Epigenetic inactivation of T-box transcription factor 5, a novel tumor suppressor gene, is associated with colon cancer

T-box transcription factor 5 (TBX5) is a member of a phylogenetically conserved family of genes involved in the regulation of developmental processes. The function of TBX5 in cancer development is largely unclear. We identified that TBX5 was preferentially methylated in cancer using methylation-sensitive arbitrarily primed PCR. We aim to clarify the epigenetic inactivation, biological function and clinical significance of TBX5 in colon cancer. Promoter methylation was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. Cell proliferation was examined by cell viability assay and colony formation assay, apoptosis by flow cytometry and cell migration by wound-healing assay. TBX5 target genes were identified by cDNA microarray analysis. Cox regression model and log-rank test were used to identify independent predictors of prognosis. TBX5 was silenced or downregulated in 88% (7/8) colon cancer cell lines, but was expressed in normal colon tissues. Loss of gene expression was associated with promoter methylation. The biological function of TBX5 in human colon cancer cells was examined. Re-expression of TBX5 in silenced colon cancer cell lines suppressed colony formation (P<0.001), proliferation (P<0.001), migration and induced apoptosis (P<0.01). Induction of apoptosis was mediated through cross-talk of extrinsic apoptosis pathway, apoptotic BCL2-associated X protein and Granzyme A signaling cascades. TBX5 suppressed tumor cell proliferation and metastasis through the upregulation of cyclin-dependent kinase inhibitor 2A, metastasis suppressor 1 and downregulation of synuclein gamma and metastasis-associated protein 1 family member 2. TBX5 methylation was detected in 68% (71/105) of primary colon tumors. Multivariate analysis showed that patients with TBX5 methylation had a significantly poor overall survival (P=0.0007). In conclusion, we identified a novel functional tumor suppressor gene TBX5 inactivated by promoter methylation in colon cancer. Detection of methylated TBX5 may serve as a potential biomarker for the prognosis of this malignancy. © 2010 Macmillan Publishers Limited All rights reserved.link_to_subscribed_fulltex

Identification of microrna-135b in stool as a potential noninvasive biomarker for colorectal cancer and adenoma

Purpose: Detecting microRNA (miRNA) in stool is a novel approach for colorectal cancer (CRC) screening. This study aimed to identify stool-based miRNA as noninvasive biomarkers for detection of CRC and adenoma. Experimental Design: A miRNA expression array covering 667 human miRNAs was performed on five pairs of CRC and two pairs of advanced adenoma tissues. The most upregulated miRNAs were validated in 40 pairs of CRC tissues, 16 pairs of advanced adenoma tissues, and 424 stool samples, including 104 CRCs, 169 adenomas, 42 inflammatory bowel diseases (IBD), and 109 healthy controls. miRNA levels were followed-up after removal of lesions. Results: In an array analysis, miR-31 and miR-135b were the most upregulated miRNAs in CRC and advanced adenoma as compared with their adjacent normal tissues (>13-fold increase). In stool samples, level of miR-135b was significantly higher in subjects with CRC (P < 0.0001) or adenomas (P < 0.0001), but not in patients with IBD compared with controls. miR-135b showed a significant increasing trend across the adenoma to cancer sequence (P < 0.0001). Levels of miR-31 were not significantly different among groups. The sensitivity of stool mR-135b was 78% for CRC, 73% for advanced adenoma, and 65% for any adenoma, respectively, with a specificity of 68%. No significant difference in the miR-135b level was found between proximal and distal colorectal lesions. Stool miR-135b dropped significantly upon removal of CRC or advanced adenoma (P < 0.0001). Conclusion: Stool-based miR-135b can be used as a noninvasive biomarker for the detection of CRC and advanced adenoma. ©2014 AACR.link_to_subscribed_fulltex

Metabolic Signatures of Kidney Yang Deficiency Syndrome and Protective Effects of Two Herbal Extracts in Rats Using GC/TOF MS

Kidney Yang Deficiency Syndrome (KDS-Yang), a typical condition in Chinese medicine, shares similar clinical signs of the glucocorticoid withdrawal syndrome. To date, the underlying mechanism of KDS-Yang has been remained unclear, especially at the metabolic level. In this study, we report a metabolomic profiling study on a classical model of KDS-Yang in rats induced by hydrocortisone injection to characterize the metabolic transformation using gas chromatography/time-of-flight mass spectrometry. WKY1, a polysaccharide extract from Astragalus membranaceus and Lycium barbarum, and WKY2, an aqueous extract from a similar formula containing Astragalus membranaceus, Lycium barbarum, Morinda officinalis, Taraxacum mongolicum, and Cinnamomum cassia presl, were used separately for protective treatments of KDS-Yang. The changes of serum metabolic profiles indicated that significant alterations of key metabolic pathways in response to abrupt hydrocortisone perturbation, including decreased energy metabolism (lactic acid, acetylcarnitine), lipid metabolism (free fatty acids, 1-monolinoleoylglycerol, and cholesterol), gut microbiota metabolism (indole-3-propionic acid), biosynthesis of catecholamine (norepinephrine), and elevated alanine metabolism, were attenuated or normalized with different degrees by the pretreatment of WKY1 or WKY2, which is consistent with the observations in which the two herbal agents could ameliorate biochemical markers of serum cortisone, adrenocorticotropic (ACTH), and urine 17-hydroxycorticosteroids (17-OHCS)

Polysaccharide from <em>Lentinus edodes</em> Inhibits the Immunosuppressive Function of Myeloid-Derived Suppressor Cells

<div><p>Reversing the function of immune suppressor cells may improve the efficacy of cancer therapy. Here, we have isolated a novel polysaccharide MPSSS (577.2 Kd) from <em>Lentinus edodes</em> and examined its effects on differentiation and function of myeloid-derived suppressor cells (MDSCs). MPSSS is composed of glucose (75.0%), galactose (11.7%), mannose (7.8%), and xylose (0.4%). In vivo, it inhibits the growth of McgR32 tumor cells, which is correlated with a reduced percentage of MDSCs in peripheral blood. In vitro, it induces both morphological and biophysical changes in MDSCs. Importantly, MPSSS up-regulates MHC II and F4/80 expression on MDSCs, and reverses their inhibition effect on CD4⁺ T cells in a dose-dependent manner. The mechanism study shows that MPSSS may stimulate MDSCs through a MyD88 dependent NF-κB signaling pathway. Together, we demonstrated for the first time that MPSSS stimulates the differentiation of MDSCs and reverses its immunosuppressive functions, shedding new light on developing novel anti-cancer strategies by targeting MDSCs.</p> </div

MPSSS activated Ly6C^high CD11b⁺, but not Ly6C^int CD11b⁺ cells.

<p>(A) Ly6C^high CD11b⁺ cells (P2) and Ly6C^int CD11b⁺ cells (P3) were purified from splenocyte (P1) of McgR32 tumor-bearing mice. (B) Ly6C^high CD11b⁺ cells and Ly6C^int CD11b⁺ cells with MPSSS stimulation for 72 hours were viewed with bright-field (original magnification ×100). (C & D) Supernatants of Ly6C^high CD11b⁺ cells and Ly6C^int CD11b⁺ cell cultures were used for determination of the cytokines, including TNF-α, IFN-γ, MCP-1 and IL-10. Quantitative data are presented as means ± SD and are analyzed by one-way ANOVA. Multiple comparisons between groups are performed using the S-N-K method. *<i>P</i><0.05.</p

MPSSS inhibited tumor growth and reduced MDSC numbers.

<p>(A) C57BL/6 mice were injected subcutaneously with 1×10⁶ McgR32 cellsand 8 day later, with MPSSS or PBS intraperitoneally once every two days. Tumor volume was recorded as the mean ± SD and each group contained 3–4 mice and was analyzed by one-way ANOVA for repeated measures data. (B) At the indicated days after tumor cell injection, peripheral blood was taken for MDSC detection by flow cytometry, Quantitative data are expressed as mean ± SD and analyzed by Student's <i>t</i> tests. *<i>P</i><0.05, **<i>P</i><0.01. (C) A representative flow cytometry analysis was shown for the staining of CD11b and Gr1.</p

Composition and structure analysis of MPSSS.

<p>(A) MPSSS appeared as a single peak in Sepharose CL-6B chromatography, indicating its high degree of purity. (B) The monosaccharides derived from MPSSS were detected by HPLC. (C) Analytical results of the composition of MPSSS. (D) Results of ¹³C NMR detection of MPSSS. (E) Analytical results of the MPSSS polysaccharide basic structure unit.</p

MPSSS promoted functional recovery of splenocytes following MDSC-induced suppression.

<p>(A) [³H]-thymidine or (B) CFSE-labeled splenocytes were co-cultured with purified MDSCs isolated from McgR32 tumor-bearing mice and stimulated with or without ConA or MPSSS as indicated. Results were presented as mean ± SD of triplicate samples from one representative experiment and were analyzed by one-way ANOVA. Comparisons between these groups were performed using the S-N-K method. **<i>P</i><0.01.</p

MPSSS activated MDSCs through a MyD88-dependent NF-κB signal pathway.

<p>Purified MDSCs from control C57BL/6 and MyD88^−/− tumor-bearing mice were stimulated with MPSSS for different times as indicated. Levels of P-p65, and P-IκB in total cell lysates were determined by Western blotting. The expression of β-actin served as a control. The image shown was representative for 3 independent experiments.</p

MPSSS promoted differentiation of MDSCs.

<p>(A) FACS-sorted splenic MDSCs from McgR32 tumor-bearing mice with or without MPSSS stimulation were viewed with bright-field illumination, (original magnification ×200) and (B) after Wright’s staining for nuclei (original magnification ×200). (C) Electrical impedance measured using a real-time cell electronic sensing (RT-CES) system. (D) All adherent splenocytes from McgR32 tumor-bearing mice were cultured with or without MPSSS for 72 hours and the expression of MHC I, MHC II, F4/80, CD11c or CD86 on MDSCs were analyzed by FACS. (E) Immunofluorescent staining of F4/80 on MPSSS-treated MDSCs (original magnification ×100). (F) Purified MDSCs were stimulated with MPSSS for 4 hours, and the expression of CXCL9, IL-6, or TGF-β mRNA was determined by RT-PCR. Results were given relative to β-actin mRNA and are presented as means ± SD of triplicate samples from one representative experiment. Data is analyzed with Student's <i>t-</i>tests. *<i>P</i><0.05, **<i>P</i><0.01.</p