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.

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.

Sterol regulatory element-binding protein 1 inhibitors decrease pancreatic cancer cell viability and proliferation.

Sterol regulatory element-binding protein1 (SREBP1) is a key regulatory factor that controls lipid homeostasis. Overactivation of SREBP1 and elevated lipid biogenesis are considered the major characteristics in malignancies of prostate cancer, endometrial cancer, and glioblastoma. However, the impact of SREBP1 activation in the progression of pancreatic cancer has not been explored. The present study examines the effect of suppression of SREBP1 activation by its inhibitors like fatostatin and PF429242 besides analyzing the impact of inhibitory effects on SREBP1 downstream signaling cascade such as fatty acid synthase (FAS), hydroxymethylglutaryl-CoA reductase (HMGCoAR), stearoyl-CoA desaturase-1 (SCD-1), and tumor suppressor protein p53 in MIA PaCa-2 pancreatic cancer cells. Both fatostatin and PF429242 inhibited the growth of MIA PaCa-2 cells in a time and concentration-dependent manner with maximal inhibition attained at 72 h time period with IC50 values of 14.5 µM and 24.5 µM respectively. Detailed Western blot analysis performed using fatostatin and PF429242 at 72 h time point led to significant decrease in the levels of the active form of SREBP1 and its downstream signaling proteins such as FAS, SCD-1 and HMGCoAR and the mutant form of tumor suppressor protein, p53, levels in comparison to the levels observed in vehicle treated control group of MIA PaCa-2 pancreatic cells over the same time period. Our in vitro data suggest that SREBP1 may contribute to pancreatic tumor growth and its inhibitors could be considered as a potential target in the management of pancreatic cancer cell proliferation.

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.

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.

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.

Impact of Hybrid-polar Histone Deacetylase Inhibitor m-Carboxycinnamic Acid bis-Hydroxyamide on Human Pancreatic Adenocarcinoma Cells.

BACKGROUND: Histone deacetylase inhibitors (HDACIs) have got immense importance as promising drugs for cancer treatment as these inhibitors regulate cellular differentiation, gene expression, cell cycle arrest and apoptosis. The current study investigates the effect of the hybrid-polar HDACI m-carboxycinnamic acid bishydroxyamide (CBHA) on the growth of human pancreatic adenocarcinoma cells, using the cell line MIA PaCa- 2 as an in vitro model. METHODS: Following CBHA treatment of the MIA PaCa-2 cells, we characterized the effect of CBHA by in vitro cytotoxicity evaluation, clonogenic assay, cell cycle analysis, immunoblotting for soluble and insoluble fractions of tubulin, immunofluorescence and caspase-3 assay. RESULTS: We observed that the histone deacetylase inhibitor CBHA markedly impaired growth of the pancreatic cancer cells by resulting in dose-dependent G2/M arrest, disruption of microtubule organization, induction of caspase-mediated apoptosis and in vitro suppression of HDAC6. Our study also shows that inhibition of HDAC6 by CBHA induced acetylation of α-tubulin. CONCLUSION: Together, our findings show that CBHA can be a potential plausible therapeutic that could be exploited for pancreatic cancer therapy.

Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin.

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was isolated from a soil sample and was identified by morphological, biochemical and 16S rRNA sequence. Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 °C. The enzyme exhibited maximum reaction rate (V max) at 74.65 U/mg and Michaelis-Menten constant (K m) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT assay and Acridine orange-ethidium bromide staining. This approach of in-situ allicin generation suggests a novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent allicin.

Metabolic Consequences of LDHA inhibition by Epigallocatechin Gallate and Oxamate in MIA PaCa-2 Pancreatic Cancer Cells.

Lactate dehydrogenase A (LDHA) is the enzyme that converts pyruvate to lactate and oxidizes the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+. Several human cancers including the pancreas display elevated expression of LDHA. Because of its essential role in cancer metabolism, LDHA has been considered to be a potential target for cancer therapy. Recently, we have shown that a green tea extract significantly down-regulated LDHA in HPAF-II pancreatic cancer cells using global proteomics profiling. The present study is to investigate how EGCG, a major biological active constituent of green tea, targets the metabolism of human pancreatic adenocarcinoma MIA PaCa-2 cells. We compared the effect of EGCG to that of oxamate, an inhibitor of LDHA, on the multiple metabolic pathways as measured by extracellular lactate production, glucose consumption, as well as intracellular aspartate and glutamate production, fatty acid synthesis, acetyl-CoA, RNA ribose and deoxyribose. Specific metabolic pathways were studied using [1, 2-13C2]-d-glucose as the single precursor metabolic tracer. Isotope incorporations in metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). We found that the EGCG treatment of MIA PaCa-2 cells significantly reduced lactate production, anaerobic glycolysis, glucose consumption and glycolytic rate that are comparable to the inhibition of LDHA by oxamate treatment. Significant changes in intracellular glucose carbon re-distribution among major glucose-utilizing macromolecule biosynthesis pathways in response to EGCG and oxamate treatment were observed. The inhibition of LDHA by EGCG or oxamate impacts on various pathways of the cellular metabolic network and significantly modifies the cancer metabolic phenotype. These results suggest that phytochemical EGCG and LDHA inhibitor oxamate confer their anti-cancer activities by disrupting the balance of flux throughout the cellular metabolic network.

The effects of secretory clusterin on oxidative damage in MIA PaCa-2 cells treated by gemcitabine and preliminary mechanism of resistance to gemcitabine / 中国癌症杂志

Background and purpose: Gemcitabine (GEM) is a first-line chemotherapy drug for pancreatic cancer. With the emergence of clinical drug resistance, the efficacy of chemotherapy has been greatly reduced, while the expression of secretory clusterin (sCLU) was closely related to chemotherapy resistance in multiple tumors. This study aimed to explore the effects of secretory clusterin on oxidative damage in MIA PaCa-2 cells treated by GEM and preliminary mechanism of resistance to GEM. Methods: MIA PaCa-2 was exposed to GEM and sCLU intervened groups with different concentrations (0, 0.63, 1.25, 2.50, 5.00 and 10.0 μg/mL) for 24 hours. The intervened concentration of GEM was 5.4 μmol/L. The inhibition rates of cell proliferation were determined by CCK-8. Cell reactive oxygen species (ROS) was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. Superoxide dismutase (SOD) activity and catalase (CAT) activity were measured by their corresponding assay kits respectively. Results: Compared with the negative control group (0 μg/mL), the inhibition rates of the GEM groups and sCLU intervened groups were significantly increased (P＜0.05) in a distinct dose-effect manner. At a low concentration of 0.63 μg/mL, the inhibition rates of the GEM groups were higher than those of the sCLU intervened groups, while the trend was reversed in high concentration range. Compared with the negative control group (0 μg/mL), the intracellular ROS levels, SOD and CAT activity of the GEM and sCLU intervened groups significantly increased (P＜0.05). ROS levels presented a distinct dose-effect relationship while the SOD and CAT activities increased first and then decreased along with the increase of GEM concentrations. The ROS levels of the GEM group were lower than those of the sCLU intervened group at the same dose (P＜0.05). The SOD activities of the GEM group were higher than those of the sCLU intervened group, while the CAT activities were opposite at the concentrations of 5.00 and 10.00 μg/mL (P＜0.05). Conclusion: GEM exposure can inhibit the growth of MIA PaCa-2 cells. After GEM exposure, the ROS levels, SOD and CAT activity of MIA PaCa-2 cells can be changed by sCLU intervention. GEM resistance could be regulated by sCLU through oxidative damage effect.