Human Pancreatic MIA PaCa-2 Cancer Research Articles

New Theonellapeptolides from Indonesian Marine Sponge Theonella swinhoei as Anti-Austerity Agents

We reported three new members of the theonellapeptolide family from theonellapeptolide II series, namely theonellapeptolides IIb (1), IIa (2), IIc (3), and three known members-IId (4), IIe (5), and Id (6)-from Kodingarengan marine sponge Theonella swinhoei collected in Makassar, Indonesia. The structures of tridecadepsipeptides 1-3, including the absolute configurations of their amino acids, were determined by the integrated NMR and tandem MS analyses followed by Marfey's analysis. To the best of our knowledge, 1 and 2 are the first theonellapeptolide-type compounds to have a valine residue with D configuration at residue position 6. The isolated theonellapeptolide-type compounds 1-6 showed selective cytotoxic activity against human pancreatic MIA PaCa-2 cancer cells in a nutrient-deprived medium. Among them, the most potent preferential cytotoxicity was observed in new theonellapeptolide IIc (3) and known IId (4), IIe (5), and Id (6).

A Metabolic Change towards Fermentation Drives Cancer Cachexia in Myotubes.

Cachexia is a disorder associated with several pathologies, including cancer. In this paper, we describe how cachexia is induced in myotubes by a metabolic shift towards fermentation, and the block of this metabolic modification prevents the onset of the cachectic phenotype. Cachectic myotubes, obtained by the treatment with conditioned medium from murine colon carcinoma cells CT26, show increased glucose uptake, decreased oxygen consumption, altered mitochondria, and increased lactate production. Interestingly, the block of glycolysis by 2-deoxy-glucose or lactate dehydrogenase inhibition by oxamate prevents the induction of cachexia, thus suggesting that this metabolic change is greatly involved in cachexia activation. The treatment with 2-deoxy-glucose or oxamate induces positive effects also in mitochondria, where mitochondrial membrane potential and pyruvate dehydrogenase activity became similar to control myotubes. Moreover, in myotubes treated with interleukin-6, cachectic phenotype is associated with a fermentative metabolism, and the inhibition of lactate dehydrogenase by oxamate prevents cachectic features. The same results have been achieved by treating myotubes with conditioned media from human colon HCT116 and human pancreatic MIAPaCa-2 cancer cell lines, thus showing that what has been observed with murine-conditioned media is a wide phenomenon. These findings demonstrate that cachexia induction in myotubes is linked with a metabolic shift towards fermentation, and inhibition of lactate formation impedes cachexia and highlights lactate dehydrogenase as a possible new tool for counteracting the onset of this pathology.

Abstract 2765A: Co-treatment of human pancreatic cancer MiaPaCa-2 Cells with PNC-27 and Gemzar

Abstract Novel anti-cancer PNC-27 peptide, derived from the p53 binding domain of MDM2, induces necrosis of human pancreatic MiaPaCa-2 cancer cells but not normal cells in vitro, in a dose dependent fashion. Recent studies support a membranolytic mechanism of action for the peptide, initiated by binding of peptide monomers to the cells’ plasma membrane that subsequently aggregate to form transmembrane pores. Gemzar, on the other hand, is an established, non-selective chemotherapeutic used to treat pancreatic cancer. As a nucleoside analog, Gemzar inhibits DNA replication, resulting in cell cycle arrest, and ultimately apoptosis. Speculation regarding whether the two drugs, with different mechanisms, could produce a combined killing effect if administered simultaneously is investigated. Cell proliferation assays (MTT), cell cytotoxicity assays (LDH) and confocal microscopy were used to study the susceptibility of the MiaPaCa-2 cells to the co-treatment of PNC-27 and Gemzar. To determine the working concentration of PNC-27 that would result in pore formation but would not be cytotoxic to the cells, a dose response assay with serial dilutions was conducted. Results revealed that while MiaPaCa-2 cells treated with PNC-27 in DMEM have an LD50 = 150 ug/mL, the working concentration that would enable minimal pore formation without cell death was determined to be ∼10 ug/mL. Through confocal microscopy, it was then determined that propidium iodide (PI) (668.4 Da), a marker for dead cells and that is impermeable for intact cells, would nevertheless be able to enter the cell cytoplasm via PNC-27 pores. The results found PI staining in the cytoplasm but complete absence of nuclear staining. This observation not only indicates the sustained viability of the cells treated with 10 ug/mL PNC-27, but also supports the hypothesis that PI entered the cells through the PNC-27 induced pores. To explore a combined cytotoxic effect of Gemzar and PNC-27, MTT assays were carried out with Gemzar concentrations between 0 uM-100 uM for either 24 or 48 hours, in presence or absence of PNC-27. Our results demonstrate that co-treatment of MiaPaCa-2 cells with PNC-27 and Gemzar leads to a greater cytotoxic effect than with either Gemzar or PNC-27 alone. Additionally, the 48hr treatment resulted in a greater cytotoxic effect than did the 24hr condition. Our results indicate that the increased permeability of the cancer cells to Gemzar via the PNC-27-induced pores may allow for a more rapid mechanism of entry for Gemzar. Due to the selectivity of PNC-27, more Gemzar is able to target the cancer cells which have compromised membrane integrity caused by PNC-27. Therefore, co-treatment with Gemzar may allow for a dose reduction of Gemzar due to increased accessibility of the chemotherapeutic to its site of action. By reducing the dose of Gemzar required to achieve a significant cytotoxic effect, the severity of the side-effects of the drug experienced by patients may also be reduced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2765A. doi:1538-7445.AM2012-2765A

Hypoxia activates heparanase expression in an NF-κB dependent manner

Hypoxia was shown to increase tumor cell invasion into the extracellular matrix in vitro. This result suggests that heparanase (Hpa), one of the key enzymes involved in tumor invasion and metastasis, may be regulated by hypoxia. RT-PCR, Western blot and Matrigel invasive assays were used to study the regulation of Hpa under hypoxia in human pancreatic MIA PaCa-2 cancer cells. Compared with those in normoxia (20% O2), Hpa mRNA, protein and enzymatic activity levels, were up-regulated by a reduction in the oxygen level (1% O2). Invasion by tumor cells into the extracellular matrix was found to be significantly enhanced. A reduction in Hpa protein levels was observed when nuclear factor kappaB (NF-kappaB) activation was blocked by pyrrolidine dithiocarbamate. The levels of Hpa were also reduced when Hpa was inhibited by an Hpa-specific antisense oligonucleotide. The MMP-9 mRNA, protein and gelatinase B activity levels in supernatants decreased significantly when Hpa was inhibited. We conclude that up-regulation of Hpa by hypoxia is NF-kappaB-dependent in MIA PaCa-2 cells and inhibition of Hpa reduces MMP-9 activity. This reduction in MMP-9 activity may be an important mechanism in tumor metastasis.

Period is involved in the proliferation of human pancreatic MIA-PaCa2 cancer cells by TNF-α

Recent studies have found alterations to clock genes in several forms of cancer. Period (Per) gene plays an important role in the circadian system, the cell cycle, the induction of apoptosis, and DNA damage. However, the functions of Per in pancreatic cancer have not yet been elucidated. Here, we show that tumor necrosis factor-alpha (TNF-alpha) suppressed the expression of both Per1 and Per3 in MIA-PaCa2 cells, a human pancreatic cancer cell line. The levels of these proteins were 10% lower in the cells treated with 10 ng/mL TNF-alpha. Cell proliferation showed a 15%, 14%, and 16% decrease at 0.4, 2.0, and 10 ng/mL of TNF-alpha, respectively. In MTS-assays, MIA-PaCa2 cells transfected with siRNA against Per1 showed a 19% reduction in proliferation. However, the knockdown of Per3 did not significantly inhibit cell proliferation. The results suggest Per1 to be involved in the inhibition of the proliferation of MIA-PaCa2 cells by TNF-alpha.

Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth.

Geranylgeranyltransferase I inhibitors (GGTIs) are presently undergoing advanced preclinical studies and have been shown to disrupt oncogenic and tumor survival pathways, to inhibit anchorage-dependent and -independent growth, and to induce apoptosis. However, the geranylgeranylated proteins that are targeted by GGTIs to induce these effects are not known. Here we provide evidence that the Ras-like small GTPases RalA and RalB are exclusively geranylgeranylated and that inhibition of their geranylgeranylation mediates, at least in part, the effects of GGTIs on anchorage-dependent and -independent growth and tumor apoptosis. To this end, we have created the corresponding carboxyl-terminal mutants that are exclusively farnesylated and verified that they retain the subcellular localization and signaling activities of the wild-type geranylgeranylated proteins and that Ral GTPases do not undergo alternative prenylation in response to GGTI treatment. By expressing farnesylated, GGTI-resistant RalA and RalB in Cos7 cells and human pancreatic MiaPaCa2 cancer cells followed by GGTI-2417 treatment, we demonstrated that farnesylated RalB, but not RalA, confers resistance to the proapoptotic and anti-anchorage-dependent growth effects of GGTI-2417. Conversely, farnesylated RalA but not RalB expression renders MiaPaCa2 cells less sensitive to inhibition of anchorage-independent growth. Furthermore, farnesylated RalB, but not RalA, inhibits the ability of GGTI-2417 to suppress survivin and induce p27(Kip1) protein levels. We conclude that RalA and RalB are important, functionally distinct targets for GGTI-mediated tumor apoptosis and growth inhibition.

Inhibitor of nuclear factor-kappaB induction by cAMP antagonizes interleukin-1-induced human macrophage-colony-stimulating-factor expression.

We have recently reported that interleukin-1alpha (IL-1alpha) can induce human macrophage colony-stimulating factor (M-CSF) expression through nuclear factor kappaB (NF-kappaB) activation, and treatment of human pancreatic MIA PaCa-2 cancer cells with forskolin or cAMP attenuated the NF-kappaB activation as well as M-CSF expression. In this study, we have further investigated the mechanism of cAMP attenuation. MIA PaCa-2 cells were incubated with forskolin or dibutyryl-cAMP and then stimulated with IL-1 for 1 h. Cell lysates were immunoprecipitated by anti-inhibitory kappaB (IkappaB) kinase-beta (IKKbeta) antibody and the immune complex assayed for kinase activity using recombinant inhibitor of NF-kappaB (IkappaBalpha) as substrate. The levels of IKKbeta in the respective cellular proteins were measured by subsequent Western blot. The results show that the level of IKK protein remains constant in the presence of cAMP, forskolin and/or IL-1, whereas IKK activity was robustly stimulated by IL-1. Nonetheless, dibutyryl-cAMP or forskolin did not affect the IKK activation induced by IL-1. This experiment suggests that elevated cAMP has no effect on IKK activity. IkappaBalpha protein level decreased markedly in IL-1-treated cells compared with the untreated. By contrast, cells treated with cAMP or forskolin possessed discernibly higher IkappaBalpha levels. In addition, we observed that forskolin potentiated and prolonged the IL-1-induced IkappaBalpha mRNA levels, whereas it did not stabilize the IkappaBalpha mRNA message. Wholly, these studies indicate that elevated cAMP antagonizes IL-1-induced M-CSF transcription by up-regulating IkappaBalpha gene induction and its consequent attenuation of NF-kappaB activation.

Inhibitor of nuclear factor-κB induction by cAMP antagonizes interleukin-1-induced human macrophage-colony-stimulating-factor expression

We have recently reported that interleukin-1α (IL-1α) can induce human macrophage colony-stimulating factor (M-CSF) expression through nuclear factor κB (NF-κB) activation, and treatment of human pancreatic MIA PaCa-2 cancer cells with forskolin or cAMP attenuated the NF-κB activation as well as M-CSF expression. In this study, we have further investigated the mechanism of cAMP attenuation. MIA PaCa-2 cells were incubated with forskolin or dibutyryl-cAMP and then stimulated with IL-1 for 1h. Cell lysates were immunoprecipitated by anti-inhibitory κB (IκB) kinase-β (IKKβ) antibody and the immune complex assayed for kinase activity using recombinant inhibitor of NF-κB (IκBα) as substrate. The levels of IKKβ in the respective cellular proteins were measured by subsequent Western blot. The results show that the level of IKK protein remains constant in the presence of cAMP, forskolin and/or IL-1, whereas IKK activity was robustly stimulated by IL-1. Nonetheless, dibutyryl-cAMP or forskolin did not affect the IKK activation induced by IL-1. This experiment suggests that elevated cAMP has no effect on IKK activity. IκBα protein level decreased markedly in IL-1-treated cells compared with the untreated. By contrast, cells treated with cAMP or forskolin possessed discernibly higher IκBα levels. In addition, we observed that forskolin potentiated and prolonged the IL-1-induced IκBα mRNA levels, whereas it did not stabilize the IκBα mRNA message. Wholly, these studies indicate that elevated cAMP antagonizes IL-1-induced M-CSF transcription by up-regulating IκBα gene induction and its consequent attenuation of NF-κB activation.