Epothilone B-based 3-in-1 polymeric micelle for anticancer drug therapy

Triple negative breast cancer (TNBC) is characterized by the lack of of estrogen, progesterone and HER2/ErbB2 receptors. It is a highly heterogeneous class of breast cancer and transcriptomics has recently been used to define 6 major subtypes of TNBC. We studied a panel of 15 TNBC cell lines using a chemical biology approach where we measure the responses to 306 approved and investigational oncology drugs. Clustering of cell lines based on their overall drug responses resulted in a strikingly different grouping compared to the gene expression derived one, highlighting that the current TNBC subtyping is not easily converted to differential sensitivities to drugs. To further evaluate the nature of the drug responses and to differentiate between their cell growth and cytotoxic effects, we multiplexed the standard cell viability readout in the cell line screening with detection of cytotoxicity. This simple multiplexed readout identified several drug classes that previously had been assumed to be cytotoxic based on strong effects on cell viability (cell numbers) while they in fact showed no or a very heterogeneous effect on cytotoxicity. Drug classes exhibiting this type of response included mTOR inhibitors, cyclin-dependent kinase inhibitors (eg. alvociclib), mitotic inhibitors (eg. paclitaxel) as well as proteasome inhibitors (eg. bortezomib) and RNA synthesis inhibitors (eg. dactinomycin). Further investigation of these drug classes showed that their static effects were reversible and in some cases the cells even overcame the inhibitory effect in the presence of the drug in a matter of a few days. Given the non-toxic responses to major classes of anticancer compounds such as mTOR inhibitors, we performed combination screens with these compounds to identify other drugs with which they may synergize to promote cancer cell specific killing. Surprisingly, we instead found that mTOR inhibitors had an antagonistic effect on the activity of many other cancer drugs such as different cytotoxic and antimitotic drugs, tyrosine kinase inhibitors, HDAC inhibitors and PARP inhibitors, suggesting that combining these classes of drugs may be counterproductive also in the clinic. We also found out that accessing a cytotoxic readout allowed us to identify effective synergistic drug combination concentrations that were not seen in cell viability readouts. For example, these synergistic toxic combination responses were seen in DU4475 cells when the MEK inhibitor trametinib was combined either with the PARP inhibitor iniparib or with the broad spectrum tyrosine kinase inhibitor ponatinib. In conclusion, multiplexed cell viability cell death readouts in drug sensitivity testing yields novel critical information on single drug and drug combination activities and liabilities. With this we were able to conclude that antimitotic, mTOR, CDK, proteasome and metabolic inhibitors have a heterogeneous cytotoxic effect across the panel of TNBC cell lines in contrast to their homogenous effect on metabolic inactivation. Citation Format: Prson Gautam, Leena Karhinen, Agnieszka Szwajda, Sawan Kumar Jha, Bhagwan Yadav, Tero Aittokallio, Krister Wennerberg. Identification of subgroups of triple negative breast cancer cells with selective responses to mTOR, CDK, mitotic and proteasome inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-01.

Modulation of drug resistance by α-tubulin in paclitaxel-resistant human lung cancer cell lines

Editorial: Signalling pathways in anti-cancer drug resistance.

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

Cervical cancer is one of the most leading causes of women death. Currently, paclitaxel is still one of the main therapeutic regimens for cervical cancer patients. However, some patients developed to be paclitaxel-resistant. Hence, studies to find out the novel strategies to resolve this problem are important. Generating resistant cancer cell lines can be utilized as the potent tool to evaluate the efficacy of any therapeutic agent toward cancer drug-resistant problems. Current studies describing the methods to establish chemoresistance are lacking. Moreover, study in Indonesia conducting chemoresistance in cell line is limited. This study was aimed to elaborate the characteristics of HeLa cells during generation of paclitaxel-resistant cervical cancer cells. The parental HeLa cells were exposed to an escalating concentration of paclitaxel for a long time period. Subsequently, cells were divided into two groups for the evaluation of resistance characteristics. The values of inhibitory concentration 50 (IC50) and inhibitory concentration 90 (IC90) were analyzed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Our data showed that the longer exposing periods of paclitaxel, the higher IC50 and IC90 values of HeLa cells are. IC90 of paclitaxel in HeLa Pac RB was increased from 69 pM, 440 pM, 2,561 pM and 10,337 pM on 0th, 1st, 2nd, 3rd and 4th months, respectively. Interestingly, the resistant cells were recovered to be paclitaxel-sensitive when they were not being continuously exposed to paclitaxel. In addition, the paclitaxel resistant cells become less sensitive against 5-FU but not doxorubicin, cisplatin and etoposide. We were able to generate cervical cancer HeLa paclitaxel-resistant cell line. These cell line could potentially be utilized for further studies in order to understand the molecular mechanisms of drug resistance in cervical cancer and as a tool for cancer drug discovery.Keywords: cervical cancer, drug resistant cell line, paclitaxel resistant cells, stepwise escalating concentration.

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.

Effect of combined use of nonionic surfactant on formation of oil-in-water microemulsions

BackgroundThe development of drug resistance is a major cause of cancer therapy failures. To inhibit drug resistance, multiple drugs are often treated together as a combinatorial therapy. In particular, synergistic drug combinations, which kill cancer cells at a lower concentration, guarantee a better prognosis and fewer side effects in cancer patients. Many studies have sought out synergistic combinations by small-scale function-based targeted growth assays or large-scale nontargeted growth assays, but their discoveries are always challenging due to technical problems such as a large number of possible test combinations.MethodsTo address this issue, we carried out a medium-scale optical drug synergy screening in a non-small cell lung cancer cell line and further investigated individual drug interactions in combination drug responses by high-content image analysis. Optical high-content analysis of cellular responses has recently attracted much interest in the field of drug discovery, functional genomics, and toxicology. Here, we adopted a similar approach to study combinatorial drug responses.ResultsBy examining all possible combinations of 12 drug compounds in 6 different drug classes, such as mTOR inhibitors, HDAC inhibitors, HSP90 inhibitors, MT inhibitors, DNA inhibitors, and proteasome inhibitors, we successfully identified synergism between INK128, an mTOR inhibitor, and HDAC inhibitors, which has also been reported elsewhere. Our high-content analysis further showed that HDAC inhibitors, HSP90 inhibitors, and proteasome inhibitors played a dominant role in combinatorial drug responses when they were mixed with MT inhibitors, DNA inhibitors, or mTOR inhibitors, suggesting that recessive drugs could be less prioritized as components of multidrug cocktails.ConclusionsIn conclusion, our optical drug screening platform efficiently identified synergistic drug combinations in a non-small cell lung cancer cell line, and our high-content analysis further revealed how individual drugs in the drug mix interact with each other to generate combinatorial drug response.

To investigate the expression profiles of 86 miRNAs in paclitaxel-resistant prostate cancer cell lines and to identify the genes that have a role in the development of drug resistance. Three prostate cancer cell lines, androgen-dependent VCaP, androgen-independent PC-3 and DU-145, were used to obtain paclitaxel-resistant cells by progressively increasing the concentration of paclitaxel in the culture medium. Viability assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and sulforhodamine B were used to assess the cell resistance level and cytotoxic effects of paclitaxel treatment. Total RNA was isolated from both prostate cancer cell lines and their resistant versions, and cDNA samples were reverse transcribed from total RNA. Selected target genes of miRNAs that showed differences in expression and were estimated to be effective on drug resistance mechanism were analyzed with western blot analysis. Expression study of 86 miRNAs by RT-PCR demonstrated that several of the miRNAs were expressed at different levels in paclitaxel-resistant cells compared to wild-type cells. Moreover, the expression profiles of these miRNAs varied among different prostate cancer cell line types, with 13 miRNAs being up-regulated in the resistant cells. Among these, miR-200b-3p, miR-34b-3p and miR-375 exhibited a marked up-regulation. Further, miR-100-5p showed a prominent increase in paclitaxel-resistant VCaP-R and DU145-R cells. Western blot and RT-PCR studies showed that only the LARP1 and CCND1 genes were over-expressed up to 2-5 times in all paclitaxel-resistant cell lines compared to the other investigated genes. In this study, the three paclitaxel-resistant prostate cancer cell lines examined showed remarkably different miRNA expression profiles.

Despite favorable responses to initial chemotherapy, drug resistance is a major cause limiting chemotherapeutic efficacy in many advanced cancers. However, mechanisms that drive drug-specific resistance in chemotherapy for patients with advanced cancers are still unclear. Here, we report a unique role of death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) associated with paclitaxel resistance in cervical cancer cells. Interestingly, DRAK1 protein level was markedly decreased in paclitaxel-resistant cervical cancer cells without affecting its mRNA expression, which resulted in an increase in tumor necrosis factor receptor-associated factor 6 (TRAF6) expression, as well as an activation of TRAF6-mediated nuclear factor-kappa B (NF-κB) signaling cascade, thereby promoting tumor progression. DRAK1 depletion markedly increased the chemotherapeutic IC50 values of paclitaxel in cervical cancer cells. Ectopic expression of DRAK1 inhibited growth of paclitaxel-resistant cervical cancer cells in vitro and in vivo. Furthermore, DRAK1 was markedly underexpressed in chemoresistant cervical cancer patient tissues compared with chemosensitive samples. We found that DRAK1 protein was destabilized through K48-linked polyubiquitination promoted by the Cullin scaffold protein 3 (CUL3) / speckle-type POZ (poxvirus and zinc finger protein) protein (SPOP) E3 ubiquitin ligase in paclitaxel-resistant cells. Collectively, these findings suggest that DRAK1 may serve as a potential predictive biomarker for overcoming paclitaxel resistance in cervical cancer.

Chemotherapy is the mainstay of systemic treatment for triple negative breast cancer (TNBC); however, the development of drug resistance limits its effectiveness. Therefore, we investigated the underlying mechanism for drug resistance and potential approaches to overcome it for a more effective treatment for TNBCs. Using a pulse-stimulated selection strategy to mimic chemotherapy administration in the clinic, we developed a new paclitaxel-resistant MDA-MB-231 cell line and analyzed these cells for changes in autophagy activity, and the role and mechanisms of the increased autophagy in promoting drug resistance were determined. We found that the pulse-stimulated selection strategy with paclitaxel resulted in MDA-MB-231 variant cells with enhanced resistance to paclitaxel. These resistant cells were found to have enhanced basal autophagy activity, which confers a cytoprotective function under paclitaxel treatment stress. Inhibition of autophagy enhanced paclitaxel-induced cell death in these paclitaxel-resistant cells. We further revealed that up-regulated autophagy in resistant cells enhanced the clearance of damaged mitochondria. Last, we showed that the paclitaxel-resistant cancer cells acquired cross resistance to epirubicin and cisplatin. Together, these results suggest that combining autophagy inhibition with chemotherapy may be an effective strategy to improve treatment outcome in paclitaxel-resistant TNBC patients.

In Vitro Antimyeloma Effects of Inhibitors of the Heat Shock Protein 70 (Hsp70) Molecular Chaperone.

Paclitaxel resistance is a significant clinical problem. In the current study, we evaluated antiproliferative effects of a tubulin binding agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2-7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage and DNA fragmentation. Immunoblot analysis shows that survivin protein expression was depleted in resistant cells. BPR0L075 induced cell death in the paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms, supported by the absence of caspase-3 cleavage and ineffectiveness of caspase-3 inhibitor in protecting the resistant cells from BPR0L075 induced cell death. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a promising antimitotic agent in controlling taxane-resistant ovarian cancer cells. Citation Format: Xinli Liu, Weimin Sun, Xiaolei Wang, Erxi Wu. Tubulin binding agent BPR0L075 induces iitotic catastrophe in paclitaxel-resistant ovarian cancer cells that is independent of caspase-3 activation. [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 4500. doi:10.1158/1538-7445.AM2013-4500

Ovarian cancer is currently the most lethal gynecologic malignancy in developed countries, and paclitaxel is a cornerstone in the treatment of this malignancy. Unfortunately, the efficacy of paclitaxel is limited by the development of drug resistance. Clinical paclitaxel resistance is often associated with ABCB1 (MDR1) overexpression, and in vitro paclitaxel resistance typically demonstrates overexpression of the ABCB1 gene. In this study, we demonstrate that paclitaxel-resistant cell lines overexpress both ABCB1 and ABCB4 (MDR3). To evaluate the role of these transporters in paclitaxel-resistant ovarian cancer cells, small interference RNAs (siRNAs) were used to target ABCB1 and ABCB4 RNA in the paclitaxel-resistant SKOV-3TR and OVCAR8TR ovarian cancer cell lines. Treatment of these lines with either chemically synthesized siRNAs or transfection with specific vectors that express targeted siRNAs demonstrated decreased mRNA and protein levels of ABCB1 or ABCB4. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays of siRNA-treated cells demonstrated 7- to 12.4-fold reduction of paclitaxel resistance in the lines treated with the synthesized siRNA of ABCB1 and 4.7- to 7.3-fold reduction of paclitaxel resistance in the cell lines transfected with siRNA of ABCB1 expressing vectors. ABCB4 siRNA-treated cell lines showed minor reduction in paclitaxel resistance. These results indicate that siRNA targeted to ABCB1 can sensitize paclitaxel-resistant ovarian cancer cells in vitro and suggest that siRNA treatment may represent a new approach for the treatment of ABCB1-mediated drug resistance.

An increasing number of studies have confirmed that microRNAs (miRNAs) are involved in various biological processes, including tumor growth and drug resistance. MiR-4284 has been proved to be abnormally regulated in several cancers, but the function of miR-4284 in ovarian carcinoma (OC) is unclear. Paclitaxel resistance is a key obstacle in OC treatment. Here, the role of miR-4284 in cell sensitivity to paclitaxel in OC was investigated. Two OC cell lines (SKOV3ip1 and HeyA8) were utilized for the establishment of paclitaxel-resistant cell lines. Reverse transcription-quantitative PCR (RT-qPCR) was applied to analyze the levels of miR-4284 and potential mRNAs in OC cell lines. Western blotting was performed to evaluate the levels of DNA meiotic recombinase 1 (DMC1) protein and cell cycle-associated proteins. Identification of the relationship between miR-4284 and DMC1 was achieved by luciferase reporter assay. CCK-8 and flow cytometry assays were utilized for evaluating the impact of miR-4284 on the malignant characteristics of paclitaxel-resistant OC cells. MiR-4284 was upregulated in paclitaxel-resistant OC cell lines and correlated with an adverse prognosis in OC patients. Depletion of miR-4284 suppressed cell proliferation and cell cycle progression of paclitaxel-resistant OC. MiR-4284 targeted DMC1 which was downregulated in paclitaxel-resistant cells and reversed the inhibitory influence of miR-4284 silencing on the malignant characters of paclitaxel-resistant OC cells. MiR-4284 targets DMC1 to suppress sensitivity to paclitaxel in human OC cells.