Human papillomavirus up-regulates MMP-2 and MMP-9 expression and activity by inducing interleukin-8 in lung adenocarcinomas.

Human papillomavirus (HPV) infection is associated with non-smoking female lung cancer. Our previous report demonstrated that HPV 16 promotes lung tumor cell progression by up-regulating interleukin-17 (IL-17). IL-17 and its downstream signaling mediator, interleukin-8 (IL-8), have been implicated to modulate a variety of pro-angiogenic factors and play important roles in tumor angiogenesis and metastasis. Accordingly, we hypothesized that HPV infection may potentiate tumorigenic and metastatic characteristics of the infected cells through IL-8. The goal of the present study was to determine whether HPV infection in lung adenocarcinoma cells can promote the expression of IL-8 and metalloproteinases (MMPs) to make the transformed cells equipped with angiogenic and metastatic characteristics. The expression of IL-8 and MMPs in HPV 16 E6-transfected H1299 cells was analyzed to examine the hypothesis. HPV 16 E6 up-regulates pro-angiogenic MMP-2 and MMP-9 through inducing IL-8 expression in lung cancer cells. The results indicate that, in addition to cell proliferation-related machinery, HPV infection promotes the expression and activities of angiogenic and metastatic molecules in lung adenocarcinoma cells. The cytokines induced by HPV infection may work together to confer the malignant and tumorigenic potentials on the infected cells by promoting machineries of growth, angiogenic and metastatic characteristics.

Trends of mycobacterial clinical isolates in Taiwan.

Non-tuberculous mycobacteria (NTM) can cause chronic pulmonary infection, however, NTM infection is generally overlooked. This retrospective study analyzed the frequencies of Mycobacterium tuberculosis complex (MTBC) and NTM clinical isolates from 99 200 specimens of patients suspected with pulmonary mycobacterial infection in Taiwan from 2002-2007. A total of 8024 mycobacterial isolates, including 5349 MTBC and 2675 NTM, were obtained from the 99 200 specimens in the study period. The overall mycobacterial isolation rate was 8.09% (8024/99 200), and the overall MTBC and NTM isolation rate was 5.39% (5349/99 200) and 2.7% (2675/99 200), respectively. Notably, the prevalence of NTM isolates among the identified mycobacteria strains was increased 2.6 fold from 2002 (17.54%, 147/838) to 2007 (45.80%, 659/1439). The frequencies of MTBC and NTM isolates showed a reciprocal trend: the NTM isolation rates were steadily increasing while the overall mycobacterial isolation rates remained stable over the study period. Our results suggest that the diagnosis, identification and susceptibility tests for NTM should be standardized and integrated in clinical routines, for providing the information of NTM infection and prescribing clinical treatment in a more precise and efficient way to reduce the increasing NTM in the studied area.

Up-regulation of interleukin-17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer.

BACKGROUND: Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)-17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line. METHODS: IL-17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL-17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6-transfected H1299 human NSCLC cells. RESULTS: Immunohistochemical data showed that 48.1% of lung tumors had IL-17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL-17 with IL-6 (P < .001) and IL-17 with Mcl-1 (P < .001) expression were also observed. Cell growth rate was increased, and IL-17/Mcl-1 expression levels were elevated in HPV 16 E6-transfected H1299 cells. The transfected E6 oncoproteins can significantly up-regulate expression levels of IL-17 and antiapoptotic protein Mcl-1. CONCLUSIONS: The study suggests that HPV infection-induced IL-17 levels can stimulate Mcl-1 expression through the PI3K pathway and promote lung tumor cell progression through a p53- and IL-6-independent pathway.

Association of interleukin-4 promoter polymorphisms in Taiwanese patients with type 2 diabetes mellitus.

Many factors have been implicated in the onset of type 2 diabetes mellitus (T2DM). Recently, immune response and inflammation were suggested to play certain roles in the development and complications of T2DM. The aim of this study is to investigate the putative correlation between the promoter polymorphisms of interleukin-4 (IL-4), one of the immune-regulatory type 2 helper T-cell cytokines, and T2DM. Genomic DNA from 425 Taiwanese T2DM patients and 148 nondiabetic control study subjects were extracted, and their IL-4 promoter polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Both of the distribution of IL-4 C-589T (P = .013) and C-34T (P = .05) genotypes were significantly different between T2DM patients and control subjects. Significant association between IL-4 C-589T alleles (P = .002) and T2DM, as well as C-34T alleles and T2DM (P =.024), was also identified. In addition, a statistically significant association between homologous IL-4 -589 C/C genotype and lower circulatory high-density lipoprotein cholesterol levels was observed. Our results suggested that IL-4 promoter polymorphisms are associated with T2DM. A significant association between IL-4 -589 C/C genotype and lower circulatory high-density lipoprotein cholesterol level was observed as well. The above results suggested that IL-4 may participate in lipid metabolism and diabetic susceptibility.

Elevated circulatory MMP-2 and MMP-9 levels and activities in patients with rheumatoid arthritis and systemic lupus erythematosus.

OBJECTIVES: Matrix metalloproteinases (MMPs) are suggested to play important roles in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study is to examine the MMPs expressions and activities in Taiwanese RA and SLE patients. DESIGN AND METHODS: Levels and activities of plasma MMP-2 and MMP-9 were investigated by enzyme-linked immunosorbent assay and zymography, respectively. RESULTS: MMP-2 levels in control subjects, RA and SLE patients were 146.1+/-34.2, 194.0+/-24.2 and 208.9+/-75.9 ng/mL respectively, and for MMP-9 were 51.4+/-57.1, 567.7+/-313.1 and 208.7+/-105.5 ng/mL respectively. Both MMP-2 and MMP-9 levels and activities from all patients were significantly higher than that from control subjects. CONCLUSIONS: MMP-2 levels in both patients groups were approximately 1.3-1.4 folds higher than that in control subjects, notably, MMP-9 levels were 11- and 4-folds significantly higher, respectively, in RA and SLE patients. The results which MMP-2 and MMP-9 levels and activities are significantly elevated support the involvement of MMPs proteins in these autoimmune disorders.

Ionic versus nonionic contrast media solvents used with an epirubicin-based agent for transarterial chemoembolization of hepatocellular carcinoma.

PURPOSE: To compare the stability of epirubicin-iodized oil emulsions prepared with ionic or nonionic contrast medium and to compare the efficacy of these emulsions in a prospective, randomized, controlled trial of transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Epirubicin-iodized oil emulsions prepared with ionic and nonionic contrast media was evaluated for stability with light microscopy and magnetic resonance imaging. One hundred ninety-seven patients with inoperable HCC were randomized to receive TACE with epirubicin, prepared either with ionic (control group, n = 99) or nonionic (experimental group, n = 98) contrast medium. Tumor response was graded according to iodized oil retention (grade 1 = >90% retention, grade 2 = 50%-90% retention, and grade 3 = <50% retention), as characterized with computed tomography. Survival probabilities were calculated with the Kaplan-Meier method. RESULTS: The epirubicin-iodized oil emulsions prepared with ionic contrast medium were less stable, exhibiting rapid separation of the oil and aqueous phases, compared with emulsions prepared with nonionic medium. Ninety-one patients in the control group and 87 in the experimental group underwent follow-up CT. Thirty-seven of the 91 patients in the control group (41%) had grade 1 tumors, 41 (45%) had grade 2 tumors, and 13 (14%) had grade 3 tumors. Forty-eight of the 87 patients in the experimental group (55%) had grade 1 tumors, 22 (25%) had grade 2 tumors, and 17 (20%) had grade 3 tumors. The number of patients with grade 1 tumors was significantly higher in the experimental group than in the control group (P = .02); however, there was no difference in patient survival (P = .94). CONCLUSIONS: Epirubicin-iodized oil emulsions prepared with nonionic contrast medium are more stable and are associated with lower tumor grade in patients with inoperable HCC. The choice of solvent, however, does not appear to have an effect on patient survival.

Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3.

Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of (359)serine and (835)serine of 14-3-3beta binding sites to alanine, which slightly reduces the interaction with 14-3-3beta, does not influence the PTPN3 effect. In contrast, mutation of the invariant (842)cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3beta, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3beta, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression.

Increased expression of matrix metalloproteinase-2 in oral cells after short-term stimulation and long-term usage of areca quid.

BACKGROUND AND PURPOSE: Arecoline, an areca quid (AQ) component, has been shown to inhibit the secretion and activity of matrix metalloproteinase-2 (MMP-2) in fibroblast cultures. This study assessed whether MMP-2 expression was inhibited in the saliva samples and tumor specimens of oral tumor patients with a long-term history of AQ consumption. The net effect of crude AQ extract (AQE) on MMP-2 expression by oral cells was also investigated. METHODS: Western blot analysis, zymography, and reverse transcriptase-polymerase chain reaction were used to detect MMP-2 protein and mRNA in saliva and tumor samples, as well as in the conditioned media (CM) of oral cell cultures. RESULTS: The level of MMP-2 protein was significantly higher in the saliva samples of 12 oral tumor patients who had a minimum 10-year AQ-consuming history than in those of 12 non-AQ-using healthy controls (p < 0.05). MMP-2 mRNA was expressed in 26 of 28 oral squamous cell carcinoma (OSCC) specimens. MMP-2 protein was also detectable in the tested OSCC homogenates. Short-term stimulation with 10% AQE increased the secretion of MMP-2 protein in the CM of oral epidermoid carcinoma cell Meng-1 (an OSCC cell line) and oral fibroblasts. CONCLUSIONS: MMP-2 expression is elevated rather than inhibited in most oral tumor patients with long-term AQ usage. Short-term AQE stimulation also increases the secretion of MMP-2 by oral epithelial cells and fibroblasts. Our results suggest that AQ consumption may promote oral tumor progression through the induction of MMP-2 secretion.

Areca quid chewing enhances the expression of salivary matrix metalloproteinase-9.

BACKGROUND AND PURPOSE: The effects of areca quid (AQ) consumption on salivary matrix metalloproteinases (MMPs) which may participate in tumor invasion and metastasis remains unclear. This study assessed the change in salivary MMP-9 protein levels 2 hours after 5-minute AQ chewing stimulation (AQCS) in non-AQ users and the expression profile of this proteinase in saliva and tumor specimens of oral squamous cell carcinoma (OSCC) patients with a history of AQ use. METHODS: MMP-9 transcript was analyzed by reverse transcription-polymerase chain reaction. MMP-9 protein level was measured by both Western blot and gelatin zymography. RESULTS: The protein level of salivary MMP-9 was 3.1- to 8.9-fold enhanced 2 h after AQCS in 3 healthy volunteers as revealed by Western blot and zymography. As a control, gum chewing did not significantly change salivary MMP-9 protein level. Expression of MMP-9 transcript was found in 25 of 28 OSCC specimens and significantly correlated with cervical lymph node metastasis (p = 0.037). All of the 8 tested OSCC tissue homogenate samples available and all 12 saliva samples from 12 oral tumor outpatients were positive for MMP-9 protein. CONCLUSIONS: Elevation of MMP-9 may be one of the net effects of AQCS in vivo, which may play a role in the pathogenesis of oral mucosal lesions. Furthermore, the association of MMP-9 expression with neck-lymph-node metastasis may imply a significant role of MMP-9 in the progression of OSCC among patients with a history of AQ use in Taiwan.

Overexpression of Rac-1 small GTPase binding protein in oral squamous cell carcinoma.

PURPOSE: Oral squamous cell carcinoma (OSCC) is a fairly common malignancy in Taiwan and most countries of South Asia due to the popularity of areca quid use. In vitro studies have indicated that Rac proteins, a member of ras-related small GTPase protein family, can regulate cytoskeletal structures and activate signaling cascade and have the potential to transform cultured cells. However, Rac alteration during oral carcinogenesis in vivo has yet to be shown. The present study was conducted to investigate the importance of Rac-1 in oral tumorigenesis in vivo. PATIENTS AND METHODS: Expression level of Rac-1 protein and mRNA in OSCC together with noncancerous match tissue (NCMT) was explored using immunohistochemistry and reverse transcription-polymerase chain reaction analysis. RESULTS: The immunoreactivity of Rac-1 was shown in a significantly higher fraction of OSCC (74%) relative to the 48% in NCMT (P =.03). Eight of 14 (57%) available tissue pairs also showed overexpression of Rac-1 mRNA in OSCC than that in NCMT. The Rac-1 immunoreactivity did not differ significantly in accord with clinicopathologic parameters including areca quid use. However, 3 of 4 recurrent OSCC studied lacked the Rac-1 immunoreactivity. CONCLUSIONS: Our results presented novel findings: Rac-1 overexpression is a frequent occurrence in OSCC, highlighting the involvement of GTPase elements in the neoplastic growth of OSCC.

Areca nut extract treatment elicits the fibroblastoid morphological changes, actin re-organization and signaling activation in oral keratinocytes.

BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.

Intercellular calcium waves mediate preferential cell growth toward the wound edge in polarized hepatic cells.

During liver regeneration, hepatocytes sense the damage and initiate proliferation of the quiescent cells through poorly understood mechanisms. Here, we have used cultured hepatic cells to study the roles played by intercellular calcium in mediating wound-healing processes. Well-differentiated and polarized Hep-G2 cells repaired an experimentally induced wound by induction of cell divisions. The resulting cellular growth did not occur evenly across the healing cell lawn; instead, proliferations were three times more active within 150-200 microm from the wound edge than further away; this periwound preferential cell growth was not observed in the poorly differentiated and/or nonpolarized cells. We have provided experimental evidence demonstrating that the wounding procedure itself could elicit a propagating calcium wave, and interestingly, blocking this injury-associated intercellular calcium communication could effectively inhibit the biased cell growth along the margin of the wound. A photolithography-based patterned cell culture system was employed to help delineate the mechanisms underlying this type of calcium signaling. In conclusion, our results suggested that intercellular communications via propagating calcium waves coordinate regenerative cell proliferations in response to hepatic tissue losses.

Chromosomal changes in betel-associated oral squamous cell carcinomas and their relationship to clinical parameters.

To investigate the chromosomal imbalances that occur in oral carcinoma associated primarily with betel use and their clinical implications, we performed chromosomal analysis using comparative genomic hybridization on 47 patients with this disease. The most common gains of chromosome arms were 8q, 9q and 11q, and the most frequent losses were of chromosomal arms 3p and 4q. The clinical parameters significantly associated with the numbers of chromosomal imbalances per tumor were the age of the patients and nodal metastasis. The preliminary findings of a lower incidence of loss of 4q and gain of 8q in betel-associated tumors compared to non-betel-associated tumors might provide insight into the carcinogenic effect of betel. Deletion of 3p and the gain of 11q alterations were more prevalent in carcinomas with lymph node metastasis than in node-negative tumors, indicating possible loci for metastasis suppressor or metastasis enhancing genes, respectively. Losses of 3p and 4q and gain of 9q were associated with poor outcome for the patients. These data demonstrated that the frequent aberrations in 4q and 9q sites can be used as novel prognostic predictors.

Multiple molecular alterations of FHIT in betel-associated oral carcinoma.

To determine the alterations of the FHIT (fragile histidine triad) gene in oral squamous cell carcinoma (OSCC), this study examined mutation, promoter methylation, mRNA transcription, and protein expression of FHIT in OSCC associated mostly with the use of betel and/or tobacco. Analyses of the coding exons (exons 5-9) identified a deletion of one base in intron 4 in one tumour and a deletion of exon 7 in two tumours. Using bisulphite genomic sequencing, 28% of the informative subjects exhibited promoter methylation. An aberrant FHIT transcript spanning from exon 3 to exon 10, which was verified by RT-PCR analysis, was identified in 36% of the OSCC subjects, 50% of the oral pre-invasive lesions, and 5% of the non-cancerous match tissue. An abnormal immunohistochemical level of Fhit was detected in 41% of OSCC subjects. A statistically significant association was found between aberrant transcription of the FHIT gene and an abnormal level of Fhit immunoreactivity. The results indicated that alteration of FHIT is a frequent occurrence in OSCC and thus suggests that the aberrance in FHIT transcription could be an early event of oral carcinogenesis.

Resistance to tamoxifen-induced apoptosis is associated with direct interaction between Her2/neu and cell membrane estrogen receptor in breast cancer.

Overexpression of Her2/neu is implicated in the development of resistance to the antiestrogen tamoxifen (TAM) that exerts its inhibitory effect through interaction with estrogen receptor (ER). Whereas Her2/neu and ER are believed to be important cell survival/death factors in human breast cancer cells, if and how they interact to confer resistance to hormone therapy is not known. This prompted us to investigate whether modulation of the effect of TAM occurs via the Her2/neu pathway and whether targeting the interaction between the Her2/neu pathway and the ER pathway is beneficial. There are 2 forms of ER that are localized to the cell membrane and to the nucleus. For the first time, we found that Her2/neu directly interacts with ER at the cell membrane. We then investigated the role of Her2/neu overexpression in the regulation of the cell membrane ER pathway in TAM-resistant breast cancer cells and the nature of this interaction in apoptotic signaling. Relief of TAM resistance was associated with Her2/neu downregulation and ER upregulation. TAM-induced apoptosis occurred immediately after dissociation of Her2/neu from cell membrane ER. These results demonstrate a novel mechanism by which Her2/neu regulates the cell membrane ER-coupled apoptosis and the possible involvement of the Her2/neu in TAM resistance of breast cancer cells. Moreover, the antiproliferative activity of TAM should rely on the integration between the signal transduction from the cell membrane ER and the gene regulation by the nuclear ER. Coordinated modulation on the cell membrane ER/Her2/neu pathway and the nuclear ER/RAR pathway may provide a new approach for treatment of ER-positive, Her2/neu overexpressing breast cancer.