Wound Healing Migration Assays Research Articles

Activation of mitochondrial-associated apoptosis signaling pathway and inhibition of PI3K/Akt/mTOR signaling pathway by voacamine suppress breast cancer progression

BackgroundBreast cancer is one of the malignant tumors with the highest morbidity and mortality rate. Numerous efficient anti-breast cancer drugs are being derived from the development of natural products. Voacamine (VOA), a bisindole alkaloid isolated from Voacanga africana Stapf, possesses various pharmacological and biological activities. PurposeIn this study, we investigated the efficacy of VOA against breast cancer cells and elucidated the underlying mechanisms in vitro and in vivo. MethodsHuman breast cancer cell line MCF-7 and mouse breast cancer cell line 4T1 were used to study the underlying anti-cancer mechanisms of VOA. The proliferation was detected by MTT, colony formation, cell proliferation and wound-healing migration assays. Flow cytometry was utilized to determine the level of reactive oxygen species (ROS) cell-cycle, apoptosis and mitochondrial membrane potential. The target proteins were analyzed by Western blot. Molecular docking was performed and scored by AutoDock. Subcutaneous cancer models in mice were established to evaluate the anticancer effects in vivo. ResultOur results demonstrated that VOA selectively suppressed breast cancer MCF-7 and 4T1 cells proliferation with IC50 values of 0.99 and 1.48 μM, and significantly inhibited the migration and colony formation of tumor cells. Furthermore, the cell cycle was arrested in the S phase with the decreased expression levels of CDK2, Cyclin A and Cyclin E. Additionally, exposure to VOA dose-dependently brought about dose-dependently the loss of mitochondrial membrane potential (Δψm) and amassment of reactive oxygen species (ROS), resulting in the initiation of the intrinsic apoptotic pathway. Western blot analysis unveiled that VOA significantly activated mitochondrial-associated apoptosis and obviously suppress the PI3K/Akt/mTOR pathway via modulation of related protein expression levels in both tumor cell lines. In tumor-bearing mouse models, administration of VOA dose-dependently inhibited the tumor growth without causing apparent toxicities. ConclusionThese findings revealed the novel properties of VOA in promoting apoptosis of breast cancer cells by activating mitochondrial-associated apoptosis signaling pathway and inhibiting PI3K/Akt/mTOR signaling pathway and significantly decreasing tumor size without detecting appreciable toxicity. In summary, the present results demonstrated VOA could be an encouraging drug candidate to cure breast cancer, exhibiting an effective method to exploit unique drugs from natural components.

Exosomes derived from stem cells of human deciduous exfoliated teeth inhibit angiogenesis in vivo and in vitro via the transfer of miR-100-5p and miR-1246

BackgroundAnti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism.MethodsExosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism.ResultsThe results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression.ConclusionsThese results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.

Upregulation of ADAMDEC1 correlates with tumor progression and predicts poor prognosis in non-small cell lung cancer (NSCLC) via the PI3K/AKT pathway.

BackgroundADAM‐like decysin‐1 (ADAMDEC1) has been reported to play an important role in the pathogenesis of multiple diseases, including cancers. However, its biological role in non‐small cell lung cancer (NSCLC) remains largely unknown. Here, we aimed to investigate the biological functions and potential mechanism of ADAMDEC1 in NSCLC.MethodsWe verified ADAMDEC1 as a DEG by a comprehensive strategy of TCGA and GEO datasets miming and computational biology. Relative levels of ADAMDEC1 in NSCLC tissues and the adjacent peritumoral tissues were identified by qRT‐PCR, WB and IHC staining. The biological function of ADAMDEC1 was determined by CCK8, EdU, colony formation assay, apoptosis, wound healing migration and transwell invasion assays. Then, an in vivo tumor formation assay was conducted to explore the effects of ADAMDEC1 on tumor growth.ResultsThe mRNA and protein expression levels of ADAMDEC1 were upregulated in NSCLC tissues and cell lines. ADAMDEC1 expression was associated with clinicopathological characteristics and overall survival of patients with NSCLC. Knockdown of ADAMDEC1 could decrease proliferation and colony forming ability of NSCLC cells, and promoted cell apoptosis, whereas ADAMDEC11 overexpression has opposite effects in NSCLC cells both in vivo and in vitro. Furthermore, we identified ADAMDEC1 accelerates NSCLC progression via activation of the PI3K/ AKT pathway.ConclusionWe verified that ADAMDEC1 promotes the progression of NSCLC via the PI3K/AKT pathway. These findings showed the potential of ADAMDEC1 to be used for therapeutic approaches in NSCLC.

Abstract P3-06-04: Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer

Abstract Introduction: The Notch and Wnt signaling pathways play critical role in mammary development and tumourigenesis. The epithelia-mesenchymal transition (EMT) is an important process of breast cancer development. Growing evidence shows that Glycogen Synthase Kinase 3 Beta (GSK 3β) is considered as an EMT suppressor of its downregulation of protein snail, a zinc-finger transcriptional repressor that regulates epithelia marker E-cadherin. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. In mammalian cell, GSK3β has been show to physically bind and phosphorylate the intracellular domain of two Notch paralogues. But the relationship between Notch3,GSK3β and EMT in breast cancer still unclear. Intriguingly, we found that knockdown of Notch3 in luminal subtype of breast cancer cells MCF-7 downregulated GSK3β and promoted EMT. In contrast, overexpression of Notch3 in triple-negative cell lines MDA-MB-231 upregulated GSK3βand inhibited EMT. Mechanistically, our results revealed that Notch3 specifically binds to the CSL binding element of the GSK3β promoter and trans-activates its expression. Furthermore, Notch3 and GSK3β expression were positively associated in human breast cancer. Moreover, the mRNA expression of both Notch3 and GSK3βpredicted a better Overall-Survival for breast cancer patients. Our findings demonstrate that Notch3 inhibits breast cancer cells EMT via trans-activating GSK3β expression. Methods: 1. Western blot and/or qRT-PCR were performed to study the expressions of Notch3,GSK3β, β-catenin and EMT related-molecules in breast cancer cell line. 2. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays were carried out to investigate whether Notch3 bound to GSK3β promoter and mediatesGSK3β expression on transcriptional level. 3. Wound healing assay, migration and invasion assays were performed to analyze the effect of Notch3 and GSK3β on breast cancer cell lines migration abilities. 4. The association of Notch3 and GSK3β expression in human breast cancer samples and correlation between Notch3 or GSK3β expression and patient survival in a cohort of human breast cancer patients in Kaplan Meier Plotter database were analyzed. Results: 1. Notch3 up-regulates GSK3β expression in mRNA and protein levels. 2. Notch3 regulates GSK3β expression by binding to the CSL antisense binding site (TTCCCA) positions -308bp to -303bp in the GSK3β promoter. 3. Notch3 intracellular domain (N3ICD) down-regulates the expression of Vimentin, β-catenin, but up-regulates E-cadherin expression. 4. Notch3 inhibits the ability of migration and invasion in breast cancer cell lines. 5. Notch3 and GSK3β expression were positively associated in human breast cancer. The mRNA expression of both Notch3 and GSK3β predicted a better Overall-Survival for breast cancer patients. Summary: We have identified a positive correlation between Notch3 and GSK3β in breast cancer and confirmed a role for the Notch3- GSK3β axis in inhibiting EMT. In addition, we explored in detail and first clarified the possible molecular mechanism of regulating this axis. Our results provide new insights into the complex regulation of breast cancer proliferation and tumorigenesis. We believe that the Notch3- GSK3β axis may be a prospective prognostic predictor for breast cancer. Citation Format: Chen Wei-Ling, Yong-Qu Zhang, Wen-He Huang, Min Chen, Yao-Chen Li, Guo-Jun Zhang. Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-04.

The KDM6A-SPARCL1 axis blocks metastasis and regulates the tumour microenvironment of gastrointestinal stromal tumours by inhibiting the nuclear translocation of p65.

It is urgent to explore the pathogenic mechanism of gastrointestinal stromal tumours (GISTs). KDM6A, a histone demethylase, can activate gene transcription and has not been reported in GISTs. SPARCL1 may serve as a metastasis marker in GIST, but the molecular mechanism remains to be further explored. This study aimed to explore the biological function and molecular mechanism of KDM6A and SPARCL1 in GIST. CCK-8, live cell count, colony formation, wound-healing and Transwell migration and invasion assays were employed to detect the cell proliferation, migration and invasion. A xenograft model and hepatic metastasis model were used to assess the role of KDM6A and SPARCL1 in vivo. KDM6A inhibited the proliferation, migration and invasion of GIST cells. Mechanistically, KDM6A promotes the transcription of SPARCL1 by demethylating histone H3 lysine trimethylation and consequently leads to the inactivation of p65. SPARCL1 affected the metastasis of GIST cells in a mesenchymal-epithelial transition- and matrix-metalloproteinase-dependent manner. SPARCL1 knockdown promoted angiogenesis, M2 polarisation and macrophage recruitment by inhibiting the phosphorylation of p65. Moreover, KDM6A and SPARCL1 inhibited hepatic metastasis and macrophage infiltration in vivo. Our findings establish the critical role of the KDM6A-SPARCL1-p65 axis in restraining the malignancy of GIST.

Hinokiflavone Inhibits Growth of Esophageal Squamous Cancer By Inducing Apoptosis via Regulation of the PI3K/AKT/mTOR Signaling Pathway.

BackgroundGlobally, esophageal cancer ranks as the seventh most common cancer. Esophageal squamous cell carcinoma (ESCC) is one of its major histological types. ESCC accounts for the vast majority of cases in China, and the mortality rate is high. Cisplatin, the standard adjuvant chemotherapy drug for ESCC, has a modest response rate due to the development of drug resistance. Hinokiflavone (HF) is a natural biflavonoid compound with anti-melanoma activity. However, its anti-tumor effect on ESCC and the underlying mechanisms remain largely unknown.MethodsThe ESCC cell lines KYSE150 and TE14 were used. The cell counting kit-8 assay and flow cytometry analysis, along with colony formation, EdU, wound healing, and Transwell migration assays, were performed to assess cell characteristics (viability, migration, invasion, and apoptosis) following treatment with HF. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), western blotting, and molecular docking were used to investigate the pathways potentially modulated by HF. In vivo anti-tumor effects of HF were also investigated using a mouse xenograft model.ResultsOur findings revealed that HF inhibited ESCC cell proliferation. Hoechst 33342 staining, annexin V-FITC/PI staining, and western blotting confirmed that HF causes caspase-dependent apoptosis. KEGG pathway enrichment analysis and western blotting indicated that the PI3K/AKT/mTOR pathway played an important role in the process of HF-induced apoptosis. Furthermore, HF effectively impaired the migration and invasion abilities of KYSE150 cells and downregulated the expression of the matrix metalloproteinases (MMP) MMP2 and MMP9. HF inhibited tumor growth and exhibited minimal toxicity in the organs of the KYSE150 xenograft model.ConclusionThis is the first study to demonstrate the inhibition of ESCC growth and progression by HF. The underlying mechanism is through blocking the PI3K/AKT/mTOR signaling pathway, thereby inhibiting cell proliferation and inducing apoptosis. HF can be used as a complementary/alternative agent for ESCC therapy.

High expression of TRIM24 predicts worse prognosis and promotes proliferation and metastasis of epithelial ovarian cancer

BackgroundTripartite Motif-Containing 24 (TRIM24) is a member of the tripartite motif family. TRIM24 is claimed aberrantly activated in a number of cancers, such as breast cancer, prostate cancer and lung cancer. However, the expression of TRIM24 in epithelial ovarian cancer (EOC) and its relationship with prognosis remain unclear. In this study, we investigated the expression pattern and underlying clinical significance of TRIM24 in EOC.ResultsData from Oncomine and immunohistochemistry of tissue samples demonstrated that TRIM24 expression was obviously elevated in ovarian carcinoma compared with normal ovary tissues. Elevated TRIM24 expression was closely correlated with serum CA-125 (P = 0.0294), metastasis (P = 0.0022), FIGO (International Federation of Gynecology and Obstetrics) stage (P = 0.0068) and Ki-67 level (P = 0.0395). Kaplan–Meier survival analysis found that TRIM24 expression increased inversely with the clinical prognosis of patients with EOC. Moreover, colony formation and CCK-8 assays showed that TRIM24 promoted EOC cell growth, and tumorigenic experiments in nude mice showed that TRIM24 knockdown inhibited tumor growth in vivo. The Spearman’s correlations revealed that the expression of TRIM24 was significantly correlated with levels of Ki-67 (P = 0.01), at a correlation coefficient of 0.517. Wound-healing and transwell migration assays demonstrated TRIM24 facilitated cell migration. Mechanism studies showed that TRIM24 could promote the phosphorylation level of Akt and the process of EMT.ConclusionOur results confirmed that TRIM24 could predict poor prognosis of EOC patients and promote tumor progression by regulating Akt pathway and EMT. TRIM24 may be used as a new prognostic marker for EOC and may provide a new strategy for targeted therapy of epithelial ovarian cancer.

A Novel IGLC2 Gene Linked With Prognosis of Triple-Negative Breast Cancer.

BackgroundImmunoglobulin-related genes are associated with the favorable prognosis of triple-negative breast cancer (TNBC) patients. We aimed to analyze the function and prognostic value of immunoglobulin lambda constant 2 (IGLC2) in TNBC patients.MethodsWe knocked down the gene expression of IGLC2 (IGLC2-KD) in MDA-MB-231 cells to evaluate the proliferation, migration, and invasion of tumors via 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, wound healing, and transwell cell migration assay respectively. Relapse-free survival (RFS) and distant metastasis-free survival (DMFS) analyses were conducted using the KM plotter online tool. The GSE76275 data set was used to analyze the association of IGLC2 and clinical characteristics. A pathway enrichment analysis was conducted using the next-generation sequencing data of wild-type and IGLC2-KD MDA-MB-231 cells.ResultsThe low gene expression of IGLC2 was related to unfavorable RFS, DMFS. The high expression of IGLC2 was exhibited in the basal-like immune-activated (BLIA) TNBC molecular subtype, which was immune-activated and showed excellent response to immune therapy. IGLC2 was positively correlated with programmed death-ligand 1 (PD-L1) as shown by Spearman correlation (r = 0.25, p < 0.0001). IGLC2 had a strong prognostic effect on lymph node-negative TNBC (RFS range: 0.31, q value= 8.2e-05; DMFS = 0.16, q value = 8.2e-05) but had no significance on lymph node-positive ones. The shRNA-mediated silencing of IGLC2 increased the proliferation, migration, and invasion of MDA-MB-231 cells. The results of pathway enrichment analysis showed that IGLC2 is related to the PI3K-Akt signaling pathway, MAPK signaling pathway, and extracellular matrix–receptor interaction. We confirmed that MDA-MB-231 tumor cells expressed IGLC2, subverting the traditional finding of generation by immune cells.ConclusionsIGLC2 linked with the proliferation, migration, and invasion of MDA-MB-231 cells. A high expression of IGLC2 was related to favorable prognosis for TNBC patients. IGLC2 may serve as a biomarker for the identification of TNBC patients who can benefit the most from immune checkpoint blockade treatment.

Long non-coding ribonucleic acid ATP2B1-AS1 modulates endothelial permeability through regulating the miR-4729-IQGAP2 axis in diabetic retinopathy.

ABSTRACTAims/IntroductionMounting evidence shows that long non‐coding RNAs (lncRNAs) are important to modulate the biological process of diabetic retinopathy (DR). We aimed to investigate the role of lncRNAs in DR and elucidate the exact mechanism.Materials and MethodsReal‐time quantitative polymerase chain reaction was carried out to distinguish the lncRNA ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1‐AS1) expression in DR patients and HG‐treated human retinal endothelial cells (HRECs). Dual‐luciferase reporter system was used to verify that ATP2B1‐AS1 could act as a microRNA (miR)‐4729 sponge, and miR‐4729 could bind to 3′UTR of IQ motif‐containing GTPase‐activating protein 2 (IQGAP2). Cell proliferation assay, wound healing migration assay, transwell assay, tube formation assay and immunofluorescence were used to investigate cell proliferation, migration and angiogenesis in HRECs.ResultsThe present results showed that ATP2B1‐AS1 was downregulated in DR patients and high‐glucose‐induced HRECs. In gain‐ and loss‐of‐function assays, ATP2B1‐AS1 overexpression could significantly reduce cell proliferation, migration, angiogenesis and permeability induced by high glucose in vitro. Additionally, we carried out dual‐luciferase reporter experiments to determine that ATP2B1‐AS1 could act as a miR‐4729 sponge. ATP2B1‐AS1 overexpression could rescue miR‐4729 mimics and short hairpin RNA‐IQGAP2 induced cell proliferation, migration and angiogenesis in HRECs.ConclusionsThe present study showed that ATP2B1‐AS1 acted as a miR‐4729 sponge to regulate IQGAP2 reducing high‐glucose‐induced endothelial dysfunction in DR.

Royal Jelly and trans-10-Hydroxy-2-Decenoic Acid Inhibit Migration and Invasion of Colorectal Carcinoma Cells.

SUMMARYResearch backgroundAcquisition of migratory potential is pivotal for cancer cells, enabling invasion and metastasis of colorectal carcinoma. Royal jelly and its bioactive component trans-10-hydroxy-2-decenoic acid (10H2DA) showed remarkable antimetastatic potential, but the molecular mechanism underlying this activity is unclear.Experimental approachIdentification and quantification of 10H2DA in royal jelly originating from Serbia was done by HPLC method. Cytotoxicity of 10H2DA was measured by tetrazolium dye MTT test in concentration range 1-500 μg/mL after 24 and 72 h. Its effect on the collective and single-cell migration was measured by wound healing and transwell migration assays. Invasive potential of cancer cells was evaluated by a transwell method modified with collagen. Immunofluorescence was used for migratory and invasive protein expression, while the gene expression of these markers was evaluated by quantitative real time polymerase chain reaction (qRT-PCR). All assays were applied on human colorectal carcinoma HCT-116 and SW-480 cell lines and, except for MTT, evaluated after 24 h of treatment with two selected concentrations of royal jelly and 10H2DA.Results and conclusionsAccording to HPLC, the mass fraction of 10H2DA in royal jelly was 0.92% (m/m). Treatment with 10H2DA showed no cytotoxic effect; however, significant inhibitory potential of royal jelly and 10H2DA on the motility and invasiveness of colorectal cancer cells was observed. More pronounced effect was exerted by 10H2DA, which significantly suppressed collective cell migration and invasiveness of SW-480 cells, as well as single- and collective cell migration and invasive potential of HCT-116 cell line. Treatments increased epithelial markers E-cadherin and cytoplasmic β-catenin in HCT-116 cells, thus stabilizing intercellular connections. In SW-480 cells, 10H2DA increased E-cadherin on protein and gene level, and suppressed epithelial-mesenchymal transition (EMT) markers. In both cell lines, treatments induced significant suppression of promigratory/proinvasive markers: N-cadherin, vimentin and Snail on protein and gene level, which explains decreased migratory and invasive potential of HCT-116 and SW-480 cells.Novelty and scientific contributionOur study presents new findings and elucidation of royal jelly and 10H2DA molecular mechanism that underlies their antimigratory/antiinvasive activity on colorectal cancer cells. These findings are shown for the first time indicating that these natural products are a valuable source of anticancer potential and should be reconsidered for further antitumour therapy.

Cerium-Containing Bioactive Glasses Promote In Vitro Lymphangiogenesis.

The lymphatic system is crucial for the regeneration of many tissues due to its fundamental role in immune cell trafficking, protein transport, and tissue homeostasis maintenance. Strategies stimulating lymphangiogenesis can provide new therapeutic approaches for tissue repair and regeneration (e.g., chronic wound healing). Here, we explored the effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on lymphangiogenesis. The results showed that the extracts of Ce-MBGNs (1, 5, or 10 wt/v%) were non-cytotoxic toward lymphatic endothelial cells (LECs), while they enhanced the proliferation of LECs. Moreover, as evidenced by the scratch wound healing and Transwell migration assays, conditioned media containing the extract of Ce-MBGNs (1 wt/v%) could enhance the migration of LECs in comparison to the blank control and the media containing vascular endothelial growth factor-C (VEGF-C, 50 ng/mL). Additionally, a tube-formation assay using LECs showed that the extract of Ce-MBGNs (1 wt/v%) promoted lymphatic vascular network formation. Western blot results suggested that Ce-MBGNs could induce lymphangiogenesis probably through the HIF-1α/VEGFR-3 pathway. Our study for the first time showed the effects of Ce-MBGNs on stimulating lymphangiogenesis in vitro, highlighting the potential of Ce-MBGNs for wound healing.

Integrin α6 overexpression promotes lymphangiogenesis and lymphatic metastasis via activating the NF-κB signaling pathway in lung adenocarcinoma.

It has been reported that tumor-associated lymphangiogenesis plays an important role in lymph node metastasis and contributes to the poor survival of lung adenocarcinoma (LUAD) patients. As yet, however, the molecular mechanism underlying LUAD-associated lymphangiogenesis has remained elusive. Immunohistochemistry (IHC) was used to determine the expression of integrin subunit alpha 6 (ITGA6) and the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) in clinicopathologically characterized LUAD specimens. The effect of ITGA6 overexpression on lymphangiogenesis and lymphatic metastasis was examined by tube formation, scratch wound-healing, and cell migration assays in vitro and a popliteal lymph node metastasis model in vivo. Mechanistically, overexpression of ITGA6 and activation of NF-κB signaling were examined by real-time PCR, ubiquitination and dual-luciferase reporter assays. Finally, high ITGA6 expression in LUAD tissue samples was related to copy number variation (CNV) using the TCGA database. We found that ITGA6 overexpression correlated with microlymphatic vessel density in LUAD specimens (p < 0.01). Importantly, by using a popliteal lymph node metastasis model, we found that ITGA6 upregulation significantly enhanced lymphangiogenesis and lymphatic metastasis in vivo (p < 0.05). In addition, we found that ITGA6 overexpression enhanced the capability of A549 and H1299 LUAD cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs). Mechanistically, we found that ITGA6 sustained NF-κB activity via binding and promoting K63 polyubiquitination of TNF receptor-associated factor 2 (TRAF2). Finally, CNV analysis revealed ITGA6 amplification of 27.5% in the LUAD tissue samples in the TCGA database. Taken together, our results uncover a plausible role for ITGA6 in mediating lymphangiogenesis and lymphatic metastasis and may provide a basis for targeting ITGA6 to treat LUAD lymphatic metastasis.

MiR-3918 Inhibits Tumorigenesis of Glioma via Targeting EGFR to Regulate PI3K/AKT and ERK Pathways.

Increasing evidence has demonstrated the miRNAs' action in cancerogenesis and tumor progression. Here, we explored the role and underlying mechanism of miR-3918 during glioma malignancy. miR-3918 and EGFR expression was detected in glioma tissues and tissues by RT-qPCR. The proliferative and migratory rate of glioma cells was assessed through CCK8 and Scratch wound-healing migration assay. Xenograft tumor mouse models were established for in vivo verification. A series of bioinformatics analysis coupled with luciferase reporter assays verified the targeted binding between miR-3918 and EGFR. Expression analyses demonstrated that miR-3918 was poorly expressed in glioma tissues while EGFR abundantly expressed. MiR-3918 overexpression impaired the proliferative and migratory capacities of glioma cells by inactivating PI3K/AKT and ERK pathways. Meanwhile, miR-3918 overexpression also retarded the growth of glioma xenograft. Mechanically, miR-3918 targeted EGFF which was further validated by the correlation of miR-3918 and EGFR expression in glioma tissues. When overexpressed, EGFR can restore the inactivated PI3K/AKT and ERK pathways caused by miR-3918 and influence the glioma cell proliferation and migration. Our findings are the first report that miR-3918/EGFR axis arrested the tumorigenesis of glioma via regulating PI3K/AKT and ERK pathways.

Anticancer effects of Piper nigrum extract against cholangiocarcinoma cells

Objectives: Piper nigrum (PN) is well known for its cytotoxic and pharmacological benefits in several cancer cells. However, there is less documented evidence about its cytotoxic efficacy against cholangiocarcinoma (CCA). The roles of PN extract in two CCA cells, KKU-100, and KKU-M452, were evaluated. Materials and Methods: Viability was determined by sulforhodamine B and cell cycle distribution. Apoptotic effects were examined by flow cytometry after staining with Annexin V-FITC and Propidium idodide, JC-1, and Dichlorodihydrofluorescein (DCF-DA) staining. Migration was studied by Wound healing and Matrigel migration assay. Results: The results indicated that PN treatment significantly inhibited cells viability and cell replication by dose-and time-dependent both of two CCA cells. Growth was decreased by detecting the cell cycle arrest at G0/G1 phase in KKU-100 cells and S to G2/M phase in KKU-M452 cells. PN extract markedly induced cancer cells apoptosis after treatment for 24 h by loss of mitochondrial membrane potential and increasing of reactive oxygen species production. Furthermore, a significant reduction of migration was observed in both two cells, KKU-100 cells was suppressed the migratory ability more than KKU-M452 cells. Conclusion: The data demonstrated that PN extract directly suppresses proliferation and inhibits cells migration in two CCA cells. Finally, PN may be useful for CCA treatment.

Effects of Low-Intensity Pulsed Ultrasound on the Migration and Homing of Human Amnion–Derived Mesenchymal Stem Cells to Ovaries in Rats With Premature Ovarian Insufficiency

Premature ovarian insufficiency (POI) can cause multiple sequelae and is currently incurable. Mesenchymal stem cell (MSC) transplantation might provide an effective treatment method for POI. However, the clinical application of systemic MSC transplantation is limited by the low efficiency of cell homing to target tissue in vivo, including systemic MSC transplantation for POI treatment. Thus, exploration of methods to promote MSC homing is necessary. This study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the migration and homing of transplanted human amnion–derived MSCs (hAD-MSCs) to ovaries in rats with chemotherapy-induced POI. For LIPUS treatment, hAD-MSCs were exposed to LIPUS or sham irradiation. Chemokine receptor expressions in hAD-MSCs were detected by polymerase chain reaction (PCR), Western blot, and immunofluorescence assays. hAD-MSC migration was detected by wound healing and transwell migration assays. Cyclophosphamide-induced POI rat models were established to evaluate the effects of LIPUS on the homing of systemically transplanted hAD-MSCs to chemotherapy-induced POI ovaries in vivo. We found that hAD-MSCs expressed chemokine receptors. The LIPUS promoted the expression of chemokine receptors, especially CXCR4, in hAD-MSCs. SDF-1 induced hAD-MSC migration. The LIPUS promoted hAD-MSC migration induced by SDF-1 through SDF-1/CXCR4 axis. SDF-1 levels significantly increased in ovaries induced by chemotherapy in POI rats. Pretreating hAD-MSCs with LIPUS increased the number of hAD-MSCs homing to ovaries in rats with chemotherapy-induced POI to some extent. However, the difference was not significant. Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation reduced ovarian injuries and improved ovarian function in rats with chemotherapy-induced POI. CXCR4 antagonist significantly reduced the number of hAD-MSCs- and LIPUS-pretreated hAD-MSCs homing to POI ovaries, and further reduced their efficacy in POI treatment. According to these findings, pretreating MSCs with LIPUS before transplantation might provide a novel, convenient, and safe technique to explore for improving the homing of systemically transplanted MSCs to target tissue.

CXCR4 Knockdown Via CRISPR/CAS9 in a Tumor-Associated Macrophage Model Decreases Human Breast Cancer Cell Migration.

IntroductionBreast cancer is the leading cause of cancer-related deaths in women worldwide with the majority of deaths due to metastasis. The development of metastasis is closely related to the tumor microenvironment where tumor-associated macrophages (TAMs) are the main immune cell component playing a crucial role in tumor migration. Key players in tumor progression, metastasis and survival are the receptor CXCR4 and its ligand CXCL12. CXCR4 is expressed in multiple cell types including macrophages and breast cancer cells. Many studies have focus on the role of CXCR4 expressed in breast cancer cells.MethodsIn this study, we investigated the role of CXCR4 expressed in TAMs on breast cancer cell migration by reducing CXCR4 expression via CRISPR-CAS9 system in differentiated THP-1 cells (a TAMs model).ResultsAccording to wound healing migration assay, MCF7 cancer cells co-cultured with genetically edited dTHP-1 cells have a lower migration rate as compared to MCF7 cancer cells co-cultured with unedited and dTHP-1 cells.ConclusionThe study demonstrates the role of CXCR4 on breast cancer cell migration through TAM-cancer cell crosstalk.

New benzoxazole derivatives as potential VEGFR-2 inhibitors and apoptosis inducers: design, synthesis, anti-proliferative evaluation, flowcytometric analysis, and in silico studies

A new series of benzoxazole derivatives were designed and synthesised to have the main essential pharmacophoric features of VEGFR-2 inhibitors. Cytotoxic activities were evaluated for all derivatives against two human cancer cell lines, MCF-7 and HepG2. Also, the effect of the most cytotoxic derivatives on VEGFR-2 protein concentration was assessed by ELISA. Compounds 14o, 14l, and 14b showed the highest activities with VEGFR-2 protein concentrations of 586.3, 636.2, and 705.7 pg/ml, respectively. Additionally, the anti-angiogenic property of compound 14b against human umbilical vascular endothelial cell (HUVEC) was performed using a wound healing migration assay. Compound 14b reduced proliferation and migratory potential of HUVEC cells. Furthermore, compound 14b was subjected to further biological investigations including cell cycle and apoptosis analyses. Compound 14b arrested the HepG2 cell growth at the Pre-G1 phase and induced apoptosis by 16.52%, compared to 0.67% in the control (HepG2) cells. The effect of apoptosis was buttressed by a 4.8-fold increase in caspase-3 level compared to the control cells. Besides, different in silico docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

Placenta-specific 8 (PLAC8) mediates inflammation and mobility of the hPDLCs via MEK/ERK signaling pathway

BackgroundPlacenta-specific 8 (PLAC8) is reported to regulate cellular functions in the progression of various diseases. However, its role in periodontitis is still unclear. MethodsHuman periodontal ligament cells (hPDLCs) were treated with lipopolysaccharide of Porphyromonas Gingivalis (LPS-PG) to mimic periodontitis in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression levels and western blot was for protein levels. Wound healing and transwell migration assays were performed to assess the cell mobility of hPDLCs. Both mRNA and protein levels of inflammatory cytokines including IFN-γ, IL-17, TNF-α, IL-4, IL-10 and IL-13 were accessed to evaluated process of periodontitis in vitro. Furthermore, the protein expressions of mitogen-activated protein kinase kinase (MEK), extracellular regulated protein kinase (ERK) and their phosphorylated products quantified by western blotting assay were determined to confirm the activation of the MEK/ERK signaling pathway. ResultsThe microarray analysis results showed that PLAC8 was most significantly downregulated in periodontium samples of patients with periodontitis, which participates in blood coagulation and integrin-mediated signaling pathway. PLAC8 was also markedly downregulated in the LPS-PG-treated hPDLCs. Moreover, overexpression of PLAC8 ameliorated inflammation and promoted cell mobility of LPS-PG-treated hPDLCs, while inhibition of PLAC8 exhibited the opposite effects. MEK/ERK was selected based on analyses of the protein-protein interaction (PPI) network as the potential signaling pathway interacted with PLAC8, and PLAC8 showed regulatory function on activation of the MEK/ERK pathway. Additionally, U0126, the inhibitor of MEK, abrogated the effects of PLAC8 on inflammation and cell mobility of LPS-PG-treated hPDLCs. ConclusionOverexpression of PLAC8 protected hPDLCs from dysfunction of inflammation and cell mobility via activating MEK/ERK pathway, indicating a novel therapeutic target for periodontitis.

Characteristic and Otopathogenic Analysis of a Vibrio alginolyticus Strain Responsible for Chronic Otitis Externa in China.

Vibrio alginolyticus, a Gram-negative rod bacterium found in marine environments, is known to cause opportunistic infections in humans, including ear infections, which can be difficult to diagnose. We investigated the microbiological and otopathogenic characteristics of a V. alginolyticus strain isolated from an ear exudate specimen obtained from a patient with chronic otitis externa to provide a basis for the future diagnosis of V. alginolyticus-associated infections. The identification of V. alginolyticus was accomplished using a combination of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), classical biochemical identification methods, and the use of Vibrio-selective media and advanced molecular identification methodologies. Antimicrobial susceptibility testing revealed that the strain was resistant to ampicillin and sensitive to β-lactam, aminoglycosides, fluoroquinolones, and sulfonamide antibiotics. The potential otopathogenic effects of V. alginolyticus were determined through the performance of cell viability, cell apoptosis, and cell death assays in tympanic membrane (TM) keratinocytes and HEI-OC1 cells treated with V. alginolyticus-conditioned medium using cell-counting kit (CCK)-8 assay, a wound-healing migration assay, Annexin V/propidium iodide (PI) flow cytometric analysis, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL staining). The results indicated that the identified V. alginolyticus strain exerts cytotoxic effects on keratinocytes and HEI-OC1 cells by inhibiting cell proliferation and migration and inducing apoptosis and cell death. To evaluate the ototoxicity of V. alginolyticus, the cell density and morphological integrity of hair cells (HCs) and spiral ganglion neurons (SGNs) were analyzed after exposing cochlear organotypic explants to the bacterial supernatant, which revealed the pre-dominant susceptibility and vulnerability of HCs and SGNs in the basal cochlear region to the ototoxic insults exerted by V. alginolyticus. Our investigation highlights the challenges associated with the identification and characteristic analysis of the Vibrio strain isolated in this case and ultimately aims to increase the understanding and awareness of clinicians and microbiologists for the improved diagnosis of V. alginolyticus-associated ear infections and the recognition of its potential otopathogenic and ototoxic effects.

MiR-616-5p Promotes Invasion and Migration of Bladder Cancer via Downregulating NR2C2 Expression.

BackgroundMicroRNAs, small non-coding RNA molecules with about 22 nucleotides in length, play a significant role in the development of bladder cancer. Previous studies found that miR-616-5p could promote the progress of cancers. However, its role in bladder cancer remains unclear. In the study, we aimed to demonstrate how miR-616-5p impacts the invasion and migration of bladder cancer and its potential downstream targets.MethodsFirstly, qRT-PCR was used to detect the expression of miR-616-5p in normal bladder uroepithelial cell lines and bladder cancer cell lines. Then, chamber–transwell invasion and wound healing migration assays were used to detect the roles of miR-616-5p and NR2C2 in invasion and migration. Subsequently, Western blot was used to evaluate the regulation effects of miR-616-5p and NR2C2. Finally, luciferase assays were performed to manifest the mechanism of miR-616-5p and NR2C2 regulation.ResultsWe found that miR-616-5p was upregulated in bladder cancer, and it could promote the invasion and migration of bladder cancer in vitro. Moreover, we demonstrated that NR2C2 was a downstream target of miR-616-5p. miR-616-5p could inhibit the expression of NR2C2 by binding to the 3′UTR of NR2C2 mRNA. Importantly, patients with a high expression of NR2C2 showed better prognoses in bladder cancer.ConclusionsThis study identifies that miR-616-5p can promote bladder cancer progression via altering the expression of NR2C2. Therefore, identifying miR-616-5p expression levels might be a useful strategy for developing potential therapeutic targets in bladder cancer.