for the treatment of a variety of cancers including breast. Currently, no biomarker is available to predict which patients will benefit from paclitaxel treatment. Paclitaxel studies in cells focus primarily on relatively high concentrations of the drug, which cause cells to die following long-term mitotic arrest. The clinically relevant concentration of paclitaxel is currently unknown, although it is widely speculated to be in the low nM range. Cells exposed to low concentrations of paclitaxel complete mitosis with minimal delay, but frequently contain multipolar spindles. In chromosomally stable breast cancer cells, low concentrations of paclitaxel are sufficient to increase aneuploidy within two days of exposure. These data suggest that the mechanism of cell death due to low dose paclitaxel is likely to be quite different from the mechanism of cell death caused by high dose paclitaxel. They also predict that chromosomally instable (CIN) cells may be more sensitive to cell death caused by low doses of paclitaxel. CIN cells have an existing level of chromosomal missegregation, which is likely to be exacerbated by treatment with low doses of paclitaxel. Since missegregation of large numbers of chromosomes per division causes rapid cell death, these cells may be more likely to die after low dose paclitaxel treatment as a result of losing both copies of a vital chromosome. Chromosomally stable cells, which do not normally missegregate chromosomes with each division, would be more resistant to death following paclitaxel treatment than CIN cells. These data suggest that CIN may be used as a biomarker to determine which tumors are most likely to be sensitive to treatment with clinically relevant concentrations of paclitaxel. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3064. doi:1538-7445.AM2012-3064