Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.
Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins that facilitate water transport across biological membranes. The genome of Arabidopsis encodes 35 aquaporin genes with 13 homologs in the plasma membrane intrinsic protein (PIP) subgroup. However, the function of each individual aquaporin isoform and the integrated function of plant aquaporins under various physiological conditions remain unclear. As a step toward understanding the aquaporin function in plants under various environmental stimuli, the expressions of a gene family encoding 13 PIPs in Arabidopsis thaliana under various abiotic stress conditions including drought, cold, and high salinity, or abscisic acid (ABA) treatment were investigated by a quantitative real-time reverse transcription-PCR analysis. Several PIP genes were predominantly expressed either in the roots or in the flowers. The expressions of both the highly expressed aquaporins including PIP1;1, PIP1;2, and PIP2;7 and the weakly expressed aquaporins such as PIP1;4, PIP2;1, PIP2;4, and PIP2;5 were modulated by external stimuli. The analyses of our data revealed that only the PIP2;5 was up-regulated by cold treatment, and most of the PIP genes were down-regulated by cold stress. Marked up- or down-regulation in PIP expression was observed by drought stress, whereas PIP genes were less-severely modulated by high salinity. The responsiveness of each aquaporin to ABA were different, implying that the regulation of aquaporin expression involves both ABA-dependent and ABA-independent signaling pathways. Together, our comprehensive expression profile of the 13 members of the PIP gene family provides novel basis to allocate the stress-related biological function to each PIP gene.
BackgroundTumor-associated macrophages (TAM) play an important role in tumor microenvironment. Particularly, M2 macrophages contribute to tumor progression, depending on the expression of NF-κB. Tumor-derived exosomes can modulate tumor microenvironment by transferring miRNAs to immune cells. Epigallocatechin gallate (EGCG) has well known anti-tumor effects; however, no data are available on the influence of EGCG on communication with cancer cells and TAM.MethodsMurine breast cancer cell lines, 4T1, was used for in vivo and ex vivo studies. Exosome was extracted from EGCG-treated 4T1 cells, and the change of miRNAs was screened using microarray. Tumor cells or TAM isolated from murine tumor graft were incubated with exosomes derived from EGCG-treated and/or miR-16 inhibitor-transfected 4T1 cells. Chemokines for monocytes (CSF-1 and CCL-2), cytokines both with high (IL-6 and TGF-β) and low (TNF-α) expression in M2 macrophages, and molecules in NF-κB pathway (IKKα and Iκ-B) were evaluated by RT-qPCR or western blot.ResultsEGCG suppressed tumor growth in murine breast cancer model, which was associated with decreased TAM and M2 macrophage infiltration. Expression of chemokine for monocytes (CSF-1 and CCL-2) were low in tumor cells from EGCG-treated mice, and cytokines of TAM was skewed from M2- into M1-like phenotype by EGCG as evidenced by decreased IL-6 and TGF-β and increased TNF-α. Ex vivo incubation of isolated tumor cells with EGCG inhibited the CSF-1 and CCL-2 expression. Ex vivo incubation of TAM with exosomes from EGCG-treated 4T1 cells led to IKKα suppression and concomitant I-κB accumulation; increase of IL-6 and TGF-β; and, decrease of TNF-α. EGCG up-regulated miR-16 in 4T1 cells and in the exosomes. Treatment of tumor cells or TAM with exosomes derived from EGCG-treated and miR-16-knock-downed 4T1 cells restored the above effects on chemokines, cytokines, and NF-κB pathway elicited by EGCG-treated exosomes.ConclusionsOur data demonstrate that EGCG up-regulates miR-16 in tumor cells, which can be transferred to TAM via exosomes and inhibits TAM infiltration and M2 polarization. We suggest a novel mechanism by which EGCG exerts anti-tumor activity via regulation of TAM in tumor microenvironment.
Abstract. Many natural anti-DNA antibodies (Abs) have the ability to translocate across the plasma membrane and localize in the nucleus of mammalian cells, frequently leading to cytotoxicity to cells. Herein, we report detailed intracellular trafficking routes and cytotoxicity in HeLa cells for a single chain variable fragment (scFv) Ab, 3D8, which is an anti-DNA Ab capable of hydrolyzing both DNA and RNA. The intracellular penetration of 3D8 scFv occurred by caveolae/lipid raft endocytosis. The time-course chasing experiments revealed that 3D8 scFv escaped directly from the caveosome into the cytosol and remained in the cytosol without further trafficking into endosomes, lysosomes, endoplasmic reticulum, Golgi, or nucleus. The cytosolically localized 3D8 scFv maintained its nuclease activity to hydrolyze cellular RNAs, mainly mRNAs, eventually triggering apoptotic cell death. Our results demonstrate that 3D8 scFv has a unique intracellular trafficking route of localizing in the cytosol, thereby exhibiting cytotoxicity due to its nuclease activity.
Programmed cell death (PD)-1/PD-1 ligand-1 (PD-L1)-targeted therapy has emerged as a promising therapeutic strategy for lung cancer. However, whether EML4-ALK regulates PD-L1 expression in lung cancer remains unknown. A total of 532 pulmonary adenocarcinomas (pADCs), including 58 -translocated tumors, were immunohistochemically evaluated for PD-L1 and PD-1. H23 (PD-L1) and H2228 (PD-L1) cells were transfected with or ALK short interfering RNAs and used to investigate the alterations in PD-L1 expression. PD-L1 expression was detected in 81% of-translocated pADCs; this value was significantly higher than those of pADCs with mutation, mutation or lacking or mutation ( <0.005 for all). Moreover, -translocated pADC with PD-L1 expression showed significantly higher numbers of tumor-infiltrating PD-1 cells. ALK knockdown or inhibition (crizotinib treatment) in H2228 cells downregulated PD-L1 expression. Transfection of H23 cells with enhanced PD-L1 expression, which was compromised by crizotinib treatment. This ALK-dependent upregulation of PD-L1 expression was mediated by STAT3 and hypoxia-inducible factor (HIF)-1α under normoxia and hypoxia. Furthermore, EML4-ALK enhanced HIF-1α expression through increasing transcription and decreasing ubiquitination of HIF-1α. In-translocated pADC tissues, significant positive correlations between PD-L1 and nuclear HIF-1α ( < 0.05) or pSTAT3 expression levels (<0.005) were observed. Among patients with -translocated pADC, strong PD-L1 expression was significantly associated with shorter progression-free ( = 0.001) and overall survival ( = 0.002) after crizotinib treatment. Collectively, our findings demonstrate that derived pADCs increase PD-L1 expression via HIF-1α and/or STAT3, thus providing a rationale for PD-1/PD-L1 pathway-targeted therapy in-translocated lung cancer.
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