Prognostic value of tumor-infiltrating lymphocytes in DCIS: a meta-analysis.

BACKGROUND: Tumor infiltrating lymphocytes (TILs) have been shown to be associated with the prognosis of breast ductal carcinoma in situ (DCIS). In this systematic review and meta-analysis, we investigated the role of TILs and TIL subsets in predicting the recurrence risk of DCIS. METHOD: PubMed, Medline, Web of Science, Embase and Cochrane were searched to identify publications investigating the prognostic role of TILs in DCIS. After study screening, data extraction and risk of bias assessment, a meta-analysis was performed to assess the association between TILs (total TILs, CD4+, CD8+, FOXP3+, PD-L1+ TILs) and the risk of DCIS recurrence. RESULTS: A pooled analysis indicated that dense stromal TILs in DCIS were associated with a higher recurrence risk (HR 2.11 (95% CI 1.35-3.28)). Subgroup analysis showed that touching TILs (HR 4.73 (95% CI 2.28-9.80)) was more precise than the TIL ratio (HR 1.49 (95% CI 1.11-1.99)) in estimating DCIS recurrence risk. Moreover, the prognostic value of TILs seemed more suitable for patients who are diagnosed with DCIS and then undergo surgery (HR 2.77, (95% CI 1.26-6.07)) or surgery accompanied by radiotherapy (HR 2.26, (95% CI 1.29-3.95)), than for patients who receive comprehensive adjuvant therapies (HR 1.16, (95% CI 1.35-3.28)). Among subsets of TILs, dense stromal PD-L1+ TILs were valuable in predicting higher recurrence risk of DCIS. CONCLUSION: This systematic review and meta-analysis suggested a non-favorable prognosis of TILs and stromal PD-L1+ TILs in DCIS and indicated an appropriate assessment method for TILs and an eligible population.

ATF4 promotes lung cancer cell proliferation and invasion partially through regulating Wnt/ß-catenin signaling.

Activating transcription factor 4 (ATF4) is a member of the cAMP response element binding (CREB) protein family and has been reported to participate in cancer progression; however, its molecular mechanism is not fully understood. In this study, we investigated the function of ATF4 in non-small cell lung cancer and its molecular regulation. We detected cytoplasmic and nuclear ATF4 expression in lung cancer A549, H1299, and LK2 cells, and the total expression of ATF4 was higher than that in HBE cells (p < 0.05). Higher nuclear ATF4 expression was detected in all these cells compared to cytoplasmic ATF4 expression (p < 0.05). Overexpression of ATF4 in A549 cells significantly promoted cancer cell growth and invasion (p < 0.05). Expression of Wnt signaling molecules, including ß-catenin, MMP7, and cyclin D1, and the activity of canonical Wnt signaling were also significantly promoted by ATF4 (p < 0.05). ICG001, a canonical Wnt signaling inhibitor that selectively inhibits ß-catenin/ cyclic adenosine monophosphate response element binding protein (CBP) interaction, significantly inhibited cancer cell invasion and Wnt signaling. The function of ATF4 was also significantly inhibited by ICG001 (p < 0.05). However, compared to treatment with ICG001, the invasion ability of cancer cells treated with both ICG001 and ATF4 cDNA significantly increased (p < 0.05), which indicates that the function of ATF4 was not dependent only on Wnt/ß-catenin signaling. The function of ATF4 in the regulation of ß-catenin expression was not significantly affected by ICG001 (p > 0.05). The function of ATF4 to promote the activity of Wnt/ß-catenin signaling in cancer cells was abolished by treatment with ICG001 (p > 0.05). These results indicate that ATF4 may contribute to lung cancer progression at least partly by regulating Wnt/ß-catenin signaling.

Pinewood outperformed bamboo as feedstock to prepare biochar-supported zero-valent iron for Cr6+ reduction.

In this work, pinewood and bamboo were pyrolyzed at 600 °C to prepare PBC and BBC-supported zerovalent iron (ZVI), respectively. Raman spectra suggested PBC was more intensively carbonized than BBC as indicated by higher ID/IG ratio. XRD and TEM confirmed nanoscaled ZVI was well dispersed in PBC but soldered in chain-structure in BBC. Maximal chromate (Cr(VI)) sorption capacity followed the order of PBC/ZVI (5.93 g kg-1)>BBC/ZVI (3.61 g kg-1)>BBC (3.55 g kg-1)>PBC (2.59 g kg-1). Desorption and XPS of four Cr-spent sorbents suggested reduction accounted for 79-88% of overall Cr(VI) detoxification. Greater Cr(VI) reduction of BBC than PBC indicated greater tendency of BBC to donate electrons. However, Cr(VI) reduction by PBC/ZVI was 1.7 times greater than BBC/ZVI, corresponding to greater electron transfer of PBC/ZVI (2.5 µA e-) than BBC/ZVI (0.5 µA e-). Thus, PBC is more conducible to transfer electrons as evidenced by Tafel and Amperometric analyses. Demineralization of pristine BC enhanced the difference between PBC/ZVI and BBC/ZVI regarding Cr(VI) reduction, suggesting the dominant role of biopolymers in biomass in terms of electron transfer capacity. Three model biopolymers were compared which indicated lignin-BC had lower electron transfer rates than cellulose-BC and hemicellulose-BC. BC prepared by lignin extracted from pinewood exhibited higher corrosion rate and lower electrical resistance than that from bamboo. Thus, unfavorable lignin in bamboo compromised electron transfer of BBC and Cr(VI) reduction by BBC/ZVI.

miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly.

Krüppel-like factor 5 (KLF5) takes part in the pathologic processes of many types of cancer; however, its expression and roles in the biological behavior of gastric cancer remain unknown. TargetScan suggested that miR-145-5p is the predicted effective and conserved microRNA (miRNA) that binds to KLF5 through its 3'-untranslated region (UTR). We investigated the expression of KLF5 and miR-145-5p messenger RNA (mRNA) in gastric cancer and then analyzed its role in the biological behavior of gastric cancer cells. Our results indicated that KLF5 expression was detected by immunohistochemistry in 39.7% of the gastric cancer cases and was increased compared with that of the corresponding noncancerous normal mucosa (0.01 < p < 0.05). The poorly differentiated subtype showed positive KLF5 expression, whereas the differentiated subtype showed negative KLF5 expression (p < 0.05). Dual-luciferase reporter assay suggested KLF5 3'-UTR was the direct target of miR-145-5p. Compared with the differentiated gastric cancer, miR-145-5p was downregulated in undifferentiated gastric cancer (p < 0.05). The downregulation of KLF5 expression and differentiation of MGC-803 and BGC-823 caused by siKLF5 or miR-145-5p mimic transfection. Our results indicated that miR-145-5p/KLF5 3'-UTR affected the differentiation of gastric cancer. miR-145-5p was able to promote gastric cancer differentiation by targeting KLF5 3'-UTR directly. Our data suggest a novel mechanism for cancer differentiation and a new facet to the role of miR-145-5p/KLF5 in gastric cancer.

CUL7 promotes cancer cell survival through promoting Caspase-8 ubiquitination.

The Cullin 7 (CUL7) gene encodes a member of the cullin family of E3 ubiquitin ligases. Accumulated evidence suggests that CUL7 is oncogenic. However, the mechanism by which CUL7 improves cancer cell survival has not been fully elucidated. Here, we reported that CUL7 confers anti-apoptotic functions by interacting with Caspase-8. CUL7 prevents Caspase-8 activation by promoting Caspase-8 modification with non-degradative polyubiquitin chains at K215. CUL7 knockdown sensitized cancer cells to TRAIL-induced apoptosis in vitro and in nude mice. These results suggest that CUL7 limits extrinsic apoptotic signaling by promoting Caspase-8 ubiquitination.

Azotobacter bryophylli sp. nov., isolated from the succulent plant Bryophyllum pinnatum.

A Gram-stain-negative, aerobic, nitrogen-fixing bacterium, designated strain L461T, was isolated from leaves of Bryophyllum pinnatum growing at the South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated it as a member of the genus Azotobacter closely related to Azotobacter beijerinckii JCM 20725T (97.82 % similarity) and Azotobacter chroococcum ATCC 9043T (97.34 %). Its major fatty acid components were C16 : 1 ω9c and C16 : 0. Its predominant isoprenoid quinone was Q-9. Its major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, phospholipid and one unknown lipid. Its DNA G+C content was 64.9 mol% (Tm). DNA-DNA relatedness values between strain L461T and the reference strains of A. beijerinckii and A. chroococcum were 46.43 and 28.23 %, respectively. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting, and comparison of cellular fatty acids distinguished strain L461T from the closely related Azotobacter species. Based on these data, the novel species Azotobacter bryophylli sp. nov. is proposed, with the type strain L461T (=KCTC 62195T=GDMCC 1.1250T).

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy.

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her-2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her-2(+ ) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog-phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PTEN-PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.

Silencing of SOCS-1 and SOCS-3 suppresses renal interstitial fibrosis by alleviating renal tubular damage in a rat model of hydronephrosis.

Our study was performed to elucidate how SOCS-1/3 silencing suppresses renal interstitial fibrosis (RIF) by alleviating renal tubular damage in rat models affected by hydronephrosis. Male Wistar rats were randomly selected to establish hydronephrosis rat model, after which all rats were classified into normal, model, negative control (NC), siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups. The levels of urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) were detected. ELISA was performed to determine levels of cystatin (CysC), ß2-microglobulin (ß2-MG), interleukin (IL)-6, and tumor necrosis factor (TNF)-α. RT-qPCR and Western blotting were used for mRNA and protein expressions of SOCS-1, SOCS-3, α-smooth muscle actin (α-SMA), and transforming Growth Factor (TGF)-ß1. Compared with the normal group, the levels of Scr, BUN, urine protein, NAG, CysC, ß2-MG, IL-6, and TNF-α were increased in other groups, as well as elevated mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA, and TGF-ß1. The siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups were found with decreased levels of Scr, BUN, urine protein, NAG, CysC, ß2-MG, IL-6, and TNF-α, as well as mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA, and TGF-ß1, including positive rates of SOCS-1 and SOCS-3 proteins in comparison with the model and NC groups. In comparison with the siRNA-SOCS-1 and siRNA-SOCS-3 groups, the siRNA-SOCS-1 + siRNA-SOCS-3 group exhibited decreased levels of Scr, BUN, urine protein, NAG, CysC, ß2-MG, IL-6, and TNF-α. Our study demonstrated that silencing of SOCS-1/3 may suppress RIF by alleviating the renal tubular damage in rat models affected by hydronephrosis.

ZNF326 promotes a malignant phenotype of breast cancer by interacting with DBC1.

The biological role and underlying mechanism of action of zinc-finger protein 326 (ZNF326) in malignant tumors, including breast cancer, are still not clear. In this study, we detected high expression of ZNF326 in breast cancer specimens (60/111, 54.1%) and breast cancer cell lines (7/7); the expression level of ZNF326 was inversely associated with advanced pTNM stage (P = 0.002), positive lymph node metastasis (P = 0.004), poor prognosis in patients with breast cancer (P = 0.0097), and ER/PR/Her2 status (P = 0.013). Meanwhile, the ectopic expression of ZNF326 significantly upregulated MMP7, EMT-related proteins (Snail and Slug), and cell cycle-related proteins (cyclinA2 and cyclinB1); downregulated E-cadherin expression; and promoted the proliferation and invasiveness of breast cancer cells both in vivo and in vitro. Mechanistically, co-immunoprecipitation and immunofluorescence assays both demonstrated that ZNF326 interacted with deleted in breast cancer-1 (DBC1) in breast cancer cells. Additionally, DBC1 knockdown eliminated the up-regulation of MMP7, EMT-related proteins, and cell cycle-related proteins as well as the enhanced proliferation and invasiveness induced by ZNF326. Therefore, we concluded that ZNF326 is highly expressed in breast cancer, is associated with poor prognosis, and plays a vital role in promoting the malignant phenotype of breast cancer cells by interacting with DBC1.

Oncogenic potential of TSTA3 in breast cancer and its regulation by the tumor suppressors miR-125a-5p and miR-125b.

TSTA3 participates in enzyme metabolism and affects glycosylation processes, and abnormal glycosylation influences the malignant transformation of cells and tumor development. However, studies have not examined the molecular biological function of TSTA3 in breast cancer (BC). The expression of TSTA3 was examined in BC tissues and cell lines. Kaplan-Meier survival tests and Cox regression were used to analyze prognosis. TSTA3 depletion was used to analyze cell function. The upstream miRNAs of TSTA3 were predicted, and the downstream target gene was analyzed using a RT2 Profiler™ PCR array. Our results show that TSTA3 was highly expressed in BC tissues and cells and was correlated with poor survival. The expression of TSTA3 was correlated with the TNM status (P < 0.01) and served as an independent prognostic factor (P = 0.041). TSTA3-siRNA decreased cell invasion and proliferation in vitro. miR-125a-5p and miR-125b are upstream targets of TSTA3, and a PCR array revealed that TSTA3 affects the CXCR4-CXCL12 genes. The findings suggest that miR-125a-5p/miR-125b suppress the expression of TSTA3, which controls cell proliferation and invasion by regulating CXCR4 expression. In conclusion, a high expression of TSTA3 exerts a proto-oncogenic effect during carcinogenesis and serves as an independent molecular marker for BC patients.

Characteristics and mechanisms of acrylate polymer damage to maize seedlings.

Superabsorbent acrylate polymers (SAPs) have been widely used to maintain soil moisture in agricultural management, but they may cause damage to plants, and the mechanisms are not well understood. In this study, seed germination, soil pot culture, hydroponic experiments, and SAPs degradation were conducted to investigate damage characteristics and mechanisms associated with SAPs application. The Results showed that SAPs inhibited maize growth and altered root morphology (irregular and loose arrangement of cells and breakage of cortex parenchyma), and the inhibitory effects were enhanced at higher SAPs rates. After 1h SAP hydrogels treatment, root malondialdehyde (MDA) content was significantly increased, while superoxide dismutase (SOD) and catalase (CAT) content were significantly decreased. Hydroponics experiment indicated that root and shoot growth was inhibited at 2.5mgL(-1) acrylic acid (AA), and the inhibition was enhanced with increasing AA rates. This effect was exacerbated by the presence of Na(+) at a high concentration in the hydrogels. Release and degradation of AA were enhanced at higher soil moisture levels. A complete degradation of AA occurred between 15 and 20 days after incubation (DAI), but it took longer for Na(+) concentration to decrease to a safe level. These results indicate that high concentration of both AA and Na(+) present in the SAPs inhibits plant growth. The finding of this study may provide a guideline for appropriate application of SAPs in agriculture.

Overexpression of SMARCA5 correlates with cell proliferation and migration in breast cancer.

SMARCA5 partners with RSF-1 to compose the RSF complex, which belongs to the ISWI family of chromatin remodelers. Recent studies referred that SMARCA5 was overexpressed in some malignant tumors. However, expression pattern and biological roles of SMARCA5 in breast cancer have not been examined. In the present study, we found that SMARCA5 was overexpressed in breast cancer specimens by immunohistochemistry. Significant association was observed between SMARCA5 overexpression and TNM stage (p = 0.0199), tumor size (p = 0.0066), high proliferation index (p = 0.0366), and poor overall survival (p = 0.0141). SMARCA5 overexpression also correlated with Rsf-1 expression levels (p = 0.0120). Furthermore, colony formation assay and Matrigel invasion assay showed that knockdown of SMARCA5 expression in MDA-MB-231 and MDA-MB-435s cell lines with high endogenous expression decreased cell proliferation and cell invasion. Flow cytometry showed knockdown of SMARCA5-arrested cell cycle. Further analysis of cell cycle and invasion-related molecules showed that SMARCA5 downregulated cyclin A, MMP2 expression and upregulated p21 expression. In conclusion, our study demonstrated that SMARCA5 was overexpressed in human breast cancers and correlated with poor prognosis. SMARCA5 contributes to breast cancer cell proliferation and invasion.

Impact of LPL gene rs283 polymorphism on exercise-induced changes in metabolism of obese adolescents and the regulatory mechanisms behind it.

NEW FINDINGS: What is the central question of this study? We investigated whether the LPL gene rs283 polymorphism affects exercise-induced changes in body composition and lipid and glucose metabolism in obese adolescents and whether it is functional. What is the main finding and its importance? Chinese obese adolescents of Han nationality with the GG genotype of the rs283 polymorphism were more sensitive to exercise-induced reduction of the body fat percentage, insulin resistance and plasma triglyceride levels. The G allele can significantly increase reporter gene expression level, which may be the molecular reason for the difference in exercise-induced parameter changes among obese adolescents. The aim of this investigation was to explore the association between the rs283 polymorphism located in the lipoprotein lipase (LPL) gene and exercise-induced changes in body composition and lipid and glucose metabolism in obese adolescents and to probe into the molecular regulatory mechanisms. Fifty-five obese adolescents of Han nationality underwent aerobic training for 4 weeks. Body composition and lipid and glucose metabolic parameters were tested before and after the training. The rs283 polymorphism was genotyped by PCR-restriction fragment length polymorphism, and association analysis with the weight-reducing effect was performed. The regulatory mechanisms of the rs283 polymorphism were explored through the dual-luciferase reporter assay. Exercise-induced change rates were as follows: the change in body fat percentage of GG genotype groups was 3.37 ± 1.60, significantly higher than that of GA genotype groups (2.09 ± 1.53, P < 0.01); the change in the homeostasis model assessment of insulin resistance was 0.52 ± 0.13, obviously higher than that of GA genotype groups (0.44 ± 0.10, P < 0.05); and the change in triglyceride was 51.91 ± 6.56, much higher than that of GA genotype groups (47.06 ± 5.36, P < 0.01). The relative luciferase activity of the reporter gene in recombinant vector carrying the G allele was 2.67 ± 0.22, markedly higher than that in recombinant vector carrying the A allele (1.63 ± 0.03, P < 0.01). Chinese obese adolescents of Han nationality with GG genotype of the rs283 polymorphism were more sensitive to exercise-induced parameter changes. The G allele can improve reporter gene expression level, indicating the effects of rs283 on gene expression.

Tumor-associated mesothelial cells are negative prognostic factors in gastric cancer and promote peritoneal dissemination of adherent gastric cancer cells by chemotaxis.

Peritoneal dissemination is highly frequent in gastric cancer. Damage to human peritoneal mesothelial cell (HPMC) barriers provokes gastric cancer peritoneal dissemination (GCPD), the key events during GCPD, is characterized by fibroblastic development. In this study, we have studied the association between fibroblast activation protein (FAP) expression in peritoneum and the pathological features of the primary tumor. The clinical prognosis of gastric cancer patients was evaluated according to FAP expression. In a gastric cancer cell-HPMC co-culture system, expression of E-cadherin, α-smooth muscle actin, and FAP were evaluated by Western blotting. Gastric cancer cell migration and adhesion to HPMC were also assayed. Our results showed positive peritoneal staining of FAP in 36/86 cases (41.9 %), which was associated with a higher TNM stage in primary gastric cancer and higher incidence of GCPD (both p<0.05). Survival analysis showed FAP expression was an independent prognostic factor of poor survival (p=0.02). Peritoneum of FAP-positive expression exhibited a distinct fibrotic development and expressed higher level of the mesenchymal marker α-SMA, which was confirmed by the in vitro Western blot assay. In HPMC and gastric cancer cell adherence assay, SGC-7901 cells preferentially adhered to TA-HPMC at different cell densities (both p<0.05). Additionally, SGC-7901 cells were more prone to chemotaxis by FAP-expressed tumor-associated-human peritoneal mesothelial cells (TA-HPMC) compared with HPMC co-cultured with normal gastric glandular epithelial cells in a time-dependent manner (both p<0.05). Our study indicated a positive correlation between peritoneum FAP expression and GCPD. FAP-expressed TA-HPMC might be an important cellular component and instigator of GCPD.

Armc8 expression was elevated during atypia-to-carcinoma progression and associated with cancer development of breast carcinoma.

Armadillo repeat-containing protein 8 (Armc8) is a key factor to regulate cell membrane adhesion complex through promoting α-catenin degradation. However, its expression and function in human malignant tumors are largely unknown. Here, we present our study investigating Armc8 expression in tumor and non-tumor breast tissues including 45 normal epithelia, 53 lesions of hyperplasia with or without dysplasia, 22 benign tumors, and 92 carcinomas including 28 carcinomas in situ and 64 infiltrating carcinomas using immunohistochemistry (IHC) and Western blotting study. Armc8 expression was detected mainly in the cytoplasm with occasional membrane immunostaining. The positive rate of Armc8 expression in normal breast epithelia (8.9%, four out of 45) was very low. No significant difference was found between Armc8 expression in usual ductal hyperplasia (UDH) (11.1%, two out of 18), benign breast tumors including intraductal papilloma (10.0%, one out of 10) and fibroadenoma (8.3%, one out of 12), and normal breast epithelia (p>0.05). Elevated expression of Armc8 was found in breast epithelia with dysplasia (24.0%, six out of 25) compared to that in normal breast epithelia, UDH, and benign breast tumors (p<0.05). Armc8 expression in breast carcinoma including breast carcinoma in situ (10/28, 35.7%), infiltrating ductal carcinoma (60.7%, 34/56), and infiltrating lobular carcinoma (50.0%, 4/8) was higher than that in normal breast epithelia, UDH, benign breast tumors, and breast epithelia with dysplasia (p<0.05). The highest expression of Armc8 was found in infiltrating breast carcinoma (59.4%, 38/64) compared to all the other breast tissues. Higher Armc8 expression was found to be linked to lymph node metastasis and advanced tumor-node-metastasis (TNM) stages (III+IV) in infiltrating breast carcinoma (p<0.05). We further confirmed Armc8 expression in breast epithelial cell line MCF10A and breast carcinoma cell lines including MCF-7, MDA-MB-231, and ZR751 using Western blotting and immunofluorescent study. These results indicate that the elevated expression of Armc8 may be involved in carcinogenesis including atypia-to-carcinoma progression and cancer development of breast carcinoma.

CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis.

CXCR4 and its ligand CXCL12 can promote the proliferation, survival, and invasion of cancer cells. They have been shown to play an important role in regulating metastasis of breast cancer to specific organs. High CXCR4 expression was also correlated to poor clinical outcome. Previous study also showed that tumor cells express a high level of CXCR4 and that tumor metastasis target tissues (lung, liver, and bone) express high levels of the ligand CXCL12, allowing tumor cells to directionally migrate to target organs via a CXCL12-CXCR4 chemotactic gradient. However, the exact mechanisms of how CXCR4 and CXCL12 enhance metastasis and/or tumor growth and their full implications on breast cancer progression are unknown. Yet it is likely that chemokine receptor signaling may provide more than just a migrational advantage by also helping the metastasized cells establish and survive in secondary environments. In this study, we investigated CXCR4 and CXCL12 expression in breast cancer and analyzed its association with clinicopathological factors by immunohistochemistry first. Then, we detected the mRNA and protein expression of CXCR4 and CXCL12 in breast cancer cell lines by Western blot and RT-PCR. The MDA-MB-231 has CXCR4 expression and very weak CXCL12 expression. So, we constructed the functional CXCL12 expression in MDA-MB-231 using a gene transfection technique. Further experiments were conducted to evaluate the effect of CXCL12 transfection on the biological behaviors of MDA-MB-231. The cell proliferation of MDA-MB-231-CXCL12 was accessed by MTT assay; the apoptosis was analyzed by an AnnexinV-FITC/propidium iodide double staining of flow cytometry method; and the cell invasive ability was examined by Matrigel invasion assay. Immunohistochemical analysis showed the co-expression of CXCR4 and CXCL12 correlated with lymph node metastasis and TNM stage (p < 0.01). It suggested that the chemokine CXCL12 and its sole ligand CXCR4 play important role in the malignance of breast cancer. To gain a deeper insight into it, we picked CXCR4-expressing cells MDA-MB-231 to be transfected with CXCL12 stably. The decreased cellular proliferation, increased apoptosis, and invasive ability were found in MDA-MB-231 with successful CXCL12 transfection (p < 0.05). Our findings underlined the CXCL12-CXCR4 axis correlated tightly with breast cancer metastasis. CXCL12-CXCR4 axis can increase the invasion and apoptosis of MDA-MB-231 simultaneously. These data strongly support the hypothesis that CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis. Our findings could have significant implications in terms of breast cancer aggressiveness and the effectiveness of targeting the receptors and downstream signaling pathways for the treatment of breast cancer.

Transforming growth factor-beta1 signaling blockade attenuates gastric cancer cell-induced peritoneal mesothelial cell fibrosis and alleviates peritoneal dissemination both in vitro and in vivo.

Peritoneal dissemination is the most frequent metastatic pattern of advanced gastric cancer and the main cause of death in gastric cancer patients. Transforming growth factor-beta1 (TGF- ß1), one of the most potent fibrotic stimuli for human peritoneal mesothelial cells, has been shown to play an important role in this process. In this study, we investigated the effect of TGF- ß1 signaling blockade in gastric cancer cell (GCC)-induced human peritoneal mesothelial cell (HPMC) fibrosis. HPMCs were cocultured with the high TGF- ß1 expressing GCC line SGC-7901 and various TGF- ß1 signaling inhibitors or SGC-7901 transfected with TGF-ß1-specific siRNA. HPMC fibrosis was monitored on the basis of morphology. Expression of the epithelial cell marker, E-cadherin, and the mesenchymal marker, α-smooth muscle actin (α-SMA), was evaluated by Western blotting and immunofluorescence confocal imaging. GCC adhesion to HPMC was also assayed. In nude mouse tumor model, the peritoneal fibrotic status was monitored by immunofluorescent confocal imaging and Masson's trichrome staining; formation of metastatic nodular and ascites fluid was also evaluated. Our study demonstrated that GCC expressing high levels of TGF-ß1 induced HMPC fibrosis, which is characterized by both upregulation of E-cadherin and downregulation of α-SMA. Furthermore, HPMC monolayers fibrosis was reversed by TGF- ß1 signaling blockade. In vivo, the TGF- ß1 receptor inhibitor SB-431542 partially attenuated early-stage gastric cancer peritoneal dissemination (GCPD). In conclusion, our study confirms the significance of TGFß1 signaling blockade in attenuating GCPD and may provide a therapeutic target for clinical therapy.

The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa shift peritoneal milky spot macrophages towards an M1 phenotype to dampen peritoneal dissemination.

Peritoneal dissemination (PD) of tumor cells is the most frequent pattern of gastric cancer recurrence and the leading cause of death. Peritoneal milky spots are deemed as the site of origin of gastric cancer PD wherein the main cellular components are macrophages. A vaccine derived from the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has exhibit strong immune modulatory properties. In the present study, we tested the hypothesis whether the PA-MSHA vaccine activated peritoneal milky spot macrophages (PMSM) in a manner that would attenuate PD. It was observed that PA-MSHA activated PMSM towards a classical activation phenotype via a toll-like receptor4/9-dependent mechanism, which increased interleukin-12 levels and promoted the expression of co-stimulatory and antigen-presenting molecules like CD80, CD86, and MHC-II (P < 0.05). In addition, PA-MSHA-treated PMSM exhibited strong nonspecific antitumor effects in both contact-dependent and contact-independent modes of action (P < 0.05). In mice treated with PA-MSHA before inoculating gastric cancer cells, we noted alleviated PD toward the untreated mice. In conclusion, our findings demonstrated that PA-MSHA can stimulate PMSM towards an M1 phenotype and that activated PMSM inhibit gastric cancer growth and PD both in vitro and in vivo. The results of the current study provide a mechanistic insight that is relevant to the potential application of PA-MSHA in the treatment of gastric cancer-mediated PD.

Influence of different hypoxia models on metastatic potential of SGC-7901 gastric cancer cells.

Gastric cancers are one of the most common gastrointestinal tumors with proclivity to metastasis. Hypoxia is an important feature in solid tumor microenvironment; accumulating evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seems to provide an advantage to tumor growth. However, mechanisms of intermittent hypoxia regulating gastric cancer metastatic potential remain to be elucidated. In the present study, we established the continuous and intermittent gastric cancer hypoxia models, and we compared the effects of these models on gastric cancer cell metastatic potential. Hypoxia-inducible factor-1α (HIF-1α) and hypoxia target protein/genes expression in response to different hypoxia models were analyzed by Western blot, immunofluorescence, and real-time PCR assays. Gastric cancer cell migration and invasion ability were analyzed by wound healing and Boyden chamber assay. Colony formation and tumor spheroid formation assays were used to assess the ability of self-renewal. Stem-related protein OCT4 and HIF-1α expression were also analyzed by confocal immunofluorescence in single tertiary generation spheroid. We demonstrated that the intermittent hypoxia model upregulated expression of both HIF-1α and hypoxia target protein/genes in a time-dependent manner compared with that of SGC-7901 cells cultured under normoxia. Remarkably, HIF-1α protein was more prone to being located in the nucleus of SGC-7901 cells under conditions of intermittent hypoxia. Gastric cancer cells' migration and invasion ability were significantly enhanced after hypoxic culture; moreover, intermittent hypoxia-conditioned SGC-7901 cells exhibited higher invasiveness than the continuous hypoxia-conditioned SGC-7901 cells. Gastric cancer stem/progenitor cell subpopulation was also enlarged which indicated an enhanced self-renewal ability in hypoxic cultured SGC-7901 cells. Our study emphasizes the significance of hypoxia in regulating gastric cancer cell metastasis potential. Compared with continuous hypoxia, intermittent hypoxia is a more effective and potent hypoxic stimulus. These results provide a new insight to investigate the hypoxic microenvironment within solid tumors.

Downregulated inhibitor of growth 3 (ING3) expression during colorectal carcinogenesis.

BACKGROUND & OBJECTIVES: ING3 (inhibitor of growth protein 3) overexpression decreased S-phase cell population and colony-forming efficiency, and induced apoptosis at a p53-mediated manner. The aim of this study was to investigate the clinicopathological and prognostic significance of ING3 expression in colorectal carcinogenesis and subsequent progression. METHODS: ING3 expression was examined by immunohistochemistry on tissue microarray containing colorectal non-neoplastic mucosa (NNM), adenoma and adenocarcinoma. Colorectal carcinoma tissue and cell lines were studied for ING3 expression by Western blot or RT-PCR. RESULTS: ING3 mRNA was differentially expressed in Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480, SW620 and WiDr cells. Carcinomas showed significantly lower ING3 expression than matched NNM at mRNA level (P< 0.05), but not at protein level. Immunohistochemically, ING3 expression was significantly decreased from NNM, adenoma to adenocarcinoma (P< 0.05). ING3 expression was not correlated with age, sex, tumour size, depth of invasion, lymphatic or venous invasion, lymph node metastasis, tumour- node- metastasis staging or differentiation. Kaplan-Meier analysis indicated that ING3 protein expression was not associated the prognosis of the patients with colorectal carcinoma (P< 0.05). INTERPRETATION & CONCLUSIONS: Our study showed that downregulated ING3 expression might play an important role in colorectal adenoma-adenocarcinoma sequence. Further studies are required to understand the mechanism.