Taxol-resistant Cancer Cells Research Articles

Combination of ruthenium (II) polypyridyl complex Δ-Ru1 and Taxol enhances the anti-cancer effect on Taxol-resistant cancer cells through Caspase-1/GSDMD-mediated pyroptosis

Taxol is the first-line drug for cancer treatment. However, tumor resistance to Taxol is still a significant challenge in clinical practice. Here, we studied the synergistic effect of a ruthenium (II) polypyridyl complex, Δ-[Ru(bpy)2(HPIP)](ClO4)2(Δ-Ru1, where bpy = 2,2′-bipyridine, HPIP = 2-(2-hydroxyphenyl) imidazo[4,5-f] [1, 10] phenanthroline) combined with Taxol (Δ-Ru1 & Taxol) on Taxol resistant cervical cancer HeLa cell line (HeLa/Taxol). The results of the Chou-Talalay and Synergyfinder analytical test show that the Δ-Ru1 & Taxol combination has a synergistic effect in HeLa/Taxol cells. Especially, vesicles structures were observed on the membranes of HeLa/Taxol cells treated with Δ-Ru1 & Taxol, accompanied by cell swelling, which characterizes pyroptosis. Furthermore, the release of Interleukin-1β (IL-1β) and Lactate Dehydrogenase (LDH) were increased. At the same time, the activation and cleavage of Caspase-1 and Gasdermin D (GSDMD), the key molecules of the pyroptosis pathway, were detected. In addition, the Δ-Ru1 & Taxol combination had a synergistic anti-tumor effect in the mice model and could effectively inhibit tumor growth and significantly reduce the side effects on the lungs and the neuroethology of Taxol. Taken together, the Δ-Ru1 & Taxol combination can induce cell death through Caspase-1/GSDMD-mediated pyroptosis to enhance the therapeutic effect on HeLa/Taxol cells. This study provides a novel idea for the combined application of ruthenium complexes and other drugs, which may be utilized to overcome cancer drug resistance.

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest

mTOR is a serine/threonine kinase and a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in cells arrested in mitosis. Expression of a non-phosphorylatable raptor mutant reactivates mTORC1 and significantly reduces cytotoxicity of the mitotic poison Taxol. This effect is mediated via degradation of PDCD4, a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A is able to enhance the effects of Taxol and restore sensitivity in Taxol-resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in the death versus slippage decision during mitotic arrest and may be exploited clinically to treat tumors resistant to anti-mitotic agents.

Role of the TLR4-androgen receptor axis and genistein in taxol-resistant ovarian cancer cells

Toll-like receptor 4 (TLR4) is often overexpressed in taxol-resistant cancer cells. Here we used whole-genome transcriptomic analysis to identify 787 upregulated genes in SKOV3 ovarian carcinoma cells that ectopically express TLR4. Using chromatin immunoprecipitation enrichment analysis, we observed that 27.8% of the TLR4-upregulated genes identified were androgen receptor (AR)-regulated genes. Accordingly, AR expression was induced in taxol-resistant SKOV3 cells overexpressing TLR4, whereas depletion of TLR4 by shRNA repressed AR expression. Activation of AR by androgens or silencing of AR using shRNA also regulated expression of AR-related genes. We found that expression of DCDC2, ANKRD18B, ALDH1A1, c14orf105, ITGBL1 and NEB was overexpressed in taxol-resistant cells, suggesting the involvement of these AR-related genes in taxol resistance. Pathway enrichment analysis confirmed that the expression of several upregulated genes enriched in steroid biosynthesis pathways was inducible by androgens, supporting the results of previous studies. We also observed that genistein inhibits AR activation, leading to suppression of AR-driven genes and reduced taxol resistance in ovarian cancer cells. Overall, we identified six TLR4- and AR-regulated genes involved in taxol resistance. Our results reveal that the TLR4/AR axis plays a critical role in taxol resistance and that genistein is a candidate compound to limit chemoresistance and improve cancer treatment in ovarian cancer.

Timosaponin AIII, a steroidal saponin, exhibits anti-tumor effect on taxol-resistant cells in vitro and in vivo

Timosaponin AIII (TAIII), a steroidal saponin isolated from the rhizome of Anemarrhena asphodeloides, exerted cytotoxic effect in many cancer cell lines. However, the effect of TAIII on resistant tumor cancer cells was unclear. In this study, MTT assay showed that TAIII exhibited significant cytotoxicity against A549/Taxol and A2780/Taxol cells in vitro. Annexin V-FITC/PI staining revealed that TAIII induced apoptosis in A549/T and A2780/T cells. Furthermore, Western blot analysis demonstrated that TAIII inhibited the expressions of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR) as well as Ras, Raf, mitogen-activated protein kinase (MEPK), extracellular regulated protein kinases (ERK) in two taxol-resistant cancer cell lines. Besides, in vivo studies demonstrated that TAIII inhibited tumor growth in a nude mouse xenograft model. Additionally, TAIII (2.5 and 5 mg/kg) also down-regulated the protein expressions of PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways in vivo. Taken together, these findings demonstrated that TAIII exhibited significant anti-tumor effect on taxol-resistant cells.

Taxol Analogues Exhibit Differential Effects on Photoaffinity Labeling of β-Tubulin and the Multidrug Resistance Associated P-Glycoprotein.

Several next-generation taxanes have been reported to possess high potency against Taxol-resistant cancer cell lines overexpressing βIII-tubulin and/or P-glycoprotein (P-gp), both of which are involved in drug resistance. Using a photoaffinity Taxol analogue, 2-( m-azidobenzoyl)taxol, two potent next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibited distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources that differ in β-tubulin isotype composition. They also specifically inhibited photolabeling of P-gp, and the inhibitory effect correlated well with the steady-state accumulation of [3H]vinblastine in a multidrug resistant (MDR) cell line, SKVLB1. Several microtubule-stabilizing agents (MSAs)-resistant cell lines from the human ovarian cancer cell line Hey were isolated, and their MDR1 and βIII-tubulin levels determined. Distinct potencies of the two taxanes against different MSA-resistant cells expressing unique levels of MDR1 and βIII-tubulin were found. Cytotoxicity assays, done in the presence of verapamil, indicated that SB-T-1214 is a substrate, although not as good as Taxol, for P-gp. The mechanisms involved in drug resistance are multifactorial, and the effectiveness of new Taxol analogues depends on the interaction between the drugs and all possible targets; in this case the two major cellular targets are β-tubulin and P-gp.

Pentamethinium salts as ligands for cancer: Sulfated polysaccharide co-receptors as possible therapeutic target

A series of pentamethinium salts with benzothiazolium and indolium side units comprising one or two positive charges were designed and synthesized to determine the relationships among the molecular structure, charge density, affinity to sulfated polysaccharides, and biological activity. Firstly, it was found that the affinity of the pentamethinium salts to sulfated polysaccharides correlated with their biological activity. Secondly, the side heteroaromates displayed a strong effect on the cytotoxicity and selectivity towards cancer cells. Finally, doubly charged pentamethinium salts possessing benzothiazolium side units exhibited remarkably high efficacy against a taxol-resistant cancer cell line.

Single-Cell Transcriptome Analyses Reveal Taxol Resistant Subpopulations in Esophageal Squamous Cancer Cells

Taxol resistance is a main problem in the chemotherapy for many different cancers, including esophageal squamous cell carcinoma (ESCC). The functional heterogeneity of gene expression within the taxol resistant cancer cells correlates with its taxol resistance. However, the mechanisms of taxol resistance remains poorly understood. Single-cell RNA-seq can provide comprehensive gene expression profiles in single cancer cells, which will allow us to gain better understanding the mechanism responsible for taxol resistance in ESCC. Through low dose and long-term treatment of taxol, we successfully induced taxol resistance in ESCC cell line KYSE 30. Gene expression profiles were measured utilizing population RNA-seq. Furthermore, single-cell RNA-seq was utilized to investigate the heterogeneity of taxol resistant cancer cells. 37 single cells from KYSE 30 cells and 73 single cells from taxol resistant KYSE 30 cells were subjected to single-cell RNA-seq for gene expression profiling. Except previously reported cell cycles, cell death, and cell proliferation, we found that genes in cell movement are significantly up-regulated in taxol resistant cancer cells. In canonical pathways, decreased EIF2 signaling pathway and increased epithelial adherens junctions are functionally involved in taxol resistance. With single-cell RNA-seq, taxol resistant cancer cells were clustered into two major subclones, which shown unexpected progressive dynamics of taxol resistance. Comparison of these two subpopulations, we found 307 specific genes in subclone 1 and 85 specific genes in subclone 2. The subclone 1 specific genes which were significantly enriched in the biosynthesis of amino acids and cell-cell adhesion, can be identified in population RNA-seq and single-cell RNA-seq, while the subclone 2 specific genes which were enriched in the RNA splicing and RNA stabilization, only can be identified in the single-cell RNA-seq. Single-cell RNA-seq reveals taxol resistance subpopulations in the resistant cancer cells. And the subclone specific genes may function as a real taxol resistance related genes, which could provide putative targets against drug-resistant for clinical cancer therapy.

Sulforaphane-cysteine induces apoptosis by sustained activation of ERK1/2 and caspase3 in human glioblastoma U373MG and U87MG cells.

We previously demonstrated that sulforaphane (SFN) inhibited invasion via sustained activation of ERK1/2 in human glioblastoma cells. However, sulforaphane-cysteine (SFN-Cys), an analog of SFN, enriched in plasma with longer half-life, had more potentiality to induce apoptosis. Here we investigated the molecular mechanisms of SFN-Cys-induced apoptosis in human glioblastoma U373MG and U87MG cells. Cell viability assay showed that SFN-Cys inhibited cell viability in a dose-dependent manner. Cell morphology observation also showed SFN-Cys increased the phenotype of cell death in a dose-dependent manner. Furthermore, flow cytometry assay showed that SFN-Cys induced apoptosis significantly in a dose-dependent manner in both cell lines. Furthermore, western blot analysis demonstrated that SFN-Cys induced activation of ERK1/2 in a sustained manner and the activation contributed to upregulation of Bax/Bcl-2 ratio and cleaved caspase3, and these results can be reversed by the ERK1/2 blocker PD98059. Our results showed that SFN-Cys induced cell apoptosis via sustained activation of ERK1/2 and the ERK1/2 mediated signaling pathways such as activation of caspase3 and apoptosis-related proteins, thus indicating that SFN-Cys might be a more promising therapeutic agent versus SFN to resist glioblastoma cells, especially in Taxol-resistant cancer cells.

LC-MS Based Sphingolipidomic Study on A2780 Human Ovarian Cancer Cell Line and its Taxol-resistant Strain

Drug resistance elicited by cancer cells continue to cause huge problems world-wide, for example, tens of thousands of patients are suffering from taxol-resistant human ovarian cancer. However, its biochemical mechanisms remain unclear. Sphingolipid metabolic dysregulation has been increasingly regarded as one of the drug-resistant mechanisms for various cancers, which in turn provides potential targets for overcoming the resistance. In the current study, a well-established LC-MS based sphingolipidomic approach was applied to investigate the sphingolipid metabolism of A2780 and taxol-resistant A2780 (A2780T) human ovarian cancer cell lines. 102 sphingolipids (SPLs) were identified based on accurate mass and characteristic fragment ions, among which 12 species have not been reported previously. 89 were further quantitatively analyzed by using multiple reaction monitoring technique. Multivariate analysis revealed that the levels of 52 sphingolipids significantly altered in A2780T cells comparing to those of A2780 cells. These alterations revealed an overall increase of sphingomyelin levels and significant decrease of ceramides, hexosylceramides and lactosylceramides, which concomitantly indicated a deviated SPL metabolism in A2780T. This is the most comprehensive sphingolipidomic analysis of A2780 and A2780T, which investigated significantly changed sphingolipid profile in taxol-resistant cancer cells. The aberrant sphingolipid metabolism in A2780T could be one of the mechanisms of taxol-resistance.

Synergistic cytotoxicity of cisplatin and Taxol in overcoming Taxol resistance through the inhibition of LDHA in oral squamous cell carcinoma.

The development of chemoresistance in patients represents a major challenge in cancer treatment. Lactate dehydrogenase-A (LDHA) is one of the principle isoforms of LDH that is expressed in breast tissue, controlling the conversion of pyruvate to lactate and also playing a significant role in the metabolism of glucose. The aim of this study was to identify whether LDHA was involved in oral cancer cell resistance to Taxol and whether the downregulation of LDHA, as a result of cisplatin treatment, may overcome Taxol resistance in human oral squamous cells. The OECM-1 oral epidermal carcinoma cell line was used, which has been widely used as a model of oral cancer in previous studies. The role of LDHA in Taxol and cisplatin resistance were investigated and the synergistic cytotoxicity of cisplatin and/or Taxol in oral squamous cells was analyzed. Cell viability was analyzed by MTT assay, LDHA expression was analyzed by western blot analysis and siRNA tranfection was performed to knock down LDHA expression. The present study results showed that decreased levels of LDHA were responsible for the resistance of oral cancer cells to cisplatin (CDDP). CDDP treatments downregulated LDHA expression, and lower levels of LDHA were detected in the CDDP-resistant oral cancer cells compared with the CDDP-sensitive cells. By contrast, the Taxol-resistant cancer cells showed elevated LDHA expression levels. In addition, small interfering RNA-knockdown of LDHA sensitized the cells to Taxol, but desensitized them to CDDP treatment, while exogenous expression of LDHA sensitized the cells to CDDP, but desensitized them to Taxol. The present study also revealed the synergistic cytotoxicity of CDDP and Taxol for killing oral cancer cells through the inhibition of LDHA. This study highlights LDHA as a novel therapeutic target for overcoming Taxol resistance in oral cancer patients using the combined treatments of Taxol and CDDP.

YAP-induced resistance of cancer cells to antitubulin drugs is modulated by a Hippo-independent pathway.

Although antitubulin drugs are used widely to treat human cancer, many patients display intrinsic or acquired drug resistance that imposes major obstacles to successful therapy. Mounting evidence argues that cancer cell apoptosis triggered by antitubulin drugs relies upon activation of the cell-cycle kinase Cdk1; however, mechanistic connections of this event to apoptosis remain obscure. In this study, we identified the antiapoptotic protein YAP, a core component of the Hippo signaling pathway implicated in tumorigenesis, as a critical linker coupling Cdk1 activation to apoptosis in the antitubulin drug response. Antitubulin drugs activated Cdk1, which directly phosphorylated YAP on five sites independent of the Hippo pathway. Mutations in these phosphorylation sites on YAP relieved its ability to block antitubulin drug-induced apoptosis, further suggesting that YAP was inactivated by Cdk1 phosphorylation. Notably, we found that YAP was not phosphorylated and inactivated after antitubulin drug treatment in taxol-resistant cancer cells. Our findings suggest YAP and its phosphorylation status as candidate prognostic markers in predicting antitubulin drug response in patients.

Abstract 2130: Overcoming chemotherapy resistance by targeting LDHA mediated glycolysis

Abstract Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2130. doi:10.1158/1538-7445.AM2011-2130

MicroRNA-125b Confers the Resistance of Breast Cancer Cells to Paclitaxel through Suppression of Pro-apoptotic Bcl-2 Antagonist Killer 1 (Bak1) Expression

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

Paclitaxel Nanocrystals for Overcoming Multidrug Resistance in Cancer

Here we described a paclitaxel (PTX) nanocrystal formulation using d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) as the sole excipient for overcoming multidrug resistance (MDR), a key challenge in current cancer therapy. To the best of our knowledge, it is the first report on PTX nanocrystals which can reverse MDR. TPGS serves as a surfactant to stabilize the nanocrystals and a P-gp inhibitor to reverse MDR. The size and morphology of the nanocrystals were studied by transmission electron microscopy, and the crystalline structure was determined by powder X-ray diffraction. An in vitro drug release profile showed that the nanocrystals exhibited sustained release kinetics compared to Taxol, which is the clinical paclitaxel formulation. The cytotoxicity and antitumor efficacy in xenograft models were also investigated. It is demonstrated that PTX/TPGS nanocrystals have significant advantages over Taxol in achieving better therapeutic effect in Taxol-resistant cancer cells both in vitro and in vivo, which was also confirmed by apoptosis assays. We envision that further development of this type of nanocrystal will provide a novel strategy for drug delivery and multidrug resistance treatment.

Natural Product Celastrol Destabilizes Tubulin Heterodimer and Facilitates Mitotic Cell Death Triggered by Microtubule-Targeting Anti-Cancer Drugs

BackgroundMicrotubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy.Methodology and Principal FindingsWe report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death.Conclusions and SignificanceWe provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.

Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol

BackgroundTaxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.ResultsTaxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.ConclusionLDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Anti‐apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells

TWIST, a basic helix-loop-helix transcription factor, has been reported to be associated with development and progression of human cancer. Recently, over expression of TWIST is found in cancer patients with shorter survival and poor response to chemotherapy. Previously, we found that upregulation of TWIST was responsible for the development of acquired resistance to taxol in a nasopharyngeal carcinoma (NPC) cell line, HNE1-T3 (Wang et al., Oncogene, 2004;24:274). In this study, we investigated the underlying molecular mechanisms responsible for the TWIST-mediated taxol resistance. By comparison of the parental HNE1 and its derivative HNE1-T3 cell lines, we found that the resistance to taxol in HNE1-T3 cells was associated with suppression of taxol-induced apoptosis evidenced by decreased expression of Bak and Bax and increased Bcl-2, as well as inhibition of PARP and caspase cleavage and DNA ladder formation. However, there was no correlation between taxol sensitivity and alterations on G2/M cell cycle distribution, suggesting that the TWIST-induced taxol resistance is mediated through protection against apoptosis but not mitotic arrest. Analysis of additional 8 NPC cell lines showed that upregulation of TWIST was associated with resistance to microtubule disrupting agents, especially taxol, and inactivation of TWIST through small RNA interference led to increased sensitivity to taxol-induced cell death. Subsequent studies also demonstrated that the TWIST-mediated taxol resistance may be regulated through its positive involvement with the Akt pathway. Our findings suggest an underlying molecular mechanism responsible for the TWIST-mediated chemodrug resistance and suggest a target for overcoming taxol resistance in cancer cells.

Survivin Deregulation in β-Tubulin Mutant Ovarian Cancer Cells Underlies Their Compromised Mitotic Response to Taxol

Taxol is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Taxol, the development of drug resistance hampers its clinical applicability. Herein we report that beta-tubulin mutant, Taxol-resistant ovarian cancer cells exhibit defective mitotic response to Taxol, even at high concentrations that are sufficient to trigger apoptosis. This mitotic response-defective phenotype is independent of p53 status. We have found that survivin, the mitosis regulator and inhibitor of apoptosis protein, is deregulated in these Taxol-resistant cancer cells; Taxol fails to induce survivin levels and survivin phosphorylation in these cells, in contrast to their parental drug-sensitive counterparts. Exogenous expression of wild-type survivin is able to restore the mitotic response of the resistant cells to Taxol treatment. On the other hand, exogenous expression of dominant-negative survivin abrogates the Taxol-induced mitotic response in drug-sensitive cancer cells. We have also found that overexpression of the mitotic kinase Cdk1, which phosphorylates survivin, is unable to restore the Taxol-induced mitotic response in the resistant cells. Our results show the importance of survivin for the mitotic response in the context of Taxol resistance and provide novel insights into the mechanisms of mitotic arrest and apoptosis induced by microtubule-targeting agents.