Β-tubulin Protein Levels Research Articles

Moroidin, a Cyclopeptide from the Seeds of Celosia cristata That Induces Apoptosis in A549 Human Lung Cancer Cells.

Interference of microtubule dynamics with tubulin-targeted drugs is a validated approach for cancer chemotherapy. Moroidin (1) is an Urticaceae-type cyclopeptide having a potent inhibitory effect on purified tubulin polymerization. So far, moroidin has not been chemically synthesized, and its effect on cancer cells remains unknown. Herein, the cyclopeptide moroidin was isolated and identified from the seeds of Celosia cristata, and a revised assignment of its NMR data was presented. For the first time, moroidin (1) was demonstrated as having cytotoxic effects for several cancer cells, especially A549 lung cancer cells. The cellular evidence obtained showed that moroidin disrupts microtubule polymerization and decreases β-tubulin protein levels, but is not as potent as colchicine. Molecular docking indicated that 1 has a high binding potential to the vinca alkaloid site on tubulin. Moreover, moroidin arrested A549 cells in the G2/M phase and induced cell apoptosis. The intrinsic mitochondrial pathway and AKT were involved in the moroidin-induced cell apoptosis. In addition, moroidin (1) inhibited the migration and invasion of A549 cells at sublethal concentrations.

Alteration of Neural Stem Cell Functions in Ataxia and Male Sterility Mice: A Possible Role of β-Tubulin Glutamylation in Neurodegeneration.

Ataxia and Male Sterility (AMS) is a mutant mouse strain that contains a missense mutation in the coding region of Nna1, a gene that encodes a deglutamylase. AMS mice exhibit early cerebellar Purkinje cell degeneration and an ataxic phenotype in an autosomal recessive manner. To understand the underlying mechanism, we generated neuronal stem cell (NSC) lines from wild-type (NMW7), Nna1 mutation heterozygous (NME), and Nna1 mutation homozygous (NMO1) mouse brains. The NNA1 levels were decreased, and the glutamylated tubulin levels were increased in NMO1 cultures as well as in the cerebellum of AMS mice at both 15 and 30 days of age. However, total β-tubulin protein levels were not altered in the AMS cerebellum. In NMO1 neurosphere cultures, β-tubulin protein levels were increased without changes at the transcriptional level. NMO1 grew faster than other NSC lines, and some of the neurospheres were attached to the plate after 3 days. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1 neurospheres and that the neuronal differentiation potentials were reduced in NMO1 cells compared to NME or NMW7 cells. These results demonstrate that the AMS mutation decreased the NNA1 levels and increased glutamylation in the cerebellum of AMS mice. The observed changes in glutamylation might alter NSC properties and the neuron maturation process, leading to Purkinje cell death in AMS mice.

FAM172A affects cell proliferation and apoptosis not by targeting β-tubulin in HepG2 cells.

BackgroundMicrotubules pull chromosomes apart during cell mitosis and take part in cell division, Inhibiting the formation of spindle microtubules during mitosis has become one of the current anti-tumor research strategies. Earlier studies have found that the family with sequence similarity 172, member A (FAM172A) can significantly inhibit the proliferation of human colorectal cancer cell line LOVO cells and promote apoptosis. The purpose of this study was to investigate the biological effects of FAM172A on liver cancer cells and the interaction mechanism with tubulin.MethodsUse STRING software predicted the interactions between FAM172A and β-tubulin, and verify by immunoprecipitation. Real-Time qPCR was used to determine the expression levels of β-tubulin in liver cancer cell line HepG2, western blot was performed to detect protein expression levels. Immunofluorescence experiment to detect the distribution, shape and the dynamic behavior of depolymerization-aggregation of β-tubulin in cells. MTT, wound healing and Transwell assay were employed to determine cell proliferation, migration and invasion respectively. Flow cytometry was conducted to determine cell cycle and apoptosis.ResultsThere is no interactions between FAM172A and β-tubulin. We determined that when FAM172A was up-regulated or down-regulated, the mRNA and protein levels of β-tubulin did not change significantly (P>0.05). Furthermore, the distribution, shape of β-tubulin in cells, and the dynamic behavior of depolymerization-aggregation was not affected. After FAM172A overexpression, the migration and invasion of HepG2 cells were significantly inhibited (P<0.05), the cell proliferation was also significantly inhibited (P<0.05) and was time-dependent. The HepG2 cells had apparent S phase arrest and apoptosis (P<0.05). After interfering with FAM172A, the opposite result will appear.ConclusionsThe results show that FAM172A may be a new tumor suppressor gene, which has a specific role in cell cycle control and cell proliferation, but the specific mechanism of action has not been explained in this study and needs further exploration.

Protective effect of calpeptin on acrylamide-induced microtubule injury in sciatic nerve

The present study aimed to investigate the protective effect and mechanism of calpeptin (CP) on acrylamide (ACR)-induced microtubule (MT) injury in the sciatic nerve of rats. All rats were divided into four groups (control, CP, ACR, and ACR + CP):1 ml/kg saline, 200 μg/kg CP, 30 mg/kg ACR, and 30 mg/kg ACR plus 200 μg/kg CP were administered to the corresponding rats for 4 weeks through intraperitoneal injection. Body weight and neurobehavioral indicators were measured weekly and α-tubulin, β-tubulin, and other concerned proteins were estimated by western blotting and immunohistochemistry. At 4 weeks, decreased body weight, increased gait scores, increased hindlimb splay, and decreased time of fall of ACR rats were observed compared with those of control rats. All these mentioned changes were restored in the ACR + CP group compared with the ACR group. After 4 weeks of administration, western blotting and immunohistochemistry revealed significant increase in the protein levels of β-tubulin, calpain I, calpain II, Tau, microtubule-associated protein 2 (MAP2), protein kinase C, and cyclin-dependent kinase 5 in the ACR group compared with the control group; these increases were significantly lower in the ACR + CP group than in the ACR group. Furthermore, histopathological examination revealed loose arrangement, disorganised structure, uneven density, and exfoliated perineurium in the ACR group, and CP administration improved these changes significantly. The present results suggest that CP has an intervening effect on ACR-induced MT injury. A possible mechanism is that calpain maintains the stability of MTs by regulating the metabolism of Tau and MAP2.

Mitochondrial activity in different regions of the brain at the onset of streptozotocin-induced diabetes in rats.

Diabetes affects a variety of tissues including the central nervous system; moreover, some evidence indicates that memory and learning processes are disrupted. Also, oxidative stress triggers alterations in different tissues including the brain. Recent studies indicate mitochondria dysfunction is a pivotal factor for neuron damage. Therefore, we studied mitochondrial activity in three brain regions at early type I-diabetes induction. Isolated mitochondria from normal hippocampus, cortex and cerebellum revealed different rates of oxygen consumption, but similar respiratory controls. Oxygen consumption in basal state 4 significantly increased in the mitochondria from all three brain regions from diabetic rats. No relevant differences were observed in the activity of respiratory complexes, but hippocampal mitochondrial membrane potential was reduced. However, ATP content, mitochondrial cytochrome c, and protein levels of β-tubulin III, synaptophysin, and glutamine synthase were similar in brain regions from normal and diabetic rats. In addition, no differences in total glutathione levels were observed between normal and diabetic rat brain regions. Our results indicated that different regions of the brain have specific metabolic responses. The changes in mitochondrial activity we observed at early diabetes induction did not appear to cause metabolic alterations, but they might appear at later stages. Longer-term streptozotocin treatment studies must be done to elucidate the impact of hyperglycemia in brain metabolism and the function of specific brain regions.

Calpeptin is neuroprotective against acrylamide-induced neuropathy in rats

The aim of this study is to explore the potent neuroprotective effect of calpeptin (CP) on neuron damage induced by acrylamide (ACR) and its mechanism. Behavioural indicators such as hind limb splay, rota-rod performance, and gait analysis were assessed weekly to evaluate neurobehavioural changes after ACR and/or CP administration. The histopathological alterations and the changes of μ-calpain, m-calpain, microtubule-associated protein 2 (MAP2), and α-tubulin and β-tubulin protein levels in spinal cord were determined. Results showed that after administration of 30 mg/kg ACR, decreased body weight, attenuated neurobehavioural function, injury of motor neuron, increased protein levels of m-calpain and β-tubulin, suppressed MAP2 protein level, and no significant changes of μ-calpain and α-tubulin protein levels were observed compared with the control group rats. After administration of 200 μg/kg CP, partially restored body weight and neurobehavioural function, improvement of motor neuron injury, decreased protein levels of m- calpain and β-tubulin, and reversed effects of MAP2 protein level were observed compared with the ACR group rats. Our results suggested that CP alleviates neuropathy induced by ACR in rats. The calpain’s overactivation causes the degrading of MAP2 and eventually leads to the destruction of microtubules (MTs), which may be one of the mechanisms of cytoskeletal damage induced by ACR.

Induction of morphological and functional differentiation of human neuroblastoma cells by miR-124

Neuroblastoma is the most common extracranial solid tumour in children, and differentiation is considered its most appropriate therapy. In this work, we studied effects of miR-124 overexpression on differentiation in M17 cell line as a model of neuroblastoma cancer. Influence of miR-124 overexpression on differentiation in M17 cells was studied. M17 cells were infected with lentivirus that contained miR-124 precursor sequence and followed for 2 weeks to differentiate. Ectopic expression of miR-124 in M17 cells changed the shape of spherical undifferentiated cells to cells with extended neurites that formed neuronal networks. Overexpression of MiR-124 respectively increased the expression level of markers of β-Tubulin III, MAP2, SYN, NF-M and Nestin by 16-, 5-, 4-, 2.3- and 2-folds at the messenger RNA level. MiR-124 overexpression also increased the protein levels of β-Tubulin III and MAP2. Moreover, exogenous expression of miR-124 significantly increased the intracellular calcium in differentiated M17 cells. Since miR-124 is naturally expressed in neuronal cells and is downregulated in neuroblastoma cancer cells, differentiation with this type of microRNA can be a novel treatment for neuroblastoma cancer.

Bis-cyclopropane analog of disorazole C1 is a microtubule-destabilizing agent active in ABCB1-overexpressing human colon cancer cells.

The novel, chemically stabilized disorazole analog, (-)-CP2-disorazole C1 (1) displayed potent anti-proliferative activity against a broad-spectrum of human colorectal cancer cells. HCT15 and H630R1 cell lines expressing high basal levels of the ABCB1 protein, known to cause multi-drug resistance, were also sensitive to growth inhibition by 1 but were resistant to both vincristine and docetaxel, two commonly used microtubule inhibitors. Compound 1 exhibited strong inhibition of tubulin polymerization at a level comparable to vincristine. In addition, treatment with 1 resulted in decreased protein levels of β-tubulin but not α-tubulin. An analysis of cellular proteins known to interact with microtubules showed that 1 caused decreased expression of c-Myc, APC, Rb, and additional key cellular signaling pathways in CRC cells. Treatment with compound 1 also resulted in G2/M cell cycle arrest and induction of apoptosis, but not senescence. Furthermore, endothelial spheroid sprouting assays demonstrated that 1 suppressed angiogenesis and can, therefore, potentially prevent cancer cells from spreading and metastasizing. Taken together, these findings suggest that the microtubule disruptor 1 may be a potential drug candidate for the treatment of mCRC.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Down-regulation and Covalent Binding at Cysteine 303 of β-Tubulin

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.

Short-term memory in the heart: A road map for channel trafficking required

Short-term memory in the heart: A road map for channel trafficking required