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Objective: Natural killer (NK) cells are critical immune cells for acute myeloid leukemia (AML) targeting. However, little is known about the relationship between using checkpoint inhibitors and heat shock protein 70 (Hsp70) as NK cell activators to control AML. Therefore, the study aims to find the best formulation of Hsp70, human PD-1 (Programmed cell death protein 1) blocker, and interleukin 15 (IL-15) to activate NK cells against AML. Materials and Methods: In this experimental study, the NK cells were isolated from mononuclear cells (MNCs) by using magnetic activation cell sorting (MACS) and were activated using the different combinations of Hsp70, PD-1 blocker, and IL-15 and then followed by immunophenotyping, functional assays to estimate their killing potential, and evaluation of expression pattern of PRF1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A and B. Results: The expression of PD-1 was significantly (P<0.05) reduced after NK cell activation by the different formulas of IL-15, Hsp70, and PD-1 blocker. The expression of NKG2A in the treated NK cells was reduced particularly in the IL-15 (P<0.01) and IL-15+PD-1 blocker (P<0.05) groups. The addition of Hsp70 increased its expression. The cytotoxic effect of NK cells increased in all groups, especially in IL-15+PD-1 blocker besides increasing interferon-gamma (IFN-γ), Granzymes, and perforin expression (P<0.05). All IL-15+PD-1 blocker group changes were associated with the upregulation of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing, and down-regulation of PIK3CB, AKT-1, Granzymes, and Perforin (P<0.05). Conclusion: We suggested the combination of IL-15 and PD-1 blocker could enhance the killing potential of AMLNK cells. Moreover, Hsp70 in combination with IL-15 and PD-1 blocker interferes activation of AML-NK cells through unknown mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expression patterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development. Materials and Methods: In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression. Results: Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression. Conclusion: We suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs. [ABSTRACT FROM AUTHOR]

Objective: Cadherin-1 (CDH1) plays an important role in the metastasis, while expression of this protein is under control of epigenetic changes on its gene promoter. Therefore we evaluated both DNA methylation (DNAmet) and histone modification marks of CDH1 in prostate cancer stem like cells (PCSLCs). Materials and Methods: In this experimental study, we isolated PCSLCs using cell surface marker and prostaspheroid formation, respectively. The cells isolated from both methods were characterized and then the levels of H3K4me2, H3K27me3, H3K9me2/3 and H3K9ac as well as DNAmet were assessed in CDH1 promoter of the isolated cells. Results: The CD44+ CD49hi cells were not validated as PCSLCs. However, prostaspheres overexpressed stemness related genes and had higher ability of invasion potential, associated with reduction in CDH1 expression. Epigenetic status analysis showed that CDH1 promoter was hypo-methylated. Histone modifications of H3K9ac and H3K4me3 were significantly reduced, in parallel with an increased level of H3K27me3. Conclusion: Our results suggest that slight decrease of DNAmet of the CpG island in CDH1 promoter does not significantly contribute to the change of CDH1 expression. Therefore, histone modifications are responsible in repressing CDH1 in PCSLCs. [ABSTRACT FROM AUTHOR]

Objective: Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods: In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133 and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results: Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A compared to other groups (P<0.05). Conclusion: Although CD133+ derived melanoma cells represented stemness features, our findings demonstrated that spheroid culture could be more effective method to enrich melanoma stem cells. [ABSTRACT FROM AUTHOR]

Objective: Gastric cancer (GC) is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcription-3 (STAT3) signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell (ESCs) pluripotency. Here, we have investigated the activation status of STAT3 in GC stem-like cells (GCSLCs). Materials and Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, characterized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation. Results: Spheroid cells showed higher potential for spheroid formation than the parental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition (EMT) related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated (P<0.05), but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel (DTX) when compared with parental cells (P<0.05) according to the MTS assay. Although immunostaining and Western blotting showed expression of the STAT3 protein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 (p-STAT3) in spheroid structures relative to the parent cells according to flow cytometry analysis (P<0.05). Conclusion: The present findings point to STAT3 over activation in GCSLCs. Complementary experiments are required to extend the role of STAT3 in stemness features and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy. [ABSTRACT FROM AUTHOR]