Microarray-based detection and expression analysis of drug resistance in an animal model of peritoneal metastasis from colon cancer.

Chemotherapy drugs efficiently eradicate rapidly dividing differentiated cells by inducing cell death, but poorly target slowly dividing cells, including cancer stem cells and dormant cancer cells, in the later course of treatment. Prolonged exposure to chemotherapy results in a decrease in the proportion of apoptotic cells in the tumour mass. To investigate and characterize the molecular basis of this phenomenon, microarray-based expression analysis was performed to compare tHcred2-DEVD-EGFP-caspase 3-sensor transfected C-26 tumour cells that were harvested after engraftment into mice treated with or without 5-FU. Peritoneal metastasis was induced by intraperitoneal injection of C-26 cells, which were subsequently reisolated from omental metastatic tumours after the mice were sacrificed by the end of the 10th day after tumour injection. The purity of reisolated tHcred2-DEVD-EGFP-caspase 3-sensor-expressing C-26 cells was confirmed using FLIM, and total RNA was extracted for gene expression profiling. The validation of relative transcript levels was carried out via real-time semiquantitative RT‒PCR assays. Our results demonstrated that chemotherapy induced the differential expression of mediators of cancer cell dormancy and cell survival-related genes and downregulation of both intrinsic and extrinsic apoptotic signalling pathways. Despite the fact that some differentially expressed genes, such as BMP7 and Prss11, have not been thoroughly studied in the context of chemoresistance thus far, they might be potential candidates for future studies on overcoming drug resistance.

Surface Modification Strategies for Chrysin-Loaded Iron Oxide Nanoparticles to Boost Their Anti-Tumor Efficacy in Human Colon Carcinoma Cells.

Enhancing nanoparticles' anti-cancer capabilities as drug carriers requires the careful adjustment of formulation parameters, including loading efficiency, drug/carrier ratio, and synthesis method. Small adjustments to these parameters can significantly influence the drug-loading efficiency of nanoparticles. Our study explored how chitosan and polyethylene glycol (PEG) coatings affect the structural properties, drug-loading efficiency, and anti-cancer efficacy of Fe3O4 nanoparticles (NPs). The loading efficiency of the NPs was determined using FTIR spectrometry and XRD. The quantity of chrysin incorporated into the coated NPs was examined using UV-Vis spectrometry. The effect of the NPs on cell viability and apoptosis was determined by employing the HCT 116 human colon carcinoma cell line. We showed that a two-fold increase in drug concentration did not impact the loading efficiency of Fe3O4 NPs coated with PEG. However, there was a 33 Å difference in the crystallite sizes obtained from chitosan-coated Fe3O4 NPs and drug concentrations of 1:0.5 and 1:2, resulting in decreased system stability. In conclusion, PEG coating exhibited a higher loading efficiency of Fe3O4 NPs compared to chitosan, resulting in enhanced anti-tumor effects. Furthermore, variations in the loaded amount of chrysin did not impact the crystallinity of PEG-coated NPs, emphasizing the stability and regularity of the system.

A Systematic Review and Bayesian Network Meta-Analysis on the Effect of Different Anticoagulants on the Prophylaxis of Post-Thrombotic Syndrome after Deep Venous Thrombosis.

BACKGROUND: Postthrombotic syndrome (PTS) has a major impact on the quality of life after deep venous thrombosis (DVT). From clinical practice and related trials, anticoagulants show potential for reducing the occurrence and alleviating the symptoms of PTS. METHODS: A systematic review and Bayesian network meta-analysis (NMA) were conducted by combing the literature from the databases of MEDLINE, Embase, Web of Science, Cochrane Libraries, and ClinicalTrials, through a variety of medical subject headings (Mesh) and PTS keywords. With regard to PTS prophylaxis, all anticoagulant-related randomized controlled trials (RCTs) and observational studies were assessed. The network model was conducted through the R software, and further comparisons were conducted using the Bayesian hierarchical random effects model. The odds ratio (OR) and the corresponding 95% CI were calculated for analysis. RESULTS: Data from two RCTs and nine non-randomized studies meeting the selection criteria were included in the Bayesian analysis model, which incorporated seven anticoagulants. Edoxaban (OR: 0.42, 95% CI: 0.18-1.0) and rivaroxaban (OR: 0.54, 95% CI: 0.38-0.76) were significantly more effective than warfarin in the prevention of PTS (Villalta score ≥ 5). A subgroup analysis based on the severity of PTS showed that rivaroxaban was more effective than warfarin, with OR: 0.59, 95% CI: 0.41-0.84 (Villalta score 5 to 14) and OR: 0.48, 95% CI: 0.22-0.9 (Villalta score ≥ 15, ulceration), respectively. Edoxaban had the highest probability (80.1%) of providing preventive benefits for PTS. For mild/moderate and severe PTS, rivaroxaban provided the highest benefits in preventing PTS (89.3% and 85.6%, respectively). CONCLUSION: Edoxaban demonstrated a better prophylactic effect on PTS (Villalta score > 5), while rivaroxaban displayed a better effect against mild/moderate (Villalta score 5 to 14) and severe PTS (Villalta score ≥ 15, ulceration).

Association of miR-149 T>C and miR-196a2 C>T Polymorphisms with Colorectal Cancer Susceptibility: A Case-Control Study.

The principal aim of the current study was to investigate the relationship between miR-149 T>C (rs2292832) and miR-196a2 C>T (rs11614913) small non-coding RNA polymorphisms and the risk of developing CRC in the Azerbaijani population. The study included 120 patients diagnosed with CRC and 125 healthy individuals. Peripheral blood samples were collected from all the subjects in EDTA tubes and DNA extraction was performed by salting out. Polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. While comparing without gender distinction no statistical correlation was found between the heterozygous TC (OR = 0.66; 95% CI = 0.37-1.15; p = 0.142), mutant CC (OR = 1.23; 95% CI = 0.62-2.45; p = 0.550), and mutant C (OR = 1.03; 95% CI = 0.72-1.49; p = 0.859) alleles of the miR-149 gene and the CT (OR = 1.23; 95% CI = 0.69-2.20; p = 0.485), mutant TT (OR = 1.29; 95% CI = 0.67-2.47; p = 0.452), and mutant T (OR = 1.17; 95% CI = 0.82-1.67; p = 0.388) alleles of the miR-196a2 gene and the risk of CRC. However, among women, miR-149 TC (OR = 0.43; 95% CI = 0.19-1.01; p = 0.048) correlated with a reduced risk of CRC, whereas miR-196a2 CT (OR = 2.77; 95% CI = 1.13-6.79; p = 0.025) correlated with an increased risk of CRC. Our findings indicated that miR-149 T>C (rs2292832) might play a protective role in the development of CRC in female patients, whereas the miR-196a2 (rs11614913) polymorphism is associated with an increased risk of CRC in women in the Azerbaijani population, highlighting the importance of gender dimorphism in cancer etiology.

Analysis of Inflammation-Related Genes in Patients with Stanford Type A Aortic Dissection.

Background: Aortic dissection (AD) is a life-threatening cardiovascular disease. Pathophysiologically, it has been shown that aortic wall inflammation promotes the occurrence and development of aortic dissection. Thus, the aim of the current research was to determine the inflammation-related biomarkers in AD. Methods: In this study, we conducted differentially expressed genes (DEGs) analysis using the GSE153434 dataset containing 10 type A aortic dissection (TAAD) and 10 normal samples downloaded from the Gene Expression Omnibus (GEO) database. The intersection of DEGs and inflammation-related genes was identified as differential expressed inflammation-related genes (DEIRGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for DEIRGs. We then constructed the protein-protein interaction (PPI) network using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and identified hub genes using the Cytoscape plugin MCODE. Finally, least absolute shrinkage and selection operator (LASSO) logistic regression was used to construct a diagnostic model. Results: A total of 1728 DEGs were identified between the TAAD and normal samples. Thereafter, 61 DEIRGs are obtained by taking the intersection of DEGs and inflammation-related genes. The GO indicated that DEIRGs were mainly enriched in response to lipopolysaccharide, in response to molecules of bacterial origin, secretory granule membrane, external side of plasma, receptor ligand activity, and signaling receptor activator activity. KEGG analysis indicated that DEIRGs were mainly enriched in cytokine-cytokine receptor interaction, TNF signaling pathway, and proteoglycans in cancer. We identified MYC, SELL, HIF1A, EDN1, SERPINE1, CCL20, IL1R1, NOD2, TLR2, CD69, PLAUR, MMP14, and HBEGF as hub genes using the MCODE plug-in. The ROC indicated these genes had a good diagnostic performance for TAAD. Conclusion: In conclusion, our study identified 13 hub genes in the TAAD. This study will be of significance for the future development of a preventive therapy of TAAD.

MTHFR Gene C677T Polymorphism (rs1801133) and Susceptibility to Colorectal Polyps in an Azerbaijani Population.

BACKGROUND: Understanding the relationships between the methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, colorectal polyps, and CRC risk can aid in advancing personalized medicine approaches in CRC prevention. The aim of the current study is to identify the association of C677T polymorphism of the MTHFR gene with the risk of colorectal polyps in the Azerbaijani population. METHODS: This study included 125 patients with colon polyps and 155 healthy individuals as a control group. DNA was extracted from venous blood samples obtained from patients and healthy individuals, and the results were analyzed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and agarose gel electrophoresis. RESULTS: Wild-type, heterozygote, and homozygous mutant were revealed within 69 (55.2%), 49 (39.2%), and 7 (5.6%) patients and within 100 (64.5%), 45 (29%), and 10 (6.5%) healthy controls, respectively. However, no significant statistical associations were observed between CT and TT genotypes, dominant (CC vs. CT + TT) and recessive (CC + CT vs. TT) models, and the mutant T allele and disease risk. There were also no significant differences between patients and controls regarding age, sex, smoking and alcohol use. CONCLUSION: Our research did not reveal any significant association between the MTHFR C677T polymorphism and susceptibility to colorectal polyps in the Azerbaijan population.

Overview of Physicochemical Properties of Nanoparticles as Drug Carriers for Targeted Cancer Therapy.

The advent of nanotechnology has brought about revolutionary innovations in biological research techniques and medical practice. In recent years, various "smart" nanocarriers have been introduced to deliver therapeutic agents specifically to the tumor tissue in a controlled manner, thereby minimizing their side effects and reducing both dosage and dosage frequency. A large number of nanoparticles have demonstrated initial success in preclinical evaluation but modest therapeutic benefits in the clinical setting, partly due to insufficient delivery to the tumor site and penetration in tumor tissue. Therefore, a precise understanding of the relationships betweenthe physicochemical properties of nanoparticles and their interaction with the surrounding microenvironment in the body is extremely important for achieving higher concentrations and better functionality in tumor tissues. This knowledge would help to effectively combine multiple advantageous functions in one nanoparticle. The main focus of the discussion in this review, therefore, will relate to the main physicochemical properties of nanoparticles while interacting within the body and their tuning potential for increased performance.

TP53 Codon 72 Polymorphism and the Risk of Colorectal Cancer in an Azerbaijani Population.

BACKGROUND: We aimed to evaluate the association between the TP53 Arg72Pro gene polymorphism and risk of colorectal cancer in an Azerbaijani population. METHODS: A total of 141 patients with colorectal cancer and 150 gender- and age-matched controls were involved in the study. The genotypes of the TP53 gene Arg72Pro polymorphism were detected by polymerase chain reaction-based restriction fragment length polymorphism analysis. RESULTS: We found that the heterozygous genotypes Arg/Pro (odds ratio, 1.128; 95% CI, 0.657-1.937) and mutant Pro/Pro (odds ratio, 1.274; 95% CI, 0.648-2.504) were more frequent in colorectal cancer patients compared to healthy controls. The frequency of the mutant Pro allele (odds ratio, 1.122; 95% CI, 0.809-1.554) was revealed in 47.5% of colorectal cancer patients and in 44.7% of healthy controls. There was no association observed between TP53 Arg72Pro polymorphism and risk of colorectal cancer in an Azerbaijani population (P > .05). CONCLUSION: Our findings indicate a lack of relationship between TP53 Arg72Pro polymorphism and risk of colorectal cancer. Furthermore, we have found no statistical differences in the frequency of genotype and allele by sex, age, histological grade, tumor stage, smoking status, and alcohol consumption in this study.

Investigation of the DNMT3B -579 G>T Promoter Polymorphism in Patients with Colorectal Cancer in an Azerbaijani Population.

OBJECTIVE: The main aim of the present study was to determine the clinical significance of the DNA methyltransferase 3B (DNMT3B) gene -579 G>T polymorphism in colorectal cancer (CRC) patients. METHODS: A total of 140 patients with CRC and 164 healthy individuals were included in the study. According to the manufacturer's instructions, DNA was isolated from blood, and genotypes were determined on agarose gel by the PCR-RFLP method. Genotype confirmation was performed using Sanger sequencing in randomly selected samples. RESULTS: When comparing the case and control groups, heterozygous GT (OR=0.53; 95% CI=0.32-0.88), under the dominant model (OR=0.53; 95% CI=0.33-0.87), and the mutant T allele (OR=0.71; 95% CI=0.51-0.98) were statistically associated with a reduced risk of CRC. However, when the age, pathological tumor grade and stage, smoking habit, and alcohol consumption were compared, no significant relationship was determined (P>0.05). Furthermore, among males, heterozygous GT was associated with a reduced risk of CRC (OR=0.40; 95% CI=0.19-0.84). CONCLUSION: Our study highlighted that the -579 G>T polymorphism of the DNMT3B gene plays a protective role against CRC development.

Downregulation of SPARC Is Associated with Epithelial-Mesenchymal Transition and Low Differentiation State of Biliary Tract Cancer Cells.

BACKGROUND: Biliary tract cancers (BTCs) have a poor prognosis. BTCs are characterized by a prominent desmoplastic reaction which possibly contributes to the aggressive phenotype of this tumor. The desmoplastic reaction includes excessive production and deposition of extracellular matrix proteins such as periostin, secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, as well as accumulation of α-smooth muscle actin-positive cancer-associated fibroblasts and immune cells, secreting growth factors and cytokines including transforming growth factor (TGF)-ß. In the present study, we investigated the expression of SPARC in BTC as well as its possible regulation by TGF-ß. METHODS: Expression levels of Sparc, TGF-ß1 and its receptor ALK5 were evaluated by quantitative real-time PCR in 6 biliary tract cell lines as well as 1 immortalized cholangiocyte cell line (MMNK-1). RNAs from tumor samples of 7 biliary tract cancer patients were analyzed for expression of Sparc, TGF-ß type II receptor (TbRII) as well as Twist and ZO-1. MMNK-1 cells were stimulated with TGF-ß for 24 h, and Sparc, ZO-1 and E-Cadherin expressions were determined. The presence of SPARC protein was analyzed by immunohistochemistry in tumor specimens from 10 patients. RESULTS: When comparing basal Sparc transcript levels in diverse BTC cell lines to MMNK-1 cells, we found that it was strongly downregulated in all cancer cell lines. The remaining expression levels were higher in highly differentiated cell lines (CCSW1, MZChA1, MZChA2 and TFK-1) than in less differentiated and undifferentiated ones (BDC, SKChA1). Expression of Sparc in BTC patient samples showed a significant positive correlation with expression of the epithelial marker ZO-1. In contrast, the mesenchymal marker Twist and the TbRII showed a trend of negative correlation with expression of Sparc in these samples. TGF-ß exposure significantly downregulated Sparc expression in MMNK-1 cholangiocytes in vitro in parallel to downregulation of epithelial markers (E-Cadherin and ZO-1). Finally, SPARC immunostaining was performed in 10 patient samples, and the correlation between absence of SPARC and survival times was analyzed. CONCLUSIONS: These data imply that a decrease in SPARC expression is correlated with dedifferentiation of BTC cells resulting in enhanced EMT being possibly mediated by TGF-ß. Thereby SPARC levels might be a marker for individual prognosis of a patient, and strategies aiming at inhibition of SPARC downregulation might have potential for new future therapies.

Fluorescent resonance energy transfer imaging of VEGFR dimerization.

Activation of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) and 2 (VEGFR2/KDR) involves receptor dimerization. Formation of VEGFR dimer has so far not been visualized in single intact cells. In the present study we describe different optical assays which can be used to observe dimerization of VEGFR1 and VEGFR2. Bimolecular fluorescence complementation (BIFC) assays confirmed homo,- and heterodimerization of transfected receptors. Fluorescence resonance energy transfer (FRET) techniques in living and fixed CHO-K1 cells allowed observation of VEGFR1 homodimer,- and VEGFR1 and VEGFR2 heterodimer formation after ligand stimulation. After inhibition of ligand binding by the VEGFA JH121 antibody VEGFR1 homodimerization was completely abolished. Under the same conditions, cells transfected by VEGFR1 and VEGFR2 maintained relevant receptor heterodimerization. These techniques to monitor VEGFR1 and VEGFR2 homo- and heterodimerization in living and fixed cells may help in the search for new angiogenesis-directed inhibitors of VEGFR dimerization.

Combination of the EGFR tyrosine kinase inhibitor AG1478 and 5-FU: no synergistic effect on EGFR phosphorylation, cell proliferation and apoptosis induction.

The combination of a fluoropyrimidine with receptor tyrosine kinase inhibitors in tumor treatment has been proposed to enhance their therapeutic efficiency. The synergism of such a combination in the treatment of colorectal carcinoma is equivocal although the epidermal growth factor receptor (EGFR) is frequently overexpressed in the tumors. We used human colorectal SW 480 cells, to analyze the EGFR phosphorylation levels. We showed that incubation of cells with 5-fluorouracil (5-FU) does not influence EGFR phosphorylation of tyrosine 1173 and the overall phosphorylation after stimulation. Inhibition of EGFR phosphorylation with AG1478 reduced cell proliferation compared to 5-FU. Cells exhibited highest apoptosis rates with 5-FU. AG1479 inhibited cell proliferation more potently than 5-FU alone. Apoptosis rates after incubation with AG1478 alone and AG1478 in combination with 5-FU were significantly lower than apoptosis induction by 5-FU alone. Therefore, no synergism of both substances can be demonstrated. This experimental data argues against the combination of EGFR-inhibitors with 5-FU in a clinical setting.

Treatment of experimental pancreatic cancer by doxorubicin-, mitoxantrone-, and irinotecan-drug eluting beads.

BACKGROUND AND AIMS: Peritoneal carcinomatosis is a common cause of death in pancreatic cancer patients. In this metastatic stage of the disease, few patients show a sustained response to therapy. In the palliative situation, targeted and compartment restricted delivery of drugs offers the opportunity to focus drugs directly to the tumor site, which is a prerequisite for avoiding toxic side effects. Here, we demonstrate the therapeutic efficiency of biocompatible polyvinyl-alcohol hydrogel drug eluting beads (DEBs) containing doxorubicin, mitoxantrone and irinotecan in vitro and in vivo in a syngenic model of experimental pancreatic cancer. METHODS: Panc02 murine pancreatic carcinoma cells were exposed to doxorubicin, mitoxantrone and irinotecan DEBs and free compounds. The effect on cell proliferation and apoptosis induction was compared. Using this cell line, peritoneal carcinomatosis was induced in C57 black6 mice. Mortality, tumor load and therapy-associated weight loss were compared after treatment of tumor-bearing mice with DEBs or free compounds. RESULTS: In vitro treatment with DEBs decreases tumor cell proliferation and induces apoptosis. The effect is less pronounced than with corresponding doses of the free drug. Repeated applications of the free drugs in vivo, however, induce significantly higher lethality and weight loss than corresponding doses of DEBs. No relevant differences in antitumoral activity were observed. Using computer tomography and HE-histology after subcutaneous and intraperitoneal injection of radiopaque beads no systemic spread of the beads could be found. CONCLUSION: DEBs show the advantage of delivering potent cytotoxic activity to the intraperitoneal tumor manifestation while maintaining a low systemic toxicity.

Doxorubicin and mitoxantrone drug eluting beads for the treatment of experimental peritoneal carcinomatosis in colorectal cancer.

We investigated the therapeutic efficiency of sulfonate-modified polyvinyl alcohol beads loaded with doxorubicin, irinotecan or mitoxantrone in vitro and in vivo in a model of experimental peritoneal carcinomatosis (PC). In vitro, cell proliferation was efficiently impaired by doxorubicin drug eluting bead (DEB) treatment while mitoxantrone DEBs were less effective than. Irinotecan showed little effect for both DEBs and free drug. Apoptosis was not different between free mitoxantrone and the DEB form while more apoptosis induction was observed in cells incubated with free doxorubicin and irinotecan. Experimental PC was produced in mice. The therapeutic efficiency of either mitoxantrone and doxorubicin DEB or free drugs were compared. Mice were treated either once on day 12 or by 3 repetitive applications on days 7, 10 and 12. Mice treated by DEBs showed less weight loss and mortality. Therapeutic effect was determined by measuring tumor volume and tumor load on the day 15 after tumor inoculation. For the single application on the day 12, an advantage could be observed for the free drugs. After 3 repeated injections of both free and mitoxantrone DEB no difference in tumor load or tumor volume could be observed. Least tumor load and tumor volume was observed in mice that received 3 repeated injections of doxorubicin DEB. No animal survived 3 injections of free doxorubicin. We conclude that bead encapsulation of chemotherapeutic drugs may show the advantage of less toxicity in peritoneal spread of colorectal cancer.

Quantitative imaging of apoptosis commitment in colorectal tumor cells.

We have studied caspase-3 activation by combined DNA damage induction and EGFR kinase inhibition in order to identify potential EGFR-mediated survival signals conferring resistance to apoptosis in human colorectal tumor cells. The onset of apoptosis was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1, enabling us to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy or fluorescence correlation spectroscopy. Both optical approaches provide parameters quantitatively reporting the ratio between cleaved and uncleaved sensor, thereby facilitating the comparison of caspase-3 activation between different cells. Using these methods, we show that EGFR kinase inhibitors sensitize colorectal SW-480 tumor cells for 5-fluorouracil-induced apoptosis, indicating that EGFR-mediated survival signaling contributes to apoptosis resistance via its intrinsic kinase activity.