Silver nanoparticles modify the hypothalamic-pituitary-interrenal axis and block cortisol response to an acute stress in zebrafish, Danio rerio.

The present study aimed at assessing the effects of exposure to silver nanoparticle (AgNP) and a subsequent acute stress on the expression of various genes involved in the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish, Danio rerio. The fish were exposed to 0 (Control), 0.1 (LC), 0.4 (MC), and 1.2 (HC) mg Ag/L (as AgNP) over a 2-week period, followed by an acute air exposure stress. The whole body cortisol and the expression of selected genes in the fish brain and kidney were analyzed, before and after the acute stress. The results showed that AgNP increased basal cortisol levels and the expression of corticotropin releasing factor, prohormone convertase 1, pro-opiomelanocortin, and melanocortin 2 receptor; however, it suppressed/inhibited whole body cortisol, brain corticotropin releasing factor responses, pro-opiomelanocortin, and the kidney melanocortin 2 receptor responses to the acute stress. AgNP down-regulated the expression of the steroidogenic acute regulatory protein, but it intensified the gene expression in response to the acute stress. Before the acute stress, LC treatment exhibited an up-regulation in Cytochrome P450-11A-1 expression, but MC and HC treatments induced down-regulation. After the acute stress, the AgNP-exposed fish exhibited decreased Cytochrome P450-11A-1 expressions, compared with the Control. Exposure to AgNP significantly increased Cytochrome P450-11B expression. However, after the acute stress, LC treatment exhibited an up-regulation, but MC and HC treatments exhibited down-regulation in the Cytochrome P450-11B gene expression. In conclusion, AgNP suppressed cortisol response to stress, which appears to be a consequence of alterations in the HPI axis at the transcriptomic levels.

Fabrication of nano-decorated ZnO-fibrillar chitosan exhibiting a superior performance as a promising replacement for conventional ZnO.

In this research, bioactive nano-hybrids based on the nano-fibrillar chitosan-ZnO (NF-CS-ZnO) were synthesized to diminish the toxicity of ZnO-NPs. The successful formation of nano-hybrids was confirmed by FT-IR, UV-Vis, and FE-SEM analyses, showing a uniform spherical ZnO-NPs with an average diameter of 20-30 nm, homogeneously dispersed on NF-CS. The obtained results demonstrated a remarkable antibacterial activity of NF-CS-ZnO-0.6 nano-hybrid against E. coli and S. aureus and, interestingly, no cytotoxic on normal cells (even at a high concentration of 100 µg/mL). Furthermore, NF-CS hybridization efficiently decreased the up-regulation in Cas3, Cas9, and Il6 of inspected fishes compared to the ZnO-NPs. Histopathological examination revealed hepatocyte necrosis in the fish exposed to ZnO-NPs and hyperemia exposed to NF-CS-ZnO-0.6 nano-hybrid. Finally, NF-CS efficiently improved the bio-safety and bactericidal activity of ZnO-NPs; therefore, NF-CS-ZnO nano-hybrid is prominently recommended as a talented low-toxicity antibacterial agent replacement of conventional ZnO-NPs for use in different applications.

Design and characterization of keratin/PVA-PLA nanofibers containing hybrids of nanofibrillated chitosan/ZnO nanoparticles.

In this paper, designing electrospun composite nanofibers containing poly (lactic acid) (PLA) and keratin/poly (vinyl alcohol) (K/PVA) as the major components and natural nanofibrillated chitosan (CHNF)/ZnO nanoparticles (ZnONPs) (CSZ) combination as the nanofiller ingredient, has been investigated. PLA solution from one syringe and K/PVA from another one with incorporation of CHNF (CS), CSZ (2:1), (1:1) and (1:2) were electrospun and produced nanofibers were formed on the rotating collector. Addition of CHNF and ZnONPs amounts in CSZ combination resulted in reduction of the diameter of nanofibers. The highest hydrophilicity was reported for K/PVA/CS-PLA/CS sample with the contact angle of about 43 ± 3°. AFM results for K/PVA-PLA, K/PVA/CS-PLA/CS and K/PVA/CSZ(2:1)-PLA/CSZ(2:1), K/PVA/CSZ(1:2)-PLA/CSZ(1:2) samples indicated that the surface roughness factor for these nanofibers was about 708, 277, 378 and 658 nm, respectively. DSC analysis for K/PVA/CSZ(1:2)-PLA/CSZ(1:2) structure exhibited that the peaks related to the melting points of PLA and PVA shifted to higher temperatures. Overally, K/PVA/CSZ(2:1)-PLA/CSZ(2:1) nanofiber with diameter of 352.50 ± 31 nm, contact angle of 48 ± 3°, tensile strength of 0.96 ± 0.18 MPa is suggested as a proper wound healing scaffold that has highest antibacterial as well as potential to increase cell proliferation.

Enhanced radiotherapy efficacy of breast cancer multi cellular tumor spheroids through in-situ fabricated chitosan-zinc oxide bio-nanocomposites as radio-sensitizing agents.

Overwhelming evidence has shown that three-dimensional multicellular tumor spheroids (MCTSs) as a mimic of in-vivo tumor can accurately exhibit cellular responses to treatments. So, we compared the capability of pure zinc oxide nanoparticles (ZnO-NPs) and chitosan-ZnO bio-nanocomposites (CS-ZnO BNCs) for enhancing the radiosensitization of MDA-MB-231 breast cancer cells (BCCs) in the 3D-MCTSs model. ZnO-NPs and CS-ZnO BNCs were synthesized by a facile co-precipitation method. FE-SEM images revealed that the uniform spherical ZnO-NPs with an average diameter of 35 nm were successfully dispersed on chitosan. MDA-MB-231 MCTSs which were formed in a non-adherent culture plate, possessed functional features of in-vivo tumor. The priority of such culture method to conventionally used 2D monolayer (or parental) cell culture is the mimicking of tumor microenvironment. The toxicity of CS-ZnO BNCs and ZnO-NPs against the MDA-M-231 BCCs was evaluated using MTT-colorimetric assay, which demonstrated superior biocompatibility of CS-ZnO BNCs compared to pure ZnO-NPs (even at high concentration of 100 µg/mL). Survival fraction analysis of cells under clinical X-ray irradiation (6 MV) showed that MCTSs had a higher radioresistance compared to parental cells. Besides, the clonogenic potential of irradiated MCTSs was significantly decreased by the addition of CS-ZnO BNCs similar to that of monolayer cells. The sensitivity enhancement ratios (SER) for MCTSs and monolayer cells were calculated 1.5 and 1.63, respectively. Further, tracking of radiobiological properties and apoptosis induction of MCTSs showed that CS-ZnO BNCs not only could lead to the creation of higher radiation-induced complex DNA break and apoptosis death in MCTSs, but also weakened DNA repair mechanisms. It was found that non-toxic concentration of CS-ZnO BNCs has promising potential to enhance radiosensitivity of resistant-MCTSs as a superior in-vitro tumor model. So, CS-ZnO BNCs can be a prominent candidate for overcoming the resistance of BCCs to radiotherapy.

Efficacy and safety of rituximab therapy in patients with systemic sclerosis disease (SSc): systematic review and meta-analysis.

The clinical benefits of rituximab in systemic sclerosis (SSc) are still contentious. The present meta-analysis aimed to systematically assess rituximab's safety and efficacy profile in SSc patients. A systematic online query was performed in PubMed, Scopus, Web of Science, and Embase. The studies on the application of rituximab for patients with SSc were reviewed comprehensively for over two years. In terms of efficacy profile, mRSS, MS, LVEF, sPAP, FVC, DLCO, TLC, FEV, DAS, severity activity, HAQ-DI and SF36 were assessed for organ involvement and quality of life. The level of biological and immunological markers was also evaluated in SSc patients treated with RTX. In total, 24 studies met the criteria. Although they did not have a high quality, they were free from heterogeneity and publication bias. The pooled results revealed a long-term improvement in mRSS and MS. HAQ-DI was improved to 0.78 after 12 months, and DAS was significantly reduced to 0.33, 0.23, and 0.24 following 6, 12, and 24 months of treatment, respectively (p = 0.00 for both parameters). The rest of the parameters remained stable over time in patients with SSc. The pooled analysis of these patients demonstrated that the induction of death, cancer, infection, and infusion were 9, 5, 18 and 10%, respectively. Based on the pooled results of this meta-analysis, rituximab improves skin score and disease indices and stabilizes organ involvement in SSc patients. Rituximab seems to possess reasonable safety, similar to previous data from other autoimmune diseases.

Reducing cytotoxicity of poly (lactic acid)-based/zinc oxide nanocomposites while boosting their antibacterial activities by thymol for biomedical applications.

In the present study, ternary blends based on poly (lactic acid)/poly (ε-caprolactone)/thermoplastic starch were prepared at different concentrations of synthesized zinc oxide nanoparticles (ZnO-NPs) and thymol. The sizes of ZnO-NPs with an average diameter of about 30-50 nm were detected by FE-SEM analysis. Moreover, the effect of ZnO-NPs and thymol on morphological, FT-IR spectrum, UV absorption, thermal stability, cytotoxicity, and antibacterial properties of neat blend was investigated. TGA analysis showed that the addition of ZnO-NPs and/or thymol diminished thermal stability of the system. Incorporating ZnO-NPs improved antibacterial activities of the neat blend, but MTT-assay and AO fluorescent staining test results depicted a decrease in cell viability to less than 20% by the addition of 5 wt% ZnO-NPs. In such a condition, the addition of thymol to the nanocomposites exhibited a dose-dependent increase in cell survival mostly due to thymol antioxidant properties. Interestingly, the antibacterial performance of compounds was also improved by the presence of thymol. Therefore, the obtained nanocomposites have potential to extend applications of innovative biomedical devices for future research in which both high cell viability and superior antibacterial properties are needed such as an antibacterial wound healing film.

Assessment of left ventricular torsion in long axis view in patients with chronic stenosis of coronary arteries.

BACKGROUND: Left ventricular torsion is one of the most important biomechanical parameters of heart that routinely is measured in short axis view. A review of the literature has indicated that assessment of left ventricular torsion in short axis view has some limitations. In the present study, we evaluated whether torsion angle assessment in long axis view can be used as a diagnostic biomechanical marker in patients with coronary artery disease (CAD). METHODS: We assessed 20 males and 15 females who suffered from CAD and 24 healthy males and females. Two dimensional echocardiography images were scanned in cine loop format position throughout four cardiac cycles at basal and apical levels in the long axis view (4CH). Peak torsion angle in long axis view was obtained by speckle tracking method under block matching algorithm. RESULTS: In long axis view, peak torsion angle and time of peak torsion angle were similar in female (34.87±5.8˚, 287±18ms) and males (33.26±5.60˚, 295 22ms) while they were significantly decreased and increased in CAD patients (female: 24.91±3.5˚, 345±26ms and male: 24.15±2.16˚, 358±24 ms) in comparison to healthy subjects. The peak torsion angle reduction in CAD patients was a subsequent of reduced rotation angle of basal and apical levels (P<0.001). CONCLUSION: The results showed that sex difference did not influence torsion angle of the left ventricle. It is concluded that left ventricular torsion assessment in long axis view has the potential to distinct patients with CAD from healthy subjects in routine echocardiography evaluation.

Efficient co-cultivation of human fibroblast cells (HFCs) and adipose-derived stem cells (ADSs) on gelatin/PLCL nanofiber.

In this study, we investigated whether the nanofibers produced by natural-synthetic polymers can probably promote the proliferation of co-cultured adipose-derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L-lactide co-ɛ-caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real-time polymerase chain reaction were applied to assess the proliferation of co-cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor-basic and transforming growth factor-ß1 were also measured in culture medium of co-cultured ADSs/HDCs using enzyme-linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co-culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non-coated plate. Also, synthesis of collagen and cytokines secretion of co-cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non-coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co-cultured ADSs/HFCs in wound healing process.

Investigating the best strategy to diminish the toxicity and enhance the antibacterial activity of graphene oxide by chitosan addition.

The main objective of this work was to find a way to increase the bio-applicability of graphene oxide (GO) nanoparticles. In this way, various kinds of graphene oxide-chitosan (GO-CS) nano-hybrids were synthesized through attachment of different kinds of chitosan (CS) structures with GO. Subsequently, they were assessed in terms of structural characterization, antibacterial activity and cytotoxicity to obtain a hybrid structure representing the highest bactericidal and biocompatibility performance. Our results revealed that the single-layer GO and also three different kinds of GO-CS nano-hybrid structures (pristine powder, spherical and nano-fibrilar network structures) were successfully synthesized. Antibacterial activity results indicated superior antibacterial activity of nano-hybrids compared to the pure GO. In addition, it was observed that the attachment of CS to GO interestingly reduced the cytotoxicity effect of GO and even caused cell proliferation in some samples. Furthermore, the antibacterial and bio-safety properties of different hybrids were compared and suggestive mechanisms for their particular performances were proposed.

Identification of the crucial parameters regarding the efficacy of ribavirin therapy in Crimean-Congo haemorrhagic fever (CCHF) patients: a systematic review and meta-analysis.

OBJECTIVES: Recently, ribavirin has been suggested as a therapeutic approach in Crimean-Congo haemorrhagic fever (CCHF) patients; however, there are controversial findings about its efficacy. In the current study, a meta-analysis was systematically performed to assess the effectiveness of ribavirin administration regarding CCHF patient survival and to explore the most important influential parameters for its efficacy. METHODS: All of the outcomes of the clinically studied CCHF patients who were treated with ribavirin were included in the meta-analysis. RESULTS: Overall, 24 studies met our criteria. Although the studies did not have high quality there was no heterogeneity and publication bias across studies. The results indicated that the administration of ribavirin to CCHF patients significantly decreased the mortality rate (by 1.7-fold) compared with those who did not receive this medication. Furthermore, it was found that the prescription of ribavirin in the initial phase of disease was more effective, and a delay in the start of treatment resulted in a 1.6-fold increase in mortality rate. In addition, interventional therapy resulted in an ∼2.3-fold reduction in the mortality rate of those who received ribavirin along with corticosteroids compared with those who were treated with ribavirin monotherapy. CONCLUSIONS: This meta-analysis reveals that ribavirin should be considered as a crucial antiviral drug in the therapeutic approach used for CCHF patients, especially in early phases of the disease. Additionally, it seems that the administration of corticosteroids alongside ribavirin can play an effective role in alleviation of the disease status, particularly in haemorrhagic phases.

The most effective polymorphisms of paraoxonase-1 gene on enzyme activity and concentration of paraoxonase-1 protein in type 2 diabetes mellitus patients and non-diabetic individuals: A systematic review and meta-analysis.

INTRODUCTION: Many studies have evaluated the association of paraoxonase-1 (PON1) gene polymorphisms with enzyme activity and concentration in type 2 diabetes mellitus (T2DM). However, the exact impact of these polymorphisms is not still obvious. Hence, we conducted a systematic review and meta-analysis to clarify the association of PON1 polymorphisms with its enzyme characteristics in T2DM patients and non-diabetic individuals. METHODS: We searched electronic databases including PubMed, Web of Science, Embase and Scopus for publications by April 2018. The pooled response ratio (rr) for the association and their corresponding 95% confidence intervals (CIs) were calculated using a fixed-effect model. RESULTS: Fifteen relevant studies fulfilled our inclusion criteria. The results showed a 1.25-fold increase in total PON1 activity in non-diabetic group against T2DM patients (p-value = 0.024). Also, only Q192R and L55M polymorphisms had sufficient studies to be included in the meta-analysis. All three genotypes of Q192R polymorphism showed significantly different activities between the study groups with the highest pooled effect size for RR genotype (rrQQ < rrQR < rrRR) while this difference was seen only in LL genotype of L55M polymorphism. Therefore, Q192R polymorphism was more correlated with type 2 diabetes mellitus. In case of concentration, there was no significant differences between two groups (p-value = 0.897). CONCLUSION: Current meta-analysis suggested that the observed difference of total PON1 activity was due to the different activity of various genotypes of PON1 enzyme in case of L55M and Q192R polymorphisms so that LL and RR genotypes had the most important role in the establishment of mentioned difference.

Facile and rapid in-situ synthesis of chitosan-ZnO nano-hybrids applicable in medical purposes; a novel combination of biomineralization, ultrasound, and bio-safe morphology-conducting agent.

In this study, zinc oxide nanoparticle (ZnO-NPs) and also chitosan­zinc oxide (CS-ZnO-NPs) nano-hybrid were synthesized by a rapid ultrasound assisted co-precipitation method. The morphology, chemical bonding, crystal structure, UV absorption, toxicity and antibacterial properties of the CS-ZnO-NPs and ZnO-NPs were characterized. The FE-SEM (field emission scanning electron microscopy) micrographs and XRD (X-ray diffraction) analysis revealed that the used technique led to the preparation of homogeneous, ultra-thin (thickness of 20-30 nm) and highly pure ZnO sheets for the both kinds of nanoparticles. The obtained results also demonstrated a superior performance of CS-ZnO-NPs hybrid rather than ZnO-NPs in terms of antibacterial activity, cell viability and UV absorption. It was deduced that the designed biomineralization technique was a very fast and successful strategy to provide a ZnO hybrid with elevated bacterial growth inhibition and bio-safety. Furthermore, the experimental data of antibacterial analyses were compared with the curves obtained from modified Gompertz model and good accordance was observed.

Combination of gold nanoparticles with low-LET irradiation: an approach to enhance DNA DSB induction in HT29 colorectal cancer stem-like cells.

PURPOSE: High-linear energy transfer (high LET) irradiation has significant cytotoxic effects on different cancerous stem-like cells (CSLCs) such as colorectal CSLCs. A review of the literature has indicated that the presence of gold nanoparticles (GNPs) enables low-LET irradiation to produce highly non-homogeneous dose distributions like high-LET irradiation. The purpose of this study was to evaluate the radioresponsiveness of HT29 colorectal CSLCs under low-LET irradiation (X-ray) and in the presence of GNPs. METHODS: Radioresponsiveness was evaluated using the ϒ-H2AX foci formation assay, the clonogenic assay, the cell cycle progression assay and analyses of radiobiological parameters. RESULTS: In the presence of GNPs, the survival fraction of HT29 CSLCs was significantly reduced and caused significant changes in the radiobiological parameters after irradiation. In addition, ϒ-H2AX assay showed that in the presence of GNPs, the persistent DNA double-strand breaks were significantly increased in irradiated HT29 CSLCs. The relative biological effectiveness value of GNPs with X-rays was about 1.6 for HT-29 CSLCs at the 10% of cell survival fraction (D10 level) when compared to X-rays alone. CONCLUSION: Therefore, the combination of GNPs with X-ray irradiation has the potential to kill HT29 CSLCs greater than the X-ray alone, and may be considered as an alternative for high-LET irradiation.

The most reliable surface marker for the identification of colorectal cancer stem-like cells: A systematic review and meta-analysis.

Several surface markers have been proposed for the identification and characterization of colorectal cancer stem-like cells (CR-CSLCs). However, their reliability in CR-CSLCs identification remains controversial. This study evaluated the correlation between all candidate surface marker's expression and CSLCs properties (tumorigenicity) through monitoring in vivo tumor incidence and final tumor volume. PubMed, Web of Science, and Scopus databases were systematically searched until November 2017. A total of 27 studies were found that met the inclusion criteria for cluster of differentiation 133 (CD133) and CD44 markers. Results indicated that either CD133 or CD44 positive cells caused about twofold increase in tumor volume compared with the negative cells (p < 0.05). In two groups of cells derived from primary tumors and cell lines, CD133 + cells had 25 and 1.45 times higher tumor incidence potential than CD133 - cells, respectively ( p < 0.05). Also, cohort evaluation showed that CD133 overexpression at protein level is a marker of poor overall survival in colorectal cancer (CRC) patients. While CD44 + cells displayed twofold tumorigenicity compared with the negative cells ( p < 0.05), combination of CD44 and CD133 showed about sevenfold tumorigenicity potential ( p < 0.05). In conclusion, the present meta-analysis suggests that CD133 is a robust biomarker to identify primary tumor CSLCs and can be proposed as a prognostic marker of CRC patient whereas it should be used with caution in cell lines. It seems to be more reliable to use CD133 in combination with CD44 as target biomarkers for the isolation of CR-CSLCs in both cell line and primary tumor cells populations.

Using of keratin substrate for enrichment of HT29 colorectal cancer stem-like cells.

Eradication of cancer stem-like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high-throughput testing of more effective treatments. In this study, spheroid-like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin-based substrate as a bio-mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio-/chemoresistance of HT29 cells. Moreover, cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog, Oct4, and c-MYC were increased. These data suggest that keratin can potentially be used for spheroid-like structure formation and enrichment of HT29 CSLCs. In addition, it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α2 ß1 integrin signaling pathway. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1264-1271, 2019.

Gold nanoparticles in combination with megavoltage radiation energy increased radiosensitization and apoptosis in colon cancer HT-29 cells.

PURPOSE: Gold nanoparticles (GNP) act as a radiosensitizer in radiation therapy. However, recent studies have shown contradictory evidence in terms of radiosensitization in the presence of GNP combined with X-ray megavoltage energy (MV) on different cell types. In this study, the effect of GNP on radiosensitization enhancement of HT-29 human colorectal cancer cells at MV X-ray energy was evaluated. MATERIALS AND METHODS: The cytotoxicity and radiosensitization of GNP were evaluated in HT-29 human colorectal cancer cells by MTS-assay and multiple MTS-assay, respectively. Cellular uptake was assayed using graphite furnace atomic absorption spectrometry (GFAAS). Apoptosis and cell cycle progression were determined by an Annexin V-FITC/propidium iodide (PI) kit and PI/RNase solution with flow cytometry, respectively. RESULTS: Results showed that the cell viability of the HT-29 cells was not influenced by exposure to different concentrations of GNP (10-100 µM). GNP alone did not affect the cell cycle progression and apoptosis. In contrast, GNP, in combination with radiation (9 MV), induced more apoptosis. The interaction of GNP with MV energy resulted in a significant radiosensitization enhancement compared with irradiation alone. CONCLUSION: It was concluded that GNP may work as bio-inert material on HT-29 cancer cells and their enhancement of radiosensitization may be due to increase in the absorbed irradiation dose.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Culturing in serum-free culture medium on collagen type-I-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell.

BACKGROUND: A sub-population of tumor cells termed cancer stem cells (CSCs) has an important role in tumor initiation, progression, and recurrence. Selecting a suitable procedure for isolation and enrichment of CSCs is the biggest challenge in the study of CSCs. In the present study, the role of the combination of stem cell culture medium and collagen type-I was evaluated for successful isolation and enrichment of HT-29 CSCs. MATERIALS AND METHODS: HT-29 cells were cultured in serum-containing medium (parental culture medium: Medium + 10% fetal bovine serum) and serum-free medium (stem cell culture medium); both on collagen-coated plates. Spheres forming ability and CD133 expression, as a potential marker of colorectal CSCs, were evaluated in two culture mediums. RESULTS: The results show spheroids usually give rise completely within 15 days in the stem cell culture medium on the collagen-coated plate. CD133 expression in spheroid cells (84%) is extensively higher than in parental cells (25%). Moreover, relative to parental cells, spheroid cells were more radioresistance. CONCLUSION: Finding of this study suggested that CSCs derived from colon cancer cell line (HT-29) can be propagated and form colonospheres in serum-free culture medium on collagen type-I. According to maintenance of their original phenotype in these conditions, it seems serum-free culture medium on collagen type-I is a suitable way to drug screening of HT-29 CSCs.

Multiple MTS Assay as the Alternative Method to Determine Survival Fraction of the Irradiated HT-29 Colon Cancer Cells.

A multiple colorimetric assay has been introduced to evaluate the proliferation and determination of survival fraction (SF) of irradiated cells. The estimation of SF based on the cell-growth curve information is the major advantage of this assay. In this study, the utility of multiple-MTS assay for the SF estimation of irradiated HT-29 colon cancer cells, which were plated before irradiation, was evaluated. The SF of HT-29 colon cancer cells under irradiation with 9 MV photon was estimated using multiple-MTS assay and colony assay. Finally, the correlation between two assays was evaluated. Results showed that there are no significant differences between the SF obtained by two assays at different radiation doses (P > 0.05), and the survival curves have quite similar trends. In conclusion, multiple MTS-assay can be a reliable method to determine the SF of irradiated colon cancer cells that plated before irradiation.