Anti-cancer effect of toosendanin and its underlying mechanisms.

Toosendanin (TSN) is a triterpenoid purified from the medicinal herb Melia toosendan Sieb. et Zucc and has been used as an insecticide for decades. Recent studies have attracted increasing interest of TSN due to its novel anti-cancer effect in diverse cancer models. The broad spectrum anti-cancer activity suggests that TSN inhibits multiple pathways/targets that are critical for cancer cell survival and proliferation. Our recent study indicated that TSN has anti-cancer effect in glioblastoma through induction of estrogen receptor ß (ERß) and p53. This review highlights the anti-cancer efficacy of TSN and provides proof-of-principle insight into the underlying mechanisms.

ZEB1-mediated vasculogenic mimicry formation associates with epithelial-mesenchymal transition and cancer stem cell phenotypes in prostate cancer.

The zinc finger E-box-binding homeobox 1 (ZEB1) induced the epithelial-mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid-Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1-induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT-related and CSC-associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p-Src527 level but not p-Src416 level, while ZEB1 knockdown also down-regulated the level of p-Src527 in PC3 and DU-145 cells. PP2 treatment also significantly reduced the expression of VE-cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.

Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry.

Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future.

Notch signaling regulates CXCR4 expression and the migration of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been used to repair injured tissues through immune-suppression and/or cell replace mechanisms. However, a significant barrier to MSC therapy is insufficient MSC engraftment in injured tissues after systemic administration. Here, we report that cell surface, total protein, and mRNA levels of CXCR4 were significantly increased in MSCs when Notch signaling was interrupted by γ-secretase inhibitor (GSI) or knockout of the transcription factor RBP-J, which mediates signaling from all four mammalian Notch receptors. The GSI-treated or RBP-J deficient MSCs showed stronger migration toward stromal cell-derived factor-1α (SDF-1α) than that of the control. In a mouse hepatic ischemia/reperfusion model, RBP-J deficient MSCs migrated into the injured liver tissues at a significantly higher efficiency than that of the control MSCs. Mice transfused with RBP-J deficient MSCs showed reduced liver damage. Therefore, Notch signaling regulates MSC migration and function, at least partially via the modulation of CXCR4 expression.

[Effects of Wenyang Shengjing Decoction containing serum on the estradiol secretion of Leydig cells of sterile rats of shen-yang deficiency].

OBJECTIVE: To study the effects of Wenyang Shengjing Decoction (WSD) containing serum on the estradiol (E2) secretion, the synthesized cytochrome P450 aromatase (P450arom) activities, as well as the expression of its encode gene CYP19 in Leydig cells of male sterile rats of adenine induced Shen-yang deficiency (SYD). METHODS: Experimental rats were randomly divided into 4 groups, i.e., the normal control group, the high, middle, and low dose WSD groups, 5 in each group. The normal saline, low, middle, and high dose WSD were respectively given to rats of all groups for 10 successive days. Blood was drawn from rats' heart 2 h after the last gastrogavage. The serum was separated after centrifuge. Leydig cells isolated and purified from SYD rats were primary cultured in vitro and divided into 5 groups in random, i. e., the blank control group, the model group, the high, middle, and low dose WSD groups (1.2, 1.0, and 0.8 g/mL, respectively). The content of E2 released in the culture supernate was determined by radioimmunoassay. The P450arom activity was detected by tritium release assay. Meanwhile, the mRNA and protein expressions of CYP19 were analyzed using fluorescent quantitative PCR and Western blot respectively. RESULTS: Compared with the blank control group, the E2 secretion of the supernate of Leydig cells obviously decreased in the model groups, accompanied with the inhibition of P450arom activities, significant decreased protein and mRNA expressions of CYP19 (P < 0.01, P < 0.05). Compared with the model group, after intervened by WSD containing serum, the E2 secretion in the Leydig cells could be significantly increased, the P450arom activities up-regulated, the CYP19 expressions up-regulated at the protein and mRNA levels partially in a dose-dependent manner (P < 0.01, P < 0.05). CONCLUSIONS: WSD containing serum could effectively elevate the E2 secretion in Leydig cells, which might be partially achieved through up-regulating P450arom activities and enhancing the gene expression of CyP19. This might be one of its mechanisms of action for treating male infertility of SYD.

[TGF-beta1 reduces connexin43-mediated gap junctional intercellular communication in rat Leydig cells].

OBJECTIVE: To observe the effects of TGF-beta on the expression of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) in rat Leydig cells, and investigate the association of its effects on Leydig cells with its ability of changing GJIC. METHODS: Primarily cultured purified Leydig cells were divided into a blank control group, a positive control group (treated with the GJIC inhibitor Carbenoxolone), and four TGF-beta1 groups (treated with TGF-beta1 at the concentration of 1, 2, 5 and 10 ng/ml, respectively, for 20 hours). The localization and expression of Cx43 were detected by immunofluorescence and Western blot, and the changes in GJIC analyzed by FRAP assay. RESULTS: Cx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells. TGF-beta1 significantly elevated the expression of Cx43 in the cytoplasm, but caused no evident change in the membrane. Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-beta1 as compared with that of the blank control group (P < 0.05). After 20 hours of treatment with TGF-beta1 at 5 ng/ml, the fluorescence intensity of Leydig cells was markedly reduced (P < 0.01), with a mean fluorescence recovery rate of merely (43.58 +/- 1.87)%. CONCLUSION: TGF-beta1 could significantly down-regulate GJIC between adjacent Leydig cells, and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.

Successful robot-assisted partial nephrectomy for giant renal hilum angiomyolipoma through the retroperitoneal approach: A case report.

BACKGROUND: Giant renal angiomyolipomas (AMLs) may lead to complications including flank pain, hematuria, hypertension, retroperitoneal hemorrhage and even death. Giant AMLs which grow around renal hilar vessels and the ureter are rare. Most previous reports on the treatment of giant renal AMLs have focused on open surgery or a transperitoneal approach, with few studies on the retroperitoneal approach for large AMLs. We here report a case of giant renal hilum AML successfully treated with robot-assisted laparoscopic nephron sparing surgery the retroperitoneal approach, with a one-year follow-up. CASE SUMMARY: A 34-year-old female patient was diagnosed with renal AML 11 years ago and showed no discomfort. The tumor gradually increased in size to a giant AML over the years, which measured 63 mm × 47 mm ×90 mm and was wrapped around the right hilum. Therefore, a robotic laparoscopic partial nephrectomy (LPN) via the retroperitoneal approach was performed. The patient had no serious postoperative complications and was discharged soon after the operation. At the one-year follow-up, the patient's right kidney had recovered well. CONCLUSION: Despite insufficient operating space via the retroperitoneal approach, LPN for giant central renal AMLs can be completed using a well-designed procedure with the assistance of a robotic system.

Negative regulation of endogenous protein kinase Calpha on the dynamic change of carbachol-induced intracellular calcium response in different melanoma cells.

Regulations of intracellular protein kinase C (PKC) on carbachol (CCh)-induced intracellular calcium ([Ca(2+)]i) responses were investigated in different stages of melanoma cells. We found that CCh (1 mM) significantly increased [Ca(2+)]i with 6-, 4-, 4-, and 25-folds intensities in WM793B, 451Lu, SK-MEL-5, and A2058 melanoma cells, respectively. Pretreatment of phorbol 12, 13-dibutyrate (PDBu, 2 microM), an activator of intracellular PKC, significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5, and A2058 cells. RT-PCR data showed that mRNA levels of PKCalpha were 12-, 4-, 6-, and 0.9-folds higher in above four melanoma cells. Short interfering RNA (siRNA) targeting to PKCalpha in WM793B cells enhanced CCh-induced peak calcium reactions. Present data indicated that CCh-induced [Ca(2+)]i responses were dynamically changed in different stages of melanoma progression. Moreover, intracellular PKCalpha activated by exogenous agonist and expressed through endogenous gene transcription negatively regulated CCh-induced calcium responses. The functional analysis on the relationship between CCh-induced calcium response and endogenous PKCalpha expression might be helpful to predict the development of melanoma.

Tanshinone IIA and Astragaloside IV promote the angiogenesis of mesenchymal stem cell-derived endothelial cell-like cells via upregulation of Cx37, Cx40 and Cx43.

Tanshinone IIA (Tan IIA) and Astragaloside IV (AGS-IV) were used as therapeutic treatments for coronary heart diseases (CHDs) in ancient China. However, the underlying mechanisms mediating the effects of Tan IIA and AGS-IV in angiogenesis remain unknown. In the present study, mesenchymal stem cells (MSCs) were induced to differentiate into endothelial cell (EC)-like cells in vitro and the effects of Tan IIA and/or AGS-IV on the functions of these cells, including cell proliferation and tube formation, were assessed. Compared with the single-agent groups (Tan IIA or AGS-IV only), combined-agent (Tan IIA and AGS-IV) treatment significantly enhanced the proliferation and tube formation capacity of EC-like cells. In addition, the expression of connexin 37 (Cx37), Cx40 and Cx43 in the combined-agent group was significantly increased compared with the single-agent groups. Furthermore, enhanced gap junctional intercellular communication (GJIC) was identified in the combined-agent group, as evidenced by increased dye transfer in scrape-loading dye transfer assays. In conclusion, Tan IIA and AGS-IV may promote the angiogenesis of EC-like cells by upregulating the expression of Cx37, Cx40 and Cx43 and enhancing GJIC function. The results of the present study may provide experimental evidence for the clinical application of Tan IIA and AGS-IV as a treatment for CHDs.

Erratum: Tanshinone IIA and Astragaloside IV promote the angiogenesis of mesenchymal stem cell-derived endothelial cell-like cells via upregulation of Cx37, Cx40 and Cx43.

[This corrects the article DOI: 10.3892/etm.2017.5636.].

[Regulatory effect of Wenyang Whengling Decoction on Smads expressions in testis of sterile rats with Shen-yang deficiency].

OBJECTIVE: To study the localization and distribution of expressions of Smads (mother against dpp), the intracellular signal transduction molecules in the transforming growth factor-beta (TGF-beta) family, in the testis of male sterile rats with Shen-yang deficiency induced by adenine and to observe the effect of Wenyang Shengjing Decoction (WSD) on these expressions. METHODS: Rats were randomly divided into the control group, the model group and the WSD group. Localization and distribution of Smad 1, Smad 2 and Smad 4 expressions were detected by immunohistochemistry SABC and semi-quantitative RT-PCR and analyzed statistically by image analysis system; the contents of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were detected by radioimmunoassay; and the weights of body, testis and epididymis, as well as sperm number of rats were also measured. RESULTS: Smad 1 and Smad 2 were expressed in cytoplasm of all levels of spermatogenic cells in rats' testis with their positive immuno-responsive substance locating in the cytoplasm, and positive Smad 2 expression could also be found in cytoplasm of Sertoli's cell, but both of them showed negative response in Leydig's cell; Smad 4 was positively expressed in cytoplasm of Leydig's cell but showed negative response in spermatogenic cell and Sertoli's cell. Compared with the normal control, Smad 1 expression was lower (P < 0.05), but Smad 2 and Smad 4 were higher in the model group (both P < 0.05), these abnormal changes could be reversed by WSD treatment (all P < 0.05). Compared with the normal control group, the body weight, sperm number and serum T level were lower, and levels of FSH and LH were higher (all P < 0.05) in the model group, which could all be improved by WSD (P < 0.05); the weights of testis and epididymis were unchanged in all groups (P > 0.05). CONCLUSION: WSD could not only increase the sperm number through elevating serum T level and decreasing the levels of FSH and LH, but also by way of regulating Smads genes expression to adjust the levels of sex hormones, promote the production of sperm directly or indirectly, so as to treat male infertility.

Validation of DAB2IP methylation and its relative significance in predicting outcome in renal cell carcinoma.

We have recently reported tumor suppressive role of DAB2IP in RCC development. In this study, We identified one CpG methylation biomarker (DAB2IP CpG1) located UTSS of DAB2IP that was associated with poor overall survival in a cohort of 318 ccRCC patients from the Cancer Genome Atlas (TCGA). We further validated the prognostic accuracy of DAB2IP CpG methylation by pyrosequencing quantitative methylation assay in 224 ccRCC patients from multiple Chinese centers (MCHC set), and 239 patients from University of Texas Southwestern Medical Center at Dallas (UTSW set) by using FFPE samples. DAB2IP CpG1 can predict the overall survival of patients in TCGA, MCHC, and UTSW sets independent of patient age, Fuhrman grade and TNM stage (all p<0.05). DAB2IP CpG1 successfully categorized patients into high-risk and low-risk groups with significant differences of clinical outcome in respective clinical subsets, regardless of age, sex, grade, stage, or race (HR: 1.63-7.83; all p<0.05). The detection of DAB2IP CpG1 methylation was minimally affected by ITH in ccRCC. DAB2IP mRNA expression was regulated by DNA methylation in vitro. DAB2IP CpG1 methylation is a practical and repeatable biomarker for ccRCC, which can provide prognostic value that complements the current staging system.

[Expression of Smad1 and Smad5 in the testis of infertile rats with kidney-yang deficiency].

OBJECTIVES: To study the expression of Smad1 and Smad5 in the testis of infertile rats with adenine-modeled kidney-yang deficiency and the pathological mechanism of infertility with kidney-yang deficiency, attempting to obtain experimental evidence for the prevention and treatment of male infertility. METHODS: Forty-eight 60 d male SD rats were divided randomly into 6 groups with 8 in each: 7 d, 14 d and 21 d kidney-yang deficiency groups, and 7 d, 14 d and 21 d control groups. The experimental rats had been fed with adenine (300 mg/kg) and the expression levels of Smad1 and Smad5 were measured with immunohistochemical SABC method at the 7th, 14th and 21st day. RESULTS: Smad1 immunoreactivity was mainly located in the spermatogonia, spermatocytes and spermatids, and the reactive substance distributed in cytoplasm with negative nuclei. Sertoli cells and Leydig cells were negative. Compared with the control, the expression level of Smad1 was decreased significantly at the 21st day (P < 0.05), but with no significant difference at the 7th and 14th day (P > 0.05). Smad5 immunoreactivity was mainly located in the spermatogonia and spermatocytes, and the reactive substance distributed in cytoplasm with negative nuclei. Compared with the control, the expression level of Smad5 was not significantly different at the 7th day (P > 0.05). The expression of Smad5 was negative at the 14th and the 21st day. CONCLUSION: The weaker expression of Smad1 and no expression of Smad5 may be one of the pathological mechanisms of infertility with adenine-modeled kidney-yang deficiency.

A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma.

Clear cell renal cell carcinomas (ccRCCs) display divergent clinical behaviours. Molecular markers might improve risk stratification of ccRCC. Here we use, based on genome-wide CpG methylation profiling, a LASSO model to develop a five-CpG-based assay for ccRCC prognosis that can be used with formalin-fixed paraffin-embedded specimens. The five-CpG-based classifier was validated in three independent sets from China, United States and the Cancer Genome Atlas data set. The classifier predicts the overall survival of ccRCC patients (hazard ratio=2.96-4.82; P=3.9 × 10(-6)-2.2 × 10(-9)), independent of standard clinical prognostic factors. The five-CpG-based classifier successfully categorizes patients into high-risk and low-risk groups, with significant differences of clinical outcome in respective clinical stages and individual 'stage, size, grade and necrosis' scores. Moreover, methylation at the five CpGs correlates with expression of five genes: PITX1, FOXE3, TWF2, EHBP1L1 and RIN1. Our five-CpG-based classifier is a practical and reliable prognostic tool for ccRCC that can add prognostic value to the staging system.

Bifunctional pH-sensitive Zn(ii)-curcumin nanoparticles/siRNA effectively inhibit growth of human bladder cancer cells in vitro and in vivo.

To overcome drug resistance, the combination of two or more therapeutic strategies with different mechanisms has received much attention in recent years. In this study, a common approach has been used to process curcumin and Zn2+ into colloidal dispersions known as "nanoparticles", which are cheap and easy to prepare with high reproducibility. This novel vehicle has good biocompatibility and high cellular uptake for simultaneously delivering the curcumin drug and siRNA into tumor cells. Complexation of Zn2+ with curcumin enhances the aqueous solubility of the hydrophobic drug curcumin and further improves the cellular uptake and bioavailability. The acid-labile coordination Zn(ii)-O bond in Zn(ii)-curcumin drug nanoparticles (Zn(ii)-Cur NPs) can respond to tumor intracellular acidic pH environments to release curcumin, and promoting acid-triggered intracellular drug release. The positively charged Zn(ii)-Cur NPs can efficiently deliver siRNA into human bladder cancer cells, protect siRNA against enzymatic degradation, and facilitate the escape of loaded siRNA from the endosome into the cytoplasm, which successfully downregulates the targeted EIF5A2 oncogene and consequently inhibits cancer cell growth in vitro and in vivo. Proliferation and migration of cancer cells are inhibited by silencing the expression of EIF5A2 and increasing the ratio of pro-apoptotic BAX to anti-apoptotic BCL-2. In vitro and in vivo experiments have demonstrated that bifunctional Zn(ii)-Cur NPs/siEIF5A2 can combine chemotherapy with gene therapy to afford higher therapeutic efficacy than the individual therapeutic protocols.

TGF-ß1 regulation of estrogen production in mature rat Leydig cells.

BACKGROUND: Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-ß1 (TGF-ß1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-ß1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-ß1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultured Leydig cells were incubated in the presence of recombinant TGF-ß1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-ß1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-ß1, and E2 treatment in turn restored the inhibition of TGF-ß1 on GJIC. CONCLUSIONS: Our results indicate, for the first time in adult rat Leydig cells, that TGF-ß1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.

[Therapeutic effect on essential hypertension treated with combined therapy of acupuncture and medication].

OBJECTIVE: To explore the mechanism of acupuncture combined with medication for treatment of essential hypertension (EH). METHODS: Sixty cases of EH were randomly divided into a combined acupuncture and medication group (group A) and a medication group (group B), 30 cases in each one, treated with acupuncture in combination with oral administration of Felodipine, and simple oral administration of Felodipine respectively. Before and after treatment, the changes of blood pressure, and the contents of E-selectin (Es), iNOS and eNOS were determined. RESULTS: After treatment, the blood pressure declined in either group. The total effective rate in group A was 86.7% (26/30), which was superior to that of 73.3% (22/30) in group B. After treatment, the plasma Es and iNOS contents in two groups decreased as compared with those before treatment (both P < 0.01), of which, plasma Es content in group A decreased apparently as compared with group B (P < 0.01). After treatment, the content of plasma eNOS increased as compared with that before treatment in group A (P < 0.01). CONCLUSION: The mechanism of acupuncture on anti-blood pressure probably relies on the improvements in vascular endothelial cellular function so that Es, iNOS and eNOS expression can be recovered to normal level and ultimately blood pressure is adjusted.

The anti-arthritic effects of synthetic melittin on the complete Freund's adjuvant-induced rheumatoid arthritis model in rats.

Bee venom (BV) has been used for millennia in Chinese traditional medicine to treat rheumatoid arthritis (RA). However, its components and mechanism remain unclear, which has hampered its development and application for the treatment of RA. In this study, we examined the anti-arthritis effects of melittin, which composes nearly 50% of the dry weight of whole BV, on the complete Freund's adjuvant-induced (CFA-induced) RA model in rats. The RA animal models were treated with solutions of BV, melittin, and saline by injection into a specific acupoint (Zusanli). The BV and melittin treatments statistically diminished the thickness of the arthroses in the injected side of the paw, compared to the saline treatment. Melittin therapy also significantly reduced arthritis-induced nociceptive behaviors, as assessed by the thermal hyperalgesia test. In addition, CFA-induced Fos expression in the superficial layer of the lumbar spinal cord was significantly suppressed by the BV and melittin treatments, compared to the saline treatment. These results indicate that melittin is an effective anti-arthritis component of whole bee venom, making it a promising candidate as an anti-arthritis drug.

[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].

AIM: To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method. METHODS: PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin. RESULTS: After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained. CONCLUSION: Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.