Metabolite secretions of Lactobacillus plantarum YYC-3 may inhibit colon cancer cell metastasis by suppressing the VEGF-MMP2/9 signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is a major clinical challenge, and the gut microbiome plays important roles in the occurrence and metastasis of CRC. Lactobacillus and their metabolites are thought to be able to suppress the growth of CRC cells. However, the antimetastatic mechanism of Lactobacillus or their metabolites toward CRC cells is not clear. Therefore, the aim of this study was to assess the inhibitory mechanism of cell-free supernatants (CFSs) of L. rhamnosus GG, L. casei M3, and L. plantarum YYC-3 on metastasis of CRC cells. RESULTS: YYC-3 CFS showed the highest inhibitory effect on CRC cell growth, invasion and migration, and inhibited MMP2, MMP9, and VEGFA gene and protein expression, and protein secretion. Furthermore, it suppressed the activities of MMPs by gelatin zymography. Moreover, the effective compounds in these CFSs were analyzed by Q Exactive Focus liquid chromatography-mass spectrometry. CONCLUSIONS: Our results showed that metabolite secretions of YYC-3 may inhibited cell metastasis by downregulating the VEGF/MMPs signaling pathway. These data suggest that treatment of CRC cells with metabolites from L. plantarum YYC-3 may reduce colon cancer metastasis.

Cord blood leptin DNA methylation levels are associated with macrosomia during normal pregnancy.

BACKGROUND: We previously demonstrated an association between placental leptin (LEP) methylation levels and macrosomia without gestational diabetes mellitus (non-GDM). This study further explored the association between LEP methylation in cord blood and non-GDM macrosomia. METHOD: We carried out a case-control study of 61 newborns with macrosomia (birth weight ≥4000 g) and 69 newborns with normal birth weight (2500-3999 g). Methylation in the LEP promoter region was mapped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Average cord blood LEP methylation levels were lower in macrosomia newborns than in control newborns (P < 0.001). Eleven CpG sites were associated with macrosomia. Multivariate logistic regression revealed that low LEP methylation levels [adjusted odds ratio (AOR) = 2.84, 95% confidence interval (CI): 1.72-4.17], high pre-pregnancy body mass index (AOR = 7.44, 95% CI: 1.99-27.75), long gestational age (AOR = 3.18, 95% CI: 1.74-5.79), high cord blood LEP concentration (AOR = 2.25, 95% CI: 1.34-3.77), and male newborn gender (AOR = 3.91, 95% CI: 1.31-11.69) significantly increased the risk of macrosomia. CONCLUSIONS: Lower cord blood LEP methylation levels and certain maternal and fetal factors are associated with non-GDM macrosomia.

The In Vitro Protective Role of Bovine Lactoferrin on Intestinal Epithelial Barrier.

The intestinal epithelial barrier plays a key protective role in the gut lumen. Bovine lactoferrin (bLF) has been reported to improve the intestinal epithelial barrier function, but its impact on tight junction (TJ) proteins has been rarely described. Human intestinal epithelial crypt cells (HIECs) were more similar to those in the human small intestine, compared with the well-established Caco-2 cells. Accordingly, both HIECs and Caco-2 cells were investigated in this study to determine the effects of bioactive protein bLF on their growth promotion and intestinal barrier function. The results showed that bLF promoted cell growth and arrested cell-cycle progression at the G2/M-phase. Moreover, bLF decreased paracellular permeability and increased alkaline phosphatase activity and transepithelial electrical resistance, strengthening barrier function. Immunofluorescence, western blot and quantitative real-time polymerase chain reaction revealed that bLF significantly increased the expression of three tight junction proteins-claudin-1, occludin, and ZO-1-at both the mRNA and protein levels, and consequently strengthened the barrier function of the two cell models. bLF in general showed higher activity in Caco-2 cells, however, HIECs also exhibited desired responses to barrier function. Therefore, bLF may be incorporated into functional foods for treatment of inflammatory bowel diseases which are caused by loss of barrier integrity.

Mesenchymal stem cells enhance the metastasis of 3D-cultured hepatocellular carcinoma cells.

BACKGROUND: Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined. METHODS: In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated. RESULTS: It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-ß receptor inhibitor SB431542 was added into the co-culture system. CONCLUSIONS: Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-ß.

Potential effect of matrix stiffness on the enrichment of tumor initiating cells under three-dimensional culture conditions.

Cancer stem cell (CSC) or tumor initiating cell (TIC) plays an important role in tumor progression and metastasis. Biophysical forces in tumor microenvironment have an important effect on tumor formation and development. In this study, the potential effect of matrix stiffness on the biological characteristics of human head and neck squamous cell carcinoma (HNSCC) TICs, especially the enrichment of HNSCC TICs, was investigated under three-dimensional (3D) culture conditions by means of alginate gel (ALG) beads with different matrix stiffnesses. ALG beads with soft (21 kPa), moderate (70 kPa) and hard (105 kPa) stiffness were generated by changing alginate concentration. It was found that significant HNSCC TIC enrichment was achieved in the ALG beads with moderate matrix stiffness (70 kPa). The gene expression of stemness markers Oct3/4 and Nanog, TIC markers CD44 and ABCG2 was enhanced in cells under this moderate (70 kPa) stiffness. HNSCC TIC proportion was also highly enriched under moderate matrix stiffness, accompanying with higher tumorigenicity, metastatic ability and drug resistance. And it was also found that the possible molecular mechanism underlying the regulated TIC properties by matrix stiffness under 3D culture conditions was significantly different from 2D culture condition. Therefore, the results achieved in this study indicated that 3D biophysical microenvironment had an important effect on TIC characteristics and alginate-based biomimetic scaffolds could be utilized as a proper platform to investigate the interaction between tumor cells and 3D microenvironment.

Role of three-dimensional matrix stiffness in regulating the chemoresistance of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) was the most common primary liver cancer, and its resistance to anti-tumor drugs often caused the death of patients suffering with HCC. Matrix stiffness was reported to be closely related to tumor chemoresistance; however, the relationship between HCC drug resistance and three-dimensional (3D) matrix stiffness is still unclear at present. In this study, alginate gel (ALG) beads with controllable matrix stiffness were used to mimic tumor tissue rigidity, and the role of 3D matrix stiffness in regulating the chemoresistance of HCC cells was investigated by using these ALG beads. It was found that HCC cells in ALG beads with 105 kPa stiffness had highest resistance to paclitaxel, 5-FU, and cisplatin. Although the mechanism was still uncovered, ABC transporters and endoplasmic reticulum stress-related molecules were highly expressed in ALG bead-encapsulated HCC cells compared with two-dimensional-cultured cells, which suggested a very complex mechanism underlying HCC drug resistance in 3D culture conditions. In addition, to mimic the specific stiffness of HCC tumor tissue, or other tumor tissues in vivo, response surface methodology (RSM) was used to build up a prediction mathematical model so that ALG beads with desired matrix stiffness could be prepared by simply changing three factors: molecular weight, G content, and alginate concentration.

Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expression levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research.

The effect of electrostatic microencapsulation process on biological properties of tumour cells.

Microencapsulation is one of the promising strategies to develop a three-dimensional in vivo tumour-mimic model in cancer research. Although previous studies have shown that tumour cells grow well during the microencapsulated culture, it is still not clear whether the electrostatic encapsulation process has an important effect on cellular characteristics. In this study, we investigated cellular response against non-physiological stress factors existing in the electrostatic microencapsulation process, such as the high-voltage electrostatic field, suspension and nutrition-free status. Our results showed that these non-physiological stress factors did not significantly induce cellular apoptosis, and did not affect cellular adhesion and viability. Furthermore, no change was found about invasion and drug resistance of the tumour cells. The normal endoplasmic reticulum function might play a role in maintaining biological properties during the electrostatic microencapsulation process.

Placental mtDNA copy number and methylation in association with macrosomia in healthy pregnancy.

INTRODUCTION: Fetal growth and development depend on metabolic energy from placental mitochondria. However, the impact of placental mitochondria on the occurrence of macrosomia remains unclear. We aimed to explore the association between macrosomia without gestational diabetes mellitus (non-GDM) and changes in placental mitochondrial DNA (mtDNA) copy number and methylation. METHODS: Fifty-four newborns with macrosomia and 54 normal birthweight controls were enrolled in this study. Placental mtDNA copy number and mRNA expression of nuclear genes related to mitochondrial replication or ATP synthesis-related genes were measured by real-time quantitative polymerase chain reaction (qPCR). Methylation levels of the non-coding regulatory region D-loop and ATP synthesis-related genes were detected by targeted bisulfite sequencing. RESULTS: Newborns with macrosomia had lower placental mtDNA copy number and higher methylation rates of the CpG15 site in the D-loop region (D-CpG15) and CpG6 site in the cytochrome C oxidase III (COX3) gene (COX3-CpG6) than normal birth weight newborns. After adjusting for potential covariates (gestational age, prepregnancy BMI, and infant sex), decreased placental mtDNA copy number (adjusted odds ratio [aOR] = 2.09, 95% confidence interval [CI] 1.03-4.25), elevated methylation rate of D-CpG15 (aOR = 2.06, 95% CI 1.03-4.09) and COX3-CpG6 (aOR = 2.13, 95% CI 1.08-4.20) remained significantly associated with a higher risk of macrosomia. DISCUSSION: Reduced mtDNA copy number and increased methylation levels of specific loci at mtDNA would increase the risk of macrosomia. However, the detailed molecular mechanism needs further identification.

Skin Allografting Activates Anti-tumor Immunity and Suppresses Growth of Colon Cancer in Mice.

INTRODUCTION: The tumor cells could escape from the immune elimination through the immunoediting mechanisms including the generation of immunosuppressive or immunoregulative cells. By contrast, allograft transplantation could activate the immune system and induce a strong allogenic response. The aim of this study was to investigate the efficacy of allogenic skin transplantation in the inhibition of tumor growth through the activation of allogenic immune response. METHODS: Full-thickness skin transplantation was performed from C57BL/6 (H-2b) donors to BALB/c (H-2d) recipients that were receiving subcutaneous injection of isogenic CT26 colon cancer cells (2 × 106 cells) at the same time. The tumor size and pathological changes, cell populations and cytokine profiles were evaluated at day 14 post-transplantation. RESULTS: The results showed that as compared to non-transplant group, the allogenic immune response in the skin-grafting group inhibited the growth of tumors, which was significantly associated with increased numbers of intra-tumor infiltrating lymphocytes, increased populations of CD11c+MHC-classII+CD86+ DCs, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD19+ B cells, as well as decreased percentage of CD4+CD25+Foxp3+ T cells in the spleens. In addition, the levels of serum IgM and IgG, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were significantly higher within the tumor in skin transplant groups than that in non-transplant group. CONCLUSIONS: Allogenic skin transplantation suppresses the tumor growth through activating the allogenic immune response, and it may provide a new immunotherapy option for the clinical refractory tumor treatment.

Development of a test strip for rapid detection of lactoperoxidase in raw milk.

Traditional methods for detecting lactoperoxidase (LP) are complex and time-consuming, so a test strip was made based on the enzymatic reaction principle to enable quick and convenient detection of LP in raw milk. In this study 0.1 mol/L citric acid (CA)/0.2 mol/L disodium hydrogen phosphate (NaP) buffer solution (pH 5.0), 22 mmol/L 3,3',5,5'-tetramethylbenzidine (TMB), 0.6 mmol/L hydrogen peroxide (H2O2), and 0.5% Tween-20 or 0.3% cetyltrimethyl ammonium bromide (CTAB) were optimal for preparing a quick, sensitive, and accurate LP test strip. The coefficient of variation (CV) of the estimated LP concentrations ranged from 2.47% to 6.72% and the minimum LP concentration detected by the test strip was 1-2 mg/L. Estimates of active LP in sixteen raw milk samples obtained using the test strip or the TMB method showed a good correlation (r=0.9776). So the test strip provides a quick, convenient, and accurate method for detecting the LP concentration of raw milk.

Enrichment of cancer stem cell-like cells by culture in alginate gel beads.

Cancer stem cells (CSCs) are most likely the reason of cancer reoccurrence and metastasis. For further elucidation of the mechanism underlying the characteristics of CSCs, it is necessary to develop efficient culture systems to culture and expand CSCs. In this study, a three-dimensional (3D) culture system based on alginate gel (ALG) beads was reported to enrich CSCs. Two cell lines derived from different histologic origins were encapsulated in ALG beads respectively and the expansion of CSCs was investigated. Compared with two-dimensional (2D) culture, the proportion of cells with CSC-like phenotypes was significantly increased in ALG beads. Expression levels of CSC-related genes were greater in ALG beads than in 2D culture. The increase of CSC proportion after being cultured within ALG beads was further confirmed by enhanced tumorigenicity in vivo. Moreover, increased metastasis ability and higher anti-cancer drug resistance were also observed in 3D-cultured cells. Furthermore, we found that it was hypoxia, through the upregulation of hypoxia-inducible factors (HIFs) that occurred in ALG beads to induce the increasing of CSC proportion. Therefore, ALG bead was an efficient culture system for CSC enrichment, which might provide a useful platform for CSC research and promote the development of new anti-cancer therapies targeting CSCs.