Baicalein targets STMN1 to inhibit the progression of nasopharyngeal carcinoma via regulating the Wnt/ß-catenin pathway.

BACKGROUNDS: Nasopharyngeal carcinoma is a common malignancy in the head and neck. Baicalein has been reported to exert the anticancer effects on various cancers. In this study, our aim was to explore the function of baicalein in the development of nasopharyngeal carcinoma and further investigate the potential underlying mechanisms. METHODS: Cell Counting Kit (CCK)-8 assay, EdU assay, sphere formation assay, flow cytometry, and transwell invasion assay were conducted to determine cell proliferation, stemness, apoptosis, and invasion, respectively. Western blot was performed to examine the protein levels of PCNA, MMP9, STMN1, ß-catenin, and Wnt3A. The mRNA level of STMN1 was assessed using real-time quantitative polymerase chain reaction (RT-qPCR). Xenograft tumor model was carried out to evaluate the effects of baicalein on tumor growth in vivo. Immunohistochemistry (IHC) assay was used to detect the levels of PCNA, MMP9, and STMN1 in tumor tissues from mice. RESULTS: Baicalein significantly induced cell apoptosis and impeded cell proliferation, invasion, and stemness of nasopharyngeal carcinoma cells. STMN1 was highly expressed in nasopharyngeal carcinoma, and baicalein could directly downregulate STMN1 expression. STMN1 knockdown hampered the progression of nasopharyngeal carcinoma cells. Moreover, the effects of baicalein on cell proliferation, stemness, invasion, and apoptosis in nasopharyngeal carcinoma cells were harbored by STMN1 overexpression. Baicalein regulated STMN1 to inhibit the activation of the Wnt/ß-catenin pathway. SKL2001, an agonist of the Wnt/ß-catenin pathway, could reverse the effects of STMN1 knockdown on the progression of nasopharyngeal carcinoma. In addition, baicalein markedly impeded tumor growth in vivo. CONCLUSION: Baicalein regulated the STMN1/Wnt/ß-catenin pathway to restrain the development of nasopharyngeal carcinoma.

STMN1 promotes cell malignancy and bortezomib resistance of multiple myeloma cell lines via PI3K/AKT signaling.

BACKGROUND: This study investigates the biological functions of Stathmin1 (STMN1) involving drug resistance and cell proliferation in multiple myeloma (MM) and its related mechanisms. METHODS: Bone marrow aspirates were collected from 20 MM patients, and the bone marrow mononuclear cells (BMMCs) were separated by Ficoll-Hypaque density gradient centrifugation. Blood samples of 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy donors were collected. Normal plasma cells sorted from the peripheral blood of MGUS patients and healthy subject as controls. Two bortezomib (BTZ)-resistant MM cell lines were established, namely NCI-H929/BTZ and KM3/BTZ cells, and then transfected with lentiviruses packaging sh-STMN1 to knock down STMN1 level in BTZ-resistant cells. Expression of STMN1 was assessed by RT-qPCR and western blotting. CCK-8 assays were performed to assess 50% growth inhibition (IC50) values. Green fluorescent protein in BTZ-resistant cells infected with lentiviruses was observed by fluorescence microscopy. Cell viability, proliferation, cell cycle, and apoptosis were evaluated through MTT assays, colony formation assays, flow cytometry analyses, and TUNEL staining. RESULTS: STMN1 was upregulated in MM cells and bone marrow aspirates of MM patients. Additionally, STMN1 depletion attenuated BTZ resistance in MM cells. Moreover, downregulation of STMN1 limited the malignant phenotypes of BTZ-resistant cells. Mechanistically, the PI3K/Akt signaling was inactivated by STMN1 downregulation in BTZ-resistant cells. CONCLUSION: STMN1 silencing inhibits cell proliferation and BTZ resistance in MM by inactivating the PI3K/Akt signaling.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells.

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ ß-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

The Impact of Eribulin on Stathmin Dynamics and Paclitaxel Sensitivity in Ovarian Cancer Cells.

Eribulin, an inhibitor of microtubule dynamics, is used for treating breast cancers and sarcomas. The microtubule-destabilizing protein stathmin may modulate the antiproliferative activity of eribulin on breast cancer cells and leiomyosarcoma cells. The antitumor activity of eribulin in ovarian cancers has not been fully explored, so the present study aimed to determine the antitumor efficacy of eribulin and the involvement of stathmin in ovarian cancers. In a xenograft model of ovarian cancer, eribulin treatment reduced the tumor weight, which was accompanied by an increased level of phosphorylated stathmin. Eribulin stimulated the phosphorylation of stathmin in cultured cancer cell lines. The eribulin-induced phosphorylation of stathmin was inhibited by treatment with FTY720, an activator of protein phosphatase 2A (PP2A), and eribulin downregulated the expression of PP2A subunits. Furthermore, stathmin knockdown abrogated the inhibitory effects of eribulin on cell viability. Eribulin enhanced the antiproliferative effects of paclitaxel and concomitantly decreased stathmin expression. These results suggest that eribulin-induced phosphorylation of stathmin, mediated in part by PP2A downregulation, reduces stathmin activity and enhances the antiproliferative effects of paclitaxel in ovarian cancer. Collectively, the results of this study indicate that eribulin may suppress the proliferation of ovarian cancer cells partly by regulating the activity of stathmin.

Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration.

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.

Stathmin potentiates vinflunine and inhibits Paclitaxel activity.

Cell biology and crystallographic studies have suggested a functional link between stathmin and microtubule targeting agents (MTAs). In a previous study we showed that stathmin increases vinblastine (VLB) binding to tubulin, and that conversely VLB increases stathmin binding to tubulin. This constituted the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and revealed a new mechanism of action for VLB. The question remained if the observed interaction was specific for this drug or represented a general phenomenon for all MTAs. In the present study we investigated the binding of recombinant stathmin to purified tubulin in the presence of paclitaxel or another Vinca alkaloid, vinflunine, using Isothermal Titration Calorimetry (ITC). These experiments revealed that stathmin binding to tubulin is increased in the presence of vinflunine, whereas no signal is observed in the presence of paclitaxel. Further investigation using turbidity and co-sedimentation showed that stathmin inhibited paclitaxel microtubule-stabilizing activity. Taken together with the previous study using vinblastine, our results suggest that stathmin can be seen as a modulator of MTA activity and binding to tubulin, providing molecular explanation for multiple previous cellular and in vivo studies showing that stathmin expression level affects MTAs efficiency.

Stathmin is key in reversion of doxorubicin resistance by arsenic trioxide in osteosarcoma cells.

Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. Numerous patients are unable to be cured due to the development of resistance of the osteosarcoma cells to chemotherapeutic drugs. Therefore, reversion of drug resistance is urgently required for the treatment of osteosarcoma. Arsenic trioxide (As2O3) is an active ingredient in Traditional Chinese Medicine, but the therapeutic potential of As2O3 in osteosarcoma remains largely unexplored. The current study investigated the effects of As2O3 on MG63 osteosarcoma cells using a cell proliferation assay, flow cytometric analysis of the cell cycle and cell apoptosis, reverse transcription polymerase chain reaction to detect stathmin mRNA expression levels and western blot analysis to detect the stathmin protein expression levels. As2O3 and doxorubicin (ADM) combination treatment markedly inhibited cell proliferation in ADM-resistant MG63 (MG63/dox) osteosarcoma cells, clearly induced G2/M phase cell cycle arrest and increased the number of apoptotic MG63/dox cells. Furthermore, stathmin expression was found to be downregulated in MG63/dox cells and was sensitive to ADM treatment. Additional investigation revealed that the downregulation of stathmin expression in MG63/dox cells by stathmin small interfering RNA significantly enhanced the reversion of ADM resistance in MG63/dox by As2O3. The data indicated that As2O3 reversed ADM resistance in MG63/dox cells through downregulation of stathmin and may be a potential drug for the treatment of ADM-resistant osteosarcoma.

Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.

Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.

Effect of Stathmin decoy-oligodeoxynucleotides on the proliferation and differentiation of precartilainous stem cells.

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.