Alteration in the expression of the chemotherapy resistance­related genes in response to chronic and acute hypoxia in pancreatic cancer.

Pancreatic cancer is currently one of the least curable types of human cancer and remains a key health problem. One of the most important characteristics of pancreatic cancer is its ability to grow under hypoxic conditions. Hypoxia is associated with resistance of cancer cells to radiotherapy and chemotherapy. It is a major contributor to pancreatic cancer genetic instability, which local and systemic resistance that may result in poor clinical outcome. Accordingly, identifying gene expression changes in cancer resistance genes that occur under hypoxic conditions may identify a new therapeutic target. The aim of the present study was to explore the association between hypoxia and resistance to chemotherapy and determine the alteration in the expression of cancer resistance-related genes in the presence of hypoxia. Pancreatic cancer cells (PANC-1) were exposed to 8 h hypoxic episodes (<1% oxygen) three times/week for a total of 20 episodes (chronic hypoxia) or 72 h hypoxic episodes twice/week for a total of 10 episodes (acute hypoxia). The alterations in gene expression were examined using reverse transcription-quantitative PCR array compared with normoxic cells. Chemoresistance of hypoxic cells toward doxorubicin was assessed using MTT cell proliferation assay. Both chronic and acute hypoxia induced chemoresistance toward doxorubicin in PANC-1 pancreatic cancer cell line. The greatest changes occurred in estrogen Receptor Alpha Gene (ESR1) and ETS Like-1 protein (ELK1) pathways, in nucleic transcription factor Peroxisome proliferator-activated receptors (PPARs) and in a cell cycle inhibitor cyclin dependent kinase inhibitor 1A (CDKN1A). The present study demonstrated that exposing cells to prolonged hypoxia results in different gene expression changes involving pleotropic pathways that serve a role in inducing resistance in pancreatic cancer.

Differential miRNA expression of hypoxic MCF7 and PANC-1 cells.

Background: Hypoxia plays a critical role in the tumor microenvironment by affecting cellular proliferation, metabolism, apoptosis, DNA repair, and chemoresistance. Since hypoxia provokes a distinct shift of microRNA, it is important to illustrate the relative contribution of each hypoxamiR to cancer progression. Aims: The present study aims to shed light on the hypoxamiRs that are involved in pancreatic and breast cancer progression to highlight novel targets for the development of new therapies. Methods: For 20 cycles, MCF7 breast cancer cells and PANC-1 pancreatic cancer cells were subjected to chronic cyclic hypoxia, which consisted of 72 hours of hypoxia followed by 24 hours of reoxygenation. After 10 and 20 cycles of hypoxia, miRNA expression alterations were profiled using RT-PCR array and further analyzed using a visual analytics platform. The MTT cell proliferation assay was used to determine hypoxic cells' chemoresistance to doxorubicin. Results: Under chronic cyclic hypoxia, hypoxic PANC-1 cells have a comparable doubling time with their normoxic counterparts, whereas hypoxic MCF7 cells show a massive increase in doubling time when compared to their normoxic counterparts. Both hypoxic cell lines developed EMT-like phenotypes as well as doxorubicin resistance. According to the findings of miRNet, 6 and 10 miRNAs were shown to play an important role in enriching six hallmarks of pancreatic cancer in the 10th and 20th cycles of hypoxia, respectively, while 7 and 11 miRNAs were shown to play an important role in enriching the four hallmarks of breast cancer in the 10th and 20th cycles of hypoxia, respectively. Conclusions: miR-221, miR-21, miR-155, and miR-34 were found to be involved in the potentiation of hypoxic PANC-1 hallmarks at both the 10th and 20th cycles, while miR-93, miR-20a, miR-15, and miR-17 were found to be involved in the potentiation of hypoxic MCF7 hallmarks at both the 10th and 20th cycles. This variation in miRNA expression was also connected to the emergence of an EMT-like phenotype, alterations in proliferation rates, and doxorubicin resistance. The chemosensitivity results revealed that chronic cyclic hypoxia is critical in the formation of chemoresistant phenotypes in pancreatic and breast cancer cells. miR-181a and let-7e expression disparities in PANC1, as well as miR-93, miR-34, and miR-27 expression disparities in MCF7, may be associated with the formation of chemoresistant MCF7 and PANC-1 cells following 20 cycles of chronic cyclic hypoxia. Indeed, further research is needed since the particular mechanisms that govern these processes are unknown.

P2X and P2Y receptor antagonists reduce inflammation in ATP-induced microglia.

Background: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuro-excitatory substances in the microglia. The P2X4, P2X7 and P2Y12 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known if blocking P2X4, P2X7 and P2Y12 receptors is associated with the expression and the release of interleukin-1B (IL-1ß), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) in cultured neonatal spinal cord microglia. Objective: For this reason, we examined the effects of P2X4, P2X7 and P2Y12 antagonists on the expression and the release of IL-1ß, IL-6, and TNF-α in ATP-stimulated microglia. Methods: In this study, we observed the effect of A-740003, PSB-12062 and MRS 2395 (P2X4, P2X7 and P2Y12 receptors antagonist, respectively), on the expression and release of IL-1ß, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results: ATP induced the increased expression of IL-1ß, IL-6 and TNF-α at the level of messenger RNA (mRNA). ATP-evoked increase in IL-1ß, IL-6 and TNF-α mRNA expression was inhibited by the P2X4 receptor antagonist A-740003 or P2X7 receptor antagonist PSB-12062, respectively. Similarly, ATP-evoked release of IL-1ß, IL-6 and TNF-α was inhibited by A-740003 and PSB-12062. Furthermore, ATP-evoked increased expression of Iba-1, IL-1ß, IL-6 and TNF-α mRNA, and release of IL-1ß, IL-6 and TNF-α were nearly all blocked after co-administration of A-740003 plus PSB-12062. Finally, ATP-evoked increased gene expression and release of IL-1ß, IL-6 and TNF-α were also inhibited by MRS 2395 (P2Y12 antagonist). Conclusion: These observations suggest a new clue for therapeutic strategies to treat the neuro-inflammation.

Dihydrotestosterone Induces Chemo-Resistance of Triple-Negative Breast MDA-MB-231 Cancer Cells Towards Doxorubicin Independent of ABCG2 and miR-328-3p.

BACKGROUND: Androgens potentially have an important role in the biology of breast cancer, particularly triple-negative breast cancer (TNBC). Androgen receptor (AR) may offer a novel therapeutic strategy, including the use of microRNA (miRNA) molecules. We have previously shown that AR agonist, dihydrotestosterone (DHT), increases the expression of miR-328-3p in the TNBC MDA-MB-231 cells. One target of the latter miRNA is ATP-binding cassette subfamily G member 2 (ABCG2), which modulates the chemo-response of cancer cells by pumping out xenobiotics. OBJECTIVE: Using MDA-MB-231 cells as a model system for TNBC, we hypothesized that DHT would induce cell sensitivity towards doxorubicin via increasing levels of miR-328-3p and, consequently, reducing ABCG2 levels. METHODS: Chemo-response of cells towards doxorubicin, tamoxifen, and mitoxantrone was evaluated using cell viability MTT assay. Cells were transfected with both miR-328-3p mimic or antisense molecules. Real-time PCR was utilized to assess RNA levels and immunoblotting was performed to investigate levels of ABCG2 protein. PCR arrays were used to assess changes in the expression of drug response regulatory genes. RESULTS: Contrary to our hypothesis, treating MDA-MB-231 cells with DHT no effect towards tamoxifen or mitoxantrone, increased cell resistance towards doxorubicin was noted, concomitant with decreased expression of ABCG2. This under-expression of ABCG2 was also found in MCF-7 and MDA-MB-453 cells treated with DHT. Although miR-328-3p decreased ABCG2 mRNA and protein levels, the miRNA did not alter the chemo-response of cells towards doxorubicin and did not affect DHT-induced chemo-resistance. AR activation slightly decreased the expression of 5 genes, including insulin-like growth factor 1 receptor that may explain the mechanism of DHT-induced chemo-resistance of cells. CONCLUSION: DHT regulates chemo-response via a mechanism independent of ABCG2 and miR-328-3p.

In vitro anti-inflammatory and antioxidant activities of ZnFe2 O4 and CrFe2 O4 nanoparticles synthesized using Boswellia carteri resin.

The development of plant-based nano-materials is considered an eco-friendly technology because it does not involve hazardous chemicals. In this study, bimetallic ZnFe2 O4 and CrFe2 O4 nanoparticles were synthesized using an aqueous extract of Boswellia carteri resin. Synthesized ZnFe2 O4 and CrFe2 O4 nanoparticles were characterized by UV-Vis spectroscopy, FTIR, XRD, and HR-TEM. The anti-inflammatory activity was investigated in LPS-stimulated RAW 264.7 macrophages, whereas antioxidant activity was examined using a Hydrogen Peroxide Scavenging Activity Assay, Nitric Oxide Scavenging Activity Assay, and ABTS Radical Scavenging Assay. ZnFe2 O4 and CrFe2 O4 nanoparticles demonstrated a moderate scavenger of H2 O2 with IC50 values; 87.528 ± 8 µg/ml and 146.4468 ± 12 µg/ml, respectively. While they exhibited a strong scavenger of NO with IC50 values; 4.01 ± 0.7 µg/ml and 4.01 ± 0.7µg/ml, respectively. Interestingly, ZnFe2 O4 and CrFe2 O4 nanoparticles revealed an excellent anti-inflammatory activity by dose-dependently suppressing mRNA expressions of IL-1b, IL-6, and TNF-α. Also, ZnFe2 O4 and CrFe2 O4 nanoparticles suppress the protein expression of TNF-α. Together, our results proved that phyto-mediated ZnFe2 O4 and CrFe2 O4 nanoparticles using Boswellia carteri resin have great potential in biomedical applications such as anti-inflammatory and antioxidant. PRACTICAL APPLICATIONS: Our phyto-synthesized chromium iron oxide bimetallic nanoparticles (NPs) have shown a novel and potent anti-inflammatory activity, with remarkable biosafety toward tested macrophages. Zinc iron oxide bimetallic NPs exhibited anti-inflammatory effect with a lesser extent compared to the former, with moderate cytotoxicity against tested macrophages. Both zinc and chromium iron oxide NPs exhibited an equivalent antioxidant activity. Our resin-capped chromium iron oxide NPs are suggested to be a competing nonsteroidal anti-inflammatory agent; it is further recommended to establish advanced animal studies to confirm their biosafety, stability, and anti-inflammatory activity accompanied with the antioxidant activity.

Determining the Relative Gene Expression Level of Hypoxia Related Genes in Different Cancer Cell Lines.

OBJECTIVE: This study aims to identify the changes in the expression of hypoxia-inducible genes in seven different cancer cell lines that vary in their oxygen levels in an attempt to identify hypoxia biomarkers that can be targeted in therapy. Profiling of hypoxia inducible-gene expression of these different cancer cell lines can be used as baseline data for further studies. METHODS: Human cancer cell lines obtained from the American Type Culture Collection were used; MCF7 breast cancer cells, PANC-1 pancreatic cancer cells, PC-3 prostate cancer cells, SHSY5Y neuroblastoma brain cancer cells, A549 lung cancer cells, and HEPG2 hepatocellular carcinoma. In addition, we used the MCF10A non-tumorigenic human breast epithelial cell line as a normal cell line. The differences in gene expression were examined using real-time PCR array (PAHS- 032Z, Human Hypoxia Signaling Pathway PCR Array) and analyzed using the ΔΔCt method. RESULTS: Almost all hypoxia-inducible genes showed a PO2-dependent up- and down-regulated expression. Noticeable gene expression differences were identified. The most important changes occurred in the HIF1α and NF-KB signaling pathways targeted genes and in central carbon metabolism pathway genes such as HKs, PFKL, and solute transporters. CONCLUSION: This study identified possible hypoxia biomarkers genes such as NF-KB, HIF1α, HK, PFKL, and PIM1 that were expressed in all hypoxic cells. Pleiotropic pathways that play a role in inducing hypoxia directly, such as HIF1 α and NF-kB pathways, were upregulated. In addition, genes expressed only in the severe hypoxic liver and pancreatic cells indicate that severe and intermediate hypoxic cancer cells vary in their gene expression. Gene expression differences between cancer and normal cells showed the shift in gene expression profile to survive and proliferate under hypoxia.

Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production.

OBJECTIVE: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. METHODS: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using a rat aortic ring assay and in vivo using a rat excision wound model. RESULTS: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulated the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline- treated group. This effect was comparable to that induced by MEBO, the positive control. CONCLUSION: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

Synthesis, Characterization and Biological Evaluation of Metal Adamantyl 2-Pyridylhydrazone Complexes.

Four new complexes derived from adamantly containing hydrazone (APH) ligand with Cu(II) (1), Co(II) (2), Ni(II) (3) and Zn(II) (4), have been synthesized and characterized using different physicochemical methods. The structure of the ligand APH and its copper complex 1 have been established by single-crystal X-ray diffraction direct methods, which reveal that complex 1 has distorted square-pyramidal geometry. Complexes 1-4 are screened against seven human cancer cell lines namely, breast cancer cell lines (MCF7, T47D, MDA-MB-231), prostate cancer cell lines (PC3, DU145) and the colorectal cancer cell line Coco-2, for their antiproliferative activities. Complex 1 has shown a promising anticancer activity compared to the other ones. The structural and spectroscopic analysis of APH and its complexes are confirmed by DFT calculations.

Associations of CYP2A6 Gene Polymorphism with Smoking Status Among Jordanians: Gender-Related Differences.

BACKGROUND: Cytochrome P450 2A6 enzyme (CYP2A6), an essential hepatic enzyme involved in the metabolism of drugs, is responsible for a major metabolic pathway of nicotine. Variation in the activity of polymorphic CYP2A6 alleles has been implicated in inter-individual differences in nicotine metabolism. AIMS: The objective of the current study was to assess the association between the smoking status and the cytochrome P450 2A6 enzyme (CYP2A6) genotype in Jordanians. METHODS: In the current study, 218 (117 Male and 101 female) healthy unrelated Jordanian volunteers were recruited. CYP2A6*1B, CYP2A6*4 and CYP2A6*9 were determined and correlated with subject smoking status. RESULTS: *1A/*1A was the most common genetic polymorphism in the overall study population, with no significant frequency differences between smokers and non-smokers. When the population was divided according to gender, only male smokers showed a significant correlation between genotype and smoking status. Considering the CYP2A6*9 genotype, the results showed differences in distribution between smokers and non-smokers, but only women showed a significant association between CYP2A6*9 allele genotype and smoking status. CONCLUSION: The results of this study show that there is a significant association between CYP2A6*9 genotype and smoking status. They also show that CYP2A6 genotype is significantly influenced by gender.

The effect of cycling hypoxia on MCF-7 cancer stem cells and the impact of their microenvironment on angiogenesis using human umbilical vein endothelial cells (HUVECs) as a model.

BACKGROUND: Breast cancer is the most common type of cancer among females. Hypoxia mediates cancer hallmarks and results from reduced oxygen level due to irregularities in tumor vascularization or when the tumor size prevents oxygen diffusion and triggers angiogenesis to compensate for low oxygen. Cancer stem cells (CSCs) are a rare subpopulation, able to self-renew and to give rise to tumor-initiating cells. It is proposed that CSCs' secretions help to recruit endothelial cells via angiogenic factors to establish tumor vascularization. In the tumor microenvironment, the effect of hypoxia on CSCs and the impact of their secretions on triggering angiogenesis and tumor vascularization remain questionable. In this study, three-dimensional (3D) CSCs derived from MCF-7 were directly exposed to repetitive long-term cycles of hypoxia to assess its effect on CSCs and then to evaluate the role of the hypoxic CSCs' (CSCsHYP) secretions in angiogenesis using (HUVECs) as a model for tumor neovascularization response. METHODS: CSCs derived from MCF-7 cell-line were expanded under repetitive, strictly optimized, long-term/continuous and intermittent hypoxic shots for almost four months to assess hypoxic effect on CSCs, sorted based on CD44+/CD24- biomarkers. Hypoxic phenotype of CSCsHYP was evaluated by assessing the acquired chemoresistance using MTT assay and elevated stemness properties were assessed by flow cytometry. To evaluate the effect of the secretions from CSCsHYP on angiogenesis, HUVECs were exposed to CSCsHYP conditioned-medium (CdM)-in which CSCs had been previously grown-to mimic the tumor microenvironment and to assess the effect of the secretions from CSCsHYP on the HUVECs' capability of tube formation, migration and wound healing. Additionally, co-culture of CSCsHYP with HUVECs was performed. RESULTS: CSCsHYP acquired higher chemoresistance, increased stemness properties and obtained greater propagation, migration, and wound healing capacities, when compared to CSCs in normoxic condition (CSCsNOR). HUVECs' tube formation and migration abilities were mediated by hypoxic (CSCs) conditioned media (CdM). DISCUSSION: This study demonstrates that chemoresistant and migrational properties of CSCs are enhanced under hypoxia to a certain extent. The microenvironment of CSCsHYP contributes to tumor angiogenesis and migration. Hypoxia is a key player in tumor angiogenesis mediated by CSCs.

An insight into the whole transcriptome profile of four tissue-specific human mesenchymal stem cells.

Aim: Variations in the clinical outcomes using mesenchymal stem cells (MSCs) treatments exist, reflecting different origins and niches. To date, there is no consensus on the best source of MSCs most suitable to treat a specific disease. Methods: Total transcriptome analysis of human MSCs was performed. MSCs were isolated from two adult sources bone marrow, adipose tissue and two perinatal sources umbilical cord and placenta. Results: Each MSCs type possessed a unique expression pattern that reflects an advantage in terms of their potential therapeutic use. Advantages in immune modulation, neurogenesis and other aspects were found. Discussion: This study is a milestone for evidence-based choice of the type of MSCs used in the treatment of diseases.

Genotoxicity of cisplatin and carboplatin in cultured human lymphocytes: a comparative study.

Cisplatin and carboplatin are integral parts of many antineoplastic management regimens. Both platinum analogues are potent DNA alkylating agents that robustly induce genomic instability and promote apoptosis in tumor cells. Although the mechanism of action of both drugs is similar, cisplatin appears to be more cytotoxic. In this study, the genotoxic potential of cisplatin and carboplatin was compared using chromosomal aberrations (CAs) and sister-chromatid exchange (SCE) assays in cultured human lymphocytes. Results showed that cisplatin and carboplatin induced a significant increase in CAs and SCEs compared to the control group (p<0.01). Levels of induced CAs were similar in both drugs; however, the magnitude of SCEs induced by cisplatin was significantly higher than that induced by carboplatin (p<0.01). With respect to the mitotic and proliferative indices, both cisplatin and carboplatin significantly decreased mitotic index (p<0.01) without affecting the proliferative index (p>0.05). In conclusion, cisplatin was found to be more genotoxic than carboplatin in the SCE assay in cultured human lymphocytes, and that might explain the higher cytotoxicity of cisplatin.

Dihydrotestosterone regulates expression of CD44 via miR-328-3p in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype that lacks effective targeted therapeutics strategy and has poor prognosis. Targeting androgen receptor (AR) in TNBC is thought to be a promising approach. We hypothesized that AR, functioning as a transcription factor, controls cell behavior via regulating the expression of microRNA molecules (miRNAs). The expression of 84 breast cancer-specific miRNAs in MDA-MB-231 cells, a highly invasive TNBC model system, was investigated using PCR arrays following treatment of cells with 5α-dihydrotestosterone (DHT). The expression of 33 miRNAs was changed by more than 2 folds including miR-328-3p, which was up-regulated by 13 folds. Transfection of cells with either miR-328-3p mimic or anti-sense molecules decreased cell motility. DHT-mediated effect on the expression and function of CD44, a target of miR-328-3p, was investigated. CD44 expression and cell adhesion to hyaluronic acid (HA) were down-regulated when cells were treated with DHT or transfection with a miR-328-3p mimic. On the other hand, the AR antagonist, bicalutamide, or transfection of cells with miR-328-3p anti-sense molecules had the opposite effect. Cells transfected with miR-328-3p anti-sense molecules reduced the negative effect of DHT on CD44 expression and cell adhesion to HA. In addition, DHT further reduced the expression of CD44 and cell adhesion to HA in cells transfected with miR-328-3p mimic. These results strongly suggest that miRNAs can mediate AR regulation of breast cancer cells and that AR controls the expression of CD44 via miRNA-dependent and independent mechanisms.

Ligand-based computational modelling of platelet-derived growth factor beta receptor leading to new angiogenesis inhibitory leads.

Platelet derived growth factor beta receptor (PDGFR- ß) plays an important role in angiogenesis. PDGFR-ß expression is correlated with increased vascularity and maturation of blood vessels in cancer. Pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis were combined to explore the structural requirements for ligand-PDGFR-ß recognition using 107 known PDGFR-ß inhibitors. Genetic function algorithm (GFA) coupled to k nearest neighbor (kNN) and multiple linear regression (MLR) analysis were employed to generate predictive QSAR models based on optimal combinations of pharmacophores and physicochemical descriptors. The successful pharmacophores were complemented with exclusion spheres to optimize their receiver operating characteristic curve (ROC) profiles. The QSAR models and their associated pharmacophore hypotheses were validated by identification and experimental evaluation of new angiogenesis inhibitory leads retrieved from the National Cancer Institute (NCI) structural database. Two hits illustrated low micromolar IC50 values in two distinct anti-angiogenesis bioassays.