Chiara Costanzo

Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism

Gemcitabine (GEM, 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl-L-cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.

Trichostatin A, an inhibitor of histone deacetylases, strongly suppresses growth of pancreatic adenocarcinoma cells

In cells with an altered p53 gene, the expression of p21WAF1/CIP1, a potent inhibitor of cyclin‐dependent kinases, can be induced by histone deacetylase (HDAC) inhibitors via a p53‐independent pathway, which may play a critical role in arrest of cell growth. Accordingly, HDAC inhibitors such as trichostatin A (TSA) have potential utility in pancreatic cancer, as most of these tumors possess mutations in p53, which in fact is the main cause of chemoresistance to 5‐fluorouracil. We have analyzed the effect of TSA on the proliferation of nine pancreatic adenocarcinoma cell lines, all containing a mutated p53 gene. TSA strongly inhibited the cellular growth of all these cell lines at submicromolar concentrations. The cellular mechanisms underlying this effect consisted of cell cycle arrest at the G2 phase and apoptotic cell death. The expression of p21WAF1/CIP1 normally induced at the transcriptional level by p53 was also strongly activated by TSA. These findings suggest that inhibitors of HDAC may represent a novel therapeutic strategy for treatment of pancreatic cancer.

Hyaluronic acid-coated liposomes for active targeting of gemcitabine

The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine lipophilic prodrug [4-(N)-lauroyl-gemcitabine, C12GEM]. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors. For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800 Da (12 disaccharidic units) and 12,000 Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation. Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44(+)) and that the uptake increases with increase in the polymer molecular weight. Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes. Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand.

Trichostatin A enhances the response of chemotherapeutic agents in inhibiting pancreatic cancer cell proliferation

Pancreatic cancer is an aggressive neoplasia, and standard chemotherapies are by and large ineffective. The purpose of this work was to get a comprehensive preclinical study on the ability of anticancer drug combinations that best inhibit growth of pancreatic adenocarcinoma cells. We evaluated the in vitro growth inhibition of ten pancreatic cancer cell lines to gemcitabine and 5-fluorouracil, newer generation cytotoxic agents (oxaliplatin, irinotecan), targeted therapy (gefitinib) and a histone deacetylase (HDAC) inhibitor (trichostatin A). Cells were treated with the single drug alone and all pairwise drug association. Our results demonstrate that TSA can effectively increase the drug sensitivity of all the cell lines studied. The association of TSA and irinotecan determines an increase in growth inhibition on the highest percentage of cell lines (80%). Our findings may represent an experimental basis for potential clinical application of HDAC inhibitors, in particular in association with drugs used in cancer clinical treatment, supporting the idea that HDAC inhibitors could act as sensitizers for chemotherapy.

Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells.

The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.

UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells.

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.

Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.

Targeting gemcitabine containing liposomes to CD44 expressing pancreatic adenocarcinoma cells causes an increase in the antitumoral activity

Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.

MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-α, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2′-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions –666 to –426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.

SEQUENCES RELATED TO THE OX PANCREATIC RIBONUCLEASE CODING REGION IN THE GENOMIC DNA OF MAMMALIAN-SPECIES

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.