3D Matrigel Research Articles

Abstract LB-014: Pirfenidone as a new potential therapy for malignant mesothelioma

Abstract The purpose of this study was to examine pirfenidone (PFD) as a potential new therapy for malignant pleural mesothelioma. PFD is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis (IPF). PFD reduces fibrous tissue formation e.g. by downregulating the production of fibroblasts and procollagens and inhibiting TGFß, TNFα and PDGF pathways. Since mesothelioma shares characteristics with IPF alongside with the activation of PI3K/Akt/mTOR, EGF, PDGF, HGF, VEGF, FGF and TGF-β pathways, we assessed the effect of PFD on mesothelioma cells in vitro and in vivo. In vitro studies were carried out using mesothelioma cell lines (JL-1, H2052, AB12) and primary mesothelioma cells. Cells treated with PFD and/or other agents (cisplatin, pemetrexed, MK-2206, PD98059, MG132) were subjected to cell viability assays, Transwell migration assays and 3D collagen or Matrigel growth and invasion assays. TGFβ/BMP, MAPK/Erk and Akt pathway activities were studied using luciferase reporter assays and human phospho-kinase array and validated with ELISA, RT-qPCR and Western blotting. For in vivo xenograft studies mice were injected subcutaneously with primary human mesothelioma cells expressing luciferase marker. Mice were treated with pirfenidone daily for 60 days and tumor growth was followed by bioluminescent imaging. Proliferation was studied by IHC. Differential gene expression in treated vs. untreated tumors was analyzed using RNA sequencing. We demonstrated that PFD treatment reduced significantly mesothelioma cell proliferation, migration and invasive growth in 3D in a concentration-dependent manner. The inhibitory effect of PFD increased when combined with cisplatin treatment in vitro. We also observed reduction of Akt and MAPK/Erk pathway activities and increase in BMP signaling in PFD treated tumor cells. Preliminary analyses of in vivo experiment revealed differential expression of multiple genes in pirfenidone treated vs. untreated tumors associated with a shift to more anti-fibrotic tumor microenvironment in PFD treated tumors. Expression profiles are currently under closer investigation. We identified pirfenidone as an inhibitor of mesothelioma invasive growth and showed that it has anti-fibrotic effects to tumor microenvironment. Since mesotheliomas are often inherently chemoresistant in fibrotic microenvironment, the use of pirfenidone could aid in overcoming chemoresistance by loosening ECM barrier for better drug penetration into the tumor tissue. Based on the current research we suggest that pirfenidone should be subjected to more intensive study as potential adjuvant-therapy for malignant mesotheliomas. Citation Format: Chang Li, Veronika Rezov, Emmi I. Joensuu, Ville Vartiainen, Mikko Rönty, Miao Yin, Marjukka Myllärniemi, Katri Koli. Pirfenidone as a new potential therapy for malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-014.

Abstract 3155: Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion

Abstract While Hippo pathway signaling undoubtedly plays an important role in cancer, the role of its effector YAP1 as an oncogene or tumor suppressor is context dependent. The precise conditions that support the tumor-promoting or tumor-suppressing roles of YAP1 remain largely undefined. While YAP1 can drive oncogenic programs through its nuclear function as a transcriptional coactivator of TEAD and other transcription factors, previous findings from our lab and others suggest that loss of YAP1 function can also have tumor promoting consequences. One well-characterized role of YAP1 is that of a nuclear relay of signals triggered by mechanical forces that lead to activation of Rho GTPases. Our new studies using a combination of genetic, biochemical, and microscopy techniques provide evidence that YAP1 is not only activated by Rho, but can also regulate Rho activation and the actin cytoskeleton. Knockdown of YAP1 in MCF10A immortalized mammary epithelial cells resulted in increased lamellipodia formation in culture as seen by time-lapse microscopy, and increased protrusive, invasive activity in 3D Matrigel culture. YAP1 knockdown also induced a scattering phenotype, characterized by cell dissociation from the leading edge of a wounded monolayer. While E-cadherin protein levels were unaffected by YAP1 knockdown, adherens junction maturation at cell-cell junctions was compromised. These morphological alterations were associated with decreased levels of active RhoA, as well as a decrease in downstream myosin light chain phosphorylation. Our results indicate loss of YAP1 decreases active RhoA, leading to reduced cell-cell adhesion and increased protrusive cellular behavior. Our findings raise the possibility that YAP1 is not only a Rho-sensor, but also mediates a feedforward regulatory loop to maintain Rho-regulated cytoskeletal structures, and that loss of YAP1 through genetic deletion in tumor cells could promote invasive behavior. Citation Format: Natalie M. Hendrick, Marcin P. Iwanicki, Carman Man-Chung Li, Duyen Amy Bui, Laura M. Selfors, Joan S. Brugge. Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3155.

PO-462 Investigating the potential anti-cancer properties of a novel src family kinase inhibitor in triple negative breast cancer

IntroductionThe Src Family Kinases (SFKs) are key mediators in various oncogenic pathways and are commonly activated in many forms of cancer. Of the nine members of SFKs, increasing reports suggest aberrant activation of Lyn and Fyn in breast cancer. Bioinformatics analysis reveals that Lyn and Fyn expression are strongly associated with the claudin-low TNBC patient group. Due to the current lack of targeted therapy for the TNBC subtype, it becomes worthwhile to investigate the potential of Lyn and Fyn as novel therapeutic targets. While preclinical studies involving current SFK inhibitors were promising, subsequent clinical trials have shown limited clinical benefits. In this project, we developed a novel in-house SFK inhibitor (Drug X) that specifically targets Lyn and Fyn kinases.Material and methodsPatient data analysis, Ligand-based computation, Proteome Profiler Array, in vivo mouse Pharmacokinetics and Pharmacodynamics studies, Bioinformatics analysis, Affymetrix Microarray, Secretome Analysis, Cell viability assays, Flow cytometry, Western blot, qPCR, Immunofluorescence, Transwell assays, 3D Matrigel Assays, and Tube-formation assay.Results and discussionsProteome Profiler Array results validated the ligand-based target prediction analyses of Drug X in vitro. Mouse studies show that Drug X is well-tolerated and exhibited significant tumour burden load reduction. Pharmacokinetics of Drug X was characterised. Overall, Drug X showed moderate systemic clearance, high volume of distribution and moderate oral bioavailability. Downstream functional studies were performed, and Drug X was found to induce apoptosis via the intrinsic pathway. Also, Drug X could down-regulate the mesenchymal properties of mesenchymal-like TNBC cell lines via the modulation of key EMT-related genes. Preliminary in vitro and in vivo rat aortic ring assay data also suggested that Drug X could harbour potent anti-angiogenic effects.ConclusionBased on in vitro and in vivo experiments, preliminary findings have supported that Drug X could potentially be a promising novel drug candidate for TNBC treatment and on-going efforts are being made to further elucidate the underlying mechanisms of Drug X in modulating the key oncogenic processes – namely from the angle of apoptosis, EMT and angiogenesis.

MiR-29c plays a suppressive role in breast cancer by targeting the TIMP3/STAT1/FOXO1 pathway

BackgroundmiR-29c has been associated with the progression of many cancers. However, the function and mechanism of miR-29c have not been well investigated in breast cancers.MethodsReal-time quantitative PCR was used to assess expression of miR-29c and DNMT3B mRNA. Western blot and immunochemistry were used to examine the expression of DNA methyltransferase 3B (DNMT3B) protein in breast cancer cells and tissues. The functional roles of miR-29c in breast cancer cells such as proliferation, migration, invasion, colony formation, and 3D growth were evaluated using MTT, transwell chambers, soft agar, and 3D Matrigel culture, respectively. In addition, the luciferase reporter assay was used to check if miR-29c binds the 3′UTR of DNMT3B. The effects of miR-29c on the DNMT3B/TIMP3/STAT1/FOXO1 pathway were also examined using Western blot and methyl-specific qPCR. The specific inhibitor of STAT1, fludarabine, was used to further check the mechanism of miR-29c function in breast cancer cells. Studies on cell functions were carried out in DNMT3B siRNA cell lines.ResultsThe expression of miR-29c was decreased with the progression of breast cancers and was closely associated with an overall survival rate of patients. Overexpression of miR-29c inhibited the proliferation, migration, invasion, colony formation, and growth in 3D Matrigel while knockdown of miR-29c promoted these processes in breast cancer cells. In addition, miR-29c was found to bind 3′UTR of DNMT3B and inhibits the expression of DNMT3B, which was elevated in breast cancers. Moreover, the protein level of TIMP3 was reduced whereas methylation of TIMP3 was increased in miR-29c knockdown cells compared to control. On the contrary, the protein level of TIMP3 was increased whereas methylation of TIMP3 was reduced in miR-29c-overexpressing cells compared to control. Knockdown of DNMT3B reduced the proliferation, migration, and invasion of breast cancer cell lines. Finally, our results showed that miR-29c exerted its function in breast cancers by regulating the TIMP3/STAT1/FOXO1 pathway.ConclusionThe results suggest that miR-29c plays a significant role in suppressing the progression of breast cancers and that miR-29c may be used as a biomarker of breast cancers.

Towards Resolving the Pro- and Anti-Tumor Effects of the Aryl Hydrocarbon Receptor.

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later, more lethal stages of some cancers when chronically activated by endogenous ligands. However, other studies have suggested that, under some circumstances, the AHR can oppose tumor aggression. Resolving this apparent contradiction is critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA, shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors) approaches were used to confirm the hypothesis that AHR inhibition reduces human cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden chambers), migration (scratch wound assay) and metastasis (human cancer cell xenografts in zebrafish). Furthermore, these assays were used for a head-to-head comparison between AHR antagonists and agonists. AHR inhibition or knockdown/knockout consistently reduced human ER−/PR−/Her2− and inflammatory breast cancer cell invasion, migration, and metastasis. This was associated with a decrease in invasion-associated genes (e.g., Fibronectin, VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously associated with decreased tumor aggression. Paradoxically, AHR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3′-diindolylmethane) similarly inhibited irregular colony formation in Matrigel and blocked metastasis in vivo but accelerated migration. These data demonstrate the complexity of modulating AHR activity in cancer while suggesting that AHR inhibitors, and, under some circumstances, AHR agonists, may be useful as cancer therapeutics.

The effects of restricted glycolysis on stem-cell like characteristics of breast cancer cells.

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells.

Overexpressing TPTE2 (TPIP), a homolog of the human tumor suppressor gene PTEN, rescues the abnormal phenotype of the PTEN-/- mutant.

One possible approach to normalize mutant cells that are metastatic and tumorigenic, is to upregulate a functionally similar homolog of the mutated gene. Here we have explored this hypothesis by generating an overexpressor of TPTE2 (TPIP), a homolog of PTEN, in PTEN−/− mutants, the latter generated by targeted mutagenesis of a human epithelial cell line. Overexpression of TPTE2 normalized phenotypic changes associated with the PTEN mutation. The PTEN−/−-associated changes rescued by overexpressing TPTE2 included 1) accelerated wound healing in the presence or absence of added growth factors (GFs), 2) increased division rates on a 2D substrate in the presence of GFs, 3) adhesion and viability on a 2D substrate in the absence of GFs, 4) viability in a 3D Matrigel model in the absence of GFs and substrate adhesion 5) loss of apoptosis-associated annexin V cell surface binding sites. The results justify further exploration into the possibility that upregulating TPTE2 by a drug may reverse metastatic and tumorigenic phenotypes mediated in part by a mutation in PTEN. This strategy may also be applicable to other tumorigenic mutations in which a homolog to the mutated gene is present and can substitute functionally.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330–1057) of Solo binds to the C-terminal region (1451–1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

A novel monoclonal antibody targeting a novel epitope of VE‑cadherin inhibits vasculogenic mimicry of lung cancer cells.

Vascular endothelial cadherin (VE‑cadherin) was first found in vascular endothelial cells to maintain normal vascular structures and regulate endothelial cell permeability by homology adhesion. New evidence indicates that certain invasive tumor cells also express VE‑cadherin, which is involved in vasculogenic mimicry to provide a blood supply required for tumor growth and metastasis. EC1 and EC3 domains of VE‑cadherin were reported to be important for intercellular homology adhesion. In the present study, a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of VE‑cadherin was generated and the binding epitope was identified as peptide LDREVVPWYNLTVEA in the EC4 domain. This antibody inhibited proliferation and capillary-like structure formation of lung cancer Glc‑82 cells in 3D Matrigel culture invitro. This effect was mediated by the inhibition of AKT phosphorylation. Our results suggested that the EC4 domain participates in VE‑cadherin clustering and the antibody targeting the EC4 domain of VE‑cadherin may be a promising anti‑vasculogenic mimicry agent for cancer treatment.

Conditional reprogrammed human limbal epithelial cells represent a novel in vitro cell model for drug responses

In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.

Cultivation of Hepatic Stellate Cells in 3D ECM to Simulate Physiological and Fibrotic Conditions

BackgroundHepatic stellate cells, the major providers of hepatic retinoids and extracellular matrix (ECM), reside in the space of Disse filled with loose basement like ECM, and flanked by hepatocytes and sinusoidal endothelial cells. Upon hepatic injury, stellate cells are activated to express multiple matrix metalloproteinases (MMPs) and to mobilize retinoids for wound healing. In chronic injury, the quiescent stellate cells undergo trans‐differentiation into myofibroblast like cells, marked by loss of retinoids, expression interstitial ECM as well as smooth muscle actin (alpha‐SMA), and gain of contractibility. Although knowledge has been evolved substantially, two fundamental questions remain largely unknown as to how, in normal liver, HSCs maintain quiescence, and how, by injury, HSCs are activated within the ECM milieu.ResultsTo recapitulate the sub‐sinusoid microenvironment we embedded rat primary stellate cells in three‐dimensional ECM (3D ECM). Cultured on plastic, the stellate cells activated spontaneously, showing loss of vitamin A droplets and expression of alpha‐SMA. However, in 3D Matrigel, stellate cells retained retinoid droplets, in association with elevated level of lecithin retinoid acyltransferase (LRAT), a key enzyme for retinol esterification. In 3D ECM, stellate cells were arrested at G0/G1 phase, while on plastic the cells underwent cell cycling and stopped at G2/M phase. We then reconstituted the microenvironment of sinusoids by introducing Kupffer cells, the major hepatic inflammatory cells, into 3D ECM with HSCs. Activated Kupffer cells triggered subsequent activation of stellate cells, evidenced by expression of multiple MMPs, buildup of stress fibers, and loss of retinoids. The Kupffer cell‐mediated activation of stellate cells was blocked by interleukin‐1 receptor antagonist, demonstrating interleukin‐1 as a major injury signal from Kupffer cells conducting stellate cell activation and trans‐differentiation. Furthermore, within 3D ECM, but not on the plastic, interleukin‐1 signal was greatly amplified by stellate cells in an autocrine manner, which may exacerbate liver injury by expressing MMPs in the space of Disse. The myofibroblastic cells derived from the 3D ECM culture were contractile in response to endothelin.ConclusionThe 3D ECM culture provides a model to simulate key physiological functions of stellate cells in normal liver, and also to recapitulate patho‐physiological responses in liver injury. The method may also be used in cell‐based drug screening to identify components targeting liver failure and fibrosis.Support or Funding InformationNational Institutes of Health (NIH) Grants R01s, DK069418‐05 and AR051558, and the Natural Science Foundation of ChinaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P5-09-03: Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D matrigel matrix environment

Abstract Within the same tumor microenvironment phenotypic and functional heterogeneity arise among cancer cells as a consequence of genetic change, environmental differences, and reversible epigenetic changes in cellular properties. However, it is thought that cancer stem cells are drivers of drug resistance and metastasis. Individual tumor cells growing in culture also display heterogeneity in their intrinsic ability to progress and metastasize. It remains unclear whether intrinsic and extrinsic heterogeneity contribute to the emergence of distinct progressive phenotypes that contribute more to cancer stem cells to disseminate. To this study, we have examined the ability of matrigel to stimulate complex cell behavior that is a consequence of its heterogeneous composition. We have observed that mixing matrigel with metastatic triple negative breast cancer MDA-MB-231 cells, which mimic in vivo tumor microenvironment, around 80-90% cells created network like structures resembling a clinical phenotype known as vascular mimicry (VM) and around 10-20% cells form spheroids. BT549 another triple-negative breast cancer cells also responded similarly, forming cellular networks and spheroids when mixing with matrigel. Since CD44, a marker of epithelial-to-mesenchymal transition has shown enhances tumor aggressiveness by promoting cell plasticity, we decided to examine CD44 expression in MDA-MB-231 cells grown in 3D matrigel matrix environment. We have observed that VM forming cells are showing CD44 positive staining compared to spheroid forming cells which showed negative staining in formaldehyde-fixed 3D matrigel culture of MDA-MB-231 cells, while both group of cells stained positive for VEGFC. Next, we sought to isolate two phenotypically different groups of cells (VM and Tumorsphere forming cells) from the 3D matrigel culture by using microscopic suction procedure for gene expression analysis by qPCR. Our gene expression data suggested that VM forming cells have more expression of VM inducer genes such as CD44 and HIF1α compared to spheroid forming cells isolated from the same 3D matrigel culture of MDA-MB-231 cells. Spheroid forming cells express significant level of endothelial cell adhesion marker, CD31 compared to VM forming cells. Epigenetic mechanisms mediated suppression of tumor suppressors or anti-angiogenesis marker genes are hall mark of VM formation and cancer progression, we examine whether re-expression of those genes with Entinostat (MS-275), a selective inhibitor of class I histone deacetylase (HDAC) can abolish VM structures in 3D matrigel cell culture. Data suggested that MS-275 treatment in 3D culture drastically reduced VM structure by epigenetically re-expression of anti-angiogenic genes; SERPINF1, THBS1 and THBS2 and tumor suppressor genes; APC, PTEN and p21. While MS-275 treatment also downregulated Vimentin, VEGF-A and CD44. Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional response in 3D matrigel environment. Epigenetically re-expression of anti-angiogenic gene expression could be a mechanism to control VM formation in triple-negative breast cancer cells. Citation Format: Maiti A, Takabe K, Hait NC. Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D matrigel matrix environment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-09-03.

Isolation and Expansion of Multipotent Progenitors from Human Trabecular Meshwork

To expand multi-potent progenitors from human trabecular meshwork (TM), we have created a new optimized method on two-dimensional (2D) followed by three–dimensional (3D) Matrigel in modified embryonic stem cell medium supplemented with 5% fetal bovine serum (MESCM + 5% FBS). The expanded TM cells were small cuboidal cells expressing TM markers such as AQP1, MGP, CHI3L1, and AnkG, embryonic stem cell (ESC) markers such as Oct4, Sox2, Nanog, and ABCG2, and neural crest (NC) markers such as p75NTR, FOXD3, Sox9, Sox10, and MSX1. Although expanded cells lost expression of these markers after passage, the cells regained the markers when Passage 2 cells were seeded on 3D Matrigel through activation of canonical BMP signaling. Such restored progenitors could differentiate into corneal endothelial cells, adipocytes, and chondrocytes but not keratocytes or osteocytes. Therefore, we have concluded that human TM harbors multipotent progenitors that can be effectively isolated and expanded using 2D Matrigel in MESCM + 5% FBS. This unique in vitro model system can be used to understand how TM is altered in glaucoma and whether such TM progenitor cells might one day be used for treating glaucoma or corneal endothelial dysfunction.

Utilization of Reactive Oxygen Species Targeted Therapy to Prolong the Efficacy of BRAF Inhibitors in Melanoma.

BRAF mutations occur in about 50% of melanoma patients. FDA approved BRAF and MEK inhibitors have improved the prognosis of patients with BRAF mutations. However, all responders develop resistance typically within one year of treatment. Recent observations demonstrate that BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells. A100, identified from a library screen, is a ROS-activated prodrug that self-cyclizes into a stable bicyclic ring and causes DNA double strand breaks. We proposed to examine if ROS activated therapy will inhibit tumor growth and evade resistance to BRAF inhibitors. In this study, the BRAF inhibitor dabrafenib was used to generate resistant cell lines (A375DR, SK-MEL-24DR and WM-115DR). Flow cytometry experiments showed that ROS levels are increased in these dabrafenib-resistant cells as compared to parental cells, assessed by both the H2DCFDA and MitoSOX assays. Furthermore, we observed that resistant cells had increased levels of the mitochondrial enzymes SOD2 and PRDX1, which function to reduce ROS levels in the mitochondria. We found that A100 sensitized the resistant melanoma cells to dabrafenib and induced DNA damage. Co-treatment of both A100 and dabrafenib significantly suppressed in vitro cell proliferation and three- dimensional (3D) matrigel growth. This study suggests that the combination of A100 with a BRAF inhibitor could be a potential strategy to treat melanoma patients with BRAF mutations.

Apoptotic Effects of Temozolomide and Naturopathic Agents upon Glioblastoma Cells

Temozolomide (TMZ), thymoquinone (TMQ), epigallocatechin gallate (EPIGAL) and staurosporine (STAURO) were used as apoptotic inducing agents acting upon the U87-MG (ATCC, HTB15), LN18 (ATCC, CRL2610) and U118-MG (ATCC, HTB14) glioblastoma multiforme (GBM) cell lines. TMZ is the current drug of choice for primary treatment and adjuvant therapy for recurrent GBM. TMQ and EPIGAL are naturopathic agents, while STAURO is a well-studied apoptotic-inducing agent. The degree and time course of apoptosis were measured by flow cytometry techniques capable of detecting changes in mitochondrial function using the fluorescent dye MitoTracker Deep Red. Phosphatidylserine exposure and plasma membrane permeability were detected simultaneously using violet fluorescent reactive dye (VFRD) in combination with AnnexinV-488. The apoptotic effectiveness of the inducing agents TMZ, TMQ, EPIGAL and STAURO were compared with their ability to inhibit invasiveness and degrade Class I major histocompatibility complex (MHC) determinants. Invasiveness was measured in vitro by the 3D matrigel spheroid invasion assay. The density of the class 1 MHC determinants was measured by flow cytometry. Although TMZ is widely used for the chemotherapeutic treatment of GBM, it was determined that the time course for TMZ induced apoptosis was slower than those of TMQ, EPIGAL and STAURO. Unexpectedly, TMZ was ineffective in its ability to inhibit in vitro invasiveness and did not degrade the class I MHC determinants as effectively as the other apoptotic inducing agents. The findings raise the question of whether in vitro assays of apoptosis and invasiveness are the best measures of the effectiveness of chemotherapeutic agents for the primary treatment and adjuvant therapy of recurrent GBM. The findings also point to the in vivo complexity of the efficacy of chemotherapeutic agents whereby preserving the components of natural and acquired immune mechanisms may be more important than the rapid apoptotic effects of the chemotherapeutic agent.

Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models

Survivin, a member of inhibitor of apoptosis (IAP) protein family, is a multifunctional protein expressed in most cancers. In addition to inhibition of apoptosis, it regulates proliferation and promotes migration. Its presence and function in cells is strongly regulated via transcription factors, intracellular localization, and degradation. We analyzed the presence of survivin at protein level in various culture environments and under activation of Src tyrosine kinase in epithelial canine kidney MDCK cells in order to elucidate factors controlling survivin ‘lifespan’. We used untransformed and temperature sensitive ts-Src MDCK cells as a model and forced them to grow in suspension (1D), in 2D on hard and soft surfaces and in soft 3D Matrigel environment with or without EGTA. In addition, we tested the effect of stressful conditions by cultivating the cells in the presence of an anti-cancer drug and a generator of reactive oxygen species (ROS), piperlongumine (PL) with or without an antioxidant, N-acetylcysteine (NAC). We could confirm that inhibition of apoptosis and simultaneous downregulation of survivin in MDCK cells required both intact cell–cell junctions, trans-interactions of E-cadherin and soft 3D matrix environment. In ts-Src-transformed MDCK cells, survivin was upregulated as soon as the cell–cell junctions were disintegrated. ROS generation with PL-induced cell death of ts-Src MDCK cells concomitantly with survivin downregulation. NAC rescued the ts-Src MDCK cells from ROS-induced apoptosis without upregulation of survivin resulting in a situation resembling untransformed MDCK cells in 3D environment and E-cadherin delineating the lateral cell walls.

Hepatogenic Differentiation Capacity of Human Wharton's Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

BackgroundHuman Wharton’s jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development.Materials and MethodsHWJMSCs were extracted from human Wharton’s jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry.ResultsAccording to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19.ConclusionsCo-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Structural determinant of BST-2-mediated regulation of breast cancer cell motility: a role for cytoplasmic tail tyrosine residues.

There is now irrefutable evidence that overexpression of the innate immunity protein―BST-2, in breast cancer cells is implicated in tumor growth and progression. The cellular mechanisms that control BST-2-mediated effect in tumor progression involve enhancement of cancer cell motility―migration/invasion. However, the distinct structural elements of BST-2 that mediate breast cancer cell motility remain unknown. Here, we used various motility assays and different variants of BST-2 to examine the cellular and structural mechanisms controlling BST-2-mediated cell motility. We show that BST-2 silencing in various cancer cell lines inhibits cell motility. Restoration of BST-2 expression using construct expressing wild type BST-2 rescues cell motility. Mutational analysis identifies the cytoplasmic tail of BST-2 as a novel regulator of cancer cell motility, because cell motility was significantly abrogated by substitution of the BST-2 cytoplasmic tail tyrosine residues to alanine residues. Furthermore, in a spheroid invasion model, BST-2-expressing tumor spheroids are highly invasive inside 3D Matrigel matrices. In this model, the spreading distance of BST-2-expressing spheroids was significantly higher than that of BST-2-suppressed spheroids. Collectively, our data reveal that i) BST-2-expressing breast cancer cells in spheroids are more motile than their BST-2-supressed counterparts; ii) BST-2 cytoplasmic tail regulates non-proteolytic (migration) and proteolytic (invasion) mechanisms of breast cancer cell motility; and iii) replacement of the tyrosine residues at positions 6 and 8 in the cytoplasmic tail of BST-2 with alanine residues inhibits cell motility.

Estradiol promotes epithelial-to-mesenchymal transition in human benign prostatic epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is involved in pathogenesis of human benign prostatic hyperplasia (BPH). Estrogenic signaling pathways may stimulate the induction of EMT. However, the details of estradiol (E2) and estrogen receptors (ERs) effects on EMT, as well as E2-induced modulation of benign prostatic epithelial cell phenotype in vitro have not been completely clarified. The effects of E2 on EMT markers and cytokeratins (CKs) expression were evaluated in benign epithelial cell lines BPH-1 and RWPE-1, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model using hanging drop technique or 3D Matrigel model. ER antagonist, ICI182,780, was used to confirm the regulatory effects of E2 on EMT and phenotypic modulation. In 3D culture, immunohistochemical stainings were performed to detect the specific phenotype of cells that underwent EMT in acinar-like spheroids formed by RWPE-1. To illustrate the exact function of ERs in E2-induced EMT and phenotypic modulation, specific short interfering RNAs (siRNAs), and agonists were used to knockdown or activate individual ERs, respectively. E2-induced EMT was observed both in 2D and 3D culture, with related regulation of EMT markers expression at both mRNA and protein level. In addition, E2 down-regulated luminal cell type markers CK18 and CK8 and up-regulated basal cell type markers CK5 and CK14. E2 also increased intermediate type markers CK15 and CK17, while it attenuated CK19 in 3D culture. ICI182,780 blocked E2-induced EMT and cell phenotypic switching. In 3D Matrigel culture, Vimentin was co-expressed with ERα and CK17, as well as with SMemb, which is related to cell status switching and proliferation. Knockdown of ERα but not GPR30 inhibited EMT, while ERβ knockdown facilitated EMT process. Knockdown of ERα blocked E2-induced EMT both in RWPE-1 and BPH-1. MRNA expression of EMT markers was stimulated by ERα-specific agonist PPT and inhibited by ERβ-specific agonist DPN. Estrogenic effect mediated by ERα can promote EMT. E2 is also an inductive factor of cell phenotypic switching. Cell type modulation is associated with E2-induced EMT in benign prostatic epithelial cells. Taken together the results support a contribution of estrogens to the pathogenesis of BPH in elderly men.

P-REX1 amplification promotes progression of cutaneous melanoma via the PAK1/P38/MMP-2 pathway

P-REX1 (PIP3-dependent Rac exchange factor-1) is a guanine nucleotide exchange factor that activates Rac by catalyzing exchange of GDP for GTP bound to Rac. Aberrant up-regulation of P-REX1 expression has a role in metastasis however, copy number (CN) and function of P-REX1 in cutaneous melanoma are unclear. To explore the role of P-REX1 in melanoma, SNP 6.0 and Exon 1.0 ST microarrays were assessed. There was a higher CN (2.82-fold change) of P-REX1 in melanoma cells than in melanocytes, and P-REX1 expression was significantly correlated with P-REX1 CN. When P-REX1 was knocked down in cells by P-REX1 shRNA, proliferation, colony formation, 3D matrigel growth, and migration/invasiveness were inhibited. Loss of P-REX1 inhibited cell proliferation by inhibiting cyclin D1, blocking cell cycle, and increased cell apoptosis by reducing expression of the protein survivin. Knockdown of P-REX1 expression inhibited cell migration/invasiveness by disrupting P-REX1/RAC1/PAK1/p38/MMP-2 pathway. Assessment of patient tumors and disease outcome demonstrated lower distant metastasis-free survival among AJCC stage I/II/III patients with high P-REX1 expression compared to patients with low P-REX1 expression. These results suggest P-REX1 plays an important role in tumor progression and a potential theranostic target.