Taxol Treatment Research Articles

Microtubules play a crucial role in regulating actin organization and cell initiation in cotton fibers.

Dynamic organization of actin and microtubule cytoskeletons directs a distinct expansion behavior of cotton fiber initiation from cell elongation. Cotton fibers are highly elongated single cells derived from the ovule epidermis. Although actin and microtubule (MT) cytoskeletons have been implicated in cell elongation and secondary wall deposition, their roles in fiber initiation is poorly understood. Here, we used fluorescent probes and pharmacological approaches to study the roles of these cytoskeletal components during cotton fiber initiation. Both cytoskeletons align along the growth axis in initiating fibers. The dorsal view of ovules shows that unlike the fine actin filaments (AFs) in nonfiber cells, the AFs in fiber cells are dense and bundled. MTs are randomized in fiber cells and well-ordered in nonfiber cells. The pharmacological experiments revealed that the depolymerization of AFs and MTs assisted fiber initiation. Both AF stabilization and depolymerization inhibited fiber elongation. In contrast, the proper depolymerization of MTs promoted cell elongation, although the MT-stabilizing drug consistently resulted in a negative effect. Notably, we found that the organization of AFs was correlated with MT dynamics. Stabilizing the MTs by taxol treatment promoted the formation of AF bundles (in fiber initials) and transversely aligned AFs (in elongating fibers), whereas depolymerizing the MTs by oryzalin treatment promoted the fragmentation of AFs. Collectively, our data indicates that MTs plays a crucial role in regulating AF organization and early development of cotton fibers.

CRIF1-CDK2 Interface Inhibitors Enhance Taxol Inhibition of the Lethal Triple-Negative Breast Cancer.

Simple SummaryThis study reported our most recent results for targeting triple-negative breast cancer (TNBC) with a low survival rate, using CR6-interacting factor 1–cyclin-dependent kinase 2 (CRIF1–CDK2) interface inhibitors, by inhibiting the resistance to taxol treatment. Presently, over 50% of TNBC patients become resistant to chemotherapy and, to date, no solution is available. The combined treatment, using CRIF1–CDK2 interface inhibitors with chemotherapy, provides an unprecedented strategy against the deadly TNBC.Paclitaxel (taxol), a chemotherapeutic agent, remains the standard of care for the lethal triple-negative breast cancer (TNBC). However, over 50% of TNBC patients become resistant to chemotherapy and, to date, no solution is available. CR6-interacting factor 1 (CRIF1) is reported to act as a negative regulator of the cell cycle by interacting with cyclin-dependent kinase 2 (CDK2). In our study, two selective CRIF1–CDK2 interface inhibitors were used to investigate whether they could exert anti-proliferative activity on the TNBC cell lines. When combined with taxol treatment, these two inhibitors can advance the cells from G0/G1 to S and G2/M phases, producing irreparable damage to the cells, which then undergo apoptosis. Moreover, they enhanced the reduction in cell proliferation induced by taxol in TNBC cells, thereby improving sensitivity to taxol in these cell lines. Importantly, the inhibitors did not regulate the cell cycle in normal cells, indicating their high selectivity towards TNBC cells. Overall, the resistance to the anti-proliferative effects induced by taxol can be significantly reduced by the combined treatment with selective CRIF1–CDK2 interface inhibitors, making a conceptual advance in the CDK-related cancer treatment.

An active RUNX1-ID1/ID3 axis governs differentiation and chemoresistance of cancer stem cell population in epithelial ovarian cancer cells

Progression, relapse, and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell (CSC) population. This enigmatic subpopulation of cancer cells has therefore emerged as promising therapeutic candidate. We earlier reported enrichment of CSC-like side population (SP) with increasing resistance towards Cisplatin and Paclitaxel either alone or in combination in epithelial ovarian cancer (EOC) cells. This SP population is a small proportion of the total population of cancer cells characterised with high expression of drug transporters, a unique feature of stem cells and thereby can be isolated through their efflux properties of DNA binding dyes. While the bulk non-SP (NSP) population of the cancer cells lack overexpression of the drug transporters and thus can be identified as the dye containing population. In this study, we show that increased expression of Runt related transcription factor 1 (RUNX1) maintains undifferentiated state of CSC-like SP cells through upregulation of inhibitors of DNA binding/differentiation genes (ID1 and ID3) in late cisplatin-paclitaxel resistant cells. Higher RUNX1 expression was found to correlate with decreased median overall survival and disease-free survival in The Cancer Genome Atlas (TCGA) data set of high grade serous ovarian cancer (HGSOC) patients. The protein-protein interaction network analysis of 397 upregulated genes in RUNX1-high samples of TCGA data show significant enrichment of pathways known to negatively regulate CSC differentiation. Intriguingly RUNX1 inhibition not only induces CSC differentiation but also downregulates anti-apoptotic protein BCL2 in both SP and NSP cells and potentiates cytotoxic effects of Cisplatin-Paclitaxel in chemoresistant EOC cells. Inhibition of BCL2 through Venetoclax treatment, a small molecule BH3 mimic, sensitized these cells to platinum taxol treatment. Altogether, our data reveal new regulatory roles by RUNX1 to modulate CSC differentiation via ID1 and ID3 and to promote chemoresistance through BCL2 upregulation.

Effects and mechanisms of FBXO31 on Taxol chemoresistance in esophageal squamous cell carcinoma

Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC.

Up-regulation of GSTT1 in serous ovarian cancer associated with resistance to TAXOL / carboplatin

Serous ovarian cancer (SOC) is the most common women cancer and the leading cause of cancer-related mortality among the gynaecological malignancies. Although effective chemotherapeutics combined with surgery are developed for the treatment, the five-year survival rate is unsatisfactory due to chemoresistance. To overcome this shortcoming of chemotherapy, we established taxol and carboplatin resistant SOC cell lines for the understandings of the molecular and cellular mechanisms of chemoresistance. Here, we found that these chemoresistant cell lines showed less viability and proliferation, due to more cells arrested at G0/G1 phase. Glutathione-S-transferases-theta1 (GSTT1) was significantly upregulated in these chemoresistant cells, along with other chemoresistant genes. Meanwhile, GSTT1 expression was also significantly upregulated in the SOC patient tissues after taxol treatment, indicating this upregulation was physiologically relevant to chemotherapy. Further, suppression of GSTT1 expression by shRNA in SOC cell lines led to more sensitivity to drug treatment, through increasing divided cells and promoting cell death. Moreover, the expression of DNA topoisomerase 1 (Topo I) was in synergy with that of GSTT1 in the chemoresistant cells, and GSTT1 can bind to Topo I in vitro, which suggested GSTT1 could function through DNA repair mechanism during chemoresistance. In summary, our data imply that GSTT1 may be a potential biomarker or indicator of drug resistance in serous ovarian cancer.

Radix Tetrastigma Extracts Enhance the Chemosensitivity in Triple-Negative Breast Cancer Via Inhibiting PI3K/Akt/mTOR-Mediated Autophagy

ObjectiveDrug resistance in tumors is one of the major factors that leads to chemotherapy failure. This study aims to investigate the effect of Radix Tetrastigma extracts (RTEs) on Taxol-induced autophagy and the chemosensitivity against drug resistance in triple-negative breast cancer (TNBC). MethodsTaxol-resistant MDA-MB-468 (MDA-MB-468/Taxol) cells were induced and treated with RTEs and/or Taxol. Mice were subcutaneously inoculated with MDA-MB- 468/Taxol cells to establish xenograft models. The associated protein levels were measured by western blotting. Flow cytometry, CCK-8 and EdU assay were performed to detect cell apoptosis, viability, and proliferation, respectively. ResultsIn MDA-MB-468/Taxol cells, RTEs & Taxol treatment increased cell apoptosis, reduced cell viability and proliferation, up-regulated anti-autophagy marker LC3I/LC3II ratio, and enhanced mTOR level. With RTEs & Taxol treatment, mTOR silencing downregulated LC3I/LC3II ratio, increased cell viability and proliferation, and reduced cell apoptosis, while mTOR overexpression showed the opposite results. PI3K inhibitor reduced AKT and mTOR levels, and the effects on cell activities were similar to the results of mTOR silencing. After RTEs & Taxol injection, xenograft tumor was smaller, and AKT, mTOR, LC3I/LC3II ratio and apoptotic marker cleaved caspase-3 were increased. ConclusionRTEs enhanced the chemosensitivity of resistant TNBC cells to Taxol through inhibiting PI3K/Akt/mTOR-mediated autophagy. MicroRTEs exerted anti-tumor effects in various cancers, and this study determined its role in TNBC. Taxol-resistant MDA-MB-468 cells were induced and xenograft models were established. We found that RTEs inhibited autophagy of MDA-MB-468/Taxol cells and reduced tumor growth. Inhibition of PI3K/Akt/mTOR pathway promoted autophagy of MDA-MB-468/Taxol cells. We may provide a new potential strategy for TNBC treatment.

Tracing of intracellular pH in cancer cells in response to Taxol treatment

ABSTRACT Genetically encoded pH-sensors are the promising instrument for intracellular pH (pHi) registration. In tumor tissue the reversed pH gradient is known to be the important hallmark of cancer and regulator of tumor response on chemotherapy. However the effect of chemotherapeutic drugs on the pHi of tumor cells is largely unknown.Here we using genetically encoded ratiometric pH-sensor SypHer2 were able to monitor pHi in vitro in cell monolayer and tumor spheroids and in vivo in tumor xenografts. In tumor cell monolayer different pHi dynamic was revealed in the dying cell and division-arrested surviving cells. The treatment effect of taxol varied in monolayer and tumor spheroids and pHi changes were able to reflect these difference. The tend to pHi decrease in respect to taxol in vivo matched with results obtained for the cell monolayer. Also in both cases the cell cycle-arrest was the main treatment effect in contrast to tumor spheroid, where the cell death was the primary result.These findings elucidate the significance of pHi in the mechanisms of taxol action on cervical cancer cells and will be valuable for development of new approaches for cancer treatment.

PERK activation by CCT020312 chemosensitizes colorectal cancer through inducing apoptosis regulated by ER stress

Endoplasmic reticulum (ER) stress is a significant mechanism for chemoresistance to colorectal cancer (CRC) treatment. The RNA-like endoplasmic reticulum kinase (PERK) is critical for ER stress induction. In the present study, we attempted to explore whether PERK activator CCT020312 (CCT) could be effective for CRC treatment, and reveal the underlying mechanisms. We first found that CCT dose- and time-dependently reduced CRC cell proliferation. Importantly, it markedly improved the chemosensitivity of CRC cells that were drug-sensitive or -resistant to taxol treatment, as evidenced by the significantly decreased cell viability. Moreover, CCT at the non-toxic concentration exhibited obviously synergistic effects with taxol to induce apoptosis and cell cycle arrest in G2/M phase in vitro. In addition, we showed that CCT alone considerably induced ER stress in CRC cells through a dose- and time-dependent fashion. Meanwhile, CCT combined with taxol caused significant ER stress through improving phosphorylated PERK, eukaryotic translation initiation factor 2α (eIF2ɑ), C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78). More studies showed that the interaction between PERK and GRP78 was a potential target for CCT to perform its regulatory events. Intriguingly, PERK knockdown markedly abolished the regulatory role of CCT and taxol cotreatments in cell proliferation suppression and apoptosis induction, indicating the importance of PERK for CCT to perform its anti-cancer bioactivity. Our in vivo experiments confirmed that CCT plus taxol dramatically reduced tumor growth in CRC xenografts. Together, all these results suggested that promoting PERK activation by CCT may be an effective therapeutic strategy to improve CRC to taxol treatment.

MicroRNA-424-5p enhances chemosensitivity of breast cancer cells to Taxol and regulates cell cycle, apoptosis, and proliferation.

Combination therapy has been considered as a potential method to overcome the BC chemoresistance. MicroRNAs (miRs) have been suggested as a therapeutic factor in the combination therapy of BC. This project aimed at examining the possible activity and molecular function of miR-424-5p and Taxol combination in the human BC cell line. MDA-MB-231 cells were treated with miR-424-5p mimics and Taxol, in a combined manner or separately. We used the MTT test for assessing the cell proliferation. In addition, flow-cytometry was used for evaluating apoptosis and cell-cycle. Expression levels of underlying molecular factors of miR-424-5p were assessed using western-blotting and qRT-PCR. The obtained results demonstrated that miR-424-5p repressed BC cell proliferation and sensitized these cells to Taxol treatment through the induction of apoptosis. Further investigations showed that miR-424-5p might increase BC chemosensitivity through the regulation of apoptosis-related factors including P53, Caspase-3, Bcl-2, and Bax as well as the proliferation-related gene c-Myc. Moreover, miR-424-5p restoration in combination with Taxol treatment decreased the colony formation by regulating Oct-4 and led to G2 arrest via modulating Cdk-2 expression. Western-blotting demonstrated that miR-424-5p may perform its anti-chemoresistance role by regulating the PD-L1 expression and controlling PTEN/PI3K/AKT/mTOR. Overall, the upregulation of miR-424-5p was indicated to upregulate the sensitivity of BC cells to treatment with Taxol. MiR-424-5p might regulate the chemosensitivity of the BC cell line by modulating PD-L1 and controlling the PTEN/mTOR axis. Therefore, the combination of miR-424-5p with Taxol would represent a novel procedure to treat against BC.

Fbf1 regulates mouse oocyte meiosis by influencing Plk1

Fas binding factor 1 (Fbf1) is one of the distal appendage proteins in the centriole, located at its distal and proximal ends. It influences the duplication and separation of centrosomes, thereby affecting the progression of the cell cycle during mitosis. However, the function of Fbf1 in meiosis has remained unclear. To explore the role of Fbf1 in the in vitro maturation of mouse oocyte, immunofluorescence staining was used to examine the Fbf1 location in the oocyte and their phenotype after protein deletion. Western blot was used to examine the protein abundance. This study showed that mouse oocytes express Fbf1 which locates at the spindle poles and around the microtubules. Through taxol and nocodazole treatment, and microinjection of siRNA, it was demonstrated that Fbf1 had an important role in the spindle assembly and chromosome separation during mouse oocyte meiosis In particular, microinjection of Fbf1-siRNA resulted in severe abnormalities in the spindle and chromosome arrangement, decreased aggregation of microtubules, disrupted the first oocyte meiosis, and the extrusion of the first polar body. Furthermore, in the Fbf1-siRNA group, there was reduced expression of Plk1 and its agglutination at the spindle poles, along with retarded chromosome segregation due to the activation of the spindle assembly checkpoint (SAC) component BubR1. These results indicate that Fbf1 may function in microtubule depolymerization and agglutination, control the microtubule dynamics, spindle assembly and chromosome arrangement and, thus, influence the mouse oocyte meiotic maturation.

Evaluation of metabolomics behavior of human colon cancer HT29 cell lines treated with ionic liquid graviola fruit pulp extract

Ethnopharmacological relevanceMedicinal plants have been used by indigenous people across the world for centuries to help individuals preserve their wellbeing and cure diseases. Annona muricata L. (Graviola) which is belonging to the Annonaceae family has been traditionally used due to its medicinal abilities including antimicrobial, anti-inflammatory, antioxidant and cancer cell growth inhibition. Graviola is claimed to be a potential antitumor due to its selective cytotoxicity against several cancer cell lines. However, the metabolic mechanism information underlying the anticancer activity remains limited. Aim of the studyThis study aimed to investigate the effect of ionic liquid-Graviola fruit pulp extract (IL-GPE) on the metabolomics behavior of colon cancer (HT29) by using an untargeted GC-TOFMS-based metabolic profiling. Materials and methodsMultivariate data analysis was used to determine the metabolic profiling, and the ingenuity pathway analysis (IPA) was used to predict the altered canonical pathways after treating the HT29 cells with crude IL-GPE and Taxol (positive control). ResultsThe principal components analysis (PCA) identified 44 metabolites with the most reliable factor loading, and the cluster analysis (CA) separated three groups of metabolites: metabolites specific to the non-treated HT29 cells, metabolites specific to the treated HT29 cells with the crude IL-GPE and metabolites specific to Taxol treatment. Pathway analysis of metabolomic profiles revealed an alteration of many metabolic pathways, including amino acid metabolism, aerobic glycolysis, urea cycle and ketone bodies metabolism that contribute to energy metabolism and cancer cell proliferation. ConclusionThe crude IL-GPE can be one of the promising anticancer agents due to its selective inhibition of energy metabolism and cancer cell proliferation.

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P>0.05). The 2.0μM taxol treatment improved chromosome configuration (P<0.05) with no effect on microtubule distribution compared with the control group (P>0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P<0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP<0.05;. bP<0.001: Different superscripts within a column indicate a significant difference.

Confidence intervals by constrained optimization-An algorithm and software package for practical identifiability analysis in systems biology.

Practical identifiability of Systems Biology models has received a lot of attention in recent scientific research. It addresses the crucial question for models' predictability: how accurately can the models' parameters be recovered from available experimental data. The methods based on profile likelihood are among the most reliable methods of practical identification. However, these methods are often computationally demanding or lead to inaccurate estimations of parameters' confidence intervals. Development of methods, which can accurately produce parameters' confidence intervals in reasonable computational time, is of utmost importance for Systems Biology and QSP modeling. We propose an algorithm Confidence Intervals by Constraint Optimization (CICO) based on profile likelihood, designed to speed-up confidence intervals estimation and reduce computational cost. The numerical implementation of the algorithm includes settings to control the accuracy of confidence intervals estimates. The algorithm was tested on a number of Systems Biology models, including Taxol treatment model and STAT5 Dimerization model, discussed in the current article. The CICO algorithm is implemented in a software package freely available in Julia (https://github.com/insysbio/LikelihoodProfiler.jl) and Python (https://github.com/insysbio/LikelihoodProfiler.py).

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

Aurovertin B exerts potent antitumor activity against triple-negative breast cancer in vivo and in vitro via regulating ATP synthase activity and DUSP1 expression.

Aurovertin B, a natural compound from Calcarisporium arbuscular, exhibits potent antiproliferative activity particularly against triple-negative breast cancer cells (TNBC), while having less cytotoxicity on normal breast cell MCF10A. However, very little is known about the in vivo antitumor activity of aurovertin B and the possible mechanism of the selective effect on triple-negative breast cancer cells. In this study, flow cytometry and DAPI staining analysis showed that aurovertin B treatment in human triple-negative breast cancer cell MDA-MB-231 could induce more apoptotic cells than taxol treatment group. Furthermore, the present study also revealed that aurovertin B induced apoptosis was due to regulation of ATP synthase activity rather than changes in gene expression. Interestingly, the cancer genome atlas (TCGA) data analysis implied that the expression level of DUSP1, a member of the dual-specificity phosphatases, was highly downregulated in breast tissue of TNBC patients compared with their adjacent normal tissues. Real-time PCR and western blot analyses further demonstrated that aurovertin B could dramatically increase mRNA and protein expression levels of DUSP1 in MDA-MB-231 cells but not in MCF10A cells. The potent anti-tumor activity of aurovertin B was further verified in a human MDA-MB-231 xenograft mouse model.

Aurora B Kinase Promotes CHIP-Dependent Degradation of HIF1α in Prostate Cancer Cells.

Hypoxia is a major factor in tumor progression and resistance to therapies, which involves elevated levels of the transcription factor HIF1α. Here, we report that prostate tumor xenografts express high levels of HIF1α and show greatly enhanced growth in response to knockdown of the E3 ligase CHIP (C-terminus of Hsp70-interacting protein). In multiple human prostate cancer cell lines under hypoxia, taxol treatment induces the degradation of HIF1α, and this response is abrogated by knockdown of CHIP, but not by E3 ligase VHL or RACK1. HIF1α degradation is accompanied by loss of function, evidenced by reduced expression of HIF1α-dependent genes. CHIP-dependent HIF1α degradation also occurs in cells arrested in mitosis by nocodazole instead of taxol. Mitotic kinase Aurora B activity is required for taxol-induced HIF1α degradation. Purified Aurora B directly phosphorylates HIF1α at multiple sites, and these modifications enhance its polyubiquitination by CHIP in a purified reconstituted system. Our results show how activation of Aurora B promotes CHIP-dependent degradation of HIF1α in prostate cancer cells. This new knowledge may affect the use of mitotic kinase inhibitors and open new approaches for treatment of hypoxic prostate tumors.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

The role of Caspase-1/GSDMD-mediated pyroptosis in Taxol-induced cell death and a Taxol-resistant phenotype in nasopharyngeal carcinoma regulated by autophagy.

Taxol has been widely used as a first-line chemotherapeutic agent for the treatment of advanced nasopharyngeal carcinoma (NPC). However, acquired drug resistance has caused great difficulties in clinical treatment. Pyroptosis is a newly discovered programmed cell death pathway, and Caspase-1 and gasdermin D (GSDMD) play key roles in driving canonical pyroptosis. Increasing evidence suggests that pyroptosis is associated with the development of cancer; however, the function and mechanism of pyroptosis in NPC remain obscure. In this study, we observed that Taxol treatment caused pyroptotic cell death, along with activation of Caspase-1 and maturation of IL-1β, as well as cleavage of GSDMD, which is the canonical pyroptosis executor. Furthermore, Taxol-induced pyroptotic cell death could be suppressed by Caspase-1 inhibitor (Z-YVAD-FMK) and GSDMD knockout. Moreover, NPC parental cells demonstrated higher levels of pyroptosis than Taxol-resistant cells, and pyroptosis mediated by Caspase-1/GSDMD suppression induced by a Caspase-1 inhibitor and GSDMD knockout could induce a Taxol-resistant phenotype in vitro and in vivo. By transfecting an siRNA targeting Beclin-1 into NPC Taxol-resistant cells, we discovered that autophagy could negatively regulate pyroptosis by inhibiting Caspase-1/GSDMD activation. Taken together, our results indicated that Caspase-1/GSDMD mediated Taxol-induced pyroptosis and a Taxol-resistant phenotype in NPC cell lines, which may be regulated by autophagy. Thus, we provide novel insight into the mechanisms of Taxol-induced cell death and a promising approach to improve the therapeutic outcomes of patients with advanced NPC.

Interfering cellular lactate homeostasis overcomes Taxol resistance of breast cancer cells through the microRNA-124-mediated lactate transporter (MCT1) inhibition

BackgroundBreast cancer, the most common invasive cancer of women, is a malignant neoplasm and the second main cause of cancer death. Resistance to paclitaxel (Taxol), one of the frequently used chemotherapy agents for breast cancer, presents a major clinical challenge. Recent studies revealed that metabolic alterations of cancer cells play important roles in chemo-resistance.Materials and methodsIn this study, Human breast cancer cells, BT474, SKBR3 and MCF7 were used to study the causal relationship between the lactate exporter, MCT1 (SLC16A1)-modulated glucose metabolism and Taxol resistance of breast cancer cells. Taxol resistant breast cancer cells were established. The intracellular lactate and extracellular lactate levels as well glucose uptake and oxygen consumption were measured. MicroRNA-124 expressions were detected by qRT-PCR from both breast cancer patient samples and breast cancer cells. Target of miR-124 was predicted and verified by Western blot and luciferase assay. An xenograft mice model was established and evaluated for the in vivo tumor therapeutic effects of MCT1 inhibitor plus microRNA-124 treatments.ResultsLow toxic Taxol treatments promoted cellular glucose metabolism and intracellular lactate accumulation with upregulated lactate dehydrogenase-A (LDHA) and MCT1 expressions. By establishing Taxol resistant breast cancer cell line, we found Taxol resistant cells exhibit upregulated LDHA and MCT1 expressions. Furthermore, glucose consumption, lactate production and intracellular ATP were elevated in Taxol resistant MCF7 cells compared with their parental cells. The miR-124, a tumor suppressive miRNA, was significantly downregulated in Taxol resistant cells. Luciferase assay and q-RT-PCR showed MCT1 is a direct target of miR-124 in both breast cancer cell lines and patient specimens. Moreover, co-treatment of breast cancer cells with either MCT1 inhibitor or miR-124 plus Taxol led to synergistically cytotoxic effects. Importantly, based on in vitro and in vivo results, inhibition of MCT1 significantly sensitized Taxol resistant cells. Finally, rescue experiments showed restoration of MCT1 in miR-124 overexpressing cells promoted Taxol resistance.ConclusionsThis study reveals a possible role of miRNA-214-mediated Taxol resistance, contributing to identify novel therapeutic targets against chemoresistant breast cancers.

Chemokine-triggered microtubule polymerization promotes neutrophil chemotaxis and invasion but not transendothelial migration.

Microtubules (MTs) are critically involved in the transport of material within cells, but their roles in chemotactic leukocyte motility and effector functions are still obscure. Resting neutrophils contain few MTs assembled in an MT organizing center (MTOC) behind their multilobular nuclei. Using a probe of real-time tubulin polymerization, SiR-tubulin, we found that neutrophils elongated their MTs within minutes in response to signals from the two prototypic chemotactic peptides, CXCL1 and fMLP. Taxol, a beta-tubulin binding and MT stabilizing drug, was found to abolish this CXCL1- and fMLP-stimulated MT polymerization. Nevertheless, taxol treatment as well as disruption of existing and de novo generated MTs did not impair neutrophil protrusion and squeezing through IL-1β-stimulated endothelial monolayers mediated by endothelial deposited CXCL1 and neutrophil CXCR2. Notably, CXCL1-dependent neutrophil TEM was not associated with neutrophil MT polymerization. Chemokinetic neutrophil motility on immobilized CXCL1 was also not associated with MT polymerization, and taxol treatment did not interfere with this motility. Nevertheless, and consistent with its ability to suppress MT polymerization induced by soluble CXCL1 and fMLP, taxol treatment inhibited neutrophil chemotaxis toward both chemotactic peptides. Taxol treatment also suppressed CXCL1- and fMLP-triggered elastase-dependent neutrophil invasion through collagen I barriers. Collectively, our results highlight de novo chemoattractant-triggered MT polymerization as key for neutrophil chemotaxis and elastase-dependent invasion but not for chemotactic neutrophil crossing of inflamed endothelial barriers.