Anti-Inflammatory and Anti-Adipocyte Dysfunction Effects of Ficus lindsayana Latex and Root Extracts.

Low-grade chronic inflammation and adipocyte dysfunction are prominent risk factors of insulin resistance and type 2 diabetes mellitus (T2DM) in obesity. Thus, prevention of inflammation and adipocyte dysfunction could be one possible approach to mitigate T2DM development. Several Ficus species have been used in traditional medicine for ameliorating inflammation and T2DM. Our previous studies reported biological effects of Ficus lindsayana including antioxidant, anti-cancer, and anti-α-glucosidase activities. Further, this study therefore investigated whether F. lindsayana latex (FLLE) and root (FLRE) extracts inhibit inflammation-stimulated insulin resistance in adipocytes and inflammation in macrophages. FLLE and FLRE (200 µg/mL) had no significant cytotoxicity for macrophages, adipocytes, and blood cells (PBMCs and RBCs). FLRE had a total flavonoid content about three times higher than FLLE, while both had similar levels of total phenolic content. FLRE showed higher abilities than FLLE in suppressing inflammation in both macrophages and adipocytes and reversing the inflammation-induced insulin resistance in adipocytes. In TNF-α-induced adipocytes, FLRE significantly improved insulin-induced glucose uptake and insulin-suppressed lipolysis, while FLLE only significantly improved glucose uptake. Moreover, FLRE and FLLE remarkably reduced chemoattractant (MCP-1) but improved adipogenic (PPARγ and CEBPα) gene expression, leading to the promotion of adipogenesis and the suppression of insulin resistance. In LPS-induced macrophages, FLRE, but not FLLE, significantly inhibited LPS-induced NO production. Moreover, FLRE significantly reduced LPS-stimulated iNOS, COX-2, IL-1ß, IL-6, and TNF-α gene expression. These results may provide the potential data for the development of this plant, especially the root part, as an alternative medicine, functional ingredient, or food supplement for the prevention of inflammation and obesity-associated insulin resistance, as well as T2DM.

The Toxicological Assessment of Anoectochilus burmannicus Ethanolic-Extract-Synthesized Selenium Nanoparticles Using Cell Culture, Bacteria, and Drosophila melanogaster as Suitable Models.

Selenium nanoparticles (SeNPs) are worthy of attention and development for nutritional supplementation due to their health benefits in both animals and humans with low toxicity, improved bioavailability, and controlled release, being greater than the Se inorganic and organic forms. Our previous study reported that Anoectochilus burmannicus extract (ABE)-synthesized SeNPs (ABE-SeNPs) exerted antioxidant and anti-inflammatory activities. Furthermore, ABE could stabilize and preserve the biological activities of SeNPs. To promote the ABE-SeNPs as supplementary and functional foods, it was necessary to carry out a safety assessment. Cytotoxicity testing showed that SeNPs and ABE-SeNPs were harmless with no killing effect on Caco2 (intestinal epithelial cells), MRC-5 (lung fibroblasts), HEK293 (kidney cells), LX-2 (hepatic stellate cells), and 3T3-L1 (adipocytes), and were not toxic to isolated human PBMCs and RBCs. Genotoxicity assessments found that SeNPs and ABE-SeNPs did not induce mutations in Salmonella typhimurium TA98 and TA100 (Ames test) as well as in Drosophila melanogaster (somatic mutation and recombination test). Noticeably, ABE-SeNPs inhibited mutation in TA98 and TA100 induced by AF-2, and in Drosophila induced by urethane, ethyl methanesulfonate, and mitomycin c, suggesting their anti-mutagenicity ability. This study provides data that support the safety and anti-genotoxicity properties of ABE-SeNPs for the further development of SeNPs-based food supplements.

Phytosome Supplements for Delivering Gymnema inodorum Phytonutrients to Prevent Inflammation in Macrophages and Insulin Resistance in Adipocytes.

Gymnema inodorum (GI) is a leafy green vegetable found in the northern region of Thailand. A GI leaf extract has been developed as a dietary supplement for metabolic diabetic control. However, the active compounds in the GI leaf extract are relatively nonpolar. This study aimed to develop phytosome formulations of the GI extract to improve the efficiencies of their phytonutrients in terms of anti-inflammatory and anti-insulin-resistant activities in macrophages and adipocytes, respectively. Our results showed that the phytosomes assisted the GI extract's dispersion in an aqueous solution. The GI phytocompounds were assembled into a phospholipid bilayer membrane as spherical nanoparticles about 160-180 nm in diameter. The structure of the phytosomes allowed phenolic acids, flavonoids and triterpene derivatives to be embedded in the phospholipid membrane. The existence of GI phytochemicals in phytosomes significantly changed the particle's surface charge from neutral to negative within the range of -35 mV to -45 mV. The phytosome delivery system significantly exhibited the anti-inflammatory activity of the GI extract, indicated by the lower production of nitric oxide from inflamed macrophages compared to the non-encapsulated extract. However, the phospholipid component of phytosomes slightly interfered with the anti-insulin-resistant effects of the GI extract by decreasing the glucose uptake activity and increasing the lipid degradation of adipocytes. Altogether, the nano-phytosome is a potent carrier for transporting GI phytochemicals to prevent an early stage of T2DM.

Stabilization of Antioxidant and Anti-Inflammatory Activities of Nano-Selenium Using Anoectochilus burmannicus Extract as a Potential Novel Functional Ingredient.

Anoectochilus burmannicus is an orchid that contains phenolic compounds and exhibits antioxidant and anti-inflammation properties. This study aimed to investigate whether its ethanolic extract (ABE) can be used as a reducing agent and/or a stabilizer of nano-selenium (SeNP) synthesis. SeNPs exhibited higher antioxidant activity than ABE-SeNPs. In contrast, ABE-SeNP (4 µM Se) had greater anti-inflammatory activity in LPS-induced macrophages than SeNPs. Interestingly, ABE acted as a stabilizer for SeNPs by preventing particle aggregation and preserving its antioxidant activity after long-term storage (90 days). Moreover, after the freeze-drying process, ABE-SeNPs could be completely reconstituted to suspension with significantly stable antioxidant and anti-inflammatory activities compared to freshly prepared particles, suggesting the cryoprotectant and/or lyoprotectant role of ABE. The present study shows the potential of ABE as an effective stabilizer for nanoparticles and provides evidence for the development of ABE-SeNPs as a food supplement or novel functional ingredient for health benefits.

Protective effect of Anoectochilus burmannicus extracts and its active compound, kinsenoside on adipocyte differentiation induced by benzyl butyl phthalate and bisphenol A.

Benzyl butyl phthalate (BBP) and bisphenol-A (BPA) are obesogens that have been reported to be associated with obesity. Inhibition of their adipogenic activity could decrease the risk of obesity-related metabolic disorders. This study hypothesized that Anoectochilus burmannicus ethanolic extract (ABE) which has been previously reported its anti-inflammation property and its known active compound, kinsenoside (Kin) abrogate BBP- and BPA-induced adipogenesis. ABE and Kin markedly suppress both BBP- and BPA-stimulated adipogenesis with different modulations on adipogenic-gene expression including C/EBPα, PPARγ, adiponectin, and leptin in 3T3-L1. BBP induced C/EBPα, adiponectin, and leptin mRNA expressions and slightly increased PPARγ mRNA level, whereas BPA markedly induced PPARγ and adiponectin mRNA levels. ABE significantly decreased the expression of C/EBPα and leptin, but not PPARγ and adiponectin in the BBP-treated cells. In the BPA-treated cells, ABE significantly decreased the mRNA expression of C/EBPα and PPARγ, but not adiponectin and leptin. Interestingly, Kin significantly overcame BBP- and BPA-induced C/EBPα, PPARγ, adiponectin, and leptin expressions. This study first provides evidence to support the health benefits of this plant, especially for people exposed to obesogens. Besides, this finding would encourage the conservation and culture of this orchid for development as an economic plant and healthy food.

Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions. AIM OF THE STUDY: The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety. MATERIALS AND METHODS: The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster. RESULTS: The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic. CONCLUSION: Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1ß, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.

Inactivation of AKT/NF­κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor­κB (NF­κB) signaling has often been associated with the induction of non­small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF­κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF­κB activity in TNF­α­activated 293 cells stably transfected with an NF­κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF­κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu­1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro­apoptotic caspase­3 and poly (ADP­ribose) polymerase, as well as the reduction of anti­apoptotic proteins Bcl­xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF­κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase­3, but decreased Bcl­xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated­AKT and phosphorylated­NF­κB, whereas co­treatment with ECL inhibited the cisplatin­induced phosphorylation of AKT and NF­κB, leading to an increased sensitization effect on cisplatin­induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF­κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

Ginger Extract Promotes Telomere Shortening and Cellular Senescence in A549 Lung Cancer Cells.

Replicative senescence, which is caused by telomere shortening from the end replication problem, is considered one of the tumor-suppressor mechanisms in eukaryotes. However, most cancers escape this replicative senescence by reactivating telomerase, an enzyme that extends the 3'-ends of the telomeres. Previously, we reported the telomerase inhibitory effect of a crude Zingiber officinale extract (ZOE), which suppressed hTERT expression, leading to a reduction in hTERT protein and telomerase activity in A549 lung cancer cells. In the present study, we found that ZOE-induced telomere shortening and cellular senescence during the period of 60 days when these A549 cells were treated with subcytotoxic doses of ZOE. Using assay-guided fractionation and gas chromatography/mass spectrometry analysis, we found that the major compounds in the active subfractions were paradols and shogaols of various chain lengths. The results from studies of pure 6-paradol and 6-shogaol confirmed that these two compounds could suppress hTERT expression as well as telomerase activity in A549 cells. These results suggest that these paradols and shogaols are likely the active compounds in ZOE that suppress hTERT expression and telomerase activity in these cells. Furthermore, ZOE was found to be nontoxic and had an anticlastogenic effect against diethylnitrosamine-induced liver micronucleus formation in rats. These findings suggest that ginger extract can potentially be useful in dietary cancer prevention.

Anti-inflammatory and anti-insulin resistance activities of aqueous extract from Anoectochilus burmannicus.

This study investigated biological activities including antioxidative stress, anti-inflammation, and anti-insulin resistance of Anoectochilus burmannicus aqueous extract (ABE). The results showed abilities of ABE to scavenging DPPH and ABTS free radicals in a dose-dependent manner. Besides, ABE significantly reduced nitric oxide (NO) production in the lipopolysaccharide (LPS)-treated RAW 264.7 via inhibition of mRNA and protein expressions of nitric oxide synthase (iNOS). The LPS-induced mRNA expressions of cyclooxygenase-2 (COX-2) and interleukin 1ß (IL-1ß) were suppressed by ABE. Moreover, ABE exerted anti-insulin resistance activity as it significantly improved the glucose uptake in tumor necrosis factor (TNF)-α treated 3T3-L1 adipocytes. In addition, ABE at the concentration of up to 200 µg/mL was not toxic to human peripheral blood mononuclear cells (PBMCs) and did not induce mutations. Finally, the results of our study suggest the potential use of A. burmannicus as anti-inflammatory, anti-insulin resistance agents, or food supplement for prevention of chronic diseases.

The effect of IGF-I receptor blockade for human esophageal squamous cell carcinoma and adenocarcinoma.

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and tumor development, and this pathway has not been well studied in human esophageal carcinomas. Esophageal cancer is one of the human cancers with the worst prognosis and has two main histologies: squamous cell carcinomas (ESCC) and adenocarcinoma (EAC). Previously, we have reported that detection of the IGF axis may be useful for the prediction of recurrence and poor prognosis of ESCC. We have also shown the successful therapy for several gastrointestinal cancers using recombinant adenoviruses expressing dominant negative IGF-IR (ad-IGF-IR/dn). The aim of this study is to develop potential targeted therapeutics to IGF-IR and to assess the effect of IGF-IR blockade in both of these types of esophageal cancer. We determined immunohistochemical expression of IGF-IR in a tissue microarray. We then assessed the effect of IGF-IR blockade on signal transduction, proliferation, apoptosis, and motility. Ad-IGF-IR/dn, a tyrosine kinase inhibitor, BMS-536924, and adenovirus expressing shRNA for IGF-IR were used. IGF-IR expression was common in both tumor types but not in normal tissues. IGF-IR was detected in metastatic sites at similar levels compared to the primary site. IGF-IR inhibition suppressed proliferation and colony formation in both cancers. IGF-IR blockades up-regulated both stress- and chemotherapy-induced apoptosis and reduced migration. Although IGF-IR/dn blocked ligand-induced activation of Akt-1 mainly, BMS-536924 effectively blocked both activation of Akt and MAPK. The IGF axis might play a key role in tumor progression of esophageal carcinomas. The IGF-IR targeting strategies might thus be useful anticancer therapeutics for human esophageal malignancies.

Ginger extract inhibits human telomerase reverse transcriptase and c-Myc expression in A549 lung cancer cells.

The rhizome of ginger (Zingiber officinale Roscoe) has been reputed to have many curative properties in traditional medicine, and recent publications have also shown that many agents in ginger possess anticancer properties. Here we show that the ethyl acetate fraction of ginger extract can inhibit the expression of the two prominent molecular targets of cancer, the human telomerase reverse transcriptase (hTERT) and c-Myc, in A549 lung cancer cells in a time- and concentration-dependent manner. The treated cells exhibited diminished telomerase activity because of reduced protein production rather than direct inhibition of telomerase. The reduction of hTERT expression coincided with the reduction of c-Myc expression, which is one of the hTERT transcription factors; thus, the reduction in hTERT expression might be due in part to the decrease of c-Myc. As both telomerase inhibition and Myc inhibition are cancer-specific targets for cancer therapy, ginger extract might prove to be beneficial as a complementary agent in cancer prevention and maintenance therapy.

Targeting for insulin-like growth factor-I receptor with short hairpin RNA for human digestive/gastrointestinal cancers.

BACKGROUND AND AIMS: Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling plays important parts in both the tumorigenicity and progression of digestive/gastrointestinal malignancies. In this study, we sought to test the effectiveness of a practical approach to blocking IGF-IR signaling using RNA interference delivered by recombinant adenoviruses. METHODS: We constructed a recombinant adenovirus expressing short hairpin RNA targeting IGF-IR (shIGF-IR) and assessed its effect on signal transduction, proliferation, and survival in digestive/gastrointestinal cancer cell lines representing colorectal, gastric, and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. We analyzed the effects of shIGF-IR alone and with chemotherapy in vitro and in nude mouse xenografts, as well as on insulin signaling and hybrid receptor formation between IGF-IR and insulin receptor. RESULTS: shIGF-IR blocked expression and autophosphorylation of IGF-IR and downstream signaling by the IGFs, but not by insulin. shIGF-IR suppressed proliferation and carcinogenicity in vitro and up-regulated apoptosis in a dose-dependent fashion. shIGF-IR augmented the effects of chemotherapy on in vitro growth and apoptosis induction. Moreover, the combination of shIGF-IR and chemotherapy was highly effective against tumors in mice. shIGF-IR reduced hybrid receptor formation without effect on expression of insulin receptor. CONCLUSIONS: shIGF-IR may have therapeutic utility in human digestive/gastrointestinal cancers, both alone and in combination with chemotherapy.

Insulin-like growth factor-I receptor blockade reduces the invasiveness of gastrointestinal cancers via blocking production of matrilysin.

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. We have previously shown significant therapeutic activity for recombinant adenoviruses expressing dominant-negative insulin-like growth factor-I receptor (IGF-IR/dn), including suppression of tumor invasion. In this study, we sought to evaluate the mechanism of inhibition of invasion and the relationship between IGF-IR and matrix metalloproteinase (MMP) activity in GI carcinomas. We analyzed the role of IGF-IR on invasion in three GI cancer cell lines, colorectal adenocarcinoma, HT29; pancreatic adenocarcinoma, BxPC3 and gastric adenocarcinoma, MKN45, using a modified Boyden chamber method and subcutaneous xenografts in nude mice. The impact of IGF-IR signaling on the expression of MMPs and the effects of blockade of matrilysin or IGF-IR on invasiveness were assessed using recombinant adenoviruses, a tyrosine kinase inhibitor NVP-AEW541 and antisense matrilysin. Invasive subcutaneous tumors expressed several MMPs. IGF-IR/dn reduced the expression of these MMPs but especially matrilysin (MMP-7). Insulin-like growth factor (IGF) stimulated secretion of matrilysin and IGF-IR/dn blocked IGF-mediated matrilysin induction in three GI cancers. Both IGF-IR/dn and inhibition of matrilysin reduced in vitro invasion to the same degree. NVP-AEW541 also reduced cancer cell invasion both in vitro and in murine xenograft tumors via suppression of matrilysin. Thus, blockade of IGF-IR is involved in the suppression of cancer cell invasion through downregulation of matrilysin. Strategies of targeting IGF-IR may have significant therapeutic utility to prevent invasion and progression of human GI carcinomas.

Insulin-like growth factor-I receptor blockade by a specific tyrosine kinase inhibitor for human gastrointestinal carcinomas.

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. In this study, we sought to evaluate the effect of a new tyrosine kinase inhibitor of IGF-IR, NVP-AEW541, on the signal transduction and the progression of GI carcinomas. We assessed the effect of NVP-AEW541 on signal transduction, proliferation, survival, and migration in four GI cancer cells: colorectal adenocarcinoma HT29, pancreatic adenocarcinoma BxPC3, esophageal squamous cell carcinoma TE1, and hepatoma PLC/PRF/5. The effects of NVP-AEW541 alone and with chemotherapy were studied in vitro and in nude mouse xenografts. We also analyzed the effects of NVP-AEW541 on insulin signals and hybrid receptor formation between IGF-IR and insulin receptor. NVP-AEW541 blocked autophosphorylation of IGF-IR and both Akt and extracellular signal-regulated kinase activation by IGF but not by insulin. NVP-AEW541 suppressed proliferation and tumorigenicity in vitro in a dose-dependent manner in all cell lines. The drug inhibited tumor as a single agent and, when combined with stressors, up-regulated apoptosis in a dose-dependent fashion and inhibited mobility. NVP-AEW541 augmented the effects of chemotherapy on in vitro growth and induction of apoptosis. Moreover, the combination of NVP-AEW541 and chemotherapy was highly effective against tumors in mice. This compound did not influence hybrid receptor formation. Thus, NVP-AEW541 may have significant therapeutic utility in human GI carcinomas both alone and in combination with chemotherapy.

Insulin-like growth factor-I receptor as a marker for prognosis and a therapeutic target in human esophageal squamous cell carcinoma.

Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for tumorigenicity and progression of many tumors but this pathway has not been well studied as a prognostic factor or potential therapeutic target in esophageal squamous cell carcinoma (ESCC). In this paper, the association between the expression of IGF-Ir and IGF-II ligand and prognosis was investigated immunohistochemically in 100 surgically resected ESCC. We then assessed the therapeutic effect of blocking IGF receptor signaling using dominant negative IGF-Ir (IGF-Ir/dn) in ESCC in vitro. Expression of IGF-Ir and IGF-II were detected in 60 and 50% of tumors, respectively, and were associated with invasion depth, metastasis, advanced tumor stage and recurrence. Patients with tumors expressing both IGF-Ir and IGF-II had a significantly shorter survival than those expressing either alone or neither in both single and multivariate analysis. IGF-Ir/dn suppressed proliferation and motility as well as upregulating chemotherapy-induced apoptosis through blocking ligand-induced Akt activation. We propose that detection of IGF-Ir/IGF-II in ESCC may be useful for the prediction of recurrence and poor prognosis and for selecting patients for IGF-Ir-targeted therapy. Therapeutic blockade of IGF-Ir may be a useful anticancer therapeutic for ESCC.

Adenovirus expressing shRNA to IGF-1R enhances the chemosensitivity of lung cancer cell lines by blocking IGF-1 pathway.

RNA interference is a phenomenon whereby small double-stranded RNA knocks down the expression of a sequence-specific gene. Double-stranded siRNA transfection, as currently used, is considered to have transient and low transfection efficiency. We constructed an adenoviral vector-based short hair-pin(sh)RNA system to overcome the limitations of the genetic blockade of IGF-1R, one of most important cancer therapy targets. We constructed three different IGF-1R specific shRNAs (612, 801, and 3425) and generated three ad-shIGF-1Rs using BD Adeno-X expression system. We assessed the effect of ad-shIGF-1R on signal transduction, induction of apoptosis, and in vitro tumorigenicity of lung cancer cell lines. Western blot and FACS assays demonstrated that endogenous IGF-1R expression was efficiently suppressed after transduction of lung cancer cell lines with the three different ad-shIGF-1Rs. IGF-1R blockade by ad-shIGF-1R inhibited ligand induced phosphorylation of pAkt and pErk, and ad-shIGF-1R effectively blocked the in vitro tumorigenicity of lung cancer cell lines. Moreover, the transduction of a human lung cancer cell line with ad-IGF-1R(3425) enhanced chemosensitivity to anticancer drugs. We conclude that the adenoviral vector-based approach to the RNA interference of IGF-1R induced effective IGF-1R silencing in lung cancer cell lines as manifested by effective blocking of the downstream pathway of IGF-1R and by an antitumor effect. We believe that this system can be usefully applied to other cancer targets.

Association of matrilysin-2 (MMP-26) expression with tumor progression and activation of MMP-9 in esophageal squamous cell carcinoma.

Expression of matrilysin-2, matrix metalloproteinase (MMP)-26, has been implicated in the progression of several types of human cancer. Matrilysin-2 has been reported to be a physiological and pathological activator of pro-MMP-9. The aim of this study was to examine matrilysin-2 expression and determine whether it is correlated with progression of human esophageal squamous cell carcinoma (ESCC). Semi-quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, zymography and an in vitro invasion assay were performed. Matrilysin-2 mRNA expression was undetectable or only faintly detected in non-tumor tissues, but its overexpression was detected in 24 of the 50 ESCC tissues. Matrilysin-2 overexpression was significantly correlated with depth of invasion, lymph node metastasis and an advance in pathological tumor node metastasis (pTNM) stage. Sections with immunostaining signals in >10% of carcinoma cells at the invasive front, which were observed in 46 of 100 cases, were judged to be positive for matrilysin-2 expression. Matrilysin-2 expression was significantly correlated with depth of invasion, lymph node and distant metastasis, advance in pTNM stage and recurrence. Expression of matrilysin-2 was significantly correlated with nuclear beta-catenin expression and MMP-9 expression. Patients with matrilysin-2-positive cancer had significantly shorter overall and disease-free survival periods than did those with matrilysin-2-negative cancer. Matrilysin-2 expression retained its significant predictive value for overall and disease-free survival in multivariate analysis. Moreover, patients with concomitant expression of matrilysin-2 and MMP-9 had the worst prognosis. Zymography revealed that matrilysin-2 expression was significantly correlated with expression of active MMP-9 in ESCC tissues. Matrilysin-2-transfected TE-1 ESCC cells showed active MMP-9 activity and were more invasive in vitro compared with mock-transfected TE-1 cells. The results of this study suggest that matrilysin-2, the expression of which is closely correlated with nuclear beta-catenin expression and active MMP-9 activity, plays a key role in the progression of ESCC.

Association of trypsin expression with tumour progression and matrilysin expression in human colorectal cancer.

Overexpression of the matrix serine protease (MSP) trypsin has been implicated in tumour growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathological and prognostic significance of trypsin expression in colorectal cancer. This study analysed the association between immunohistochemically detected trypsin expression in colorectal cancer and clinicopathological characteristics, and investigated whether trypsin is a predictor of recurrence and/or survival. Trypsin immunoreactivity was more intense at the invasive front than in the superficial part of the tumour. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 48 cases (48%), were judged to be positive for trypsin. Trypsin positivity was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advanced pathological tumour-node-metastasis (TNM) stage, and recurrence. Patients with trypsin-positive carcinoma had significantly shorter overall and disease-free survival periods than did those with trypsin-negative carcinoma. Trypsin retained its significant predictive value for overall and disease-free survival in multivariate analysis that included conventional clinicopathological factors. It is well known that trypsin activates matrilysin (matrix metalloproteinase-7), which plays an important role in colorectal cancer progression. Patients with concordant overexpression of trypsin and matrilysin at the invasive front, in which they were often co-localized, had the worst prognosis. Trypsinogen-1-transfected HCT116 colon cancer cells showed not only trypsin activity, but also active matrilysin activity and were more invasive in vitro than mock-transfected HCT116 cells. These results suggest that trypsin plays a key role in the progression of colorectal cancer. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence in and poor prognosis of colorectal cancer patients.

Differential involvement of the hypermethylator phenotype in hereditary and sporadic colorectal cancers with high-frequency microsatellite instability.

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair occurs in the majority of hereditary nonpolyposis colorectal cancers (HNPCCs) and in a subset of sporadic malignant tumors. Clinicopathologic and genotypic features of MSI-H colorectal tumors in HNPCC patients and those in sporadic cases are very similar but not identical. Correlation between the MSI phenotype and aberrant DNA methylation has been highlighted recently. A strong association between MSI and CpG island methylation has been well characterized in sporadic colorectal cancers with MSI-H but not in those of hereditary origin. To address the issue, we analyzed hereditary and sporadic colorectal cancers for aberrant DNA methylation of target genes using methylation-specific polymerase chain reaction. DNA methylation of the MLH1, CDKN2A, MGMT, THBS1, RARB, APC, and p14ARF genes was found in 0%, 23%, 10%, 3%, 73%, 53%, and 33% of 30 MSI-H cancers in HNPCC patients and in 80%, 55%, 23%, 23%, 58%, 35%, and 50% of 40 sporadic colorectal cancers with MSI-H, respectively. Cases showing methylation at three or more loci of six genes other than MLH1 were defined as CpG island methylator phenotype-positive (CIMP +), and 23% of HNPCC tumors and 53% of sporadic cancers with MSI-H were CIMP+ (P = 0.018). Differences in the extent of CpG island methylation, coupled with the differential involvement of several genes by methylation, in HNPCC tumors and sporadic MSI-H colorectal cancers may be associated with diverging developmental pathways in hereditary and sporadic cancers despite similar MSI-H phenotypes.

Analysis of gene expression in human colorectal cancer tissues by cDNA array.

BACKGROUND: The development and progression of cancer are accompanied by complex changes in patterns of gene expression. The purpose of this study was to clarify the relevance of macroarray analysis of human colorectal cancer tissues. METHODS: Hybridization of cDNA macroarray filters on which 550 genes had been spotted was performed with biotin-labeled cDNA targets that were prepared from mRNA extracted from 20 pairs of colorectal cancer and corresponding noncancerous tissues. Expression of differentially expressed genes was further studied by semiquantitative RT-PCR. RESULTS: Fourteen (2.5%) of the 550 genes were differentially expressed and up- or downregulated in cancer tissues by at least threefold compared with matched noncancerous tissues in 10 or more of the 20 patients. The genes that were upregulated in cancer tissues were associated with transcription, cell cycle, growth factor receptor, cell adhesion, extracellular matrix-degrading enzymes, and angiogenesis, and the downregulated genes were those involved in apoptosis and immune recognition. Semiquantitative RT-PCR analysis of these differentially expressed genes gave results consistent with those by cDNA array analysis. CONCLUSIONS: Although the macroarray used in this study contained only a small number of genes, our results support the feasibility and usefulness of this approach to study variation in gene expression patterns in human colorectal cancer tissues. The results also suggest the possibility of a diagnostic application of cDNA macroarrays in daily clinical settings.