Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients.

BACKGROUND: The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment. METHODS: We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity. RESULTS: CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development. CONCLUSION: CEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.

An inhibitor of Bcl-2 family proteins induces regression of solid tumours.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Bexarotene (LGD1069, Targretin), a selective retinoid X receptor agonist, prevents and reverses gemcitabine resistance in NSCLC cells by modulating gene amplification.

Acquired drug resistance is a major obstacle in cancer therapy. As for many other drugs, this is also the case for gemcitabine, a nucleoside analogue with activity against non-small cell lung cancer (NSCLC). Here, we evaluate the ability of bexarotene to modulate the acquisition and maintenance of gemcitabine resistance in Calu3 NSCLC models. In the prevention model, Calu3 cells treated repeatedly with gemcitabine alone gradually developed resistance. However, with inclusion of bexarotene, the cells remained chemosensitive. RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3-GemR), a gene known to be involved in gemcitabine resistance. In addition, the expression of genes surrounding the chromosomal location of rrm1 was increased, suggesting that resistance was due to gene amplification at the chr11 p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 gene copy number was increased over 10-fold. Correspondingly, fluorescence in situ hybridization analysis of metaphase chromosomes showed an intrachromosomal amplification of the rrm1 locus. In the therapeutic model, bexarotene gradually resensitized Calu3-GemR cells to gemcitabine, reaching parental drug sensitivity after 10 treatment cycles. This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors generated from the resistant cells did not respond to gemcitabine but were growth inhibited when bexarotene was added to the cytotoxic agent. The data indicate that bexarotene can resensitize gemcitabine-resistant tumor cells by reversing gene amplification. This suggests that bexarotene may have clinical utility in cancers where drug resistance by gene amplification is a major obstacle to successful therapy.

Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

Crystal structure of HsEg5 in complex with clinical candidate CK0238273 provides insight into inhibitory mechanism, potency, and specificity.

HsEg5 is an important mitotic kinesin responsible for bipolar spindle formation at early mitosis. A rich body of evidence shows that inhibition of HsEg5 can result in mitotic arrest followed by cellular apoptosis. Recently identified HsEg5 inhibitor, CK0238273, exhibits potent antitumor activity and is currently in clinical trial. Here we report the cocrystal structure of the motor domain of HsEg5 in complex with CK0238273 at a 2.15 A resolution. Compared to the previously published HsEg5-Monastrol complex structure, CK0238273 shares the same induced-fit pocket with similar allosteric inhibitory mechanism. However, CK0238273 shows better fitting to the binding pocket with 65% increase of hydrophobic interaction area than that of Monastrol. Some unique hydrophilic interactions were also observed mostly between the phenyl ring and 8-chloro on quinazolinone of CK0238273 with ARG221 and GLY217. We believe that the combination of these interactions defines the superior potency and specificity of CK0238273.

Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

Identification of a small molecule that induces mitotic arrest using a simplified high-content screening assay and data analysis method.

High-content screening has emerged as a new and powerful technique for identifying small-molecule modulators of mammalian cell biology. The authors describe the development and execution of a high-content screen to identify small molecules that induce mitotic arrest in mammalian cancer cells. Many widely used chemotherapeutics, such as Taxol and vinblastine, induce mitotic arrest, and the creation of new drugs that also induce mitotic arrest may have tremendous therapeutic value. In their screen, the authors employed a simple DNA stain (DAPI) and a sensitive nonparametric statistical test to identify compounds from an internal collection of approximately 13,000 high-quality lead-like small molecules. Subsequent analysis of 1 active compound indicated that it induces mitotic arrest, assessed using a high-content phosphohistone H3 detection assay, and caused cell proliferation defects in multiple cancer cell lines. The active compound, a quinazolinone originating from a natural product-like subset of the screened compounds, is active in cells at approximately 500 nM and appears to act by inhibiting the polymerization of tubulin.

Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins.

Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank. Chimeric proteins Bcl-2(1) and Bcl-2(2) derived from the above sequences, although similar in structure, showed different binding affinities to Bak and Bad BH3 peptides (Petros et al., 2001). In this study, we show that the Bcl-2(1) sequence in normal and tumor human tissue samples differs from M13994 and M14745, and contains P59, T96, R110, S117 and G237. The actual sequence in the binding pocket matches the Bcl-2-Ig fusion sequence X06487, originally identified in a t(14:18) translocation of the Bcl-2 gene, associated with follicular lymphoma. The possible effects of the observed amino acid differences compared to M13994 and M14745 were investigated by combining structural data with fluorescence anisotropy. G110R substitution confers on Bcl-2(1) substantially increased binding affinity to Bak, Bad and Bax BH3 peptides, demonstrating that R110 is a key contributor to the BH3 binding affinity of Bcl-2. Although NMR structure did not predict R110 involvement in binding to these BH3 peptides, fluorescence anisotropy data clearly points to a critical role for this residue in binding to pro-apoptotic Bcl-2 family members.

Novel one-pot total syntheses of deoxyvasicinone, mackinazolinone, isaindigotone, and their derivatives promoted by microwave irradiation.

[reaction: see text]. Total syntheses of deoxyvasicinone (1), mackinazolinone (2), and 8-hydroxydeoxyvasicinone (3) via novel microwave-assisted domino reactions, as well as a novel three-component one-pot total synthesis of isaindigotone (5) promoted by microwave irradiation, are reported. The efficient reaction process enabled us to rapidly access related natural product derivatives and to identify a new class of cytotoxic agents.

Characterization of the anti-apoptotic mechanism of Bcl-B.

Bcl-B protein is an anti-apoptotic member of the Bcl-2 family protein that contains all the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal transmembrane domain. Our previous results showed that Bcl-B binds Bax and suppresses apoptosis induced by over-expression of Bax; however, Bcl-B does not bind or suppress Bak. To explore the molecular basis for the differential binding and suppression of Bax and Bak, we studied the BH3 dimerization domains of Bax and Bak. Chimeric mutants of Bax and Bak were generated that swapped the BH3 domains of these pro-apoptotic proteins. Bcl-B associated with and blocked apoptosis induced by mutant Bak containing the BH3 domain of Bax, but not mutant Bax containing the BH3 domain of Bak. In contrast, Bcl-X(L) protein bound and suppressed apoptosis induction by Bax, Bak and both BH3-domain chimeras. A strong correlation between binding and apoptosis suppression was also obtained using a series of alanine substitutions spanning the length of the Bax BH3 domain to identify critical residues for Bcl-B binding. Conversely, using structure-based modelling to design mutations in the BH3-binding pocket of Bcl-B, we produced two Bcl-B mutants (Leu86-->Ala and Arg96-->Gln) that failed to bind Bax and that also were unable to suppress apoptosis induced by Bax over-expression. In contrast, other Bcl-B mutants that still bound Bax retained protective activity against Bax-induced cell death, thus serving as a control. We conclude that, in contrast with some other anti-apoptotic Bcl-2-family proteins, a strong correlation exists for Bcl-B between binding to pro-apoptotic multidomain Bcl-2 family proteins and functional apoptosis suppression.

Human Chk1 expression is dispensable for somatic cell death and critical for sustaining G2 DNA damage checkpoint.

Mammalian Chk1 is an essential kinase for embryonic development and plays an important role in the cellular response to DNA damage. However, it remains unclear whether inhibition of Chk1 induces apoptosis in somatic cells. The uncertainty has become a critical issue for rationale design of Chk1 mechanism-based anticancer drugs. Here we show that Chk1 small interfering RNA (siRNA) effectively eliminates Chk1 protein expression without altering the cell cycle profile or inducing apoptosis in various human cancer cell lines under normal conditions. In the presence of DNA-damaging agents, however, Chk1 siRNA alone is sufficient to abrogate the DNA damage-induced G(2) checkpoint and significantly enhance apoptosis. Cell cycle kinetic profiles show that abrogation of G(2) arrest is mediated through shortening of the checkpoint. We also demonstrate that Chk1 siRNA enhances DNA damage-induced apoptosis in p53-deficient cancer cell lines and augments the growth inhibition conferred by DNA-damaging agents. These findings imply that Chk1 inhibitors will have low cytotoxicity on their own and can enhance the efficacy of DNA-damaging drugs.

Biological activity of A-289099: an orally active tubulin-binding indolyloxazoline derivative.

In this report, we describe the antitumor activity of A-289099, an indolyloxazoline derivative with antimitotic activity. A-289099 decreased the proliferation of a variety of cells with EC(50) values ranging from 5.1 to 12.8 nM in a P-glycoprotein-independent manner. In cultured cells, microtubules depolymerized in a time- and dose-dependent manner when treated with A-289099. In competition-binding assays, A-298099 competed with [(3)H]colchicine for binding to tubulin (K(i) = 0.65 micro M); however, it did not compete with [(3)H]paclitaxel or [(3)H]vincristine. There was an accumulation of cells in G(2)-M after treatment with A-289099 for 8 h and a subsequent increase in a subdiploid population and an increase in caspase-3 activity, indicative of apoptosis after treatment for 24 and 48 h. The antitumor activities of A-289099 were evaluated using the syngeneic M5076 murine reticulum sarcoma flank tumor model. Animals size-matched for established tumors ( approximately 350 mm(3)) were dosed p.o. (50 mg/kg every day) for 11 days starting on day 10 postinoculation. Tumors from A-289099-treated animals regressed throughout the 11-day dosing period with a percentage of the average treated-tumor-volume divided by the average vehicle-control-tumor-volume (% T/C) value of 11% after treatment for 7 days. Examination of tumor sections revealed an increase in internucleosomal DNA fragmentation or cell death within the central core after drug-treatment. A decrease in the perfusion of tumors was observed after drug-treatment that was localized primarily to the central core and closely associated with the regions of cell death. In summary, our findings indicate A-289099 is a promising, orally active tubulin-binding compound with antitumor activity in vivo.

Cloning and Characterization of the Rat Gene Encoding GAP-43.

GAP-43 is a gene expressed only in the nervous system. The protein product is believed to be important to neuronal growth and plasticity. Most, and likely all, neurons express high levels of GAP-43 during periods of neurite elongation. To initiate studies of GAP-43 gene regulation we have cloned the rat gene encoding GAP-43. The GAP-43 gene includes three exons. The first exon encodes only the amino terminal 10 amino acids, which corresponds to the membrane targeting domain of GAP-43. The second exon encodes a putative calmodulin binding domain and a protein kinase C phosphorylation site. The 5'-flanking sequence is unusual in that it lacks CAAT or TATA elements, and directs RNA transcription initiation from several sites. Some of the transcription start sites are used to a different degree in the central and peripheral nervous systems.

Farnesyltransferase inhibition: a novel method of immunomodulation.

Farnesyltransferase inhibitors (FTIs) are anticancer compounds that inhibit Ras GTPases. Since Ras GTPases play key roles in T cell activation and function, we hypothesized that FTIs have immunomodulatory properties and are potential antirejection agents. An investigation was performed on a potent FTI to evaluate this hypothesis in the in vitro setting. The in vitro effects of the FTI A-228839 were evaluated. Lectin- or antigen presenting cell (APC)-induced lymphocyte proliferation in the presence of A-228839 was measured. The effects of A-228839 on 1E5 T cell polarity were assessed by microscopy. Intracellular calcium ([Ca(2+)](i)) kinetics of lectin-activated lymphocytes was monitored by flow cytometry. The effects of A-228839 on peripheral blood mononuclear cell (PBMC) cytokine production was assessed by a cytometric bead array method. Activation-induced apoptosis was measured with an annexin V staining assay.A-228839 inhibited lectin-induced proliferation (IC(50)=0.24+/-0.11 microM). The inhibitory effects of A-228839 on lectin induced lymphocyte proliferation were additive to those of CsA. A-228839 was more effective in inhibiting APC-induced T cell proliferation (IC(50)=0.10+/-0.09 microM). A-228839 significantly disrupted the polarized shape of 1E5 T cells at physiologic concentrations. A-228839 altered PBMC baseline [Ca(2+)](i) but did not affect [Ca(2+)](i) kinetics during lectin-induced lymphocyte activation. A-228839 inhibited lymphocyte Th1 cytokine production at submicromolar levels and promoted apoptosis in lectin-activated lymphocytes.A-228839 potently inhibits lymphocyte activation and function. Our results suggest that FTIs may represent a new class of clinically useful immunomodulatory agents. A-228839 has potent in vitro immunomodulatory properties that warrant in vivo evaluation as an antirejection agent.

Chkl binds and phosphorylates BAD protein.

Chk1 (checkpoint kinase 1) is a serine-threonine kinase that is critical for G2/M arrest in response to DNA damage. Chk1 phosphorylates Cdc25C at serine-216, a major regulatory site, in response to DNA damage. Furthermore, Chk1 also phosphorylates Cdc25A on serine 123 which accelerates its degradation through the ubiquitin-proteasome pathway and arrests cells in late G2-phase after DNA damage. In the present study, we demonstrated that Chk1 phosphorylates pro-apoptotic protein BAD (Bcl-2/Bcl-XL-Antagonist, causing cell Death) in vitro. In vitro phosphorylation analysis with various mouse BAD peptides has revealed two phosphorylation sites for Chk1 at serine-155 and serine-170. When wild-type and mutant BAD (S155A) constructs were transfected into 293T cells, an association between BAD and Chk1 was observed by co-immunoprecipitation. In addition, there was an increase in the phosphorylation of serine-155 following DNA damage by adriamycin treatment. Our results suggest that Chk1 associates with BAD and phosphorylates the BAD protein at serine-155. Taken together, our results suggest that Chk1 may inactivate BAD by associating with and phosphorylating residues critical for BAD function in response to DNA damage.

Identification of polymorphisms associated with hypertriglyceridemia and prolonged survival induced by bexarotene in treating non-small cell lung cancer.

BACKGROUND: Bexarotene was evaluated in treating advanced non small cell lung cancer (NSCLC) in two phase III trials. Although a significant survival benefit was not observed for the overall bexarotene-treated population (617 patients), a third of bexarotene-treated patients who developed high-grade hypertriglyceridemia exhibited significantly longer survival. PATIENTS AND METHODS: In order to identify genomic polymorphisms that could serve as potential predictive biomarkers for response and improved survival in NSCLC patients, DNA samples extracted from plasma archived from 403 patients were genotyped using Affymetrix 500K whole genome SNP arrays and/or Sequenom iPLEX™ assays. RESULTS: Fourteen SNPs were identified on nine loci that showed significant associations with high-grade hypertriglyceridemia induced by bexarotene. Four such single nucleotide polymorphisms (SNPs) reside on the region upstream of solute carrier family 10, member 2 (SLC10A2), and one SNP is located close to lymphocyte cytosolic protein 1 (LCP1), whose expression correlated with the activity of bexarotene in tumor cells. CONCLUSION: We identified novel polymorphisms exhibiting significant association with bexarotene induced hypertriglyceridemia, implicating their potential in predicting bexarotene-improved survival response.

Indoloprodigiosins from the C-10 bipyrrolic precursor: new antiproliferative prodigiosin analogs.

The condensation of the C-10 methoxybipyrrole precursor (3) of prodigiosin with indoles and a related pyrrole derivative yields novel analogs of prodigiosin. Biological evaluation of these products revealed compounds that inhibit cancer cell proliferation from 50 nM to 50 microM.

Privileged structure-based quinazolinone natural product-templated libraries: identification of novel tubulin polymerization inhibitors.

A focused quinazolinone natural product-templated library was designed and synthesized. Compounds from this privileged structure-based library were identified as antimitotic agents acting through destabilization of tubulin polymerization. The results suggested that 2 could be a privileged substructure.

A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.