Maintaining essential microtubule bundles in meter-long axons: a role for local tubulin biogenesis?

Axons are the narrow, up-to-meter long cellular processes of neurons that form the biological cables wiring our nervous system. Most axons must survive for an organism's lifetime, i.e. up to a century in humans. Axonal maintenance depends on loose bundles of microtubules that run without interruption all along axons. The continued turn-over and the extension of microtubule bundles during developmental, regenerative or plastic growth requires the availability of α/ß-tubulin heterodimers up to a meter away from the cell body. The underlying regulation in axons is poorly understood and hardly features in past and contemporary research. Here we discuss potential mechanisms, particularly focussing on the possibility of local tubulin biogenesis in axons. Current knowledge might suggest that local translation of tubulin takes place in axons, but far less is known about the post-translational machinery of tubulin biogenesis involving three chaperone complexes: prefoldin, CCT and TBC. We discuss functional understanding of these chaperones from a range of model organisms including yeast, plants, flies and mice, and explain what is known from human diseases. Microtubules across species depend on these chaperones, and they are clearly required in the nervous system. However, most chaperones display a high degree of functional pleiotropy, partly through independent functions of individual subunits outside their complexes, thus posing a challenge to experimental studies. Notably, we found hardly any studies that investigate their presence and function particularly in axons, thus highlighting an important gap in our understanding of axon biology and pathology.

TUBB3 is associated with PTEN, neuroendocrine differentiation, and castration resistance in prostate cancer.

BACKGROUND: Tubulin-ß3 encoded by the Tubulin-ß3 (TUBB3) gene is a microtubule protein. Previous studies have shown that TUBB3 expression is upregulated in castration-resistant prostate cancer (CaP) and is involved in taxane resistance. However, the biological mechanism of TUBB3 involvement in the progression to castration-resistant CaP is not fully elucidated. This study aimed to analyze the expression and function of TUBB3 in localized and metastatic CaP. METHODS: TUBB3 expression was determined using immunohistochemistry in localized and metastatic CaP. We also investigated the association between TUBB3, phosphatase and tensin homolog (PTEN), and neuroendocrine differentiation and examined the involvement of TUBB3 in new antiandrogen drugs (enzalutamide and apalutamide) resistance in metastatic CaP. RESULTS: In 155 cases of localized CaP, immunohistochemistry showed that 5 (3.2%) of the CaP cases were positive for tubulin-ß3. Kaplan-Meier analysis showed that high expression of tubulin-ß3 was associated with poor prostate-specific antigen recurrence-free survival after radical prostatectomy. In 57 cases of metastatic CaP, immunohistochemistry showed that 14 (25%) cases were positive for tubulin-ß3. Tubulin-ß3 expression was higher in metastatic CaP than in localized CaP. High tubulin-ß3 expression was correlated with negative PTEN expression. TUBB3 expression was increased in neuroendocrine CaP based on several public databases. PTEN knockout decreased the sensitivity to enzalutamide and apalutamide in 22Rv-1 cells. TUBB3 knockdown reversed the sensitivity to enzalutamide and apalutamide in PTEN-CRISPR 22Rv-1 cells. High expression of tubulin-ß3 and negative expression of PTEN were significantly associated with poor overall survival in metastatic CaP treated with androgen deprivation therapy. CONCLUSIONS: These results suggest that TUBB3 may be a useful predictive biomarker for survival and play an essential role in antiandrogen resistance in CaP.

Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice.

The five tubulin-binding cofactors (TBC) are involved in tubulin synthesis and the formation of microtubules. Their importance is highlighted by various diseases and syndromes caused by dysfunction or mutation of these proteins. Posttranslational modifications (PTMs) of tubulin promote different characteristics, including stability-creating subpopulations of tubulin. Cell- and time-specific distribution of PTMs has only been investigated in the organ of Corti in gerbils. The aim of the presented study was to investigate the cell type-specific and time-specific expression patterns of TBC proteins and PTMs for the first time in murine cochleae over several developmental stages. For this, murine cochleae were investigated at the postnatal (P) age P1, P7 and P14 by immunofluorescence analysis. The investigations revealed several profound interspecies differences in the distribution of PTMs between gerbil and mouse. Furthermore, this is the first study to describe the spatio-temporal distribution of TBCs in any tissue ever showing a volatile pattern of expression. The expression analysis of TBC proteins and PTMs of tubulin reveals that these proteins play a role in the physiological development of the cochlea and might be essential for hearing.

TUBB3 Is Associated with High-Grade Histology, Poor Prognosis, p53 Expression, and Cancer Stem Cell Markers in Clear Cell Renal Cell Carcinoma.

BACKGROUND: ßIII-Tubulin, encoded by the TUBB3 gene, is a microtubule protein. Several studies have shown that overexpression of TUBB3 is linked to poor prognosis and is involved in taxane resistance in some cancers. OBJECTIVE: The aim of this study was to analyze the expression and function of TUBB3 in clear cell renal cell carcinoma (ccRCC). METHODS: The expression of TUBB3 was determined using immuno-histochemistry in ccRCC specimens. The effects of TUBB3 knockdown on cell growth and invasion were evaluated in RCC cell lines. We analyzed the interaction between TUBB3, p53, cancer stem cell markers, and PD-L1. RESULTS: In 137 cases of ccRCC, immunohistochemistry showed that 28 (20%) of the ccRCC cases were positive for TUBB3. High TUBB3 expression was significantly correlated with high nuclear grade, high T stage, and N stage. A Kaplan-Meier analysis showed that high expression of TUBB3 was associated with poor overall survival after nephrectomy. In silico analysis also showed that high TUBB3 expression was correlated with overall survival. Knockdown of TUBB3 suppressed cell growth and invasion in 786-O and Caki-1 cells. High TUBB3 expression was associated with CD44, CD133, PD-L1, and p53 in ccRCC. We generated p53 knockout cells using the CRISPR-Cas9 system. Western blotting revealed that p53 knockout upregulated the expression of TUBB3. CONCLUSION: These results suggest that TUBB3 may play an oncogenic role and could be a potential therapeutic target in ccRCC.

Direct interaction of metastasis-inducing S100P protein with tubulin causes enhanced cell migration without changes in cell adhesion.

Overexpression of S100P promotes breast cancer metastasis in animals and elevated levels in primary breast cancers are associated with poor patient outcomes. S100P can differentially interact with nonmuscle myosin (NM) isoforms (IIA > IIC > IIB) leading to the redistribution of actomyosin filaments to enhance cell migration. Using COS-7 cells which do not naturally express NMIIA, S100P is now shown to interact directly with α,ß-tubulin in vitro and in vivo with an equilibrium Kd of 2-3 × 10-7 M. The overexpressed S100P is located mainly in nuclei and microtubule organising centres (MTOC) and it significantly reduces their number, slows down tubulin polymerisation and enhances cell migration in S100P-induced COS-7 or HeLa cells. It fails, however, to significantly reduce cell adhesion, in contrast with NMIIA-containing S100P-inducible HeLa cells. When taxol is used to stabilise MTs or colchicine to dissociate MTs, S100P's stimulation of migration is abolished. Affinity-chromatography of tryptic digests of α and ß-tubulin on S100P-bound beads identifies multiple S100P-binding sites consistent with S100P binding to all four half molecules in gel-overlay assays. When screened by NMR and ITC for interacting with S100P, four chemically synthesised peptides show interactions with low micromolar dissociation constants. The two highest affinity peptides significantly inhibit binding of S100P to α,ß-tubulin and, when tagged for cellular entry, also inhibit S100P-induced reduction in tubulin polymerisation and S100P-enhancement of COS-7 or HeLa cell migration. A third peptide incapable of interacting with S100P also fails in this respect. Thus S100P can interact directly with two different cytoskeletal filaments to independently enhance cell migration, the most important step in the metastatic cascade.

ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression.

Acquired chemoresistance is a critical issue for advanced bladder cancer patients during long-term treatment. Recent studies reveal that a fraction of tumor cells with enhanced tumor-initiating potential, or cancer stem-like cells (CSCs), may particularly contribute to acquired chemoresistance and recurrence. Thus, CSC characterization will be the first step towards understanding the mechanisms underlying advanced disease. Here we generated long-term patient-derived cancer cells (PDCs) from bladder cancer patient specimens in spheroid culture, which is favorable for CSC enrichment. Pathological features of bladder cancer PDCs and PDC-dependent patient-derived xenografts (PDXs) were basically similar to those of their corresponding patients' specimens. Notably, CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), a critical enzyme that synthesizes retinoic acid (RA), was abundantly expressed in PDCs. ALDH1A1 inhibitors and shRNAs repressed both PDC proliferation and spheroid formation, whereas all-trans RA could rescue ALDH1A1 shRNA-suppressed spheroid formation. ALDH inhibitor also reduced the in vivo growth of PDC-derived xenografts. ALDH1A1 knockdown study showed that tubulin beta III (TUBB3) was one of the downregulated genes in PDCs. We identified functional RA response elements in TUBB3 promoter, whose transcriptional activities were substantially activated by RA. Clinical survival database reveals that TUBB3 expression may associate with poor prognosis in bladder cancer patients. Moreover, TUBB3 knockdown was sufficient to suppress PDC proliferation and spheroid formation. Taken together, our results indicate that ALDH1A1 and its putative downstream target TUBB3 are overexpressed in bladder cancer, and those molecules could be applied to alternative diagnostic and therapeutic options for advanced disease.

A Synthetic Snake-Venom-Based Tripeptide Protects PC12 Cells from the Neurotoxicity of Acrolein by Improving Axonal Plasticity and Bioenergetics.

The synthetic peptide p-BTX-I is based on the native peptide (formed by glutamic acid, valine and tryptophan) isolated from Bothrops atrox venom. We have previously demonstrated its neuroprotective and neurotrophic properties in PC12 cells treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Now, we have investigated the neuroprotective effects and mechanisms of p-BTX-I against the toxicity of acrolein in PC12 cells. Studies have demonstrated that acrolein might play an important role in the etiology of Alzheimer's disease (AD), which is characterized by neuronal and synaptic loss. Our results showed that not only acrolein reduced cell differentiation and cell viability, but also altered the expression of markers of synaptic communication (synapsin I), energy metabolism (AMPK-α, Sirt I and glucose uptake), and cytoskeleton (ß-III-tubulin). Treatment with p-BTX-I increased the percentage of differentiation in cells treated with acrolein and significantly attenuated cell viability loss, besides counteracting the negative effects of acrolein on synapsin I, AMPK-α, Sirt I, glucose uptake, and ß-III-tubulin. Additionally, p-BTX-I alone increased the expression of apolipoprotein E (apoE) gene, associated with the proteolytic degradation of ß-amyloid peptide aggregates, a hallmark of AD. Taken together, these findings demonstrate that p-BTX-I protects against acrolein-induced neurotoxicity and might be a tool for the development of novel drugs for the treatment of neurodegenerative diseases.

Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

Upregulation of MAPK10, TUBB2B and RASL11B may contribute to the development of neuroblastoma.

The present study aimed to investigate genes and transcription factors (TFs) that may contribute to neuroblastoma (NB) development. The GSE78061 dataset that included 25 human NB cell lines and four retinal pigment epithelial cell lines was used to analyze differentially expressed genes (DEGs) between groups. Functional enrichment analysis and protein­protein interaction (PPI) network analysis were performed for the identified DEGs. Additionally, submodule analysis and TF­target regulatory networks were conducted. The relative mRNA expression levels of mitogen­activated protein kinase 10 (MAPK10), tubulin ß 2B class IIb (TUBB2B), RAS like family 11 member B (RASL11B) and integrin subunit α 2 (ITGA2) in the NB cell line SH­SY5Y were compared with retinal pigment epithelial cell lines. A set of 386 upregulated and 542 downregulated DEGs were obtained. Upregulated DEGs were significantly associated with the 'neuron migration' and 'dopaminergic synapse signaling' pathways, whereas, downregulated DEGs were primarily involved in 'focal adhesion' such as ITGA2 and ITGA3. In the PPI networks analyzed, MAPK10, dopa decarboxylase (DDC), G protein subunit γ 2 (GNG2), paired like homeobox 2B (PHOX2B), TUBB2B, RASL11B, and ITGA2 were hub genes with high connectivity degrees. Additionally, PHOX2B was predicted to be a TF regulating TUBB2B in the regulatory network. The expressions of MAPK10, TUBB2B, RASL11B and ITGA2 were detected by reverse transcription­quantitative polymerase chain reaction in the NB cell line SH­SY5Y, and were consistent with the present bioinformatics results, suggesting that MAPK10, DDC, GNG2, PHOX2B, TUBB2B, RASL11B, ITGA2 and ITGA3 may contribute to NB development. Additionally, the present study identified a novel significant association between the increased expression levels of MAPK10, TUBB2B and RASL11B, and NB cells.

Tubulin heterogeneity regulates functions and dynamics of microtubules and plays a role in the development of drug resistance in cancer.

Microtubules, composed of αß-tubulin heterodimers, exhibit diverse structural and functional properties in different cell types. The diversity in the microtubule structure originates from tubulin heterogeneities, namely tubulin isotypes and their post-translational modifications (PTMs). These heterogeneities confer differential stability to microtubules and provide spatial cues for the functioning of the cell. Furthermore, the altered expressions of tubulin isotypes and PTMs are prominent factors for the development of resistance against some cancer drugs. In this review, we summarize our current knowledge of the tubulin isotypes and PTMs and how, together, they control the cellular functions of the microtubules. We also describe how cancer cells use this tubulin heterogeneity to acquire resistance against clinical agents and discuss existing attempts to counter the developed resistance.

Senescence as a main mechanism of Ritonavir and Ritonavir-NO action against melanoma.

The main focus of this study is exploring the effect and mechanism of two HIV-protease inhibitors: Ritonavir and Ritonavir-nitric oxide (Ritonavir-NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir-NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of ß-galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir-NO treatment only in metastatic B16F10 cells, ranking it as a late-response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton-as we observed diminished expression of vinculin, α-actin, and ß-tubulin. Permanent inhibition of S6 protein by Ritonavir-NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO-modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.

Expression of γ-tubulin in non-small cell lung cancer and effect on patient survival.

INTRODUCTION: It has been reported that overexpression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor aggressiveness in a variety of human malignancies. We have shown that γ-tubulin expression and cellular distribution pattern is also altered in non-small cell lung cancer (NSCLC) (Histol. Histopathol. 2012; 27: 1183-1194). In the present study we examined the relationship between γ-tubulin expression and patient overall survival (OS). MATERIAL AND METHODS: Immunohistochemistry was performed, with well-characterized anti-γ-tubulin antibodies, on 109 formalin-fixed, paraffin-embedded NSCLC specimens (p-TNM stage I-III). γ-Tubulin labeling indexes (LIs) were determined, and the association of γ-tubulin expression with clinicopathological parameters was evaluated. To analyze OS rates according to γ-tubulin LIs, patients were categorized into three groups: those with low (0-30%), intermediate (31-69%) or high (70-100%) γ-tubulin LI. Association of clinicopathological parameters and γ-tubulin with survival were examined using univariate and multivariate Cox regression analysis. RESULTS: No statistically significant association was seen between γ-tubulin overexpression and histological type, tumor differentiation, p-TNM stage and adenocarcinoma subtyping. Longer survival was observed in the high γ-tubulin LI group of patients with p-TNM stages II+III when compared to intermediate or low γ-tubulin LI groups, but the difference was not statistically significant (p=0.066). On the other hand, when combined low and intermediate γ-tubulin LI groups (p-TNM stages II+III) where compared to high γ-tubulin LI group, statistically significant longer survival was observed in high γ-tubulin group (p=0.021). CONCLUSION: Our findings suggest that level of γ-tubulin expression may have an impact on patient survival at more advanced NSCLC stages.

Expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues and adjacent normal tissues and their clinical significance.

PURPOSE: To investigate the expressions of class III ß-tubulin (TUBB3), nucleotide excision repair cross-complementary gene 1 (ERCC1) and P-glycoprotein (P-gp) in ovarian cancer tissues and adjacent normal tissues and their clinical significance. METHODS: Ovarian cancer patients undergoing surgical resection at the Department of Oncology of the Affiliated Hospital of Shandong Medical College from March 2012 to May 2016 were enrolled in this study, from which 166 cases of pathologically confirmed cancer tissues and 50 cases of adjacent normal tissues were collected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues and adjacent normal tissues, and their relationships with ovarian cancer clinical stage and grade of pathological differentiation were analyzed. RESULTS: The expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues were significantly higher than those in adjacent normal tissues (p<0.05). The later the clinical stage of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). The lower the pathological differentiation grade of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). TUBB3, ERCC1 and P-gb were positively correlated with clinical stage and pathological differentiation grade. CONCLUSION: TUBB3, ERCC1 and P-gp are involved in the occurrence and development of ovarian cancer and can be used as important indexes judging the severity of ovarian cancer, providing a reference for the occurrence and development of the disease in ovarian cancer patients in clinical practice.

Docosahexaenoic acid protection in a rotenone induced Parkinson's model: Prevention of tubulin and synaptophysin loss, but no association with mitochondrial function.

Rotenone, a classic mitochondrial complex I inhibitor, leads to dopaminergic neuronal death resulting in a Parkinson's-like-disease. Docosahexaenoic acid (DHA) has shown neuroprotective effects in other experimental models of Parkinson's disease, but its effect on the rotenone-induced parkinsonism is still unknown. We tested whether DHA in vivo exerts a neuroprotective effect on rotenone-induced parkinsonism and explored the mechanisms involved, including mitochondrial function and ultrastructure as well as the expression of tubulin and synaptophysin. We pretreated eighty male Wistar rats with DHA (35 mg/kg/day) for seven days and then administered rotenone for eight days. We then measured rearing behavior, number of dopaminergic neurons, tyrosine hydroxylase content, tubulin and synaptophysin expression, mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential, ATP production activity and mitochondrial ultrastructure. We found that in vivo DHA supply exerted a neuroprotective effect, evidenced by decreased dopaminergic neuron cell death. Although we detected rotenone induced mitochondrial ultrastructure alterations, these were not associated with mitochondrial dysfunction. Rotenone had no effect on mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential or ATP production activity. DHA also prevented a rotenone-induced decrease in tubulin and synaptophysin expression. Our results support the neuroprotective effect of DHA on rotenone-induced parkinsonism, and a possible effect on early stage Parkinson's disease. This protective effect is not associated with mitochondrial function improvement, but rather with preventing loss of tubulin and synaptophysin, proteins relevant to synaptic transmission.

Design, Synthesis and Screening of 4,6-Diaryl Pyridine and Pyrimidine Derivatives as Potential Cytotoxic Molecules.

A new series of pyridine and pyrimidine derivatives is designed and synthesized as potential antitumor molecules. The tested compounds show promising in vitro cytotoxic activity against HL-60 cell line as eight compounds: 4, 6, 11, 13, 14, 15, 18 and 21 exhibit potent cytotoxic activity in sub-micromolar concentration higher than the combretastatin A4 (CA-4). Compound 21 shows a cytotoxic activity 5-fold more potent than CA-4 on HL-60 cells. DNA-Flow cytometry cell cycle analysis and annexin-V assay on HL-60 cells show that compounds 4, 18 and 21 exhibit potent cell growth inhibition, cell cycle arrest at G2/M phase and pro-apoptotic inducing activities. The percentage inhibition assay of ß-tubulin polymerization on HL-60 cells shows that the antitumor activity of the tested compounds appears to correlate well with its ability to inhibit ß-tubulin polymerization. In addition, enzyme-linked immunosorbene assay (ELlSA) measurement for compound 21 shows apoptotic inducing activities through significant up regulation of p53, Bax/Bcl-2 ratio and caspase-3 proteins parallel to down regulation of the level of survivin proteins.

The inhibitory effect of kokusaginine on the growth of human breast cancer cells and MDR-resistant cells is mediated by the inhibition of tubulin assembly.

The emergence of multidrug resistance (MDR) is a significant challenge in breast carcinoma chemotherapy. Kokusaginine isolated from Dictamnus dasycarpus Turcz. has been reported to show cytotoxicity in several human cancer cell lines including breast cancer cells MCF-7. In this study, kokusaginine showed the potent inhibitory effect on MCF-7 multidrug resistant subline MCF-7/ADR and MDA-MB-231 multidrug resistant subline MDA-MB-231/ADR. Kokusaginine markedly induced apoptosis in a concentration-dependent manner in MCF-7/ADR cells. Furthermore, kokusaginine reduced P-gp mRNA and protein levels, and suppressed P-gp function especially in MCF-7/ADR cells. In addition, kokusaginine showed to inhibit tubulin assembly and the binding of colchicine to tubulin by binding directly to tubulin and affects tubulin formation in vitro. Taken together, these results support the potential therapeutic value of kokusaginine as an anti-MDR agent in chemotherapy for breast carcinoma.

Expression of Class III Beta-tubulin Predicts Prognosis in Patients with Cisplatin-resistant Bladder Cancer Receiving Paclitaxel-based Second-line Chemotherapy.

BACKGROUND/AIM: Class III beta-tubulin (TUBB3) expression is recognized as a predictive marker for chemosensitivity to cisplatin- and taxane-based chemotherapies in various malignancies. The aim of this study was to clarify the predictive value of TUBB3 expression for the anticancer effects of first-line cisplatin-based chemotherapy and second-line paclitaxel-based chemotherapy in patients with urothelial cancer (UC). PATIENTS AND METHODS: We reviewed 116 patients with UC (90 with bladder cancer and 27 with upper urinary tract cancer) treated with first-line cisplatin-based chemotherapy. Among them, 42 patients received a paclitaxel-based regimen as second-line chemotherapy for advanced cisplatin-resistant UC. TUBB3 expression was evaluated using immunohistochemistry, and survival analyses were performed using Kaplan-Meier survival curves and multivariate Cox proportional hazard analysis. RESULTS: TUBB3 was mainly detected in the cytoplasm of cancer cells, and 64 patients (55.2%) were judged as having positive TUBB3 expression. TUBB3 expression was significantly associated with tumour grade (p<0.001). TUBB3 expression was not associated with time to progression after first-line cisplatin-based chemotherapy. However, positive expression of TUBB3 was significantly associated with unfavourable overall survival in patients receiving second-line paclitaxel-based chemotherapy (p=0.021). In addition, a multivariate analysis model including T-stage and metastasis at the beginning of second-line therapy and regimen showed that TUBB3 expression was an independent predictor of poorer survival (hazard ratio(HR)=3.44, 95% confidential interval(CI)=1.15-10.33, p=0.027). CONCLUSION: TUBB3 expression was identified as a useful predictive factor for survival after second-line paclitaxel-based therapy in patients with cisplatin-resistant UC. Our results are useful for determining treatment strategies for such patients.

Preliminary investigation about the expression of tubulin in platelets from patients with iron deficiency anemia and thrombocytosis.

OBJECTIVE: In order to inquire into the pathogenesis of increased platelet counts in peripheral blood of patients with iron deficiency anemia (IDA), the phenomenon of thrombocytosis was confirmed, and then the expression of tubulin within platelets from IDA patients was investigated. METHODS: Peripheral blood samples were collected from 79 patients with IDA and were divided into 2 groups, group of IDA with normal platelet counts (34 cases), and group of IDA with increased platelet counts (thrombocytosis) (45 cases). Additionally, 45 peripheral blood samples from healthy volunteers were enrolled as a group of healthy controls. Count of platelets in peripheral blood was detected by means of LH-780 hematology analyzer and hemocytometer under a microscope respectively, and analyzed statistically. RESULTS: There was no statistical difference between platelet counts detected by LH-780 hematology analyzer and hemocytometer under a microscope (P > .05). The mean fluorescence intensity (MFI) of both α-tubulin and ß-tubulin within platelets from IDA patients with thrombocytosis was significantly less than that from healthy volunteers and IDA patients with normal platelet counts (P < .01), and there was no statistical difference between the latter two groups (P > .05). CONCLUSION: Some patients with IDA are accompanied by thrombocytosis, from which the expression of α-tubulin and ß-tubulin within platelets reduced obviously compared with those with normal platelet counts and healthy controls respectively. It is implied that downregulation of tubulin probably is a part of the pathogenesis leading to increased platelet counts in IDA.

High expression of class III ß-tubulin has no impact on functional cancer cell growth inhibition of a series of key vinblastine analogs.

Clinical association studies have implicated high expression of class III ß-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III ß-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3-10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III ß-tubulin unlike the taxanes. Most significant are the results with 4-6, a subset of 20' amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2-2-fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux.

Overexpression of lamin B1 induces mitotic catastrophe in colon cancer LoVo cells and is associated with worse clinical outcomes.

Lamins are the major components of the nuclear lamina and play important roles in many cellular processes. The role of lamins in cancer development and progression is still unclear but it is known that reduced expression of lamin B1 has been observed in colon cancer. Thus, the aim of the present study was to elucidate the influence of LMNB1 upregulation on colon cancer cell line after treatment with 5-FU. The results indicate, that overexpression of LMNB1 induced dose-dependent cell death mainly by mitotic catastrophe pathway. Furthermore, after upregulation of this intermediate protein, lower expression of lamin A/C was observed. Moreover, we observed an increase in fluorescence intensity of nuclear ß-catenin and decrease in cell-cell interaction area, that was connected with inhibition of colon cancer cells migration. We present the reorganization of actin filament and ß-tubulin, because these cytoskeletal proteins are directly or indirectly linked with lamins, and analyzing publicly available mRNA data we show that patients with overexpression of LMNB1 are characterized by lower survival rates within the first 30 months from diagnosis. Summarizing our results, upregulation of LMNB1 induce mitotic catastrophe and only small percentage of apoptosis. Moreover, we showed inhibition of cell migration and promotion of cell-cell contact as a results of direct and indirect regulation of ß-catenin, lamin A/C, actin and tubulin. However, it is possible that mitotic catastrophe cells in patients with colorectal cancer may be a reservoir of the cells responsible for faster disease progression, and further investigations are necessary to confirm this hypothesis.