Scratch Migration Assays Research Articles

Circ-DONSON promotes malignant progression of glioma through modulating FOXO3.

The aim of this study was to investigate the expression level of circ-DONSON in glioma and to explore its effect on glioma metastasis and the underlying mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine circ-DONSON expression in 40 paired glioma tumor tissues and adjacent tissues. Meanwhile, the relation between circ-DONSON level and clinical parameters of glioma and the prognosis of patients was analyzed. The expression of circ-DONSON in glioma cell lines was analyzed by qRT-PCR as well. In addition, circs-DONSON silencing model was constructed in glioma cell lines. Cell counting kit-8 (CCK-8), cell scratch, and transwell migration assays were performed to investigate the effect of circ-DONSON on biological functions of glioma cells. Finally, the interplay between FOXO3 and circ-DONSON was explored. QRT-PCR results revealed that the expression level of circ-DONSON in glioma tumor tissues was remarkably higher than that of adjacent tissues, and the difference was statistically significant (p<0.05). Compared with patients with low expression of circ-DONSON, significantly higher prevalence of lymph node or distant metastasis and worse prognosis were observed in patients with high expression of circ-DONSON (p<0.05). The proliferation and migration abilities of glioma cells in circ-DONSON silenced group were remarkably suppressed when compared with NC group (p<0.05). Additionally, FOXO3 expression was remarkably down-regulated in glioma cell lines and tissues. FOXO3 expression was negatively correlated with circ-DONSON expression. In addition, cell reverse experiment demonstrated that circ-DONSON and FOXO3 can regulate each other, thereby together affecting the malignant progression of glioma. Circ-DONSON was remarkably associated with lymph node or distant metastasis, as well as poor prognosis of patients with glioma. Furthermore, it promoted the metastasis of glioma cells via regulating FOXO3.

Bioactive chemical constituents from Curcuma caesia Roxb. rhizomes and inhibitory effect of curcuzederone on the migration of triple-negative breast cancer cell line MDA-MB-231

Rhizomes of Curcuma caesia are traditionally used to treat cancer in India. The aim is to isolate chemical constituents from C. caesia rhizomes through bioassay-guided fractionation. The extract, hexanes and chloroform fractions showed effect on MCF-7 and MDA-MB-231cells in cell viability assay. The chromatographic separation afforded germacrone (1), zerumbone (2), furanodienone (3), curzerenone (4), curcumenol (5), zederone (6), curcumenone (7), dehydrocurdione (8) from hexanes fraction and curcuminol G (9), curcuzederone (10), (1S, 10S), (4S,5S)-germacrone-1 (10), 4-diepoxide (11), wenyujinin B (12), alismoxide (13), aerugidiol (14), zedoarolide B (15), zedoalactone B (16), zedoarondiol (17), isozedoarondiol (18) from chloroform fraction. This is first report of compounds 2, 9–13, 15–18 from C. caesia. The study demonstrated compounds 1–4 and 10 are the bioactive compounds. The effect of curcuzederone (10) on MDA-MB-231 cell migration showed significant inhibition in scratch and Transwell migration assays. The results revealed that curcuzederone could be a promising drug to treat cancer.

DDIS-34. HIGH-THROUGHPUT DRUG SCREENING OF FDA-APPROVED ANTINEOPLASTIC DRUGS FOR THE TREATMENT OF AGGRESSIVE MENINGIOMAS

Abstract Currently, we are facing several challenges in the treatment of meningiomas: only subtotally removable tumors, high rate of recurrence in higher-grade meningiomas, malignancy, and lack of effective chemotherapeutic agents for aggressive or inoperable tumors. For these reasons, there is an urgent need for more successful treatments for aggressive meningiomas. Therefore, we used 147 FDA-approved antineoplastic drugs on two different meningioma cell lines, including the genetically modified cell line Ben-Men-1 (WHO°I) and the newly established anaplastic meningioma cell line NCH93 (WHO°III). The impact of the drugs on proliferation was assessed in a high-throughput 386-well format with CellTiter-Glo (Promega) and further validation was done by crystal-violet assay. Subsequent screening of 147 FDA-approved drugs resulted in the identification of potential 5 drugs, including Bortezomib, Carfilzomib, Omacetaxine, Paclitaxel, and Ponatinib. Dose-curve analysis revealed IC50 values of these drugs in the Ben-Men-1 and NCH93 cell lines as follows: Bortezomib 16.2 and 5.7 nM, Carfilzomib 4.8 and 2.6 nM, Omacetaxine and 5.0 and 8.9 nM, Paclitaxel 2.6 and 1.9 µM, and Ponatinib 278 and 206 nM. Inhibitor dosage of 10xIC50 values and higher lead to an average inhibition of Ben-Men-1 and NCH93 cells by up to 90% on day 2 (P&lt; .001). The impact of the antineoplastic agents on the cell cycle was analyzed by flow cytometry. Percentages of sub-G1 phase were significantly elevated with increasing drug concentrations of all five tested compounds (P&lt; .001). To assess the effects of the drugs on migration scratch assay was performed. Drug concentrations of 10xIC50 values resulted in an inhibition of migration in Ben-Men-1 cells for Bortezomib, Carfilzomib, and Omacetaxine by 49%, 30%, and 23%, respectively (P&lt; .05). In conclusion, we identified 5 promising drugs for the treatment of aggressive meningiomas by applying a high-throughput drug screening of 147 FDA-approved antineoplastic drugs.

Increased cell size, structural complexity and migration of cancer cells acquiring fibroblast organelles by cell-projection pumping.

We recently described a hydrodynamic mechanism for cytoplasmic transfer between cells, termed cell-projection pumping (CPP). Earlier image analysis related altered SAOS-2 osteosarcoma cell morphology, to what we now recognize as CPP uptake of fibroblast cytoplasm. We here examine SAOS-2 phenotype following co-culture with human dermal fibroblasts (HDF) in which organelles were pre-labelled with a fluorescent lipophilic marker. Fluorescence activated cell sorting (FACS) analysis was performed of HDF and SAOS-2, cultured either alone or together. FACS forward scatter is proportionate to cell size, and increased for SAOS-2 with high levels of HDF fluorescence uptake (p < 0.004). FACS side scatter is proportionate to internal cell complexity, and increased in SAOS-2 with increasing uptake of HDF fluorescence (p < 0.004), consistent with uptake of HDF organelles. Scratch migration assays revealed that HDF migrated more quickly than SAOS-2 in both isolated cell culture, and following co-culture (p < 0.004). Notably, SAOS-2 with high levels of HDF labelling migrated faster compared with SAOS-2 with low HDF labelling (p < 0.008). A slight and unconvincing reduction in SAOS-2 proliferation was seen (p < 0.02). Similar results were obtained in single additional experiments with A673 and H312 cancer cells. Forward and side scatter results suggest organellar transfer by CPP increases cancer cell morphological diversity. This may contribute to histological pleomorphism relevant to cancer diagnosis and prognosis. Also, increased migration of sub-populations of cancer cells with high CPP organellar uptake, may contribute to invasion and metastasis in-vivo. We thus suggest relevance of CPP to cancer diagnosis and progression.

Apoptotic Induction and Anti-Migratory Effects of Rhazya Stricta Fruit Extracts on a Human Breast Cancer Cell Line.

Rhazya stricta is a medicinal plant that is widely used in Saudi folklore medicine for treatment of various diseases. R. stricta fruit powder was sequentially extracted with n-hexane, chloroform, ethyl acetate, and methanol using a Soxhlet extractor. The cytotoxic effects of these fractions on human breast cancer cells (MDA-MB-231 and MCF-7) and non-tumorigenic control cells (MCF-10A) were evaluated via cell viability measurements, microscopy, gene expression, and migration assays. Moreover, the effect of the most promising extract on 7,12-dimethyl-benz[a]anthracene (DMBA)-induced breast cancer was investigated in rats. The promising extract was also subjected to gas chromatography–mass spectrometry. Fruit extracts of R. stricta were significantly cytotoxic toward all tested cell lines, as demonstrated by MTT and LDH assays. Treatment of MDA-MB-231 cells with fruit ethyl acetate fraction (RSF EtOAc) increased expression 11of P53, Bax and activation of caspase 3/7. A cell migration scratch assay demonstrated that extracts at non-cytotoxic concentrations exerted a potent anti-migration activity against the highly invasive MDA-MB-231 cell line. Moreover, RT-PCR results showed that RSF EtOAc significantly downregulated MMP-2 and MMP-9 expression, which play an important role in breast cancer metastasis. Histological studies of breast tissue in experimental animals showed a slight improvement in tissue treated with fruit ethyl acetate extract. GC-MS chromatogram showed thirteen peaks with major constituents were camphor, trichosenic acid and guanidine. Our current study demonstrates that fruit extracts of R. stricta are cytotoxic toward breast cancer cell lines through apoptotic mechanisms.

Gallic acid loaded poloxamer gel as new adjuvant strategy for melanoma: A preliminary study

The present study describes the production and characterization of poloxamer gels containing the antioxidant molecule gallic acid. The gels were particularly designed in order to obtain a formulation suitable for administration on the skin to treat melanoma. The polymer concentration was selected after rheological characterization and determination of gel transition temperature. In order to study the gallic acid diffusion, in vitro experiments were performed using Franz cells associated to different membranes. As first approach the gallic acid diffusion was evaluated through synthetic membranes, such as cellulose, nylon, polycarbonate, polytetrafluoroethylene, polyvinylidene fluoride and the commercial Strat-M® membrane. The membranes were employed separately or in association and compared to stratum corneum epidermis membranes, in order to find a system able to reproduce the gallic acid diffusion through the skin. Selected membranes were used for studying gallic acid diffusion from poloxamer gel. It was found that the diffusion of gallic acid was dramatically influenced by the type of membrane, both in the case of the aqueous solution or poloxamer gel. Scratch wound healing and migration assays conducted on human keratinocytes and melanoma cells demonstrated the ability of gallic acid loaded gel to inhibit cellular migration, suggesting its potential as adjuvant strategy for melanoma.

TGF-\u03b21-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts

HSP47 is required for the production of collagen and serves an important role in tissue remodeling, a pathophysiologic mechanism of chronic rhinosinusitis (CRS). We investigated the relationship between HSP47 expression and tissue remodeling in CRS. We also determined the underlying molecular mechanisms of TGF-β1-induced HSP47 and extracellular matrix (ECM) production in nasal fibroblasts. HSP47, α-SMA, fibronectin, and collagen type I expression levels were measured using real-time PCR, western blotting, and immunofluorescence staining. Fibroblast migration was analyzed using scratch and transwell migration assays. Contractile activity was measured with a collagen gel contraction assay. HSP47 is increased in patients with CRS without nasal polyps. TGF-β1 induced HSP47 expression in nasal fibroblasts. Myofibroblast differentiation and ECM production, which are induced by TGF-β1, were inhibited by siHSP47. We also confirmed that the Smad2/3 signaling pathway is involved in TGF-β1-induced HSP47 expression in nasal fibroblasts. In a functional assay, TGF-β1-enhanced migration and contraction ability were inhibited by HSP47 knockout. Glucocorticoid reversed the stimulatory effects of TGF-β1 on HSP47 expression and ECM production in nasal fibroblasts and ex vivo organ cultures. HSP47 expression is involved in TGF-β1-induced myofibroblast differentiation and ECM production through the Smad2/3 signaling pathway, which might contribute to tissue remodeling in chronic rhinosinusitis.

Autologous Micro-Fragmented Adipose Tissue as Stem Cell-Based Natural Scaffold for Cartilage Defect Repair

Osteoarthritis (OA) poses a tough challenge worldwide. Adipose-derived stem cells (ASCs) have been proved to play a promising role in cartilage repair. However, enzymatic digestion, ex vivo culture and expansion, with significant senescence and decline in multipotency, limit their application. The present study was designed to obtain micro-fragmented adipose tissue (MFAT) through gentle mechanical force and determine the effect of this stem cell-based natural scaffold on repair of full-thickness cartilage defects. In this study, ASCs sprouted from MFAT were characterized by multi-differentiation induction and flow cytometry. Scratch and transwell migration assays were operated to determine whether MFAT could promote migration of chondrocytes in vitro. In a rat model, cartilage defects were created on the femoral groove and treated with intra-articular injection of MFAT or PBS for 6 weeks and 12 weeks (n = 12). At the time points, the degree of cartilage repair was evaluated by histological staining, immunohistochemistry and scoring, respectively. Two unoperated age-matched animals served as native controls. ASCs derived from MFAT possessed properties to differentiate into adipocytes, osteocytes and chondrocytes, with expression of mesenchymal stem cell markers (CD29, 44, 90) and no expression of hematopoietic markers (CD31, 34, 45). In addition, MFAT could significantly promote migration of chondrocytes. MFAT-treated defects showed improved macroscopic appearance and histological evaluation compared with PBS-treated defects at both time points. After 12 weeks of treatment, MFAT-treated defects displayed regular surface, high amount of hyaline cartilage, intact subchondral bone reconstruction and corresponding formation of type I, II, and VI collagen, which resembled the normal cartilage. This study demonstrates the efficacy of MFAT on cartilage repair in an animal model for the first time, and the utility of MFAT as a ready-to-use therapeutic alternative to traditional stem cell therapy.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the β3 subunit of voltage-gated calcium channels (Cavβ3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cavβ3-deficient mice (Cavβ3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cavβ3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cavβ3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cavβ3-deficient mice or cells might also be applicable for specific molecules other than Cavβ3.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

KLF4 overexpression decreases the viability, invasion and migration of papillary thyroid cancer cells.

Kruppel-like factor 4 (KLF4) has been implicated in a number of different types of cancer; however, the role of KLF4 in papillary thyroid cancer remains elusive. The present study aimed to investigate the role of KLF4 in papillary thyroid cancer and its potential underlying molecular mechanisms. The expression of KLF4 in thyroid tumor tissue and adjacent non-cancerous tissues were detected via immunohistochemistry and western blotting. The papillary thyroid cancer cell line, KTC1, was transfected with viruses carrying KLF4 overexpression vectors. The relative expression of KLF4, E-cadherin, N-cadherin, Vimentin, matrix metalloproteinase (MMP)2, MMP9 and collagen was detected via quantitative-PCR. The viability of KTC1 cells was detected using a cell counting kit-8 assay at 24, 48 and 72 h. Cell invasion was examined via a transwell invasion assay. Cell migration was examined via a scratch migration assay at 0 and 24 h. Compared with adjacent non-cancerous tissues, the expression of KLF4 was significantly lower in thyroid tumor tissues. The expression of KLF4 in KTC1 cells were significantly increased compared with the blank or negative control groups. The expression of N-cadherin, MMP2, MMP9 and collagen was significantly decreased in the KLF4 overexpression group. The viability of KTC1 cells was markedly decreased in KLF4 overexpression group at 24, 48 and 72 h when compared with the blank or negative control groups. The invasion of KTC1 cells in the KLF4 overexpression group was markedly decreased. Compared with the negative control group, the KTC1 cell migration in the KLF4 overexpression group was markedly decreased at 24 h. The expression of KLF4 was also significantly lower in thyroid tumor tissues. The cell viability, tumor invasion and migration ability and expression levels of N-cadherin, MMP2, MMP9 and collagen in papillary thyroid cancer cells were markedly decreased with KLF4 overexpression.

472 MicroRNA-130a: a tumor suppressive miRNA in cutaneous squamous cell carcinoma

Cutaneous Squamous Cell Carcinoma (cSCC) is a malignant neoplasm of the skin resulting from the accumulation of somatic mutations due to solar radiation. It is one of the fastest increasing malignancies that represents a particular problem among immunosuppressed individuals. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein-coding genes at the posttranscriptional level. Global analysis of miRNA expression in human cSCC identified miR-130a as one of the miRNAs that downregulated in cSCC and in this study we investigated its function in the disease in in vitro and in vivo disease models. The decreased expression of miR-130a in cSCCs was confirmed by qPCR and LNA in situ hybridization. We found that the expression of miR-130a is regulated by HRas proto-oncogene. To address the role of miR-130a in vivo, we engineered a stable miR-130a cSCC cell line and performed tumor xenograft experiments in immunodeficient mice. We observed that miR-130a suppressed cancer growth on mice from 4 weeks after injection and onwards significantly. The tumors were harvested at week 6 and we observed that tumors formed by miR-130a-overexpressing SCC cells weighed significantly less compared with controls. We investigated the function of miR-130a in cSCC and found that ectopic overexpression of miR-130a in two different SCC cell lines (A431 and UT-SCC-7) resulted in the suppression of their self-renewal capacity as determined by colony formation and sphere formation assay. MiR-130a also inhibited cell motility of SCC cells as demonstrated by scratch and Transwell migration assays as well as invasion capacity through Matrigel in Transwell invasion assays. Mechanistically, we verified ACVR1, a TGF-β superfamily member, as a direct target of miR-130a. These results could contribute to the understanding of the molecular processes of neoplastic transformation in epidermal keratinocytes which could lead to novel therapeutic target for cSCC.

Anti-tumor effect of chitin oligosaccharide plus cisplatin in vitro and in vivo.

BackgroundLung cancer is one of the most common malignant tumors in human beings, and cisplatin is a widely used chemotherapy drug, but its clinical application is limited because of its dose-dependent toxicity and drug resistance. Chitin is known to have various biological activities including anti-tumor, but the insoluble feature in common solvents greatly restricts its application. Chitin oligosaccharide is a small water-soluble molecule degraded from chitin without any toxic effect.MethodsChitin oligosaccharide was adopted to investigate the effects on lung adenocarcinoma A549 cells and tumor xenografts of nude mice. The experiments were divided into control group, chitin oligosaccharide group, cisplatin group and combination group. MTS assay, cell scratch test and migration assay were used to observe the proliferation and migration of A549 cells, and Western blot was used to detect the expression levels of caspase8, caspase3 and BAK. Ki67 and P53 expressions of tumor xenografts were detected to explore the effects of drugs on tumor prognosis.ResultsThe results in vitro showed that chitin oligosaccharides could inhibit the proliferation and migration of A549 cells, and the effect was superior to chitin oligosaccharide or cisplatin when combined with cisplatin. Chitin oligosaccharide plus cisplatin up-regulated the expression level of caspase8 and caspase3, while had minor influence on the expression level of BAK. In vivo experiments showed that chitin oligosaccharide plus cisplatin could down-regulate the expression level of Ki67, while had minor influence on the expression level of P53.ConclusionThe study demonstrated that chitin oligosaccharide plus cisplatin had positive synergistic effects, and it is possible to improve the prognosis of lung adenocarcinoma patients by up-regulating the expression level of caspase8, caspase3 and down-regulating the expression level of Ki67.

Up-regulation of TIMP-3 and RECK decrease the invasion and metastasis ability of colon cancer

Background and study aimsAlthough the function of microRNA21 (miR-21) in the invasion and metastasis of colon cancer has been extensively studied, the mechanisms of invasion and migration related pathways between and its targets are still not elucidated. This study explored the mechanisms of the pathway between miR-21 and the target genes in vitro and in vivo. Materials and methodsWe transfected pmiRZip21 or Leti3 into colon cancer cells. The levels of miR-21 expression, mRNA transcription and protein of target genes were analysed by TaqMan microRNA assays, RT-PCR and western blot, respectively. Scratch migration and trans-well assays were used to evaluate metastasis and invasion. To build a subcutaneous tumour animal model, detect the level of miR-21 and the target genes and then identify the mechanisms in vivo. ResultsMiR-21 expression levels in colon cancer cells transfected with pmiRZip21 in vivo or in vitro were decreased (P < 0.05). The mRNA and protein levels of TIMP-3 and RECK were up-regulated after inhibiting miR-21 in vitro and in vivo (P < 0.05), but those of BMPR-II and PCDH17 were not. In pmiRZip21-transfected colon cancer cells, invasion and migration were significantly decreased both in vitro and vivo (P < 0.05). ConclusionsUp-regulation of TIMP-3 and RECK, by inhibiting miR-21 expression can decrease tumour invasion and metastasis ability in vitro and in vivo, and has potential as a possible target site in anti-tumour therapy. More effects in vivo have to be investigated in further research.

SNORD89 promotes stemness phenotype of ovarian cancer cells by regulating Notch1-c-Myc pathway

BackgroundOvarian cancer is the leading cause of death in gynecological cancer. Cancer stem cells (CSCs) contribute to the occurrence, progression and resistance. Small nucleolar RNAs (SnoRNAs), a class of small molecule non-coding RNA, involve in the cancer cell stemness and tumorigenesis.MethodsIn this study, we screened out SNORNAs related to ovarian patient’s prognosis by analyzing the data of 379 cases of ovarian cancer patients in the TCGA database, and analyzed the difference of SNORNAs expression between OVCAR-3 (OV) sphere-forming (OS) cells and OV cells. After overexpression or knockdown SNORD89, the expression of Nanog, CD44, and CD133 was measured by qRT-PCR or flow cytometry analysis in OV, CAOV-3 (CA) and OS cells, respectively. CCK-8 assays, plate clone formation assay and soft agar colony formation assay were carried out to evaluate the changes of cell proliferation and self-renewal ability. Scratch migration assay and trans-well invasion analysis were used for assessing the changes of migration and invasion ability.ResultsHigh expression of SNORD89 indicates the poor prognosis of ovarian cancer patients and was associated with patients’ age, therapy outcome. SNORD89 highly expressed in ovarian cancer stem cells. The overexpression of SNORD89 resulted in the increased stemness markers, S phase cell cycle, cell proliferation, invasion and migration ability in OV and CA cells. Conversely, these phenomena were reversed after SNORD89 silencing in OS cells. Further, we found that SNORD89 could upregulate c-Myc and Notch1 expression in mRNA and protein levels. SNORD89 deteriorates the prognosis of ovarian cancer patients by regulating Notch1-c-Myc pathway to promote cell stemness and acts as an oncogene in ovarian tumorigenesis. Consequently, SNORD89 can be a novel prognostic biomarker and therapeutic target for ovarian cancer.

Honokiol inhibits breast cancer cell metastasis by blocking EMT through modulation of Snail/Slug protein translation

Honokiol (HNK), an active compound isolated from traditional Chinese medicine Magnolia officinalis, has shown potent anticancer activities. In the present study, we investigated the effects of HNK on breast cancer metastasis in vitro and in vivo, as well as the underlying molecular mechanisms. We showed that HNK (10-70 μmol/L) dose-dependently inhibited the viability of human mammary epithelial tumor cell lines MCF7, MDA-MB-231, and mouse mammary tumor cell line 4T1. In the transwell and scratch migration assays, HNK (10, 20, 30 μmol/L) dose-dependently suppressed the invasion and migration of the breast cancer cells. We demonstrated that HNK (10-50 μmol/L) dose-dependently upregulated the epithelial marker E-cadherin and downregulated the mesenchymal markers such as Snail, Slug, and vimentin at the protein level in breast cancer cells. Using a puromycin incorporation assay, we showed that HNK decreased the Snail translation efficiency in the breast cancer cells. In a mouse model of tumor metastasis, administration of HNK (50 mg/kgevery day, intraperitoneal (i.p.), 6 times per week for 30 days) significantly decreased the number of metastatic 4T1 cell-derived nodules and ameliorated the histological alterations in the lungs. In addition, HNK-treated mice showed decreased Snail expression and increased E-cadherin expression in metastatic nodules. In conclusion, HNK inhibits EMT in the breast cancer cells by downregulating Snail and Slug protein expression at the mRNA translation level. HNK has potential as an integrative medicine for combating breast cancer by targeting EMT.

Depletion of PRDM9 enhances proliferation, migration and chemotaxis potentials in human periodontal ligament stem cells

ABSTRACT Purpose Periodontal ligament mesenchymal stem cells (PDLSCs) are important for periodontal tissue regeneration, but how these cells are regulated remains unclear. PRDM (PRDI-BF1 and RIZ homology domain containing) genes play key roles in cell proliferation and differentiation. The present study aimed to investigate the role of one PRDM gene, PRDM9, in the proliferation, migration and chemotaxis potential of PDLSCs. Materials and methods Cell proliferation was examined on the basis of the cell doubling time, cell counting kit-8 (CCK8) assays, and flow cytometry analysis of the cell cycle. Gene expression was detected by Western blotting and real-time RT-PCR. Scratch migration and Transwell chemotaxis assays were used to analyse cell migration and chemotaxis abilities. Microarray analysis and ChIP assays were used to examine the downstream genes of PRDM9 and the corresponding mechanism. Results The results showed that knock-down of PRDM9 enhanced cell proliferation by promoting cell cycle progression and rapid transition from the G1 to S phase via downregulation of p21 and p27 and upregulation of cyclin E. Additionally, depletion of PRDM9 increased the migration and chemotaxis potential of PDLSCs. Microarray results showed that 13 genes, including IGFBP5, IFI44L, and POSTN, were upregulated and 34 genes, including PIP, were downregulated after the depletion of PRDM9. Furthermore, we observed that the depletion of PRDM9 promoted the transcription of IGFBP5 by increasing H3K4me3 methylation in the IGFBP5 promoter. Conclusion These discoveries indicated that depletion of PRDM9 increased the cell proliferation, migration and chemotaxis potential of PDLSCs and revealed important downstream genes.

&lt;p&gt;Long noncoding RNA FEZF1-AS1 promotes the motility of esophageal squamous cell carcinoma through Wnt/β-catenin pathway&lt;/p&gt;

Background: Long noncoding RNAs (lncRNAs), a class of noncoding RNA nucleotides >200 bp, has been demonstrated to play vital role in the development of cancer. FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) has been reported as an lncRNA which acts as a tumor-promoting effect in some cancers. However, the role of it in esophageal squamous cell carcinoma (ESCC) and its potential regulatory mechanism was unclear now.Methods: qRT-PCR was used to detect the levels of FEZF1-AS1 and mRNA CTNNB1 (β-catenin) in ESCC tissues and cells. Cell transfection experiments were used to knock down or overexpress the level of FEZF1-AS1 in EC1 and EC9706 cell lines. WST-1 assays, cell cycle assays, scratch wound assays, migration, and invasion assays were used to evaluate the function of FEZF1-AS1 in ESCC progression.Results: FEZF1-AS1 was remarkably upregulated in ESCC tissues and cell lines. Silencing of FEZF1-AS1 significantly inhibited the migration and invasion of ESCC cells, while overexpression of FEZF1-AS1 notably accelerated ESCC migration and invasion. Meanwhile, the levels of FEZF1-AS1 had no effect on ESCC cell proliferation and cell cycle. We also found that β-catenin was upregulated in ESCC tissues, and the level of it was positively correlated with the expression of FEZF1-AS1. Silencing of FEZF1-AS1 could decrease the mRNA and protein level of β-catenin, while overexpression FEZF1-AS1 could lead to the contrary.Conclusion: Our results suggested that the expression of lncRNA FEZF1-AS1 played an important role in ESCC progression, especially the motility of the tumor. FEZF1-AS1 may provide us with a new sight for ESCC treatment.

Sea cucumber extract TBL-12 inhibits the proliferation, migration, and invasion of human prostate cancer cells through the p38 mitogen-activated protein kinase and intrinsic caspase apoptosis pathway.

Sea cucumber is a kind of nutritious echinoderm that has multiple biological activities, including antioxidant, antibacterial, and antitumor activities. However, there is no extensive study on the antitumor effect of sea cucumber extract on prostate cancer (PCa). TBL-12 is a new sea cucumber extract. In this study, we investigated the in vivo anti-PCa effect of TBL-12 and its in vitro effects on the proliferation, apoptosis, migration, and invasion of the human PCa cell lines LNCaP, 22RV1, PC-3, and DU145, and evaluated its possible mechanisms. Cell proliferation was analyzed by cell counting kit-8 and colony formation assays. Scratch migration assay and transwell invasiveness assay were used to observe TBL-12 effect on the migration and invasion of PCa cells. Matrix metalloproteinase 2 (MMP-2) and MMP-9 expression and enzymatic activity was determined by Western blot analysis, quantitative reverse-transcription polymerase chain reaction, and gelatin zymography. Apoptosis level was detected by flow cytometry analysis. Western blot analysis was used to analyze p38 mitogen-activated protein kinase (MAPK) and apoptosis pathways. Angiogenic array analysis was used to explore autocrine and paracrine growth factors in PCa cell lines. Xenograft tumor model was built to observe the in vivo anticancer effect. TBL-12 could significantly inhibit tumor growth in xenograft PCa mice in vivo, and dramatically inhibit the proliferation, colony formation, migration, and invasiveness of PCa cells in vitro (P < 0.05 and P < 0.001). The expression and enzyme activity of MMP-2 and MMP-9 were significantly suppressed by TBL-12 ( P < 0.01), and decreased phosphorylation level of p38 in PCa cells was detected ( P < 0.001). Furthermore, TBL-12 could reinforce the MMP-2/MMP-9 inhibitory effect of SB203580, a specific inhibitor of the p38 MAPK pathway ( P < 0.05). Besides, TBL-12 could induce the apoptosis of PCa cells by activating caspase-9, caspase-7, and poly(ADP-ribose) polymerase and suppressing survivin, and inhibit the secretion of angiogenin, angiopoietin-2, and vascular endothelial growth factor in PCa cells. Sea cucumber extract TBL-12 could suppress the proliferation and metastasis of human PCa cells by inhibiting MMP-2 and MMP-9 via blocking the p38 MAPK pathway, inducing apoptosis through intrinsic caspase apoptosis pathway and inhibiting the secretion of angiogenic factors. Our findings may be of importance and significance for the research and clinical applications of sea cucumber extract in PCa treatment.

The ERH gene regulates migration and invasion in 5637 and T24 bladder cancer cells

BackgroundThis study aimed to determine whether the enhancer of the rudimentary homolog (ERH) gene regulates cell migration and invasion in human bladder urothelial carcinoma (BUC) T24 cells and the underlying mechanism.MethodsFirst, we knocked down ERH in BUC T24 and 5637 cells by shRNA and then used wound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays to determine the migration and invasion ability after ERH was knocked down. Moreover, we used gene expression profiling chip analysis and further functional experiments to explore the possible mechanism through which ERH knockdown downregulated metastasis ability in T24 cells.ResultsWound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays all showed that the metastasis ability was significantly inhibited in human BUC T24 and 5637 cells with ERH knockdown. A gene expression profiling chip analysis in T24 cells showed that the MYC gene may be an important downstream target of the ERH gene, and the functional experiments showed that MYC is a functional target of ERH in BUC T24 cells.ConclusionERH knockdown could inhibit the metastasis of BUC T24 cells in vitro and in vivo. This study further explored the mechanism of the ERH gene in the metastasis of the T24 human bladder cancer cell line and found that ERH may regulate MYC gene expression. The results of this research provide a basis for the clinical application of ERH as a potential target for BUC treatment.