[Effect of jianpi-jiedu formula on tumor angiogenesis-relevant genes expression in colorectal cancer].

OBJECTIVE: To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer.
 Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis.
 Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein.
 Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.

Curcumin decreases acid sphingomyelinase activity in colon cancer Caco-2 cells.

Curcumin has been shown to inhibit cell growth and induce apoptosis in colon cancer cells. The metabolism of sphingomyelin has implications in the development of colon cancert. We examined whether curcumin affects the enzymes that hydrolyse sphingomyelin in Caco-2 cells. The cells were cultured in both monolayer and polarized conditions and stimulated with curcumin. The activities of sphingomyelinases were determined. Sphingomyelin and its hydrolytic products were analysed by thin layer chromatography. The changes of acid sphingomyelinase protein were examined by Western blotting. We found that curcumin reduced the hydrolytic capacity of the cells against choline-labelled sphingomyelin, associated with a mild increase of cellular sphingomyelin in the cells. Analysis of the hydrolytic products revealed that the activity was derived from acid sphingomyelinase not from phospholipase D. The curcumin-induced reduction of acid SMase required more than 8 h stimulation. Western blotting showed reduced acid sphingomyelinase protein after curcumin stimulation. The inhibitory effect was more potent in monolayer cells than in polarised cells. No changes of other sphingomyelinases were identified. In the concentrations inhibiting acid sphingomyelinase, curcumin inhibited DNA synthesis and induced cell death. In conclusion, curcumin inhibits acid sphingomyelinase and the effect might be involved in its antiproliferative property against colon cancer cells.

In vitro and in vivo effects of unsaturated fatty acids on Schistosoma mansoni and S. haematobium lung-stage larvae.

Incubation of Schistosoma mansoni lung-stage larvae in 90% corn oil for 6 hr was shown to elicit exposure of their, otherwise masked, apical membrane antigens to binding of anti-schistosome antibodies in the indirect membrane immunofluorescence test (IF). The possibility that unsaturated fatty acids (FA) are responsible for this effect was herein supported by IF data on ex vivo lung-stage larvae of S. mansoni and S. haematobium incubated for 1/2-2 hr with 80% corn oil, 50% olive oil, or 10-20 microM arachidonic acid. Treatment with unsaturated FA followed by filipin staining for cholesterol visualization indicated that unsaturated FA do not induce exposure of schistosomular surface membrane antigens via extraction of surface membrane cholesterol. Evidence using inhibitors and stimulators of neutral sphingomyelinase suggested that unsaturated FA perhaps activate worm tegument-bound neutral sphingomyelinase, leading to sphingomyelin hydrolysis and changes in surface membrane fluidity. Larval apical membrane antigens are, thus, allowed to diffuse freely in the plane of the membrane and bind specific antibodies in IE Excessive sphingomyelin hydrolysis might explain why high FA concentrations or long incubation periods eventually lead to larval death. The significant decrease (P < 0.01) in S. mansoni and increase (P < 0.02) in S. haematobium worm recovery in BALB/c mice given unsaturated FA-high and -poor diets, respectively, indicated these findings have in vivo relevance and led to the proposal that unsaturated FA likely plays a role in natural attrition of S. mansoni and S. haematobium lung-stage larvae.