Significant Change In Proliferation Research Articles

Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages.

Mesenchymal stem cells (MSCs) have been extensively studied for therapeutic potential, due to their regenerative and immunomodulatory properties. Serial passage and stress factors may affect the biological characteristics of MSCs, but the details of these effects have not been recognized yet. To investigate the effects of stress factors (high glucose and severe hypoxia) on the biological characteristics of MSCs at different passages, in order to optimize the therapeutic applications of MSCs. In this study, we investigated the impact of two stress conditions; severe hypoxia and high glucose on human adipose-tissue derived MSCs (hAD-MSCs) at passages 6 (P6), P8, and P10. Proliferation, senescence and apoptosis were evaluated measuring WST-1, senescence-associated beta-galactosidase, and annexin V, respectively. Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control, while the extent of senescence did not change significantly under stress conditions. At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6. Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage, while no change in apoptosis was observed. On the other hand, MSCs cultured under hypoxia showed decreased senescence, increased apoptosis and no significant change in proliferation when compared to the same conditions at P8. These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages, and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.

The anticancer potential of chemical constituents of Moringa oleifera targeting CDK-2 inhibition in estrogen receptor positive breast cancer using in-silico and in vitro approches

Most of the breast cancers are estrogen receptor-positive recurring with a steady rate of up to 20 years dysregulating the normal cell cycle. Dinaciclib is still in clinical trials and considered as a research drug against such cancers targeting CDK2.The major goal of this study was to identify the potential inhibitors of CDK-2 present in Moringa oleifera for treating hormonal receptor positive breast cancers. For this purpose, in silico techniques; molecular docking, MM-GBSA and molecular dynamics simulations were employed to screen Moringa oleifera compounds and their anticancer potential was determined against CDK-2 protein targets. Among 36 compounds of Moringa oleifera reported in literature, chlorogenic acid (1), quercetin (2), ellagic acid (3), niazirin (4), and kaempferol (5) showed good affinity with the target. The interaction of the compounds was visualized using PYMOL software. The profiles of absorption, distribution, metabolism, excretion (ADME) and toxicity were determined using SWISS and ProTox II webservers. The MTT assay was performed in-vitro using MCF-7 cancer cell lines to validate the anticancer potential of Moringa oleifera leaf extract.MTT assay results revealed no significant change in proliferation of Mcf-7 cells following 24 h treatment with fraction A (petroleum ether). However, significant antiproliferative effect was observed at 200 µg/mL dose of fraction B (ethyl acetate) and cell viability was reduced to 40%.In conclusion, the data suggested that all the compounds with highest negative docking score than the reference could be the potential candidates for cyclin dependent kinase-2 (CDK-2) inhibition while ellagic acid, chlorogenic acid and quercetin being the most stable and potent inhibitors to treat estrogen receptor positive breast cancer targeting CDK-2. Moreover, the data suggested that further investigation is required to determine the optimum dose for significant antiproliferative effects using in-vivo models to validate our findings of in-silico analysis.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 & SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change >2 was observed in the DHT treated cells (p< 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p< 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change>3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p< 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p< 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-02.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Pathogenic RUNX1 C-Terminal Truncation Mutations Contribute to Differentiation Block and Result in Unique Changes in Transcription and Chromatin Organization

The transcription factor RUNX1 is one of the most frequently mutated genes in hematological disorders, including familial platelet disorder (FPD), predisposition to myeloid dysplastic syndromes (MDS) and acute myeloid leukemia (AML). In addition to a large number of mutations in the N-terminal DNA binding domain (RHD) of RUNX1, 27% of all hematopoietic mutations lie within the C-terminal region beyond the RHD. Previous studies have mainly focused on loss of function RHD mutations while C-terminal mutations are not well characterized and lack "hotspot" mutations. The majority of mutations in the C-terminal region are frameshift and nonsense mutations rather than missense. Some of these mutations are thought to be subject to degradation by nonsense-mediated decay (NMD), emulating loss of RUNX1. Here we demonstrate that RUNX1 C-terminal truncation mutations represent an alternative means of RUNX1 pathogenesis beyond loss of RUNX1 and RHD mutations. To examine the prevalence of RUNX1 C-terminal mutations we first analyzed publicly available data (COSMIC - Sanger Institute) and identified 247 RUNX1 nonsense/frameshift mutations C-terminal of the RHD in patients with blood disorders. Importantly, 76% of these mutations are predicted to escape Nonsense Mediated Decay (NMD) and produce protein products. To study this class of RUNX1 mutation, we introduced the pathogenic RUNX1R320* mutation into the endogenous RUNX1 locus of the human blood cell line K562 using CRISPR-Cas9. We experimentally validated that the RUNX1R320* transcript is not subject to NMD and is expressed at significantly higher levels than the wild-type at both the RNA and protein level. The resulting truncated protein retains the DNA binding RHD but lacks C-terminal domains for binding to other cofactors. To identify biological changes caused by the RUNX1R320* mutation we examined effects on proliferation, differentiation, and DNA damage. Although no significant change in proliferation was observed, megakaryocytic differentiation was impaired. After TPA treatment RUNX1R320* cells failed to fully differentiate and retained high levels of the erythroid marker CD235a. Furthermore, RUNX1R320* showed sensitivity to DNA damage after treatment with etoposide while cell death via apoptosis was not significantly affected. Together these data suggest that C-terminal truncation RUNX1 contributes to differentiation block and an increase in DNA damage sensitivity while evading apoptotic cell signaling pathways. As RUNX1 functions as a master transcriptional regulator of hematopoiesis, we examined how RUNX1 truncations alter transcription and if the effects emulate RUNX1 loss of function mutations by performing RNA-seq with parental, RUNX1 knockdown, and RUNX1R320* knock-in cells. We first identified 2880 significantly differentially expressed genes (DEGs) between wild-type and RUNX1R320*. When compared to RUNX1 knockdown data, we found that the majority of RUNX1R320* DEGs (2052) were unique to RUNX1R320*. This demonstrates that RUNX1R320* results in different transcriptional perturbations than the loss of RUNX1. We next asked whether changes in chromatin confirmation attributed to the observed transcriptional alterations. To study this the newly developed technology Global RNA Interactions with DNA by deep sequencing (GRID-seq) was performed on wild-type and RUNX1R320* cells. Combining these data with H3K27ac and H3K4me3 ChIP-seq data to identify active enhancers and promoters, respectively, we detected 2,891 significantly altered enhancer-promoter (E-P) pairs. Focusing on DEGs with GRID-seq detected enhancer RNA (eRNA), we conducted Gene Ontology (GO) analysis and found enrichment in pathways of chronic myeloid leukemia, regulation of hematopoietic stem cell differentiation, and cell division, revealing the effects of RUNX1R320* on key hematopoietic and myeloid pathways. In summary, we show that RUNX1 C-terminal truncation mutants can escape NMD, contribute to differentiation block, and increase sensitivity to DNA damage. Furthermore, RUNX1R320* affects transcription in a manner distinct from RUNX1 loss. In our GRID-seq analysis, we identify changes in enhancer-promoter interactions that affect key hematological pathways. Our findings demonstrate that pathogenic RUNX1 C-terminal mutations disrupt hematopoietic cell function, transcription, and chromatin in a distinctive fashion.

Lamin A/C Downregulation Alters Prostate Cancer Cellular Phenotypes

Nuclear lamina proteins are associated with the structural stability of cells and the anchoring and localization of heterochromatin. The downregulation of lamin A/C has been shown to contribute to cellular deformity, genome instability, and affect the ability of cells to differentiate. Therefore, evaluating lamins in prostate cancer progression may uncover important mechanisms for coordinated regulation of cellular processes, thereby highlighting potential vulnerabilities that may be targeted therapeutically to alter the clinical course of the disease. Previous multiplex fluorescent immunolabeling experiments showed a substantial fraction of prostate cancers displaying dramatically diminished lamin A/C expression in the cancer cells compared to the benign prostate epithelial (luminal and basal) cell populations. In this study, we investigate how downregulation of lamin A/C directly affects cellular characteristics associated with metastasis. By knocking down lamin A/C expression (~50%) with siRNAs targeting lamin A/C, we tested how the modulation of lamin A/C directly affected cell proliferation, clonogenic survival, and cell invasion in three prostate cancer cell lines (PC3, DU145, and CWR22Rv1). Compared to a scrambled siRNA control, all three cell lines showed no significant change in proliferation and clonogenic survival when lamin A/C was downregulated. While PC3 and DU145 showed no significant change in invasion potential, lamin A/C knockdown in CWR22Rv1 resulted in a decreased invasion potential of 22.8% compared to controls. We are conducting further experiments to fully elucidate the effects of lamin A/C downregulation on these metastasis‐relevant cellular properties. Overall, these findings will help to further understand the significance of alterations in the nuclear lamina protein's involvement in metastatic progression, and to further validate lamin dysregulation measurements as a potential biomarker to predict clinical outcomes in prostate cancer.

The influence of oxygen deprivation and donor age on the effect of statins on human mesenchymal stromal cells

To date, no study evaluated the effect of oxygen deprivation together with statins pretreatment on human mesenchymal stromal cells (MSCs). The aim of our study was to establish the influence of atorvastatin and rosuvastatin on MSC proliferation and cytotoxicity in different oxygenic conditions. Human MSCs isolated from the bone marrow (n = 12) were incubated with statins. The proliferation rate and cytotoxic effect were evaluated in normoxic (21 %O2) and hypoxic (2%O2) conditions, also in relation to donor age. The treatment with atorvastatin was associated with significantly higher proliferation rate compared to control, both in hypoxic (19 % median increase) and normoxic conditions (20 %), p = 0.02 and p = 0.04, respectively. Atorvastatin had no significant cytotoxic effect on MSCs. Treatment with rosuvastatin in hypoxia resulted in significantly higher proliferation rate (15 %, p = 0.02) comparing to control with no significant cytotoxicity. In atmospheric oxygen concentration, rosuvastatin was associated with no significant change in proliferation and higher cytotoxicity compared to untreated control (p = 0.042 and p = 0.015, for 0.04 μM and 1 μM solutions respectively). There were no differences in the effect of statins on MSC from young donors vs. aged donors. These results suggest that statins could support MSC-based therapy of acute myocardial infarction.

The estrogen receptor-alpha S118P variant does not affect breast cancer incidence or response to endocrine therapies.

Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

Extracellular Vesicles (EVs) Shape the Leukemic Microenvironment

BackgroundExtracellular Vesicles (EVs) compose a naturally occurring, heterogeneous group of membrane-bound, nano-sized particles shed by all cells. Depending on cellular type, physiological state, and mode of secretion some harbor potent regenerative properties while others have the propensity to induce disease. Human bone marrow mesenchymal stem cell (MSC)-derived EVs harbor regenerative potential. Our own studies have shown MSC-EVs are able to mitigate radiation damage to bone marrow, and to reverse the malignant phenotype in prostate and colorectal cancer. On the contrary, EVs isolated from neoplastic cells induce a neoplastic phenotype in non-cancerous cells. Leukemic EVs also potentiate the phenotypic change of healthy MSCs into cancer associated fibroblasts. The role of EVs within the leukemic microenvironment may provide insight for therapeutic advances. We hypothesize that EVs derived from normal MSCs inhibit the growth of nascent acute myelogenous leukemia (AML), and that the predominant EV population changes during leukemia progression. Neural network machine learning will allow us to capture these changes in order to build predictive models.MethodsKasumi AML cells lines were seeded with various concentrations of MSC-derived EVs. Vesicles were isolated using an established differential centrifugation technique, and co-cultured with Kasumi. To study cellular proliferation we employed a fluorescence-based method for quantifying viable cells (CyQuant). We also investigated the modes of death (apoptosis vs necrosis) EVs may induce on AML via a three die fluorescent system. Fluorescence intensities were normalized to control wells containing non-EV treated cells alone.Our neural network achieved 90.16% classification accuracy with cell culture data, and was tasked to classify the similarity of patient samples to the AML-derived EVs.ResultsProliferation of AML cells after one day of co-culture with 2.6E8 &1.3E10 MSC-EVs respectively was inhibited in a dose dependent manner: with 2.6E8 EVs leading to ~ 15% reduction in growth, and 1.3E10 EVs leading to ~60% reduction when normalized to non-EV treated controls.3 days of co-culture with similar doses resulted in ~40% and ~80% reduction in proliferation when normalized to control.At day 6 of co-culture growth was inhibited by ~80% at both EV concentrations.At multiple time points hMSC-EV treated AML cells showed a significantly higher proportion of apoptosis. Cellular necrosis was negligible.There was no statistically significant change in proliferation of MSC exposed to MSC-derived EVs when compared to non-EV treated controls.There was also no statistically significant change in proliferation of AML cells exposed to AML-derived EVs.Samples of AML, Chronic Myelomonocytic Leukemia, and Multiple Myeloma entered into our machine alogrithm were calculated to be 100%, 100%, and 66%, respectively, similar to the AML-derived EVs.Summary/ConclusionMSC-derived EVs inhibits the proliferation of the AML cell line in vitro via an apoptotic mechanism. This effect is seen as early as one day of co-culture and persists out to three, and six days, implicating an miRNA-mediated mechanism that has been discussed in previous works. We have also shown that when cells are exposed to their own EV there is no change or (in the case of leukemia) a statistically insignificant increase in proliferation. We feel this is perhaps a model of how a normal marrow works to suppress early cancer. As leukemia develops the cross-talk between AML and its microenvironment, via direct EV mediated effects, alters the MSCs to promote a survival signal favoring AML growth. This restructuring is EV mediated. Future work involves the capacity of AML-derived EVs to alter the phenotype of normal marrow towards a pro-leuekmic phenotype. This also includes the use of AML mouse models to further investigate the therapeutic potential MSC-derived EVs may have as single or adjunct therapy; as well as to study potential cellular mediators that may be involved in EV-direceted AML progression. Lastly we endeavor to employ machine learning networks to characterize and predict the dynamic restructuring the EV milieu undergoes as leukemia progresses. Capturing these alteration will allow the creation of reliable predictive models that will have direct inferences on clinical diagnosis and prognosis. DisclosuresNo relevant conflicts of interest to declare.

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.

3D bioprinter applied picosecond pulsed electric fields for targeted manipulation of proliferation and lineage specific gene expression in neural stem cells

Objective. Picosecond pulse electric fields (psPEF) have the potential to elicit functional changes in mammalian cells in a non-contact manner. Such electro-manipulation of pluripotent and multipotent cells could be a tool in both neural interface and tissue engineering. Here, we describe the potential of psPEF in directing neural stem cells (NSCs) gene expression, metabolism, and proliferation. As a comparison mesenchymal stem cells (MSCs) were also tested. Approach. A psPEF electrode was anchored on a customized commercially available 3D printer, which allowed us to deliver pulses with high spatial precision and systematically control the electrode position in three-axes. When the electrodes are continuously energized and their position is shifted by the 3D printer, large numbers of cells on a surface can be exposed to a uniform psPEF. With two electric field strengths (20 and 40 kV cm−1), cell responses, including cell viability, proliferation, and gene expression assays, were quantified and analyzed. Main results. Analysis revealed both NSCs and MSCs showed no significant cell death after treatments. Both cell types exhibited an increased metabolic reduction; however, the response rate for MSCs was sensitive to the change of electric field strength, but for NSCs, it appeared independent of electric field strength. The change in proliferation rate was cell-type specific. MSCs underwent no significant change in proliferation whereas NSCs exhibited an electric field dependent response with the higher electric field producing less proliferation. Further, NSCs showed an upregulation of glial fibrillary acidic protein (GFAP) after 24 h to 40 kV cm−1, which is characteristic of astrocyte specific differentiation. Significance. Changes in cell metabolism, proliferation, and gene expression after picosecond pulsed electric field exposure are cell type specific.

Abstract 1516: BAT-1 up-regulation decreases cell invasion and migration in vitro

Abstract Prostate cancer is the most common cancer in men in the United States and is the third cause of cancer related deaths in men. In Puerto Rico, prostate cancer is the most common type of cancer and the leading cause of cancer death in men. Approximately one out of seven men will be diagnosed with prostate cancer in his lifetime. The current available biomarkers are unable to predict malignant outcomes such as recurrence. Thus, there is a critical demand for the development of innovative diagnostic and prognostic tools for the management of prostate cancer. In the current study, we evaluated the biological role of BAT-1 in prostate cancer. Preliminary data from patients who had prostate cancer recurrence identified that HLA-B associated transcript 1 (BAT-1) was down-regulated in patients with prostate cancer recurrence when compared with non-recurrent patients. We up-regulated BAT-1 in androgen independent PC3 and androgen dependent 22RV1 prostate cancer cell lines using a BAT-1 recombinant protein. In vitro assays were performed to measure proliferation, migration and invasion. Proliferation assays using MTS showed no significant change in BAT-1 recombinant protein concentrations ranging from 0.25-2.0μg/mL at 12, 24 and 48hrs for PC3 and 22RV1 cells. Wound healing assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased migration at 6, 12hrs and 24hrs in PC3 cells when compared to control. Boyden assay showed that cells treated with BAT-1 recombinant protein at 0.25μg/mL and 2.0μg/mL significantly decreased invasion in PC3 and 22RV1 cells at 24hrs when compared to control. Our results showed that BAT-1 up-regulation decreased migration and cell motility. Cells did not show a significant change in proliferation. These results were expected due to previous data showing that BAT-1 down-regulation increased cell migration and invasion, suggesting that BAT-1 expression promotes aggressiveness in prostate cancer recurrence. A possible mechanism of action for BAT1 is the modulation of cell migration and invasion. Citation Format: Aileen M. Garcia-Vargas, Maria Sanchez, Magaly Martinez-Ferrer. BAT-1 up-regulation decreases cell invasion and migration in vitro [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 1516.

Alpha‐2c Adrenergic Receptor Promotes the Malignant Phenotype of Colon Cancer Cells

Cancer remains one of the leading causes of death worldwide, with colorectal cancer (CRC) being the third most common type. Accumulating evidence shows that stress plays a pivotal role in the malignant phenotype of CRC, by promoting hallmarks of CRC including cellular proliferation, migration, invasion, and angiogenesis. During stressful situations, the sympathetic nervous system is activated, causing an elevation in levels of catecholamines, including epinephrine which elicits its effects mainly by activating adrenoceptors. Interestingly, however, the role of alpha2C adrenergic receptor (a2c‐AR) in CRC malignancy remains obscure. Here, we proposed that activation of a2c‐AR potentiates the malignant phenotype of human CRC cells. Treatment of human colon cancer cell lines, SW480 and HT‐29, with increasing concentrations of a2‐AR specific agonist (UK 14,304; 10, 30 or 100 nM) for different time points (24, 48, or 72 hours) did not cause a significant change in proliferation. However, using monolayer scratch assay, a significant increase in migration was observed after treatment with UK 14,304 (100 nM) for 8 and 12 hours in both cell lines. This was further supported by a transwell migration assay where UK 14,304 increased cell migration. Pretreatment with a specific a2c‐AR antagonist (JP 1302; 100 nM) abolished the UK 14,304‐induced cell migration. This increase in migratory capacity was concomitant with increased Rho activation evident by translocation of Rho from cytoplasm to the membrane as well as via a Rhotekin‐binding activation assay. Moreover, treatment with UK 14,304 evoked actin reorganization, an element known to be a prerequisite for cell migration. The induced Rho activation and actin cytoskeletal rearrangement could be mediated by increased oxidative stress since treatment with UK 14,304 increased ROS production. Importantly, ROS is known to induce migration of these cells. Furthermore, adhesion of colon cancer cells to TNF‐alpha‐activated HUVECs was promoted by UK 14,304. This UK14,304‐induced adhesion was followed by a dramatic increase in transendothelial migration of SW480 through HUVECs, clearly indicating that a2c‐AR activation may promote intra‐ or extravasation. Because breaking the endothelial cell barrier requires matrix digestion, we measured the levels of matrix metalloproteinases (MMPs). Importantly, stimulation of a2c‐ARs by UK14,304 increased the production of MMP2 and MMP9, and JP 1302 abolished this UK14,304‐induced production. Taken together, our data suggests that a2c‐AR potentiates the migratory and invasive capacities and hence the malignant phenotype of human colon cancer cells. Therefore, a2c‐AR may represent a novel target in the treatment of CRC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methylation of mitochondrial DNA displacement loop region regulates mitochondrial copy number in colorectal cancer.

It is not established whether de-methylation of the displacement loop (D-loop) region if mitochondrial DNA (mtDNA) directly influences mtDNA copy number and further alters the cell cycle, apoptosis and cell proliferation in colorectal cancer. The current study employed cell viability assays, cell cycle analysis, and mtDNA methylation analysis using 5 colorectal cancer cell lines. The present results demonstrated that 5-aza-2′-deoxycytidine (5-AZA), a DNA hypomethylating agent, significantly increased proliferation of Lovo and Colo-205 colorectal cancer cell lines. In Colo-205 cells, the proportion of G0/G1 phase cells was increased following 5-AZA treatment. Additionally, the apoptosis rate in Colo-205 cells was decreased by 5-AZA treatment. Compared with their controls, a significantly higher mtDNA copy number was observed in Colo-205 and Lovo cells following 5-AZA treatment. Notably, the Colo-205 and Lovo cells had relatively higher methylation levels at the 4 and 6th/7th CpG sites of D-loop region, respectively, compared with the levels at the corresponding sites following 5-AZA treatment. However, in HCT116, SW480, LS-174T, and HT-29 cells, 5-AZA treatment did not induce a significant change in proliferation, cell cycle, apoptosis and mtDNA copy number. Demethylation at the 4 and 6th/7th CpG sites of the D-loop region of HCT116, SW480, LS-174T and HT-29 cells was not observed following 5-AZA treatment. In conclusion, de-methylation of specific sites on CpG islands of D-loop promoter may lead to the elevation of mtDNA copy number in colorectal cancer, triggering alterations in biological behaviors, including increased cell proliferation, reduced apoptosis and a relative cell cycle arrest in G0/G1 phase.

High extracellular pressure promotes gastric cancer cell adhesion, invasion, migration and suppresses gastric cancer cell differentiation.

Slightly increased pressure stimulates tumor cell adhesion and proliferation. In the present study, we aimed to evaluate the effects of high pressure on gene expression and the biological behavior of gastric cancer cells. After incubation for 30min at 37˚C under ambient and increased pressure, one portion of SGC7901 cells was used for cell proliferation and apoptosis assays, cell cycle analysis, adhesion invasion or migration assays. The other portion of cells was harvested for detection of matrix metalloproteinase-2 (MMP-2), inhibitor of DNA binding-1 (ID1), sonic Hedgehog (SHH) and E-cadherin expression by western blotting or RT-PCR. In addition, we investigated the effects of high pressure on SGC7901 cell ultrastructure by transmission electron microscopy. We found that the adhesion fold under increased pressure of 760 and 1,520mmHg was 2.39±1.05 (P<0.05) and 2.47±0.85 (P<0.01) as compared with the control, respectively. The invasion fold was 3.42±2.06 (P<0.05) and 5.13±2.49 (P<0.01) as compared with the control, respectively. The migration was 1.65±0.20 (P<0.001) and 2.53±0.50 (P<0.001) as compared with the control, respectively. At increased pressure, MMP-2 and ID1 expression increased significantly, while the expression of SHH decreased significantly. However, we did not find significant change in proliferation, apoptosis, cell cycle or ultrastructure of the SGC7901 cells under high pressure. In conclusion, high pressure promoted the adhesion, invasion and migration of SGC7901 cells. Moreover, the present study suggests that the pressure-augmented invasion and migration may be related to the increase in MMP-2 expression. Moreover, high pressure may suppress SGC7901 cell differentiation, which may result from the change in SHH and ID1 expression.

Abstract 2718: Genomic and microRNAome subtraction identifies pathogenic viral sequences in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of pancreatic cancer. It constitutes about 90% of tumors of the exocrine pancreas. The aggressive nature of PDAC along with its poor diagnostic markers contributes to a high lethality of this disease. Studies from other cancer entities implicate the role of viruses in tumor development. However, there have been no established reports about virus(es) associated with pancreatic cancer. The present study identified a virus by genomic and digital microRNAome subtraction between healthy and PDAC patients, suggesting that this virus may play a role in carcinogenesis. Experimental procedures: Representational difference analysis (RDA) was utilized to perform an experimental genome-wide search for DNA sequences that occur in PDAC but not in normal tissues. Ten healthy and ten PDAC tissue DNA samples were used for the generation of representations and subtractive hybridization. The difference products (DPs) so obtained were paired end sequenced on the Illumina MiSeq platform. The sequencing data were aligned against the viral RefSeq database. We also did in silico subtraction of microRNA content (miRNA-seq) of PDAC samples. Eight PDAC samples were analyzed for viral sequences using relevant RefSeq databases. The two approaches (RDA and miRNA-seq) were pursued in parallel. RT-qPCR analysis and the in vitro functional characterization of viral sequences in PDAC cell lines were respectively performed using hydrolysis probes and second generation microRNA mimic systems. Stable cell lines expressing viral microRNA and luciferase were produced using lentivirus for mice in vivo studies. Results and Summary: We found DNA sequences of a virus in the difference products of RDA whose signature was also implicated in the microRNA analysis. Further, using RT-qPCR, we identified that a particular microRNA from this virus is expressed at significantly higher levels in PDAC samples than in normal tissue. This observation was also verified by droplet digital PCR analysis. Non-metastatic PDAC cell lines overexpressing viral microRNA showed significant invasion against relevant controls, interestingly with no significant change in proliferation. We are currently analyzing the functional consequences of these viral sequences and their involvement in carcinogenesis. Conclusion: From two different robust approaches, these findings strongly indicate viral sequences associated with PDAC that could affect pathways relevant to tumor development. Citation Format: Mohanachary Amaravadi, Agnes Hotz-Wagenblatt, Sandeep Kumar Botla, Pouria Jandaghi, Mehdi Manoochehri, Nathalia Giese, Markus W. Büchler, Andrea S. Bauer, Jörg D. Hoheisel. Genomic and microRNAome subtraction identifies pathogenic viral sequences in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2015-2718

Papillary thyroid cancer–derived exosomes contain miRNA-146b and miRNA-222

BackgroundWith the increasing diagnosis of indolent papillary thyroid cancer (PTC), the task of identifying those likely to suffer from recurrence is becoming ever more challenging. MicroRNA (miRNA/miR) in the circulation has been demonstrated as potential biomarkers of recurrence in PTC. This study aimed to investigate in vitro if extracellular miRNAs are contained in exosomes, and their potential effect on other cells. MethodsTPC-1 (PTC) and NTHY (normal thyroid follicular) cell lines were treated with exosome isolates and conditioned medium (CM), both containing miR-146b and miR-222. The changes in proliferation over a 72-h period of TPC-1 and NTHY were compared. Student t-test and analysis of variance were used for significance testing, and P < 0.05 was considered significant. ResultsExosomes derived from TPC-1 cells were demonstrated to contain miR-146b and miR-222 in relative abundance. These exosomes caused a negative proliferative effect on both TPC-1 and NTHY cells. Exosomes derived from NTHY cells did not exert a significant proliferative effect on either cell line. CM from both cell types caused an initial increase in TPC-1 proliferation at 24 h. No significant change in proliferation was seen with NTHY cells when treated with either of the CM. ConclusionsThe results showed that PTC cells overexpress miR-146b and miR-222 in exosomes; and that factors released by both normal thyroid and PTC cells alter proliferation of other cells in a complex manner. The intercellular interactions were likely conferred in part by exosomal miRNA, which can potentially be developed as biomarkers of PTC recurrence.

Targeting CXCR4 with CTCE-9908 inhibits prostate tumor metastasis

BackgroundCXCL12/CXCR4 transactivation of epidermal growth factor family receptors in lipid raft membrane microdomains on cell surface is thought to mediate tumor growth and subsequent development of metastatic disease. CTCE-9908 is a known inhibitor of CXCR4. Herein, we tested the efficacy of CTCE-9908 in inhibiting prostate cancer cell growth, invasion, and metastasis.MethodsWe used a panel of in vitro assays utilizing human prostate cancer cell lines and an in vivo orthotopic prostate cancer model to assess the anti-tumoral activity of CTCE-9908.ResultsWe demonstrated that (a) CTCE-9908 treatment resulted in no significant change in the growth of PC-3 and C4-2B cells; (b) 50 μg/ml of CTCE-9908 inhibited the invasive properties of PC-3 cells; (c) 25 mg/kg of CTCE-9908 did not alter primary tumor growth but it did significantly reduce total tumor burden in the animal including the growth of prostate and soft tissue metastases to lymph node and distant organ tissues. Histological analysis showed that CTCE-9908 treatment resulted in tumor necrosis in primary prostate tumors and no significant change in proliferation of tumor cells as measured by Ki-67 staining; (d) CTCE-9908 inhibited the tumor angiogenesis as measured by CD34 positive vessels in tumors.ConclusionsThese data suggest that CXCR4 inhibition by CTCE-9908 decreases the invasion potential in vitro, which then translated to a reduction of tumor spread with associated reduction in angiogenesis. Hence, CTCE-9908 may prove to be an efficacious novel agent to prevent and treat the spread of metastatic prostate cancer.

Expression of lymphocyte-derived growth hormone (GH) and GH-releasing hormone receptors in aging rats

In the present study, we show that higher levels of lymphocyte GH are expressed in spleen cells from aging animals compared to young animals. Further, leukocytes from primary and secondary immune tissues and splenic T and B cells from aging rats all express higher levels of GHRH receptors compared to younger animals. Bone marrow and splenic T cells express the highest levels of GHRH receptor in aging animals. Spleen cells from aging animals showed no significant change in proliferation or GH induction after treatment with GHRH. Taken together, the data for the first time show alterations in GH synthesis and expression of the GHRH receptor on cells of the immune system that may play a role in the immune response in aging.

Proliferation of fibroblasts and endothelial cells is enhanced by treatment with Phyllocaulis boraceiensis mucus.

Previously, mucus of some molluscs has been studied as a potential source of new natural compounds capable of inducing cell proliferation and of remodelling tissue. Here, the focus of the study is possible use of mucus released by Phyllocaulis boraceiensis - a compound inducing cell proliferation and enhancing collagen synthesis in dermal fibroblasts and inducing proliferation human endothelial cell cultures. Fibroblasts treated with P. boraceiensis mucus at concentrations below 0.012 μg/μl developed high rates of proliferation, as evaluated using MTT assay; the proliferative effect was dose-dependent. Production and secretion of extracellular matrix components and collagen type I fibres were enhanced after 24 h of treatment, revealing a hormesis effect, biphasic dose response - low dose for proliferation yet toxic at high dose. No significant change in proliferation was observed in treated endothelial cells and production of lipid polyunsaturated free radicals was low in both cell types. Treatment with P. boraceiensis mucus produced pronounced changes in fibroblast cell number and morphology, and in quantities of well-ordered collagen deposition. These results support the premise that Phyllocaulis boraceiensis mucus demonstrates proliferative properties in cells involved in the healing process.