Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the ataxia telangiectasia and RAD3-related (ATR) kinase.

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells, it reduced ATR to 40 % of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells.

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, induced cell apoptosis in 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 activities at 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/ß, and E-cadherin. Nevertheless, results also show PW06 decreased p-Akt, mTOR, NF-κB, p-GSK3ß, ß-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.

Tetrahydrobenzothiophene derivatives ameliorate Mia PaCa-2 cell progression and induces apoptosis via inhibiting EGFR2 tyrosine kinase signal.

A series of new thiophene analogues with acarbonitrile-basedmoiety were designed and synthesized via structural optimization. The conjugates were assessed for their in-vitro cytotoxic activity against a human pancreatic cancer cell line (Mia PaCa-2) and among them compound 5b showed IC50 value of 13.37 ± 2.37 µM. The compounds 5b (20 µM & 25 µM) and 7c (30 & 35 µM) also showed reduced clonogenicity, enhanced ROS and decreased mitochondrial membrane potential in Mia PaCa-2 cells. Treatment with these compounds also increased apoptotic population as evident with the double staining assay. Among the evaluated series, compounds 5b, 5g, 7c, and 9a attained a greater inhibitory potency than first generation's reversible EGFR inhibitor, Gefitinib. EGFR2 enzyme inhibitory studies revealed that 5b efficiently and arbitrarily suppressed the development of EGFR2 dependent cells and inhibited the enzymatic activity with an IC50 value of 0.68 µM; interestingly, the most effective molecule 5b with N-methyl piperazine substitution, has 1.29-fold greater potency than well-known EGFR inhibitor Gefitinib and increased Gefitinib's anti-growth impact with 2.04 folds greater against Mia PaCa-2. The in-vitro studies were validated with in-silico docking studies wherein compounds 5b and 7c exhibited binding energies of -8.2 and -7.4 Kcal/mol respectively. The present study reveals that tetrahydrobenzothiophene based analogues could be a promising lead for the evolution of potent chemo preventives over pancreatic cancer.

Determination of the effects of fusaric acid, a mycotoxin, on cytotoxicity, gamma-H2AX, 8-hydroxy-2 deoxyguanosine and DNA repair gene expressions in pancreatic cancer cells.

Pancreatic cancer has a poor prognosis and is an important public health problem for developing countries. Oxidative stress plays an important role in cancer initiation, progression, proliferation, invasion, angiogenesis and metastasis. For this reason, one of the important strategic targets of new cancer therapeutics is to drive cancer cells into apoptosis through oxidative stress. In nuclear and mitochondrial DNA, 8-hydroxy-2'-deoxyguanosine and gamma-H2AX (γ-H2AX) are used as important oxidative stress biomarkers. Fusaric acid (FA) is a mycotoxin that mediates toxicity produced by Fusarium species and exhibits anticancer effects in various cancers via inducing apoptosis, cell cycle arrest, or other cellular mechanisms. The aim of this study was to determine the effects of fusaric acid on cytotoxic and oxidative damage in MIA PaCa-2 and PANC-1 cell lines. In this context, dose and time dependent cytotoxic effect of fusaric acid was determined by XTT method, mRNA expression levels of genes related to DNA repair were determined by RT-PCR, and its effect on 8-hydroxy-2'-deoxyguanosine and γ-H2AX levels was revealed by ELISA assay. According to XTT results, fusaric acid inhibits cell proliferation in MIA PaCa-2 and Panc-1 cells in a dose- and time-dependent manner. IC50 doses were determined as 187.74 µM at 48 h in MIA PaCa-2 cells and 134.83 µM at 48 h in PANC-1 cells, respectively. γ-H2AX and 8-OHdG changes were not found significant in pancreatic cancer cells. The mRNA expression levels of DNA repair-related genes NEIL1, OGG1, XRCC and Apex-1 change with exposure to fusaric acid. This study contributes to the therapeutic approaches to be developed for pancreatic cancer and demonstrates the potential of fusaric acid as an anticancer agent.

Aspergillus niger CJ6 extract with antimicrobial potential promotes in-vitro cytotoxicity and induced apoptosis against MIA PaCa-2 cell line.

Nowadays, the increased number of multidrug-resistant strains among pathogens is a severe public health concern and cancer is posing a great threat for humans. These problems should be tackled with the development of novel and broad-spectrum antimicrobials from microbial origin. During the present study, the bioactive secondary metabolites from Aspergillus niger CJ6 were extracted, characterized; their biological properties were evaluated by subjecting them for antimicrobial, antifungal and anticancer activities. The potent isolate Aspergillus niger CJ6 with nucleotide sequence of 959 base pairs showed antagonistic activity against fungal pathogens in dual culture. The chemical profiling of crude ethyl acetate extract indicated the presence of various bioactive molecules belonging to phenolic, hydrocarbons, and phthalate derivative classes. In antimicrobial activity, the crude extract displayed increasing activity with increased concentration; the highest activity observed against Shigella flexneri with 15 ± 1.0, 19 ± 0.5, 20 ± 1.0 and 24 ± 1.0 mm zones of inhibition at 25, 50, 75 and 100 µl concentrations. The MTT assay illustrated deformed cells of MIA PaCa-2 cell line in in-vitro cytotoxic activity; outflow of cell matrix and membrane rupture; the IC50 of 90.78 µg/ml suggested moderate potential of extract to prevent cancer cell growth. The apoptosis/necrosis study by flow cytometer exhibited 8.98 ± 0.85% early and 73 ± 0.7% of late apoptotic population with 3.8 ± 1.1% necrotic cells; only 14.22 ± 0.6% of healthy cells suggested the increased apoptosis inducing capacity of Aspergillus niger CJ6 crude extract. The outcomes of this study persuade further exploration on the identification, purification and development of novel bioactive agents that could help battle fatal diseases in humans.

Tracing 2D Growth of Pancreatic Tumoroids Using the Combination of Image Processing Techniques and Mini-Opto Tomography Imaging System.

Objectives: In this study, we aimed to trace the 2D growth development of tumoroids produced with MIA PaCa-2 pancreatic cancer cells at different time points. Methods We cultured 3 different tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations and calculated the growth rate of the tumoroids with their images acquired at 9 imaging time points by mini-Opto tomography imaging system applying image processing techniques. We used the metrics contrast-to-noise ratio (CNR), peak signal-to-noise ratio (PSNR), and mean squared error (MSE) to analyze the distinguishability of the tumoroid structure from its surroundings, quantitatively. Additionally, we calculated the increase of the radius, the perimeter, and the area of 3 tumoroids over a time period. Results In the quantitative assessment, the bilateral and Gaussian filters gave the highest CNR values (ie, Gaussian filter: at each of 9 imaging time points in range of 1.715 to 15.142 for image set-1). The median filter gave the highest values in PSNR in the range of 43.108 to 47.904 for image set-2 and gave the lowest values in MSE in the range of 0.604 to 2.599 for image set-3. The areas of tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations were 1.014 mm2, 1.047 mm2, and 0.530 mm2 in the imaging time point-1 and 33.535 mm2, 4.538 mm2, and 2.017 mm2 in the imaging time point-9. The tumoroids with 0.5%, 0.8%, and 1.5% agarose concentrations grew up to times of 33.07, 4.33, and 3.80 in area size over this period, respectively. Conclusions The growth rate and the widest borders of the different tumoroids in a time interval could be detected automatically and successfully. This study that combines the image processing techniques with mini-Opto tomography imaging system ensured significant results in observing the tumoroid's growth rate and enlarging border over time, which is very critical to provide an emerging methodology in vitro cancer studies.

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer.

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.

A potent ion channel blocker, hydroquinidine, exhibits strong anti-cancer activity on colon, pancreatic, and hepatocellular cancer cells.

BACKGROUND: Despite recent advances in drug discovery, cancer is still one of the most lethal health problems worldwide. In most cases, standard therapy methods and multi-modal treatments fail, and new therapeutic approaches are required. Ion channels are essential in multiple cellular processes regulating cell division, differentiation, and death. Recent studies on ion-channel modulators emphasize their potential to suppress tumor growth. In that regard, we reasoned that an underinvestigated potassium channel modulator, Hydroquinidine (HQ), may exhibit an anti-carcinogenic activity. METHODS AND RESULTS: HQ's potential as an anti-neoplastic compound was examined using colony formation assay, wound healing assay, soft agar assay, and Annexin-V assay in the colon, pancreatic, and hepatocellular carcinomas. Our findings unveiled a remarkable anti-cancer activity of HQ by decreasing colony-forming ability, migration capacity, tumorigenicity, and proliferation and stimulating cellular death. HQ significantly reduced the formed colonies and tumorigenicity for all cells. It displayed a significant anti-migrative effect on hepatocellular carcinoma cells and promoted apoptosis in pancreatic and liver cancer cells. The altered gene expression profile upon HQ treatment was in accordance with observed cellular effects. Cells incubated with HQ downregulated the genes acting in cell division and survival, whereas the expression level of genes functioning in cell cycle arrest and apoptosis was elevated. CONCLUSION: Our data indicate HQ's competency to limit cancer growth and suggest its utilization as a novel potent anti-carcinogenic agent. Future studies are necessary to provide new insights into the HQ action mechanism and to evaluate its capacity in in-vivo.

Generation of the novel anti-FXYD5 monoclonal antibody and its application to the diagnosis of pancreatic and lung cancer.

Despite recent advances in cancer treatments, pancreatic cancer has a dismal prognosis globally. Early detection of cancer cells and effective treatments for recalcitrant tumors are required, but the innovative therapeutic tools remain in development. Cancer-specific antigens expressed only on cancer cells may help resolve these problems, and antibodies to such antigens have potential in basic research and clinical applications. To generate specific antibodies that bind to proteins expressed on the surface of pancreatic cancer cells, we immunized mice with human pancreatic cancer MIA PaCa-2 cells, and isolated a hybridoma that produces a monoclonal antibody (mAb), named 12-13.8. This antibody was applied to molecular biological experiments such as immunocytochemistry, immunoblotting, flow cytometry, and immunoprecipitation. In addition, we showed that mAb 12-13.8 could accumulate in tumors, through in vivo experiments using cancer-bearing mice. Immunohistochemical staining of pancreatic and lung tumor tissues indicated that the increase of the staining strength by mAb 12-13.8 positively and inversely correlated with the patients' cancer recurrence and survival rate, respectively. We identified the FXYD5 protein as the target protein of mAb 12-13.8, by a human protein array screening system. The FXYD5 protein is overexpressed in various types of cancer and is modified by O-linked glycosylation. We confirmed the binding of the FXYD5 protein to mAb 12-13.8 by using FXYD5-knockout MIA PaCa-2 cells, and detailed epitope mapping identified amino acid residues 45-52 as the minimal peptide sequence. Our results indicate that mAb 12-13.8 could be a valuable tool for FXYD5 studies, and useful in diagnostic and drug delivery applications for cancer patients.

Nickel Complexes Bearing ONS Chelating Ligands: A Promising Contender for In Vitro Cytotoxicity Effects on Human Pancreatic Cancer MIA-PaCa-2 Cells.

The highly chronic human pancreatic cancer cell is one of the major reasons for cancerous death. Nickel complexes are recently gaining interest in anticancer activities on different types of cancer cells. Hence, in this study, we synthesized and characterized a series of ONS donor ligands [2-HO-C6H4-CH═N-(C6H4)-SH] (L1), [2-OH-3-OMe-C6H3-CH═N-(C6H4)-SH] (L2), [2-OH-3,5-(C(Me)3)2-C6H2-CH═N-(C6H4)-SH] (L3), [2-OH-C6H4-CH═N-(C6H4)-SMe] (L4), [2-OH-3-OMe-C6H3-CH═N-(C6H4)-SMe] (L5), [2-OH-3,5-(C(Me)3)2-C6H2-CH═N-(C6H4)-SMe] (L6) and their Ni(II) metal complexes [(MeOH)Ni(L1-L1-4H)] (1), [(MeOH)Ni(L2-L2-4H)] (2), [(MeOH)Ni(L3-L3-4H)] (3), [(L4-H)2Ni] (4), [(L5-H)2Ni] (5), and [(L6-H)2Ni] (6). The single-crystal X-ray diffraction data of complexes 1 and 4 were collected to elucidate the geometry around the metal center. The anticancer activity of complexes 1-6 was investigated on human pancreatic cancer cell line MIA-PaCa-2, which revealed that complexes 4 and 6 were the most significantly effective in decreasing the cell viability of cancer cells at the lowest dose. The structure parameters obtained from single-crystal X-ray diffraction data are found to be in good agreement with the data from density functional theory and Hirshfeld surface analysis for complex 1.

Metabolites from the plant endophytic fungus Penicillium sp. CPCC 401423 and their cytotoxic activity against MIA PaCa-2 cells.

Twenty-two metabolites were isolated from Penicillium sp. CPCC 401423 cultured on rice. The structures of all compounds were elucidated mainly by MS and NMR analysis as well as the necessary CD experimental evidence, of which penicillidione A (1), penicillidione B (2), (E)-4-[(4-acetoxy-3-methyl-2-butenyl)oxy]phenylacetic acid (3), (S)-2-hydroxy-2-{4-[(3-methyl-2-butenyl)oxy]phenyl} (4), (S)-4-(2,3-dihydroxy-3-methyl-butoxy)phenylacetic acid (5), (E)-4-[(3-carboxy-2-butenyl)oxy]benzoic acid (6), (Z)-4-[(4-hydroxy-3-methyl-2-butenyl)oxy]benzoic acid (7), open-cycled N-demethylmelearoride A (12), and penostatin M (16) were identified as new compounds. The cytotoxic activity against human pancreatic carcinoma cell line MIA PaCa-2a was detected. Among them, compounds 13-15 and 22 displayed significant cytotoxicity against MIA-PaCa-2 cells with IC50 values of 8.9, 36.5, 31.8, and 22.3 µM, respectively (positive control gemcitabine IC50 65.0 µM).

Efficacy of HMJ-38, a new quinazolinone analogue, against the gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cells.

Gemcitabine is frequently utilized to treat pancreatic cancer. The purpose of our study was to create a gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cell line (MIA-GR100) and to evaluate the anti-pancreatic cancer efficacy of HMJ-38, a new quinazolinone analogue. Compared to their parental counterparts, MIA-PaCa-2, established MIA-GR100 cells were less sensitive to gemcitabine. MIA-GR100 cell viability was not affected by 10, 50 and 100 nM gemcitabine concentrations. HMJ-38 reduced MIA-GR100 cell growth and induced autophagy and apoptosis. When stained with monodansylcadaverine (MDC), acridine orange (AO), and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), MIA-GR100 cells shrunk, punctured their membranes, and produced autophagy vacuoles and apoptotic bodies. Combining chloroquine (CQ) and 3-methyladenine (3-MA) with HMJ-38 dramatically reduced cell viability, indicating that autophagy function as a cytoprotective mechanism. MIA-GR100 cells treated with both z-VAD-FMK and HMJ-38 were much more viable than those treated with HMJ-38 alone. HMJ-38 promotes apoptosis in MIA-GR100 cells by activating caspases. Epidermal growth factor receptor (EGFR) is one of HMJ-38's principal targets, as determined via in silico target screening with network prediction. HMJ-38 also inhibited EGFR kinase activity and EGFR-associated signaling in MIA-GR100 cells. HMJ-38 may be an effective chemotherapeutic adjuvant for gemcitabine-resistant pancreatic cancer cells, in which it induces an antitumor response.

Potential of Gold Nanoparticle in Current Radiotherapy Using a Co-Culture Model of Cancer Cells and Cancer Associated Fibroblast Cells.

Many cancer therapeutics are tested in vitro using only tumour cells. However, the tumour promoting effect of cancer associated fibroblasts (CAFs) within the tumour microenvironment (TME) is thought to reduce cancer therapeutics' efficacy. We have chosen pancreatic ductal adenocarcinoma (PDAC) as our tumor model. Our goal is to create a co-culture of CAFs and tumour cells to model the interaction between cancer and stromal cells in the TME and allow for better testing of therapeutic combinations. To test the proposed co-culture model, a gold nanoparticle (GNP) mediated-radiation response was used. Cells were grown in co-culture with different ratios of CAFs to cancer cells. MIA PaCa-2 was used as our PDAC cancer cell line. Co-cultured cells were treated with 2 Gy of radiation following GNP incubation. DNA damage and cell proliferation were examined to assess the combined effect of radiation and GNPs. Cancer cells in co-culture exhibited up to a 23% decrease in DNA double strand breaks (DSB) and up to a 35% increase in proliferation compared to monocultures. GNP/Radiotherapy (RT) induced up to a 25% increase in DNA DSBs and up to a 15% decrease in proliferation compared to RT alone in both monocultured and co-cultured cells. The observed resistance in the co-culture system may be attributed to the role of CAFs in supporting cancer cells. Moreover, we were able to reduce the activity of CAFs using GNPs during radiation treatment. Indeed, CAFs internalize a significantly higher number of GNPs, which may have led to the reduction in their activity. One reason experimental therapeutics fail in clinical trials relates to limitations in the pre-clinical models that lack a true representation of the TME. We have demonstrated a co-culture platform to test GNP/RT in a clinically relevant environment.

Abietane diterpenes from Abies spectabilis and their anti-pancreatic cancer activity against the MIA PaCa-2 cell line.

An ethanolic extract of the stem of Abies spectabilis exhibited strong cytotoxicity against MIA PaCa-2 human pancreatic cancer cells preferentially under nutrient-deprived conditions. Therefore, phytochemical investigation of this bioactive extract was carried out, and that led the isolation of ten compounds (1-10) including a new abietane-type diterpene (1). The structure of the new compound (1) was elucidated by combined spectroscopic techniques, including HRFABMS, NMR and quantum ECD calculation. All the isolated compounds were evaluated for their efficacy against MIA PaCa-2 human pancreatic cancer cell line by employing an anti-austerity strategy. Among the tested compounds, dehydroabietinol (5) displayed the most potent activity with a PC50 value of 6.6 µM. Dehydroabietinol (5) was also found to retard the MIA PaCa-2 cell migration under normal nutrient-rich conditions displaying its anti-metastatic potential. Investigation on the mechanism suggested that dehydroabietinol (5) is an inhibitor of the key cancer cell survival Akt/mTOR/autophagy signaling pathway.

Evaluation of Integrin αvß3 Expression in Murine Xenograft Models: [68Ga]Ga-DOTA-C(RGDfK) PET Study with Immunohistochemical Confirmation.

Tumor blood flow (TBF) is related to drug delivery and hypoxia, both of which can impact the efficacy of anti-cancer therapies. Although integrin αvß3 expression is related to tumor angiogenesis, it remains unclear whether the degree of angiogenesis affects TBF. This study aimed to evaluate the expression of integrin αvß3 in mouse tumor models using [68Ga]Ga-DOTA-c(RGDfK) peptide positron emission tomography (PET) and immunohistochemical staining. PET studies were conducted using mouse C6 glioma models and MIA PaCa-2 (n = 6 each). The [68Ga]Ga-DOTA-c(RGDfK) peptide was injected via the tail vein (2.17 ± 0.28 MBq), and 10 min static PET scans were performed. Immunohistochemical analysis was conducted using an integrin αVß3 antibody. [68Ga]Ga-DOTA-c(RGDfK) peptide PET revealed higher uptake of the radiotracer in C6 gliomas than in MIA PaCa-2 tumors. The mean standardized uptake value was significantly higher in C6 gliomas (0.35 ± 0.058) than in MIA PaCa-2 tumors (0.17 ± 0.045). Histological analysis revealed intense integrin αVß3 expression in the C6 gliomas, whereas the MIA PaCa-2 tumors had low expression levels. This study showed that the expression of integrin αvß3 can be differentiated by the [68Ga]Ga-DOTA-c(RGDfK) peptide, suggesting the potential applicability of this peptide in the evaluation of the relationship between angiogenesis and TBF.

Investigation of Nano-Bio Interactions within a Pancreatic Tumor Microenvironment for the Advancement of Nanomedicine in Cancer Treatment.

Pancreatic cancer is one of the deadliest types of cancer, with a five-year survival rate of only 10%. Nanotechnology offers a novel perspective to treat such deadly cancers through their incorporation into radiotherapy and chemotherapy. However, the interaction of nanoparticles (NPs) with cancer cells and with other major cell types within the pancreatic tumor microenvironment (TME) is yet to be understood. Therefore, our goal is to shed light on the dynamics of NPs within a TME of pancreatic origin. In addition to cancer cells, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were examined in this study due to their important yet opposite roles of suppressing tumor growth and promoting tumor growth, respectively. Gold nanoparticles were used as the model NP system due to their biocompatibility and physical and chemical proprieties, and their dynamics were studied both quantitatively and qualitatively in vitro and in vivo. The in vitro studies revealed that both cancer cells and CAFs take up 50% more NPs compared to NFs. Most importantly, they all managed to retain 70-80% of NPs over a 24-h time period. Uptake and retention of NPs within an in vivo environment was also consistent with in vitro results. This study shows the paradigm-changing potential of NPs to combat the disease.

Galectin-3 Expression in Pancreatic Cell Lines Under Distinct Autophagy-Inducing Stimulus.

Hypoxia and nutrient deprivation are responsible for inducing malignant behavior in neoplastic cells. In these conditions, metabolic stress leads the cells to enhance their autophagic flux and to activate key molecules for homeostasis maintenance. Galectin-3 (Gal-3) is upregulated in pancreatic cancer and it is activated under the hypoxic atmosphere. We aimed to analyze the most effective autophagic-inducing conditions in pancreatic ductal adenocarcinoma cells and the effect exerted under these conditions in association with hypoxia on the Gal-3 expression. Gal-3 and the microtubule-associated protein light chain 3 beta (LC3) were accessed through western blot and immunofluorescence. Degradative vacuole quantification was analyzed by transmission electronic microscopy, and inhibition of Gal-3 was performed using siRNA. According to the analyses, the most effective conditions in the inducement of autophagy for PANC-1 and MIA PaCa-2 cells were nutritional deprivation and complete amino acid/glucose deprivation, respectively. PANC-1 cells presented higher Gal-3 when they were submitted to 24 h of nutritional deprivation alone and simultaneously nutritional and oxygen deprivation. Inhibition of Gal-3 causes a decrease of LC3 levels in all experimental conditions. These results confirm that Gal-3 is modulated by microenvironment factors and the possibility of Gal-3 participating in an adaptive response from PDAC cells to extreme conditions.

Synergistic Anticancer Effects of Gemcitabine with Pitavastatin on Pancreatic Cancer Cell Line MIA PaCa-2 in vitro and in vivo.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with an overall 5-year survival rate of 9.3%, and this malignancy is expected to become the second leading cause of cancer-related death by 2030. Gemcitabine resistance develops within weeks of PDAC patient's chemotherapeutic initiation. Statins, including pitavastatin, have been indicated to have anticancer effects in numerous human cancer cell lines. Thus, in this study, we hypothesized that a combination of gemcitabine and pitavastatin may have a greater anticancer effect than gemcitabine alone on the human pancreatic carcinoma cell line MIA PaCa-2. METHODS: The anticancer effects of gemcitabine with pitavastatin were evaluated using human MIA PaCa-2 cell line in vitro and in vivo Balb/c murine xenograft tumor model. Cell viability was assessed with CCK-8, and cell migration was stained by crystal violet. Cell cycle distribution, apoptosis and mitochondrial membrane potential were examined by flow cytometry. Activation of drug transporters (hENTs, hCNTs), intracellular drug activating (dCK) and inhibition of inactivating enzymes (RRMs) pathways were assessed by Western blotting analysis. Molecular mechanisms and signaling pathways of apoptosis, necrosis and autophagy also were assessed by Western blotting. RESULTS: We observed that gemcitabine and pitavastatin synergistically suppressed the proliferation of MIA PaCa-2 cells through causing sub-G1 and S phase cell cycle arrest. Activation of apoptosis/necrosis was confirmed by annexin V/propidium iodide double staining, which showed increasing levels of active caspase 3, cleaved poly(ADP-ribose) polymerase and the RIP1-RIP3-MLKL complex. Moreover, gemcitabine-pitavastatin-mediated S phase arrest downregulated cyclin A2/CDK2 and upregulated p21/p27 in MIA PaCa-2 cells. Furthermore, this combination improved drug cellular metabolism pathway, mitochondria function and activated autophagy as part of the cell death mechanism. In vivo, gemcitabine-pitavastatin effectively inhibited tumor growth in a nude mouse mode of Mia PaCa-2 xenografts without observed adverse effect. CONCLUSION: Combined gemcitabine-pitavastatin may be an effective novel treatment option for pancreatic cancer.

EZH2 Downregulation Augments the Effect of Irradiation in Reducing Pancreatic Cancer Cell Proliferation in vitro.

OBJECTIVE: Pancreatic ductal carcinoma has a 5-year survival rate of 9%. This makes it the 4th leading cause of cancer-related death in the United States. Advanced-stage diagnosis and limited treatment options contribute to poor prognosis. Thus, there is an urgent need for new approaches to treatment. Enhancer of Zeste 2 (EZH2), a catalytic subunit of the multi-protein histone methyltransferase, known as the polycomb repressive complex, has been implicated in carcinogenesis. E2H2's downregulation has been shown to have a therapeutic effect in B cell lymphomas. MATERIALS AND METHODS: We examined the effect of EZH2 downregulation in combination with irradiation on the proliferation and apoptosis of pancreatic cancer cells (PANC-1 and MIA PaCa-2) in vitro. EZH2 downregulation was accomplished by treatment of cells with small interfering RNA (siRNA) or EPZ. To do this, cell survival was assessed over a 96 hr (short-term) by ATP measurement and immunohistochemical assessment of Phosphohistone 3 (PHH3), Ki-67 and CC3 over two weeks (long-term) by clonogenic assay. RESULTS: EZH2 downregulation resulted in the decreased proliferation of PANC-1 and MIA PaCa-2 cells within short-term assays with maximal reduction at 72 hr. Irradiation reduced cell proliferation beginning at 96 hr and continued over the long-term. Irradiation with EZH2 downregulation reduced cell proliferation between 48 and 72 hr. This combined treatment reduced cell proliferation by 3 to 14% as compared to those treated with irradiation alone at two weeks. PANC-1 and MIA PaCa-2 cells exhibited similar responses to EZH2 downregulation and irradiation, but to different degrees. siRNA or EPZ were equally effective in EZH2 downregulation. CONCLUSIONS: EZH2 downregulation in combination with irradiation reduces PANC-1 and MIA PaCa-2 cell proliferation more than irradiation alone. This study affirms the role of EZH2 downregulation for radiosensitization in pancreatic cancer treatment.

The CCR5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, and novel therapeutic strategies are urgently needed. Recently, expression of the C-C chemokine receptor 5 (CCR5) and its ligands has been found to play an important role in cancer progression and metastasis. In this study, we blocked the CCR5 receptor by the FDA approved antagonist maraviroc (MVC) in Suit2-007 and MIA-PaCa-2 human PDAC cells. The treatment significantly inhibited their proliferation and induced apoptosis of exposed cells as evidenced by caspases activation and increased Bax levels. Moreover, MVC inhibited the cell cycle by down regulating the proteins of the complexes of cyclin dependent kinase (CDK) 4/6 - Cyclin D and CDK2 - Cyclin E, as well as by increasing the protein levels of CDK inhibitors p18, p21 and p27. In line with this, MVC caused significant retardation of Suit2-007 cells growing in a PDAC liver metastasis xenograft model (p < 0.05). These results suggest that maraviroc could be a promising treatment strategy for PDAC patients with liver metastases.