Smooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemia.

Objective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays. Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice. Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.

CS2164, a novel multi-target inhibitor against tumor angiogenesis, mitosis and chronic inflammation with anti-tumor potency.

Although inhibitors targeting tumor angiogenic pathway have provided improvement for clinical treatment in patients with various solid tumors, the still very limited anti-cancer efficacy and acquired drug resistance demand new agents that may offer better clinical benefits. In the effort to find a small molecule potentially targeting several key pathways for tumor development, we designed, discovered and evaluated a novel multi-kinase inhibitor, CS2164. CS2164 inhibited the angiogenesis-related kinases (VEGFR2, VEGFR1, VEGFR3, PDGFRα and c-Kit), mitosis-related kinase Aurora B and chronic inflammation-related kinase CSF-1R in a high potency manner with the IC50 at a single-digit nanomolar range. Consequently, CS2164 displayed anti-angiogenic activities through suppression of VEGFR/PDGFR phosphorylation, inhibition of ligand-dependent cell proliferation and capillary tube formation, and prevention of vasculature formation in tumor tissues. CS2164 also showed induction of G2/M cell cycle arrest and suppression of cell proliferation in tumor tissues through the inhibition of Aurora B-mediated H3 phosphorylation. Furthermore, CS2164 demonstrated the inhibitory effect on CSF-1R phosphorylation that led to the suppression of ligand-stimulated monocyte-to-macrophage differentiation and reduced CSF-1R+ cells in tumor tissues. The in vivo animal efficacy studies revealed that CS2164 induced remarkable regression or complete inhibition of tumor growth at well-tolerated oral doses in several human tumor xenograft models. Collectively, these results indicate that CS2164 is a highly selective multi-kinase inhibitor with potent anti-tumor activities against tumor angiogenesis, mitosis and chronic inflammation, which may provide the rationale for further clinical assessment of CS2164 as a therapeutic agent in the treatment of cancer.

Indole diketopiperazines from endophytic Chaetomium sp 88194 induce breast cancer cell apoptotic death.

Diketopiperazines are important secondary metabolites of the fungi with variety bioactivities. Several species belonging to genus Chaetomium produce compounds of this class, such as chetomin. To identify new antitumor agents, secondary metabolites of fungus Chaetomium sp 88194 were investigated and three new indole diketopiperazines, Chaetocochins G (1), Oidioperazines E (2) and Chetoseminudin E (3), along with two known compounds Chetoseminudins C (4) and N-acetyl-ß-oxotryptamine (5), were obtained. Chaetocochins G and Chetoseminudin E were recrystallized in CHCl3 containing a small amount of MeOH, and their structures with absolute configuration were established by spectroscopic data interpretation and single-crystal X-ray diffraction analysis. The absolute configuration of Oidioperazines E was defined by comparing of experimental and calculated electronic circular dichroism spectra. These isolates were also evaluated the anticancer activity, and Chaetocochins G displayed more potent cytotoxicity in MCF-7 cells than the common chemotherapeutic agent (5-fluorouracil) associated with G2/M cell cycle arrest. More importantly, Chaetocochins G induced cell apoptotic death via caspase-3 induction and proteolytic cleavage of poly (ADP-ribose) polymerase, concomitantly with increased Bax and decreased Bcl-2 expression. Our findings suggested that indole diketopiperazines from endophytic Chaetomium sp 88194 may be potential resource for developing anti-cancer reagents.

Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis.

Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains high concentrations of flavonoids. The flavonoids are mainly represented by rutin, anthocyanins and proanthocyanins in tartary buckwheat. R2R3-type MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthetic pathway. In this study, two TF genes, FtMYB1 and FtMYB2, were isolated from F. tataricum and characterized. The results of bioinformatic analysis indicated that the putative FtMYB1 and FtMYB2 proteins belonged to the R2R3-MYB family and displayed a high degree of similarity with TaMYB14 and AtMYB123/TT2. In vitro and in vivo evidence both showed the two proteins were located in the nucleus and exhibited transcriptional activation activities. During florescence, both FtMYB1 and FtMYB2 were more highly expressed in the flowers than any other organ. The overexpression of FtMYB1 and FtMYB2 significantly enhanced the accumulation of proanthocyanidins (PAs) and showed a strong effect on the target genes' expression in Nicotiana tabacum. The expression of dihydroflavonol-4-reductase (DFR) was upregulated to 5.6-fold higher than that of control, and the expression level was lower for flavonol synthase (FLS). To our knowledge, this is the first functional characterization of two MYB TFs from F. tataricum that control the PA pathway.

New norclerodane diterpenoids from the tubers of Dioscorea bulbifera.

Phytochemical investigations of the tubers of Dioscorea bulbifera L. resulted in the isolation of nine norclerodane diterpenoids, including two new compounds, diosbulbins N (1) and P (3), a new naturally occurring compound, diosbulbin O (2), and six known ones, diosbulbins A-D, F and G (4-9). Their structures were established by spectroscopic and chemical methods. The absolute stereochemistry of 1 was determined by a modified Mosher's method, and the absolute configuration of 2 was determined by a single-crystal X-ray diffraction analysis with CuKα irradiation. Compounds 1-3 were evaluated for in vitro cytotoxicity against five human cancer cell lines.

[Effect of Shugan Jianpi Recipe on LXRα/FAS signaling pathway mediated hepatocyte fatty deposits in NAFLD rats].

OBJECTIVE: To explore the effect of Shugan Jianpi Recipe (SJR) on LXRα/FAS signaling pathway mediated hepatocyte fatty deposits in nonalcoholic fatty liver disease (NAFLD) rats. METHODS: Totally 75 SPF grade male SD rats were randomly divided into 5 groups, i.e., the normal control group, the model group, the Shugan Recipe (SR) treatment groups, the Jianpi Recipe (JR) treatment group, and the SJR group. Except rats in the normal control group, the NAFLD rat model was duplicated using high fat diet (HFD). SR (Chaihu Shugan Powder) was administered to rats in the SR group. JR (Shenlin Baizhu Powder) was administered to rats in the JR group. SJR (Chaihu Shugan Powder plus Shenlin Baizhu Powder) was administered to rats in the SJR group. Changes of liver fat were analyzed using automatic biochemical analyzer. Liver cells were separated by low-speed centrifugation. Their activities and purities were identify using Typan blue and flow cytometry (FCM). Expression levels of LXRα and FAS mRNA in hepatocytes detected by Real-time quantitative PCR. Expression levels of LXRα and FAS protein were detected by Western blot. RESULTS: (1) Pathological results showed in the model group, hepatocytes were swollen with nucleus locating at the cell edge after oil red O staining; unequal sized small vacuoles could be seen inside cytoplasm. Some small vacuoles merged big vacuoles. All these indi- cated a NAFLD rat model was successfully established by high fat diet. Pathological structural changes could be impaired to some degree in all medicated groups, especially in the SR group. (2) Compared with the normal control group, expression levels of LXRα and FAS genes and proteins obviously increased in the model group (P < 0.01). Compared with the model group, their expression levels were obviously down-regulated in the JR group and the SR group (P < 0.01, P < 0.05). CONCLUSIONS: LXRα/FAS signaling pathway was an important signaling pathway for mediating lipid metabolism disorders of NAFLD rats. SJR could make hepatocyte fatty deposits tend to repair by adjusting the LXRα/FAS signaling pathway in NAFLD rats, which might be one of important mechanisms for SJR to prevent and cure NAFLD.

[Construction and tobacco transformation of COR and BBE genes hairpin RNA vector of Papaver somniferum].

The gene expressions of codeinone reductase (COR) and berberine bridge enzyme (BBE) in Papaver somniferum were blocked by RNA hairpin of RNA interference (RNAi). The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.

[Correlation between XRCC2 and XRCC5 single nucleotide polymorphisms and drug-sensitivity of human lung cancer cells].

OBJECTIVE: To study the associations between XRCC2 and XRCC5 single nucleotide polymorphisms (SNPs) and chemosensitivity of human lung cancer cells. METHODS: Specimens of human lung cancer were collected from 150 patients, 120 males and 30 females, aged 58.1, during operation. MTT method was used to detect the sensitivity of the lung cancer cells to cisplatin (DDP) and carboplatin (CBP), and the patients were genotyped for polymorphisms in XRCC2 and XRCC5 genes. The polymorphisms of XRCC2 41657T C41657T and XRCC5 G74582A were detected by polymerase chain reaction-restriction fraction length polymorphism (PCR-RFLP), and the polymorphisms of XRCC2 G4234C and XRCC5 C74468A were detected by primer-introduced restriction analysis- polymerase chain reaction (PIRA-PCR). The correlation of these polymorphisms of these polymorphisms with the drug-sensitivity of lung cancer cells was analyzed. RESULTS: The sensitive rates of the lung cancer cells to CBP and DDP in the patients with XRCC2 41657T allele (C/T + T/T genotype) were 70.2% and 66.7% respectively, both significantly higher the those in the patients with C/C genotype [53.7% (chi(2) = 3.97, P = 0.046) and 49.5% (chi(2) = 4.25, P = 0.039) respectively]. The sensitivity levels to CBP and DDP in the patients with at least one T allele was 2.06 times (pathological type adjusted OR = 2.06, 95% CI = 1.02 - 4.18) and 2.07 times (pathological type adjusted OR = 2.07, 95% CI = 1.04 - 4.14) higher than those in the patients with C/C genotype. There was no significant difference in the distribution of genotypes of XRCC2 G4234C C allele (C/C + G/C) and G/C genotype between the sensitive group and resistant groups (chi(2) = 0.09, P = 0.766 for CBP and chi(2) = 1.63, P = 0.202 for DDP). The sensitivity rate to DDP of the patients with 41657T/4234G haplotype was 2.28 times as high as that of the patients with 41657T/4234C (OR = 2.18, 95% CI = 1.15 - 4.12). The sensitivity rates to CBP and DDP of the tumor tissues from the patients with the XRCC5 G74582A G allele (A/G + G/G), were 57.9% and 61.8% respectively, and those of the patients with the A/A genotype were 62.2% and 50.0% respectively. There was no significant differences in the genotype distribution between the sensitive and resistant groups (chi(2) = 0.28, P = 0.594 for CBP, and chi(2) = 2.13, P = 0.144 for and DDP). CONCLUSION: The XRCC2 C41657T SNP is associated with the sensitivity to CBP and DDP. The sensitive rate to the drugs of the cancer cells with the combination of C/T + T/T genotype is higher than that with C/C genotype. In the subject with 41657T/4234G the sensitivity to the drugs of the cancer cells is higher than that with 41657C/4234G haplotypes. The XRCC5 G74582A and C74468A SNPs are not associated with the sensitivity to CBP and DDP.

[Study on the origin of H/RS cell and their biological behavior in Hodgkin lymphoma by using multiple mark techniques].

OBJECTIVE: To investigate the apoptosis-related genes and protein expression patterns in relation to classical Hodgkin lymphomas (CHL) and the origin of H/RS cell. METHODS: Sixty-two cases of CHL were retrieved from Shanxi Tumor Hospital files. An ABC method was used to detect the expression of bcl-2, CD3, CD20, CD30, CD15 and CD10, a double immunohistochemical method to study the H/RS cells P53 expression, a double immunohistochemical ABC-DNA end labeling technique to detect the apoptosis, a double immunohistochemical ABC- in situ hybridization technique to detect the expression of kappa mRNA and lambda mRNA, and a multiple mark techniques to detect the distribution of background non-neoplastic T and B cells. RESULT: Of 62 CHL, 14 (22.58%) were p53 positive and 35 (56.45%) bcl-2 positive. Apoptosis was found in the background non-neoplastic cells in all of the cases, but in H/RS cells in only 10 of 62 cases. There was a significant reverse correlation between bcl-2 expression and apoptosis in H/RS cells (P = 0.02). CD30 positive H/RS cells were observed in all cases, whereas CD15 positive in only 41 cases, and CD20 positive in 8 cases. None was positive for CD3, MPO, bcl-6, CD10, kappa RNA and lambda RNA in H/RS cells. The H/RS cells were surrounded by non-neoplastic T cells looked like a rosette and the outer periphery was B cells. CONCLUSIONS: The H/RS cell of classical Hodgkin lymphoma has a great variety of B lineage markers. The characteristic distributions of T, B and H/RS cells may serve as a reference for the diagnosis. Multiple marker technique is able to highlight the critical cells, and facilitate the study of H/RS cells. Abnormal expression of P53 may not play a major role in CHL. Over expression of bcl-2 may be linked to blockage of apoptosis in CHL.