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β3-Tubulin production in human cancer cell lines. (A) CHO (lane 1), MCF7 (lane 2), HeLa (lane 3), and DU145 (lane 4) cells were lysed and analyzed on western blots with antibodies to β3-tubulin and actin. (B) The indicated cell lines were stained with antibodies specific for α-tubulin and β3tubulin. Note the heterogeneity of the cells with respect to β3tubulin staining. Arrows for DU145 indicate the rare presence of cells that are β3 negative. Bar, 50 μm.
Source publication
Class III β-tubulin (β3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested β3's effect on cell migration. Expression of β3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclita...
Contexts in source publication
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... a control, we used CHO cells that were previously shown to express β1, β4b, and β5 tubulin, but not β3 [22,23]. As shown in Figure 1A, CHO cells (lane 1) failed to react with the antibody; but MCF7 (lane 2), HeLa (lanfe 3), and DU145 (lane 4) cells, derived from human breast, cervical, and prostate tumors respectively, showed the presence of varying amounts of β3. Immunofluorescence microscopy ( Figure 1B) confirmed the presence of β3-tubulin in the microtubules and further indicated that the staining varied greatly on a cell-to-cell basis. ...
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... shown in Figure 1A, CHO cells (lane 1) failed to react with the antibody; but MCF7 (lane 2), HeLa (lanfe 3), and DU145 (lane 4) cells, derived from human breast, cervical, and prostate tumors respectively, showed the presence of varying amounts of β3. Immunofluorescence microscopy ( Figure 1B) confirmed the presence of β3-tubulin in the microtubules and further indicated that the staining varied greatly on a cell-to-cell basis. For example, we estimated that only 25% of the cells in the MCF7 cell population were positive for β3; whereas HeLa was 40% positive. ...
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... example, we estimated that only 25% of the cells in the MCF7 cell population were positive for β3; whereas HeLa was 40% positive. Only the DU145 cells appeared mostly uniform for β3 production, but even in that cell line, there were some negative cells (arrows, Figure 1B). We also examined K562 and KB3 cells but found no evidence for β3 production by either western blot analysis or by immunofluorescence microscopy (data not shown). ...
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... MCF7 cells migrate much more slowly than HeLa (5 versus 22 µm/h), cells were fixed and stained for fluorescence microscopy at different time points (7 h for HeLa and 24 h for MCF7) that corresponded to an approximate 30% closing of the wound. As anticipated, the β3-producing cells in both cell lines became enriched at the edge of the wound (Supplementary Figure 1A). To quantify the results, β3 positive and negative cells at the edge of the wound were counted in 20 microscopic fields chosen at random in each of 3 independent experiments. ...
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... quantify the results, β3 positive and negative cells at the edge of the wound were counted in 20 microscopic fields chosen at random in each of 3 independent experiments. The data confirmed that there was an increased percentage of β3- producing cells at the leading edge of the wound when paclitaxel was present (Supplementary Figure 1B) and indicated that β3 expression could be playing a significant role in the inability of microtubule targeted drugs to halt the migration of aggressive tumor cells. ...
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... tumor cell lines are notoriously heterogeneous and the cells frequently differ by multiple genetic changes that could influence their response to paclitaxel treatment. Also, the cells were not uniform in their level of β3 production (see Figure 1B). Thus, cells with low production that might be sufficient to provide resistance could potentially be scored as β3-negative and thereby complicate the quantification of experiments such as those shown in Supplementary Figure 1. ...
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... the cells were not uniform in their level of β3 production (see Figure 1B). Thus, cells with low production that might be sufficient to provide resistance could potentially be scored as β3-negative and thereby complicate the quantification of experiments such as those shown in Supplementary Figure 1. To eliminate these ambiguities, we turned to HAβ3-5, a cloned CHO cell line, created by transfection with HA-tagged β3-tubulin under the control of a tetracycline regulated promoter, that exhibits homogenous expression of the β3 isotype when the cells are grown in the absence of the antibiotic [15]. ...
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... eliminate these ambiguities, we turned to HAβ3-5, a cloned CHO cell line, created by transfection with HA-tagged β3-tubulin under the control of a tetracycline regulated promoter, that exhibits homogenous expression of the β3 isotype when the cells are grown in the absence of the antibiotic [15]. The parental CHO cells used to generate this cell line were shown to have no detectable expression of β3 by antibody binding ( Figure 1A), 2D gel analysis [23], or sequencing of expressed cDNAs for β-tubulin [22]. By combining HAβ3-5 with wild-type cells, we created a wound healing experiment similar to that shown in Supplementary Figure 1A but with cells that were more clearly positive A transwell assay was used to measure the migration of cells from the upper to the lower chamber over a 6 h period at various paclitaxel concentrations. ...
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... parental CHO cells used to generate this cell line were shown to have no detectable expression of β3 by antibody binding ( Figure 1A), 2D gel analysis [23], or sequencing of expressed cDNAs for β-tubulin [22]. By combining HAβ3-5 with wild-type cells, we created a wound healing experiment similar to that shown in Supplementary Figure 1A but with cells that were more clearly positive A transwell assay was used to measure the migration of cells from the upper to the lower chamber over a 6 h period at various paclitaxel concentrations. The graph was generated by defining the number of cells that migrated to the lower chamber at 0 nM paclitaxel as 100% and expressing the data for the other concentrations relative to the zero control. ...
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Citations
... Alteration of growth of MTs, in turn, could be explained by increased expression of β-tubulin III after EMT described elsewhere [33][34][35][36] and confirmed in our study (Supplementary Fig. S2e). However, the effect of elevated expression of β-tubulin III on MT dynamics remained uncertain and the results of direct measurements show that increased dynamics of MTs containing β-tubulin III is observed in some cancer cells (A435) 29,37 while it is impeded in other types cells and in vitro 38,39 . We found that not only MT growth is stimulated by EMT but also the spatial organization of the MT array is "improved"-it becomes more radial, especially at cell edges. ...
Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially cytoskeleton dynamics are poorly described. Addressing the question we induced EMT in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there using immunostaining and life cell imaging of cells transfected with microtubule and focal adhesion markers. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We next analyzed three components of the cytoskeleton: microtubules, stress fibers and focal adhesions. The following changes were observed after EMT in cultured cells: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules.
... The mechanism of PTX resistance in tumor cells is mainly related to increased expression of efflux protein and b3tubulin. [45][46][47] To explore whether NO/PTX increases the sensitivity of PTX against HCC cells, we examined the expression of Pgp, b3-tubulin and apoptosis-related proteins by western blot. The results showed that compared with the PTX group, NO/PTX reduced the expression of P-gp, b3-tubulin, BCL-2 and nuclear NF-kB, while increasing the expression of caspase-3 and cleaved caspase-3 in Bel-7402 cells (Fig. 7A and B). ...
The objective of this study was to investigate the anticancer activities of biodegradable polymeric micelles composed of monomethoxy poly(ethylene glycol), polylactic acid, and nitric oxide (mPEG-PLA-NO) loaded with paclitaxel (PTX) as a nanomedicine delivery system. We aimed to compare the anticancer effects of these NO/PTX micelles with PTX alone and elucidate their mechanism of action. We evaluated the impact of NO/PTX and PTX on cell viability using Cell Counting Kit-8 (CCK8) assays conducted on the Bel-7402 liver cancer cell line. Additionally, we employed H22 xenografted mice to assess the in vivo tumor growth inhibitory activity of NO/PTX. To examine the cytotoxicity of NO/PTX, the intracellular levels of reactive oxygen species (ROS), and the expression of ferroptosis-related proteins, we conducted experiments in the presence of the ferroptosis inhibitor ferrostatin-1 (Fer-1) or the ROS inhibitor N-acetyl cysteine (NAC). Furthermore, we investigated the expression of endoplasmic reticulum stress (ERS) and apoptosis-associated proteins. Our results demonstrated that NO/PTX exhibited enhanced anticancer effects compared to PTX alone in both Bel-7402 cells and H22 xenografted mice. The addition of Fer-1 or NAC reduced the anticancer activity of NO/PTX, indicating the involvement of ferroptosis and ROS in its mechanism of action. Furthermore, NO/PTX modulated the expression of proteins related to ERS and apoptosis, indicating the activation of these cellular pathways. The anticancer effects of NO/PTX in liver cancer cells were mediated through the induction of ferroptosis, pyroptosis, ERS, and apoptosis-associated networks. Ferroptosis and pyroptosis were activated by treatment of NO/PTX at low concentration, whereas ERS was induced to trigger apoptosis at high concentration. The superior anti-tumor effect of NO/PTX may be attributed to the downregulation of a multidrug resistance transporter and the sensitization of cells to PTX chemotherapy. In summary, our study highlights the potential of mPEG-PLA-NO micelles loaded with PTX as a nanomedicine delivery system for liver cancer treatment. The observed enhancement in anticancer activity, combined with the modulation of key cellular pathways, provides valuable insights into the therapeutic potential of NO/PTX in overcoming resistance and improving treatment outcomes in liver cancer patients.
... Tau competes with PTX to inhibit its role in promoting microtubule aggregation, thereby causing resistance to PTX. 13 Altered expression levels of specific isotype microtubule proteins can also lead to PTX resistance. 14 Because of its non-ionizing and noninvasive properties, high-intensity focused ultrasound (HIFU) is a relatively safe treatment. 15,16 Since the repeated treatment of HIFU is possible and the complication occurrence rate is low, HIFU has become an attractive option for breast cancer therapy. ...
Background
Paclitaxel (PTX) is an important oncologic chemotherapeutic agent against breast cancer, but breast cancer patients develop significant resistance to PTX during chemotherapy. Alterations in tubulin and associated proteins have been implicated in resistance to PTX. High-intensity focused ultrasound (HIFU) induces deep tumor penetration of anti-tumor agents in solid tumors.
Methods
We investigated the influence of HIFU on the anti-tumor activities of PTX in breast cancer. Both in vivo and in vitro experiments were performed in this research: mice were treated with 2 mg/Kg PTX through tail vein injection, while breast cancer cells were treated with 400 nM PTX. Cell viability was analyzed through Cell Counting Kit-8. Cell apoptosis was evaluated through Annexin-V/PI Apoptosis Analysis Kit. The activities of catalase (CAT) and superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were evaluated by relative commercial kits.
Results
HIFU enhanced PTX-inhibited breast cancer cell viability and PTX-induced cell apoptosis. Simultaneous treatment of HIFU and PTX decreased the activities of CAT and SOD and increased the concentration of MDA. In mice bearing MDA-MB-231 tumors, the treatment of HIFU and PTX significantly decreased tumor size, increased body weight and elevated animal survival. HIFU enhanced the distribution of PTX in tumor tissues.
Conclusion
The performance of HIFU promoted the distribution of PTX and enhanced its anti-tumor activities in breast cancer.
... Tumor cell migration is a crucial process for cancer cell dissemination and metastasis that is controlled by extracellular matrix (ECM) remodeling, and dynamic reorganization of cell adhesions with neighboring cells and with the underlying connective tissue (29,30). Paclitaxel inhibited numbers of tumor cell migration by suppressing microtubule dynamics (31,32). Our study first showed that Tan-1 could inhibit the migration of ovarian cancer cells. ...
Background:
Paclitaxel is a widely used clinical first line chemotherapy drug for ovarian carcinoma. Tanshinone I (Tan-I) is one of the vital fat-soluble components, which derived from Chinese herbal medicine, Salvia miltiorrhiza Bunge. Herein, we evaluated whether Tan-I could enhance the efficacy of ovarian cancer to chemotherapy of Paclitaxel.
Methods:
Ovarian cancer cells A2780 and ID-8 were exposed with Tan-I (4.8 µg/mL), Paclitaxel (0.1 µg/mL), or Tan-I combination with Paclitaxel for 24 hours. The cell proliferation was analyzed by CCK8 and EdU staining. Cell apoptosis was analyzed by the TUNEL assay and flow cytometry. The protein levels were determined by western blot. Cell migration was analyzed by Transwell and wound healing. Cell senescence was analyzed by senescence-associated b-galactosidase staining. Antitumor activity was analyzed by a subcutaneous tumor xenograft model of human ovarian cancer in nude mice. The protein expression and apoptosis level of tumor tissues were analyzed by immunohistochemistry and TUNEL staining.
Results:
Tan-I treatment significantly elevated the Paclitaxel-cause reduction of A2780 and ID-8 cell proliferation and cell migration. Tan-I combination with Paclitaxel promotes apoptosis of cancer cells by promoting Bax expression and Bcl-2 expression. Besides, Tan-I treatment can notably increase Paclitaxel-inducing cell senescence by promoting DNA damage and senescence-associated proteins such as p21 and p16. Furthermore, the result of the transplanted tumor model indicated that Tan-I combination with Paclitaxel could inhibit tumor growth in vivo by inhibiting cell proliferation and inducing cell apoptosis.
Conclusions:
Natural compound Tan-I enhances the efficacy of ovarian cancer to Paclitaxel chemotherapy. The results will help to supply the potential clinical use of ovarian carcinoma cells.
... Distinct from the other six beta-tubulin isotypes in the human genome, β3-tubulin has a unique molecular structure that facilitates its binding to factors involved in the oxidative stress and nutrient deprivation response, thereby bolstering the survival of stressed cells [23]. For example, induced expression of β3-tubulin promotes resistance to the widely used chemotherapeutic agent, paclitaxel, in Hela and MCF-7 cell lines [24]. Furthermore, increased levels of β3-tubulin expression have been linked to malignant transformation and migration of melanocytes [17]. ...
... Further studies are required to elucidate the mechanistic role of the α-and βtubulins isotypes in the treatment and prevention of melanoma progression. Olmsted, University of Rochester, USA) was performed as previously described [24]: briefly, cells were seeded in twelve-well plates at a density of 150,000 cells per well and transfected with 1 μg plasmid DNA using lipofectamine 2000 (catalog no. P/N52887, Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. ...
... A375 cells were transfected with EGFP-MAP-4 (provided by Dr. Joanna Olmsted, University of Rochester, USA) as previously described [24]. MTs dynamics analysis was performed as previously published [38]: briefly, MTs +ends were tracked using ImageJ software (Manual tracking plugin, https://imagej.nih.gov/ij/plugins/track/track.html). ...
Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (β3-tubulin), one of seven β-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of β3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of β3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of β3-tubulin in melanoma.
... When the initial concentration of Si[PTX-NC]s was increased, this resulted in a modest increase in drug loading within the cells ranging from 2.57 -3.81 μg PTX/ million NSCs (Figure 4b). Based on our previous work, NSCs accumulated at ovarian tumors after IP injection within 24 hours 15 PTX is a very potent microtubule stabilizer and can inhibit cell migration at doses well below their IC 50 27 . In order to evaluate if this was a concern, the migration capability of NSCs loaded with Si[PTX-NC]s was investigated using a Boyden Chamber migration assay. ...
Ovarian cancer is commonly diagnosed only after it has metastasized to the abdominal cavity (stage III). While the current standard of care of intraperitoneal (IP) administration of cisplatin and paclitaxel (PTX) combination chemotherapy has benefit, patient 5-year survival rates are low and have not significantly improved in the last decade. The ability to target chemotherapy selectively to ovarian tumors while sparing normal tissue would improve efficacy and decrease toxicities. We have previously shown that cisplatin-loaded nanoparticles (NPs) loaded within neural stem cells (NSCs) are selectively delivered to ovarian tumors in the abdominal cavity following IP injection, with no evidence of localization to normal tissue. Here we extended the capabilities of this system to also include PTX delivery. NPs that will be loaded into NSCs must contain a high amount of drug by weight but constrain the release of the drug such that the NSCs are viable after loading and can successfully migrate to tumors. We developed silica coated PTX nanocrystals (Si[PTX-NC]) meeting these requirements. Si[PTX-NC] were more effective than uncoated PTX-NC or Abraxane for loading NSCs with PTX. NSCs loaded with Si[PTX-NC] maintained their migratory ability and, for low dose PTX, were more effective than free PTX-NC or Si[PTX-NC] at killing ovarian tumors in vivo. This work demonstrates that NSC/NP delivery is a platform technology amenable to delivering different therapeutics and enables the pursuit of NSC/NP targeted delivery of the entire preferred chemotherapy regimen for ovarian cancer. It also describes efficient silica coating chemistry for PTX nanocrystals that may have applications beyond our focus on NSC transport.
... It is well known that cancer cells demonstrate profound intra-and interline variation after prolonged exposure to antimitotic drugs [76]. One of the interesting questions about MT dynamics is that mammalian cells might have MTs composed of several β-tubulin isotypes [77][78][79], while composition of MTs predicts cellular response to anti-microtubule drugs [80,81]. Heterogeneous response to taxol was directly observed in a population of HeLa cells [81]. ...
... One of the interesting questions about MT dynamics is that mammalian cells might have MTs composed of several β-tubulin isotypes [77][78][79], while composition of MTs predicts cellular response to anti-microtubule drugs [80,81]. Heterogeneous response to taxol was directly observed in a population of HeLa cells [81]. Nocodazole 100 BS-C-1 9.2 ± 0.76 3.0 ± 0.27 −67 Rhodaminelabeled tubulin [68] (continued) ...
Microtubules (MTs) are dynamic components of the cytoskeleton playing an important role in a large number of cell functions. Individual MTs in living cells undergo stochastic switching between alternate states of growth, shortening and attenuated phase, a phenomenon known as tempered dynamic instability. Dynamic instability of MTs is usually analyzed by labeling MTs with +TIPs, namely, EB proteins. Tracking of +TIP trajectories allows analyzing MT growth in cells with a different density of MTs. Numerous labs now use +TIP to track growing MTs in a variety of cell cultures. However, heterogeneity of MT dynamics is usually underestimated, and rather small sampling for the description of dynamic instability parameters is often used. The strategy described in this chapter is the method for repetitive quantitative analysis of MT growth rate within the same cell that allows minimization of the variation in MT dynamics measurement. We show that variability in MT dynamics within a cell when using repeated measurements is significantly less than between different cells in the same chamber. This approach allows better estimation of the heterogeneity of cells' responses to different treatments. To compare the effects of different MT inhibitors, the protocol using normalized values for MT dynamics and repetitive measurements for each cell is employed. This chapter provides detailed methods for analysis of MT dynamics in tissue cultures. We describe protocols for imaging MT dynamics by fluorescent microscopy, contrast enhancement technique, and MT dynamics analysis using triple color-coded display based on sequential subtraction analysis.
... Microtubule function is also modulated through differential expression of αand β-tubulin isoforms. The different isoforms have different characteristics and this can alter the binding of MAPs or drugs to microtubules (Ganguly et al., 2011). In mammalian cells, microtubules are nucleated from the centrosome which is surrounded by a mass of pericentriolar proteins. ...
There is mounting evidence that schizophrenia and depression are polar extremes of the same processes. The neuronal microtubule cytoskeleton is regulated through the actions of microtubule-associated proteins, the centrosome, tubulin posttranslational modifications and differential expression of tubulin isoforms. It is beginning to be thought that they may be important in the development of consciousness itself. It is proposed here that cytoskeletal tensegrity architecture plays a pivotal role in the etiology of psychiatric conditions. If the subcellular architecture is too tense, schizophrenia, if to loose, depression. This review focuses on the role of the microtubule cytoskeleton in schizophrenia, bipolar disorder and depression. Schizophrenia appears to be a disorder of microtubule architecture, depression one of intracellular transport along microtubules and bipolar disorder shares aspects of both schizophrenia and depression. Both current and potential treatments for these disorders target the microtubule cytoskeleton.
... βIII-tubulin-mediated Taxol ® resistance has been associated with reduced effects on microtubule dynamic instability [61]. It has also been demonstrated that βIII-tubulin counteracts Taxol ® 's inhibition of cell migration [62]. The relative binding affinities of MSAs have been analyzed by studying their inhibition of [ 3 H]2-m-AzTax photoaffinity labeling of tubulins containing distinct β-tubulin isotype content. ...
Taxol®, an antitumor drug with significant activity, is the first microtubule stabilizing agent described in the literature. This short review of the mechanism of action of Taxol® emphasizes the research done in the Horwitz’ laboratory. It discusses the contribution of photoaffinity labeled analogues of Taxol® toward our understanding of the binding site of the drug on the microtubule. The importance of hydrogen/deuterium exchange experiments to further our insights into the stabilization of microtubules by Taxol® is addressed. The development of drug resistance, a major problem that arises in the clinic, is discussed. Studies describing differential drug binding to distinct β-tubulin isotypes are presented. Looking forward, it is suggested that the β-tubulin isotype content of a tumor may influence its responses to Taxol®.
... Mechanistically, microtubules that are enriched in heterodimers formed by βIII-tubulin are unstable polymers and the overexpression of βIII-tubulin increases the rate of microtubule detachment from microtubule organizing centers, and reduces the stability of microtubule networks (45,46). Our data are in agreement with these studies, as inducible expression of βIII-tubulin in response to miR-34a overexpression correlated with microtubule destabilization, cell cycle arrest and apoptosis. ...
MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintains normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma; however, the molecular mechanisms underlying miR-34a activity in osteosarcoma are not well-defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in osteosarcoma. Levels of miR-34a were attenuated in human osteosarcoma cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pull-down assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3′-untranslated region. Overexpression of miR-34a in osteosarcoma cells suppressed STMN1 expression and reduced cell growth in vitro. Restoration of miR-34a led to microtubule destabilization and increased βIII-tubulin expression, with corresponding G1–G2 phase cell-cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated βIII-tubulin expression in osteosarcoma cells, suggesting that miR-34a indirectly affects Sp1. Validating the coordinating role of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in osteosarcoma cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates βIII-tubulin, leading to disruption of the microtubule network and cell death.
Implications: The miR-34a/STMN1/βIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy. Mol Cancer Res; 15(7); 953–64. ©2017 AACR.
