Pancreatic cancer patients frequently show hyperglycemia, but it is uncertain whether hyperglycemia stimulates pancreatic cancer cells. We have investigated whether excess glucose induces hypoxia-inducible factor-1α (HIF-1α) and stimulates glucose metabolism and cell migration in pancreatic cancer cells. We studied wild-type (wt) MiaPaCa2 pancreatic cancer cells and a MiaPaCa2 subline (namely si-MiaPaCa2) that had HIF-1α-specific small interfering RNA. Wt-MiaPaCa2 cells are known to be HIF-1α-positive in hypoxia and HIF-1α-negative in normoxia, whereas si-MiaPaCa2 cells are devoid of HIF-1α in both normoxia and hypoxia. We incubated these cells with different amounts of glucose and determined HIF-1α mRNA and protein by real-time polymerase chain reaction and western blotting. We determined glucose consumption, lactate production and intracellular hexokinase-II and ATP to assess glucose metabolisms and determined pyruvate dehydrogenase kinase-1, reactive oxygen species and fumarate to assess mitochondrial activities. Further, we studied cell migration using a Boyden chamber. Excess glucose (16.7-22.2mM) increased HIF-1α in hypoxic wt-MiaPaCa2 cells. HIF-1α expression increased ATP contents and inhibited mitochondrial activities. Extracellular glucose and hypoxia stimulated glucose metabolisms independent of HIF-1α. Excess glucose stimulated the migration of wt- and si-MiaPaCa2 cells in both normoxia and hypoxia. Thus, glucose stimulated cell migration independent of HIF-1α. Nevertheless, hypoxic wt-MiaPaCa2 cells showed greater migrating ability than their si-MiaPaCa2 counterparts. We conclude that (1) excess glucose increases HIF-1α and ATP in hypoxic wt-MiaPaCa2 cells, (2) extracellular glucose and hypoxia regulate glucose metabolisms independent of HIF-1α and (3) glucose stimulates cell migration by mechanisms that are both dependent on HIF-1α and independent of it.

SummaryStudies of diabetes and hepatocellular carcinoma (HCC) yielded inconsistent findings. This meta‐analysis was conducted to examine the association between diabetes and risk of HCC. Studies were identified by searching PUBMED and MEDLINE database up to February 2011. Pooled risk estimates were calculated using the random‐effects model. Potential sources of heterogeneity were explored by subgroup analyses. A total of 17 case‐control studies and 32 cohort studies were included in the meta‐analysis. The combined risk estimate of all studies showed a statistically significant increased risk of HCC prevalence among diabetic individuals (RR = 2.31, 95% CI: 1.87–2.84). The pooled risk estimate of 17 case‐control studies (OR = 2.40, 95% CI: 1.85–3.11) was slightly higher than that from 25 cohort studies (RR = 2.23, 95% CI: 1.68–2.96). Metformin treatment was potentially protective. On the contrary, long duration of diabetes and sulfonylureas or insulin treatment possibly increase HCC risk. Also meta‐analysis of 7 cohort studies found a statistically significant increased risk of HCC mortality (RR = 2.43, 95% CI: 1.66–3.55) for individuals with (versus without) diabetes. This meta‐analysis shows that diabetes is associated with moderately increased risk of HCC prevalence, as well as HCC mortality. Considering the rapidly increasing prevalence of diabetes mellitus, the study underlines the need for cancer prevention in diabetic individuals. Further investigation is needed to focus on the potential mechanism for the pathogenesis of HCC and the link between HCC and different types, severity, treatment and duration of diabetes. Copyright © 2011 John Wiley & Sons, Ltd.

The epidermal growth factor receptor (EGFR) is expressed in a variety of human solid tumors, including malignant mesothelioma. EGFR has been implicated in regulation of cell proliferation, survival, angiogenesis, and metastasis, making it an ideal target for drug development. ZD1839 (gefitinib) and OSI-774 (erlotinib) are new, low-molecular-weight, EGFR-selective tyrosine kinase (TK) inhibitors, whereas CI-1033 is a pan-EGFR family TK inhibitor. In the present study, we used ZD1839, OSI-774, and CI-1033 and investigated the effect of these drugs on proliferation, migration, and matrix metalloprotease (MMP) production in three malignant mesothelioma cell lines (M14K, ZL34, and SPC212). Using [3H]thymidine incorporation, DNA synthesis assay, we found that all three drugs inhibited transforming growth factor-alpha (TGF-alpha)-induced cellular proliferation in a dose-dependent manner. In addition, all three drugs induced apoptosis in ZL34 cells as determined by flow cytometry using annexin-V staining. Furthermore, all three drugs inhibited TGF-alpha-induced cell migration (chemotaxis) in a dose-dependent manner as determined by Boyden chamber assay. TGF-alpha-induced MMP-9 production was also inhibited in a dose-dependent manner as determined by gelatin zymography in three cell lines tested. In conclusion, our study demonstrates inhibitory effectiveness of EGFR-TK inhibitors in malignant mesothelioma cells and suggests that these drugs may be an effective treatment strategy for malignant mesothelioma.

4535 The epidermal growth factor receptor (EGFr) and insulin-like growth factor receptors (IGF-Ir and IGF-IIr) are expressed on a variety of human solid tumors, including malignant mesothelioma and have been implicated in regulation of cell proliferation, survival, angiogenesis and metastasis. Previous studies have also suggested that EGFr may be involved in asbestos-induced the pathogenesis of malignant mesothelioma. CI-1033 is a pan-EGFr-family tyrosine kinase inhibitor. In the present study, we have examined the anti-proliferative effects of CI-1033 in three malignant mesothelioma cell lines (M14K, ZL34 and SPC212) which represent epithelial, fibrous and mixed histological subtypes, respectively. Using 3H-thymidine incorporation DNA synthesis assay, we found that CI-1033 inhibited TGF-α, IGF-I and IGF-II induced cellular proliferation in a dose-dependent manner. In addition, CI-1033 induced apoptosis in ZL34 cells. Since our previous study have shown that ligands of EGFr, IGF-Ir and IGF-IIr stimulated cell migration and secretion of MMPs in malignant mesothelioma cells, we have further evaluated whether CI-1033 has inhibitory effect of cell migration and MMPs production induced by TGF-α or/and IGF-I, IGF-II. Our results show that CI-1033 inhibit TGF-a induced cell migration (chemotaxis) in a dose-dependent manner in three tested cell lines as determined by Boyden chamber assay. In contrast, CI-1033 failed to inhibit IGF-I and IGF-II induced chemotaxis. Furthermore, CI-1033 inhibited the activity of MMP-9 induced by TGF-α in a dose-dependent manner in all tested cell lines as determined by gelatin zymography. The level of MMP-2 activity was not affected by the drug treatment. In conclusion, our study demonstrates inhibitory effectiveness of CI-1033 in malignant mesothelioma cells and suggests that this may be an effective treatment strategy for malignant mesothelioma.

BACKGROUND:Chemotaxis is defined as directional cell movement of cells towards concentration gradients of solubilized attractants, whereas chemokinesis is defined as random cell movement in the absence of chemoattractant gradients. Since tumor cell motility plays an important role in the process of tumor invasion and metastasis, we investigated these two distinct motile behaviors in highly invasive tumor, malignant mesothelioma.MATERIALS AND METHODS:Chemotaxis and chemokinesis of mesothelioma cells were assayed using Boyden chambers fitted with filters coated with collagen type IV and different growth factors and cytokines were used as chemoattractants.RESULTS:We found that growth factors such as epidermal growth factor, transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor-like growth factor, beta-cellulin, insulin-like growth factor-I, insulin-like growth factor-II and stem cell factor stimulated directional (chemotactic) and/or random (chemokinetic) motility in all mesothelioma cell lines tested, whereas none of acidic fibroblast growth factor, basic fibroblast growth factor, granulocyte-macrophage colony-stimulating factor or interleukin-6 induced migration in the same mesothelioma cells.CONCLUSION:These findings provide evidence that: (i) multiple growth factors can induce chemotaxis and chemokinesis in malignant mesothelioma cell lines, and (ii) may contribute to our understanding of the highly invasive behavior of malignant mesotheliomas in vivo.

BACKGROUND:Hyaluronan is one of the main components of the extracellular matrix. It is synthesized at the cell plasma membrane by specific hyaluronan synthases (HAS). Although a large number of studies have described hyaluronan in pleural effusion from malignant mesothelioma, the source of hyaluronan in malignant mesothelioma has been subject to controversy.MATERIALS AND METHODS:The mRNA expression of all three HAS in malignant mesothelioma cells was studied using RT-PCR. The hyaluronan production in culture medium of malignant mesothelioma cells was also examined using high-performance liquid chromatography (HPLC).RESULTS:We found that 9/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-1, while 10/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-2 and HAS-3. In addition, we demonstrated hyaluronan in the culture medium of 6 out of 10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells.CONCLUSION:Our results show that malignant mesothelioma cells express all three HAS and synthesize hyaluronan. The expression of HAS isoforms and hyaluronan in malignant mesothelioma cells in cultures and previous observations by other investigators indicate that these cells are, at least in part, responsible for hyaluronan synthesis in vivo.

Growth factors secreted by either host or tumour cells play a major role in tumour cell progression. Besides stimulating cell division, growth factors may also stimulate cell migration and modulate matrix metalloprotease (MMP) production. MMPs are enzymes involved in a variety of physiological and pathological processes including tumour cell invasion and metastasis. We have previously shown that different growth factors regulate the motile behaviour of human lung cancer cell lines. In order to further advance our knowledge of the role the different growth factors play in lung cancer, we investigated their effect on two key enzymes belonging to the MMP family of enzymes, namely MMP-9 and MMP-2. Serum-free cultures of three human non-small cell lung cancer cell lines were exposed to five different growth factors: insulin-like growth factor I (IGF I) and II (IGF II), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and stem cell factor (SCF). The expression of MMP-9 and MMP-2 in growth factor-treated and untreated cell lines was evaluated using gelatine zymography and quantified using computer-assisted image analyses. We found heterogeneous expression and activity of MMP-9 and MMP-2 in all three lung cancer cell lines. The most important finding in our study is that HGF and EGF are capable of stimulating the conversion of MMP-9 from a latent to an active form in human large cell lung cancer cell line U-1810 [corrected]. IGF I, IGF II, HGF and EGF stimulated an enhanced expression and activity of the latent form of MMP-2 and MMP-9. SCF did not enhance MMP activity in any of the cell lines tested. Our previous studies have shown that IGF I, IGF II, HGF, EGF and SCF induce migration of human non-small cell lung cancer cells in the presence of extracellular matrix (ECM) components. In the present study we show that growth factors can also enhance the expression of MMP's in these cells. Taken together these results indicate that certain growth factors may promote invasiveness through their ability to induce not only cell migration, but also by enhancing the expression and activity of matrix degrading MMP-2 and MMP-9.

Human lung tumors express different types of growth-factor receptors and corresponding ligands that might modulate several biological functions such as proliferation, differentiation, adhesion, and chemotaxis. In the present study, we have investigated the expression of different growth-factor receptors and their ligands in 5 established human lung-cancer cell lines. Using RT-PCR, we found that IGF-II/mannose-6-phosphate (M6P), c-met, EGF and c-kit receptors are expressed in 5/5 human lung-cancer cell lines. In order to investigate the biological function of these receptors, we performed Boyden-chamber assays using various growth factors as chemo-attractants. Human non-small-cell-lung-cancer cells (non-SCLC) migrated to recombinant human (rh)IGF I and IGF II at concentrations ranging from 1 to 1000 ng/ml, to HGF at 10 to 100 ng/ml, to EGF at 1 to 100 ng/ml and SCF at 1 to 50 ng/ml. In addition, we performed Boyden-chamber assays using U-1810-, U-1752- and Wart-derived serum-free conditioned medium as chemo-attractants. Serum-free conditioned medium stimulated migration of producer cells in a dose-dependent manner. The autocrine motility stimulating effect of U-1810-derived serum-free conditioned medium could be inhibited by 50% in the presence of neutralizing ahIGF-II antibodies in the assay, suggesting a possible autocrine motility loop in vitro. Int. J. Cancer 82:338–345, 1999. © 1999 Wiley-Liss, Inc.

Hepatocyte growth factor (HGF) and its receptor c-met are present in several human tissues but their expression in mesothelial cells has not been examined. In this study, we have investigated the expression of HGF and c-met in normal human mesothelial cells and 11 human malignant mesothelioma cell lines. Using RT-PCR and Western blotting we found that HGF is produced by 3/11 mesothelioma cell lines whereas c-met is expressed in 11/11 mesothelioma cell lines. In addition, c-met expression was also found in 6/6 cell samples obtained from pleural fluids of patients with mesothelioma. In contrast, neither normal cultured mesothelial cells nor mesothelial cells obtained directly from patients without mesothelioma expressed HGF nor c-met. We have also analysed the biological function of HGF and c-met in mesothelioma cell lines. Recombinant human (rh) HGF stimulated both directional (chemotactic) and random (chemokinetic) motility in all mesothelioma cell lines tested. Furthermore, mesothelioma serum free conditioned medium containing HGF stimulated mesothelioma cell migration. This effect could be blocked in the presence of neutralizing anti-HGF monoclonal antibodies (MAbs) in the assay. Addition of HGF to mesothelioma cells cultured on collagen type IV was associated with induction of bipolar shape and protrusion of prominent pseudopodia. We have also found that rhHGF was mitogenic for mesothelioma cells. Our findings suggest that expression of HGF/c-met is involved not only in mesothelioma progression but also in its growth and migration and that c-met expression found in mesothelioma cells taken directly from patients may be of diagnostic importance.