In vitro and in vivo evaluation of the neuroprotective activity of Uncaria hirsuta Haviland.

The incidence of neurodegeneration leading to the conditions such as Alzheimer's and Parkinson's diseases are on the increase, they require the approaches that focus on protection prevention rather than treatment. Plants are rich sources of many compounds which possess medicinal properties. We sought to investigate the neuroprotective effects of Uncariahirsuta and its compounds on d-galactose-induced stress in BALB/c mice as well as 6-hydroxydopamine (6-OHDA)-induced stress in mouse nerve growth factor (mNGF)-differentiated PC12 cells. Our results demonstrate that the 95% ethanol extract of U. hirsuta reversed the d-galactose-induced learning and memory dysfunctions and decreased the malodialdehyde levels. Furthermore, the isolated compounds, 5ß-carboxystrictosidine (1) and chlorogenic acid (2), protected mNGF-differentiated PC12 cells against toxicity induced by 6-OHDA by acting as antiapoptotic agents. The 50% inhibitory concentration (IC50) for intracellular reactive oxygen species (ROS) scavenging was found to be 24.5 (for 1) and 19.7 µM (for 2), and both 1 and 2 reduced intracellular calcium levels with respective IC50 values of 46.9 and 27 µM. Interestingly, both compounds inhibited caspase 3 and 9 activities with respective IC50 values of 25.6 and 24.5 µM for 1 and 19.4 and 16.3 µM for 2. Our results identify U. hirsuta and its active compounds as potential neuroprotective agents and deserve further evaluation for drug development for neuroprotection in the future.

Ovatodiolide inhibits the oncogenicity and cancer stem cell-like phenotype of glioblastoma cells, as well as potentiate the anticancer effect of temozolomide.

BACKGROUND: Ovatodiolide (Ova), a major bioactive diterpenoid isolate of Anisomeles indica has drawn considerable attention lately as an effective anticancer agent with several published works demonstrating its tumor-inhibitory activity in various cancer types. PURPOSE: In this study, we examined the modulatory effect of Ova on the oncogenicity, proliferation, and cancer stem cell-like traits of glioblastoma (GBM) cells, as well as investigated the underlying molecular mechanism for the anticancer activity of Ova in GBM cell lines, U-87MG and GBM8401. METHODS: The antiproliferative, apoptotic, and stemness-attenuating effects of Ova were evaluated using the sulforhodamine B (SRB) colorimetric assay, western blot and fluorescent immunocytochemistry. Cell apoptosis was analyzed based on variation in the expression levels of Bcl-2 family of regulator proteins Bax, Bak, Bcl-2 and Bcl-xL. RESULTS: Ova induced the apoptosis of the U-87MG and GBM8401 cells, as well as effectively inhibited the proliferation and motility of the GBM cell lines in a dose- and time-dependent manner. Ova-induced apoptosis correlated with increased Bax/Bcl-2 ratio, while inhibition of tumor cell migration and colony formation was associated with reduced Slug, Vimentin, NCadherin and ß-catenin protein expression and increased E-Cadherin. In addition, exposure to Ova inhibited tumorsphere formation, elicited downregulation of CD44, CD133, Sox2, and Oct4, as well as correlated with dysregulation of the JAK2-STAT3 signaling pathway. Furthermore, we showed for the first time to the best of our knowledge that Ova potentiate the chemotherapeutic effect of Temozolomide. CONCLUSION: Taken together, our findings demonstrate the anticancer potential of Ova in GBM and its efficacy in the treatment of GBM as monotherapy and in combination with Temozolomide.

Antrodia cinnamomea sensitizes radio-/chemo-therapy of cancer stem-like cells by modulating microRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The discovery of many tissue-specific cancer stem cells (CSCs) continues to attract scientific attention. These CSCs are considered to be associated with chemo- and radio-resistance, and consequently, failure of conventional anticancer therapies. The recent demonstration of several microRNAs as enhancers of tumorigenicity via modulation of epithelial-mesenchymal transition and cancer stemness, makes them putative novel therapeutic target in oncology. Antrodia cinnamomea is a Chinese traditional medicine with several biological functions including anti-inflammation, antioxidant, and cancer prevention. However, the anti-CSC capability of A. Cinnamomea is not clear yet. AIM OF THE STUDY: To investigate the inhibitory effect of A. cinnamomea mycelium and extract on CSCs derived from various human cancer cell lines using our in-house therapeutics and human genome-wide miRNA screening panels. MATERIALS AND METHODS: A broad range of human cancer cell lines, including the acute monocytic leukemia (THP-1), glioblastoma multiforme (GBM 8401), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), hepatoblastoma (HepG2), colorectal adenocarcinoma (SW620), and foreskin fibroblast (HS68), were exposed to A. cinnamomea in this study. CD133+ CSCs generated from the cell lines were characterized and isolated by flow cytometry, effect of chemo- and radiotherapy was assessed using the MTT assay, while the RT-PCR and human genome wide qRT-PCR determined the differential gene expression patterns. A comparative analysis of the anticancer effect of A. cinnamomea and Cisplatin, Taxol, or irradiation was also performed. RESULTS: Our results indicated that A. cinnamomea mycelium and its ethyl acetate extracts showed anti-proliferation effects against all types of CSCs, especially the lung, breast, and head and neck squamous cell carcinoma CSCs. Furthermore, CSCs treatment with A. cinnamomea combined with irradiation or chemotherapeutics demonstrated significant anti-cancer effect. We also established an association between the CSC-inhibitory effect of A. cinnamomea and significant downregulation of several microRNAs and cancer stemness expression levels in brain and breast CSCs. More importantly, higher CD133 expression is associated with poor prognosis in glioblastoma and breast cancer patients. CONCLUSION: Herein, we demonstrate the putative role of A. cinnamomea as an effective ethnopharmacologic therapeutic agent for cancer treatment.

Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes.

Standard interventions for glioma include surgery, radiation and chemotherapies but the prognosis for malignant cases such as glioblastoma multiforme remain grim. Even with targeted therapeutic agent, bevacitumab, malignant glioma often develops resistance and recurrence. Thus, developing alternative interventions (therapeutic targets, biomarkers) is urgently required. Bruton's tyrosine kinase (Btk) has been long implicated in B cell malignancies but surprisingly it has recently been shown to also play a tumorigenic role in solid tumors such as ovarian and prostate cancer. Bioinformatics data indicates that Btk is significantly higher in clinical glioma samples as compared to normal brain cells and Btk expression level is associated with stage progression. This prompts us to investigate the potential role of Btk as a therapeutic target for glioma. Here, we demonstrate Btk expression is associated with GBM tumorigenesis. Down-regulation of Btk in GBM cell lines showed a significantly reduced abilities in colony formation, migration and GBM sphere-forming potential. Mechanistically, Btk-silenced cells showed a concomitant reduction in the expression of CD133 and Akt/mTOR signaling. In parallel, Ibrutinib (a Btk inhibitor) treatment led to a similar anti-tumorigenic response. Using xenograft mouse model, tumorigenesis was significantly reduced in Btk-silenced or ibrutinib-treated mice as compared to control counterparts. Finally, our glioma tissue microarray analysis indicated a higher Btk staining in the malignant tumors than less malignant and normal brain tissues. Collectively, Btk may represent a novel therapeutic target for glioma and ibrunitib may be used as an adjuvant treatment for malignant GBM.

Folate deficient tumor microenvironment promotes epithelial-to-mesenchymal transition and cancer stem-like phenotypes.

Clinically, serum level of folate has been negatively correlated to the stage and progression of liver cancer. Nevertheless, the functional consequence of folate deficiency (FD) in malignancy has not been fully investigated. Human hepatocellular carcinoma (HCC) cells (as study model) and other cancer types such as lung and glioma were cultured under folate deficient (FD) and folate complete (FD) conditions. Molecular characterization including intracellular ROS/RNS (reactive oxygen/nitrogen species), viability, colony formation, cancer stem-like cell (CSC) phenotype analyses were performed. In vivo tumorigenesis under FD and FC conditions were also examined. FD induced a significant increase in ROS and RNS, suppressing proliferative ability but inducing metastatic potential. Mesenchymal markers such as Snail, ZEB2, and Vimentin were significantly up-regulated while E-cadherin down-regulated. Importantly, CSC markers such as Oct4, ß-catenin, CD133 were induced while PRRX1 decreased under FD condition. Furthermore, FD-conditioned HCC cells showed a decreased miR-22 level, leading to the increased expression of its target genes including HDAC4, ZEB2 and Oct4. Finally, xenograft mouse model demonstrated that FD diet promoted tumorigenesis and metastasis as compared to their FC counterparts. Our data provides rationales for the consideration of folate supplement as a metastasis preventive measure.

Neurocytoprotective effects of the bioactive constituents of Pueraria thomsonii in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 cells.

Chronic neurodegenerative disorders are having an increasing impact on public health as human longevity increases. Parkinson's disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine-producing brain cells. Pueraria thomsonii Benth. (Fabaceae) is an herbal medicine that has traditionally been used as an antipyretic agent. In the present study, the active constituents, daidzein and genistein, were isolated from P. thomsonii. Both compounds exhibited neurocytoprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in nerve growth factor (NGF)-differentiated PC12 cells. Neither daidzein nor genistein affected 6-OHDA-induced cellular reactive oxygen species (ROS) generation according to flow cytometric analysis. Rather, they inhibited caspase-8 and partially inhibited caspase-3 activation, providing a protective mechanism against 6-OHDA-induced cytotoxicity in NGF-differentiated PC12 cells. The present results imply that daidzein and genistein may be useful in the development of future strategies for the treatment of PD.

Attenuating experimental spinal cord injury by hyperbaric oxygen: stimulating production of vasculoendothelial and glial cell line-derived neurotrophic growth factors and interleukin-10.

The present study was carried out to further examine the mechanisms underlying the beneficial effects of hyperbaric oxygen (HBO(2)) on experimental spinal cord injury (SCI). Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy + SCI + normobaric air (NBA; 21% oxygen at 1 ATA); and (3) laminectomy + SCI + HBO(2) (100% oxygen at 2.5 ATA for 2 h). Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. HBO(2) therapy was begun immediately after SCI. Behavioral tests of hindlimb motor function as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor scale was conducted on days 1-7 post-SCI. The triphenyltetrazolium chloride staining assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. Cells positive for glial cell line-derived neurotrophic nerve growth factor (GDNF) and vascular endothelial growth factor (VEGF) and cytokines in the injured spinal cord were assayed by immunofluorescence and commercial kits, respectively. It was found that HBO(2) therapy significantly attenuated SCI-induced hindlimb dysfunction, and spinal cord infarction and apoptosis, as well as overproduction of spinal cord interleukin-1beta and tumor necrosis factor-alpha. In contrast, the numbers of both GDNF-positive and VEGF-positive cells and production of spinal cord interleukin-10 after SCI were all significantly increased by HBO(2). These data suggest that HBO(2) may attenuate experimental SCI by stimulating production of GDNF, VEGF, and interleukin-10.