Abstract. Salinomycin is a monocarboxylic polyether antibiotic that has been reported to induce apoptosis in various types of cancer cells with specificity for cancer stem cells. However, its anticancer effect in colorectal cancer stem cells has never been reported. In the present study, we examined the ability of salinomycin to induce cell death in the colorectal cancer stem cell line CD44 + EpCAM + HCT-116, and we measured its in vivo tumor inhibition capacity. Salinomycin dose-dependently induced cytotoxicity in the CD44 + EpCAM + HCT-116 cells and inhibited colony formation. Salinomycin treatment was shown to induce apoptosis, as evidenced by nuclear fragmentation, an increase in the proportion of acridine orange/ethidium bromide-positive cells and an increase in the percentage of Annexin V-positive cells. Apoptosis was induced in colorectal cancer stem cells in a caspase-dependent manner, as shown by an increase in the levels of cleaved caspase-3, -8 and -9. JC-1 staining further revealed that salinomycin induced colorectal cancer cell apoptosis via the mitochondrial pathway. In addition, salinomycin treatment of xenograft mice inhibited the growth of tumors derived from the CD44 + EpCAM + HCT-116 cells. The present study demonstrated that the antibiotic salinomycin exerts an anti-colorectal cancer effect in vitro and in vivo, suggesting salinomycin as a potential drug for colorectal cancer therapy. IntroductionColorectal cancer (CRC) is the third most common malignancy worldwide, accounting for ~10% of all cancer cases and CRC is one of the most common causes of death related to gastrointestinal cancers (1-3). Although the incidence rates of colon cancer have declined somewhat, current therapies are associated with serious side-effects, high cost and recurrence rates exceeding 50%, primarily due to the development of acquired chemoresistance to conventional chemotherapeutics (4,5).Emerging data suggest that malignant tumors contain a small distinct population of cancer stem cells (CSCs), which are responsible for tumor initiation and propagation (6). Stem cell research and the cancer stem cell (CSC) hypothesis have shown that colonic stem cells or CSCs are involved in tissue regeneration and colonic carcinogenesis (7-9). Drug-resistant CSCs are thought to be one of the key causes of CRC treatment failure, and it is hypothesized that these cells are ultimately the likely cause of metastasis and tumor recurrence (10-12). Most modern treatments are ineffective against solid tumors and this may be the result of the increased resistance of CSCs (13). Therefore, it is vital to find novel therapeutic methods to eradicate CSCs and enable the development of more effective treatment protocols (14).Salinomycin is a 751-Da monocarboxylic polyether antibiotic, which was initially used to eliminate bacteria, fungi and parasites and is fed to ruminants to improve nutrient absorption and feeding efficiency (15,16). This compound is now considered an important anticancer drug candidate (17,18). It has recently been reporte...

Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

Increasing evidence supports the concept that cancer stem cells (CSCs) are responsible for cancer progression and metastasis, therapy resistance and relapse. In addition to conventional therapies for colon cancer, the development of immunotherapies targeting cancer stem cells appears to be a promising strategy to suppress tumor recurrence and metastasis. In the present study, dendritic cells (DCs) were pulsed with whole-tumor cell lysates or total RNA of CD44+ colon cancer stem cells (CCSCs) isolated from mouse colon adenocarcinoma CT-26 cell cultures and investigated for their antitumor immunity against CCSCs in vivo and in vitro. In a model of colon adenocarcinoma using BALB/c mice, a sequential reduction in tumor volume and weight was associated with an extended survival in tumor-bearing mice vaccinated with DCs pulsed with RNA or CCSC lysate. In addition, a lactate dehydrogenase assay indicated that cytotoxic T-cells derived from the treated mice exhibited strong cytotoxic activity. Additionally, an enzyme-linked immunosorbent assay revealed that the cytotoxic T-cells of the treated mice released higher levels of interferon-γ against CCSCs compared with those of the control group. In all experiments, the antitumor efficacy of the lysate-pulsed DC-treated and RNA-pulsed DC-treated groups were significantly higher compared with that of the DC-treated and control groups. The results of the present study indicated the potential use of DCs pulsed with cancer stem cell lysates as a potent therapeutic antigen to target CSCs in colon cancer. Additionally, the results provided a rationale for using lysate-pulsed DCs in vivo to eliminate residual tumor deposits in post-operative patients.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are required to combat this disease. It is well known that tumor‑suppressor microRNA (miRNA) acts as a new potential anticancer agent. Accumulating evidence showed that microRNA-145 (miR-145) is a candidate tumor suppressor miRNA. However, whether miR-145 is involved in the development and progression of MM reamins to be determined. In the present study, we investigated the therapeutic potential of synthetic miR-145 against human MM cells in vitro and in vivo. The results showed that miR-145 was reduced in MM tissues and cell lines. Enforced expression of miR-145 by transfection with miR-145 mimics inhibited cell proliferation, migration, and the invasion abilities of H929 cells. Furthermore, our results demonstrated that the enforced expression of miR-145 in H929 cells profoundly decreased the levels of p-AKT and p-PI3K, which may contribute to some extent to the inhibition of MM cell proliferation and survival. The enforced expression of miR-145 in a xenograft mouse model suppressed tumor growth. In conclusion, our findings suggested that miR-145 may act as a tumor suppressor and contributes to the progression of MM. Additionally, miR-145 mimics is a potential therapeutic agent for the treatment of MM.

Background A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter. Results We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity. Conclusion These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.

Induced pluripotent stem cells (iPSCs), derived from reprogramming of somatic cells by a cocktail of transcription factors, have the capacity for unlimited self-renewal and the ability to differentiate into all of cell types present in the body. iPSCs may have therapeutic potential in regenerative medicine, replacing injured tissues or even whole organs. In this study, we examine epigenetic factors embedded in the specific 3-dimensional intrachromosomal architecture required for the activation of endogenous pluripotency genes. Using chromatin RNA in situ reverse transcription sequencing (CRIST-seq), we identified an Oct4-Sox2 binding long noncoding RNA, referred as to Osblr8, that is present in association with pluripotency status. Osblr8 was highly expressed in iPSCs and E14 embryonic stem cells, but it was silenced in fibroblasts. By using shRNA to knock down Osblr8, we found that this lncRNA was required for the maintenance of pluripotency. Overexpression of Osblr8 activated endogenous stem cell core factor genes. Mechanistically, Osblr8 participated in the formation of an intrachromosomal looping structure that is required to activate stem cell core factors during reprogramming. In summary, we have demonstrated that lncRNA Osblr8 is a chromatin architecture modulator of pluripotency-associated master gene promoters, highlighting its critical epigenetic role in reprogramming.

Tumor hypoxia contributes to the development of resistance to chemotherapeutic drugs in several human cancer cell lines. Atovaquone, an anti-malaria drug approved by the US Food and Drug Administration, has recently demonstrated anti-cancer effects in vitro and in vivo in several cancer models. To assess the potential of atovaquone as an anti-cancer agent under hypoxia in colorectal carcinoma, EpCAM + CD44 + colon cancer stem cells were isolated from HCT-116 human colon cancer cells through magnetic-activated cell sorting. The efficacy of atovaquone on cytotoxicity, tumorsphere formation, apoptosis, invasion and cell-cycle progression under hypoxic conditions were evaluated. MTS assays indicated that atovaquone inhibited the proliferation of EpCAM + CD44 + HCT-116 cells with a half-maximal inhibitory concentration of 15 µM. Atovaquone inhibited tumorsphere formation and cell proliferation by causing cell-cycle arrest in S-phase, which induced apoptosis of EpCAM + CD44 + HCT-116 cells, as detected by Annexin V-FITC/PI double staining assays, and caused mitochondrial membrane potential depolarization, as determined by a JC-1 staining assay. Reverse transcription-quantitative PCR demonstrated increased expression of Bax and downregulation of Bcl-2. Transwell invasion assays indicated that atovaquone inhibited the invasiveness of EpCAM + CD44 + HCT-116 cells under hypoxia, which was associated with upregulation of MMP-2 and-9 and increased expression of tissue inhibitor of MMPs (TIMP)-1. Taken together, atovaquone reduced the tumorsphere formation and invasion ability of EpCAM + CD44 + HCT-116 cells, at least in part by increasing the expression of TIMP-1 and downregulating the expression of MMP-2 and-9, as well as the cells' viability by inducing cell-cycle arrest in S-phase and induction of apoptosis via the Bcl-2/Bax pathway under hypoxic conditions. Further studies are warranted to explore the mechanisms of action of atovaquone as a promising anticancer agent in the treatment of colorectal carcinoma.

We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.