Expression Of Α-tubulin Research Articles

Particulate matter (PM10) destabilizes mitotic spindle through downregulation of SETD2 in A549 lung cancer cells

Air pollution represents an environmental problem, impacting negatively in human health. Particulate matter of 10 μm or less in diameter (PM10) is related to pulmonary diseases, including lung cancer. Mitotic spindle is made up by chromosome-microtubule (MT) interactions, where SETD2 plays an important role in MT stability. SETD2 binds and activates α-TUBULIN sub-unit and promotes MT polymerization. Alongside this mechanism, the spindle assembly checkpoint (SAC) senses the adequate mitotic progression through proteins such as BUBR1, AURORA B and SURVIVIN. Alterations in MT dynamics as well as in SAC cause aneuploidy and chromosomal instability, a common phenotype in cancer cells. In this study, we evaluated the effect of PM10 in the expression and protein levels of SETD2, as well as the effect in the expression and protein levels of SAC and mitotic components involved in chromosomal segregation/mitosis, using the A549 lung cancer cell line. A549 cell cultures were exposed to PM10 (10 μg/cm2) for 24 h to evaluate the expression and protein levels of SETD2 (SETD2), TUBA1A (α-TUBULIN), CCNB1 (CYCLIN B1), BUB1B (BUBR1), AURKB (AURORA B) and BIRC5 (SURVIVIN). We observed that PM10 decreases the expression and protein levels of SETD2, α-TUBULIN and BUBR1 and increases the levels of AURORA B and SURVIVIN in A549 cells, compared with non-treated cells. PM10 also caused a decrease in mitotic index and in the percentage of cells in G2/M when compared with control group. Co-localization of SETD2/α -TUB was lower in PM10-treated cells in comparison with non-treated cells. Finally, micronuclei (MN) frequency was higher in PM10-treated cells in contrast with non-treated cells, being whole chromosomes more common in PM10-treated MN than in non-treated MN. Our results suggest that PM10 causes missegregation and aneuploidy through downregulation of SETD2 and SAC components, inducing aneuploidy and predisposing to the generation of chromosomal instability in transformed cells.

Establishment of Adenomyosis Organoids as a Preclinical Model to Study Infertility.

Adenomyosis is related to infertility and miscarriages, but so far there are no robust in vitro models that reproduce its pathological features to study the molecular mechanisms involved in this disease. Endometrial organoids are in vitro 3D models that recapitulate the native microenvironment and reproduce tissue characteristics that would allow the study of adenomyosis pathogenesis and related infertility disorders. In our study, human endometrial biopsies from adenomyosis (n = 6) and healthy women (n = 6) were recruited. Organoids were established and hormonally differentiated to recapitulate midsecretory and gestational endometrial phases. Physiological and pathological characteristics were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, and ELISA. Secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype (pan-cytokeratin, Muc-1, PAS, Laminin, and Ki67) and secretory and gestational features (α-tubulin, SOX9, SPP1, PAEP, LIF, and 17βHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-β2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17βHSD2 compared with control organoids. Our results demonstrate that organoids derived from endometria of adenomyosis patients and differentiated to secretory and gestational phases recapitulate native endometrial-tissue-specific features and disease-specific traits. Adenomyosis-derived organoids are a promising in vitro preclinical model to study impaired implantation and pregnancy disorders in adenomyosis and enable personalized drug screening.

Microtentacle Formation in Ovarian Carcinoma.

Simple SummaryOvarian cancer commonly spreads throughout the peritoneal cavity by exfoliation of malignant cells into ascites. Chemoresistance remains an important therapeutic obstacle. Microtentacles (McTNs) are microtubule-based protrusions that may influence the metastatic potential and chemoresistance profile of free-floating cells. In this study, we define the various microtentacle morphologies that can be observed in detached ovarian cancer cells, and their clustering behavior in relationship to histology, α-tubulin post-translational modifications, β-tubulin isotype, modulators of cortical stiffness, and sensitivity to clinically relevant microtubule-targeting agents. McTNs represent a new therapeutic target for this disease, and an understanding of their biology could have implications for the refinement of therapies, including intraperitoneal drug delivery. Background: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. Methods: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot β-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. Results: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. Conclusions: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

Evaluation of poly (lactic-co-glycolic acid) nanoparticles to improve the therapeutic efficacy of paclitaxel in breast cancer.

Introduction: Paclitaxel (PTX) is a cornerstone in the treatment of breast cancer, the most common type of cancer in women. However, this drug has serious limitations, including lack of tissue-specificity, poor water solubility, and the development of drug resistance. The transport of PTX in a polymeric nanoformulation could overcome these limitations. Methods: In this study, PLGA-PTX nanoparticles (NPs) were assayed in breast cancer cell lines, breast cancer stem cells (CSCs) and multicellular tumor spheroids (MTSs) analyzing cell cycle, cell uptake (Nile Red-NR-) and α-tubulin expression. In addition, PLGA-PTX NPs were tested in vivo using C57BL/6 mice, including a biodistribution assay. Results: PTX-PLGA NPs induced a significant decrease in the PTX IC50 of cancer cell lines (1.31 and 3.03-fold reduction in MDA-MB-231 and E0771 cells, respectively) and CSCs. In addition, MTSs treated with PTX-PLGA exhibited a more disorganized surface and significantly higher cell death rates compared to free PTX (27.9% and 16.3% less in MTSs from MCF-7 and E0771, respectively). PTX-PLGA nanoformulation preserved PTX's mechanism of action and increased its cell internalization. Interestingly, PTX-PLGA NPs not only reduced the tumor volume of treated mice but also increased the antineoplastic drug accumulation in their lungs, liver, and spleen. In addition, mice treated with PTX-loaded NPs showed blood parameters similar to the control mice, in contrast with free PTX. Conclusion: These results suggest that our PTX-PLGA NPs could be a suitable strategy for breast cancer therapy, improving antitumor drug efficiency and reducing systemic toxicity without altering its mechanism of action.

Bio-essential Inorganic Molecular Nanowires as a Bioactive Muscle Extracellular-Matrix-Mimicking Material.

Many physiochemical properties of the extracellular matrix (ECM) of muscle tissues, such as nanometer scale dimension, nanotopography, negative charge, and elasticity, must be carefully reproduced to fabricate scaffold materials mimicking muscle tissues. Hence, we developed a muscle tissue ECM-mimicking scaffold using Mo6S3I6 inorganic molecular wires (IMWs). Composed of bio-essential elements and having a nanofibrous structure with a diameter of ∼1 nm and a negative surface charge with high stability, Mo6S3I6 IMWs are ideal for mimicking natural ECM molecules. Once Mo6S3I6 IMWs were patterned on a polydimethylsiloxane surface with an elasticity of 1877.1 ± 22.2 kPa, that is, comparable to that of muscle tissues, the proliferation and α-tubulin expression of myoblasts enhanced significantly. Additionally, the repetitive one-dimensional patterns of Mo6S3I6 IMWs induced the alignment and stretching of myoblasts with enhanced α-tubulin expression and differentiation into myocytes. This study demonstrates that Mo6S3I6 IMWs are promising for mimicking the ECM of muscle tissues.

Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation.

Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.

Downregulation of miR‑29b‑3p promotes α‑tubulin deacetylation by targeting the interaction of matrix metalloproteinase‑9 with integrinβ1 in nasal polyps.

Matrix metalloproteinase (MMP)-9 is a key enzyme responsible for extracellular matrix degradation and contributes to the progressive histological changes observed in lower respiratory tract infections. Integrin β1 and α-tubulin are potential MMP-9-interacting proteins, and microRNA (miR)-29b-3p can regulate MMP-9 expression. MMP-9 is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNPs), regardless of its effects on miR-29b-3p, integrin β1 and α-tubulin expression. In the present study, samples from 100 patients with CRSwNPs were examined via reverse transcription-quantitative PCR to assess the mRNA expression of miR-29b-3p, and western blotting was performed to assess the protein expression of MMP-2, MMP-9, acetyl-α-tubulin, integrin β1 and tissue inhibitor of metalloproteinase 1 (TIMP-1). A dual-luciferase reporter assay was used to verify the direct binding of miR-29b-3p and MMP-2/MMP-9. Co-immunoprecipitation (Co-IP) and GST pull-down assays showed that integrin β1 and α-tubulin were MMP-9-interacting proteins. Cell viability, apoptosis and inflammatory cytokine levels were determined via a Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. miR-29b-3p expression was found to be positively correlated with MMP-2 and MMP-9 expression. Whereas, TIMP-1 expression was negatively correlated with MMP-2 and MMP-9 expression. The dual-luciferase assay revealed that miR-29b-3p targeted the 3′ untranslated region of MMP-2/MMP-9. The Co-IP and GST pull-down assays showed that MMP-9 could directly bind to integrin β1 and indirectly bind to α-tubulin. Finally, the overexpression of miR-29b-3p decreased the expression of MMP-9 and increased the levels of acetyl-α-tubulin. By contrast, the knockdown of miR-29b-3p increased the expression of MMP-9 and decreased the levels of acetyl-α-tubulin. Additionally, MMP-9 expression was found to be negatively correlated with acetyl-α-tubulin expression. Of note, the expression of integrin β1 did not change following the overexpression and knockdown of MMP-9. Finally, the overexpression of miR-29b-3p not only decreased MMP-9 expression, but also alleviated lipopolysaccharide-induced inflammation in NP69 cells. The results showed that the downregulation of miR-29b-3p promoted α-tubulin deacetylation by increasing the number of MMP-9-integrin β1 complexes in CRSwNPs, thus targeting miR-29b-3p/MMP-9 may be a potential novel strategy for the clinical treatment of CRSwNPs.

Expression Pattern of α-Tubulin, Inversin and Its Target Dishevelled-1 and Morphology of Primary Cilia in Normal Human Kidney Development and Diseases.

The spatiotemporal expression of α-tubulin, inversin and dishevelled-1 (DVL-1) proteins associated with the Wnt-signaling pathway, and primary cilia morphology were analyzed in developing kidneys (14th–38th developmental weeks), healthy postnatal (1.5- and 7-years old) and pathologically changed human kidneys, including multicystic dysplastic kidneys (MCDK), focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). The analysis was performed by double immunofluorescence, electron microscopy, semiquantitative and statistical methods. Cytoplasmic co-expression of α-tubulin, inversin and DVL-1 was observed in the proximal convoluted tubules (pct), distal convoluted tubules (dct) and glomeruli (g) of analyzed tissues. During kidney development, the overall expression of α-tubulin, inversin and DVL-1 decreased, while in the postnatal period slightly increased. The highest expressions of α-tubulin and inversin characterized dct and g, while high DVL-1 characterized pct. α-tubulin, inversin and DVL-1 expression pattern in MCDK, FSGS and CNF kidneys significantly differed from the healthy control. Compared to healthy kidneys, pathologically changed kidneys had dysmorphic primary cilia. Different expression dynamics of α-tubulin, inversin and DVL-1 during kidney development could indicate that switch between the canonical and noncanonical Wnt-signaling is essential for normal kidney morphogenesis. In contrast, their disturbed expression in pathological kidneys might be associated with abnormal primary cilia, leading to chronic kidney diseases.

Structure-activity relationship and mechanistic studies for a series of cinnamyl hydroxamate histone deacetylase inhibitors

Histone deacetylases (HDACs) are a family of enzymes that modulate the acetylation status histones and non-histone proteins. Histone deacetylase inhibitors (HDACis) have emerged as an alternative therapeutic approach for the treatment of several malignancies. Herein, a series of urea-based cinnamyl hydroxamate derivatives is presented as potential anticancer HDACis. In addition, structure–activity relationship (SAR) studies have been performed in order to verify the influence of the linker on the biological profile of the compounds. All tested compounds demonstrated significant antiproliferative effects against solid and hematological human tumor cell lines. Among them, 11b exhibited nanomolar potency against hematological tumor cells including Jurkat and Namalwa, with IC50 values of 40 and 200 nM, respectively. Cellular and molecular proliferation studies, in presence of compounds 11a-d, showed significant cell growth arrest, apoptosis induction, and up to 43-fold selective cytotoxicity for leukemia cells versus non-tumorigenic cells. Moreover, compounds 11a-d increased acetylated α-tubulin expression levels, which is phenotypically consistent with HDAC inhibition, and indirectly induced DNA damage. In vitro enzymatic assays performed for 11b revealed a potent HDAC6 inhibitory activity (IC50: 8.1 nM) and 402-fold selectivity over HDAC1. Regarding SAR analysis, the distance between the hydroxamate moiety and the aromatic ring as well as the presence of the double bond in the cinnamyl linker were the most relevant chemical feature for the antiproliferative activity of the series. Molecular modeling studies suggest that cinnamyl hydroxamate is the best moiety of the series for binding HDAC6 catalytic pocket whereas exploration of Ser568 by the urea connecting unity (CU) might be related with the selectivity observed for the cinnamyl derivatives. In summary, cinnamyl hydroxamate derived compounds with HDAC6 inhibitory activity exhibited cell growth arrest and increased apoptosis, as well as selectivity to acute lymphoblastic leukemia cells. This study explores interesting compounds to fight against neoplastic hematological cells.

HDAC6 inhibition enhances the anti-tumor effect of eribulin through tubulin acetylation in triple-negative breast cancer cells.

Improved prognosis for triple-negative breast cancer (TNBC) has plateaued and the development of novel therapeutic strategies is required. This study aimed to explore the anti-tumor effect of combined eribulin and HDAC inhibitor (vorinostat: VOR, pan-HDAC inhibitor and ricolinostat: RICO, selective HDAC6 inhibitor) treatment for TNBC. The effect of eribulin in combination with an HDAC inhibitor was tested in three TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157) and their eribulin-resistant derivatives. The expression of acetylated α-tubulin was analyzed by Western blotting for TNBC cells and immunohistochemical analyses for clinical specimens obtained from breast cancer patients who were treated with eribulin. The simultaneous administration of low concentrations (0.2μM) of VOR or RICO enhanced the anti-tumor effect of eribulin in MDA-MB-231 and Hs578T cells but not in MDA-MB-157 cells. Meanwhile, pretreatment with 5μM of VOR or RICO enhanced eribulin sensitivity in all three cell lines. Low concentration of VOR or RICO increased acetylated α-tubulin expression in MDA-MB-231 and Hs578T cells. In contrast, whereas 5μM of VOR or RICO increased the expression of acetylated α-tubulin in MDA-MB-157 cells, low concentrations did not. Eribulin increased the expression of acetylated α-tubulin in MDA-MB-231 and Hs578T cells but not in MDA-MB-157 cells. These phenomena were also observed in eribulin-resistant cells. Immunohistochemical analyses revealed that the expression of acetylated α-tubulin was increased after eribulin treatment in TNBC. HDAC6 inhibition enhances the anti-tumor effect of eribulin through the acetylation of α-tubulin. This combination therapy could represent a novel therapeutic strategy for TNBC.

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility.

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.

The Effect of Umbilical Cord Mesenchymal Stem Cells Combined with Tetramethylpyrazine Therapy on Ischemic Brain Injury: A Histological Study

BackgroundRelatively poor survival and differentiation performance of umbilical cord mesenchymal stem cells (ucMSCs) limits its application of transplantation. The purpose of this study was to investigate the combined effect of ucMSCs and tetramethylpyrazine (TMP) on the histological therapy of ischemia stroke. MethodsUsing a middle cerebral artery occlusion (MCAO) model, we sought to determine the therapeutic effects of ucMSCs combined with TMP on ischemic stroke in rats. 1 × 106 ucMSCs was intracerebral transplanted after 24 hours and TMP (50 mg/kg) was injected intraperitoneally every day. After 7 days, the brain tissues were subjected to infarct weight measurement and preparation for 2,3,5-triphenyltetrazolium chloride (TTC) staining, HE staining, and immunohistochemical analysis. ResultsThe results showed that TMP combined with ucMSCs treatment significantly decreased the neurological deficit score, as well as the cerebral infarct ratio (from 16.33±3.35 to 7.67±1.19%) compared to TMP or ucMSCs treated alone. Moreover, TMP+ucMSCs treatment improved the morphological architecture of the infarct zone, dramatically up-regulated the expression of α-tubulin and nestin, and down-regulated GFAP and IL-1 expression. ConclusionsThese data suggest that ucMSCs combined with TMP are able to exert therapeutic effects following ischemic injury by improving neurogenesis, inhibiting inflammation, and ameliorating histological damage. This may therefore be a promising future treatment for ischemic stroke.

Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models.

The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

Protective effect of EX-527 against high-fat diet-induced diabetic nephropathy in Zucker rats

High-fat diet (HFD)-induced obesity is implicated in diabetic nephropathy (DN). EX-527, a selective Sirtuin 1 (SIRT1) inhibitor, has multiple biological functions; however, its protective effect against DN is yet to be properly understood. This study was aimed to explore the protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. After 21 weeks of continually feeding HFD to the rats, the apparent characteristics of progressive DN were observed, which included an increase in kidney weight (~160%), hyperglycemia, oxidative stress, and inflammatory cytokines, and subsequent renal cell damage. However, the administration of EX-527 for 10 weeks significantly reduced the blood glucose concentration and kidney weight (~59%). Furthermore, EX-527 significantly reduced the serum concentration of transforming growth factor-β1 (49%), interleukin (IL)-1β (52%), and IL-6 in the HFD-fed rats. Overall, the antioxidant activities significantly increased, and oxidative damage to lipids or DNA was suppressed. Particularly, EX-527 significantly reduced blood urea nitrogen (81%), serum creatinine (71%), microalbumin (43%), and urinary excretion of protein-based biomarkers. Histopathological examination revealed expansion of the extracellular mesangial matrix and suppression of glomerulosclerosis following EX-527 administration. EX-527 downregulated the expression of α-SMA (~64%), TGF-β (25%), vimentin, α-tubulin, fibronectin, and collagen-1 in the kidneys of the HFD-fed rats. Additionally, EX-527 substantially reduced claudin-1 and SIRT1 expression, but increased the expression of SIRT3 in the kidneys of the HFD-fed rats. EX-527 also inhibited the growth factor receptors, including EGFR, PDGFR-β, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

Design, synthesis and antitumour and anti-angiogenesis evaluation of 22 moscatilin derivatives.

Two series of moscatilin derivatives were designed, synthesized and evaluated as anti-tumor and anti-angiogenesis agents. Most of these compounds showed moderate-to-obvious cytotoxicity against five cancer cell lines (A549, HepG2, MDA-MB-231, MKN-45, HCT116). Among these cell lines, compounds had obvious effects on HCT116. Especially for 8Ae, the IC50 was low to 0.25 μM. 8Ae can inhibit the viability and induce the apoptosis of HCT116 cells but exhibit no cytotoxic activity in noncancerous NCM460 colon cells. 8Ae can also arrest the G2/M cell cycle in HCT116 cells by inhibiting the α-tubulin expression. Zebrafish bioassay-guided screen showed the 22 moscatilin derivatives had potent anti-angiogenic activities and compound 8Ae had better activities than positive compound. Molecular docking indicated 8Ae interacted with tubulin at the affinity of -7.2 Kcal/mol. In conclusion, compound 8Ae was a potential antitumor and anti-angiogenesis candidate for further development.

Yiqi Huoxue Decoction modifies the expression of myocardial cytoskeleton-associated proteins by regulating the AMPK signaling pathway in H9c2 cells exposed to hypoxic conditions

Abstract Background In myocardial ischemia, hypoxia leads to destruction of the cytoskeleton, and especially the imbalance of microtubule polymerization-depolymerization, which seriously affects the structure and function of cardiomyocytes. We previously showed that a Yiqi Huoxue Decoction (YQHX) improves mitochondrial function and decreases anti-oxidative effects in hypoxia-induced H9c2 cell injury. Therefore, in this study we investigated whether YQHX protects against hypoxia-induced damage by decreasing damage to the cardiac cytoskeleton. Methods After reaching 70%–80% confluence, H9c2 cells were synchronized in serum-free Dulbecco's Modified Eagle Medium for 6 hours, then divided into control, model, and YQHX (100, 200, 400 μg/mL) groups, which were then grown in a hypoxic atmosphere for 12 hours. Cardiac cell viability was assessed using an xCELLigence system. The levels of lactate dehydrogenase, maleic dialdehyde, and superoxide dismutase in H9c2 cell supernatants were measured. Hoechst 33258 staining was employed to observe cardiac cell apoptosis. Confocal microscopy, immunofluorescence, and western blot analysis were performed to evaluate the protective effects of the YQHX against hypoxia-induced injury in the H9c2 cell line. Results Cells that were pretreated with YQHX were more able to maintain their microtubule structure in the early stages of hypoxia and had better myocardial fitness in response to hypoxia compared with cells that were not pretreated. However, hypoxia-induced upregulation of α-tubulin and β-tubulin expression antagonized the protective effect of YQHX (100 μg/mL). In addition, YQHX (100 μg/mL) treatment significantly upregulated MAP4 protein expression (P = .003) and downregulated p-AMPKα protein expression (P Conclusion The results indicate that YQHX plays a role in protecting against oxidative stress injury and apoptosis in H9c2 cells. Notably, our results suggested that the YQHX could mitigate the damage to the cardiac cytoskeleton and the dysregulation of AMPK-related protein signaling pathways that are induced by hypoxia.

Carbendazim induces death in alveolar epithelial cells: A comparison between submerged and at the air-liquid interface cell culture

The fungicide Carbendazim is widely used in agriculture and preservation of films and fibers. In mammals, it can promote germ cell mutagenicity, carcinogenicity, and reproductive toxicity. However, few data about the effects of this toxicant upon the respiratory system are available. In this work, we evaluated Carbendazim toxicity upon A549 alveolar cells both in monolayer and upon air-liquid interface cell system. Monolayer cell exposed to non-cytotoxic concentrations of this fungicide showed cell arrest at G2/M phase, and did not show additional alterations. On the other hand, alveolar 3D reconstructed epithelial model (air-liquid interface cell system) was characterized and exposed to IC25 of Carbendazim using the Vitrocell® Cloud 12 chamber. Expression of Active Caspase-3, α-tubulin and ROS was significantly increased after such exposure. Mitochondrial activity was also reduced after exposed to Carbendazim. The obtained results indicate that besides the environmental and reproductive toxicity concerns regarding Carbendazim exposure, pulmonary toxicity must be considered for this fungicide. In addition, we observed that the way of exposure impacts considerably on the cell response for in vitro assessment of chemicals inhalation toxicity profile.

Effect of Electroacupuncture on Spermatogenesis in Rats with Oligozoospermia of Insufficiency of Shen (Kidney) Essence Syndrome.

To assess the effect of electroacupuncture (EA) on expression of cytoskeletal proteins from Sertoli cells (SCs) and spermatogenesis in rats with oligozoospermia of insufficiency of Shen (Kidney) essence syndrome (OIKES). Twenty healthy male Sprague-Dawley rats were randomly assigned to four groups using a random number table: control, tripterygium glycosides (TG) treatment, sham and EA groups (n=5 in each group). A rat model of OIKES was established by oral gavage with TG. The EA group was treated with TG and received EA at Shenshu (BL 23) and Zusanli (ST 36) acupoints for 20 min, once daily for 30 days, while the sham group received EA at identical acupoints with skin penetration without stimulation. After 30 days, the final body weight and coefficients for the testis and epididymis were calculated and sperm parameters were measured. Immunohistochemical analyses were performed to detect expression of vimentin and α-tubulin in SCs and proliferating cell nuclear antigen (PCNA) immunoreactivity in germ cells. Apoptosis in germ cells was quantified by the transferase biotin-dUTP nick end labeling assay. Compared with the control group, the final body weight and testis/epididymis coefficients of rats in the TG-treated group were not significantly different, but the sperm count and motility were lower (P<0.05). Expressions of vimentin and α-tubulin were also significantly weaker (P<0.01). The PCNA immunoreactivity of germ cells was decreased (P=0.059), whereas the apoptotic index of germ cells was increased significantly (P<0.01). In contrast, EA at BL 23 and ST 36 acupoints significantly improved the final body weight as well as the sperm count, concentration and motility (P<0.01 or P<0.05). EA increased expression of vimentin and α-tubulin in SCs markedly, and significantly enhanced PCNA immunoreactivity with decreased apoptosis in germ cells (P<0.01 or P<0.05). EA at BL 23 and ST 36 acupoints has protective effects on spermatogenesis in rats with OIKES. This effect seems to be achieved by attenuating TG-induced disruption of cytoskeletal protein in SCs.

Stably maintained microtubules protect dopamine neurons and alleviate depression-like behavior after intracerebral hemorrhage

Mesolimbic dopamine (DA) system lesion plays a key role in the pathophysiology of depression, and our previous study demonstrated that reduced microtubule (MT) stability aggravated nigrostriatal pathway impairment after intracerebral hemorrhage (ICH). This study aimed to further investigate the occurrence regularity of depression-like behavior after ICH and determine whether maintaining MT stabilization could protect DA neurons in ventral tegmental area (VTA) and alleviate depression-like behavior after ICH. An intrastriatal injection of 20 μl of autologous blood or MT depolymerization reagent nocodazole (Noco) was used to mimic the pathology of ICH model in mice. The concentration of DA, number of DA neurons and acetylated α-tubulin (a marker for stable MT) in VTA were checked, and depression-related behavior tests were performed after ICH. A MT-stabilizing agent, epothilone B (EpoB), was administered to explore the effects of MT stabilization on DA neurons and depression-like behavior after ICH. The results showed that obvious depression-like behavior occurred at 7, 14, and 28 days (P < 0.01) after ICH. These time-points were related to significant decreases in the concentration of DA (P < 0.01) and number of DA neurons (P < 0.01) in VTA. Moreover, The decrease of acetylated α-tubulin expression after ICH and Noco injection contributed to DA neurons’ impairment in VTA, and Noco injecton also aggravate ICH-induced depression-like behaviors and DA neurons’ injury. Furthermore, EpoB treatment significantly ameliorated ICH and Noco-induced depression-like behaviors (P < 0.05) and increased the concentration of DA (P < 0.05) and number of DA neurons (P < 0.05) in VTA by increasing the level of acetylated α-tubulin. The results indicate that EpoB can protect DA neurons by enhancing MT stability, and alleviate post-ICH depressive behaviors. This MT-targeted therapeutic strategy shows promise as a bench-to-bedside translational method for treating depression after ICH.

Diosgenin restores A\u03b2-induced axonal degeneration by reducing the expression of heat shock cognate 70 (HSC70)

We previously found diosgenin, an herbal drug-derived steroid sapogenin, to be remarkably effective at restoring Aβ-induced axonal degeneration and improving memory function in model of Alzheimer’s disease (AD), 5XFAD mouse. In this study, we investigated the downstream signaling of diosgenin and explored new therapeutic targets in AD. We showed that the expression of heat shock cognate (HSC) 70 was increased in Aβ-treated neurons and in 5XFAD mice but was decreased by diosgenin treatment. In addition, knockdown of HSC70 significantly promoted axonal growth in neurons. As an association molecule of HSC70 in neurons, α-tubulin was detected by immunoprecipitation. After Aβ treatment, α-tubulin expression was greatly reduced in the degenerated axons, suggesting that a decline in α-tubulin may be one of the factors which correlates with axonal disruption in AD pathology. We hypothesized that the degradation of α-tubulin is triggered by the chaperone activity of HSC70. However, diosgenin significantly normalized the α-tubulin level, a potentially critical process for axonal formation. Our study indicated that reducing the HSC70 level is a new possible therapeutic target of axonal regeneration in AD.