Effect of Pioglitazone and Cetuximab on Colon Cancer Stem-like Cell (CCSLCs) Properties.

BACKGROUND: One of the main reasons for cancer resistance to chemotherapy is the presence of cancer stem cells (CSCs) in cancer tissues. It is also believed that CSCs are the unique originators of all tumor cells. On the other hand, the Epithelial-Mesenchymal Transition pathway (EMT) can act as the main agent of metastasis. Therefore, it is possible that targeting CSCs as well as the EMT pathway could help in cancer therapy. Considering that CSCs constitute only a small percentage of the total tumor mass, enrichment before study is necessary. In our previous study, CSCs were enriched in the human colon cancer cell line HT29 by induction of EMT. These CSC-enriched HT29 cells with mesenchymal morphology were named "HT29-shE". In the present study, these cells were used to investigate the effect of pioglitazone (Pio) and Cetuximab (Cet) in order to find CSC and EMT targeting agents. METHOD: The viability and IC50 rate of cells treated with different concentrations of Pio and Cet were evaluated using the MTT test. EMT and CSC markers and cell morphology were assessed in Pio and Cet treated and untreated HT29-shE cells using flow cytometry, realtime PCR, immunocytochemistry, and microscopic monitoring. RESULTS: The findings showed that Pio and Cet at concentrations of 250 µM and 40 µg/ml, respectively, decrease cell viability by 50%. Also, they were able to reduce the expression of CSC markers (CD133 and CD44) in the CSC enriched HT29 cell line. Furthermore, Pio and Cet could efficiently reduce the expression of vimentin as a mesenchymal marker and significantly upregulate the expression of E-cadherin as an epidermal marker of EMT and its reverse mesenchymal-- to-epithelial transition (MET). In addition, the mesenchymal morphology of HT29-shE changed into epithelial morphology after Cet treatment. CONCLUSION: Pio and Cet could inhibit EMT and reduce CSC markers in the EMT induced/CSC enriched cell line. We expect that focus on finding EMT/CSC-targeting agents like these drugs can be helpful for cancer treatment.

Menstrual Blood-Derived Mesenchymal Stem Cell Therapy for Severe COVID-19 Patients.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), was declared a global pandemic in March 2020 and resulted in more than 6 million deaths worldwide to date. Although several vaccines were produced against COVID-19 and many therapeutic protocols were developed for the management of this respiratory infection, COVID-19 pandemic has still remained an unresolved problem with the emergence of new variants of SARS-CoV-2, especially vaccine-resistant variants. Probably, end of the COVID-19 needs effective and certain treatments which were undiscovered to date. According to immunomodulatory and regenerative properties, mesenchymal stem cells (MSCs) have been considered a therapeutic approach to suppressing cytokine storm caused by SARS-CoV-2 and the treatmet of severe COVID-19. Following intravenous (IV) infusion of MSCs, cells entrap in the lung, guard alveolar epithelial cells, suppress pulmonary fibrosis and improve lung dysfunction. The human menstrual blood-derived stem cells (hMenSCs) as a novel source of MSCs are collected by noninvasive, painless, and easy way without ethical issues. MenScs are an abundant and cheap source with a high proliferation rate and differentiation ability into multiple cell lineages. Regarding immunomodulatory and anti-inflammatory properties, regenerative ability and low immunogenicity, these cells exhibit great potential in the treatment of various diseases. Some clinical trial studies have begun using MenSCs to treat severe COVID-19. According to these trials, MenSC therapy showed promising and encouraging results in treating severe COVID-19. We reviewed published clinical trials and summarized the effects of MenSC therapy on severe COVID-19 with a focus on clinical and laboratory data, immune and inflammatory factors and concluded the advantages and possible risks of this procedure.

The role of probiotics in tissue engineering and regenerative medicine.

Tissue engineering and regenerative medicine (TERM) as an emerging field is a multidisciplinary science and combines basic sciences such as biomaterials science, biology, genetics and medical sciences to achieve functional TERM-based products to regenerate or replace damaged or diseased tissues or organs. Probiotics are useful microorganisms which have multiple effective functions on human health. They have some immunomodulatory and biocompatibility effects and improve wound healing. In this article, we describe the latest findings on probiotics and their pro-healing properties on various body systems that are useable in regenerative medicine. Therefore, this review presents a new perspective on the therapeutic potential of probiotics for TERM.

Tissue engineering and regenerative medicine can design processes or products to restore, repair, or replace injured or diseased cells, tissues or organs. It contains the generation and making use of therapeutic stem cells, and engineered scaffolds for the manufacture of artificial organs. This field focuses on the development and application of new treatments to heal tissues and organs as well as repair functions lost due to damage, defects, disease or aging. The World Health Organization has described probiotics as "live microorganisms that, when administered in sufficient amounts, confer a health advantage on the host". Probiotics are found naturally in certain foods, such as kimchi and fermented yogurt. They are also found in your gut, where they partake in a type of important bodily processes, such as vitamin production, digestion, mood regulation, and immune function. Probiotics with their suitable pro-healing effects on different systems of the body can be used in regenerative medicine. Probiotic bacteria induce their beneficial effects via proven mechanisms including pathogens killing, modulating the gut microbiota, immunomodulatory effects, and anti-diabetic, anti-obesity and anti-cancer functions. Moreover, recent studies indicated that probiotics could neutralize infections caused by COVID-19. Probiotics are healthy microorganisms that exert multiple positive effects on human health, especially through the battle against pathogens and repairing different types of body tissues.

Toward in Vitro Production of Platelet from Induced Pluripotent Stem Cells.

Platelets (PLTs) are small anucleate blood cells that release from polyploidy megakaryocytes(MKs). PLT transfusion is standard therapy to prevent hemorrhage. PLT transfusion is donor-dependent way which have limitations including the inadequate donor blood supply, poor quality, and issues related to infection and immunity. Overcoming these obstacles is possible with in vitro production of human PLTs. Currently several cells have been considered as source to in vitro production of PLTs such as hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, HSCs are a limited source for PLT production and large-scale expansion of HSC-derived PLT remains difficult. Alternative sources can be ESCs which have unlimited expansion capacity. But ESCs have ethical issues related to destroying human embryos. iPSCs are considered as an ideal unlimited source for PLT production. They are able to differentiate into any cells and have the capacity of self-renewal. Moreover, iPSCs can be acquired from any donor and easily manipulated. Due to new advances in development of MK cell lines, bioreactors, feeder cell-free production and the ability of large scale generation, iPSC-based PLTs are moving toward clinical applicability and considering the minimal risk of alloimmunization and tumorigenesis of these products, there is great hopefulness they will become the standard source for blood transfusions in the future. This review will focus on how to progress of in vitro generation of PLT from stem cell especially iPSCs and some of the successful strategies that can be easily used in clinic will be described.

A Decrease in CD44 on Cell Surfaces (MKN-45 cell line) After RELA Knockout Using CRISPR/Cas9.

The NF-kB signaling pathway was introduced as a key pathway in carcinogenesis that is induced by inflammation in gastrointestinal malignancies. The RelA transcription factor is an important component of this signaling pathway. Furthermore, CD44 is implicated in the tumorigenesis and metastasis of gastric cancer. The aim of this study was to assay the effect of RELA knockout on CD44 expression in MKN45 cells. CRISPR/Cas9 was used to knock out RELA in MKN-45. The median fluorescence intensity (MFI) of CD44 before and after RELA knockout is analyzed in MKN45. The CRISPR/Cas9 vector pSpCas9 (BB)-2A-Puro (PX459) was used for gRNA cloning (two guides). The MKN-45 cell line was co-transfected. The purified co-transfected cells with puromycin were cultured and used for the RELA gene expression assay by real-time PCR. Flow cytometry was used for the analysis of the MFI of CD44+ in MKN45. The results showed that 180 nucleotide sequences between exon 2 and exon 3 of RELA were deleted in MKN45. RELA expression significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells significantly decreased (P <0.001). Knockout of RELA significantly decreased CD44 expression in MKN45 cells. It can be concluded that the NF-kB signaling pathway via RELA is related to CD44 expression and consequently the tumorigenesis of gastric cancer. More studies about this relationship are recommended.

Association study between superoxide Dismutases gene polymorphisms and development of diabetic retinopathy and cataract in Iranian patients with type two diabetes mellitus.

PURPOSE: Reactive oxygen species (ROS) plays pathological roles on development of diabetic retinopathy (DR) and cataract. Superoxide dismutases (SODs) are a set of enzymes to eliminate ROS and cell protection. Based on the diminished activities of SOD1 during DR and cataract, the polymorphisms within SOD1 gene may be associated with these disorders. This study assessed the relationship between SOD1 -251A/G (rs2070424) and SOD1 + 35A/C (rs2234694) gene polymorphisms and DR and cataract in Iranian patients with type 2 diabetes mellitus (T2DM). METHODS: Totally, 141 cases (73 patients with cataract and 68 with DR) with a at least five years history of T2DM and 119 age/gender matched persons without history of DM were included in the case and control groups, respectively. Genomic DNA was extracted from peripheral venous blood cells and genotyping of 251A/G and + 35A/C polymorphisms was done using PCR-RFLP technique. Statistical analysis was done using SPSS version 22. RESULTS: Results showed SOD1 -251A/G and + 35A/C genotype frequency were significantly associated (1.9 folds) with cataract (P = 0.045, OR = 0.524, 95% CI = 0.277-0.991 and P = 0.037, OR = 0.505, 95% CI = 0.265-.0965, respectively). Instead, no significant differences found between SOD -251A/G (P = 0.053, OR = 0.52, 95% CI = 0.276-1.014) and + 35A/C (P = 0.073, OR = 0.547, 95% CI = 0.281-1.063) polymorphisms and DR. Multivariate Logistic Regression model showed significant relationship between BMI, HDL and TC levels and independent predictors of T2DM (P < 0.05). CONCLUSIONS: Based on the results, there was significant association between SOD1 gene polymorphisms and cataract in patients with T2DM. Therefore, SOD1 gene polymorphisms might be a potential marker for increased risk of cataract in patients with T2DM.

Increasing the colon cancer cells sensitivity toward radiation therapy via application of Oct4-Sox2 complex decoy oligodeoxynucleotides.

Low sensitivity of cancer stem cells toward regular cancer therapy strategies is an important issue in the field of cancer remedy. The concept of cancer stem cell elimination has been a topic of interest in the field of molecular medicine for a long time. At the current study, it was aimed to elevate the sensitivity of cancer stem-like cells toward radiotherapy by treating with Oct4-Sox2 complex decoy oligodeoxynucleotides (ODNs). After treating HT29 and HT29-ShE cells with Oct4-Sox2 complex decoy ODNs, and analyzing the cellular uptake and localization of decoys, treated cells and control groups were subjected to irradiation by fractionated 6MV X-ray with a final dose of 2 Gy. Thereafter, the influence of radiotherapy on ODNs treated groups and control group was investigated on cell viability, cell cycle, apoptosis, colonosphere formation and scratch assay. Cellular uptake and localization assays demonstrated that decoy ODNs can efficiently be transfected to the cells and reside in subcellular compartment, where they pose their action on gene regulation. Post radiotherapy analysis indicated statistical significance in decoy ODNs treated cells by means of lower cell viability, cell cycle arrest in G2/M phase, increased cellular apoptosis, and reduced cell motility. Also, formed colonospheres were smaller in size and fewer in numbers. Considering the role of Oct4, and Sox2 transcription factors in signaling pathways of preserving stemness and inducing reverse EMT, application of decoy strategy could increase the sensitivity of cancer cells toward irradiation, which has a potential to eliminate the cancerous cells from tumors and support cancer treatment.

Role of Oct4-Sox2 complex decoy oligodeoxynucleotides strategy on reverse epithelial to mesenchymal transition (EMT) induction in HT29-ShE encompassing enriched cancer stem-like cells.

Cancer stem cells are commonly tolerant toward chemotherapy and radiotherapy. Oct4 and Sox2 transcription factors are shown to be overexpressed in various cancers. At the current research, inhibition of Oct4 and Sox2 transcription factors was performed through application of decoy oligodeoxynucleotides (ODNs) strategy via repressing stemness properties in HT29-ShE cells encompassing enriched cancer stem-like cells. Designed Oct4-Sox2 complex decoy ODNs were transfected into HT29-ShE cells with Lipofectamine reagent. At the next step, ODNs efficiency transfection and subcellular localization were determined via flow cytometry and fluorescence microscopy, respectively. Further investigations such as cell proliferation and apoptosis analysis, colonosphere formation, invasion and migration, and real-time PCR assays were also carried out. Obtained results shed light on the fact that the designed complex decoys were effectively transfected into HT29-ShE cells, and they were found to be localized in subcellular compartments. Oct4-Sox2 decoy ODNs led to decreased cell viability, arresting the cell cycle in G0/G1 phases, increasing apoptosis, inhibition of migration/invasion and colonosphere formation ability of HT29-ShE cells in comparison with control and scramble groups. Furthermore, Oct4-Sox2 complex decoy could modulate the MET process via alteration of mRNA expression of downstream genes. It could be concluded that application of Oct4-Sox2 transcription factor decoy strategy in cells with stemness potential could lead to inhibiting the cell growth and triggering differentiation. Therefore, this technique could be applied along with usual remedies (chemotherapy and radiotherapy) as high potential method for treating cancer.

Association between PPARGC1A single nucleotide polymorphisms and increased risk of nonalcoholic fatty liver disease among Iranian patients with type 2 diabetes mellitus

Background/aim: Environmental and genetic factors may play a major role in the development of nonalcoholic fatty liver disease (NAFLD) among people with obesity and type 2 diabetes mellitus. Based on the fact that PGC-1α, as the protein encoded by the PPARGC1A gene, plays a key role in energy metabolism pathways, it has been hypothesized that polymorphisms within the PPARGC1Agene may be associated with increased risks of NAFLD. Thus, this study was designed to evaluate the Gly482Ser polymorphism (rs8192678) within the PPARGC1A gene and its association with the increased risk of NAFLD in Iranian patients with type 2 diabetes. Materials and methods: A total of 145 NAFLD patients with a history of type 2 diabetes and 145 healthy control subjects were included in the study. Gly482Ser polymorphism genotyping was done using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. Results: The results showed a significant difference between the PPARGC1A Gly482Serpolymorphism in NAFLD patients and the healthy controls. Accordingly, the AA genotype and A allele were increased in the NAFLD patients when compared to the healthy controls. However, no significant correlation was observed between the Gly482Ser polymorphism and the physiological and biochemical parameters. Conclusion: Based on the results, the AA genotype, which is associated with the insertion of Ser, can be considered as a risk factor for the development of NAFLD in Iranian patients with diabetes type 2.

Investigation of specific binding of designed oligodeoxynucleotide decoys to transcription factors in HT29 cell line undergoing epithelial-mesenchymal transition (EMT).

Expression of master transcriptional regulators of stem cells (Oct4 and Sox2) is associated with mediating tumor proliferation and tumor differentiation. The main goal of this study is the investigation of specific binding of designed Oct4-Sox2 transcription factors decoy oligodeoxynucleotides (ODNs) sequence to their nucleus-extracted proteins in HT29-ShE cells containing enriched cancer stem-like cells (SCLCs). First, gene expression of Oct4, Sox2, and E-cadherin revealed the overexpression of Oct4 and Sox2 and downregulation of E-cadherin in HT29-ShE cells compared with HT29 wild-type and HT29-ShC cells. Next, Oct4-Sox2 complex decoy ODNs were designed according to their elements in the promoter region of Sox2 gene. Then, the interactions of Oct4 and Sox2 proteins to designed ODNs were evaluated in silico. Finally, DNA-protein interactions of decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Analysis of gel shift retardation assay admitted the specific binding of designed ODNs sequence to the nuclear extracted Oct4 and Sox2 proteins. The results will be a promising approach to target cancer stem cells for potential use in differentiation therapy before chemotherapy and radiotherapy of cancers.

Enrichment of cancer stem-like cells by the induction of epithelial-mesenchymal transition using lentiviral vector carrying E-cadherin shRNA in HT29 cell line.

A better understanding of cancer stem cells (CSCs) may facilitate the prevention and treatment of cancers. Epithelial-mesenchymal transition (EMT) is a process activated during invasion and metastasis of tumors. EMT induction in normal and tumor cells makes them more resistant to chemotherapy. E-cadherin is a membrane protein and plays a role in tumor invasion, metastasis, and prognosis. Downregulation of E-cadherin is a hallmark of EMT. Here, we created a model of cancer stem-like cells enrichment via EMT induction using E-cadherin downregulation in HT29 cell line using a lentiviral vector carrying shRNA. We aimed to evaluate cancer and anti-CSC chemotherapeutics screening. The markers of EMT and CSCs were assessed and compared with control cells using flow cytometry, real-time PCR, immunocytochemistry, western blot, migration assay, invasion assay, and colony formation assay. The transduced cells showed a mesenchymal morphology. High levels of EMT-related proteins were also expressed. These results confirmed that the transduced cells underwent EMT. In addition, we observed an increased population of E-cadherin-downregulated HT29 cell line among the cells expressing colon CSC markers (CD133+ and CD44+ ) after EMT induction. E-cadherin-downregulated cells were morphologically like mesenchymal cells, and the number of CD133+ - and CD44+ -cells (CSC-like cells) increased. These cells can be used as stable models to study cancer cells and screening of antitumor therapeutics.

Down-regulation of miR-122 after transplantation of mesenchymal stem cells in acute liver failure in mice model.

This study investigated the correlation between the hepatic level of miR-122 and the extent of liver tissue regeneration in CCl4 induced liver injury mice model following transplantation of menstrual blood-(MenSCs) and bone marrow-derived stem cells (BMSCs). Hepatic miR-122 levels were significantly up-regulated following administration of CCl4 (P < 0.01). The significant positive correlations were observed between hepatic miR-122 and biochemical serum markers and the severity of liver injury in histopathological assessments (P < 0.01). Following stem cell therapy, all cell treated groups showed a significant down-regulation in miR-122 that was significantly correlated with improvement in histopathological features and biochemical markers (P < 0.01). Furthermore, the hepatic level of miR-122 was lower in the MenSCs-treated group compared with the BMSCs-treated group (P < 0.01) and in HPL cells-treated groups in reference to undifferentiated cells-treated groups (P < 0.05). These data suggest that miR-122 could be used as a potential predictor of outcome of liver injury after mesenchymal stem cell transplantation.

The Pro12Ala polymorphism in the PPAR-γ2 gene is not associated with an increased risk of NAFLD in Iranian patients with type 2 diabetes mellitus.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Several studies have demonstrated a significant association between Pro12Ala polymorphism of the PPAR-γ2 gene and metabolic disorders. Therefore, this study aimed to evaluate the association of Pro12Ala polymorphism with increased risk of NAFLD in Iranian patients with type 2 diabetes mellitus. METHODS: This cross-sectional study was performed on 145 healthy control subjects and 145 NAFLD patients with a history of type 2 diabetes. Pro12Ala polymorphism genotyping was performed using PCR-restriction fragment length polymorphism (RFLP) technique with the Bs1I restriction enzyme. RESULTS: Our results demonstrated that CC and GG genotypes of Pro12Ala were found in the participants, but there was no statistically significant difference between NAFLD patients and healthy controls (P = 0.64 and χ2 = 0.21). CONCLUSION: This study suggests that Pro12Ala polymorphism of the PPAR-γ2 gene cannot be considered as a risk factor for NAFLD in the Iranian population.

Preparation and investigation of indirubin-loaded SLN nanoparticles and their anti-cancer effects on human glioblastoma U87MG cells.

Indirubin, an ingredient in traditional Chinese medicine, is considered as an anti-cancer agent. However, due to its hydrophobic nature, clinical efficiency has been limited. Drug delivery via nanotechnology techniques open new windows toward treatment of cancerous patients. Glioblastoma multiforme (GBM) is the most severe and common type of brain primary tumors. Of common problems in targeting therapies of glioblastoma is the availability of drug in tumoric tissues. In this study, Indirubin loaded solid lipid nanoparticles were prepared and their therapeutic potentials and antitumoric effects were assessed on GBM cell line (U87MG). The SLNs were prepared with Cetyl palmitate and Polysorbat 80 via high-pressure homogenization (HPH) methods in hot mode. Then, properties of SLNs including size, zeta potential, drug encapsulation efficacy (EE %) and drug loading were characterized. SLNs morphology and size were observed using SEM and TEM. The crystalinity of formulation was determined by different scattering calorimetry (DSC). The amount of drug release and antitumor efficiency were evaluated at both normal brain pH of 7.2 and tumoric pH of 6.8. The prapared SLNs had mean size of 130 nm, zeta potential of -16 mV and EE of 99.73%. The results of DSC showed proper encapsulation of drug into SLNs. Drug release assessment in both pH displayed sustain release property. The result of MTT test exhibited a remarkable increment in antitumor activity of Indirubin loaded SLN in comparison with free form of drug and blank SLN on multiform GB. This study indicated that Indirubin loaded SLNs could act as a useful anticancer drugs.