Porphyromonas gingivalis increases the invasiveness of oral cancer cells by upregulating IL-8 and MMPs

Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs.     

Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells

Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of Mr 42,000-50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 degrees C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 mM. However, CSF-1 activity was inhibited totally by the combination of 10 mM DTT and 1 mM SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of Mr 21,000-25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-beta-D-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000-40,000, and to 18,000-20,000 in the presence of mercaptoethanol. Because of the specificity of endo-beta-D-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units.     

Oleanolic acid increases the anticancer potency of doxorubicin in pancreatic cancer cells

Combination therapy is a novel cancer therapy approach that combines two or more chemotherapy drugs. This treatment modality enhances the efficacy of chemotherapy by targeting key pathways in an additive or synergistic manner. Therefore, we investigated the efficacy of combination therapy by widely used chemotherapy drug doxorubicin (DOX) and oleanolic acid (OA) to induction of apoptosis for pancreatic cancer (PC) therapy. The effects of DOX, OA, and their combination (DOX-OA) were investigated on proliferation and viability of PC cell line (PANC-1) by MTT assay. Moreover, migration and invasion of the cancer cells were evaluated by trans-well migration assay and wound healing assay. Flow cytometry and DAPI (4′,6-diamidino-2-phenylindole) staining were employed to investigate apoptosis quantification and qualification of the treated cancer cells. Finally, mRNA expression of apoptosis-related genes was assessed by quantitative real-time polymerase chain reaction. Our results demonstrated that the proliferation and metastasis potential of PC cells significantly decreased after treatment by DOX, OA, and DOX-OA. Moreover, we observed an increase in apoptosis percentage in the treated cancer cells. The apoptosis-related gene expression was modified to increase the apoptosis rate in all of the treatment groups. However, the anticancer potency of DOX-OA combination was significantly more than that of DOX and OA treatments alone. Our study suggested that DOX-OA combination exerts more profound anticancer effects against PC cell lines than DOX or OA monotherapy. This approach may increase the efficiency of chemotherapy and reduce unintended side effects by lowering the prescribed dose of DOX.     

Melanoma inhibitory activity (MIA): an important molecule in melanoma development and progression

Cutaneous malignant melanoma is the leading cause of skin cancer death in industrialized countries. Melanoma development and progression are well defined by clinical and histopathological aspects; however, detailed analysis of molecular changes is still ongoing. The protein MIA, which is strongly expressed in melanoma cells but not in melanocytes, is likely to represent a key molecule regulating melanoma progression. Consistent with this, several in vitro and in vivo model systems indicate a direct involvement of MIA in melanoma migration and invasion, with recent studies suggesting a central role for MIA in early melanoma development by regulating important melanoma-related pathways and molecules. The latest developments in MIA research are summarized in this review, which describes recently published data related to the MIA protein structure and function, the role of MIA in melanoma development and progression, and the regulation of MIA expression. Furthermore, newly discovered MIA-homologous genes are discussed.     

Vitamin D3 decreases glycolysis and invasiveness,and increases cellular stiffness in breast cancer cells

Breast cancer is one of the major causes of death in the USA. Cancer cells, including breast, have high glycolysis rates to meet their energy demands for survival and growth. Vitamin D₃ (VD₃) is important for many important physiological processes such as bone mineralization, but its anticancer role is yet to be proven. We find that VD₃ treatment significantly down-regulates glycolytic enzymes and genes and decreases glucose uptake — for both lowly metastatic MCF-7 and highly metastatic MDA-MB-231 (MB231) breast cancer cells. VD₃ also significantly decreases cell viability by inducing apoptosis — consistent with decreased expression of mammalian target of rapamycin (mTOR), which regulates glycolysis and cancer cell survival, and increases 5′ adenosine monophosphate-activated protein kinase (AMPK) activation. These changes accompany a significant reduction of cell migration and increased cell stiffness, presumably a consequence of reversal of the epithelial to mesenchymal transition resulting in increased E-cadherin, and F-actin, and reduced vimentin expression. High levels of cytoskeletal and cortical F-actin may cause high cell stiffness. VD₃-induced mechanical changes are stronger in highly metastatic MB231 than in lowly metastatic MCF-7 cells. Our results suggest therapeutic and preventive roles of VD₃ in breast cancer.     

Vimentin affects the mobility and invasiveness of prostate cancer cells

A significant proportion of prostate cancer patients treated with curative intent go on to develop advanced disease. At a fundamental biological level, very little is known about what makes the disease aggressive and metastatic. Observational pathology reports and experimental data suggest that epithelial-mesenchymal transition is involved in prostate cancer invasiveness. Here, we investigated vimentin expression of prostate cancer cells, and explored the potential mechanism of vimentin promoting prostate cancer cells invasion. Vimentin expression was not detected in well differentiated tumors or in moderately differentiated tumors, but the majority of poorly differentiated cancers (5/11 with negative bone scan, 11/14 bone with positive scan) and bone metastases (8/8) had high vimentin expression in tumor cells. Downregulation of vimentin expression in PC-3 cells by transfection with antisense-vimentin led to a significant decrease in tumor cells motility and invasive activity. Furthermore, the expression of E-cadherin was inversely associated with expression of vimentin. Our results suggest that vimentin affects prostate cancer cells motility and invasiveness.     

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) is currently an emerging target in pancreatic cancer. In this report, we describe a series of indolequinones, based on 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), and evaluate NQO1 inhibition and growth inhibitory activity in the human pancreatic MIA PaCa-2 tumor cell line. The indolequinones with 4-nitrophenoxy, 4-pyridinyloxy, and acetoxy substituents at the (indol-3-yl)methyl position were NADH-dependent inhibitors of recombinant human NQO1, indicative of mechanism-based inhibition. However, those with hydroxy and phenoxy substituents were poor inhibitors of NQO1 enzyme activity, due to attenuated elimination of the leaving group. The ability of this series of indolequinones to inhibit recombinant human NQO1 correlated with NQO1 inhibition in MIA PaCa-2 cells. The examination of indolequinone interactions in complex with NQO1 from computational-based molecular docking simulations supported the observed biochemical data with respect to NQO1 inhibition. The design of both NQO1-inhibitory and noninhibitory indolequinone analogues allowed us to test the hypothesis that NQO1 inhibition was required for growth inhibitory activity in MIA PaCa-2 cells. ES936 and its 6-methoxy analogue were potent inhibitors of NQO1 activity and cell proliferation; however, the 4-pyridinyloxy and acetoxy compounds were also potent inhibitors of NQO1 activity but relatively poor inhibitors of cell proliferation. In addition, the phenoxy compounds, which were not inhibitors of NQO1 enzymatic activity, demonstrated potent growth inhibition. These data demonstrate that NQO1 inhibitory activity can be dissociated from growth inhibitory activity and suggest additional or alternative targets to NQO1 that are responsible for the growth inhibitory activity of this series of indolequinones in human pancreatic cancer.     

Glycine-extended gastrin promotes the invasiveness of human colon cancer cells.

Colorectal cancers express significant amounts of immature glycine-extended gastrin (G-Gly) and G-Gly is able to stimulate cell proliferation in colonic cell lines and mucosa. Here we wished to investigate whether G17-Gly promote the invasiveness of LoVo human colonic cancer cells, a process which requires degradation of extracellular matrix by proteases and concomitant induction of cell migration. We confirmed that LoVo cells express gastrin and gastrin/CCK-B receptor mRNAs. We showed that these cells secrete matrix metalloproteinase (MMP)-1, -2, and -9. The function of MMP being to degrade components of extracellular matrix, they may thus favor cell migration. As compared to controls, G17-Gly (10(-7) to 10(-12) M) significantly enhanced about two to three times the LoVo cell migration through Matrigel, an artificial basement matrix barrier. Moreover, G17-Gly increased and gastrin/CCK-B receptor antagonists decreased MMP secretion in conditioned culture media of LoVo cells. Our findings show that physiological doses of incompletely processed form of gastrin induce the invasiveness of tumor cells in vitro and suggest a novel potential role for this peptide in the metastatic process of colonic cancers in vivo.     

Redd1 inhibits the invasiveness of non-small cell lung cancer cells

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Stimulation by somatostatin of dephosphorylation of membrane proteins in pancreatic cancer MIA PaCa-2 cell line

A membrane receptor and a cytosolic receptor for somatostatin were found in a human undifferentiated pancreatic cancer cell line (MIA PaCa-2). Binding of somatostatin to this membrane receptor activates dephosphorylation of a phosphotyrosyl-membrane protein whose phosphorylation was promoted by epidermal growth factor (EGF). Vanadate, a purported inhibitor of dephosphorylation, interferes with the action of somatostatin. These findings suggest a possible biochemical mechanism by which somatostatin may inhibit the growth of human pancreatic cancers.     

Sensitivity of human pancreatic carcinoma cell line (MIA PaCa-2) to Bisantrene and Theprubicin in vitro

Summary We tested the effect of Bisantrene (BS) and Theprubicin (THP-ADR) on cell growth of a human pancreatic carcinoma cell line (MIA PaCa-2). After 1 h exposure ID₅₀ of BS or THP-ADR was 3×10⁻⁷ and 5×10⁻⁸ M, respectively. Increasing the exposure time from 1 h to continuous exposure for 5d resulted in 11-fold decrease in ID₅₀ for BS and a 6-fold decrease for THP-ADR. Both drugs inhibited [¹⁴C]thymidine incorporation to the same extent and caused an accumulation of cells into G₂+M phase of the cell cycle. This work is supported by a gift, from Mr. Issam Fares and U.S. Public Health Service Grants AM 07114 and CA 19182.     

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S‐phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27‐mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27‐overexpressing cells displayed an early S‐phase arrest subsequently followed by a strongly increased sub‐G1 fraction. Apoptosis was characterized by PARP‐, CASPASE 3‐, CASPASE 8‐, CASPASE 9‐ and BIM‐ activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock‐induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27‐overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor‐targeting agents, suggesting another pro‐apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well‐established anti‐apoptotic properties of HSP27 in cancer, our study reveals novel pro‐apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.