Delta-catenin attenuates medulloblastoma cell invasion by targeting EMT pathway.

Background: Medulloblastoma is the most common pediatric malignant tumor in central nervous system. Although its prognosis has been improved enormously by the combination treatments with surgery, radiotherapy, and chemotherapy, it still could progress via invasion and distant dissemination. We aimed to investigate molecular mechanisms of medulloblastoma invasion in the current work. Methods: The gene expression profile of medulloblastoma were analyzed based on the data deposited in Gene Expression Omnibus (GEO) and filtered according to brain specific proteins in the Uniprot. Delta-catenin was identified and further analyzed about its expression and roles in the prognosis of medulloblastoma patient. The function of delta-catenin on cell invasion and migration were investigated by transwell and wound healing assay. Whether delta-catenin participates in the epithelial-mesenchymal transition (EMT) regulated invasion was also studied. Results: Delta-catenin expression was highly upregulated in tumor tissues compared to normal tissues from medulloblastoma patients in five independent, nonoverlapping cohorts. Furthermore, delta-catenin expression level was upregulated in WNT subgroup, and significantly correlated with better prognosis, and associated with metastasis through GEO database analysis. Functional assays indicated that delta-catenin inhibited medulloblastoma cell invasion and migration through regulating the key factors of EMT pathway, such as E-cadherin and vimentin. Conclusion: Delta-catenin might be a positive predictor for prognosis of medulloblastoma patients, through attenuating medulloblastoma cell invasion by inhibiting EMT pathway.

Skp2 modulates proliferation, senescence and tumorigenesis of glioma.

BACKGROUND: Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Surgery followed by radiotherapy and chemotherapy has been used as a standard strategy but yielded unsatisfactory improvements in patient survival outcomes. The S-phase kinase protein 2 (Skp2), a critical component of the E3-ligase SCF complex, has been documented in tumorigenesis in various cancer types but its role in glioma has yet to be fully clarified. In this study, we investigated the function of Skp2 in the proliferation, stem cell maintenance, and drug sensitivity to temozolomide (TMZ) of glioma. METHODS: To investigate the role of Skp2 in the prognosis of patients with glioma, we first analyzed data in databases TCGA and GTEx. To further clarify the effect of Skp2 on glioma cell proliferation, we suppressed its level in glioblastoma (GBM) cell lines through knockdown and small molecule inhibitors (lovastatin and SZL-P1-41). We then detected cell growth, colony formation, sphere formation, drug sensitivity, and in vivo tumor formation in xenograft mice model. RESULTS: Skp2 mRNA level was higher in both low-grade glioma and GBM than normal brain tissues. The knockdown of Skp2 increased cell sensitivity to TMZ, decreased cell proliferation and tumorigenesis. In addition, Skp2 level was found increased upon stem cells enriching, while the knockdown of Skp2 led to reduced sphere numbers. Downregulation of Skp2 also induced senescence. Repurposing of lovastatin and novel compound SZL-P1-41 suppressed Skp2 effectively, and enhanced glioma cell sensitivity to TMZ in vitro and in vivo. CONCLUSION: Our data demonstrated that Skp2 modulated glioma cell proliferation in vitro and in vivo, stem cell maintenance, and cell sensitivity to TMZ, which indicated that Skp2 could be a potential target for long-term treatment.

Novel histone deacetylase inhibitor N25 exerts anti-tumor effects and induces autophagy in human glioma cells by inhibiting HDAC3.

N25, a novel histone deacetylase inhibitor, was created through structural modification of suberoylanilide hydroxamic acid. To evaluate the anti-tumor activity of N25 and clarify its molecular mechanism of inducing autophagy in glioma cells, we investigated its in vitro anti-proliferative effect and in vivo anticancer effect. Moreover, we detected whether N25 induces autophagy in glioma cells by transmission electron microscope and analyzed the protein expression level of HDAC3, Tip60, LC3 in glioma samples by western blot. We additionally analyzed the protein expression level of HDAC3, Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment with N25 in glioma cells. Our results showed that the anti-tumor activity of N25 in glioma cells is slightly stronger than SAHA both in vitro and in vivo. We found that N25 induced autophagy, and HDAC3 was significantly elevated and Tip60 and LC3 significantly decreased in glioma samples compared with normal brain tissues. Nevertheless, N25 inhibited HDAC3 and up-regulated the protein expression of Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment of glioma cells with N25. In conclusion, these data suggest that N25 has striking anti-tumor activity in part due to inhibition of HDAC3. Additionally, N25 may induce autophagy through inhibiting HDAC3.

Screening Novel SAHA Derivatives as Anti-lung Carcinoma Agents: Synthesis, Biological Evaluation, Docking Studies and Further Mechanism Research between Apoptosis and Autophagy.

Four suberoylanilide hydroxamic acid (SAHA) derivatives (N34, N4I, N4B, N24) were designed and synthesized on the basis of our previous studies on N25. Assays for anti-proliferative activity and histone deacetylase (HDAC) activity were performed against human lung cancer (SPC-A-1, LTEP-a-2, NCI-H1650) and normal lung cells (MRC-5), which were compared with those of SAHA. Molecular docking was used to theoretically confirm the receptor-binding ability of N34. Ultimately, N34 was validated as the best HDAC inhibitor candidate. Furthermore, the effects of N34 on the levels of apoptosis- and autophagy-associated proteins caspase-3, caspase-9, Bcl-2 and Beclin-1 in SPC-A-1 cells were evaluated. N34 exerted more evident effects on human lung cancer than the other three SAHA derivatives did.

Synthesis, Biological Evaluation, and Computer-Aided Drug Designing of New Derivatives of Hyperactive Suberoylanilide Hydroxamic Acid Histone Deacetylase Inhibitors.

The synthesis and biological evaluation of a novel series of compounds based on suberoylanilide hydroxamic acid (SAHA) had been designed as potential histone deacetylase inhibitors (HDACis). Molecular docking studies indicated that our derivatives had better fitting in the binding sites of HDAC8 than SAHA. Compounds 1-5 were synthesized through the synthetic routes. In biological test, compounds also showed good inhibitory activity in HDAC enzyme assay and more potent growth inhibition in human glioma cell lines (MGR2, U251, and U373). A representative compound, N3F, exhibited better inhibitory effect (HDAC, IC50  = 0.1187 µm; U251, IC50  = 0.8949 µm) and lower toxicity for human normal cells (LO2, IC50  = 172.5 µm and MRC5, IC50  = 213.6 µm) compared with SAHA (HDAC, IC50  = 0.8717 µm; U251, IC50  = 8.938 µm; LO2, IC50  = 86.52 µm and MRC5, IC50  = 81.02 µm). In addition, N3F obviously increased Beclin-1 and Caspase-3 and 9 as well as inhibited Bcl-2 in U251 cells. All of our results indicated that these SAHA cap derivatives could serve as potential lead compounds for further optimization. In addition, N3F and N2E both displayed promising profile as antitumor candidates for the treatment of human glioma.

A novel suberoylanilide hydroxamic acid histone deacetylase inhibitor derivative, N25, exhibiting improved antitumor activity in both human U251 and H460 cells.

N1- (2, 5-dimethoxyphenyl)-N(8)-hydroxyoctanediamide (N25) is a novel SAHA cap derivative of HDACi, with a patent (No. CN 103159646). This invention is a hydroxamic acid compound with a structural formula of RNHCO(CH2)6CONHOH (wherein R=2, 5dimethoxyaniline), a pharmaceutically acceptable salt which is soluble. In the present study, we investigated the effects of N25 with regard to drug distribution and molecular docking, and anti-proliferation, apoptosis, cell cycling, and LD50. First, we designed a molecular approach for modeling selected SAHA derivatives based on available structural information regarding human HDAC8 in complex with SAHA (PDB code 1T69). N25 was found to be stabilized by direct interaction with the HDAC8. Anti-proliferative activity was observed in human glioma U251, U87, T98G cells and human lung cancer H460, A549, H1299 cells at moderate concentrations (0.5-30 µM). Compared with SAHA, N25 displayed an increased antitumor activity in U251 and H460 cells. We further analyzed cell death mechanisms activated by N25 in U251 and H460 cells. N25 significantly increased acetylation of Histone 3 and inhibited HDAC4. On RT-PCR analysis, N25 increased the mRNA levels of p21, however, decreased the levels of p53. These resulted in promotion of apoptosis, inducing G0/G1 arrest in U251 cells and G2/M arrest in H460 cells in a time-dependent and dose- dependent manner. In addition, N25 was able to distribute to brain tissue through the blood-brain barrier of mice (LD50: 240.840 mg/kg). In conclusion, our findings demonstrate that N25 will provide an invaluable tool to investigate the molecular mechanism with potential chemotherapeutic value in several malignancies, especially human glioma.

Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells.

O6-methylguanine DNA methyltransferase (MGMT) can remove DNA alkylation adducts, thereby repairing damaged DNA and contributing to the drug resistance of gliomas to alkylating agents. In addition, glioma stem-like cells (GSCs) have been demonstrated to be involved in the recurrence and treatment resistance of gliomas. In this study, we aimed to investigate MGMT expression and regulatory mechanisms in GSCs and the association of MGMT with temozolomide (TMZ) sensitivity. GSCs were enriched from one MGMT-positive cell line (SF-767) and 7 MGMT-negative cell lines (U251, SKMG-4, SKMG-1, SF295, U87, MGR1, and MGR2) through serum-free clone culture. GSCs from the U251G, SKMG-4G, SF295G, and SKMG-1G cell lines became MGMT-positive, but those from the U87G, MGR1G, and MGR2G cell lines remained MGMT-negative. However, all the GSCs and their parental glioma cell lines were positive for nuclear factor-κB (NF-κB). In addition, GSCs were more resistant to TMZ than their parental glioma cell lines (P < 0.05). However, there was no significant difference in the 50% inhibition concentration (IC50) of TMZ between MGMT-positive and MGMT-negative GSCs (P > 0.05). When we treated the MGMT-positive GSCs with TMZ plus MG-132 (an NF-κB inhibitor), the antitumor activity was significantly enhanced compared to that of GSCs treated with TMZ alone (P <0.05). Furthermore, we found that MGMT expression decreased through the down-regulation of NF-κB expression by MG-132. Our results show that MG-132 may inhibit NF-κB expression and further decrease MGMT expression, resulting in a synergistic effect on MGMT-positive GSCs. These results indicate that enhanced MGMT expression contributes to TMZ resistance in MGMT-positive GSCs.

Indazoles as potential c-Met inhibitors: design, synthesis and molecular docking studies.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is considered as an attractive target for small molecule drug discovery. In this study, a series of indazoles were designed, synthesized and evaluated as novel c-Met inhibitors. The results showed that the majority of the compounds exhibited significant inhibition on c-Met and compound 4d showed highest activity against c-Met with IC50 value of 0.17 µM in TR-FRET-based assay and IC50 value of 5.45 µM in cell-based assay as compared to other tested compounds. Molecular docking experiments verified the results and explained the molecular mechanism of pretty activities to c-Met.

[Study on the vasodilation effect of blood vessel and its mechanism by the extract of bu shen huo xue recipe].

OBJECTIVE: To study the effect of the extract of Bu Shen Huo Xue Recipe on blood vessel and the content of NO, NOS, cNOS and iNOS in plasma and aortic tissue. METHODS: The effect of blood vessels was studied by guinea pig mesentery blood capillary, rat aortic ring and celiac vein ring. The content of NO, NOS, cNOS and iNOS were determined by method of nitroreductase chromatometry in plasma and aortic tissue. RESULTS: The extract of Bu Shen Huo Xue Recipe could significantly increase the sum of crisscross blood capillary, improve bloodstream state and speed. The significantly vasodilating effect induced by the extract of Bu Shen Huo Xue Recipe was seen in rat aortic ring and celiac vein (based on 10 micromol/L phenylephrine-induced concentration). But there was contracting effect in denuded endothelium. The relaxations induced by the extract on blood vessels mentioned above were markedly attenuated by preincubation in organ chambers with glibenclamide (1 micromol/L), the same effect in lower dose with propranolol (3 micromol/L), the contractional effect in high dose with propranolol (3 micromol/L). The extract in high dose could significantly affect the content of NO in plasma, increase the content of NO, NOS and cNOS in aortic tissue. CONCLUSION: The mechanism of vasodilation effect of blood vessel of the extract may be related to releasing NO from endothelium of blood vessels and increasing the content of synthetic NOS and cNOS in aortic tissue.