BrdU Assay Research Articles

Assessment of Rose Bengal Photodynamic Therapy on Viability and Proliferation of Human Keratolimbal Epithelial and Stromal Cells In Vitro.

To investigate the effect of Rose Bengal photodynamic therapy (RB-PDT) on viability and proliferation of human limbal epithelial stem cells (T-LSCs), human corneal epithelial cells (HCE-T), human limbal fibroblasts (LFCs), and human normal and keratoconus fibroblasts (HCFs and KC-HCFs) in vitro. T-LSCs and HCE-T cell lines were used in this research. LFCs were isolated from healthy donor corneal limbi (n = 5), HCFs from healthy human donor corneas (n = 5), and KC-HCFs from penetrating keratoplasties of keratoconus patients (n = 5). After cell culture, RB-PDT was performed using 0.001% RB concentration and 565 nm wavelength illumination with 0.14 to 0.7 J/cm2 fluence. The XTT and the BrdU assays were used to assess cell viability and proliferation 24 h after RB-PDT. RB or illumination alone did not change cell viability or proliferation in any of the cell types (p ≥ 0.1). However, following RB-PDT, viability decreased significantly from 0.17 J/cm2 fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm2 fluence in T-LSCs (p < 0.001), HCE-T (p < 0.05), and LFCs ((p < 0.0001). Cell proliferation decreased significantly from 0.14 J/cm2 fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm2 fluence in HCFs (p < 0.05). Regarding LFCs proliferation, no values could be determined by the BrdU assay. Though RB-PDT seems to be a safe and effective treatment method in vivo, its dose-dependent phototoxicity on corneal epithelial and stromal cells has to be respected. The data and experimental parameters applied in this study may provide a reliable reference for future investigations.

Rosline promotes p21 expression to inhibit ovarian cancer cell proliferation via p53-independent pathway.

To investigate the effect of benzothiazole derivatives (Rosline) on ovarian cancer and the potential mechanism. Ovarian cancer tissues were collected clinically and immunohistochemistry was used to detect the expression of p53 and p21. Ovarian cancer cells were exposed to 0, 2.5, 5, 10 μmol/L Rosline for 24 h. 100 nmol/L Pifithrin-α pre-incubation was used to inhibit the transcriptional activity of p53. CCK-8 and BrdU assays were used to detect the effects of different concentrations of rosline on the proliferation and cell cycle of OVCAR420 and SKOV3 cells. Flow cytometry assay was used to detect cell cycle. The transcriptional and translational expression of p21 and p53 were detected by RT-qPCR and Western blot. p21 was expressed in ovarian cancer tissues without p53 expression. Rosline inhibits the proliferation of ovarian cancer cells and blocks the cell cycle progression. Meanwhile, Rosline promotes p21 expression in ovarian cancer cells at both mRNA and protein levels, but with no significant effect on p53 expression. Besides, Rosline promotes p21 expression, inhibits cell proliferation, and blocks the cell cycle via the p53-independent pathway. Rosline promoted p21 expression thereby inhibiting cell proliferation and blocks the cell cycle via p53-independent pathway.

The effect of GP-2250 on cultured virus-negative Merkel cell carcinoma cells: preliminary results.

Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells. MethodsWe employed three cell lines (MCC13, MCC14.2, MCC26) which were exposed to different GP-2250doses. GP-2250's effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression. Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied. The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells.

Effect of Smilax spp. and Phellinus linteus combination on cytotoxicity and cell proliferation of breast cancer cells

BackgroundAlthough the prevalence of breast cancer (BC) has been reduced in recent years, proficient therapeutic regimens should be further investigated with the aim of further reducing the mortality rate. To obtain more effective treatment, the present study aimed to observe the effects of PL synergistically combined with Smilax corbularia and S. glabra extracts (PSS) on BC cell lines, MCF7, T47D, MDA-MB-231, and MDA-MB-468.MethodsThe half-maximal inhibition (IC50) concentrations of PSS and PL were determined in a dose- and time-dependent manner using MTT assay. The activity of PSS and PL on anti-BC proliferation was evaluated using BrdU assay, and colony formation assay. Moreover, cell cycle analysis and apoptosis induction as a result of PSS and PL exposure were investigated using propidium iodide (PI) staining and co-staining of annexin V DY634 and PI combined flow cytometric analysis, respectively. Finally, changes in the mRNA expression of genes involved in proliferative and apoptotic pathways (MKI67, HER2, EGFR, MDM2, TNFα, PI3KCA, KRAS, BAX, and CASP8) were explored using RT-qPCR following PSS and PL treatment.ResultsThe PSS and PL extracts exhibited significant potential in BC cytotoxicity which were in were in dose- and time-dependent response. This inhibition of cell growth was due to the suppression of cell proliferation, the cell cycle arrest, and the induction of apoptosis. Additionally, an investigation of the underlying molecular mechanism revealed that PSS and PL are involved in downregulation of the MKI67, HER2, EGFR, MDM2, TNFα, and PI3KCA expression.ConclusionsThis present study has suggested that PSS and PL possess anti-BC proliferative activity mediated via the downregulation of genes participating in the relevant pathways. PSS or PL may be combined with other agents to alleviate the adverse side effects resulted from conventional chemotherapeutic drugs.

Synergism of antiproliferative effects of young green barley and chlorella water extracts against human breast cancer cells.

Breast cancer is the most common type of tumour in terms of incidence and mortality among women. In the light of recent data that has revealed the beneficial impact of increasing plant-based food consumption on the risk of breast cancer, the use of young green barley and chlorella, the chemopreventive properties of which have been previously reported, seems to be a reasonable therapeutic strategy in this type of cancer. Nevertheless, there are only a few scientific reports focused on the influence of the mentioned products on breast cancer development; thus, the aim of the study was to enrich knowledge resources in this area. The chemopreventive effect of water extracts of chlorella (CH) and young green barley (YGB) and their mixture (MIX) was investigated in human breast adenocarcinoma T47D cells and human skin fibroblasts HSF by LDH, MTT and BrdU assays. Changes in cell morphology in response to tested extracts were examined in light microscopy. Tested extracts were not toxic against HSF and did not affect their proliferation and morphology. Simultaneously, extracts increased the permeability of T47D cell membranes and inhibited their proliferation. Microscopic observation confirmed the results of biochemical assays and additionally suggested necrosis induction in T47D cells in response to tested compounds. Obtained results demonstrated that MIX induced stronger beneficial changes than their components. The study revealed the chemopreventive properties of the investigated green food products against breast cancer cells, without any side-effects in human skin fibroblasts. The beneficial properties discovered of the tested extracts on cancer cells enhanced by their concomitant administration, and in the case of antiproliferative effects YGB and CH, revealed synergism of action.

Topical effect of polyherbal flowers extract on xanthan gum hydrogel patch—induced wound healing activity in human cell lines and male BALB/c mice

Wound or injury is a breakdown in the skin’s protective function as well as damage to the normal tissues. Wound healing is a dynamic and complex phenomenon of replacing injured skin or body tissues. In ancient times the Calendula officinalis and Hibiscus rosa-sinensis flowers were extensively used by the tribal communities as herbal medicine for various complications including wound healing. But loading and delivery of such herbal medicines are challenging because it maintains their molecular structure against temperature, moisture, and other ambient factors. This study has fabricated xanthan gum (XG) hydrogel through a facile process and encapsulated C. officinalis and H. rosa-sinensis flower extract. The resulting hydrogel was characterized by different physical methods like x-ray diffractometer, UV–vis spectroscopy, Fourier transform infrared spectroscopy, SEM, dynamic light scattering, electronkinetic potential in colloidal systems (ZETA) potential, thermogravimetric differential thermal analysis (TGA-DTA), etc. The polyherbal extract was phytochemically screened and observed that flavonoids, alkaloids, terpenoids, tannins, saponins, anthraquinones, glycosides, amino acids, and a few percentages of reducing sugar were present in the polyherbal extract. Polyherbal extract encapsulated XG hydrogel (X@C–H) significantly enhanced the proliferation of fibroblast and keratinocyte cell lines in comparison to the bare excipient treated cells as determined by 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide assay. Also, the proliferation of these cells was confirmed by BrdU assay and enhanced expression of pAkt. In an in-vivo study, wound healing activity of BALB/c mice was carried out and we observed that X@C–H hydrogel showed significant result compared to the other groups (untreated, X, X@C, X@H). Henceforth, we conclude that this synthesized biocompatible hydrogel could emerge as a promising carrier of more than one herbal excipients.

MiR-199a-5p from bone marrow mesenchymal stem cell exosomes promotes the proliferation of neural stem cells by targeting GSK-3β.

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes are a promising therapeutic agent for human disease, but their effects on neural stem cells (NSCs) subject to spinal cord ischaemia-reperfusion injury (SCIRI) remain unknown. Here, we examine the impact of miR-199a-5p-enriched exosomes derived from BMSCs on NSC proliferation. We establish a rat model of aortic cross-clamping to induce SCIRI in vivo and a primary NSC model of oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate SCIRI in vitro. CCK8, EdU, and BrdU assays are performed to evaluate the proliferation of NSCs. Hematoxylin and eosin (H&E) staining is used to determine the number of surviving neurons. The Basso, Beattie, and Bresnahan (BBB) scale and inclined plane test (IPT) are used to evaluate hind limb motor function. DiO-labelled exosomes are efficiently internalized by NSCs and increase ectopic amounts of miR-199a-5p, which promotes the proliferation of NSCs. In contrast, exosomes derived from miR-199a-5p-depleted BMSCs exert fewer beneficial effects. MiR-199a-5p targets and negatively regulates glycogen synthase kinase 3β (GSK-3β) and increases nuclear β-catenin and cyclin D1 levels. miR-199a-5p inhibition reduces the total number of EdU-positive NSCs after OGD/R, but the GSK-3β inhibitor CHIR-99021 reverses this effect. In vivo, intrathecal injection of BMSC-derived exosomes increases the proliferation of endogenous spinal cord NSCs after SCIRI. In addition, more proliferating NSCs are found in rats intrathecally injected with exosomes overexpressing miR-199a-5p. In summary, miR-199a-5p in BMSC-derived exosomes promotes NSC proliferation via GSK-3β/β-catenin signaling.

ANKRD22 enhances cancer stem cell growth and cisplatin resistance in cervical cancer via NUSAP1/Wnt/β catenin pathway

Purpose: To investigate the role of ankyrin repeat domain 22 (ANKRD22) and nucleolar spindle-associated protein 1 (NUSAP1)-mediated Wnt/β-catenin pathway in cervical cancer (CC). Methods: ANKRD22 levels were evaluated in tissue samples collected from CC patients. The CC cells were transfected with an ANKRD22 silencing vector. Cell proliferation and migration were determined via colony formation, BrdU, scratch and Transwell assays. Sphere formation test was performed to determine the effect of ANKRD22 on the cancer stem cell (CSC)-like traits of CC cells. The effect of ANKRD22 on CSC-like traits was further evaluated by determining the expressions of the CSC markers, Oct4, SOX2, and Nanog, while cisplatin resistance was assessed by CCK-8 assay. The effect of ANKRD22 on the NUSAP1-mediated Wnt/β-catenin pathway was evaluated by determining the expression levels of β-catenin, matrix metalloproteinase-7, and adenomatous polyposis coli. Moreover, ANKRD22-mediated tumor growth was monitored in vivo using an animal model. Results: ANKRD22 was overexpressed in cancer tissues from CC patients (p &lt; 0.05). ANKRD22 knockdown suppressed CC cell proliferation, migration, invasion, and CSC traits, and also positively regulated Wnt/β-catenin pathway through NUSAP1 (p &lt; 0.05). However, the inhibition of ANKRD22 suppressed tumor growth in vivo through NUSAP1. In addition, the silencing of ANKRD22 alleviated cisplatin resistance in CC cells (p &lt; 0.05). Conclusion: ANKRD22 activates NUSAP1/Wnt/β-catenin pathway, and enhances CSC-like characteristics and cisplatin resistance, thus exacerbating the malignant behaviors of CC cells. Therefore, ANKRD22 could serve as a promising target for CC treatment.

Abstract LB228: Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells

Abstract Background: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinases, which is approved for HER2-positive metastatic breast cancer as second line treatment. Significant proportion of patients experience disease progression due to acquired resistance. Activation of DNA damage repair (DDR) is one of the drug-resistance mechanism, however, the impact of DDR on sensitivity to lapatinib is unclear. Thus, lapatinib resistance mechanism was explored from the standpoint of DDR activation. Methods: Acquired lapatinib-resistant (LR) SK-BR-3 cell lines were generated by continuously exposing to lapatinib, starting with 30 nmol/L and incrementally increasing to 10 μmol/L over 7 months. MTT assay was used to confirm lapatinib sensitivity. Cell cycle progression was analyzed using BrdU assay. Replication fork speed and stalled fork were analyzed by DNA fiber assay. Expression of the molecules was examined using Western blotting, immunofluorescence assay and transcriptome data analysis. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. Results: G1/S phase transition was increased and early/late S phase population was increased in SK-BR-3 LR cells. Replication fork speed was accelerated and replication stress are elevated in SK-BR-3 LR cells. p-Chk1, Rad51, Rad51B, Rad51C and XRCC3 were upregulated in SK-BR-3 LR cells. After irradiation or treatment with ATR inhibitor, γ-H2AX was continuously increased in parental cells. In contrast, γ-H2AX did not change significantly in SK-BR-3 LR cells, and Chk1 was activated after irradiation or treatment with ATR inhibitor. Tail of comet disappeared at early time point in in SK-BR-3 LR cells after induction of DNA damage compared with parental cells. These results demonstrated that DDR was activated and DNA damage repair capacity was enhanced in LR cells. Conclusion: Replication stress is elevated in SK-BR-3 LR cells. Up-regulation of molecules involved in the homologous recombination repair pathway was observed in SK-BR-3 LR cells. Moreover, DNA repair capacity was increased in SK-BR-3 LR cells. These data suggested that activation of DNA damage repair pathway caused by replication stress contributes to lapatinib resistance. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, Changyun Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB228.

Abstract 1141: The CoREST complex as a novel target to disrupt AT/RT’s abnormal epigenetic landscape

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors. Their aggressive phenotype results from a single recurring biallelic loss-of-function mutation in genes encoding components of the SWI/SNF chromatin-remodeling complex - SMARCB1 in the majority of tumors and SMARCA4 in remaining tumors. Residual SWI/SNF activity continues to inhibit EZH2 methyltransferase except at gene promoter regions where EZH2 co-localizes with the REST complex. At these sites, EZH2 increases repressive H3K27me3 marks. EZH2 co-localizes with REST on neuronal differentiation and tumor suppressor genes. As a result, this epigenetic abnormality helps maintain AT/RT in a stem cell-like state, driving its aggressive growth and therapy resistance. We study the consequences of disrupting SWI/SNF inhibition at sites of EZH2/REST co-localization to target AT/RT precisely. While there are no known interactions between the REST complex and SWI/SNF, there are interactions between REST’s primary co-factor CoREST and SWI/SNF. The CoREST complex consists of LSD1, HDAC1, and the scaffolding protein RCOR. We disrupted the CoREST complex with lentiviral knockdown of RCOR2 (kdRCOR2). KdRCOR2 reversed AT/RT’s stem cell-like state, driving neuronal differentiation and slowing AT/RT growth. Interestingly, kdRCOR1 had no impact on cell differentiation (microscopy and qRT-PCR) and had a modest impact on cell growth (MUSE cell viability assay). We next used Corin to pharmacologically target the CoREST complex in 3 AT/RT cell lines (CHLA05, CHLA06, and BT37). Corin is a bi-functional inhibitor of LSD1 and HDAC1/2, the component enzymes of the CoREST complex. Similar to kdRCOR2, Corin induced neuronal differentiation in AT/RT with increased adherence to plastic and new axonal development (microscopy). Cells also increased expression of neuronal differentiation markers identified by immunofluorescence (MAP2) and qRT-PCR (CEND1, and NEUROD2, t-test p&amp;lt;0.05), and decreased expression of stem cell markers (western blot SOX2 and LIN28). In addition, Corin reduced AT/RT cell viability and proliferation (MUSE Cell viability assay p&amp;lt;0.05; MUSE BrdU assay p&amp;lt;0.05) and increased apoptosis (MUSE Annexin V assay, p&amp;lt;0.05). In mice bearing orthotopic CHLA06 tumors, convection-enhanced delivery of a single dose of Corin increased H3K27me3 and H3K27ac, demonstrating that Corin engaged its target in vivo and induced apoptosis (western blot, PARP). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis. Targeting the CoREST complex with Corin is a promising new strategy that may translate into the clinical setting to help improve AT/RT’s dismal survival. Citation Format: Anupa Geethadevi, Marianne Collard, Nikhil Vaidya, Tyler Findlay, Kristen Malebranche, Charles Eberhart, Eric Raabe, Philip Cole, Rhoda Alani, Jeffrey Rubens. The CoREST complex as a novel target to disrupt AT/RT’s abnormal epigenetic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1141.

NONHSAG028908.3 sponges miR‑34a‑5p to promote growth of colorectal cancer via targeting ALDOA.

Colorectal cancer (CRC) is an aggressive tumor, whose development is considered to be modulated by certain long non‑coding RNAs (lncRNAs). Therefore, the aim of the present study was to investigate the regulatory mechanism of lncRNA NONHSAG028908.3 on CRC. Data from The Cancer Genome Atlas (TCGA) database revealed that NONHSAG028908.3 was increased in CRC tissues compared with normal tissues (P<0.001). The results of reverse transcription‑quantitative PCR indicated that NONHSAG028908.3 was upregulated in four types of CRC cells compared with that in NCM460, a normal colorectal cell line. MTT, BrdU, and flow cytometric assays were applied to evaluate CRC cell growth. The migratory and invasive abilities of CRC cells were detected using wound healing and Transwell assays. Silencing of NONHSAG028908.3 inhibited proliferation, migration, and invasion of CRC cells. A dual‑luciferase reporter assay demonstrated that NONHSAG028908.3 served as a sponge to combine with microRNA (miR)‑34a‑5p. MiR‑34a‑5p suppressed the aggressiveness of CRC cells. The effects induced by NONHSAG028908.3 knockdown were partly reversed by inhibition of miR‑34a‑5p. Furthermore, miR‑34a‑5p, a target of NONHSAG028908.3, modulated aldolase, fructose‑bisphosphate A (ALDOA) expression in a negative feedback manner. Suppression of NONHSAG028908.3 notably decreased ALDOA expression, which was rescued via silencing of miR‑34a‑5p. Moreover, suppression of ALDOA revealed the inhibitory action on CRC cell growth and migration. In summary, the data of the present study indicate that NONHSAG028908.3 may positively regulate ALDOA via sponging miR‑34a‑5p, thereby promoting malignant activities in CRC.

Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions.

Suprabasin (SBSN) is a secreted protein that is isolated as a novel gene expressed in differentiated keratinocytes in mice and humans. It induces various cellular processes such as proliferation, invasion, metastasis, migration, angiogenesis, apoptosis, therapy and immune resistance. The role of SBSN was investigated in oral squamous cell carcinoma (OSCC) under hypoxic conditions using the SAS, HSC‑3, and HSC‑4 cell lines. Hypoxia induced SBSN mRNA and protein expression in OSCC cells and normal human epidermal keratinocytes (NHEKs), and this was most prominent in SAS cells. The function of SBSN in SAS cells was analyzed using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT); 5‑bromo‑2'‑deoxyuridine (BrdU); cell cycle, caspase 3/7, invasion, migration, and tube formation assays; and gelatin zymography. Overexpression of SBSN decreased MTT activity, but the results of BrdU and cell cycle assays indicated upregulation of cell proliferation. Western blot analysis for cyclin‑related proteins indicated involvement of cyclin pathways. However, SBSN did not strongly suppress apoptosis and autophagy, as revealed by caspase 3/7 assay and western blotting for p62 and LC3. Additionally, SBSN increased cell invasion more under hypoxia than under normoxia, and this resulted from increased cell migration, not from matrix metalloprotease activity or epithelial‑mesenchymal transition. Furthermore, SBSN induced angiogenesis more strongly under hypoxia than under normoxia. Analysis using reverse transcription‑quantitative PCR showed that vascular endothelial growth factor (VEGF) mRNA was not altered by the knockdown or overexpression of SBSN VEGF, suggesting that VEGF is not located downstream of SBSN. These results demonstrated the importance of SBSN in the maintenance of survival and proliferation, invasion and angiogenesis of OSCC cells under hypoxia.

Effects and Mechanisms of Action of Preussin, a Marine Fungal Metabolite, against the Triple-Negative Breast Cancer Cell Line, MDA-MB-231, in 2D and 3D Cultures

Triple-negative breast cancer (TNBC) represents an aggressive subtype of breast cancer (BC) with a typically poorer prognosis than other subtypes of BC and limited therapeutic options. Therefore, new drugs would be particularly welcome to help treat TNBC. Preussin, isolated from the marine sponge-associated fungus, Aspergillus candidus, has shown the potential to reduce cell viability and proliferation as well as to induce cell death and cell cycle arrest in 2D cell culture models. However, studies that better mimic the tumors in vivo, such as 3D cell cultures, are needed. Here, we studied the effects of preussin in the MDA-MB-231 cell line, comparing 2D and 3D cell cultures, using ultrastructural analysis and the MTT, BrdU, annexin V-PI, comet (alkaline and FPG modified versions), and wound healing assays. Preussin was found to decrease cell viability, both in 2D and 3D cell cultures, in a dose-dependent manner, impair cell proliferation, and induce cell death, therefore excluding the hypothesis of genotoxic properties. The cellular impacts were reflected by ultrastructural alterations in both cell culture models. Preussin also significantly inhibited the migration of MDA-MB-231 cells. The new data expanded the knowledge on preussin actions while supporting other studies, highlighting its potential as a molecule or scaffold for the development of new anticancer drugs against TNBC.

Selective Modulation of the Keratoconic Stromal Microenvironment by FSH and LH

Keratoconus (KC) affects the corneal structure, with thinning and bulging outward into a conelike shape. Irregular astigmatism and decreased visual acuity appear during puberty and progress into the mid-30s, with unpredictable disease severity. The cause of KC is recognized as multifactorial, but remains poorly understood. Hormone imbalances are a significant modulator of the onset of KC. This study sought to investigate the role of gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in KC, using a three-dimensional, self-assembled matrix invitro model. Healthy corneal fibroblasts and human KC cells in the corneal stroma were isolated, cultured, and stimulated with stable vitamin C to promote extracellular matrix assembly. Cultures were further stimulated with 2.5 or 10 mIU/mL FSH and 5 or 35 mIU/mL LH. Samples were evaluated for cell proliferation and morphology via BrdU assay and imaging; protein expression was assessed via Western blot analysis. Proliferation was significantly greater in human KC cells compared to healthy corneal fibroblasts with LH stimulation, but no changes were found with FSH stimulation. Additionally, in sex hormone receptors, fibrotic markers, proteoglycans, and members of the gonadotropin signaling pathway were significantly changed, largely driven by exogenous LH. The impact of exogenous FSH/LH in the KC stromal microenvironment was demonstrated. These results highlight the need to further examine the role of FSH/LH in KC and in human corneal homeostasis.

Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth.

Regenerative therapy for tissues by mesenchymal stem cell (MSCs) transplantation has received much attention. The cluster of differentiation (CD)146 marker, a surface-antigen of stem cells, is crucial for angiogenic and osseous differentiation abilities. Bone regeneration is accelerated by the transplantation of CD146-positive deciduous dental pulp-derived mesenchymal stem cells contained in stem cells from human exfoliated deciduous teeth (SHED) into a living donor. However, the role of CD146 in SHED remains unclear. This study aimed to compare the effects of CD146 on cell proliferative and substrate metabolic abilities in a population of SHED. SHED was isolated from deciduous teeth, and flow cytometry was used to analyze the expression of MSCs markers. Cell sorting was performed to recover the CD146-positive cell population (CD146+) and CD146-negative cell population (CD146-). CD146 + SHED without cell sorting and CD146-SHED were examined and compared among three groups. To investigate the effect of CD146 on cell proliferation ability, an analysis of cell proliferation ability was performed using BrdU assay and MTS assay. The bone differentiation ability was evaluated using an alkaline phosphatase (ALP) stain after inducing bone differentiation, and the quality of ALP protein expressed was examined. We also performed Alizarin red staining and evaluated the calcified deposits. The gene expression of ALP, bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) was analyzed using a real-time polymerase chain reaction. There was no significant difference in cell proliferation among the three groups. The expression of ALP stain, Alizarin red stain, ALP, BMP-2, and OCN was the highest in the CD146+ group. CD146 + SHED had higher osteogenic differentiation potential compared with SHED and CD146-SHED. CD146 contained in SHED may be a valuable population of cells for bone regeneration therapy.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation.Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method.Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level.Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia.

Regenerative capacity of trophoblast stem cell-derived extracellular vesicles on mesenchymal stem cells

BackgroundHuman mesenchymal stem cells (MSCs) are therapeutic for clinical applications because of their excellent immunomodulatory and multiple lineage differentiation abilities at tissue injury sites. However, insufficient number of cells and lack of regenerative properties during in vitro expansion still limit the clinical applicability of MSC therapies. Here, we demonstrated a preconditioning strategy with trophoblast stem cell-derived extracellular vesicles (TSC-EVs) to boost the proliferation and regenerative capacity of MSCs.MethodsWe employed cell proliferation analyses such as CCK8 and BrdU assays to determine the proliferation-promoting role of TSC-EVs on MSCs. Osteogenic effects of TSC-EVs on MSCs were assessed by alkaline phosphatase (ALP) activity, calcium assays, and calvarial bone defect animal models. For skin regenerative effects, skin wound mice model was exploited to analyze wound-healing rate in this study, as well as immunofluorescence and histological staining evaluates. We also performed the small RNA profiling and RNA-sequencing analyzes to understand the cellular mechanism of TSC-EVs on MSCs.ResultsTSC-EVs significantly promoted MSC proliferation under xeno-free conditions and facilitated the therapeutic effects of MSCs, including osteogenesis, anti-senescence, and wound healing. Transcriptomic analysis also provided evidence that specific microRNAs in TSC-EVs and differentially expressed genes (DEGs) in TSC-EV-treated MSCs showed the possibility of TSC-EVs triggering the regenerative abilities of MSCs with cytokine interaction. Hence, we found that NGF/Akt signaling mediated the regenerative effects of TSC-EVs on MSCs as a particular cellular signaling pathway.ConclusionThe results of this study demonstrated the functional properties of TSC-EVs on MSCs for MSC-based therapeutic applications, suggesting that TSC-EVs may serve as a potential preconditioning source for MSC therapy in the clinical field of regenerative medicine.Graphical abstract

The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells.

The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.

Loganic acid regulates the transition between epithelial and mesenchymal-like phenotypes by alleviating MnSOD expression in hepatocellular carcinoma cells

AimsCancer metastasis is the major cause of cancer-related deaths. There are few prior studies reported on molecules targeting C-X-C chemokine receptor (CXCR) family and manganese superoxide dismutase (MnSOD). CXCRs are known to involve in angiogenesis, metastasis, cell survival and MnSOD is reported to be related in Epithelial–mesenchymal transition (EMT). Main methodsCell viability and cell proliferation were measured by MTT and BrdU assay. Protein expression level of CXCR4/7, MMP-2/9, MnSOD, and EMT markers were evaluated by Western blot analysis. mRNA levels of Snail and Occludin were analyzed by Real-time RT-qPCR. Expression of EMT markers in cells was observed by immunocytochemistry. Cell invasion and migrations were evaluated by wound healing assay and boyden chamber assay. Key findingsWe noticed that LGA abolished proliferation, invasive ability, and cellular migration. LGA down-regulated the protein levels of mesenchymal markers such as Twist, Snail, Fibronectin, and Vimentin in CXCL12-treated HCC cells. It also suppressed the gelatinolytic activity of MMP-9/2. The amplification of MnSOD increased EMT-like phenotypes but with LGA treatment, these phenotypes were markedly attenuated. The overexpression of MnSOD increased the ROS levels significantly but ROS levels were decreased upon exposure to LGA and deletion of MnSOD suppressed the levels of various mesenchymal proteins. SignificanceLGA could function as a novel anti-metastatic agent by suppressing metastasis and EMT process via attenuation of MnSOD expression in hepatocellular carcinoma cells.

P095 Newly discovered gut matrikines from Crohn’s disease intestinal tissue induce cell proliferation, activation and inflammatory response in intestinal myofibroblasts in vitro

Abstract Background The interaction between intestinal myofibroblasts (iMFBs) and extracellular matrix (ECM) changes contributes to Crohn’s disease (CD) fibrosis, but the exact mechanisms are not yet clear. Our group recently discovered a selection of matrix-derived peptides (matrikines) that specifically appear in the intestinal tissue of patients with CD fibrostenosis.1 Here, we evaluate the in vitro effect of 19 matrikines (with various bioactivity likelihood) on primary human intestinal myofibroblasts (IMFBs), key cells in CD fibrosis. Methods Primary human iMFBS (500K/mL, Passage 4, Novabiosis) were cultured in serum-free medium (SFM) (DMEM high glucose, 1% sodium pyruvate, 1% non-essential aminoacids, 1% Antibiotics-Antimycotics and 3mg Gentamicin), complete medium (SFM with 10% fetal bovine serum), PDGF-bb 10ng/mL or treated with each of the matrikines at 10, 50, 100 μg/mL for 24hours. The effect of each matrikine on cell proliferation, gene expression and migration was assessed by BrdU, qPCR and Transwell assays, respectively. RNA sequencing data was analysed using the GSEA software. Results The effect on iMFBs proliferation and viability was tested with each of the 19 matrikines at 3 different doses (Table 1). Among 9 most likely-bioactive matrikines (M1-M9) (Figure 1), M1, M4, M7, M8 and M9 induced the highest effect in cell proliferation and were selected for further experiments. Gene expression assays showed upregulation of ACTA2 with M1 or M7 and upregulation of COL1A1 with M1, M7 or M8, indicating activation of the iMFBs (Figure 2). Transwell assays also showed that M1, M4 and M8 induce iMFB cell migration. A total of 1948, 1024, 460, 570, and 2749 differentially expressed genes (DEGs) were identified in iMBFs treated with M1, M4, M7, M8 and M9 vs no treatment, showing enrichment in pathways related to “smooth muscle contraction”, “fibroblast growth factor”, “cell migration”, “collagen formation”, “elastin formation” and “ECM organization”; most of which were also enriched by complete medium and/or PDGFbb that served as positive controls. However, pathways related to “proinflammatory and profibrotic mediators” or “vascular permeability” were only enriched in iMFBs treated with M1, M4, M8 and M9 or M1 and M9, respectively (Figure 3). Conclusion A selection of newly discovered CD-specific matrikines induce activation of iMFBs as well as inflammatory response and endothelial changes, suggesting an important role and therefore their potential value as biomarkers in the progression of intestinal fibrostenosis.