Extracellular vesicle-based formulation of doxorubicin: drug loading optimization, characterization, and cytotoxicity evaluation in tumor spheroids.

Doxorubicin (DOX) is a chemotherapeutic with considerable efficacy, but its application is limited due to cardiotoxicity. Nanoparticles can improve DOX efficacy and prevent its adverse effects. Herein, DOX-loaded extracellular vesicles (DOX-EVs) were prepared using different loading methods including incubation, electroporation, and sonication in different hydration buffers to permeabilize nanostructures or desalinize DOX for improved entrapment. Different protein:drug (µg:µg) ratios of 1:10, 1:5, and 1:2, and incubation parameters were also investigated. The optimal formulation was characterized by western blotting, electron microscopy, Zetasizer, infrared spectroscopy, and release study. The cellular uptake and efficacy were investigated in MCF-7 spheroids via MTS assay, sphere formation assay (SFA), confocal microscopy, and flow cytometry. The percentage of entrapment efficiency (EE) of formulations was improved from 1.0 ± 0.1 to 22.0 ± 1.4 using a protein:drug ratio of 1:2 and sonication in Tween 80 (0.1%w/v) containing buffer. Characterization studies verified the vesicles' identity, spherical morphology, and controlled drug release properties. Cellular studies revealed the accumulation and cytotoxicity of DOX-EVs in the spheroids, and SFA and confocal microscopy confirmed the efficacy and cellular localization. Flow cytometry results revealed a comparable and amplified efficacy for DOX-EV formulations with different cell origins. Overall, the EV formulation of DOX can be applied as a promising alternative with potential advantages.

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells.

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

Religious Affiliation and Childbearing Preferences of Iranian Women.

Religious differences in fertility are one of the essential issues in health and demographic research in Western societies. However, they have received less attention in Muslim countries. This study aims to investigate Shiite and Sunni religious groups' childbirth preferences in Iranian society. It also seeks to analyze their differences based on socioeconomic and demographic factors. We used data from 1020 married women aged 18-44 surveyed as part of Iran's National Family Survey in 2018. The study was designed quantitatively with a cross-sectional approach, and samples were selected using multi-stage cluster sampling. SPSS26 was used to run logistics and Poisson regression models. The results revealed that the means (± SD) of ideal family sizes for Shiite and Sunni women were 2.6 (± 1.0) and 3.4 (± 1.3) children, respectively. Sunni women (49%) had a higher intention to have a (or another) child than Shiite women (35%). According to the multivariate analysis findings, religion alone significantly affected the ideal family size, even when other socioeconomic and demographic variables were controlled. However, it had no significant effect on the intention to have a (or another) child within the next three years. Therefore, it is possible to conclude that within the specific social structure and cultural context of Iran, religion has explanatory power regarding childbearing beliefs and values. Furthermore, regarding fertility intentions and behaviors, demographic and socioeconomic factors become more important than religion.

NK cell upraise in the dark world of cancer stem cells.

One of the obstacles in treating different cancers, especially solid tumors, is cancer stem cells (CSCs) with their ability in resistance to chemo/radio therapy. The efforts for finding advanced treatments to overcome these cells have led to the emergence of advanced immune cell-based therapy (AICBT). Today, NK cells have become the center of attention since they have been proved to show an appropriate cytotoxicity against different cancer types as well as the capability of detecting and killing CSCs. Attempts for reaching an off-the-shelf source of NK cells have been made and resulted in the emergence of chimeric antigen receptor natural killer cells (CAR-NK cells). The CAR technology has then been used for generating more cytotoxic and efficient NK cells, which has increased the hope for cancer treatment. Since utilizing this advanced technology to target CSCs have been published in few studies, the present study has focused on discussing the characteristics of CSCs, which are detected and targeted by NK cells, the advantages and restrictions of using CAR-NK cells in CSCs treatment and the probable challenges in this process.

Overcoming the UCB HSCs -Derived NK cells Dysfunction through Harnessing RAS/MAPK, IGF-1R and TGF-ß Signaling Pathways.

BACKGROUND: The natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC-derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-ß signaling pathways using IL-15, IGF-1 and SIS3 respectively. METHODS: The CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed. RESULTS: The NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 - (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with IL-15, demonstrated a higher cytotoxicity against K562 (≥ 90%; P ≤ 0.001) and SKOV3 tumor cells (≥ 65%; P ≤ 0.001) compared to IL-15/Hsp70-activated NK cells. CONCLUSIONS: The differentiation of ex vivo expanded CD34 + cells through manipulation of RAS/MAPK, IGF-1R and TGF-ß signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.

An integrated analysis to predict micro-RNAs targeting both stemness and metastasis in human gastric cancer.

BACKGROUND AND AIM: Cancer stem cells (CSCs), a subpopulation of tumor cells, assess the capacity of self-renewal, metastasis, and therapeutic resistance. Regulation of CSCs and their epithelial to mesenchymal transition (EMT) potential is one of the promising strategies to eliminate cancer or to inhibit metastasis. Micro-RNAs (miRNAs) as regulators of several cell properties, such as self-renewal, metastasis, and resistance to the drug, could be proper targets in cancer diagnosis and therapy. The aim of the present study is to select common miRNAs targeting both self-renewal and metastasis in gastric cancer. METHODS: Stemness-related and EMT-related genes were selected by literature mining. The common miRNAs targeting genes were chosen using different databases and r programming language. The expression pattern of selected miRNAs and genes was evaluated in gastrospheres-as a gastric CSC model-and gastric tumor biopsies. RESULTS: Based on the integrated analysis, six miRNAs common to both stemness and metastasis were identified. miR-200c-3p and miR-520c-3p overexpressed in MKN-45 gastrospheres and grade III tumors. In AGS spheres, however, miR-520c-3p and miR-200c-3p upregulation and miR-34a-5p downregulation were similar to grade II tumors. Interestingly, miR-200c-3p and miR-520c-3p indicated a positive correlation with OCT4 and NOTCH1 expression in grade III tumors and MKN-45 spheres. Protein-protein network revealed that the EMT acquisition can be induced by stemness activation through intermediated core-regulatory genes, including CTNNB1, CTNND1, MAML1, KAT2A, and MAML3. CONCLUSION: The upregulation of mir-200c-3p and mir-520c-3p could effect on stemness and metastasis in gastric cancer as well as gastric CSCs. Therefore, they can be used as diagnosis and prognostic factors.

Potential theranostics of circulating tumor cells and tumor-derived exosomes application in colorectal cancer.

BACKGROUND: At the present time, colorectal cancer (CRC) is still known as a disease with a high mortality rate. Theranostics are flawless scenarios that link diagnosis with therapy, including precision medicine as a critical platform that relies on the development of biomarkers particularly "liquid biopsy". Circulating tumor cells (CTCs) and tumor-derived exosomes (TDEs) in a liquid biopsy approach are of substantial importance in comparison with traditional ones, which cannot generally be performed to determine the dynamics of the tumor due to its wide restriction of range. Thus, recent attempts has shifted towards minimally noninvasive methods. MAIN TEXT: CTCs and TDEs, as significant signals emitted from the tumor microenvironment, which are also detectable in the blood, prove themselves to be promising novel biomarkers for cancer diagnosis, prognosis, and treatment response prediction. The therapeutic potential of them is still limited, and studies are at its infancy. One of the major challenges for the implementation of CTCs and TDEs which are new trends in translational medicine is the development of isolation and characterization; a standardizable approach. This review highlights and discusses the current challenges to find the bio fluids application in CRC early detection and clinical management. CONCLUSION: Taken together, CTCs and TDEs as silent drivers of metastasis can serve in the management of cancer patient treatment and it is of the upmost importance to expand our insight into this subject. However, due to the limited data available from clinical trials, further validations are required before addressing their putative application in oncology.

Glioblastoma cancer stem cell biology: Potential theranostic targets.

Glioblastoma multiforme (GBM) is among the most incurable cancers. GBMs survival rate has not markedly improved, despite new radical surgery protocols, the introduction of new anticancer drugs, new treatment protocols, and advances in radiation techniques. The low efficacy of therapy, and short interval between remission and recurrence, could be attributed to the resistance of a small fraction of tumorigenic cells to treatment. The existence and importance of cancer stem cells (CSCs) is perceived by some as controversial. Experimental evidences suggest that the presence of therapy-resistant glioblastoma stem cells (GSCs) could explain tumor recurrence and metastasis. Some scientists, including most of the authors of this review, believe that GSCs are the driving force behind GBM relapses, whereas others however, question the existence of GSCs. Evidence has accumulated indicating that non-tumorigenic cancer cells with high heterogeneity, could undergo reprogramming and become GSCs. Hence, targeting GSCs as the "root cells" initiating malignancy has been proposed to eradicate this devastating disease. Most standard treatments fail to completely eradicate GSCs, which can then cause the recurrence of the disease. To effectively target GSCs, a comprehensive understanding of the biology of GSCs as well as the mechanisms by which these cells survive during treatment and develop into new tumor, is urgently needed. Herein, we provide an overview of the molecular features of GSCs, and elaborate how to facilitate their detection and efficient targeting for therapeutic interventions. We also discuss GBM classifications based on the molecular stem cell subtypes with a focus on potential therapeutic approaches.

HSP70/IL-2 Treated NK Cells Effectively Cross the Blood Brain Barrier and Target Tumor Cells in a Rat Model of Induced Glioblastoma Multiforme (GBM).

Natural killer (NK) cell therapy is one of the most promising treatments for Glioblastoma Multiforme (GBM). However, this emerging technology is limited by the availability of sufficient numbers of fully functional cells. Here, we investigated the efficacy of NK cells that were expanded and treated by interleukin-2 (IL-2) and heat shock protein 70 (HSP70), both in vitro and in vivo. Proliferation and cytotoxicity assays were used to assess the functionality of NK cells in vitro, after which treated and naïve NK cells were administrated intracranially and systemically to compare the potential antitumor activities in our in vivo rat GBM models. In vitro assays provided strong evidence of NK cell efficacy against C6 tumor cells. In vivo tracking of NK cells showed efficient homing around and within the tumor site. Furthermore, significant amelioration of the tumor in rats treated with HSP70/Il-2-treated NK cells as compared to those subjected to nontreated NK cells, as confirmed by MRI, proved the efficacy of adoptive NK cell therapy. Moreover, results obtained with systemic injection confirmed migration of activated NK cells over the blood brain barrier and subsequent targeting of GBM tumor cells. Our data suggest that administration of HSP70/Il-2-treated NK cells may be a promising therapeutic approach to be considered in the treatment of GBM.

An integrated analysis to predict micro-RNAs targeting both stemness and metastasis in breast cancer stem cells.

Several evidences support the idea that a small population of tumour cells representing self-renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self-renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF-7, MDA-MB231, and MDA-MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness- and EMT-related genes expression. Our results determined that miR-204, -200c, -34a, and -10b contemporarily could target both self-renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up-regulation of OCT4, SOX2, KLF4, c-MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down-regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor ß pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self-renewal and metastasis potential and eradication of breast cancer.

microRNA modified tumor-derived exosomes as novel tools for maturation of dendritic cells.

Tumor-derived exosomes (TEX) are known by their immune suppression effects as well as initiation mediators in cancer progression and metastasis. Meanwhile, they are appropriate sources to induce immunity against tumor cells, as consist of tumor specific and associated antigens. The aim of the current study is modifying TEX with microRNA miR-155, miR-142, and let-7i, to enhance their immune stimulation ability and induce potent dendritic cells (DC). For this, exosomes were isolated from mouse mammalian breast cancer cell line; 4T1, and subjected to miR-155, miR-142, and let-7i by electroporation. Immature DCs were generated from mouse bone marrow in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). To mature DCs, lipopolysaccharide (LPS), TEX, and modified TEX were used. The expression level of miRNAs and their target genes (IL-6, IL-17, IL-1b, TGFß, SOCS1, KLRK1, IFNγ, and TLR4) was determined. TEX were nanovesicles with spheroid morphology which expressed CD81, CD63, and TSG101, as exosome markers, at protein level. MHCII, CD80, and CD40 as maturation markers were assessed by flow cytometry. Overexpression of miRNAs were confirmed in exosomes and mDCs. Up and downregulation of target genes confirmed the gene network in DC maturation. We found that Let-7i could efficiently induce the DC maturation, as well as miR-142 and miR-155 have enhancing effects. These findings reveal that the modified TEX would be a hopeful cell-free vaccine for the cancer treatment.

MicroRNA-203 reinforces stemness properties in melanoma and augments tumorigenesis in vivo.

One of the challenges encountered in microRNA (miRNA) studies is to observe their dual role in different conditions and cells. This leads to a tougher prediction of their behavior as gene expression regulators. miR-203 has been identified to play a negative role in the progression of malignant melanoma; however, it has been reported, with dual effect, as both an oncomiR and tumor suppressor miRNA in some malignancies, such as breast cancer, meanwhile, the role of miR-203 in melanoma stem cells or even metastatic cells is unclear. In the present study, after observation of upregulation of miR-203 in melanoma patient's serum and also melanospheres as cancer stem cells model, we examined its overexpression on the stemness potential and migration ability of melanoma cells. Our data demonstrated that the increased miR-203 level was significantly associated with significant increase in the ability of proliferation, colony and spheres formation, migration, and tumorigenesis in A375 and NA8 cells. All of these changes were associated with enhancement of BRAF, several epithelial to mesenchymal transition factors, and stemness genes. In conclusion, our results clearly determined that miR-203 could be down-regulateddownregulated in melanoma tissues but be overexpressed in melanoma stem cells. It has an important role as oncomiR and promote repopulation, tumorigenicity, self-renewal, and migration. Therefore, we suggested overexpression of miR-203 as biomarker for early detection of metastasis. However, more studies are needed to validate our data.

Neuropathological and genomic characterization of glioblastoma-induced rat model: How similar is it to humans for targeted therapy?

Glioblastoma multiforme (GBM) is a unique aggressive tumor and mostly develops in the brain, while rarely spreading out of the central nervous system. It is associated with a high mortality rate; despite tremendous efforts having been made for effective therapy, tumor recurrence occurs with high prevalence. To elucidate the mechanisms that lead to new drug discovery, animal models of tumor progression is one of the oldest and most beneficial approaches to not only investigating the aggressive nature of the tumor, but also improving preclinical research. It is also a useful tool for predicting novel therapies' effectiveness as well as side effects. However, there are concerns that must be considered, such as the heterogeneity of tumor, biological properties, pharma dynamic, and anatomic shapes of the models, which have to be similar to humans as much as possible. Although several methods and various species have been used for this approach, the real recapitulation of the human tumor has been left under discussion. The GBM model, which has been verified in this study, has been established by using the Rat C6 cell line. By exploiting bioinformatic tools, the similarities between aberrant gene expression and pathways have been predicted. In this regard, 610 common genes and a number of pathways have been detected. Moreover, while magnetic resonance imaging analysis enables us to compare tumor features between these two specious, pathological findings provides most of the human GBM characteristics. Therefore, the present study provides genomics, pathologic, and imaging evidence for showing the similarities between human and rat GBM models.

Increased expression of miR-146a, miR-10b, and miR-21 in cancer stem-like gastro-spheres.

BACKGROUND: Gastric cancer remains one of the leading causes of cancer-associated mortalities globally. Accumulating evidence support the presence of gastric cancer stem cells (CSCs) and their role in the pathogenesis and therapeutic challenges of gastric cancer. MicroRNAs (miRNAs) may be influenced by the cellular differentiative state and as critical regulators of the cellular fate in development and cancer, can modulate the behavior of CSCs too. Here, we aimed to investigate the expression relevance of three prognostic miRNAs (miR-21, miR-10b, and miR-146a) in CSCs of AGS and MKN-45 gastric cancer cell lines. METHODS: Serial sphere-forming assay in serum-free culture medium was used to enrich the cellular population with stem-like properties. Gastro-spheres were characterized by evaluating the stemness gene expression, clonogenicity, and resistance to docetaxel and cisplatin in comparison with their parental cells. The expression level of miRNAs in gastro-spheres and their parental cells was measured using quantitative reverse transcription polymerase chain reaction. RESULTS: Gastro-spheres from both cell lines exhibit stem-like properties: upregulated stemness associated genes (P < 0.05), more colonogenicity and more resistance to docetaxel (P < 0.05). MKN-45 gastro-spheres exhibited upregulated expression of miR-21 (1.8-folds), miR-10b (1.34-folds) and miR-146a (4.8-folds; P < 0.05) compared with the parental cells. AGS-derived gastro-spheres showed upregulation of miR-21 (4.7-folds; P < 0.01), miR-10b (15.2-folds; P < 0.001) and miR-146a (39.3-folds; P < 0.05). CONCLUSION: Our data exhibited upregulation of miR-21, miR-10b, and miR-146a in the stem-like gastro-spheres; however; their function in gastric CSCs remains to be verified by further experiments.

Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains.

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.

Wnt11 alters integrin and cadherin expression by ovarian cancer spheroids and inhibits tumorigenesis and metastasis.

The present study investigated the role of Wnt11 in multicellular tumor spheroid-like structures (MCTS) ovarian cancer cell proliferation, migration and invasion in vitro and in vivo tumorigenesis and metastasis in xenograft nude mice model. Moreover, samples from human serous ovarian cancer (SOC) were used to assess the association of Wnt11 with integrins and cadherins. The data showed that Wnt11 overexpressing SKOV-3 cells became more compact accompanied by increased expression of E-and N-cadherin and lower expression of EpCAM and CD44. The α5, ß2, ß3 and ß6 integrin subunits expression levels were significantly reduced in Wnt11 overexpressing cells accompanied with significantly reduced disaggregation of Wnt11 overexpressing SKOV-3 MCTS on ECM components. Moreover, Wnt11 overexpressing SKOV-3 MCTS showed decreased migration, invasion as well as no tumor growth and metastasis in vivo. We found that Wnt11 significantly and negatively correlated with ITGB2, ITGB6, and EpCAM and positively with CDH-1 in high-grade SOC specimens. Our results suggest that Wnt11 impedes MCTS attachment to ECM components and therefore can affect ovarian cancer progression.

Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells.

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self-renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34+ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway-PD0325901 (PD)-significantly reduces the expansion of CD34+ and CD34+  CD38- cells, while there is no change in the expression of stemness-related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34+ cells. Notably, when ERK pathway is blocked in UCB-MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst-forming unit-erythroid colony (BFU-E) as well as enhancement of erythroid glycophorin-A marker. These results are in total conformity with up-regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down-regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self-renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB-haematopoietic progenitor cells.

In vitro and in vivo investigation of a novel amniotic-based chitosan dressing for wound healing.

It is more than a decade that amniotic membrane has been used as a wound dressing because of its anti-inflammatory, anti-microbial, anti-fibrotic, anti-scarring properties, as well as its pain relieving and epithelialization promoting features. However, amniotic membrane had limited applications because it needs to suture in surgery, is highly fragile, firmly adhere to the wound and may cause bleeding and pain when changing the bandage. This study investigated the possibility of development of a novel amniotic-based chitosan gel dressing as a potential wound repair substrate with marked efficacy. In this experiment, amniotic gel prepared based on chitosan/PVP gel containing human amniotic membrane extract (AME-Gel) was investigated in terms of wound-healing efficacy and scar preventive effects in a rat burn model. The levels of re-epithelialization and dermal regeneration were examined by histological assessment using H&E and Masson's trichrome staining. Also, we clarified the mechanism of healing and cytokine-releasing activities of AME as well as its effect on epithelization, angiogenesis, and fibroblast growth and migration. Our results revealed that AME-Gel induces epidermal and dermal regeneration at a shorter time through formation of granulation tissue, enhancement of fibroblast proliferation, and improvement of blood capillary formation concomitant with developing collagen bundles. Therefore, AME-Gel could be considered a simple and easy to be used as a biological dressing for any type of superficial burn wounds, without any adverse effects.

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells.

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133+/CD44+ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133+/CD44+ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Effects of amniotic membrane extract and deferoxamine on angiogenesis in wound healing: an in vivo model.

OBJECTIVE: Angiogenesis, formation of new vessels from pre-existing vessels, is an essential part of wound healing. We aimed to compare amniotic membrane extract with deferoxamine in angiogenesis and to assess any synergistic effect. METHOD: We examined four groups of rats (five per group): control, deferoxamine, amniotic membrane extract, and deferocxamine and amniotic membrane extract in combination. A distal-based skin flap was created. Deferoxamine (100mg/kg), amniotic membrane extract (0.1mg/ml), and the combination of both were injected subcutaneously every other day in 10 separate points (0.1 ml at each point) in the skin flap. On day 11, the animals were euthanised for histopathological evaluation. RESULTS: Results indicated that the amniotic membrane extract raised the angiogenic markers, particularly new vessel numbers (p<0.008) and CD31+ compared with controls (p <0.003), and deferoxamine increased new vessel numbers and Von Willebrand factor (vWF) significantly compared with controls (p<0.008). There was an increase in angiogenic factors in the combined group, however, this was not statistically significant difference was observed. There was no difference between amniotic membrane extract and deferoxamine. CONCLUSION: Amniotic membrane extract or deferoxamine could be used interchangeably in angiogenesis within wound healing due to their high safety and availability.