Microtubule Inhibitors Paclitaxel Research Articles

Evaluation of neurotoxicity of anticancer drugs using nematode Caenorhabditis elegans as a model organism.

It is well established that platinum-based drugs, including oxaliplatin (L-OHP) and cisplatin (CDDP), as well as microtubule inhibitors paclitaxel (PTX) and vincristine (VCR), are associated with chemotherapy-induced peripheral neuropathy (CIPN). In this study, we examined and compared the characteristics of neuropathies induced by L-OHP, CDDP, PTX, and VCR to evaluate whether Caenorhabditis elegans (C. elegans) could serve as a model organism for human CIPN. Worms were cultured on nematode growth medium plates, and L1 larvae synchronized by gel filtration were employed. We then performed bioassays and examined motility. In the motility test, exposure was performed for 2, 24, and 48 hr, and time-dependent effects were measured for each exposure time and 24 hr after terminating exposure. Herein, we observed that L-OHP and CDDP exerted concentration-dependent effects above a certain concentration, and PTX and VCR exerted concentration-dependent negative effects in the bioassay. Motility recovered in L-OHP-, PTX-, and VCR-treated worms on terminating exposure. However, CDDP exposure tended to reduce motility even 24 hr after terminating exposure. L-OHP exposure could decrease motility 2 hr after exposure, with a trend toward recovery 24 hr after terminating drug exposure. The findings of the present study revealed that C. elegans could exhibit neuropathy characteristics suggested to be similar to those observed in humans, indicating that this organism could be a suitable model to explore human CIPN.

Lupeol and Paclitaxel cooperate in hindering hypoxia induced vasculogenic mimicry via suppression of HIF-1α-EphA2-Laminin-5γ2 network in human oral cancer.

Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1α led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1α altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1α by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5γ2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1α/EphA2/Laminin-5γ2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5γ2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC.

VERU-111: An Oral Tubulin Inhibitor That Suppresses Taxane-Sensitive and Taxane-Resistant Breast Cancer

Abstract Triple negative breast cancer (TNBC) patients have poorer overall prognosis relative to patients diagnosed with other molecular subtypes due to rapid onset of drug resistance to conventional chemotherapies and increased risk of visceral metastases. The microtubule inhibitor paclitaxel (Taxol, a taxane) is a frontline therapy for advanced breast cancer. We evaluated in TNBC models the preclinical safety and efficacy of a novel, potent, and orally bioavailable tubulin inhibitor, VERU-111, a tubulin inhibitor targeting the colchicine binding site. VERU-111 showed potent anti-proliferative and anti-migratory activity against several taxane-sensitive and taxane-resistant TNBC breast cancer cell lines. Based on these observations, taxane-resistant HER2+ cell lines were generated, and were also found to be responsive to VERU-111 treatment. In vivo, orally administered VERU-111 inhibited MDA-MB-231 tumor growth in a dose-dependent manner with antitumor potency similar to paclitaxel, and repressed metastases originating from the mammary fat pad or following tail vein injection. In contrast, in a MDA-MB-231 paclitaxel-resistant (TxR) subline, tumor growth was refractory to paclitaxel whereas VERU-111 significantly inhibited primary tumor growth and reduced lung and liver metastases. VERU-111 was then tested in a luciferase-labeled, multidrug resistant patient-derived xenograft (PDX) TNBC model. VERU-111 significantly inhibited HCI-10 PDX tumor growth and suppressed the expansion of axillary lymph node metastases present prior to initiation of therapy while suppressing lung, liver, bone and kidney metastases at study endpoint. Moreover, in contrast to paclitaxel, VERU-111 therapy did not cause a significant decrease in mouse body weight during treatment. Evaluation of efficacy of VERU-111 in taxane-sensitive and -resistant HER2+ xenograft models is in progress. Overall, we conclude that VERU-111 is a new generation orally bioavailable tubulin inhibitor that potently inhibits the growth of taxane-sensitive and taxane-resistant breast cancers with reduced adverse side effects relative to paclitaxel. Importantly, VERU-111 is well-tolerated in patients as evaluated in phase I/II clinical trials for advanced prostate cancer patients (NCT03752099). We propose that VERU-111 will be an effective second line therapy for patients with advanced breast cancer who progress on taxane-based therapeutic regimens.

Identifying fates of cancer cells exposed to mitotic inhibitors by quantitative phase imaging.

Cell cycle deregulation is a cancer hallmark that has stimulated the development of mitotic inhibitors with differing mechanisms of action. Quantitative phase imaging (QPI) is an emerging approach for determining cancer cell sensitivities to chemotherapies in vitro. Cancer cell fates in response to mitotic inhibitors are agent- and dose-dependent. Fates that lead to chromosomal instabilities may result in a survival advantage and drug resistance. Conventional techniques for quantifying cell fates are incompatible with growth inhibition assays that produce binary live/dead results. Therefore, we used QPI to quantify post-mitotic fates of G0/G1 synchronized HeLa cervical adenocarcinoma and M202 melanoma cells during 24 h of escalating-dose exposures to mitotic inhibitors, including microtubule inhibitors paclitaxel and colchicine, and an Aurora kinase A inhibitor, VX-680. QPI determined cell fates by measuring changes in cell biomass, morphology, and mean phase-shift. Cell fates fell into three groups: (1) bipolar division from drug failure; (2) cell death or sustained mitotic arrest; and (3) aberrant endocycling or multipolar division. In this proof-of-concept study, colchicine was most effective in producing desirable outcomes of sustained mitotic arrest or death throughout its dosing range, whereas both paclitaxel and VX-680 yielded dose-dependent multipolar divisions or endocycling, respectively. Furthermore, rapid completion of mitosis associated with bipolar divisions whereas prolonged mitosis associated with multipolar divisions or cell death. Overall, QPI measurement of drug-induced cancer cell fates provides a tool to inform the development of candidate agents by quantifying the dosing ranges over which suboptimal inhibitor choices lead to undesirable, aberrant cancer cell fates.

Improved Antitumor Activity of Novel Redox-Responsive Paclitaxel-Encapsulated Liposomes Based on Disulfide Phosphatidylcholine.

The microtubule inhibitor paclitaxel (PTX) is used to treat a wide range of solid tumors. Due to the poor aqueous solubility of PTX, a continuous demand for safe, efficient PTX formulations with improved antitumor activity exists. Here, we report a novel form of redox-sensitive paclitaxel (PTX)-encapsulated liposomes based on the previously developed disulfide phosphatidylcholine (SS-PC). PTX-loaded stealth liposomes (PTX/SS-LP) composed of SS-PC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000 (DSPE-PEG2000), and cholesterol were prepared using the reverse-phase evaporation method. The characterization of the PTX/SS-LP liposomes using dynamic light scattering and transmission electron microscopy confirmed theiruniform particle sizeandtypical unilamellar vesicle structure with an average bilayer thickness of approximately 4 nm. Changes in the size and morphology as well as the rapid release of PTX triggered by the addition of dithiothreitol revealed the redox sensitivity of PTX/SS-LP. Finally, evaluations in MCF-7 and A549 cells in vitro and in BALB/c mice in vivo revealed the improved anticancer efficiency, biodistribution, and safety of PTX/SS-LP compared with those of Taxol and nonredox-sensitive PTX/LP. In conclusion, PTX/SS-LP displays a redox-responsive release of paclitaxel with improved antitumor activity and has great potential as a next-generation stealth liposomal PTX delivery system.

Determination of the target proteins in chemotherapy resistant breast cancer stem cell-like cells by protein array

Breast cancer comes second among the causes of cancer deaths of women. Although new generation hormone therapy is a promising strategy, re-occurrence or emergence of drug resistance limits the success. According to the theory of cancer stem cells (CSCs); CSCs are immortal, tumor inducing and self renewing pluripotent cells and multiply as chemotherapy proceeds, making the chemotherapy inefficient. Emerging scientific reports indicate that the mechanisms of drug resistance are the main features that CSCs gain actually. Due to this fact, cancer stem cell markers should be clarified to target CSCs and this will play important role to reverse drug resistance.In this study, MCF-7/Pac, a cell line resistant to microtubule inhibitor paclitaxel and multiple drugs permanently, was used as a reference cell line for drug resistant mammary cancer. It has some properties that breast cancer stem cells possess so it is considerable to isolate breast cancer stem cell-like cells from MCF-7/Pac population. The chemotherapy resistant breast cancer stem–like (BCSC-like) cells were sorted from MCF-7/Pac population by using markers CD44, CD24 and ALDH. At the next step the proteins that are up-regulated in BCSC-like cells were determined by protein array analysis. Additionally the effect of paclitaxel on BCSC-like cell proliferation was determined.The MCF-7/Pac population contains 12.4% BCSC-like cells. The cells bearing BCSC-like cell phenotype exhibited resistance to paclitaxel. The over-expressed growth factors, MMP proteins, Frizzled proteins and IL-23 were found to be related to the BCSC-like cell proliferation. These results will guide both basic science and medical science.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Abstract B37: Overcoming resistance to chemotherapy using ZNF217 as a predictive marker and therapeutic target of breast cancer

Abstract When metastatic breast tumors do not respond to treatment (e.g., chemotherapy, endocrine therapy, and targeted therapies), ultimately the patient dies. Understanding the molecular events that cause metastatic recurrence after treatment and identifying effective second and third line therapies to treat recurrent tumors will significantly improve patient outcomes. We previously identified the transcription factor ZNF217 (human)/Zfp217 (mouse) as a prognostic marker of poor outcome in breast cancer patients. ZNF217 is overexpressed in breast and other cancers, and this overexpression promotes reduced survival, increased metastasis, and reduced response to therapy in patients and in our animal models. We found that Zfp217 overexpression promotes an increase in self-renewal capacity, invasion, and metastasis, as well as expansion of a progenitor cell population during both normal mammary development and breast cancer progression. We hypothesized that Zfp217 overexpression promotes resistance to therapies by expanding a progenitor cell population that can metastasize and remain resistant to chemotherapy. To determine experimentally if Zfp217 overexpression in vivo caused chemotherapy resistance, we used mouse orthotopic transplants of mouse breast cancer cells ± Zfp217 in syngeneic hosts. After treatment with combination chemotherapies (i.e., microtubule inhibitor epothilone B, adriamycin, and cyclophosphamide (EAC)), the mice overexpressing Znf217 developed chemoresistance with increased tumor volume and decreased percent survival compared to control mice. Treated mice that overexpressed Znf217 in their tumors had increased tumor burden compared to control animals. These data suggested that mice overexpressing Znf217 developed resistance to the EAC chemotherapy. Interestingly, the chemoresistant tumors that overexpress Zfp217 had increased numbers of progenitor cells that expressed markers of both luminal and myoepithelial cells (K8+K14+). In addition, chemotherapy treatment reduced but did not eliminate lung metastases compared to controls. Because ZNF217 is expressed at the highest levels in breast cancer patients with the worst prognosis, we aimed to identify treatments that kill chemoresistant cancer cells that overexpress ZNF217. To overcome breast cancer chemoresistance caused by ZNF217 overexpression, we identified triciribine, a nucleoside analog and AKT inhibitor, as a drug that kills cells that overexpress ZNF217. Currently triciribine is in a Phase I-II clinical trial using triciribine monophosphate in combination with the microtubule inhibitor paclitaxel in patients with breast and other cancers. The Phase I trial is completed, and the Phase II trial is recruiting patients. To model the Phase II clinical trial and to determine if Zfp217 overexpression is predictive of response to triciribine in breast cancer, we tested the efficacy of triciribine with paclitaxel and tested the order of treatment with paclitaxel and triciribine combination therapy by in vivo transplantation of mammary cancer cells ±Zfp217 overexpression. We found that the treatment order significantly impacted triciribine's therapeutic efficacy. This study directly influenced the design of the Phase II clinical trials with triciribine and paclitaxel in metastatic breast cancer patients. In addition, our animal models of chemoresistance after Zfp217 overexpression will be used to study the mechanisms of therapeutic resistance by the expansion of a progenitor cell population. Citation Format: Christopher Suarez, Joseph Sparano, Sunil S. Badve, Laurie E. Littlepage. Overcoming resistance to chemotherapy using ZNF217 as a predictive marker and therapeutic target of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B37.

Abstract 1713: BAX and BAK are preferentially activated by BIM and BID, respectively, affecting clinical chemotherapy response.

Abstract Apoptosis is a highly regulated form of cell death that is essential for maintenance of homeostasis and is controlled by the BCL-2 family of proteins. Two key members of this family, the effectors BAX and BAK, can be activated by BIM or BID, which triggers their oligomerization and permeabilization of the mitochondrial membrane, a crucial event during apoptosis. Despite their essential and distinct roles in maintaining homeostasis, BIM and BID are considered to be functionally redundant in their ability to activate BAX and BAK. However, by separately examining the activation of BAX and BAK by BIM and BID we find that these apoptosis-regulating proteins exhibit differing interaction preferences. Specifically, BIM preferentially activates BAX while BID preferentially activates BAK in murine and human cells. The preferential activation is evident when treating cells with BIM and BID BH3 peptides or recombinant, full-length proteins. Calculated EC50 values demonstrate a nearly twenty-fold decrease in the BID BH3 concentration required to activate BAK rather than BAX (0.54μM for BAK versus 9.3μM for BAX) and a three-fold decrease in the BIM BH3 concentration required to activate BAX rather than BAK (6.7μM for BAK versus 2.3μM for BAX). Based on these findings, we hypothesized that cells lacking BAK would be resistant to commonly used topoisomerase inhibitors including topotecan, etoposide and doxorubicin which can induce apoptosis via activation of BID. We confirm that BAK-/-, but not BAX-/-, cells are in fact resistant to these agents but not chemotherapies that induce apoptosis independently of BID activation including the alkylating agent cisplatin, the microtubule inhibitor paclitaxel, or the nucleoside analogue gemcitabine. Topoisomerase inhibitors are frequently used to treat ovarian cancer patients who have failed initial therapy with carboplatin and paclitaxel, yet clinical responses to these second-line therapies are varied. Importantly, we found that 23.8% of ovarian primary tumors possess a heterozygous or homozygous loss of BAK1, which encodes BAK. These patients exhibit an vastly inferior overall survival when treated with topoisomerase inhibitors in the clinic (p=0.0048) while loss of BAX had no effect on survival (p=0.9534). In addition, BAK1 loss had no effect on response to treatment with agents that were not topoisomerase inhibitors including carboplatin and paclitaxel. Thus, BIM and BID have non-overlapping roles in the induction of apoptosis via BAX and BAK which affect clinical responses to chemotherapy. Citation Format: Kristopher A. Sarosiek, John Bachman, Joan Montero, Luv Patel, Joshua J. Sims, Xiaoke Chi, David W. Andrews, Peter Sorger, Anthony Letai. BAX and BAK are preferentially activated by BIM and BID, respectively, affecting clinical chemotherapy response. [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 1713. doi:10.1158/1538-7445.AM2013-1713

Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy

Up-regulated signal transducers and activators of transcription (STAT)-mediated signaling is believed to contribute to the pathogenesis of a variety of solid and hematologic cancers. Consequently, inhibition of STAT-mediated signaling has recently been proposed as a potential new therapeutic approach to the treatment of cancers. Having shown previously that the pan-cyclin-dependent kinase inhibitor flavopiridol binds to DNA and seems to kill cancer cells via that process in some circumstances, we evaluated the hypothesis that flavopiridol might consequently disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate STAT3 downstream polypeptides, including the antiapoptotic polypeptide Mcl-1. SDS-PAGE/immunoblotting and reverse transcription-PCR were used to assess RNA and polypeptide levels, respectively. DNA cellulose affinity chromatography and a nuclear elution assay were used to evaluate the ability of flavopiridol to disrupt STAT3/DNA interactions. A STAT3 luciferase reporter assay was used to examine the ability of flavopiridol to attenuate STAT3-directed transcription. Colony-forming assays were used to assess cytotoxic synergy between flavopiridol and AG490. Flavopiridol was found to (a) disrupt STAT3/DNA interactions (DNA cellulose affinity chromatography and nuclear elution assay), (b) attenuate STAT3-directed transcription (STAT3 luciferase reporter assay), and (c) down-regulate the STAT3 downstream antiapoptotic polypeptide Mcl-1 at the transcriptional level (reverse transcription-PCR and SDS-PAGE/immunoblotting). Furthermore, flavopiridol, but not the microtubule inhibitor paclitaxel, could be combined with the STAT3 pathway inhibitor AG490 to achieve cytotoxic synergy in A549 human non-small cell lung cancer cells. Collectively, these data suggest that flavopiridol can attenuate STAT3-directed transcription in a targeted fashion and may therefore be exploitable clinically in the development of chemotherapy regimens combining flavopiridol and other inhibitors of STAT3 signaling pathways.

Macrophage podosomes assemble at the leading lamella by growth and fragmentation.

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with β-actin–ECFP and L-fimbrin–EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

Cross-resistance in the 2′,2′-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs

Gemcitabine (2′-2′-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30 000-fold dFdC-resistant variant AG6000 ( P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000–10 000-fold resistant to other deoxynucleoside analogues such as 1-β- d-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2′,2′-difluorodeoxyguanosine (dFdG) ( P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2.7-fold resistant to the lipophilic TS inhibitor AG337 ( P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) ( P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) ( P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel ( P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIα RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 ( P<0.02), mitomycin-C (MMC) ( P<0.05), cisplatin (CDDP) ( P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.