Inhibition of Survival Pathways MAPK and NF-kB Triggers Apoptosis in Pancreatic Ductal Adenocarcinoma Cells via Suppression of Autophagy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a survival rate of 4-6 months from diagnosis. PDAC is the fourth leading cause of cancer-related death in the Western world, with a mortality rate of 10 cases per 100,000 population. Chemotherapy constitutes only a palliative strategy, with limited effects on life expectancy. AIMS: To investigate the biological response of PDAC to mitogen-activated protein kinase (MAPK) and NF-kappaB (NF-kB) inhibitors and the role of autophagy in the modulation of these signaling pathways in order to address the challenge of developing improved medical protocols for patients with PDAC. METHODS: Two ATCC cell lines, MIAPaCa-2 and PANC-1, were used as PDAC models. Cells were exposed to inhibitors of MAPK or NF-kB survival pathways alone or after autophagy inhibition. Several aspects were analyzed, as follows: cell proliferation, by [(3)H]TdR incorporation; cell death, by TUNEL assay, regulation of autophagy by LC3-II expression level and modulation of pro-and anti-apoptotic proteins by Western blot. RESULTS: We demonstrated that the inhibition of the MAPK and NF-kB survival pathways with U0126 and caffeic acid phenethyl ester (CAPE), respectively, produced strong inhibition of pancreatic tumor cell growth without inducing apoptotic death. Interestingly, U0126 and CAPE induced apoptosis after autophagy inhibition in a caspase-dependent manner in MIA PaCa-2 cells and in a caspase-independent manner in PANC-1 cells. CONCLUSIONS: Here we present evidence that allows us to consider a combined therapy regimen comprising an autophagy inhibitor and a MAPK or NF-kB pathway inhibitor as a possible treatment strategy for pancreatic cancer.

Synergism of arsenic trioxide and MG132 in Raji cells attained by targeting BNIP3, autophagy, and mitochondria with low doses of valproic acid and vincristine.

We previously demonstrated that arsenic trioxide (ATO) and proteasome inhibitor MG132 synergistically induced cell death in promonocytic leukaemia cell line U937 but were antagonistic in Burkitt's lymphoma cell line Raji. Here we explore the role of autophagy, expression of BNIP3, and mitochondrial mass, in determining whether ATO and MG132 interaction can be shifted from antagonism to synergism in Raji cells. Treatment with ATO+MG132 increased the percentage of cells with collapsed mitochondrial membrane potential (MMP) in U937 cells, but had no effect in Raji cells. Mitochondria were found in cytoplasmic marginal location in U937 cells but at perinuclear location in Raji cells. ATO+MG132 increased mitochondrial mass in U937 cells but decreased it in Raji cells, while autophagy was increased in both cell lines. BNIP3 was expressed in U937 cells at cytoplasmic marginal locations and was hardly detected in Raji cells. Histone deacetylase (HDAC) inhibitor valproic acid (VPA) increased expression of BNIP3 in Raji cells at perinuclear locations. However antagonism between ATO and MG132 was increased in the presence of low doses of VPA. Addition of vincristine (VCR) blocked autophagy, while VPA+VCR treatment of Raji cells at sub-cytotoxic doses caused BNIP3 and mitochondria to redistribute to cytoplasmic peripheral location and increased mitochondrial mass. ATO+MG132 in the presence of subcytotoxic doses of VPA+VCR caused collapse of MMP in Raji cells, while interaction between ATO and MG132 shifted from antagonism to synergism. We conclude that synergism between ATO and MG132 was attained in Raji cells by disruption of the perinuclear mitochondrial cluster, blockage of selective autophagy of mitochondria (mitophagy) by VCR, increased mitochondrial mass, and upregulation of BNIP3 by VPA.

Interplay between autophagy and apoptosis in pancreatic tumors in response to gemcitabine.

Pancreatic cancer is an aggressive disease. Its incidence has increased over the last two decades. It is currently the fourth cause of death among cancers in the western world. Unfortunately, systemic chemotherapy still relies on just a few drugs which until now have produced unsatisfactory results. Gemcitabine (2'-2'-difluorodeoxycytidine) is currently the standard chemotherapy treatment at all stages of pancreatic adenocarcinoma. Survival benefit and clinical impact however remain moderate due to a high degree of intrinsic and acquired resistance. Autophagy plays an important role in cell death decision but can also protect cells from various apoptotic stimuli. We investigated the function of autophagy in pancreatic carcinoma cells, which are frequently insensitive to standard chemotherapeutic agents. Here, we demonstrate that autophagy is one of the mechanisms responsible for the refractory response of pancreatic tumors to gemcitabine. We present evidence in vitro and in vivo that proves autophagy plays a protective role in pancreatic ductal carcinoma cells, preventing them from entering the apoptotic pathway after stimulus with gemcitabine, thus contributing to treatment resistance. A better understanding of the role in the process may help in the discovery of new strategies to overcome tumor drug resistance in this aggressive disease.

Caffeic acid phenylethyl ester and MG132, two novel nonconventional chemotherapeutic agents, induce apoptosis of human leukemic cells by disrupting mitochondrial function.

The ability to modulate balance between cell survival and death is recognized for its great therapeutic potential. Therefore, research continues to focus on elucidation of cell machinery and signaling pathways that control cell proliferation and apoptosis. Conventional chemotherapeutic agents often have a cytostatic effect over tumor cells. New natural or synthetic chemotherapeutic agents have a wider spectrum of interesting antitumor activities that merit in-depth studies. In the present work, we aimed at characterizing the molecular mechanism leading to induction of cell death upon treatment of the lymphoblastoid cell line PL104 with caffeic acid phenylethyl ester (CAPE), MG132 and two conventional chemotherapeutic agents, doxorubicine (DOX) and vincristine (VCR). Our results showed several apoptotic hallmarks such as phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane, nuclear fragmentation, and increase sub-G1 DNA content after all treatments. In addition, all four drugs downregulated survivin expression. CAPE and both chemotherapeutic agents reduced Bcl-2, while only CAPE and MG132 significantly increased Bax level. CAPE and VCR treatment induced the collapse of mitochondrial membrane potential (∆ψm). All compounds induced cytochrome c release from mitochondrial compartment to cytosol. However, only MG132 caused the translocation of Smac/DIABLO. Except for VCR treatment, all other drugs increased reactive oxygen species (ROS) production level. All treatments induced activation of caspases 3/7, but only CAPE and MG132 led to the activation of caspase 9. In conclusion, our results indicate that CAPE and MG132 treatment of PL104 cells induced apoptosis through the mitochondrial intrinsic pathway, whereas the apoptotic mechanism induced by DOX and VCR may proceed through the extrinsic pathway.

The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav.) Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm) and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion.

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O2⁻) levels. Our results showed that combined arsenite+MG132 produced low levels of O2⁻ at 6h and 24h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80µM hydrogen peroxide together with arsenite+MG132 changed synergism on cell death to antagonism at all effect levels while increasing O2⁻ levels. Arsenite+hydrogen peroxide also resulted in antagonism with increased O2⁻ levels in U937 cells. In Raji cells, arsenite+MG132 also produced low levels of O2⁻ at 6h and 24h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite+CAPE showed high levels of O2⁻ production at 6h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O2⁻ levels at early time points after exposure.

Haemostatic and immune role of cellular clotting in the sipunculan Themiste petricola.

Sipunculans, a small phylum of coelomated marine worms closely related to polychaete annelids, lack a true circulatory system. We have previously shown that the sipunculan Themiste petricola can form a cellular clot, without congealing, of cell-free coelomic fluid. The clot is formed by the aggregation of large granular leukocytes (LGLs) and may serve not only haemostatic but immune functions, since dissimilar particles may become entrapped within it. We have now evaluated the capacity of a massive clot, induced in vitro by sea water contact, to stop coelomic fluid flow. We have further studied smaller clots induced on glass-slides either with or without the presence of bacteria placed for entrapment within the clot. The fate of clotting LGLs is cell death while forming a cohesive mass, although cytoplasmic and nuclear remnants are shed from the clot. These remnants and any bacteria that avoid clot entrapment or are detached from the clot are engulfed by non-clotting cells that include small granular leukocytes (SGLs) and large hyaline amebocytes (LHAs). Both cell types can be found other than in the clot but SGLs also occur around the clot edges heavily loaded with engulfed material. The cytoskeletal arrangement of SGLs evaluated with phalloidin-rhodamine correspond to motile cells and contrast with that of clotting LGLs that form a massive network of F-actin. Thus, the complementary roles between clotting LGLs and non-clotting SGLs and LHAs act a central immune strategy of Themiste petricola to deal with body wall injury and pathogen intrusion into the coelomic cavity.

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells.

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor kappaB (NF-kappaB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-kappaB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-kappaB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.

Ligaria cuneifolia flavonoid fractions modulate cell growth of normal lymphocytes and tumor cells as well as multidrug resistant cells.

Flavonoids are ubiquitous compounds present in plant extracts. They represent a major active component of the plant extract and are often known for their anti-inflammatory and anti-tumor effects. Previously, we demonstrated that Ligaria cuneifolia (R et P) Tiegh. (Loranthaceae) extracts inhibit proliferation of murine mitogen-activated lymphocytes as well as murine T leukaemia (LB) and breast tumor cells (MMT). The aim of this study was to assess the anti-proliferative and pro-apoptotic activities of three separate flavonoid fractions derived from L. cuneifolia whole extract (aqueous, methanolic and ethyl acetate) on normal and tumor cells. This was performed as a bio-guided approach leading to the isolation and identification of the active compounds responsible for the effects observed with the whole extract. Results showed that the three fractions differed in the amount and type of compounds found. Only the ethyl acetate flavonoid fraction (100 microg/ml) was able to inhibit significantly the proliferation of Con A stimulated splenocytes or LB and MMT cells. Inhibition of proliferation was mediated by apoptosis as determined by morphology and DNA hypodiploidy. The ethyl acetate fraction modified mRNA expression of IL-2, IL-10 and TGF-beta, while the methanol fraction only modified IL-10 mRNA on LB cells. Our results show that the ethyl acetate flavonoid fraction contains the most active compound/s and is the potential candidate to isolate the active compound/s responsible for the effects observed with L. cuneifolia whole extract.

Modulation of matrix metalloproteinase-9 activity by hyaluronan is dependent on NF-kappaB activity in lymphoma cell lines with dissimilar invasive behavior.

Expression and activity of matrix metalloproteinase-9 (MMP-9) as well as its relationship with hyaluronan (HA) and NF-kappaB activity were analyzed in two murine lymphoma cell lines with dissimilar migration and invasive behavior. MMP activity was evaluated by zymograms in supernatants, membrane extracts of tumor cells, and in the organs invaded by these cells. The more aggressive LBLa cell line showed MMP-9 activity in vitro, which increased after HA treatment and was blocked by anti-CD44 mAb. Such activity was not found in the less aggressive LBLc. MMP-9 and MMP-2 activity was found in organs invaded by both cell lines, although differential MMP-9 activity was observed in lung infiltrated only by LBLa cell line. NF-kappaB activation was evaluated to determine whether differential activity of MMP-9 was dependent on downstream signaling pathway, showing higher NF-kappaB activity in the more aggressive LBLa cell line. Our results showed that MMP-9 activity modulated by HA through NF-kappaB signaling pathway may be involved in the aggressive behavior of LBLa.