Exposure Of Pancreatic Cancer Cells Research Articles

Biologically optimized schedule of gemcitabine and nab-paclitaxel regimen in metastatic pancreatic adenocarcinoma.

TPS4168 Background: Metastatic pancreatic adenocarcinoma has a poor prognosis, and improvements in therapy have been challenging. Alongside efforts in developing novel agents, there is a need to optimize and maximize the benefit of currently approved drugs. Gemcitabine + nab-paclitaxel is a frequently used regimen for pancreatic adenocarcinoma. Nab-paclitaxel is albumin–bound chemotherapy; hence the role of albumin uptake is critical for its effect. Caveolae are small membrane invaginations essential for transendothelial albumin uptake. Cav-1 is the principal structural component of caveolae. Williams and colleagues have published a series of preclinical studies demonstrating that tumor cell-specific Cav-1 expression directly correlates with albumin and albumin-bound chemotherapy uptake and subsequent apoptotic response in tumor cells. In vitro studies showed that exposure of pancreatic cancer cells to Gemcitabine resulted in up-regulation of Cav-1 peaking 48 hours after gemcitabine exposure. This Cav-1 up-regulation correlated with increased temporal albumin cellular uptake. In addition, Williams and colleagues noted that exposure of pancreatic cancer cell lines to Gemcitabine resulted in a time–specific re-entry of cells into the G2/M phase (nab-paclitaxel cytotoxicity phase) between 48-60 hours after gemcitabine treatment. Collectively this data suggest that infusing nab-paclitaxel after 48 hours of gemcitabine infusion would be optimal for both increased uptake as well as increased susceptible tumor cells. We had previously shown this effect on multiple cell lines as well as mouse models. Methods: This is a phase II trial; patients will receive a standard of care chemotherapy regimen consisting of FDA-approved Gemcitabine + nab-paclitaxel with modification of the schedule to deliver nab-paclitaxel 48 hours (2 days) after gemcitabine infusions. The primary endpoint is ORR, with a null hypothesis of 20% vs. a target of 35%. Employing a 2-stage design (minimax) and assuming 80% power and a 0.05 significance level, a total of 53 patients will be required. In the first stage, if at least 7/31 patients respond to therapy, an additional 22 patients will be added for a total of 53 patients. The study will be terminated early if ≤ six patients respond in the first stage. Observation of response in at least 16/53 patients would be required to warrant further investigation of this infusion schedule of combination therapy. The secondary endpoints include the safety of the regimen schedule, Relative dose intensity, disease control rate, PFS, and OS. The trial opened to enrollment in June 2020 and is accepting patients. Clinical trial information: NCT04115163.

A Polymeric Nanogel-Based Treatment Regimen for Enhanced Efficacy and Sequential Administration of Synergistic Drug Combination in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. A combination of cisplatin (CDDP) and gemcitabine (Gem) treatment has shown favorable clinical results for metastatic disease; both are limited by toxicities and nontargeted delivery. More than 80% of PDAC aberrantly expresses the sialyl Tn (STn) antigen due to the loss of function of the core 1β3-Gal-T-specific molecular chaperone, a specific chaperone for the activity of core 1 β3-galactosyltransferase or C1GalT. Here, we report the development of polymeric nanogels (NGs) loaded with CDDP and coated with an anti-STn antigen-specific antibody (TKH2 monoclonal antibody) for the targeted treatment of PDAC. TKH2-functionalized, CDDP-loaded NGs delivered a significantly higher amount of platinum into the cells and tumors expressing STn antigens. We also confirmed that a synergistic cytotoxic effect of sequential exposure of pancreatic cancer cells to Gem followed by CDDP can be mimicked by the codelivery of CDDP-loaded NGs (NG/CDDP) and free Gem. In a murine orthotopic model of PDAC, combined simultaneous treatment with Gem and targeted NG/CDDP significantly attenuated tumor growth with no detectable acute toxicity. Altogether, these results suggest that combination therapy consisting of Gem followed by TKH2-conjugated CDDP NGs induces highly synergistic therapeutic efficacy against pancreatic cancer. Our results offer the basis for development of combination drug regimens using targeted nanomedicines to increase treatment effectiveness and improve outcomes of PDAC therapy.

Continuous exposure of pancreatic cancer cells to dietary bioactive agents does not induce drug resistance unlike chemotherapy

The repeated treatment of cancer cells with chemo- or radiotherapy induces therapy resistance, but it was previously unknown whether the same effect occurs upon continuous exposure of cancer cells to diet-derived chemopreventive agents. We elucidated this interesting question in pancreatic ductal adenocarcinoma, which is a highly aggressive cancer entity with a marked resistance toward gemcitabine and other cytotoxic drugs. The isothiocyanate sulforaphane, present in cruciferous vegetables, and the polyphenol quercetin, present in many fruits and vegetables induced apoptosis and reduced viability in gemcitabine-sensitive BxPC-3 cells but not in non-malignant ductal pancreas cells and mesenchymal stromal cells. In turn, BxPC-3 cells were treated with increasing concentrations of gemcitabine, sulforaphane or quercetin for more than 1 year and the surviving subclones Bx-GEM, Bx-SF and Bx-Q were selected, respectively. While Bx-GEM cells acquired a total resistance, Bx-SF or Bx-Q cells largely kept their sensitivity as proved by MTT assay, annexin staining and FACS analysis. The evaluation of the self-renewal-, differentiation- and migration-potential by colony formation, differentiation or migration assays demonstrated that cancer stem cell features were enriched in gemcitabine-resistant cells, but decreased in sulforaphane- and quercetin-long time-treated cells. These results were confirmed by orthotopic xenotransplantation of cancer cells to the mouse pancreas, where Bx-GEM formed large, Bx-Q small and Bx-SF cells almost undetectable tumors. An mRNA expression profiling array and subsequent gene set enrichment analysis and qRT-PCR confirmed that tumor progression markers were enriched in Bx-GEM, but reduced in Bx-SF and Bx-Q cells. This study demonstrates that the continuous exposure of pancreatic cancer cells to sulforaphane or quercetin does not induce resistance in surviving cells but reduces tumorigenicity by inhibition of tumor progression markers. These results highlight that cancer cells may not adapt to the preventive and therapeutic effects of a regular fruit- and vegetable-based diet.

Antitumor DNA‐Damaging C‐1748 is a New Inhibitor of Autophagy that Triggers Apoptosis in Human Pancreatic Cancer Cell Lines

Despite the enormous progress that has been made over the past decades in diagnosis, treatment and prevention of many types of tumors, survival rates in pancreatic cancer still remain poor. Pancreatic cancer is one of the most malignant and chemoresistant tumors and the profound mechanism supporting these phenomena is the constitutively activated prosurvival autophagy. The antitumor 1‐nitroacridine derivative C‐1748 (9‐(2′‐hydroxyethylamino)‐4‐methyl‐1‐nitroacridine) belongs to a new set of DNA‐damaging agents developed in our laboratory and is currently ready for phase I clinical trials. Here, we have uncovered a new role for C‐1748 as a potent inhibitor of autophagy in four tested pancreatic cancer cell lines: Panc‐1, MiaPaCa‐2, BxPC‐3 and AsPC‐1. C‐1748 treatment at 0.1 – 1 μM significantly reduced the typical hallmarks of autophagy, such as conversion of soluble LC3‐I protein to its lipid‐bound LC3‐II isoform or development of acidic vesicular organelles (AVOs), and importantly shifted pancreatic cancer cells into apoptotic cell death. Co‐treatment with C‐1748 and wortmannin, an early‐stage autophagy inhibitor, had a synergistic effect on autophagy inhibition and potentiated C‐1748‐induced apoptosis. In particular, compared with exposure to C‐1748 alone, the combination treatment increased time‐ and dose‐dependent cytotoxic activity of C‐1748 with the most profound effect observed in BxPC‐3 cells. Moreover, along with decrease in autophagy, co‐treatment with wortmannin significantly elevated dysfunction of mitochondria, as manifested by a drop in mitochondrial membrane potential (ΔΨm), increased caspase‐3 activation, PARP cleavage, DNA fragmentation into apoptotic bodies and accumulation of lipid droplets arising due to the interruption of autophagy. In all studied cell lines, the level of apoptotic cells after dual exposure with C‐1748 and wortmannin increased by about 20% as compared to C‐1748 alone. Surprisingly, simultaneous exposure of pancreatic cancer cells to C‐1748 in combination with the late‐stage autophagy inhibitor, chloroquine, neither significantly increased the cytotoxic activity of C‐1748 nor ameliorated its apoptosis‐inducing potential. The synergistic effect of C‐1748 and wortmannin on the inhibition of autophagy and lack of such effect when C‐1748 was co‐administered with chlroquine, suggests that C‐1748, in addition to its DNA‐damaging properties may also affects early stages of autophagy. Further studies are undergoing to uncover the role of C‐1748 as a new inhibitor of autophagy, which is especially effective against pancreatic cancer.Support or Funding InformationResearch Grant from the Polish Ministry of Science and Higher Education NR 13‐0133‐10/2011 to BB‐M, AS, EA, ZM; DoD‐W81XWH1110795 to AR‐P and UAMS TRI ABCRP funds to AR‐P

Cytotoxic effects exerted by pentachlorophenol by targeting nodal pro-survival signaling pathways in human pancreatic cancer cells

Pancreatic adenocarcinoma is one of the deadliest human solid tumors in the developed countries characterized by high resistance toward chemotherapeutic treatment. We have previously shown that silencing of the pro-survival protein kinase CK2 by RNA interference contributes to enhance the cytotoxicity of the chemotherapeutic agent 2′,2′-difluoro 2′-deoxycytidine (gemcitabine). Initial experiments showed that pentachlorophenol (PCP) inhibits CK2 and induces cell death in human pancreatic cancer cell lines. We report here evidence that exposure of this type of cells to PCP induces caspase-mediated apoptosis, inhibition of the lysosome cysteine protease cathepsin B and mitochondrial membrane depolarization. Beside cellular inhibition of CK2, the analysis of signaling pathways deregulated in pancreatic cancer cells revealed that PCP causes decreased phosphorylation levels of NF-κB/p65, suppresses its nuclear translocation and leads to activation of JNK-mediated stress response. Surprisingly, exposure to PCP results in increased phosphorylation levels of AKT at the canonical S473 and T308 activation sites supporting previous data showing that AKT phosphorylation is not predictive of tumor cell response to treatment. Taken together, our study provides novel insights into the effects induced by the exposure of pancreatic cancer cells to chlorinated aromatic compounds posing the basis for more advanced studies in vivo.

Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signalling pathway in human pancreatic cancer cells

BackgroundEpidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated.MethodsThe human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro.ResultsMetformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPKThr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPKSer485 phosphorylation and impaired AMPKThr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin.ConclusionOur results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPKThr172 activation and suppression of the insulin/IGF signalling pathways. However, hyperglycaemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile and prevent metformin from fully switching off the growth promoting signals in pancreatic cancer cells.

Garlic‐derived organosulfur compound diallyl trisulfide induces apoptosis in pancreatic cancer cells

Epidemiological observations have shown an inverse association between consumption of Allium vegetables (e.g., onions and garlic) and pancreatic cancer risk. The cancer preventive effect of Allium vegetables has been attributed to organosulfur compounds. The objective of this study was to determine the efficacy of diallyl trisulfide (DATS), a garlic‐derived organosulfur compound, against pancreatic cancer cells. Treatment with DATS inhibited the growth of MIAPaca2 and PL45 pancreatic cancer cells in a dose‐dependent manner. DATS induced apoptosis in pancreatic cancer cells as determined by cytoplasmic‐associated DNA fragmentation assay after exposure to DMSO (control) or DATS (20, 40, and 60 μmol/L) for 24 h. Exposure of pancreatic cancer cells to DATS increased levels of cleaved caspase 3 and induced proteolytic cleavage of poly‐(ADP‐ribose) polymerase (PARP). In addition, DATS treatment upregulated the expression of pro‐apoptotic Bim‐s and Bim‐L isoforms in MIAPaca 2 and PL45 cells. In conclusion, this study shows that DATS induces apoptotic cell death in pancreatic cancer cells and supports further investigation of DATS as a chemopreventive agent for pancreatic cancer.

Abstract 5156: Hyperglycemia enhances IGF-I responses and alters the AMPK activation profile that protects against metformin-induced growth inhibition in pancreatic cancer cells

Abstract Epidemiological studies have shown direct associations between type 2 diabetes (T2D) and obesity, both conditions associated with hyperglycemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients are diagnosed with either impaired glucose tolerance or new-onset T2D one or two years prior to their cancer diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin, an insulin-sensitizing and glucose lowering drug. We investigated the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin. We found that metformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions, but that hyperglycemia protected against the metformin-induced growth inhibition. The antiproliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPK(T172) together with a dose-dependent suppression of the insulin/insulin-like growth factor (IGF) signaling mediators IRS-1 and phosphorylated AKT. Furthermore, exposure to metformin during normal glucose led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose promoted a more robust IGF-I response and AKT activation, that stimulated AMPK(S485) phosphorylation and suppressed AMPK(T172) phosphorylation resulting in reduced antiproliferative and apoptotic effects by metformin. In conclusion, our results indicate that metformin has direct antitumor activities in pancreatic cancer cells involving AMPK(T172) activation and modulation of the insulin/IGF signaling pathways. However, hyperglycemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile that prevent metformin from fully switching of the growth promoting signals in pancreatic cancer cells. 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 5156. doi:1538-7445.AM2012-5156

Paradoxical cross-talk between the Stat3 and MAPK pathways in CCL25-CCR9 mediated pancreatic cancer growth and proliferation.

231 Background: Chemokine receptors have been shown to regulate the progression of several malignancies. Signal transducer and activator of transcription 3 (Stat3) may contribute to the invasive phenotype of several human cancers, but its upstream signals have not been well characterized. Our objective was to investigate the relationship between the CCL25-CCR9 axis and Stat3 signaling in pancreatic cancer cells. Methods: We exposed two established human pancreatic cancer cell lines PANC-1 and MIAPaCa-2 to the cytokine CCL25 (800 ug/uL for 20 min) and measured the activation of Stat3 (phosho-Stat3) by Western blot assay. Stattic, a small molecule Stat3 inhibitor, was used (20uM) to antagonize Stat3 signaling. We also measured activation level of extracellular signal-regulated kinase (phospho-ERK) following CCL25 exposure and used UO126 (10uM), a small molecule MEK inhibitor, to antagonize the MAPK pathway. Changes in cell proliferation were measured by CellTiter Glo Fluorescence assay. Results: Constitutive phosphorylation of Stat3 was observed in both pancreatic cancer cell lines. Exposure of MIAPaCa-2 to CCL25 further increased phospho-Stat3 levels on Western blot assay. We also observed a concomitant increase in phospho-ERK levels with exposure to CCL25 in both cell lines. Exposure of pancreatic cancer cells to CCL25 significantly increased cell proliferation. To determine the mechanism of CCR9-mediated cell proliferation, we used stattic and UO126 to specifically inhibit Stat3 and MEK activation, respectively. Interestingly, pre-treatment with stattic prior to CCL25 exposure resulted in a paradoxical enhancement of phospho-ERK levels. Conversely, inhibition of the MAPK pathway with UO126 led to a paradoxical enhancement of phospho-Stat3 levels. Conclusions: Our results demonstrate that CCL25 activates Stat3 and MAPK pathways to contribute to pancreatic cancer proliferation. We also show potential cross-talk between Stat3 and MAPK pathways, wherein antagonism of one pathway resulted in paradoxical activation of the other pathway. Our findings suggest that therapeutic targeting of downstream pathways may require a multi-drug approach.

Abstract 592: Novel IAP antagonist (822B) induces apoptosis through degradation of IAP proteins which have a BIR3 domain in human pancreatic cancer cells

Abstract Several companies recently began to develop inhibitor of apoptosis (IAP) antagonist ‘Smac/Diablo mimetic’ compounds for the treatment of cancer. IAP antagonist compounds (IACs) was, therefore, designated as being able to induce apoptosis through the inhibition of IAP proteins in cancer cells. In our study we demonstrated that the novel-in what sense is it synthetic IAP antagonist, 822B, induces apoptotic cell death through proteasomal-dependent degradation of the inhibitor of apoptosis proteins (IAPs) with a BIR3 domain. We first found that apoptotic cell death is induced by exposure of pancreatic cancer cells to 822B. This apoptotic effect increased after the knockdown of endogenous IAPs using RNA interference. Moreover, 822B-induced apoptosis results from degradation of the inhibitor of apoptosis proteins (IAPs). 822B was preferable to Samc/Diablo for binding to IAPs and it induced ubiquitination. However, we also found that degradation of XIAP is not induced in several pancreatic cancer cell types in response to 822B. Interestingly, resistance to 822B was related to phosphorylation at the ser-87 site of XIAP. This resistant effect was eliminated using the (or “an” if there's more than one . . .) XIAP mutant that was replaced with alanine at 87 amino acid site. AKT CA (constitutive active) mutant and wild type-AKT that can induce phosphorylation at the ser-87 site of XIAP also suppressed the resistant effect to 822B. In addition, mutation in the RING domain of XIAP was not observed. Mutation in the RING domain of XIAP cannot induce XIAP ubiquitination, thus suggesting that the level of phospho-XIAP and phospho-AKT may be used as an indicator for cancer therapy using 822B. These results suggest that a novel synthetic inhibitor of the apoptosis (IAP) antagonist induces apoptosis through ubiquitination of IAPs with a BIR3 domain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 592. doi:10.1158/1538-7445.AM2011-592

Inhibition of the mammalian target of rapamycin (mTOR) in advanced pancreatic cancer: results of two phase II studies

BackgroundThe phosphoinositide 3-kinase (PI3K)/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR) kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop). The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models.MethodsTwo studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN.ResultsFive patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively). One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2). Grade 4 toxicity: hyponatremia (n = 1), Grade 3: diarrhea (n = 1), cholangitis (n = 3), hyperglycemia (n = 1), fatigue (n = 1). Grade 2: pneumonia (n = 2), dehydration (n = 2), nausea (n = 2), neutropenia (n = 1), mucositis (n = 2) & rash (n = 2). Four patients were hospitalized. Progressive disease occurred in 15 and 1 was non-evaluable. Pretreatment biopsies revealed a higher pAkt/Akt ratio in tumor specimens that in nonmalignant pancreatic tissue. No such trends were noted for the other biomarkers.ConclusionsNeither study with mTOR inhibitors demonstrated objective responses or disease stability. The negative feedback loop resulting from mTOR inhibition may account for the disease progression and toxicity noted in these studies. Future strategies should aim for a broader targeting of the PI3K pathway in pancreatic cancer.Trial RegistrationTrial registration: Study A: NCT 0075647. Study B: NCT00640978

Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.

Antitumor Activity of Gemcitabine and Oxaliplatin Is Augmented by Thymoquinone in Pancreatic Cancer

Previous studies have shown biological activity of thymoquinone, an active compound extracted from Nigella sativa, in pancreatic cancer cells; however, preclinical animal studies are lacking. Here, we report, for the first time, the chemosensitizing effect of thymoquinone to conventional chemotherapeutic agents both in vitro and in vivo using an orthotopic model of pancreatic cancer. In vitro studies revealed that preexposure of cells with thymoquinone (25 mumol/L) for 48 h followed by gemcitabine or oxaliplatin resulted in 60% to 80% growth inhibition compared with 15% to 25% when gemcitabine or oxaliplatin was used alone. Moreover, we found that thymoquinone could potentiate the killing of pancreatic cancer cells induced by chemotherapeutic agents by down-regulation of nuclear factor-kappaB (NF-kappaB), Bcl-2 family, and NF-kappaB-dependent antiapoptotic genes (X-linked inhibitors of apoptosis, survivin, and cyclooxygenase-2). As shown previously by our laboratory, NF-kappaB gets activated on exposure of pancreatic cancer cells to conventional chemotherapeutic agents; interestingly, thymoquinone was able to down-regulate NF-kappaB in vitro, resulting in chemosensitization. In addition to in vitro results, here we show for the first time, that thymoquinone in combination with gemcitabine and/or oxaliplatin is much more effective as an antitumor agent compared with either agent alone. Most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in animal tumors pretreated with thymoquinone followed by gemcitabine and/or oxaliplatin. These results provide strong in vivo molecular evidence in support of our hypothesis that thymoquinone could abrogate gemcitabine- or oxaliplatin-induced activation of NF-kappaB, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics.

Loss of WISP-2/CCN5 signaling in human pancreatic cancer: A potential mechanism for epithelial-mesenchymal-transition

The objective of this study was to explore the pathophysiological relevance of WISP-2/CCN5 in progression of human pancreatic adenocarcinoma (PAC). We found WISP-2/CCN5 mRNA and protein expression was faint and sporadic in PAC and detected in only 8.7–20% of the samples with varying grades as compared to adjacent normal and chronic pancreatitis samples where expression was very high in the ducts and acini. Colocalization studies in tissue-microarray slides revealed WISP-2/CCN5 mRNA loss was associated with p53 overexpression in PAC. Like tissue samples, p53 mutant-PAC cell lines show loss of WISP-2/CCN5. Moreover, functional analysis studies demonstrate exposure of pancreatic cancer cells to WISP-2/CCN5 recombinant protein enhances mesenchymal–epithelial-transition (MET). Collectively, we suggest WISP-2/CCN5 silencing may be a critical event during differentiation and progression of PAC and mutant p53 is possibly an important player in pursuing this episode.

Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions.

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.