Quinic acid: a potential antibiofilm agent against clinical resistant Pseudomonas aeruginosa.

BACKGROUND: The biofilm state of pathogens facilitates antimicrobial resistance which makes difficult-to-treat infections. In this regard, it has been found that the compounds screened from plant extracts represent one category of the most promising antibiofilm agents. However, the antibiofilm activities and the active ingredients of plant extracts remain largely unexplored. In this background, the study is (1) to screen out the plant extracts with antibiofilm ability against Pseudomonas aeruginosa, and (2) to identify the active ingredients in the plant extracts and elucidate the underlying mechanism of the antibiofilm activities. METHODS: Micro-broth dilution method, in vitro biofilm model, LC-MS/MS analysis and P. aeruginosa-mouse infection model were adopted to assess the antibiofilm activity. GC-MS analysis was performed to detect the active ingredients in plasma. RNA-Seq, GO analysis, KEGG analysis and RT-qPCR were adopted to elucidate the underlying mechanism of antibiofilm activities against P. aeruginosa. RESULTS: Lonicerae Japonicae Flos (LJF) among 13 plants could exert significant inhibitory effects on bacterial biofilm formation, mobility and toxin release in vitro, and it could exert antibiofilm effect in vivo too. Moreover, quinic acid, as one metabolite of chlorogenic acid, was found as an active ingredient in LJF against the biofilm of P. aeruginosa. The active ingredient significantly inhibited EPS secretion in biofilm formation and maturity and could achieve synergistic antibiofilm effect with levofloxacin. It reduced the biofilm formation by regulating core targets in quorum sensing system. In GO process, it was found that the core targets were significantly enriched in multiple biological processes involving locomotion, chemotaxis and motility mediated by flagellum/cilium, which was related to KEGG pathways such as bacterial chemotaxis, oxidative phosphorylation, ribosome, biofilm formation, cyanoamino acid metabolism and quorum sensing. Finally, the binding of quinic acid with core targets rhlA, rhlR and rhlB were validated by molecular docking and RT-qPCR. CONCLUSIONS: In summary, the study verified the in vitro and in vivo antibiofilm effects of LJF against P. aeruginosa and elucidated the active ingredients in LJF and its conceivable pharmacological mechanism, indicating that quinic acid could have the potential of an antibiofilm agent against P. aeruginosa and related infections.

Cryptotanshinone-Induced p53-Dependent Sensitization of Colon Cancer Cells to Apoptotic Drive by Regulation of Calpain and Calcium Homeostasis.

Over-expression of calpains in tumor tissues can be associated with cancer progression. Thus, inhibition of calpain activity using specific inhibitors has become a novel approach to control tumor growth. In this study, the anticancer potential of cryptotanshinone in combination with calpain inhibitor had been investigated in colon cancer cells and tumor xenograft. Cryptotanshinone elicited an initial endoplasmic reticular (ER) stress response, whereas prolonged stress would result in the promotion of apoptosis. It was then discovered that cryptotanshinone could cause rapid and sustained increase in cytosolic calcium in colon cancer cells accompanied by early GRP78 overexpression, which could be attenuated by pre-treatment of the calcium chelator BAPTA-AM. Cryptotanshinone also facilitated an early increase in calpain activity, which could be blocked by BAPTA-AM or the calpain inhibitor PD150606. A dynamic interaction between GRP78 and calpain during the action of cryptotanshinone was unveiled. This together with the altered NF-[Formula: see text]B signaling could be abolished by calpain inhibitor. GRP78 knockdown increased the sensitivity of cancer cells to cryptotanshinone-evoked apoptosis and reduction of cancer cell colony formation. Such sensitization of drug action had been confirmed to be p53-dependent by using p53-mutated (HT-29) and p53-deficient (HCT116 p53-∕-) cells. The synergistic antitumor effect of cryptotanshinone and calpain inhibitor was further exhibited in vivo. Taken together, findings in this study exemplify a new chemotherapeutic regimen comprising cryptotanshinone and calpain inhibitor by regulation of calpain and calcium homeostasis. This has provided us with new insights in the search of a potential target-specific neoadjuvant therapy against colon cancer.

Therapeutic targeting of noncoding RNAs in hepatocellular carcinoma: Recent progress and future prospects.

Due to the high mortality rate and unsatisfactory treatment options available, hepatocellular carcinoma (HCC) remains one of the most common malignancies and a leading cause of cancer-associated mortality. Novel therapeutic targets for HCC are urgently required. Advanced RNA sequencing technology enables the identification of considerable amounts of noncoding RNAs (ncRNAs), including small noncoding RNAs and long noncoding RNAs, which exhibit no protein-coding activities. In this respect, ncRNAs and their regulatory processes are important factors in liver tumorigenesis. The present review focuses on the characteristics and biological roles of ncRNAs in HCC. Potential therapeutic applications of ncRNAs in HCC are also evaluated.

Vitamin D and Cancer Stem Cells in the Gastrointestinal Tract.

Vitamin D has been widely used as a dietary supplement for the prevention and treatment of bone disorders. Epidemiological and preclinical studies demonstrated the anticancer action of vitamin D in a variety of cancers including those in the gastrointestinal (GI) tract. In these studies the inhibitory action of vitamin D on cancer stem cells (CSCs) has been a focus and is also an important subject to revolutionize the therapeutic potential of vitamin D on cancer treatment. Here, we summarize the involvement of CSC markers and factors and also their signaling pathways in the development of cancers in the esophagus, stomach, colon, pancreas and also liver. It is also evidenced that vitamin D could inhibit these markers and factors and their related signaling pathways to suppress tumor progression. All these information could provide new strategies in repurposing vitamin D as therapeutic agent to inhibit cancers in the GI tract.

Interleukin-1ß increases the risk of gastric cancer through induction of aberrant DNA methylation in a mouse model.

Interleukin-1ß (IL-1ß) has a significant role in chronic gastric inflammation and manifestations of gastric diseases. The present study aimed to elucidate the specific role of IL-1ß in induction of DNA methylation using IL-1 receptor type 1 knockout (IL-1R1-/-) mice. In the present study, wild-type (WT) and IL-1R1-/- mice were injected with IL-1ß (5 µg/kg/day). Serum levels of IL-1ß, interleukin-6 (IL-6) and nitric oxide (NO) were measured by enzyme-linked immunosorbent or NO assays. E-cadherin (E-cad) methylation status and messenger (m)RNA expression of IL-1ß, IL-6, E-cad and inducible nitric oxide synthase (iNOS) were analyzed. Results from the present study indicated significantly higher IL-1ß mRNA expression (P<0.001) in WT mice compared with IL-1R1-/- mice. IL-1ß and IL-6 release was significantly increased in treated WT mice compared with IL-1R1-/- mice at 1 h, 4 h and 8 h (all P<0.005). IL-1ß release was only detected in WT mice following a second dose measured at day 3, week 1 and week 2 when compared with IL-1R1-/- mice. Promoter methylation of E-cad and a decrease in gene expression was observed in treated WT mice. mRNA expression of iNOS in WT mice was significantly increased at week 1 compared with IL-1R1-/- mice (P=0.0411). Furthermore, a significantly increased level of NO production was observed in treated WT mice (P<0.005 at 8 h and week 1; P<0.001 at 4 h and day 3) when compared with IL-1R1-/- mice. The present results indicated that IL-1ß was able to directly induce DNA methylation, which may link inflammation-induced epigenetic changes and the development of gastric diseases.

Miltirone induced mitochondrial dysfunction and ROS-dependent apoptosis in colon cancer cells.

AIMS: To study the characteristics of miltirone-induced anti-colon cancer effects. MATERIALS AND METHODS: Cell viability was detected using MTT assay. LDH (lactate dehydrogenase) leakage was detected using CytoTox96® non-radioactive cytotoxicity kit. Apoptosis was detected by FCM (flow cytometry). Caspase activation was determined by chemiluminescence or western blotting. AIF (apoptosis-inducing factor) expression in the cell fraction was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by confocal microscope. Intracellular calcium was detected by both FCM and confocal microscope. To determine the roles of ROS and Ca(2+) in the pro-apoptotic activity of miltirone, colon cancer cells were pretreated with kinds of antioxidants, dicoumarol, calpeptin or BAPTA-AM in some cases. KEY FINDINGS: Miltirone exhibited potent cytotoxicity on colon cancer cells with a better selectivity than that of dihydrotanshinone. The pro-apoptotic activity of miltirone was p53- and ROS-dependent. In detail, miltirone induced direct mitochondrial damage, including significant decrease of mitochondrial ROS, MMP, mass and increase of intracellular ROS and Ca(2+). NQO1 (quinone oxidoreductase1) was supposed to be a defender for the cytotoxicity induced by miltirone in colon cancer cells. Furthermore, miltirone induced time- and concentration-dependent translocation of AIF and activation of caspases. SIGNIFICANCE: In this study, ROS- and p53-dependent apoptosis induced by miltirone on colon cancer cells was firstly revealed. Strong positive feedback between mitochondrial dysfunction and accumulation of intracellular Ca(2+) was suggested to be the characteristic of the anti-colon cancer activity of miltirone.

Reversal of P-gp and BCRP-mediated MDR by tariquidar derivatives.

With an aim to generate non-toxic, specific and highly potent multidrug resistance (MDR) modulators, a novel series of anthranilic acid amide-substituted tariquidar derivatives were synthesized. The new compounds were evaluated for their cytotoxicity toward normal human colon fibroblasts (CCD18-Co), human gastric epithelial cell line (HFE) and primary rat liver cells, and for their ability to inhibit P-gp/BCRP-mediated drug efflux and reversal of P-gp and BCRP-mediated MDR in parental and drug-resistant cancer cell lines (LCC6 MDR1, MCF-7 FLV1000, R-HepG2, SW620-Ad300). While tariquidar is highly toxic to normal cells, the new derivatives exhibited much lower or negligible cytotoxicity. Some of the new tariquidar derivatives inhibited both P-gp and BCRP-mediated drug efflux whereas a few of them bearing a sulfonamide functional group (1, 5, and 16) are specific to P-gp. The new compounds were also found to potentiate the anticancer activity of the transporter substrate anticancer drugs in the corresponding transporter-overexpressing cell lines. The extent of resistance reversal was found to be consistent with the transporter inhibitory effect of the new derivatives. To further understand the mechanism of P-gp and BCRP inhibition, the tariquidar derivatives were found to interact with the transporters using an antibody-based UIC2 or 5D3 shift assay. Moreover, the transporters-inhibiting derivatives were found to modulate the ATPase activities of the two MDR transporters. Our data thus advocate further development of the new compounds for the circumvention of MDR.

Evaluation and SAR analysis of the cytotoxicity of tanshinones in colon cancer cells.

AIM: This study was designed to evaluate the anti-cancer actions of tanshinone I and tanshinone IIA, and six derivatives of tanshinone IIA on normal and cancerous colon cells. Structure activity relationship (SAR) analysis was conducted to delineate the significance of the structural modifications of tanshinones for improved anti-cancer action. METHOD: Tanshinone derivatives were designed and synthesized according to the literature. The cytotoxicity of different compounds on colon cancer cells was determined by the MTT assay. Apoptotic activity of the tanshinones was measured by flow cytometry (FCM). RESULTS: Tanshinone I and tanshinone IIA both exhibited significant cytotoxicity on colon cancer cells. They are more effective in p53(+/+) colon cancer cell line. It was also noted that the anti-cancer activity of tanshinone I was more potent and selective. Two of the derivatives of tanshinone IIA (N1 and N2) also exhibited cytotoxicity on colon cancer cells. CONCLUSION: The anti-colon cancer activity of tanshinone I was more potent and selective than tanshinone IIA, and is p53 dependent. The derivatives obtained by structural modifications of tanshinone IIA exhibited lower cytotoxicity on both normal and colon cancer cells. From steric and electronic characteristics point of view, it was concluded that structural modifications of ring A and furan or dihydrofuran ring D on the basic structure of tanshinones influences the activity. An increase of the delocalization of the A and B rings could enhance the cytotoxicity of such compounds, while a non-planar and small sized D ring region would provide improved anti-cancer activity.

Characterization of interleukin-1ß in Helicobacter pylori-induced gastric inflammation and DNA methylation in interleukin-1 receptor type 1 knockout (IL-1R1(-/-)) mice.

Helicobacter pylori infection induced interleukin-1ß (IL-1ß) production and is associated with aberrant DNA methylation and gastric diseases. Here, we investigated the role of IL-1ß in H. pylori-induced gastric inflammation and DNA methylation using IL-1 receptor type 1 knockout (IL-1R1(-/-)) mice, and compared the therapeutic efficacy of antimicrobial therapy with IL-1 receptor antagonist (IL-1ra). IL-1R1(-/-) and wild-type (WT) mice were infected with H. pylori for 16, 24 and 32 weeks. Infected WT mice at 24 weeks were given either antimicrobial therapy or IL-1ra. Comparing to the IL-1R1(-/-) mice, infected WT mice with functional IL-1ß signaling had higher gastritis scores, higher IL-1ß and iNOS mRNA expression, higher nitric oxide (NO) production and increased frequency of E-cadherin (E-cad) methylation at all the time points analyzed. IL-1ß release was significantly elevated in infected WT mice than normal controls at 16 weeks post-infection (p<0.005). Treatment of infected mice with antimicrobial therapy and IL-1ra significantly reduced the degree of gastritis (p<0.005; p<0.05, respectively), iNOS expression (p<0.0001; p<0.01, respectively) and NO production (both p<0.001) compared with untreated controls. Mice receiving antimicrobial therapy had significantly lower IL-1ß expression than untreated controls (p<0.0001). Both treatments reduced the incidence of E-cad methylation in infected mice compared with controls, however, no statistical significance was observed. There was no significant alteration of total DNA methyltransferase (DNMT) activity. These results demonstrated that IL-1ß played a crucial role in H. pylori-induced gastric inflammation and DNA methylation. H. pylori eradication and IL-1ra administration could ameliorate inflammatory stress.

Increased glutathione and mitogen-activated protein kinase phosphorylation are involved in the induction of doxorubicin resistance in hepatocellular carcinoma cells.

AIM: The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. METHODS: In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. RESULTS: Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. CONCLUSION: These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Helicobacter pylori induces promoter methylation of E-cadherin via interleukin-1ß activation of nitric oxide production in gastric cancer cells.

BACKGROUND: Helicobacter pylori infection causes gastric mucosal inflammatory responses, resulting in up-regulation of interleukin-1ß (IL-1ß) and overproduction of mutagenic nitric oxide (NO). The authors previously demonstrated that IL-1ß plays an important role in H. pylori-induced E-cadherin (E-cad) methylation. Here, they extend the study to investigate the downstream effect of IL-1ß on H. pylori-induced gastric inflammation and aberrant DNA methylation. METHODS: Human gastric cancer cell lines (MKN7, MKN74, and TMK-1) with and without pretreatment of IL-1 receptor antagonist (IL-1ra) were treated with IL-1ß or infected with H. pylori. Promoter methylation status of E-cad was examined by methylation-specific polymerase chain reaction (PCR). Expression of E-cad, inducible nitric oxide synthase (iNOS), and nuclear factor κB (NFκB) was assessed by quantitative reverse transcriptase PCR, Western blotting, or immunofluorescence. NO production and total DNA methyltransferase (DNMT) activity were assayed fluorometrically. RESULTS: Both IL-1ß treatment and H. pylori infection-induced E-cad methylation led to a decrease in E-cad expression at both mRNA and protein levels. Total DNMT enzymatic activity was significantly elevated in treated cells, accounting for the observed E-cad methylation induction. Increased expression of NFκB was accompanied by up-regulation of iNOS and production of NO in treated cells. Reversal of all these phenomena in cells pretreated with IL-1ra suggested H. pylori-induced E-cad methylation via IL-1ß stimulation of the NFκB transcriptional system, leading to activation of DNMT activity by NO production. CONCLUSIONS: These findings reveal a previously unknown effect of IL-1ß and NO on H. pylori-induced aberrant DNA methylation. This possible pathway indicates the role of NO in epigenetic modification that links inflammation to carcinogenesis.

[Epidermal growth factor stimulates the proliferation of human esophageal squamous cell carcinoma HKESC-1 cells by increasing COX-2 expression].

OBJECTIVE: To investigate the mechanisms responsible for epidermal growth factor (EGF)-induced proliferation of human esophageal squamous cell carcinoma cells. METHODS: (3)H-thymidine incorporation assay was used to assess the proliferation of HKESC-1 cells exposed to EGF stimulation. Enzyme immunoassay was used to measure PGE(2) release from HKESC-1 cells, and the protein levels of cyclooxygenase 1 (COX-1), COX-2, EP1 and EP2 in EGF-stimulated cells were determined by Western blotting. RESULTS: EGF upregulated COX-2 protein expression but produced no obvious effect on COX-1 protein expression in HKESC-1 cells. As a consequence of increased COX-2, EGF further enhanced cellular PGE(2) release. EGF stimulation also resulted in increased protein expression of EP2, a subtype of PGE(2) receptors. Both the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor SC-236 completely abolished EGF-induced PGE(2) release, and suppressed the mitogenic effect of EGF. CONCLUSION: EGF stimulates the proliferation of HKESC-1 cells by increasing COX-2 protein expression and PGE(2) release. Upregulated EP2 protein expression may further amplify the mitogenic action of PGE(2).

[Cisplatin induces drug resistance in human esophageal squamous carcinoma cell line EC109 by decreasing CTR1 protein expression].

OBJECTIVE: To investigate the mechanism of the development of cisplatin resistance in a human esophageal squamous carcinoma cell line. METHODS: The cytotoxicity of cisplatin in the cisplatin-resistant resistant cell line EC109/CDDP and its parental cell line EC109 was measured by MTT assay. Whole-cell cisplatin accumulation and Pt-DNA adduct formation were determined by inductively coupled plasma mass spectrometry (ICP-MS). Western blotting was used to investigate the protein expression of full length PARP, cleaved PARP, and copper transporter 1 (CTR1). RESULTS: EC109/CDDP cells was more resistant to cisplatin-induced cytotoxicity and apoptosis than EC109 cells. Compared with EC109 cells, EC109/CDDP cells exhibited less cisplatin accumulation and Pt-DNA adduct formation with also decreased CTR1 protein expression. CONCLUSION: Cisplatin induces drug resistant phenotype by decreasing the protein level of CTR1, which controls cell accumulation and cytotoxic effect of cisplatin.

Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression.

Doxorubicin is a chemotherapeutic drug widely used for the treatment of hepatocellular carcinoma but its efficacy is restricted by multidrug resistance. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors exhibit anti-cancer properties as well as abilities to overcome drug resistance. In the present study, indomethacin (a NSAID) and SC236 (a COX-2-selective inhibitor) enhanced the cytotoxicity of doxorubicin in the hepatocellular carcinoma cell line HepG2 and its drug-resistant sub-line R-HepG2. Both drugs increased the intracellular accumulation and retention of doxorubicin in vitro. The effects were not reversed by prostaglandin E(2), implicating a COX-independent mechanism. Indomethacin and SC236 partially reversed the increase in expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) induced by doxorubicin in R-HepG2 cells. In conclusion, indomethacin and SC236 increased the intracellular accumulation and retention of doxorubicin and thus its cytotoxicity in HepG2 and drug-resistant HepG2 cells. These effects, mediated through decrease in P-gp and MRP1 expression and/or direct inhibition of P-gp activity, may improve multidrug resistant-cancer chemotherapy.

Celecoxib antagonizes the cytotoxicity of cisplatin in human esophageal squamous cell carcinoma cells by reducing intracellular cisplatin accumulation.

High cyclooxygenase 2 (COX-2) expression has been reported to be clinically associated with reduced cisplatin-based therapy efficacy in esophageal cancer. However, the benefit of including COX-2-selective inhibitors in therapeutic regimens remains uncertain. Thus, we sought to determine the effects of COX inhibitors on the cytotoxicity of cisplatin and to further explore the mechanism involved in human esophageal squamous cell carcinoma cells. Among the four tested COX inhibitors [celecoxib, 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), nimesulide, and indomethacin], all of which substantially suppressed prostaglandin E(2) production to a similar extent; only the COX-2-selective inhibitors celecoxib and SC-236 antagonized cisplatin-induced cytotoxicity and apoptosis in both cisplatin-resistant cells and their wild-type counterparts. Knockdown of COX-2 by small interference RNA failed to mimic the antagonizing effects of celecoxib and SC-236, implying that their action is COX-2-independent. Further mechanistic analysis revealed that the antagonizing effect of celecoxib and SC-236 on cytotoxic action of cisplatin was associated with decreased whole-cell cisplatin accumulation and DNA platination. Reduced influx, accompanied by the reduction of protein level of copper transporter 1, accounts for decreased intracellular cisplatin accumulation. In addition, combined treatment did not elicit greater antitumor activity than cisplatin or celecoxib monotherapy in vivo in an esophageal cancer xenograft model. Collectively, these data demonstrate that celecoxib antagonizes the cytotoxicity of cisplatin by decreasing intracellular cisplatin and DNA platination. The combination treatment also shows no beneficial effect compared with cisplatin or celecoxib monotherapy in vivo. Therefore, current clinical trials with celecoxib in combination with cisplatin should be approached with caution.

Astragalus saponins modulate mTOR and ERK signaling to promote apoptosis through the extrinsic pathway in HT-29 colon cancer cells.

We have previously demonstrated that the total saponins of Astragalus membranaceus (AST) possess potential anti-tumorigenic effects in human colon cancer cells and tumor xenografts. In the present study, the proapoptotic effects of AST were investigated in native and cytokine-induced HT-29 cells to further unveil its mechanism of action. Growth-inhibitory action of AST (60 microg/ml) was demonstrated in native HT-29 cells, which was exaggerated in tumor necrosis factor (TNF) (5 ng/ml)-induced cells. These were accompanied by caspase 3 activation, cleavage of poly(ADP-ribose) polymerase and a subsequent increase in apoptotic cell numbers. Furthermore, activation of procaspase 8 indicates that the extrinsic apoptotic pathway was involved, while cleavage of Bid into t-Bid implicates cross-talk with the intrinsic apoptotic pathway. Alternatively, AST caused S and G2/M phase arrest, while in cytokine-induced cells S phase arrest was predominant. Further adding to our recent suggestion on its correlation with phosphatidylinositol 3-kinase (PI3K)-Akt signaling, we have now revealed that AST caused overexpression of PTEN and down-regulation of mammalian target of rapamycin (mTOR) expression. Nevertheless, these events were preceded by a decrease in nuclear factor-kappaB (NF-kappaB)/DNA binding activity with continuous ERK 1/2 activation. Some of these effects became more intense in cytokine-induced cells. Our findings in this study suggest that AST induces the extrinsic apoptotic cascade and causes cell cycle arrest in HT-29 cells by modulation of both mTOR and ERK signaling pathways, of which inhibition of NF-kappaB is important in the latter mechanism. Most of the above processes are more pronounced in cytokine-induced cells.

Cytotoxic acylphloroglucinol derivatives from the twigs of Garcinia cowa.

An unusual polyprenylated acylphloroglucinol derivative unsubstituted at C-2 and C-6, garcicowin A (1), together with three other new (garcicowins B-D, 2-4) and nine known analogues, was isolated and characterized from the twigs of Garcinia cowa. The structures of 1-4 were elucidated by interpretation of their spectroscopic data. The compounds isolated were evaluated for their cytotoxicity against two cancer cell lines (HT-29 and HCT116) and against normal colon cells (CCD-18Co), and the results demonstrated their selective toxicity toward the cancer cells.

Dexamethasone inhibits tumor necrosis factor-alpha-stimulated gastric epithelial cell migration.

BACKGROUND: Cell migration (restitution) occurs in the early phase of gastric ulcer healing. Tumor necrosis factor (TNF)-alpha is overexpressed at the ulcer margin and plays a physiologic role in gastric ulcer healing. Dexamethasone, which is a potent corticosteroid, delays rat gastric ulcer healing. We evaluated whether dexamethasone inhibited TNF-alpha-stimulated gastric epithelial cell migration using a rat normal gastric epithelial cell line (RGM-1). METHODS: An artificial wound model was employed to measure cell migration. Western blot was performed to evaluate the possible mechanisms. Intracellular prostaglandin E2 level was measured using an enzyme-linked immunosorbent assay. RESULTS: TNF-alpha treatment (10 ng/mL) for 12-48 hours significantly increased RGM-1 cell migration, and TNF-alpha treatment increased cyclooxygenase (COX)-2 protein expression 8 hours later and prostaglandin E2 (PGE2) synthesis 12 hours later compared with control (p < 0.05). Dexamethasone (10(-6) M) significantly inhibited the stimulatory effect of TNF-alpha on RGM-1 cell migration, which was associated with a significant decrease in COX-2 expression and PGE2 level in cells (p < 0.05). CONCLUSION: TNF-alpha plays a regulatory role in rat gastric epithelial cell migration and dexamethasone inhibited TNF-alpha-stimulated cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation.

A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID-activated gene.

Astragalus membranaceus has been used to ameliorate the side effects of antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus saponins (AST) possess anticarcinogenic and proapoptotic properties in human colon cancer cells and tumor xenograft. In this study, we identified NSAID-activated gene (NAG-1) as a potential molecular target of AST. The growth-inhibitory and proapoptotic effects of AST were assessed in a panel of human cancer cell lines. Hoechst 33342 nuclear staining, Annexin V-FITC/propidium iodide staining, Western immunoblotting, real-time PCR, luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG-1 and related transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG-1 promoting activities of AST and/or inhibitors of the PI3K-Akt pathway were also examined. AST caused overexpression of NAG-1, leading to PARP cleavage and apoptosis. The induction of NAG-1 promoter activity by the drug was associated with increased gene expression, in addition to prior increase in Egr-1 expression and DNA binding activity. AST-induced NAG-1 activation was intensified when PI3K inhibitor LY294002 or Akt inhibitor was co-treated and reversed by NAG-1 siRNA transfection. Nevertheless, the extent of NAG-1 induction could not be altered by the ERK inhibitor PD98059. Our results indicate that NAG-1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K-Akt inhibitors. The information obtained could facilitate future development of a novel target-specific chemotherapeutic agent with known molecular pathway.