Expression Profile of LGR5 and Its Prognostic Significance in Colorectal Cancer Progression.

We investigated the expression profile of leucine-rich, repeat-containing, G-protein-coupled receptor 5 (LGR5) during colorectal cancer (CRC) progression and determined the prognostic impact of LGR5 in a large cohort of CRC samples. LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 68% of 788 CRCs and was positively correlated with older age, moderately to well-differentiated cells, and nuclear ß-catenin expression. Enhanced LGR5 expression remained persistent during the adenoma-carcinoma transition, but markedly declined in the budding cancer cells at the invasive fronts, which was not due to altered wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial-mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. Notably, LGR5 positivity was an independent prognostic marker for better clinical outcomes in CRC patients. LGR5 overexpression attenuated tumor growth by decreasing ERK phosphorylation along with decreased colony formation and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony-forming and migration capacities in LoVo cells. Taken together, our data suggest a suppressive role of LGR5 in CRC progression.

Factors Associated with Osteoarthritis and Their Influence on Health-Related Quality of Life in Older Adults with Osteoarthritis: A Study Based on the 2020 Korea National Health and Nutrition Examination Survey.

Numerous studies have investigated factors associated with osteoarthritis (OA), but few have investigated their effects on psychological problems and health-related quality of life in older adults with OA. We aimed to investigate factors associated with OA and their influence on health-related quality of life in older adults with OA. Among 1394 participants aged ≥65 years, 952 and 442 were categorized into the OA and non-OA groups, respectively. Comprehensive data on demographic characteristics, medical conditions, health-related quality of life, blood test results, and nutritional intake were obtained. Univariate and multivariate logistic regression analyses were used to evaluate the odds ratio for factors associated with OA, including age (odds ratio (OR), 1.038; p = 0.020), female sex (OR, 5.692; p < 0.001), body mass index (OR, 1.108; p < 0.001), hypertension (OR, 1.451; p < 0.050), hyperlipidemia (OR, 1.725; p = 0.001), osteoporosis (OR, 2.451; p < 0.001), and depression (OR, 2.358; p = 0.041). The OA group showed a significantly lower subjective health status (p < 0.001) and higher difficulty in mobility (p < 0.001) and pain/discomfort (p = 0.010) than the non-OA group. The sleeping hours were significantly shorter in the OA group than those in the non-OA group (p = 0.013). OA was a significant contributing factor for unfavorable health-related quality of life in older adults. Controlling the factors associated with OA should be prioritized, and health-related quality of life should be monitored in older adults with OA.

Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: involvement of Ca(2+) influx.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation.

Involvement of heme oxygenase-1 in Korean colon cancer.

Heme oxygenase-1 (HO-1) catabolizes heme into carbon monoxide, biliverdin, and free iron which mediate its protective effect against oxidative stress. The aim of the present study was to determine the expression level and activity of HO-1 in Korean colon cancer tissues and cell lines. HO-1 protein expression was higher (>1.5-fold) in tumor tissues than in adjacent normal tissues in 14 of 20 colon cancer patients, and HO-1 protein expression was closely correlated with HO-1 enzyme activity in cancer tissues. Immunohistochemical data confirmed that HO-1 protein was expressed at a higher level in colon cancer tissues than in normal mucosa. Furthermore, HO-1 mRNA and protein expression and enzyme activity were higher in the colon cancer cell lines Caco-2, SNU-407, SNU-1033, HT-29, and SW-403 than in the normal fetal human colon cell line FHC. Treatment with the HO-1 inhibitor zinc protoporphyrin decreased the viability of colon cancer cell lines. These data indicate that HO-1 may serve as a clinically useful biomarker of colon cancer and as a target for anticolon cancer drugs.

Mode of primary cancer detection as an indicator of screening practice for second primary cancer in cancer survivors: a nationwide survey in Korea.

BACKGROUND: While knowledge and risk perception have been associated with screening for second primary cancer (SPC), there are no clinically useful indicators to identify who is at risk of not being properly screened for SPC. We investigated whether the mode of primary cancer detection (i.e. screen-detected vs. non-screen-detected) is associated with subsequent completion of all appropriate SPC screening in cancer survivors. METHODS: Data were collected from cancer patients treated at the National Cancer Center and nine regional cancer centers across Korea. A total of 512 cancer survivors older than 40, time since diagnosis more than 2 years, and whose first primary cancer was not advanced or metastasized were selected. Multivariate logistic regression was used to examine factors, including mode of primary cancer detection, associated with completion of all appropriate SPC screening according to national cancer screening guidelines. RESULTS: Being screen-detected for their first primary cancer was found to be significantly associated with completion of all appropriate SPC screening (adjusted odds ratio, 2.13; 95% confidence interval, 1.36-3.33), after controlling for demographic and clinical variables. Screen-detected cancer survivors were significantly more likely to have higher household income, have other comorbidities, and be within 5 years since diagnosis. CONCLUSIONS: The mode of primary cancer detection, a readily available clinical information, can be used as an indicator for screening practice for SPC in cancer survivors. Education about the importance of SPC screening will be helpful particularly for cancer survivors whose primary cancer was not screen-detected.

(1S,2S,3E,7E,11E)-3,7,11,15-Cembratetraen-17,2-olide, a cembrenolide diterpene from soft coral Lobophytum sp., inhibits growth and induces apoptosis in human colon cancer cells through reactive oxygen species generation.

We observed that (1S,2S,3E,7E,11E)-3,7,11,15-Cembratetraen-17,2-olide (LS-1), marine cembrenolide diterpene, inhibited growth and induced apoptosis in colon cancer cells via a reactive oxygen species (ROS) dependent mechanism. Treatment of HT-29 cells with LS-1 resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, sub-G1 peak accumulation, activation of Bid, caspase-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) along with the suppressive expression of B cell lymphoma-2 (Bcl-2). All these effects were significantly blocked on pretreatment with the ROS inhibitor N-acetylcysteine (NAC), indicating the involvement of increased ROS in the proapoptotic activity of LS-1. Moreover, we showed that LS-1 induced the phosphorylation of c-Jun N-terminal kinase (JNK) and dephosphorylation of p38, extracellular signal-regulated kinase (ERK), Akt, Src and signal transducer and activator of transcription (STAT)3, which were effectively attenuated by NAC. In addition, the expressions of antioxidant catalase and glutathione peroxidase were abrogated by treatment using LS-1 with or without NAC. These findings reveal the novel anticancer efficacy of LS-1 mediated by the induction of apoptosis via ROS generation in human colon cancer cells.

Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells.

The objective of this study was to elucidate the cytotoxic mechanism of Compound K, with respect to the involvement of reactive oxygen species (ROS) and the mitochondrial involved apoptosis, in HT-29 human colon cancer cells. Compound K exhibited a concentration of 50% growth inhibition (IC(50)) at 20 µg/mL and cytotoxicity in a time dependent manner. Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability. Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψ(m)). Loss of the Δψ(m) was followed by cytochrome c release from the mitochondria, resulting in the activation of caspase-9, -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage, which are the indicators of caspase-dependent apoptosis. The apoptotic effect of Compound K, exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors. This study demonstrated that Compound K-mediated generation of ROS led to apoptosis through the modulation of a mitochondria-dependent apoptotic pathway and MAPK pathway.

Prognostic Significance of EPHB2 Expression in Colorectal Cancer Progression.

BACKGROUND: A receptor tyrosine kinase for ephrin ligands, EPHB2, is expressed in normal colorectal tissues and colorectal cancers (CRCs). The aim of this study was to investigate EPHB2 expression over CRC progression and determine its prognostic significance in CRC. METHODS: To measure EPHB2 mRNA and protein expression, real-time polymerase chain reaction and immunohistochemistry were performed in 32 fresh-frozen and 567 formalin-fixed paraffin-embedded CRC samples, respectively. We further investigated clinicopathological features and overall and recurrence-free survival according to EPHB2 protein expression. RESULTS: The EPHB2 level was upregulated in CRC samples compared to non-cancerous tissue in most samples and showed a strong positive correlation with AXIN2. Notably, CD44 had a positive association with both mRNA and protein levels of EPHB2. Immunohistochemical analysis revealed no difference in EPHB2 expression between adenoma and carcinoma areas. Although EPHB2 expression was slightly lower in invasive fronts compared to surface area (p < .05), there was no difference between superficial and metastatic areas. EPHB2 positivity was associated with lymphatic (p < .001) and venous (p = .001) invasion, TNM stage (p < .001), and microsatellite instability (p = .036). Kaplan-Meier analysis demonstrated that CRC patients with EPHB2 positivity showed better clinical outcomes in both overall (p = .049) and recurrence-free survival (p = .015). However, multivariate analysis failed to show that EPHB2 is an independent prognostic marker in CRCs (hazard ratio, 0.692; p = .692). CONCLUSIONS: Our results suggest that EPHB2 is overexpressed in a subset of CRCs and is a significant prognostic marker.

Over-activation of AKT signaling leading to 5-Fluorouracil resistance in SNU-C5/5-FU cells.

Here, we investigated whether over-activation of AKT pathway is important in the resistance to 5-fluorouracil (5-FU) in SNU-C5/5-FU cells, 5-FU-resistant human colon cancer cells. When compared to wild type SNU-C5 cells (WT), SNU-C5/5-FU cells showed over-activation of PI3K/AKT pathway, like increased phosphorylation of AKT, mTOR, and GSK-3ß, nuclear localization of ß-catenin, and decreased E-cadherin. Moreover, E-cadherin level was down-regulated in recurrent colon cancer tissues compared to primary colon cancer tissues. Gene silencing of AKT1 or treatment of LY294002 (PI3 kinase inhibitor) increased E-cadherin, whereas decreased phospho-GSK-3ß. LY294002 also reduced protein level of ß-catenin with no influence on mRNA level. PTEN level was higher in SNU-C5/WT than SNU-C5/5-FU cells, whereas the loss of PETN in SNU-C5/WT cells induced characteristics of SNU-C5/5-FU cells. In SNU-C5/5-FU cells, NF-κB signaling was activated, along with the overexpression of COX-2 and stabilization of survivin. However, increased COX-2 contributed to the stabilization of survivin, which directly interacts with cytoplasmic procaspase-3, while the inhibition of AKT reduced this cascade. We finally confirmed that combination treatment with 5-FU and LY294002 or Vioxx could induce apoptosis in SNU-C5/5-FU cells. These data suggest that inhibition of AKT activation may overcome 5-FU-resistance in SNU-C5/5-FU cells. These findings provide evidence that over-activation of AKT is crucial for the acquisition of resistance to anticancer drugs and AKT pathway could be a therapeutic target for cancer treatment.

Prognostic significance of stromal GREM1 expression in colorectal cancer.

Cancer-associated fibroblasts are the dominant cell population in the cancer stroma. Gremlin 1 (GREM1), an antagonist of the bone morphogenetic protein pathway, is expressed by cancer-associated fibroblasts in a variety of human cancers. However, its biological significance for cancer patients is largely unknown. We applied RNA in situ hybridization to evaluate the prognostic value of stromal GREM1 expression in a large cohort of 670 colorectal cancers (CRCs). Overall, GREM1 expression in CRCs was lower than that of the matched normal mucosa, and GREM1 expression had a strong positive correlation with BMI1 and inverse correlations with EPHB2 and OLFM4. RNA in situ hybridization localized the GREM expression to smooth muscle cells of the muscularis mucosa and fibroblasts around crypt bases and in the submucosal space of a normal colon. In various colon polyps, epithelial GREM1 expression was exclusively observed in traditional serrated adenomas. In total, 44% of CRCs were positive for stromal GREM1, which was associated with decreased lymphovascular invasion, a lower cancer stage, and nuclear ß-catenin staining. Stromal GREM1 was significantly associated with improved recurrence-free and overall survival, although it was not found to be an independent prognostic marker in multivariate analyses. In addition, for locally advanced stage II and III CRC, it was associated with better, stage-independent clinical outcomes. In summary, CRCs are frequently accompanied by GERM1-expressing fibroblasts, which are closely associated with low lymphovascular invasion and a better prognosis, suggesting stromal GREM1 as a potential biomarker and possible candidate for targeted therapy in the treatment of CRCs.

Aurora Kinase A Is a Prognostic Marker in Colorectal Adenocarcinoma.

BACKGROUND: Aurora kinase A (AURKA), or STK15/BTAK, is a member of the serine/threonine kinase family and plays important roles in mitosis and chromosome stability. This study investigated the clinical significance of AURKA expression in colorectal cancer patients in Korea. METHODS: AURKA protein expression was evaluated by immunohistochemistry in 151 patients with colorectal adenocarcinoma using tissue microarray blocks. We analyzed the relationship between clinicopathological characteristics and AURKA expression. In addition, the prognostic significance of various clinicopathological data for progression-free survival (PFS) was assessed. Also we evaluated copy number variations by array comparative genomic hybridization and AURKA gene amplification using fluorescence in situ hybridization in colorectal carcinoma tissues. RESULTS: AURKA gene amplification was found more frequently in the 20q13.2-13.33 gain-positive group than the group with no significant gain on the AURKA-containing locus. AURKA protein expression was detected in 45% of the cases (68/151). Positive staining for AURKA was observed more often in male patients (p = .035) and distally located tumors (p = .021). PFS was shorter in patients with AURKA expression compared to those with low-level AURKA expression (p < .001). Univariate analysis revealed that AURKA expression (p = .001), age (p = .034), lymphatic invasion (p = .001), perineural invasion (p = .002), and TNM stage (p = .013) significantly affected PFS. In a multivariate analysis of PFS, a Cox proportional hazard model confirmed that AURKA expression was an independent and significant prognostic factor in colorectal adenocarcinoma (hazard ratio, 3.944; p < .001). CONCLUSIONS: AURKA could serve as an independent factor to predict a poor prognosis in Korean colorectal adenocarcinoma patients.

Interaction of DNA demethylase and histone methyltransferase upregulates Nrf2 in 5-fluorouracil-resistant colon cancer cells.

We recently reported that DNA demethylase ten-eleven translocation 1 (TET1) upregulates nuclear factor erythroid 2-related factor 2 (Nrf2) in 5-fluorouracil-resistant colon cancer cells (SNUC5/5-FUR). In the present study, we examined the effect of histone modifications on Nrf2 transcriptional activation. Histone deacetylase (HDAC) and histone acetyltransferase (HAT) were respectively decreased and increased in SNUC5/5-FUR cells as compared to non-resistant parent cells. Mixed-lineage leukemia (MLL), a histone methyltransferase, was upregulated, leading to increased trimethylation of histone H3 lysine 4, while G9a was downregulated, leading to decreased dimethylation of histone H3 lysine 9. siRNA-mediated MLL knockdown decreased levels of Nrf2 and HO-1 to a greater extent than did silencing HAT1. Host cell factor 1 (HCF1) was upregulated in SNUC5/5-FUR cells, and we observed interaction between HCF1 and MLL. Upregulation of O-GlcNAc transferase (OGT), an activator of HCF1, was also associated with HCF1-MLL interaction. In SNUC5/5-FUR cells, a larger fraction of OGT was bound to TET1, which recruits OGT to the Nrf2 promoter region, than in SNUC5 cells. These findings indicate that SNUC5/5-FUR cells are under oxidative stress, which induces expression of histone methylation-related proteins as well as DNA demethylase, leading to upregulation of Nrf2 and 5-FU resistance.

The Effect of (1S,2S,3E,7E,11E)-3,7,11,15-Cembratetraen-17,2-Olide (LS-1) from Lobophyyum sp. on the Apoptosis Induction of SNU-C5 Human Colorectal Cancer Cells.

(1S,2S,3E,7E,11E)-3,7,11,15-cembratetraen-17,2-olide (LS-1), a marine cembrenolide diterpene, has anticancer activity against colon cancer cells such as HT-29, SNU-C5/5-FU (fluorouracil-resistant SNU-C5) and SNU-C5. However, the action mechanism of LS-1 on SNU-C5 human colon cancer cells has not been fully elucidated. In this study, we investigated whether the anticancer effect of LS-1could result from apoptosis via the modulation of Wnt/ß-catenin and the TGF-ß pathways. When treated with the LS-1, we could observe the apoptotic characteristics such as apoptotic bodies and the increase of sub-G1 hypodiploid cell population, increase of Bax level, decrease of Bcl-2 expression, cleavage of procaspase-3 and cleavage of poly (ADP-ribose) polymerase in SNU-C5 cells. Furthermore, the apoptosis induction of SNU-C5 cells upon LS-1 treatment was also accompanied by the down-regulation of Wnt/ß-catenin signaling pathway via the decrease of GSK-3ß phosphorylation followed by the decrease of ß-catenin level. In addition, the LS-1 induced the activation of TGF-ß signaling pathway with the decrease of carcinoembryonic antigen which leads to decrease of c-Myc, an oncoprotein. These data suggest that the LS-1 could induce the apoptosis via the down-regulation of Wnt/ß-catenin pathway and the activation of TGF-ß pathway in SNU-C5 human colon cancer cells. The results support that the LS-1 might have potential for the treatment of human colon cancer.

Single Cavernous Hemangioma of the Small Bowel Diagnosed by Using Capsule Endoscopy in a Child with Chronic Iron-Deficiency Anemia.

Cavernous hemangiomas of the gastrointestinal tract are extremely rare. In particular, the diagnosis of small bowel hemangiomas is very difficult in children. A 13-year-old boy presented at the outpatient clinic with dizziness and fatigue. The patient was previously diagnosed with iron-deficiency anemia at 3 years of age and had been treated with iron supplements continuously and pure red cell transfusion intermittently. Laboratory tests indicated that the patient currently had iron-deficiency anemia. There was no evidence of gross bleeding, such as hematemesis or bloody stool. Laboratory findings indicated no bleeding tendency. Gastroduodenoscopy and colonoscopy results were negative. To obtain a definitive diagnosis, the patient underwent capsule endoscopy. A purplish stalked mass was found in the jejunum, and the mass was excised successfully. We report of a 13-year-old boy who presented with severe and recurrent iron-deficiency anemia caused by a cavernous hemangioma in the small bowel without symptoms of gastrointestinal bleeding.

Camptothecin suppresses expression of matrix metalloproteinase-9 and vascular endothelial growth factor in DU145 cells through PI3K/Akt-mediated inhibition of NF-κB activity and Nrf2-dependent induction of HO-1 expression.

Though camptothecin (CPT) possesses potent anti-inflammatory, immunomodulatory, anticancerous, and antiproliferative effects, little is known about the mechanism by which CPT regulates the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Therefore, the current study aimed to investigate the effects of CPT on the expression of MMP-9 and VEGF, which are important factors for the invasion of tumors. In vitro application of CPT resulted in a slight inhibition of cell proliferation and a significant reduction in the matrigel invasion of DU145 cells. Treatment with CPT also downregulated phorbol-12-myristate-13-acetate (PMA)- and tumor necrosis factor-α (TNF-α)-induced MMP-9 and VEGF expression by inhibiting nuclear factor-κB (NF-κB) activity. Downregulation of phosphoinositide 3-kinase (PI3K)/Akt phosphorylation in response to CPT was revealed as an upstream pathway regulating the expression of MMP-9 and VEGF accompanying the inhibition of NF-κB activity. We further confirmed that CPT inhibits PMA-induced MMP-9 and VEGF expression by upregulating nuclear factor-erythroid related factor-2 (Nrf2)-mediated heme oxygenase-1 (HO-1) induction. Taken together, these data indicate that CPT inhibits the invasion of cancer cells accompanied by suppression of MMP-9 and VEGF production by suppressing the PI3K/Akt-mediated NF-κB pathway and enhancing the Nrf2-dependent HO-1 pathway, suggesting that CPT may be a good candidate to inhibit MMP-9 and VEGF expression.

Involvement of glutathione and glutathione metabolizing enzymes in human colorectal cancer cell lines and tissues.

Reduced glutathione (GSH) is an abundant tripeptide present in the majority of cell types. GSH is highly reactive and is often conjugated to other molecules, via its sulfhydryl moiety. GSH is synthesized from glutamic acid, cysteine, and glycine via two sequential ATP­consuming steps, which are catalyzed by glutamate cysteine ligase (GCL) and GSH synthetase (GSS). However, the role of GSH in cancer remains to be elucidated. The present study aimed to determine the levels of GSH and GSH synthetic enzymes in human colorectal cancer. The mRNA and protein expression levels of GSH, the catalytic subunit of GCL (GCLC) and GSS were significantly higher in the following five colon cancer cell lines: Caco­2, SNU­407, SNU­1033, HCT­116, and HT­29, as compared with the normal colon cell line, FHC. Similarly, in 9 out of 15 patients with colon cancer, GSH expression levels were higher in tumor tissue, as compared with adjacent normal tissue. In addition, the protein expression levels of GCLC and GSS were higher in the tumor tissue of 8 out of 15, and 10 out of 15 patients with colon cancer respectively, as compared with adjacent normal tissue. Immunohistochemical analyses confirmed that GCLC and GSS were expressed at higher levels in colon cancer tissue, as compared with normal mucosa. Since GSH and GSH metabolizing enzymes are present at elevated levels in colonic tumors, they may serve as clinically useful biomarkers of colon cancer, and/or targets for anti-colon cancer drugs.

18ß-Glycyrrhetinic acid suppresses TNF-α induced matrix metalloproteinase-9 and vascular endothelial growth factor by suppressing the Akt-dependent NF-κB pathway.

Little is known about the molecular mechanism through which 18ß-glycyrrhetinic acid (GA) inhibits metastasis and invasion of cancer cells. Therefore, this study aimed to investigate the effects of GA on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in various types of cancer cells. We found that treatment with GA reduces tumor necrosis factor-α (TNF-α)-induced Matrigel invasion with few cytotoxic effects. Our findings also showed that MMP-9 and VEGF expression increases in response to TNF-α; however, GA reverses their expression. In addition, GA inhibited inhibitory factor kappa B degradation, sustained nuclear factor-kappa B (NF-κB) subunits, p65 and p50, in the cytosol compartments, and consequently suppressed the TNF-α-induced DNA-binding activity and luciferase activity of NF-κB. Specific NF-κB inhibitors, pyrrolidine dithiocarbamate, MG132, and PS-1145, also attenuated TNF-α-mediated MMP-9 and VEGF expression as well as activity by suppressing their regulatory genes. Furthermore, phosphorylation of TNF-α-induced phosphatidyl-inositol 3 kinase (PI3K)/Akt was significantly downregulated in the presence of GA accompanying with the inhibition of NF-κB activity, and as presumed, the specific PI3K/Akt inhibitor LY294002 significantly decreased MMP-9 and VEGF expression as well as activity. These results suggest that GA operates as a potential anti-invasive agent by downregulating MMP-9 and VEGF via inhibition of PI3K/Akt-dependent NF-κB activity. Taken together, GA might be an effective anti-invasive agent by suppressing PI3K/Akt-mediated NF-κB activity.

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1.

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE2 without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE2 production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE2, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.

Epigenetic alterations are involved in the overexpression of glutathione S-transferase π-1 in human colorectal cancers.

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These data indicate that GSTP-1 may serve as a clinically useful biomarker of colon cancer and a target for anti-colon cancer drugs.

20-O-(ß-D-glucopyranosyl)-20(S)-protopanaxadiol induces apoptosis via induction of endoplasmic reticulum stress in human colon cancer cells.

Previously, we reported that 20-O-(ß-D-gluco-pyranosyl)-20(S)-protopanaxadiol (Compound K, a meta-bolite of ginseng saponin) induces mitochondria-dependent and caspase-dependent apoptosis in HT-29 human colon cancer cells via the generation of reactive oxygen species. The aim of the present study was to elucidate the mechanism underlying apoptosis induced by Compound K with respect to endoplasmic reticulum (ER) stress in HT-29 cells. In the present study, Compound K induced apoptotic cell death as confirmed by DNA fragmentation and apoptotic sub-G1 cell population. Compound K also induced ER stress as indicated by staining with ER tracker, cytosolic and mitochondrial Ca2+ overloading, phosphorylation of protein-kinase-like endoplasmic reticulum kinase (PERK), phosphorylation of eukaryotic initiation factor-2α (eIF-2α), phosphorylation of IRE-1, splicing of ER stress-specific X-box transcription factor-1 (XBP-1), cleavage of activating transcription factor-6 (ATF-6), upregulation of glucose-regulated protein-78 (GRP-78/BiP) and CCAAT/enhancer-binding protein-homologous protein (CHOP), and cleavage of caspase-12. Furthermore, downregulation of CHOP expression using siCHOP RNA attenuated Compound K-induced apoptosis. Taken together, these results support the important role of ER stress response in mediating Compound K-induced apoptosis in human colon cancer cells.