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Objective·To analyze the effect of Escherichia coli outer membrane vesicle (E.coli-OMV) on the proliferation of 4T1 breast cancer cells in vitro and the inhibition of tumor growth in BALB/c-4T1 tumor-bearing mice in vivo.Methods·OMVs were collected from the culture supernatant of E.coli and characterized. The uptake of E.coli-OMV by 4T1 cells was detected by fluorescent label tracking method. The effect of E.coli-OMV on 4T1 cell proliferation was detected by CCK-8 method. The effect of E.coli-OMV on 4T1 cell cycle was detected by flow cytometry. The BALB/c-4T1 tumor-bearing mouse models were established by subcutaneous inoculation, and the mice were divided into E.coli-OMV group and Control group, with 10 mice in each group. The mice in the E.coli-OMV group were injected with 0.25 mg/kg E.coli-OMV every 2 d, while the mice in the Control group were injected with equal doses of PBS. The changes in body weight, 40 d survival rate, tumor volume and tumor weight of the two groups of tumor-bearing mice were observed. The pathological morphology of the tumor tissues was evaluated by hematoxylin-eosin staining (H-E staining). The expression of proliferating cell nuclear antigen (PCNA) and CyclinD1 in tumor tissues was observed by immunohistochemical staining.Results·E.coli-OMV was spherical membrane vesicle structure with a particle size of (216.00±18.30) nm, which expressed E.coli outer membrane protein A (OmpA) and OmpC. Fluorescence microscopy results showed that 4T1 cells could intake E.coli-OMV. CCK-8 results showed that the inhibitory effect of E.coli-OMV on 4T1 cells was positively correlated with time-dose. Flow cytometry results showed that E.coli-OMV arrested the growth cycle of 4T1 cells in G0/G1 phase. In vivo experiments showed that compared with the Control group, body weight of mice in the E.coli-OMV group decreased slightly after the initial injection (P=0.031), and then recovered, while 40 d survival rate increased (P=0.037). The growth of tumor volume and weight of mice in E.coli-OMV group were lower than those in the Control group (P=0.041, P=0.004). Its tumor volume inhibition rate reached 29.69%, and tumor weight inhibition rate reached 49.81%. The results of H-E staining showed that nuclear splitting images of tumor tissues of mice in the E.coli-OMV group decreased compared to the Control group (P=0.038). The results of immunohistochemical staining showed that the positive expression of PCNA and CyclinD1 in the tumor tissues of mice in the E.coli-OMV group decreased compared to the Control group (P=0.031, P=0.002).Conclusion·Both in vitro and in vivo studies show that E.coli-OMV can significantly inhibit the proliferation of 4T1 cells.

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of melanoma exosomes on fibroblast invasion and MMP2 and MMP9 expression. Methods The extracellular bodies of melanoma were extracted by ultracentrifugation. The surface markers and morphology of exosomes were identified by Western blot assay, transmission electron microscopy and particle size detection. Exosome-treated fitroblasts were observed by flow cytometry and laser confocal microscopy. After the process of ingestion, Transwell assay was used to detect the effect of melanoma exosomes on the invasive ability of fibroblasts. Western blot assay and immunocytochemistry were used to detect the effect of exosomes on the expression of MMP2 and MMP9 in fibroblasts. The effect of MMP2 and MMP9 gene levels was examined using qRT-PCR. Results The morphology of melanoma exosomes was observed as double-layered membrane, hollow, round or elliptical, and positively expressed exosome marker TSG101 and melanoma marker protein TYRP2. Compared with normal fibroblasts, the invasive ability of melanoma exosomes treated fibroblasts was significantly increased, and MMP2 and MMP9 expression were significantly up-regulated. Conclusion Melanoma exosomes may promote fibroblast invasion by inducing the expression of MMP2 and MMP9 in fibroblasts.

Abstract Background Our previous work found that mouse embryos could invade malignant cancer cells. In the process of implantation, embryo trophoblast cells express matrix metalloproteinases and the invasive ability of trophoblast cells is proportional to matrix metalloproteinase-9 protein expression. So the purpose of this study is to observe the effects of mouse embryos on human ovarian cancer cells in the co-culture environment in vitro and explore the possible mechanism of matrix metalloproteinase-9. Methods Several groups of human ovarian cancer cells HO8910PM were co-cultured with mouse embryos for different time duration, after which the effects of mouse embryos on morphology and growth behavior of HO8910PM were observed under the light microscope real-time or by H.E staining. Apoptosis was detected under laser confocal microscope by Annexin V-EGFP/PI staining in situ. Invasion ability of tumor cells was studied by transwell experiments. After matrix metalloproteinase 9 (MMP −9) activity was inhibited by MMP-9 Inhibitor I, the interaction between mouse embryos and human ovarian cancer cells HO8910PM was observed. Results Mouse embryos were able to invade co-cultured human ovarian cancer cell layer which extended in the bottom of the culture dish, and gradually pushed away tumor cells to form their own growth space. The number of apoptosis tumor cells surrounding the embryo increased under laser confocal microscope. After co-cultured with mouse embryos, tumor cells invasive ability was lowered compared with the control group. After MMP-9 activity was inhibited, the interaction between mouse embryos and HO8910PM cells had no significant difference compared with the normal MMP-9 activity group. Conclusion Mouse embryos were able to invade human ovarian cancer cells in vitro and form their own growth space, promote apoptosis of human ovarian cancer cells and lower their invasive ability. The mouse embryo was still able to invade human ovarian cancer cells after MMP-9 activity was inhibited.

Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E 2 ) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E 2 concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes. [ABSTRACT FROM AUTHOR]

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo . The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD 600 =0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs ( P =0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition (EMT)-related events in recipient cells. In endometrial epithelial cells, EMT processes are known to be involved in the development of adenomyosis. We aimed to investigate whether adenomyosis-derived extracellular vesicles (AMEVs) are able to induce an EMT process in endometrial epithelial cells. In this study, AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy, Western blot, and nanoparticle tracking. Primary endometrial epithelial cells (EECs) were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs in vitro . AMEV uptake was examined by fluorescence confocal microscopy. The invasion of EECs was confirmed by Transwell assay. Immunohistochemistry, Western blot, and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19, E-cadherin, vimentin, and zinc finger E-box-binding homeobox 1 (ZEB1). The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture, but decreased after 72 h. After co-culturing with AMEVs for 72 h, EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin, and significantly higher levels of vimentin and ZEB1. Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs. These changes may contribute to the pathogenesis and progression of adenomyosis., (© 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.)

Embryonic stem cells (ESCs) are pluripotent stem cells derived from early stage embryos. It remains unclear whether inhibiting the Wnt/β‑catenin signaling pathway using dickkopf Wnt signaling pathway inhibitor 1 (DKK1) impacts on the differentiation potential of mouse ESCs in vitro and in vivo. In the present study, immunohistochemical staining was used to measure the expression of markers of the three germ layers in ESCs and teratomas derived from ESCs. The expression of markers for the Wnt/β‑catenin signaling pathway were detected by reverse transcription‑polymerase chain reaction (RT‑qPCR). Immunohistochemistry and western blotting indicated that the expression levels of octamer‑binding transcription factor 4 in the DKK1‑treated ESC group were significantly greater compared with the control ESCs. Reduced expression levels of NeuroD and bone morphogenetic protein 4 were observed in the DKK1‑treated ESCs and teratomas derived from DKK1‑treated ESCs compared with the control group. Increased expression levels of SOX17 were observed in the DKK1‑treated ESCs compared with the control group. RT‑qPCR indicated that β‑catenin expression was significantly reduced in DKK1‑treated ESCs and teratomas derived from DKK1‑treated ESCs compared with the control groups. Western blotting indicated no alterations in the expression of GSK‑3β, however, the levels of phosphorylated‑GSK‑3β were significantly greater in the DKK1 treatment groups, while cyclin D1 and c‑Myc expression levels were significantly reduced in the DKK1 treatment groups compared with the control groups. These results suggest that inhibiting Wnt signaling in ESCs using DKK1 may promote mouse ESCs to differentiate into endoderm in vitro and in vivo, and suppress the tumorigenicity of ESCs.

Objective: To investigate the effect of high-intensity focused ultrasound (HIFU) and gemcitabine on xenograft growth in nude mice bearing human pancreatic cancer., Methods: Nude mouse models bearing subcutaneous human pancreatic cancer cell line PANC-1 xenograft were randomized into 4 groups, including a control group and 3 treatment groups subjected to treatments with HIFU, gemcitabine, or both. After the treatments, the tumor was measured on a weekly basis for 5 weeks, and the tumor growth curve was drawn. The tumor inhibition rate was calculated and the expression of vascular endothelial growth factor (VEGF) in the tumor tissue was examined by immunohistochemistry., Results: The tumor volume showed significant differences between the 3 treatment groups (P<0.01), but all significantly smaller than that in the control group; HIFU combined with gemcitabine resulted in the most obvious reduction in the tumor volume. VEGF expression in the tumor tissue was the lowest in the combined treatment group and the highest in the control group., Conclusion: HIFU therapy produces definite therapeutic effect on human pancreatic cancer in the nude mouse model, and its combination with chemotherapy is the optimal treatment modality.

Objective: To observe the clinical effect of low-intensity ultrasound in promoting uterine involution following cesarean section., Methods: A total of 122 women undergoing cesarean section were randomly selected and divided into low-intensity ultrasound treatment group (67 cases) and control group (55 cases). The women in the treatment group received daily low-intensity ultrasound treatment for 30 min 24 h after the delivery for 3 consecutive days, and the control group had no particular treatments. The descent of the uterine fundus and cessation of lochia was observed in the two groups., Results: The treatment group showed obviously greater descent of the uterine fundus with a higher rate of cessation of vaginal bleeding than the control group at 30 days postpartum. The ratio of incomplete uterine involution was significantly lower in the treatment group than in the control group., Conclusion: Low-intensity ultrasound is effective in promoting uterine contraction and uterine involution following cesarean section.

Objective: To observe the effect of ultrasound stimulation at the acupoint Guanyuan (CV 4) on follicular development in menopausal rats., Methods: Menopausal female SD rats were selected by vaginal smear examinations. The rats were subjected to ultrasound stimulation at the acupoint Guanyuan with the output power of 0.1 W, working frequency of 9 MHz, and focal length of 4.5-5 mm. Electrochemiluminescence immunoassay was used to detect the serum estrogen levels of the menopausal rats. The changes in the ovarian tissue histology and the follicle number were observed., Results: Compared with the control group, a 10-day ultrasound stimulation for 10 and 5 min daily at Guanyuan significantly increased the serum estrogen levels and the numbers of primary and secondary follicles (P<0.05) and reduced the number of atretic follicles in the menopausal rats (P<0.05)., Conclusion: Ultrasound stimulation at the acupoint Guanyuan can increase the estrogen secretion function and promote the development of follicles in menopausal rats.