PANC-1 Xenograft Research Articles

Brusatol Enhances the Chemotherapy Efficacy of Gemcitabine in Pancreatic Cancer via the Nrf2 Signalling Pathway

Although gemcitabine is the standard chemotherapy treatment for advanced pancreatic cancer, its benefits are quite limited due to prevalent chemoresistance, and the mechanism underlying gemcitabine chemoresistance remains unclear. Currently, Nrf2 has been deemed as a significant contributor to gemcitabine chemoresistance in pancreatic cancer. Brusatol is a unique inhibitor of the Nrf2 pathway, and in previous studies, we determined that brusatol exhibits the effects of growth inhibition and proapoptosis in pancreatic cancer cells. Due to these data, we speculate that brusatol can reverse gemcitabine-induced Nrf2 activation and propose that it can enhance gemcitabine efficacy in treating pancreatic cancer. In this study, we first proved that brusatol can effectively inhibit the Nrf2 signalling pathway and increase ROS accumulation in pancreatic cancer cells. Next, we demonstrated that brusatol can abrogate gemcitabine-induced Nrf2 activation in pancreatic cancer cells. In addition, we discovered that brusatol potentiates gemcitabine-induced growth inhibition and apoptosis in human pancreatic cancer cells. In nude mice with PANC-1 xenografts, treatment with a combination of brusatol and gemcitabine considerably reduced in vivo tumour growth compared with control treatment or treatment with either brusatol or gemcitabine alone. Immunohistochemical staining also showed that Nrf2 expression levels were reduced in brusatol-treated xenograft tumour tissues. In summary, our results suggest that brusatol is capable of enhancing the antitumour effects of gemcitabine in both pancreatic cancer cells and PANC-1 xenografts via suppressing the Nrf2 pathway.

Abstract A165: Novel thiosemicarbazone, Dp44mT, promotes NDRG1 to downregulate oncogenic signaling pathways in cancer

Abstract Introduction: The novel thiosemicarbazone, Dp44mT, has shown potent anticancer activity against various cancer types. The activity of this agent is, at least in part, mediated by its ability to upregulate the growth and metastasis suppressor, N-myc Downstream Regulated Gene 1 (NDRG1). NDRG1 inhibits cell proliferation, migration, and invasion by negatively regulating numerous oncogenic signaling pathways. However, the mechanisms by which NDRG1 modulates all these pathways remain to be elucidated. To examine how NDRG1 and Dp44mT affect multiple signalling pathways, we assessed their effects on the ErbB-family of receptor tyrosine kinases, namely epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), as these molecules are key regulators of downstream oncogenic signaling. Methods: PANC1 pancreatic and HT-29 colon human cancer cells were utilized to examine the effects of NDRG1 and Dp44mT on EGFR, HER2, and HER3 levels, localization, and phosphorylation in vitro. PANC1 xenografts were also utilized to examine the effects of Dp44mT on these molecules in vivo. Results: For the first time, we demonstrate that NDRG1 and Dp44mT markedly inhibit the expression, localization, and activation of EGFR, HER2, and HER3. NDRG1 also reduced activation of the MAPK-signalling pathway, which is downstream of the ErbB family of receptors. Further, the anticancer agent, Dp44mT, significantly reduced expression of EGFR, HER2, and HER3 in vivo. Conclusions: This study demonstrates for the first time that the novel thiosemicarbazone, Dp44mT, inhibits the ErbB family of proteins through its upregulation of NDRG1, providing an interesting insight into the mechanisms behind its compelling anticancer activity. Citation Format: Sharleen V. Menezes, Zaklina Kovacevic, Des Richardson. Novel thiosemicarbazone, Dp44mT, promotes NDRG1 to downregulate oncogenic signaling pathways in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A165.

Somatostatin receptor type 2 as a radiotheranostic PET reporter gene for oncologic interventions

Reporter gene systems can serve as therapy targets. However, the therapeutic use of reporters has been limited by the challenges of transgene delivery to a majority of cancer cells. This study specifically assesses the efficacy of targeting human somatostatin receptor subtype 2 (hSSTR2) with peptide receptor radionuclide therapy (PRRT) when a small subpopulation of cells bears the transgene.Methods: The hSSTR2 transgene was delivered to A549 and Panc-1tumors using the lentiviral vector, LV-hSSTR2-IRES-GFP or murine mesenchymal stem cells (mMSC)s using a retroviral vector. SSTR2 expression was assessed using Western blot and correlated to GFP fluorescence and 68Ga-DOTATOC uptake. Wild type (WT), transduced (TD), and mixed population A549 or Panc-1 xenografts were implanted in nude mice. Separate groups with A549WT and Panc-1WT tumors received intratumoral injection of SSTR2-expressing mMSCs. Tumor-bearing mice were treated with 90Y-DOTATOC or saline and evaluated with 68Ga-DOTATOC PET before and after treatment.Results: Cell studies showed a strong correlation between 68Ga-DOTATOC uptake and SSTR2 expression in A549 (p < 0.004) and Panc-1 cells (p < 0.01). 68Ga-DOTATOC PET SUVmean was 8- and 5-fold higher in TD compared to WT A549 and Panc-1 tumors, respectively (p < 0.001). After 90Y-DOTATOC treatment, 100% TD and mixed population TD xenografts showed growth cessation while the WT xenografts did not. A549WT and Panc-1WT tumors with SSTR2-expressing mMSCs treated with 90Y-DOTATOC showed significantly lower tumor volumes compared to controls (p < 0.05). 68Ga-DOTATOC PET SUVmean of treated TD tumors monotonically declined and was significantly lower than that of non-treated xenografts.Conclusions: We showed that SSTR2 delivery to a small population of cells in tumor in conjunction with PRRT is effective in tumor growth cessation. The availability of various transgene delivery methods for hSSTR2 and radiotherpaeutic somatostatin analogs highlights the direct translational potential of this paradigm in the treatment of various cancers.

Design, synthesis and biological evaluation of N-arylsulfonyl carbazoles as novel anticancer agents.

In this work, a set of structurally diverse synthetic carbazoles was screened for their anticancer activities. According to structure–activity relationship studies, carbazoles with an N-substituted sulfonyl group exhibited better anticancer activity. Moreover, compound 8h was discovered to show the most potent anticancer effects on Capan-2 cells by inducing apoptosis and cell cycle arrest in G2/M phase. Finally, the in vivo study demonstrated that 8h prevented the tumor growth in PANC-1 and Capan-2 xenograft models without apparent toxicity.

Activation of PPARα by clofibrate sensitizes pancreatic cancer cells to radiation through the Wnt/β-catenin pathway.

Radiotherapy is emerging as an important modality for the local control of pancreatic cancer, but pancreatic cancer cell radioresistance remains a serious concern. Peroxisome proliferator-activated receptor α (PPARα) is a member of the PPAR nuclear hormone receptor superfamily, which can be activated by fibrate ligands. The clinical relevance of PPARα and its biological function in pancreatic cancer radiosensitivity have not been previously described. In this study, we examined PPARα expression in tissue samples of pancreatic cancer patients. We found significantly higher expression of PPARα in pancreatic cancer tissues than in tumor-adjacent tissues and that the PPARα expression level is inversely associated with higher overall patient survival rate. We further observed that PPARα activation by its agonist clofibrate sensitizes pancreatic cancer cells to radiation by modulating cell cycle progression and apoptosis in several pancreatic cancer cell lines. Small interfering RNA-mediated PPARα silencing and PPARα blockade by the antagonist GW6471 abolish the effect of clofibrate on radiosensitization. An in vivo study showed that PANC1 xenografts treated with clofibrate are more sensitive to radiation than untreated xenografts. mRNA profiling by microarray analysis revealed that the expression of PTPRZ1 and Wnt8a, two core components of the β-catenin pathway, is downregulated by clofibrate. Chromatin immunoprecipitation analysis confirmed that clofibrate abrogates the binding of nuclear factor-κB to the PTPRZ1 and Wnt8a promoters, ultimately decreasing Wnt/β-catenin signaling activity, which is associated with radiosensitivity. Overall, we demonstrate that PPARα is overexpressed in pancreatic cancer tissues and clofibrate-mediated PPARα activation sensitizes pancreatic cancer cells to radiation through the Wnt/β-catenin pathway.

Biological nanoparticles carrying the Hmda-7 gene are effective in inhibiting pancreatic cancer in vitro and in vivo.

ObjectivesPancreatic cancer is one of the most common malignancies of the digestive system, and remains a clinical challenge. This study aimed to assess the effects of bovine serum albumin (BSA) nanoparticles carrying the hMDA-7 gene (BSA-NP-hMDA-7) in the treatment of pancreatic cancer.MethodsBSA-NP-hMDA-7 was generated by nanotechnology and gene recombination technology. A total of 5 BXPC-3 or PANC-1 pancreatic cancer cell groups were examined, including Control, BSA-NPs, Empty vector, hMDA-7 plasmid, and hMDA-7 BSA-NPs groups, respectively. Proliferation and apoptosis of cultured cells were assessed by the MTT method and flow-cytometry, respectively. In addition, pancreatic cancer models were established with both cell lines in nude mice, and the expression profiles of hMDA-7 and VEGF in cancer tissues were measured by Western blot and immunohistochemistry.ResultsBSA-NP-hMDA-7 nanoparticles were successfully generated, and significantly inhibited the proliferation of BXPC-3 and PANC-1 cells; in addition, apoptosis rates were higher in both cell lines after treatment with BSA-NP-hMDA-7 (P<0.05). Nude mouse xenograft studies indicated that treatment with BSA-NP-hMDA-7 nanoparticles resulted in decreased tumor size. Moreover, the hMDA-7 protein was found in tumor tissues after hMDA-7 gene transfection, while BSA-NP-hMDA-7 significantly suppressed VEGF expression in tumor tissues. Similar results were obtained for both BXPC-3 and PANC-1 xenograft models.ConclusionBSA nanoparticles carrying the hMDA-7 gene effectively transfected BXPC-3 and PANC-1 pancreatic cancer cells, causing reduced cell proliferation and enhanced apoptosis in vitro. In mouse xenografts, BSA-NP-hMDA-7 treatment decreased tumor size and reduced VEGF expression. These findings indicated that BSA-NP-hMDA-7 might exert anticancer effects via VEGF suppression.

CNGR-based synergistic-targeted NIR fluorescent probe for tracing and bioimaging of pancreatic ductal adenocarcinoma

Identification of fluorescent biomarkers with peptide ligand-directed receptors for diagnosis or theranostic of pancreatic ductal adenocarcinoma (PDAC) is still challenging. As potential prognostic/predictive bioimaging targets, both aminopeptidase N (APN, known as CD13) and Caveolin-1 are found as upregulation on the cell membrane surface of PDAC, in which APN is the principal receptor of the cyclic peptide cNGR (Asn-Gly-Arg, NGR) and Caveolin-1 can synergistically mediate endocytosis in this receptor-targeted process. Herein, we conjugate cNGR to dicyanomethylene-4 H -pyran (DCM) chromophore to develop a synergistic-targeted near-infrared (NIR) fluorescent probe DCM-cNGR with strongly intrinsic NIR fluorescence, stable optical performance, low cytotoxicity, and rapid accumulation in PANC-1 cells with the synergistic overexpressed APN receptor-targeted and Caveolin-1-mediated endocytosis. As demonstrated, DCM-cNGR can realize noninvasive NIR imaging for targeting PANC-1 tumor in vivo after intravenous injection into PANC-1 xenograft tumor of nude mice, making a great promise to improve the precision diagnosis and therapy of pancreatic cancer with real time tracing and bioimaging of PDAC in vitro and in vivo .

RGD-modified oncolytic adenovirus-harboring shPKM2 exhibits a potent cytotoxic effect in pancreatic cancer via autophagy inhibition and apoptosis promotion

The M2 isoform of pyruvate kinase (PKM2) is a key driver of glycolysis in cancer cells and has critical ‘non-metabolic’ functions in some cancers; however, the role of PKM2 in pancreatic cancer remains unclear. The aim of the current study was to elucidate the role of PKM2 in pancreatic cancer progression and the potential of PKM2 as a therapeutic target. In this study, we observed that PKM2 is highly expressed in patients with pancreatic cancer and is correlated to survival. Elevated PKM2 expression promoted cell proliferation, migration and tumor formation. The inhibition of cell growth by silencing PKM2 is caused by impairment of the autophagy process. To test the potential effects of downregulating PKM2 as a clinical therapy, we constructed an RGD-modified oncolytic adenovirus containing shPKM2 (OAd.R.shPKM2) to knock down PKM2 in pancreatic cancer cells. Cells transduced with OAd.R.shPKM2 exhibited decreased cell viability, and, in a PANC-1 xenograft model, intratumoral injection of OAd.R.shPKM2 resulted in reduced tumor growth. Furthermore, OAd.R.shPKM2 induced apoptosis and impaired autophagy in PANC-1 cells. Our results suggested that targeting PKM2 with an oncolytic adenovirus produced a strong antitumor effect, and that this strategy could broaden the therapeutic options for treating pancreatic cancer.

Effect of KRAS and P-STAT3 inhibition by SBT-100 on gemcitabine and pancreatic cancer growth in vivo.

e15727 Background: Over 90% of pancreatic cancers have KRAS mutations and hyper-expression of P-STAT3 oncoproteins, which if specifically targeted may help treatment of pancreatic cancers. Singh Biotechnology’s proprietary technology engineered SBT-100, a single domain antibody that is bispecific for KRAS &amp; STAT3, which can cross the cell membranes and bind to these intracellular oncoproteins. Combining this targeted therapy with an established chemotherapy, such as gemcitabine, may improve patient’s response to treatment. Methods: Human pancreatic cancer cells (PANC-1 and BX-PC3) were used. Biacore assay demonstrates SBT-100 binding to KRAS, KRAS (G12D), and STAT3. Immunoprecipitation (IP) and western blot analysis confirmed binding to STAT3 by SBT-100. Pancreatic cancer cells were treated at varying doses of SBT-100 ranging from 0µg/ml to 200µg/ml ± gemcitabine, and after 72 hours growth inhibition was determined by a MTT assay. PANC-1 tumors were grown in athymic nude mice, divided into four groups and staged to a range of 100-150mm3 before treatment. Groups were: vehicle only, SBT-100, gemcitabine, and SBT-100 &amp; gemcitabine. Animals received treatments for 14 days, then monitored for 7 days. Results: Biacore study shows SBT-100 binds KRAS with an affinity of 10-9M, KRAS (G12D) with 10-8M, and STAT3 with 10-8M. IP and western blot analysis demonstrates that SBT-100 binds P-STAT3. MTT assay demonstrates SBT-100 inhibits the growth of PANC-1 and BX-PC3 (p &lt; 0.001). In PANC1 cells a combination of SBT-100 &amp; gemcitabine demonstrates synergism in inhibiting growth of PANC-1, even at 1/8th the gemcitabine IC50 concentration. PANC-1 xenograft study demonstrates that combination therapy of SBT-100 &amp; gemcitabine is superior to either SBT-100 or gemcitabine alone. Compared to the vehicle group, SBT-100 &amp; gemcitabine is far superior (p &lt; 0.001) and gives statistically significant suppression of pancreatic cancer growth in vivo. Conclusions: Targeted therapy for KRAS and P-STAT3 expressing tumors with SBT-100 &amp; gemcitabine is synergistic for the treatment of pancreatic cancer. This study suggests that synergism maybe achieved with lower doses of gemcitabine, thereby reducing toxicity in patients.

Hydrophobic binding peptide-conjugated hybrid lipid-mesoporous silica nanoparticles for effective chemo-photothermal therapy of pancreatic cancer

Nanoparticle-based drug delivery systems are designed to reach tumor sites based on their enhanced permeation and retention effects. However, a lack of interaction of these nanoparticles with cancer cells might lead to reduced uptake in the tumors, which might compromise the therapeutic efficacy of the system. Therefore, we developed bortezomib and IR-820-loaded hybrid-lipid mesoporous silica nanoparticles conjugated with the hydrophobic-binding peptide, cyclosporine A (CsA), and referred to them as CLMSN/BIR. Upon reaching the tumor site, CsA interacts hydrophobically with the cancer cell membranes to allow effective uptake of the nanoparticles. Nanoparticles ∼160 nm in size were prepared and the stability of IR-820 significantly improved. High cellular uptake of the nanoparticles was evident with pronounced apoptotic effects in PANC-1 and MIA PaCa-2 cells that were mediated by the chemotherapeutic effect of bortezomib and the photothermal and reactive oxygen species generation effects of IR-820. An in vivo biodistribution study indicated there was high accumulation in the tumor with an enhanced photothermal effect in PANC-1 xenograft mouse tumors. Furthermore, enhanced antitumor effects in PANC-1 xenograft tumors were observed with minimal toxicity induction in the organs of mice. Cumulatively, these results indicated the promising effects of CLMSN/BIR for effective chemo-phototherapy of pancreatic cancers.

271 - Catalase as a Potential Biomarker of Pharmacological Ascorbate Cancer Therapy

Pharmacological ascorbate (P-AscH–) has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. P-AscH– can readily oxidize and deliver a high flux of H2O2, which can easily cross cell membrane and convert to more reactive species inside the cell. We hypothesize differential sensitivity of cells to P-AscH– is due to their ability to remove H2O2. The principal enzymes responsible for the elimination of H2O2 are catalase, glutathione peroxidase (GPx) and peroxiredoxins (Prx). Among these, catalase is the principal enzyme that involved in the detoxification of H2O2 at high concentration. In this study, we compared catalase levels and peroxide removal capacity in several pancreatic cancer cell lines MIA PaCa-2, AsPC-1, 403, 339 and Panc-1. Preliminary results indicated that MIA PaCa-2 cells had the lowest level of catalase and were most sensitive to P-AscH–, while Panc-1 had the highest level of catalase and were the least sensitive. In mice with pre-established xenograft pancreatic tumor, P-AscH– (4g/kg, i.p. twice daily) showed a greater inhibition of tumor growth for MIA PaCa-2 xenografts in comparison to Panc-1 xenografts. Furthermore, immunofluorescent staining of catalase in xenograft tumors also indicated that MIA PaCa-2 had lower level of catalase than Panc-1 in vivo. Taken together, catalase activity in tumors may predict which cancers will respond to P-AscH– therapy. Supported by VA Merit grant and NIH grants CA184051, CA137230, and CA169046.

Discovery of ursolic acid prodrug (NX-201): Pharmacokinetics and in vivo antitumor effects in PANC-1 pancreatic cancer

The aim of our study was to develop ursolic acid (UA) prodrugs in order to overcome UA’s weakness, which has an extremely low bioavailability. UA-medoxomil (NX-201), one of our UA prodrugs, showed an improved bioavailability about 200times better than UA in rodent model. According to in vivo test performed with PANC-1 xenograft SCID mouse model, tumor growth rate decreased dose-dependently and 100mg/kg dose of NX-201 had an anticancer effect comparable to gemcitabine. Most of all the combination of NX-201 (50mg/kg, po, daily) and gemcitabine (40mg/kg, iv, 2timesperweek) even reduced tumor size after three weeks.

The mitochondrion interfering compound NPC-26 exerts potent anti-pancreatic cancer cell activity in vitro and in vivo.

The development of novel anti-pancreatic cancer agents is extremely important. Here, we investigated the anti-pancreatic cancer activity by NPC-26, a novel mitochondrion interfering compound. We showed that NPC-26 was anti-proliferative and cytotoxic to human pancreatic cancer cells, possibly via inducing caspase-9-dependent cell apoptosis. Pharmacological inhibition or shRNA-mediated silence of caspase-9 attenuated NPC-26-induced pancreatic cancer cell death and apoptosis. Further, NPC-26 treatment led to mitochondrial permeability transition pore (mPTP) opening in the cancer cells, which was evidenced by mitochondrial depolarization, ANT-1(adenine nucleotide translocator-1)-Cyp-D (cyclophilin-D) association and oxidative phosphorylation disturbance. mPTP blockers (cyclosporin and sanglifehrin A) or shRNA-mediated knockdown of key mPTP components (Cyp-D and ANT-1) dramatically attenuated NPC-26-induced pancreatic cancer cell apoptosis. Importantly, we showed that NPC-26, at a low concentration, potentiated gemcitabine-induced mPTP opening and subsequent pancreatic cancer cell apoptosis. In vivo, NPC-26 intraperitoneal injection significantly suppressed the growth of PANC-1 xenograft tumors in nude mice. Meanwhile, NPC-26 sensitized gemcitabine-mediated anti-pancreatic cancer activity in vivo. In summary, the results of this study suggest that NPC-26, alone or together with gemcitabine, potently inhibits pancreatic cancer cells possibly via disrupting mitochondrion.

Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly.

Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors.

Short-chain C6 ceramide sensitizes AT406-induced anti-pancreatic cancer cell activity

Our previous study has shown that AT406, a first-in-class small molecular antagonist of IAPs (inhibitor of apoptosis proteins), inhibits pancreatic cancer cell proliferation in vitro and in vivo. The aim of this research is to increase AT406's sensitivity by adding short-chain C6 ceramide. We show that co-treatment of C6 ceramide dramatically potentiated AT406-induced caspase/apoptosis activation and cytotoxicity in established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells. Reversely, caspase inhibitors largely attenuated C6 ceramide plus AT406-induced above cancer cell death. Molecularly, C6 ceramide downregulated Bcl-2 to increase AT406's sensitivity in pancreatic cancer cells. Intriguingly, C6 ceramide-mediated AT406 sensitization was nullified with Bcl-2 shRNA knockdown or pretreatment of the Bcl-2 inhibitor ABT-737. In vivo, liposomal C6 ceramide plus AT406 co-administration dramatically inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) mice. The combined anti-tumor activity was significantly more potent than either single treatment. Expressions of IAPs (cIAP1/XIAP) and Bcl-2 were downregulated in Panc-1 xenografts with the co-administration. Together, we demonstrate that C6 ceramide sensitizes AT406-mediated anti-pancreatic cancer cell activity possibly via downregulating Bcl-2.

The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo

In the present study, we tested the anti-pancreatic cancer activity by AT406, a small-molecule antagonist of IAP (inhibitor of apoptosis proteins). In established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells, treatment of AT406 significantly inhibited cell survival and proliferation. Yet, same AT406 treatment was non-cytotoxic to pancreatic epithelial HPDE6c7 cells. AT406 increased caspase-3/-9 activity and provoked apoptosis in the pancreatic cancer cells. Reversely, AT406′ cytotoxicity in these cells was largely attenuated with pre-treatment of caspase inhibitors. AT406 treatment caused degradation of IAP family proteins (cIAP1 and XIAP) and release of cytochrome C, leaving Bcl-2 unaffected in pancreatic cancer cells. Bcl-2 inhibition (by ABT-737) or shRNA knockdown dramatically sensitized Panc-1 cells to AT406. In vivo, oral administration of AT406 at well-tolerated doses downregulated IAPs (cIAP1/XIAP) and inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) nude mice. Together, our preclinical results suggest that AT406 could be further evaluated as a promising anti-pancreatic cancer agent.

Phospho-Aspirin (MDC-22) Prevents Pancreatic Carcinogenesis in Mice.

Pancreatic cancer is a deadly disease with a dismal 5-year survival rate of <6%. The currently limited treatment options for pancreatic cancer underscore the need for novel chemopreventive and therapeutic agents. Accumulating evidence indicates that aspirin use is associated with a decreased risk of pancreatic cancer. However, the anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we developed phospho-aspirin (MDC-22), a novel derivative of aspirin, and evaluated its chemopreventive efficacy in preclinical models of pancreatic cancer. Phospho-aspirin inhibited the growth of human pancreatic cancer cell lines 8- to 12-fold more potently than aspirin; based on the 24-hour IC50 values. In a Panc-1 xenograft model, phospho-aspirin, at a dose of 100 mg/kg/d 5 times per week for 30 days, reduced tumor growth by 78% (P < 0.01 vs. vehicle control). Furthermore, phospho-aspirin prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. In p48-Cre;Kras(G12D) mice, cerulein treatment (6 hourly injections two times per week for 3 weeks) led to a significant increase in ductal metaplasia, replacing the majority of the exocrine compartment. Administration of phospho-aspirin 100 mg/kg/day five times per week for 21 days (starting on the first day of cerulein injection) inhibited the acinar-to-ductal metaplasia, reducing it by 87% (P < 0.01, vs. cerulein-treated control). Phospho-aspirin appeared to be safe, with the animals showing no signs of toxicity during treatment. Mechanistically, phospho-aspirin inhibited EGFR activation in pancreatic cancer, an effect consistently observed in pancreatic cancer cells, primary acinar explants and in vivo In conclusion, our findings indicate that phospho-aspirin has strong anticancer efficacy in preclinical models of pancreatic cancer, warranting its further evaluation. Cancer Prev Res; 9(7); 624-34. ©2016 AACR.

The preclinical evaluation of TIC10/ONC201 as an anti-pancreatic cancer agent

Here we evaluated the potential anti-pancreatic cancer activity by TIC10/ONC201, a first-in-class small-molecule inducer of tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL). The in vitro results showed that TIC10 induced potent cytotoxic and cytostatic activities in several human pancreatic cancer cell lines (Panc-1, Mia-PaCa2, AsPC-1 or L3.6). TIC10 activated both extrinsic (TRAIL-caspase-8-dependent) and endogenous/mitochondrial (caspase-9-dependent) apoptosis pathways in the pancreatic cancer cells. Molecularly, we showed that TIC10 inhibited Akt-Erk activation, yet induced TRAIL expression in pancreatic cancer cells. Significantly, TIC10, at a relatively low concentration, sensitized gemcitabine-induced growth inhibition and apoptosis against pancreatic cancer cells. Further, TIC10 and gemcitabine synergistically inhibited Panc-1 xenograft growth in SCID mice. The combination treatment also significantly improved mice survival. In addition, Akt-Erk in-activation and TRAIL/cleaved-caspase-8 induction were observed in TIC10-treated Panc-1 xenografts. Together, the preclinical results of the study demonstrate the potent anti-pancreatic cancer activity by TIC10, or with gemcitabine.

The effect of PI3K inhibitor LY294002 and gemcitabine hydrochloride combined with ionizing radiation on the formation of vasculogenic mimicry of Panc-1 cells in vitro and in vivo.

This research's purpose was to explore the existence of vasculogenic mimicry (VM) in both 3-D matrices of Panc-1 cells in vitro and orthotopic Panc-1 xenografts in vivo and to test the hypothesis that PI3K inhibitor LY294002 and gemcitabine hydrochloride would offer clear treatment benefit when integrated into ionizing radiation (IR) therapeutic regimens for treatment of pancreatic cancer. We explored the existence of VM in both 3-D matrices of Panc-1 cells and orthotopic Panc-1 xenografts. We subsequently investigated the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway in response to IR. LY294002 and gemcitabine hydrochloride were then evaluated for their radiosensitizing effect solely and in combination. We found that VM existed in both 3-D matrices of Panc-1 cells in vitro and orthotopic Panc-1 xenografts in vivo. The expressions of p-Akt and MMP- 2 were found to increase in response to IR. LY294002 and gemcitabine hydrochloride combined with IR better inhibited cell migration, VM formation and MMP-2 mRNA expression of Panc-1 cells in vitro, and we also proved that the novel therapeutic regimen better inhibited tumor growth, tumor metastasis and VM formation of orthotopic Panc-1 xenografts by suppressing the PI3K/MMPs/Ln-5γ2 signaling pathway in vivo. Our present study is among the first to prove the VM formation in orthotopic Panc-1 xenografts. Furthermore, our current study is also among the first to provide preliminary evidence for the use of the novel therapeutic regimen LY294002 and gemcitabine hydrochloride combined with IR for treatment of pancreatic cancer.

The anti-diabetic drug metformin inhibits pancreatic cancer cell proliferation in vitro and in vivo: Study of the microRNAs associated with the antitumor effect of metformin.

Recent studies suggest that metformin, which is a commonly used oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, yet the detailed mechanisms by which metformin affects various types of cancers, including pancreatic cancer, remain unknown. The aim of the present study was to evaluate the effects of metformin on human pancreatic cancer cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin. We used the human pancreatic cancer cell lines Panc1, PK1 and PK9 to study the effects of metformin on human pancreatic cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 5 weeks, and the expression of cell cycle-related proteins was determined. In addition, we used miRNA microarray tips to explore the differences in the levels of miRNAs in Panc1 cells and xenograft tumors treated with metformin or without. Metformin inhibited the proliferation of Panc1, PK1 and PK9 cells in vitro. This inhibition was accompanied by a strong decrease in G1 cyclins (particularly in cyclin D1) and retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor (EGFR), particularly the phosphorylation of EGFR at Tyr845, and insulin-like growth factor 1 receptor (IGF-1R) in vitro and in vivo. miRNA expression was markedly altered by the treatment with metformin in vitro and in vivo. Our results revealed that metformin inhibits human pancreatic cancer cell proliferation and tumor growth, possibly by suppressing the cell cycle-related molecules via alteration of miRNAs.