Pancreatic Carcinoma MIA PaCa-2 Research Articles

Abstract 1605: Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease that is projected to be the second leading cause of cancer-associated deaths by 2030. Owing to its intrinsic immune evasive tumor microenvironment (TME), PDAC is regarded as an immunologically “cold” tumor with a significant therapeutic challenge. Although 5-fluorouracil or gemcitabine-based chemotherapy is considered as standard of care, harnessing immunological pathways may potentially convert PDAC to a “hot” tumor, thereby providing survival advantage to patients. Lately, the use of oncolytic virus has proven to be an alternative strategy to activate the innate immune response within the TME coupled with its cytotoxic effect against the cancer cells. The FDA approval of T-VEC for the treatment of stage IIIB/IV melanoma validates the preceding argument. In this study, we have investigated the effect of herpes simplex virus type-1 (HSV-1) (VC2) overexpressing granulocyte-macrophage colony-stimulating factor (GMCSF), HSV-1VC2GM-CSF in the murine KRAS, p53, and Cre (KPC), and human MIA PaCa-2 pancreatic carcinoma cells. Following a 24h infection of the KPC and MIA PaCa-2 cells with the HSV-1VC2GM-CSF, the observed cytopathic effects (CPE) included cell rounding, detachment, and aggregation as compared to the wild-type HSV-1VC2 treated or the uninfected control cells. The CPE effect of HSV-1VC2GM-CSF was proven to be a more direct and dynamic response against these cells. In the next steps, the viral titers were determined to be 9.6E7 pfu/ml, 1.26E7 pfu/ml for HSV-1VC2 and VC2GM-CSF respectively. The infection of VC2GM-CSF 0.1, 1.0 and 10 MOI in KPC cells resulted in a significant increase in the expression of proinflammatory markers including interleukin 10 (IL-10), cyclooxygenase -2 (COX-2) and iNOS at the gene level in contrast to the uninfected controls. This was coupled with an increase in the expression of Fas ligand (FasL) in the treated cells. Intriguingly, the treated cells showed a significant upregulation of programmed death ligand (PD-L1). Interestingly, in a 3D-coculture of KPC and RAW264.7 cells the expression of PD-L1 was significantly upregulated. Based on the evidence presented here, it is speculated that VC2GM-CSF-induced proinflammatory profile in KPC and Mia PaCa-2 cells converts to an immunomodulatory one characterized by the regulation of FasL and PD-L1. The effect of VC2GM-CSF in a PDAC organoid model to understand the interplay between the tumor and the stromal cells in response to the virus is currently underway. Citation Format: Naveen Chintala Ramulu, Shilpa Thota, Brent Stanfield, Konstantin Kousoulas, Joseph Francis. Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1605.

PW06 suppresses cancer cell metastasis in human pancreatic carcinoma MIA PaCa-2 cells via the inhibitions of p-Akt/mTOR/NF-κB and MMP2/MMP9 signaling pathways in vitro.

PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one], a kind of the carbazole derivative containing chalcone moiety, inducedcell apoptosisin human pancreatic carcinoma in vitro. There is no investigation to show that PW06 inhibits cancer cell metastasis in human pancreatic carcinoma in vitro. Herein, PW06 (0.1-0.8 μM) significantly exists in the antimetastatic activities of human pancreatic carcinoma MIA PaCa-2 cells in vitro. Wound healing assay shows PW06 at 0.2 μM suppressed cell mobility by 7.45 and 16.55% at 6 and 24 hours of treatments. PW06 at 0.1 and 0.2 μM reduced cell mobility by 14.72 and 21.8% for 48 hours of treatment. Transwell chamber assay indicated PW06 (0.1-0.2 μM) suppressed the cell migration (decreased 26.67-35.42%) and invasion (decreased 48.51-68.66%). Atomic force microscopy assay shows PW06 (0.2 μM) significantly changed the shape of cell morphology. The gelatin zymography assay indicates PW06 decreased MMP2's and MMP9's activitiesat 48 hours of treatment. Western blotting assay further confirms PW06 reduced levels of MMP2 and MMP9 and increased protein expressions of EGFR, SOS1, and Ras. PW06 also increased the p-JNK, p-ERK, and p-p38. PW06 increased the expression of PI3K, PTEN, Akt, GSK3α/β, and E-cadherin. Nevertheless, results also show PW06 decreased p-Akt, mTOR, NF-κB, p-GSK3β, β-catenin, Snail, N-cadherin, and vimentin in MIA PaCa-2 cells. The confocal laser microscopy examination shows PW06 increased E-cadherin but decreased vimentin in MIA PaCa-2 cells. Together, our findings strongly suggest that PW06 inhibited the p-Akt/mTOR/NF-κB/MMPs pathways, increased E-cadherin, and decreased N-cadherin/vimentin, suppressing the migration and invasion in MIA PaCa-2 cells in vitro.

Deuterium Magnetic Resonance Imaging Using Deuterated Water-Induced 2H-Tissue Labeling Allows Monitoring Cancer Treatment at Clinical Field Strength.

An accurate and noninvasive assessment of tumor response following treatment other than traditional anatomical imaging techniques is essential. Deuterium magnetic resonance spectroscopic (MRS) imaging has been demonstrated as an alternative for cancer metabolic imaging by high-field MRI using deuterium-labeled molecules. The study aim was to use 2H tissue labeling and deuterium MRI at clinical field strength for tumor visualization and assessment of three anticancer therapies in pancreatic cancer model mice. MIA PaCa-2 pancreatic carcinoma and C26 colorectal carcinoma models of BALB/c-nu mice was prepared, and repeated deuterium MRI was performed during the first 10 days of free drinking of 30% D2O to track 2H distribution in tissues. 2H accumulation in the tumor after irradiation, bevacizumab administration, or gemcitabine administration was also measured in MIA PaCa-2-bearing mice. Confirmatory proton MRI, ex vivo metabolic hyperpolarization 13C-MRS, and histopathology were performed. The mouse's whole-body distribution of 2H was visible 1 day after drinking, and the signal intensity increased daily. Although the tumor size did not change 1 and 3 days after irradiation, the amount of 2H decreased significantly. The 2H image intensity of the tumor also significantly decreased after the administration of bevacizumab or gemcitabine. Metabolic hyperpolarization 13C-MRS, proton MRI, and 2H-NMR spectroscopy confirmed the efficacy of the anticancer treatments. Deuterium MRI at 1.5T proved feasible to track 2H distribution throughout mouse tissues during D2O administration and revealed a higher 2H accumulation in the tumor xenografts. This research demonstrated a promising successful method for preliminary assessment of radiotherapy and chemotherapy of cancer.

PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 μM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.

Disruption of the Microtubule Network and Inhibition of VEGFR2 Phosphorylation by Cytotoxic N,O-Coordinated Pt(II) and Ru(II) Complexes of Trimethoxy Aniline-Based Schiff Bases.

Platinum-based complexes are one of the most successful chemotherapeutic agents having a significant ground in cancer chemotherapy despite their side effects. During the past few decades, Ru(II) complexes have been emerging as efficient alternatives owing to their promising activities against platinum-resistant cancer. The pathway of action, lipophilicity, and cytotoxicity of a Pt or Ru complex may be tuned by varying the attached ligands, the coordination mode, and the leaving group. In this work, we report a family of Pt(II) and Ru(II) complexes (1-5) of three N,O and N,N donor-based trimethoxyanilines containing Schiff bases with the general formula [PtII(L)(DMSO)Cl], [RuII(L)(p-cymene)Cl], [RuII(L)(p-cymene)Cl]+, and [PtII(L)Cl2]. All of the complexes are characterized by different analytical techniques. 1H NMR and electrospray ionization mass spectrometry (ESI-MS) data suggest that the N,O-coordinated Pt(II) complexes undergo slower aquation compared to the Ru(II) analogues. The change of the coordination mode to N,N causes the Ru complexes to be more inert to aquation. The N,O-coordinating complexes show superiority over N,N-coordinating complexes by displaying excellent in vitro antiproliferative activity against different aggressive cancer cells, viz., triple-negative human metastatic breast adenocarcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. In vitro cytotoxicity studies suggest that Pt(II) complexes are more effective than their corresponding Ru(II) analogues, and the most cytotoxic complex 3 is 10-15 times more toxic than the clinical drugs cisplatin and oxaliplatin against MDA-MB-231 cells. Cellular studies show that all of the N,O-coordinated complexes (1-3) initiate disruption of the microtubule network in MDA-MB-231 cells in a dose-dependent manner within 6 h of incubation and finally lead to the arrest of the cell cycle in the G2/M phase and render apoptotic cell death. The disruption of the microtubule network affects the agility of the cytoskeleton rendering inhibition of tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a key step in angiogenesis. Complexes 1 and 2 inhibit VEGFR2 phosphorylation in a dose-dependent fashion. Among the Pt(II) and Ru(II) complexes, the former displays higher cytotoxicity, a stronger effect on the cytoskeleton, better VEGFR2 inhibition, and strong interaction with the model nucleobase 9-ethylguanine (9-EtG).

Synthesis, Structure, Stability, and Inhibition of Tubulin Polymerization by RuII–p-Cymene Complexes of Trimethoxyaniline-Based Schiff Bases

Four trimethoxy- and dimethoxyphenylamine-based Schiff base (L1-L4)-bearing RuII-p-cymene complexes (1-4) of the chemical formula [RuII(η6-p-cymene)(L)(Cl)] were synthesized, isolated in pure form, and structurally characterized using single-crystal X-ray diffraction and other analytical techniques. The complexes showed excellent in vitro antiproliferative activity against various forms of cancer that are difficult to cure, viz., triple negative human metastatic breast carcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. The 1H nuclear magnetic resonance data in the presence of 10% dimethylformamide-d7 or dimethyl sulfoxide-d6 in phosphate buffer (pD 7.4, containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h. Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5 molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, 1, exhibits an IC50 of approximately 10-15 μM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells. Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO ) revealed that 1 and 3 as well as L1 and L3 have a high affinity for the interface of the α and β tubulin dimer in the colchicine binding site. The immunofluorescence studies showed that 1 and 3 strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC20 or IC50 dose for 12 h. The cell cycle analysis upon treatment with 1 showed that the complexes inhibit the mitotic phase because the arrest was observed in the G2/M phase. In summary, 1 and 3 are RuII half-sandwich complexes that are capable of disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the G2/M phase, and kill the cells by an apoptotic pathway.

Cascade therapy with doxorubicin and survivin-targeted tailored nanoparticles: An effective alternative for sensitization of cancer cells to chemotherapy

Chemotherapy frequently involves combination treatment protocols to maximize tumor cell killing. Unfortunately these intensive chemotherapeutic regimes, often show disappointing results due to the development of drug resistance and higher nonspecific toxicity on normal tissues. In cancer treatment, it is critically important to minimize toxicity while preserving efficacy. We have previously addressed this issue and proposed a nanoparticle-based combination therapy involving both a molecularly targeted therapy and chemotherapeutic agent for neutralizing antiapoptotic survivin (BIRC5) to potentiate the efficacy of doxorubicin (DOX). Although the particles exhibited strong anticancer effect on the lung carcinoma A549 and the cervical carcinoma HeLa cells, there were lower-level therapeutic outcomes on the colon carcinoma HCT-116, the leukemia Jurkat and the pancreatic carcinoma MIA PaCa-2 cells. Since targeted therapies are one of the key approaches for overcoming drug resistance, tailoring the treatment of cancer cells with distinct characteristics is necessary to improve the therapeutic outcome of cancer therapy and to minimize potential pharmacokinetic interactions of drugs. In the light of this issue, this study examined whether a cascade therapy with low-dose DOX and survivin-targeted tailored nanoparticles is more effective at sensitizing HCT-116, Jurkat and MIA PaCa-2 cancer cells to DOX-chemotherapy than simultaneous combination therapy. The results demonstrated that the sequential therapy with the protocol comprising addition of the nanoparticles after incubation of cells with DOX clearly advanced the therapeutic outcome of related cancer cells, whereas the reverse protocol resulted in a reduction or delay in apoptosis, emphasizing the critical importance of formulating synergistic drug combinations in cancer therapy.

Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells.

Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated ∼3-fold enhanced in vitro activity (GI50 = 79 μM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI50 > 200 μM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 μM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

In Vivo Observation of Endothelial Cell-Assisted Vascularization in Pancreatic Cancer Xenograft Engineering.

In this study, for better understanding of patient-derived xenograft (PDX) generation, angiogenic characteristics during PDX cancerous tissue generation was investigated with different initial cell seeding conditions in the hydrogel. We monitored the angiogenic changes during the formation of in vivo cancer cell line xenografts induced by endothelial cells. Our in vivo cancer tissue formation system was designed with the assistance of tissue engineering technology to mimic patient-derived xenograft formation. Endothelial cells and MIA PaCa-2 pancreatic carcinoma cells were encapsulated in fibrin gel at different mixing configurations and subcutaneously implanted into nude mice. To investigate the effect of the initial cancerous cell distribution in the fibrin gel, MIA PaCa-2 cells were encapsulated as a homogeneous cell distribution or as a cell aggregate, with endothelial cells homogeneously distributed in the fibrin gel. Histological observation of the explanted tissues after different implantation periods revealed three different stages: isolated vascular tubes, leaky blood vessels, and mature cancerous tissue formation. The in vivo engineered cancerous tissues had leaky blood vessels with low expression of the vascular tight junction marker CD31. Under our experimental conditions, complex cancer-like tissue formation was most successful when tumorous cells and endothelial cells were homogeneously mixed in the fibrin gel. The present study implies that tumorous xenograft tissue formation can be achieved with a low number of initial cells and that effective vascularization conditions can be attained with a limited volume of patient-derived cancer tissue. Endothelial cell-assisted vascularization can be a potent choice for the effective development of vascularized cancerous tissues for studying patient-derived xenografts, cancer angiogenesis, cancer metastasis, and anticancer drugs.

Effect Of Low-Temperature Plasma On Metastatic Processes Of Solid Tumors In Vitro

Cancer metastasis is one of the leading causes of mortality in patients with solid tumors [1]. The complex metastatic process involves cell migration and remodeling of the tumor microenvironment, among other factors. The intrinsic limitations of conventional cancer therapies for solid tumors have motivated the development and application of alternative technologies. In this context, the use of low-temperature plasmas (LTPs) has been explored in vitro and in vivo with considerable success against a variety of cancers [2,3]. However, whether LTP can inhibit or promote metastasis of solid tumors is unknown. The aim of this study is to investigate the effect of direct and indirect LTP treatments on metastatic processes of glioblastoma, adenocarcinoma and pancreatic cancers. For this purpose, we used a 3-dimensional multicellular spheroid model that mimics the pathophysiological conditions and complex architecture of solid tumors [4]. Spheroids were generated using glioblastoma U87, U251 and LN229, adenocarcinoma MDA-MB-231 and human pancreatic carcinoma MIA PaCa-2 cell lines and glioblastoma primary cells GR04, GR08 and GR15. Direct and indirect plasma treatments were administered to spheroids in PBS solution using the COST plasma jet and kINPen IND. We used live cell imaging to assess cytotoxicity and spheroid morphology, microscopy to monitor cell migration and immunohistochemistry to assess extracellular matrix remodeling in response to direct and indirect LTP treatment. Our study provides insight on the application of LTP treatment as potential cancer therapy for solid tumors. Download : Download high-res image (421KB) Download : Download full-size image LN229 spheroids in PBS: (a) untreated control, directly treated with (b) kINPen IND or (c) COST jet. Outer border: total spheroid area; dark filled area: proliferative core (live cells); grey dots: dead cells.

A new HDAC inhibitor cinnamoylphenazine shows antitumor activity in association with intensive macropinocytosis.

Previous studies have shown that intensive macropinocytosis occurs in cancer cells and neutral red (NR) is noted for its capability to enter into the cell massively through a process mimetic to macropinocytosis. In addition, trans-cinnamic acid (tCA) has been found to be an inhibitor of histone deacetylase (HDAC). In the present study, cinnamoylphenazine (CA-PZ) that consists of NR and tCA moieties was synthesized and evaluated. As shown, CA-PZ massively entered into colon carcinoma HT-29 cells and pancreatic carcinoma MIA PaCa-2 cells and this entry was blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA, an inhibitor of macropinocytosis), indicating a macropinocytosis-mediated uptake. Furthermore, CA-PZ markedly increased the protein expression levels of acetyl-H3, acetyl-H4 and p21 in HT-29 cells and MIA PaCa-2 cells. CA-PZ significantly inhibited the growth of colon carcinoma HT-29 and pancreatic carcinoma MIA PaCa-2 xenografts. By in vivo imaging, CA-PZ displayed prominent accumulation in the tumor xenografts. The study indicates that the newly synthesized CA-PZ acts as an HDAC inhibitor in association with intensive macropinocytosis-mediated intracellular delivery in cancer cells. The use of neutral red for preparation of chimeric molecules with the attribute of macropinocytosis-mediated intracellular delivery might open an alternative way for development of HDAC inhibitors.

A recombinantly tailored β-defensin that displays intensive macropinocytosis-mediated uptake exerting potent efficacy against K-Ras mutant pancreatic cancer.

K-Ras mutant pancreatic cancer cells display intensive macropinocytosis, indicating that this process may be exploited in the design of anticancer targeted therapies. In this study, we constructed a macropinocytosis-oriented recombinantly tailored defensin (DF-HSA) which consists of human β-defensin-2 (DF) and human serum albumin (HSA). The macropinocytosis intensity and cytotoxicity of DF-HSA were investigated in K-Ras mutant MIA PaCa-2 cells and wild-type BxPC-3 cells. As found, the DF-HSA uptake in MIA PaCa-2 cells was much higher than that in wild-type BxPC-3 cells. Correspondingly, the cytotoxicity of DF-HSA to MIA PaCa-2 cells was more potent than that to BxPC-3 cells. In addition, the cytotoxicity of DF-HSA was much stronger than that of β-defensin HBD2. DF-HSA suppressed cancer cell proliferation and induced mitochondrial pathway apoptosis. Notably, DF-HSA significantly inhibited the growth of human pancreatic carcinoma MIA PaCa-2 xenograft in athymic mice at well tolerated dose. By in vivo imaging, DF-HSA displayed a prominent accumulation in the tumor. The study indicates that the recombinantly tailored β-defensin can intensively enter into the K-Ras mutant pancreatic cancer cells through macropinocytosis-mediated process and exert potent therapeutic efficacy against the pancreatic carcinoma xenograft. The novel format of β-defensin may play an active role in macropinocytosis-mediated targeting therapy.

Improving paclitaxel pharmacokinetics by using tumor-specific mesoporous silica nanoparticles with intraperitoneal delivery

Mesoporous silica nanoparticles (MSNs) containing paclitaxel for intraperitoneal (i.p.) delivery were developed to exploit the tumor specific accumulation of these nanocarriers after i.p. injection and the slow release of paclitaxel from the MSNs. A 3.5-fold increase in tumor cellular drug uptake was observed for the paclitaxel-loaded MSNs compared with free paclitaxel. An in vivo study using xenograft mice bearing peritoneal human pancreatic carcinoma MIA PaCa-2 demonstrated that the MSN-paclitaxel formulation, compared to free paclitaxel, exhibited a 3.2-fold increase in peritoneal cavity residence time, slower absorption into the systemic circulation with one third systemic exposure, but a 6.5-fold increase in peritoneal tumor accumulation. Tissue distribution imaging showed significantly greater accumulation of fluorescent MSNs in tumor tissues compared to other peritoneal tissues. In conclusion, intraperitoneal administration of drug-containing MSNs was effective at reducing systemic exposure and increasing the peritoneal tumor accumulation of paclitaxel.

Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation

Background and objectiveThe aim of this study was to evaluate the efficiency of proton beam irradiation in pancreatic cancer cell line MIA PaCa-2 and its role in the cell cycle, apoptosis, and formation of histone γH2AX in different reparation times (72-h follow-up). Material and methodsThe MIA PaCa-2 pancreatic carcinoma cell line was irradiated with 1.6-Gy proton beam. After irradiation, cell viability was measured colorimetrically, and the cell cycle, apoptosis, and γH2AX expression were evaluated on a FACScan cytometer. ResultsLow-dose proton beam irradiation had an effect on the MIA PaCa-2 tumor cell line already 1h after exposure, but maximal lethality was reached after 72h postirradiation with a cell viability rate of 24%. The cell cycle went into partial G1/0 arrest, and was released after 72h. The expression of γH2AX was strong and its levels were significantly elevated as late as 48h post radiation. The apoptosis levels increased with post radiation incubation time to reach 79% after 72h. ConclusionsOur data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase.

Synthesis and characterization of multifunctional hybrid-polymeric nanoparticles for drug delivery and multimodal imaging of cancer.

In this study, multifunctional hybrid-polymeric nanoparticles were prepared for the treatment of cultured multicellular tumor spheroids (MCTS) of the PANC-1 and MIA PaCa-2 pancreatic carcinoma cell lines. To synthesize the hybrid-polymeric nanoparticles, the poly lactic-co-glycolic acid core of the particles was loaded with Rhodamine 6G dye and the chemotherapeutic agent, Paclitaxel, was incorporated into the outer phospholipid layer. The surface of the nanoparticles was coated with gadolinium chelates for magnetic resonance imaging applications. This engineered nanoparticle formulation was found to be suitable for use in guided imaging therapy. Specifically, we investigated the size-dependent therapeutic response and the uptake of nanoparticles that were 65 nm, 85 nm, and 110 nm in size in the MCTS of the two pancreatic cancer cell lines used. After 24 hours of treatment, the MCTS of both PANC-1 and MIA PaCa-2 cell lines showed an average increase in the uptake of 18.4% for both 65 nm and 85 nm nanoparticles and 24.8% for 110 nm nanoparticles. Furthermore, the studies on therapeutic effects showed that particle size had a slight influence on the overall effectiveness of the formulation. In the MCTS of the MIA PaCa-2 cell line, 65 nm nanoparticles were found to produce the greatest therapeutic effect, whereas 12.8% of cells were apoptotic of which 11.4% of cells were apoptotic for 85 nm nanoparticles and 9.79% for 110 nm nanoparticles. Finally, the study conducted in vivo revealed the importance of nanoparticle size selection for the effective delivery of drug formulations to the tumors. In agreement with our in vitro results, excellent uptake and retention were found in the tumors of MIA PaCa-2 tumor-bearing mice treated with 110 nm nanoparticles.

Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

Apoptotic activity and antitumor efficacy of PEGylated TNF-related apoptosis-inducing ligand (TRAIL) in a Mia Paca-2 cell-xenografted mouse model

The purpose of this study was to demonstrate the apoptotic activity and antitumor effect of PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) in pancreatic carcinoma Mia Paca-2 cells and in Mia Paca-2 cell-xenografted mice. PEG-TRAIL was prepared using mPEG-aldehyde (Mw 5kDa). The apoptosis induced by PEG-TRAIL in Mia Paca-2 cells and in the tumors of Mia Paca-2 cell-xenografted mice was quantified by FACS analysis and using a TUNEL assay. Mia Paca-2 cell-xenografted BALB/c nu/nu mice were administered intratumoral injections of PEG-TRAIL (50μg/mouse/injection) every 3 days from day 0 (∼4 weeks after xenografting) to day 15. Tumor volumes were measured every 3 days from day 0 to day 27. PEG-TRAIL displayed obvious apoptotic activity in Mia Paca-2 cells; the FACS signal was shifted to the apoptotic area and the cells exhibited green fluorescence indicating apoptosis in the TUNEL assay. Furthermore, PEG-TRAIL was found to suppress tumors in Mia Paca-2 cell-xenografted mice (tumor volumes: 183.9±134.1 for PEG-TRAIL vs. 1827.3±264.5 mm3 for saline control). In addition, in vivo TUNEL assays of tumor tissues showed that the antitumor effect of PEG-TRAIL was due apoptosis. Our findings provide clear in vivo evidence of the antitumor potential of PEG-TRAIL in a Mia Paca-2 cell-xenografted mouse model based of pancreatic cancer.

Effect of ABTL0812, a safe dual inhibitor of mTORC1/C2 and dihydrofolate reductase, on gemcitabine and docetaxel cytotoxicity in pancreatic and lung cancer cells.

e13526 Background: We have previously described that ABTL0812 has oral anticancer properties. The action is exerted through a mechanism of action involving the inhibition of mTORC1 and mTORC2 (mammalian target of rapamycin) pathways and dihydrofolate reductase (DHFR) expression. Administration of ABTL0812 reduced tumor volume similarly or more than standards of care (SOC) in A549 or MiaPaca2 xenografts in mice. Moreover, a 28-day rat toxicity study has shown very low toxicity (NOAEL=500 mg/Kg) good bioavailability and pharmacokinetic linearity, suggesting a broad therapeutic window. A FiM phase I clinical trial in patients with advanced cancer is scheduled to start in June 2013. Methods: Cellular assays were performed in A549 lung adenocarcinoma, and MiaPaca-2 pancreatic carcinoma cells. Cell proliferation was carried out by bromodeoxyuridine (BrdU) incorporation, cell viability by MTT, and protein expression and phosphorylation by immunoblotting. Cellular apoptosis was assessed both by caspase 3 immunoblot and Hoechst 3342 nuclear staining and quantification of nuclei; and autophagy by LC3-II lipidation assay in immunoblots. All studies were performed with ABTL0812 either alone or in combination with SOC. Results: ABTL0812 potentiated the cytotoxic effects of gemcitabine and docetaxel in MiaPaCa-2 and A549 cancer cells, respectively. In MTT assays, the IC50’s of gemcitabine and docetaxel decreased 7 and 80 times in MiaPaca-2 and A549 cells, respectively, when cells were co-incubated with non-cytotoxic ABTL0812 concentrations. Analysis of the activity (Chou and Talalay’s method) revealed that ABTL0812 synergizes with gemcitabine and docetaxel to promote cytotoxicity. We also present data showing the effects of gemcitabine and docetaxel on the molecular targets of ABTL0812 such as Akt and ribosomal S6 phosphorylation, DHFR expression and cellular autophagy. Conversely, we also show the effects of ABTL0182 on cellular apoptosis induced by gemcitabine and docetaxel. Conclusions: ABTL0812 synergistically potentiates the cytotoxic effect of gemcitabine and docetaxel in pancreatic and lung cancer cell models.

Abstract 5073: Novel fusion molecule with high dual antiangiogenic and anticancer potential.

Abstract One of the most important mechanisms of tumorgenesis is formation of new blood vessels and tissue remodeling. Vascular endothelial growth factor (VEGF) is one of the most important and widely studied proangiogenic factors, that has an important role in many physiological and pathological processes. Blockade of VEGF pathway has been shown to inhibit both pathological angiogenesis and tumor growth. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) was considered as a promising anticancer agent due its remarkable ability to induce apoptosis in cancer cells without harming normal cells. However, in clinical practice TRAIL efficacy was too low to made it an effective single therapy agent. Here we present a novel fusion protein based on TRAIL/Apo2L, equipped with an antiangiogenic, effector peptide, fused to its N- terminus. The effector role plays a 7-amino acid-long peptide that binds to natural VEGF receptors and competes with their natural ligand. The proposed fusion protein AD-O51.4 consists of extracellular soluble portion of TRAIL linked to two tandemly arranged copies of the effector peptide separated by a sequence containing the motif recognized by tumor-specific proteases (MMP's, uPa). The TRAIL-targeted peptide is able to bind and sequester the VEGF receptors on malignant (cancer) and endothelial cells. As a consequence, the peptide blocks the binding of VEGF ligand, preventing formation of new vessels. In addition, a lot of literature data confirm expression of the VEGF receptors on the surfaces of various cancer cell lines. Due to the presence of VEGF receptors, those cell lines can be also targeted by our VEGF receptor-blocking peptide making them susceptible for TRAIL-induced apoptosis. In vitro, AD-O51.4 showed cytotoxic effect on various cancer cell lines at IC50 below 0.1 ng/mL and no toxic effects on normal cells. Cytotoxic activity of TRAIL alone was significantly lower in comparison to AD-O51.4. We demonstrated that TRAIL/Apo2L-VEGF-antagonist is a strong activator of caspase 3 and Bid processing. Additionally, its direct antiangiogenic activity was confirmed with the ring aortic assay and HUVEC spheroid assay. Strong antitumor activity of novel fusion molecule was also confirmed in xenograft model of multidrug resistant human uterine sarcoma MES-SA/Dx5 where our protein caused complete tumor remission and showed much higher efficacy than TRAIL alone. The similar effect was observed using human colorectal adenocarcinoma Colo205 and human pancreatic carcinoma MIA PaCa-2 xenografts, as well as an orthotropic model of human lung carcinoma NCI-H460-luc2. Citation Format: Jerzy S. Pieczykolan, Piotr Rozga, Anna Pieczykolan, Bartlomiej Zerek, Sebastian D. Pawlak, Michal Szymanik, Albert Jaworski, Marlena Galazka, Katarzyna Wiciejowska, Wojciech Strozek, Malgorzata Teska - Kaminska, Lukasz Kutner, Anna Grochot - Przeczek, Neli Kahmakowa - Trojanowska. Novel fusion molecule with high dual antiangiogenic and anticancer potential. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5073. doi:10.1158/1538-7445.AM2013-5073