Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

BACKGROUND: Recent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear. METHODS: Serum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b. RESULTS: Sphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b. CONCLUSION: The mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other's expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.

Magnolol attenuates the inflammation and enhances phagocytosis through the activation of MAPK, NF-κB signal pathways in vitro and in vivo.

Magnolol is a natural extract and the main bioactive component from Chinese medicine-Magnolia. We speculate that it's functional action might be associated with the anti-inflammatory effects of magnolol. Herein, the main purpose was to elucidate the phagocytic immune function and anti-inflammatory activities associated. The toxicity of magnolol on U937 and LO-2 cells was assayed by MTT, flow cytometry and laser scanning confocal microscope was utilized to detect the phagocytosis effect on U937 cells, C57BL/6 mice and the follow-up hematoxylin-eosin staining methods were used to evaluate its bioactivity in vivo. The results showed that magnolol had dose dependent effects on enhancement of phagocytosis ability and significantly inhibited the NO production at the concentration range from10 to 40 µM. Furthermore, Magnolol significantly reduced the gene expression and protein release of IL-1ß and TNF-α. However, the p-ERK1/2 in MAPK signaling pathway was not significantly affected by magnolol, whereas p-JNK and p-P38 were down-regulated. Magnolol also inhibited the expression of p-IκBα and p-P65 of NF-κB signaling pathways. The loss of body weight and the shorter length of colon were significantly improved in DSS-treated colitis C57BL/6 mice after the administration of magnolol. The cytokines of pro-inflammatory factors TNF-α, IL-6 and IL-1ß attenuated significantly in a concentration dependent manner. The histopathological manifestations of 5-20 mg/kg after the treatment magnolol were markedly improved in the DSS-treated mice. These findings showed that magnolol exerted an anti-inflammatory effect through immunoregulatory phagocytosis, MAPK and NF-κB signaling pathways. Our results provide experimental evidence and theory basis for research on anti-inflammatory effects for magnolol as a potentially anti-inflammatory drug candidate.

The Curcumin Analogs 2-Pyridyl Cyclohexanone Induce Apoptosis via Inhibition of the JAK2-STAT3 Pathway in Human Esophageal Squamous Cell Carcinoma Cells.

Multiple modifications to the structure of curcumin have been investigated with an aim to improve its potency and biochemical properties. Previously, we have synthesized a series of curcumin analogs. In the present study, the anticancer effect of 2-pyridyl cyclohexanone, one of the curcumin analogs, on esophageal carcinoma Eca109 and EC9706 cell lines and its molecular mechanisms were investigated. 2-Pyridyl cyclohexanone inhibited the proliferation of Eca109 and EC9706 cells by inducing apoptosis as indicated by morphological changes, membrane phospholipid phosphatidylserine ectropion, caspase 3 activation, and cleavage of poly(ADP-ribose) polymerase. Mechanistic studies indicated that 2-pyridyl cyclohexanone disrupted mitochondrial membrane potential, disturbed the balance of the Bcl-2 family proteins, and triggered apoptosis via the mitochondria-mediated intrinsic pathway. In 2-pyridine cyclohexanone-treated cells, the phosphorylation levels of JAK2 and STAT3 were dose-dependently decreased and p38 and p-ERK signals were notably activated in a dose-dependent manner. Moreover, we found that the addition of S3I-201, a STAT3 inhibitor, led to a decreased expression level of Bcl-2 in Eca109 cells. The chromatin immunoprecipitation assay demonstrated that STAT3 bound to the promoter of Bcl-2 in the Eca109 cells. Furthermore, the mutation of four STAT3 binding sites (-1733/-1723, -1627/-1617, -807/-797, and -134/-124) on the promote of Bcl-2 gene alone attenuated the transcriptional activation of STAT3. In addition, down-regulation of STAT3 resulted in less of transcriptional activity of STAT3 on Bcl-2 expression. These data provide a potential molecular mechanism of the apoptotic induction function of 2-pyridyl cyclohexanone, and emphasize its important roles as a therapeutic agent for esophageal squamous carcinoma.

Use of immune modulation by human adipose-derived mesenchymal stem cells to treat experimental arthritis in mice.

Rheumatoid arthritis is a chronic and systemic autoimmune disease characterized by inflammatory cell infiltration and joint erosion. Human adipose-derived mesenchymal stem cells (hASCs) have shown the capacity of suppressing effector T cell activation and inflammatory cytokine expression. We investigated whether hASCs play a therapeutic role in collagen-induced arthritis (CIA) by administering a single dose of hASCs in mice with established CIA. In vivo, a beneficial effect was observed following hASC infusion as shown by a marked decrease in the severity of arthritis. Human ASCs were detectable in the joints, and reduced levels of pro-inflammatory cytokines and increased levels of anti-inflammatory cytokines were observed in the sera of the hASC-treated mice. Furthermore, hASC treatment induced the expansion of regulatory T cells (Tregs) both in the peripheral blood and in the spleen tissues. In vitro, hASCs downregulated the production of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 in mouse macrophages stimulated with lipopolysaccharide and inhibited the proliferation of human primary T cells in response to mitogens. Thus hASCs represent a novel and effective therapeutic strategy for RA.

[Cloning and expression of human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells].

OBJECTIVE: To clone and express human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells. METHODS: Primers were designed based on the coding sequence for the human Oct4 cDNA. The fragment containing Oct4 gene, amplified by reverse transcription PCR (RT-PCR) from human HeLa cell cDNA as the template, was subcloned into pET-22b(+) and pcDNA3.1(+) vectors to construct the gene recombinant prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)-his-Oct4, respectively. Thereafter, the double enzymes digestion and the target gene sequencing were performed for identification. The prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)- his-Oct4 from the positive clones were respectively transformed into E.coli BL21 (DE3) and the embryo HEK293T cells for protein expression. RESULTS: DNA fragments (1100 bp) containing the coding region of Oct4 gene were obtained from RT-PCR. The recombinant expression plasmids were confirmed by enzymes digestion identification and sequencing analysis, proving both of the expression vectors were successfully constructed. SDS-PAGE analysis showed that the molecular mass of OCT4 expressed in the host E.coli BL21 (DE3) was 38 000, which was consistent with the theoretical prediction. Western blotting confirmed the expression of pcDNA3.1(+)-his-Oct4 transfected into HEK293T. CONCLUSION: Human Oct4 cDNA was cloned and its protein was successfully expressed in E.coli and HEK293T cells.

miR-150 Suppresses the Proliferation and Tumorigenicity of Leukemia Stem Cells by Targeting the Nanog Signaling Pathway.

Proliferation, a key feature of cancer cells, accounts for the majority of cancer-related diseases resulting in mortality. MicroRNAs (miRNAs) plays important post-transcriptional modulation roles by acting on multiple signaling pathways, but the underlying mechanism in proliferation and tumorigenicity is unclear. Here, we identified the role of miR-150 in proliferation and tumorigenicity in leukemia stem cells (LSCs; CD34+CD38- cells). miR-150 expression was significantly down-regulated in LSCs from leukemia cell lines and clinical samples. Functional assays demonstrated that increased miR-150 expression inhibited proliferation and clonal and clonogenic growth, enhanced chemosensitivity, and attenuated tumorigenic activity of LSCs in vitro. Transplantation animal studies revealed that miR-150 overexpression progressively abrogates tumor growth. Immunohistochemistry assays demonstrated that miR-150 overexpression enhanced caspase-3 level and reduced Ki-67 level. Moreover, luciferase reporter assays indicated Nanog is a direct and functional target of miR-150. Nanog silencing using small interfering RNA recapitulated anti-proliferation and tumorigenicity inhibition effects. Furthermore, miR-150 directly down-regulated the expression of other cancer stem cell factors including Notch2 and CTNNB1. These results provide insights into the specific biological behavior of miR-150 in regulating LSC proliferation and tumorigenicity. Targeting this miR-150/Nanog axis would be a helpful therapeutic strategy to treat acute myeloid leukemia.

miR-203 inhibits proliferation and self-renewal of leukemia stem cells by targeting survivin and Bmi-1.

Drug resistance is one of the leading causes of failed cancer therapy in the treatment of acute myeloid leukemia. Although the mechanisms of resistance are poorly understood, they may be related to the presence of leukemia stem cells (LSCs). Down-regulation of the miR-203 reportedly contributes to oncogenesis and chemo-resistance in multiple cancers. We found that miR-203 expression was down-regulated in CD34 + AML cells as compared with CD34- cells isolated from patients as well as in LSC-enriched (CD34 + CD38-) cell lines KG-1a or MOLM13. Additionally, re-expression of miR-203 led to decreased cell proliferation, self-renewal, and sphere formation in LSCs. Moreover, miR-203 was found to directly target the 3'un-translated regions of survivin and Bmi-1 mRNAs affecting proliferation and self-renewal in LSCs. In this study, we identified a novel miR-203/survivin/Bmi-1 axis involved in the regulation of biological properties of LSCs. This axis may represent a new therapeutic target for acute myeloid leukemia and a potential prognosis/diagnostic marker for LSCs therapy.

Synthesis and Evaluation of a CBZ-AAN-Dox Prodrug and its in vitro Effects on SiHa Cervical Cancer Cells Under Hypoxic Conditions.

Although doxorubicin (Dox) is widely used in clinical treatment for solid tumors, it causes many side-effects such as heart and kidney damage, bone marrow suppression, and drug resistance. Legumain is a lysosomal protease that is elevated and associated with an invasive and metastatic phenotype in a number of solid tumors. In this study, we designed and synthesized a Dox prodrug, N-benzyloxycarbonyl-Ala-Ala-Asn-Doxorubicin (CBZ-AAN-Dox), with 94% purity. Single substrate kinetic assays demonstrated hLegumain-specific enzymatic cleavage and activation of the prodrug in vitro, and this enzymatic cleavage of the prodrug substrate was more sensitive in acidic conditions, releasing more than 70% of Dox after 24 h. Treatment of tumor cells with our prodrug demonstrated a much higher IC50 value, significantly enhanced uptake of the prodrug, and considerably less cellular toxicity compared to Dox treatment alone. Our study presents a novel prodrug, CBZ-AAN-Dox, to potentially increase both the safety and efficacy of clinical treatment of tumors by exploiting the tumor's innate expression of legumain.

B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.