Withania somnifera root extract inhibits fatty acid synthesis in prostate cancer cells.

Prior research argues for a role of increased de novo fatty acid synthesis in pathogenesis of prostate adenocarcinoma, which remains a leading cause of cancer-associated mortality in American men. A safe and effective inhibitor of fatty acid synthesis is still a clinically unmet need. Herein, we investigated the effect of ethanol extract of Withania somnifera root (WRE) standardized for one of its components (withaferin A) on fatty acid synthesis using LNCaP and 22Rv1 human prostate cancer cells. Withania somnifera is a medicinal plant used in the Ayurvedic medicine practiced in India. Western blotting and confocal microscopy revealed a statistically significant decrease in protein levels of key fatty acid metabolism enzymes including ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A) in WRE-treated cells compared with solvent control. The mRNA levels of ACLY, ACC1, FASN, and CPT1A were also lower in WRE-treated cells in comparison with control. Consequently, WRE treatment resulted in a significant decrease in intracellular levels of acetyl-CoA, total free fatty acids, and neutral lipid droplets in both LNCaP and 22Rv1 cells. WRE exhibited greater potency for fatty acid synthesis inhibition at equimolar concentration than cerulenin and etomoxir. Exposure to WRE results in downregulation of c-Myc and p-Akt(S473) proteins in 22Rv1 cell line. However, overexpression of only c-Myc conferred protection against clonogenic cell survival and lipogenesis inhibition by WRE. In conclusion, these results indicate that WRE is a novel inhibitor of fatty acid synthesis in human prostate cancer cells.

In Vitro Global Gene Expression Analyses Support the Ethnopharmacological Use of Achyranthes aspera.

Achyranthes aspera (family Amaranthaceae) is known for its anticancer properties. We have systematically validated the in vitro and in vivo anticancer properties of this plant. However, we do not know its mode of action. Global gene expression analyses may help decipher its mode of action. In the absence of identified active molecules, we believe this is the best approach to discover the mode of action of natural products with known medicinal properties. We exposed human pancreatic cancer cell line MiaPaCa-2 (CRL-1420) to 34 µ g/mL of LE for 24, 48, and 72 hours. Gene expression analyses were performed using whole human genome microarrays (Agilent Technologies, USA). In our analyses, 82 (54/28) genes passed the quality control parameter, set at FDR ≤ 0.01 and FC of ≥±2. LE predominantly affected pathways of immune response, metabolism, development, gene expression regulation, cell adhesion, cystic fibrosis transmembrane conductance regulation (CFTR), and chemotaxis (MetaCore tool (Thomson Reuters, NY)). Disease biomarker enrichment analysis identified LE regulated genes involved in Vasculitis-inflammation of blood vessels. Arthritis and pancreatitis are two of many etiologies for vasculitis. The outcome of disease network analysis supports the medicinal use of A. aspera, viz, to stop bleeding, as a cure for pancreatic cancer, as an antiarthritic medication, and so forth.

Anti-proliferative and anti-cancer properties of Achyranthes aspera: specific inhibitory activity against pancreatic cancer cells.

AIMS OF THE STUDY: Achyranthes aspera (Family: Amaranthacea) is a medicinal plant used as an anti-cancer agent in ayurveda, a traditional system of medicine practiced in subcontinental India. The aim of the study was to systematically investigate the anti-proliferative properties of Achyranthes aspera leaves extracted in methanol (LE) on human cancer cells in vitro. MATERIALS AND METHODS: We tested time, dose dependent and specific anti-proliferative activity of LE by clonogenic cell survival assay on human cancer and normal epithelial cell lines in vitro. We further investigated its effect on the expression of metastatic and angiogenic genes by real time polymerase chain reaction. On silica gel column, we carried out initial fractionation analysis. RESULTS: LE exhibited time and dose dependent cytotoxicity on several tumor cells. Compared to cancer cells of colon, breast, lung and prostate origin, pancreatic cancer cells were significantly more sensitive to LE. Preliminary mechanistic studies suggested that LE selectively suppressed the transcription of metalloproteases (MMP-1 and -2), inhibitors of MMPs (TIMP-2) and angiogenic factors (VEGF-A and VEGF-B). Fractionation of LE on methanol equilibrated silica gel column resolved into three fractions of which fraction (F 3) was found to be enriched with anti-proliferative activity. CONCLUSION: Methanolic extract of Achyranthes aspera contains potent anti-proliferative compound with specific activity against pancreatic cancer. Further studies are needed to confirm the in vivo anti-tumorigenicity and subsequent chemical characterization of the active molecule(s).

Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells.

PURPOSE: This study investigated the regulated expression of collagenases (MMP-1, -8, and -13) and stromelysins (MMP-3, -10, and -11) by human corneal epithelial cells treated with IL-1 beta, TNF-alpha, and doxycycline, a medication used to treat ocular surface diseases. METHODS: Primary human corneal epithelial cell cultures were treated with IL-1 beta or TNF-alpha, with or without their corresponding inhibitors. Total RNA extracted from cells treated for 4 to 24 hours was subjected to semiquantitative RT-PCR and Northern hybridization. Conditioned media from 24-hour-treated cultures were evaluated for MMP production by ELISA and activity assays. RESULTS: Semiquantitative RT-PCR and Northern hybridization revealed that the mRNAs of MMP-1, -13, -3, -10, and -11 were dose dependently upregulated by IL-1 beta and TNF-alpha, whereas MMP-8 and -14 and tissue inhibitor of metalloproteinase (TIMP)-1 were not altered, in corneal epithelial cells. MMP ELISA and activity assays confirmed this dose-dependent increase in MMP-1, -13, -3, and -10 protein production in conditioned media by IL-1 beta and TNF-alpha. This stimulated production was inhibited by their neutralizing antibodies and by IL-1 receptor antagonist. Doxycycline suppressed stimulated MMP-1, -10, and -13 production at both the mRNA and protein levels. CONCLUSIONS: This study demonstrated that IL-1 beta and TNF-alpha upregulate collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, and -11) in human corneal epithelial cells. Doxycycline suppresses stimulated MMP-1, -13, and -10 at the mRNA and protein levels, which suggests that collagenases and stromelysins may play a role in the pathogenesis of sterile corneal ulceration and other ocular surface diseases.

An orally active Amazonian plant extract (BIRM) inhibits prostate cancer growth and metastasis.

PURPOSE: Poor efficacy of conventional chemotherapeutic drugs against metastatic hormone-refractory prostate cancer (CaP) drives patients to try "alternative medicine". The antitumor activity of one such agent, "BIRM" (biological immune response modulator; "Simple Ecuadorian Oral Solution: an extract of an Amazonian plant"), was characterized in vitro and in vivo using established CaP cell lines and a tumor model. METHODS: The cytotoxicity of BIRM in four human and one rat CaP cell line was evaluated using cell proliferation-inhibition and clonogenic survival assays. BIRM-induced apoptosis, alterations in cell cycle phase progression and inhibition of the extracellular matrix-degrading enzyme hyaluronidase were also investigated in these cells. The in vivo efficacy of BIRM was evaluated in rats with subcutaneous tumor implants of Dunning EGFP-MAT LyLu cells. The active species in BIRM were characterized by gel filtration chromatography. RESULTS: BIRM inhibited cell proliferation and clonogenic growth of the CaP cells (IC(50) about 8.0 microl/ml). It increased cell accumulation in the G(0)/G(1) phase by 33.8% and decreased the proportion of cells in S phase by 54.6%. Apoptotic cell death in BIRM-treated cells was associated with activation of cell death-associated caspases. BIRM inhibited the activity of hyaluronidase, a hyaluronic acid-degrading enzyme, at 1 microl/ml. Treatment of MAT LyLu tumor-bearing rats with BIRM by oral gavage resulted in a significant decrease in tumor incidence (50%), tumor growth rate (18.6+/-1.3 days for 1 cc tumor growth in control rats and 25.7+/-2.6 days in BIRM-treated rats), and only one out of six BIRM-treated rats versus four out of six in the control group developed lung metastasis. Three active ingredients in BIRM with a relative molecular mass (Mr) of >or=3500 were identified by ultracentrifugation and gel filtration chromatography and were found to be resistant to proteinase and heat (100 degrees C). CONCLUSION: The plant extract BIRM contains antitumor compounds of Mr >or=3500 with potent antiproliferative activity in vitro and in vivo against prostate cancer cells.

A mouse model of keratoconjunctivitis sicca.

PURPOSE: To evaluate the effects of pharmacologic inhibition of aqueous tear production and desiccating environmental stress on aqueous tear production, tear clearance, corneal epithelial permeability, and conjunctival epithelial morphology, proliferation, and conjunctival goblet cell differentiation. METHODS: Aqueous tear production was inhibited by applying transdermal scopolamine (scop) patches to the depilated midtail of female MC, CBA mice. Desiccating environmental stress was created by placing mice in a hood with a continuous airflow blower. Aqueous tear production and volume, tear clearance, and corneal barrier function were compared in four experimental groups: untreated control mice, mice placed in the blower hood, mice treated with a scop patch, and mice treated with a scop patch and blower hood (scop patch + blower). Cotton threads were used to evaluate aqueous tear production and volume. Tear clearance was assessed by fluorometric measurement of collected tear fluid 15 minutes after instillation of 1% sodium fluorescein. Corneal epithelial barrier function was assessed by fluorometric measurement of carboxyfluorescein uptake. Conjunctival morphology and goblet cell density were evaluated in stained histologic sections. Conjunctival epithelial proliferation was assessed by bromodeoxyuridine (BrdU) labeling. RESULTS: Significant decreases in cotton thread wetting and tear clearance were observed in mice treated with a scop patch (P < 0.001) or with a scop patch and blower desiccation (P < 0.001), with a greater reduction in tear clearance in the latter group. Significantly increased corneal carboxyfluorescein uptake was noted in the scop patch group compared with untreated mice (P = 0.05) and in the scop patch + blower group compared with all the other groups (P < 0.0001). Changes in conjunctival epithelial morphology and a significant decrease in conjunctival goblet cell density (P < 0.001) were observed in the scop patch + blower group compared with the untreated control group. The number of proliferating conjunctival epithelial cells was significantly greater in the scop patch + blower group. CONCLUSIONS: Pharmacologic inhibition of tear production in mice is accompanied by ocular surface epithelial changes resembling human keratoconjunctivitis sicca (KCS) that are exacerbated by desiccating environmental stress. Cholinergic stimulated tear production appears to be essential for maintaining a healthy ocular surface.

The scale-invariance of spatial patterning in a developing system.

Regulating systems, that is, those which exhibit scale-invariant patterns in the adult, are supposed, to do so on account of interactions between cells during development. The nature of these interactions has to be such that the system of positional information ("map") in the embryo also regulates. To our knowledge, this supposition regarding a regulating map has not been subjected to a direct test in any embryonic system. Here we do so by means of a simple and novel criterion and use it to examine tip regeneration in the mulicellular stage (slug) ofDictyostelium discoideum. When anterior, tip-containing fragments of slugs are amputated, a new tip spontaneously regenerates at the cut surface of the (remaining) posterior fragment. The time needed for regeneration to occur depends on the relative size of the amputated fragment but is independent of the total size of the slug. We conclude from this finding that there is at least one system underlying positional information in the slug which regulates.