Combination treatment with sorafenib and wh-4 additively suppresses the proliferation of liver cancer cells.

Sorafenib is currently used to treat hepatocellular carcinoma (HCC). However, the development of chemoresistance to sorafenib is a major limitation for sorafenib-based therapy in patients with HCC. In the present study, the effect of the combination therapy of sorafenib and wh-4 on the proliferation of liver cancer cells was investigated. The results showed that sorafenib with wh-4 additively suppressed the proliferation of liver cancer cells. The colony formation of liver cancer cells decreased significantly in response to the combination treatment of sorafenib with wh-4, and it also induced the apoptosis of liver cancer cells. Western blot analysis demonstrated decreased expression of Bcl2, and increased expression of Bax in liver cancer cells treated with a combination of sorafenib and wh-4. Moreover, the migration of liver cancer cells was inhibited. The combination treatment of sorafenib with wh-4 reduced the expression levels of ABCB1 and ABCG2 which are responsible for resistance. Finally, STAT3 overexpression abolished the proliferation inhibition effect of sorafenib with wh-4 on liver cancer cells, and sorafenib and wh-4 suppressed the proliferation of liver cancer cells by STAT3 pathway. Together, these results suggest that sorafenib-wh4 combination treatment is a potential novel therapeutic approach to suppress the proliferation of liver cancer cells.

Suppression of Esophageal Cancer Stem-like Cells by SNX-2112 Is Enhanced by STAT3 Silencing.

Many studies have demonstrated that cancer stem cells (CSCs) or tumor-initiating cells (TICs) are responsible for tumor cell proliferation, chemotherapy resistance, metastasis, and relapse in various cancers. We, and others, have previously shown that the signal transducer and activator of transcription 3 (STAT3) signaling pathway is responsible for CSCs and TICs growth. Recent reports have indicated that the heat shock protein 90 (Hsp90) is also essential for the survival of CSCs and TICs. SNX-2112 is an Hsp90 inhibitor. However, it remains unclear whether proliferation of esophageal cancer stem-like cells (ECSLCs) is suppressed by SNX-2112 with knockdown of STAT3 (shSTAT3). Here, we explored the association between SNX-2112 with shSTAT3 and the suppression of ECSLCs growth. We found that the expression level of both STAT3 and p-STAT3 was higher in clinical esophageal cancer tissue than in the adjacent normal tissue, using western blot and qPCR analysis. Furthermore, differential expression analysis demonstrated that STAT3 was overexpressed in clinical specimens. We demonstrated that SNX-2112 inhibited cancer cell proliferation, decreased ABCB1 and ABCG2 gene expression levels and reduced the colony formation capacity of ECSLCs, which was enhanced by STAT3 silencing. Flow cytometry analysis revealed that the combination of SNX-2112 and shSTAT3 significantly induced apoptosis and cell cycle arrest at G2/M phase in ECSLCs. Levels of proliferation pathway proteins, including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) which were also client proteins of Hsp90, were also reduced. In addition, SNX-2112 with shSTAT3 inhibited the proliferation of ECSLCs in vivo. Finally, STAT3 overexpression eliminated the apoptotic and antiproliferative effects of SNX-2112 on ECSLCs. Hence, these results provide a rationale for the therapeutic potential of the combination of SNX-2112 with shSTAT3 in esophageal cancer, and may indicate new targets for clinical intervention in human cancer.

The IGF2/IGF1R/Nanog Signaling Pathway Regulates the Proliferation of Acute Myeloid Leukemia Stem Cells.

Acute myeloid leukemia is an aggressive disease characterized by clonal proliferation and differentiation into immature hematopoietic cells of dysfunctional myeloid precursors. Accumulating evidence shows that CD34+CD38- leukemia stem cells (LSCs) are responsible for drug resistance, metastasis, and relapse of leukemia. In this study, we found that Nanog, a transcription factor in stem cells, is significantly overexpressed in CD34+ populations from patients with acute myeloid leukemia and in LSCs from leukemia cell lines. Our data demonstrate that the knockdown of Nanog inhibited proliferation and induced cell cycle arrest and cell apoptosis. Moreover, Nanog silencing suppressed the leukemogenesis of LSCs in mice. In addition, we found that these functions of Nanog were regulated by the insulin-like growth factor receptor (IGF1R) signaling pathway. Nanog overexpression rescued the colony formation ability of LSCs treated with picropodophyllin (PPP), an IGF1R inhibitor. By contrast, knockdown of Nanog abolished the effects of IGF2 on the colony formation ability of these LSCs. These findings suggest that the IGF2/IGF1R/Nanog signaling pathway plays a critical role in LSC proliferation.

Predicting hepatocellular carcinoma development for cirrhosis patients via methylation detection of heparocarcinogenesis-related genes.

Background: Most hepatocellular carcinoma (HCC) patients have undergone a progression from chronic hepatitis, then liver cirrhosis (LC), and finally to carcinoma. The objective of this study was to elucidate risk factors to predict HCC development for cirrhosis patients. Methods: Multiple methylated specific PCR (MSP) was applied to determine methylation status of heparocarcinogenesis-related genes in 396 tissue and plasma specimens and multivariate cox model was used to analyze the relationship between risk variables and HCC development among cirrhosis patients, followed up in a median period of 30 months. Results: Among 105 LC cases, HCC incidence rate at 30 months was 30.48% (32/105), which were statistically associated with patients' age and aberrant methylation of p16, SFRP, and LINE1 (p<0.05). Receiver operating characteristic (ROC) curve showed the overall predictive accuracy reached the highest (90.7%) if the four risk variables were concurrent to predict HCC development. Moreover, along with the growth of age from 0-40, 40-55, to 55-70 years or the increased number of aberrantly-methylated gene from 0-1 to 2-3, the HCC incidence rate of cirrhosis patients rised from 10.00%, 12.28% to 82.14% and 17.44% to 89.47%, separately. Thus, based on combined analysis with diverse age and number of aberrantly-methylated gene, 105 cases were divided into five groups and computed their respective HCC incidecne rate to categorize them into different risk groups. Of note, A significant lifting of HCC incidence rate in the high-risk group (40-55 years coupled with 2-3 aberrantly-methylated genes, 55-70 years coupled with 0-1 aberrantly-methylated gene, 55-70 years coupled with 2-3 aberrantly-methylated genes; n=33) was observed compared with the low-risk group (0-40 years coupled with 0-1 aberrantly-methylated gene, 40-55 years coupled with 0-1 aberrantly-methylated gene; (n=72) (p<0.01). Conclusions: Ultimately, high-risk cirrhosis patients with 55-over years or 2-3 aberrantly-methylated genes should be paid more attention to be regularly screened with HCC development.

[BORIS Regulates SOCS3 Expression Through Epigenetic Mechanisms in Human Hepatocellular Carcinoma Cells].

OBJECTIVE: To study the regulation of suppressor of cytokine signaling 3 (SOCS3) expression bythe brother of the regulator of the imprinted site (BORIS) in hepatocellular carcinoma cell. METHODS: The expression of SOCS3 mRNA in HCC cell lines was detected by real-time quantitative PCR (qRT-PCR). The expression of SOCS3 protein in knockdown and overexpression BORIS of HCC cell lines was tested by Western blot. The SOCS3 gene promoter methylation statusin the knockdown and overexpression BORIS of hepatocarcinoma cell lines was detected by using methylation specific PCR (MSP-PCR) method.The potential BORIS binding site of SOCS3promoter region was found by UCSC database analysis.The enrichment of BORIS in SOCS3 promoter region in endogenous high expression BORIS of HCC cells was evaluated by using chromatin immunoprecipitation (ChIP)-qPCR (ChIP-qPCR).The SOCS3 promoter region histone methylation status in the knockdown and overexpression BORIS of HCC was detected by ChIP-qPCR. RESULTS: The expression of SOCS3 mRNA in hepatocellular carcinoma cells was higher and SOCS3 protein expression was down-regulated or up-regulated in the knockdown or overexpression of BORIS mRNA hepatocarcinoma cells,so BORIS has a positive regulatory effect on SOCS3 protein expression in hepatocarcinoma cells. MSP-PCR experiments showed that the SOCS3 promoter in SMMC-7721 and HepG2 cells was unmethylated and knockdown of BORIS did not change the methylation status; the SOCS3 promoter region of Huh7 cells was methylated; after overexpression of BORIS,the SOCS3 promoter region was changed to an unmethylated state; the SOCS3 promoter was unmethylated in HCCLM3,overexpression of BORIS did not alter the methylation status. The ChIP-qPCR assay demonstrated that BORIS specifically binds to the SOCS3 promoter region in HCC cells with high expression of BORIS. Histone methylation assay indicated that knockdown of BORIS reduced BORIS enrichment in the SOCS3 promoter region, with decreasing H3K4 me2 and increasing H3K27 me3 in the region of histone,whereas the overexpress BORIS in HCC cells showed the opposite situation. CONCLUSION: BORIS plays a role of epigenetic regulationon SOCS3 gene promoter methylation and histone methylation,modulating the expression of SOCS3,and then involved in the development of hepatocellular carcinoma.

Application of multiplex methylated-specific PCR with capillary electrophoresis to explore prognostic value of TSGs hypermethylation for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant tumor that severely threatens human health. To date, early detection for HCC patients is particularly significant due to their poor survival rates even after liver resection. METHODS: Therefore, an efficient and sensitive detection method for monitoring liver cancer, multiplex methylation-specific PCR (MSP) coupled with capillary electrophoresis, is developed. RESULTS: Simulations demonstrated that the methylation status of RASSF1A, p16, SFRP1, and ELF could be detected even when DNA equaled or exceeded 12.5 ng simultaneously. Also, its accuracy for methylation detection outweighed polyacrylamide gel electrophoresis (87.5%) and agarose electrophoresis (84.3%), reaching 92.1%. Subsequently, we implemented multiplex MSP with capillary electrophoresis to investigate methylation status of the four tumor suppressor genes in tissue specimens and explore the prognostic value for HCC patients. As the data suggested, multivariate cox regression analysis revealed that the recurrence-free survival of 46 patients was greatly associated with portal vein tumor thrombus (PVTT) and p16 methylation and receiver operating characteristic (ROC) curves demonstrated that the predictive range of portal vein tumor thrombus (PVTT) combined with p16 hypermethylation was more sensitive than that of either PVTT or p16 hypermethylation alone with regard to disease recurrence in patients with HCC, which could be testified as a valuable biomarker in Clinical application. CONCLUSION: Multiplex MSP coupled with capillary electrophoresis has an excellent prospect of clinical application for monitoring early liver cancer and screening valuable biomarkers for prognosis of HCC patients.

Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

Heat-shock protein 90 (Hsp 90) acts as a molecular chaperone that maintains protein stability and regulates cell proliferation, survival, differentiation and apoptosis. The present study investigated the effect of Hsp90 inhibition on human acute myeloid leukemia (AML) cells using the novel small-molecule inhibitor SNX-2112. We found that SNX-2112 more potently inhibited KG-1a cell growth than the classical Hsp90 inhibitor 17-(2-dimethylaminoethyl)amino­17-demethoxygeldanamycin as determined by CCK-8 assay. Flow cytometry was used to examine the cell cycle, differentiation, and apoptosis, and western blotting and qRT-PCR were used to analyze the underlying mechanism. The results revealed that low concentrations of SNX-2112 arrested the cells in the G2/M phase and induced their differentiation and apoptosis, possibly by suppressing Akt and inhibitor of κB kinase, a component of the nuclear factor (NF)-κB signaling pathway. We also found that SNX-2112 increased the expression of the differentiation transcription factors PU.1 and CCAAT­enhancer-binding protein-α. Thus, SNX-2112 induced KG-1a cell differentiation, cell cycle arrest and apoptosis via modulation of Akt and NF-κB signaling, suggesting that it is a promising therapeutic agent for the treatment of AML.

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.

Episomal lentiviral vectors confer erythropoietin expression in dividing cells.

Lentiviral vectors are now widely considered as one of the most common gene delivery tools for dividing and non-dividing cells. However, insertional mutagenesis has been found in clinical trials with retroviral vectors, which poses a safety risk. The use of non-integrating lentiviral (NIL) vectors, which avoid integration, eliminates the insertional mutagenesis problem. These NIL vectors are unable to mediate stable gene delivery into dividing cells, which makes them of limited use in the clinical practice of gene therapy. In this study, we constructed a NIL vector which harbors the scaffold/matrix attachment region (S/MAR) sequence and a therapeutic gene. NIL retained episomal erythropoietin (EPO) gene expression for 74days in dividing cells both with and without selection. Furthermore, Southern blot analysis showed that the NIL vector was retained extrachromosomally in CHO cells. In conclusion, the NIL vector based on an S/MAR sequence retained the extrachromosomal expression of a therapeutic gene in dividing cells. Our results show that NIL vectors maybe a safe and effective means of gene delivery, which is of potential clinical significance.

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.

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.

The efficiency of magnetic hyperthermia and in vivo histocompatibility for human-like collagen protein-coated magnetic nanoparticles.

Magnetic hyperthermia is a promising technique for the minimally invasive elimination of solid tumors. In this study, uniform magnetite nanoparticles (MNPs) with different particle sizes were used as a model system to investigate the size and surface effects of human-like collagen protein-coated MNPs (HLC-MNPs) on specific absorption rate and biocompatibility. It was found that these HLC-MNPs possess rapid heating capacity upon alternating magnetic field exposure compared to that of MNPs without HLC coating, irrespective of the size of MNPs. The significant enhancement of specific absorption rate is favorable for larger sized nanoparticles. Such behavior is attributed to the reduced aggregation and increased stability of the HLC-MNPs. By coating HLC on the surface of certain sized MNPs, a significant increase in cell viability (up to 2.5-fold) can be achieved. After subcutaneous injection of HLC-MNPs into the back of Kunming mice, it was observed that the inflammatory reaction hardly occurred in the injection site. However, there was a significant presence of phagocytes and endocytosis after the injection of nonconjugated counterparts. The overall strategy to fabricate HLC-MNPs can serve as a general guideline to address the current challenges in clinical magnetic hyperthermia, improved biocompatibility, and enhanced heating characteristics through protein coating.

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.

EM23, a natural sesquiterpene lactone, targets thioredoxin reductase to activate JNK and cell death pathways in human cervical cancer cells.

Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants and found to have potential anticancer activities. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. In this study, we observed that EM23, a natural SL, exhibited anti-cancer activity in human cervical cancer cell lines by inducing apoptosis as indicated by caspase 3 activation, XIAP downregulation and mitochondrial dysfunction. Mechanistic studies indicated that EM23-induced apoptosis was mediated by reactive oxygen species (ROS) and the knockdown of thioredoxin (Trx) or thioredoxin reductase (TrxR) resulted in a reduction in apoptosis. EM23 attenuated TrxR activity by alkylation of C-terminal redox-active site Sec498 of TrxR and inhibited the expression levels of Trx/TrxR to facilitate ROS accumulation. Furthermore, inhibition of Trx/TrxR system resulted in the dissociation of ASK1 from Trx and the downstream activation of JNK. Pretreatment with ASK1/JNK inhibitors partially rescued cells from EM23-induced apoptosis. Additionally, EM23 inhibited Akt/mTOR pathway and induced autophagy, which was observed to be proapoptotic and mediated by ROS. Together, these results reveal a potential molecular mechanism for the apoptotic induction observed with SL compound EM23, and emphasize its putative role as a therapeutic agent for human cervical cancer.

[Segment Cloning and Expression of Human VIGILIN Gene].

OBJECTIVE: To investigate the mechanisms of interaction between high-density lipoprotein binding protein (HDLBP)-VIGILIN with other proteins, we cloned VIGILIN cDNA N, KH1-7, KH8-12, KH13-14, and C fragments separately into expression vector, and identify the expressed proteins. METHODS: The recombinant plasmid pDsred2-N1/VIGILIN was used as template to amplify VIGILIN full length, VIGILIN N terminal, KH1- 7, KH8-12, KH13-14, C terminal and recombinated them with pGEX 5X 3. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, we induced GST-VIGILIN fusion proteins on the appropriate conditions. RESULT: The recombinant plasmids of pGEX 5X 3/VIGILIN FL, pGEX 5X 3/VIGILIN N terminal, pGEX 5X 3/VIGILIN KH1-7, pGEX 5X 3/VIGILIN KH8-12, pGEX 5X 3/VIGILIN KH13-14, pGEX 5X 3/VIGILIN C terminal were constructed successfully, and induced the GST-VIGILIN fusion proteins. CONCLUSION: pGEX 5X 3/VIGILIN FL, pGEX 5X 3/VIGILIN N terminal, pGEX 5X 3/VIGILIN KH1-7, pGEX SX 3/VIGILIN KH8-12, pGEX 5X 3/ VIGILIN KH13-14, pGEX 5X 3/VIGILIN C terminal recombinant plasmids were constructed successfully, and their corresponding fusion proteins were successfully expressed.

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.

Tangeretin from Citrus reticulate Inhibits Respiratory Syncytial Virus Replication and Associated Inflammation in Vivo.

Human respiratory syncytial virus (RSV) is a common pathogen that causes pneumonia and bronchiolitis in infants and young children. Our previous study showed that tangeretin from Citrus reticulate possessed potent in vitro anti-RSV effects comparable to that of ribavirin. Therefore, in this study, we investigated the in vivo anti-RSV activity of tangeretin in 3-week-old male BALB/c mice. A plaque reduction assay and fluorescence quantitative polymerase chain reaction (FQ-PCR) showed that tangeretin inhibited RSV replication in the lung of mice. Moreover, a luminex assay indicated tangeretin relieved RSV-induced lung inflammation by attenuating interleukin (IL)-1ß secretion. Possible anti-inflammatory mechanisms of tangeretin were preliminarily explored using a RSV-infected macrophage model. A FQ-PCR, enzyme-linked immunosorbent assay (ELISA), and luciferase assay revealed that tangeretin inhibited RSV-induced inflammation by suppressing nuclear factor-κB (NF-κB) activation. This study demonstrates that tangeretin inhibited RSV replication and RSV-induced lung inflammation in vivo and may be useful in preventing and treating RSV infections and inflammation.

Sp1 and c-Myc modulate drug resistance of leukemia stem cells by regulating survivin expression through the ERK-MSK MAPK signaling pathway.

BACKGROUND: Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs), which contribute to the progression, recurrence and therapeutic resistance of leukemia. However, the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study, we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS: We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting, cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay, mutant constructs, chromatin immuno-precipitation (ChIP), quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and western blotting. The levels of Sp1, c-Myc, phospho-extracellular signal-regulated kinase (p-ERK), phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS: Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally, survivin contributes to the drug resistance of LSCs, and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically, Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover, Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway, modulating survivin levels. CONCLUSION: Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs, offering a potential new therapeutic strategy for LSCs therapy.

[Effects of CTCF on human liver cancer stem cells and cell proliferation].

OBJECTIVE: To explore the effects of CCCTC-binding factor (CTCF) on human liver cancer stem cells (HepG2) and cell proliferation of HepG2 and Nasopharyngeal carcinoma cell line (CNE1). METHODS: The pEGFP-N1/CTCF, CTCF-shRNA and GFP-shRNA plasmids were constructed and transfected into HepG2 and CNE1 cells, and RT-PCR or Western blot were performed to detect the mRNA or protein levels of CTCF. The subpopulation of CD90+ cancer stem cells in HepG2 cells transfected with CTCF-shRNA plasmid or GFP-shRNA plasmid (as transfection control) were assayed by flow cytometry with the wild type HepG2 cells as control. Proliferation of cells transfected with CTCF-overexpression or CTCF-shRNA plasmid was evaluated by MTT assay. RESULTS: The levels of both mRNA and protein of CTCF were increased in pEGFP-N1/CTCF transfected HepG2 and CNE1 cells compared to that in pEGFP-N1 transfected cells (P < 0.05), and decreased in CTCF-shRNA transfected cells compared to that in cells transfected with GFP-shRNA (P < 0.05). The results of flow cytometry demonstrated that, detection rate of CD90+ cells in cells transfected with CTCF-shRNA plasmid [(1.7330 +/- 0.4177)%] was obviously higher than that of wild-type HepG2 cells [(0.5750 +/- 0.0629)%] and cells transfected with GFP-shRNA plasmid [(0.3500 +/- 0.0866)%] (P < 0.05). The results of MTT analysis showed that, alteration of CTCF had no effect on cancer cell proliferation (P > 0.05). CONCLUSION: CTCF inhibits human liver cancer stem cells but no effect on cell proliferation.

CTCF-mediated reduction of vigilin binding affects the binding of HP1α to the satellite 2 locus.

CCCTC-binding factor (CTCF) has been implicated in numerous aspects of chromosome biology, and vigilin, a multi-KH-domain protein, participates in heterochromatin formation and chromosome segregation. We previously showed that CTCF interacts with vigilin. Here, we show that human vigilin, but not CTCF, colocalizes with HP1α on heterochromatic satellite 2 and ß-satellite repeats. CTCF up-regulates the transcription of satellite 2, while vigilin down-regulates it. Vigilin depletion or CTCF overexpression reduces the binding of HP1α on the satellite 2 locus. Furthermore, overexpression of CTCF resists the loading of vigilin onto the satellite 2 locus. Thus CTCF may regulate vigilin behavior and thus indirectly influence the binding of HP1α to the satellite 2 locus.