Propidium Iodide Cell Cycle Analysis Research Articles

Abstract 2916: Protein arginine methyltransferase 5 as a therapeutic target for KRAS mutated colorectal cancer

Abstract Introduction Protein Arginine Methyl Transferase 5 (PRMT5) is a transcription regulator for multiple cellular processes. It is being tested as a potential target/biomarker in several cancer types. Herein we analyzed the therapeutic efficacy of PRMT5 in KRAS mutated and wildtype (WT) colorectal cancer (CRC) cells. This cohort of patients (40-45%) have a worse prognosis when treated with monoclonal antibodies that have added significant therapeutic benefit for KRAS wild type CRC. Currently, there is no specific FDA approved alternate therapy guidelines for this subgroup of patients. Methodology Four CRC cell lines [HCT116 and SW620 (KRAS Mutant); and HKE3, LIM 2405 (KRAS Wildtype)] were analyzed for basal level PRMT5 expression by western blot and qPCR analysis. Next, cells were treated with PRMT5 inhibitor (EPZ015666) at 1µM and 10µM concentration for 72 hours and High-Throughput Presto Blue HS Cell Viability Assay was performed in a 96 well plate reading emission at 595 nm. Cell viability was confirmed by flow cytometry with Via Count reagent. Finally, Annexin V/7AAD Apoptosis assay and propidium Iodide cell cycle analysis were performed. ResultsBasal level of PRMT5 expression is significantly higher in KRAS mutated cells as compared to KRAS WT cells at both transcriptional and translational levels, as determined by qPCR (2.5 Fold; p=0.01) and Western Blot analysis (2.5-Fold; p=0.011).PRMT5 inhibitor treatment showed a significant reduction in cell viability in KRAS mutated cells compared to KRAS WT cells after 72 hours of treatment by High-Throughput Presto Blue HS Cell Viability Assay (at 1 µM 2.8-fold inhibition; p=0.00003 & at 10µM 2.1-fold; p=0.0025).A 5.3-fold increase in apoptosis was observed in KRAS mutated CRC cells when compared to KRAS WT cells by Annexin V/7AAD Apoptosis Assay (p=0.05) post 72 hours of 10µM inhibitor treatment.Cell cycle analysis of KRAS mutant and wild type CRC cells by flow cytometry using propidium iodide indicated a significant (p= 0.026) G2 phase arrest (7.7% increase) of the mutant cells compared to the wildtype cells when treated with 10µM PRMT5 inhibitor for 72 hours. Conclusion PRMT5 expression is significantly upregulated in KRAS mutated CRC cells when compared to KRAS WT CRC cells. While PRMT5 inhibitor treatment showed efficacy in both wildtype and mutant cells, cell cycle arrest and apoptosis are significantly upregulated for the KRAS mutant cells. In addition, significant reduction in cell viability was observed in KRAS mutant cells. PRMT5 functions by symmetrical di-methylation of several proteins including the histones. The mechanism of crosstalk between KRAS mutation and PRMT5 methylation will provide valuable insight into the disease characteristics of KRAS mutated CRC. Further analysis is in progress to delineate the molecular mechanism. Citation Format: David Shifteh, Tzuriel Sapir, Sanjay Goel, Radhashree Maitra. Protein arginine methyltransferase 5 as a therapeutic target for KRAS mutated colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2916.

Amplification of lncRNA PVT1 promotes ovarian cancer proliferation by binding to miR-140.

Gene deletion or gene amplification acts as a driving factor of onset, progress, and metastasis in various cancers, including ovarian cancers. By mining the whole genome data of ovarian cancer patients, we identify the long noncoding RNA PVT1 as the most amplified gene. Knockdown of PVT1 was then achieved using a shRNA in two ovarian cancer cell lines, and cell viability was determined by trypan blue exclusion assay, cell metabolism by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, and cell cycle alteration by propidium iodide cell cycle analysis. Potential targeting microRNAs were predicted with starBase v2.0, and direct binding of miR-140 on PVT1 was confirmed by luciferase reporter assay and microRNA pull-down assay. Evolutionary conserved transcription factor-binding site was predicted via rVista 2.0. Our results show that PVT1 was the most amplified gene in ovarian cancer patients, and it was highly correlated with poor survival outcomes. Knockdown of PVT1 caused decreased cell viability, metabolic activity, and smaller proportion of S-phase cells. PVT1 directly bound to miR-140 and acted as a microRNA sponge, while transcription of PVT1 was regulated by the transcription factor FOXO4. In conclusion, viability, metabolism, and cell cycle of ovarian cancers are regulated by the FOXO4/PVT1/miR-140 signaling pathway.

The Essentiality of Serine and Glycine for Skeletal Muscle Regeneration

Non‐essential amino acids (NEAA), serine (ser) and glycine (gly), are recognized as essential to support proliferation of some cell types, including skeletal muscle (SkM) progenitor cells (MPCs). MPCs and a well‐orchestrated myogenic program are essential for SkM regeneration following injury; impaired regeneration underlies pathological tissue remodeling and functional decline. It is appreciated that the metabolic microenvironment and nutrient availability influence the myogenic process. The objective of this research was to investigate the metabolic and cellular requirements of ser and gly for the regenerative process (MPC expansion, differentiation, and myotube formation) in vitro. Human primary MPCs (hMPCs) were cultured with and without ser/gly and markers of proliferation (live and dead staining, BrdU incorporation, Propidium Iodide cell‐cycle analysis) were assessed; markers of differentiation, and myotube formation (gene and protein expression of select myogenic regulatory factors [MRFs], myosin staining) are being investigated. Additionally, the regulation of the de novo ser synthesis pathway (SSP), ser/gly interconversion (SHMT1, SHMT2), and changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined with/without ser/gly using RNAseq, qPCR, immunoblotting, and a Seahorse bioanalyzer. Ser/gly depletion caused hMPCs to reside in a non‐proliferative state without inducing apoptosis and decreased OCR (p<0.05) but not ECAR (p=0.07), supporting a quiescent metabolic state. BrdU incorporation and cell‐cycle sorting with flow cytometry suggested that depletion of ser/gly inhibits hMPCs from leaving G1 phase. Intriguingly, proliferating hMPCs expressed all SSP enzymes (PHGDH, PSAT1, PSPH). The expression of SSP enzymes, SHMT2, ser transporters, and ATF4 (transcription factor involved in nutrient sensing) increased during ser/gly depletion suggesting ser/gly depletion is sensed by hMPCs and mechanisms to increase ser and gly availability are upregulated. Ser and gly are substrates for multiple metabolic pathways; building blocks for lipids, proteins, and nucleotides; one‐carbon donors; and help maintain cellular (epi)genetic status and redox state. Providing formate (a one‐carbon source), nucleotides, or additional glucose did not rescue proliferation. RNAseq analysis revealed an upregulation of genes involved in the glutathione metabolism pathway. In support of these findings a cell permeable version of glutathione provided a modest rescue to hMPC cell number and intracellular levels of total glutathione and the ratio of reduced to oxidized glutathione were diminished after the ser/gly depletion; glutathione may be a necessary product of ser/gly metabolism. Ongoing RNAseq analysis will allow us to identify potential mechanisms underlying the quiescent‐like state. Ongoing culture experiments will determine the effects of ser/gly depletion on differentiation and myotube formation. This research builds upon recent findings that clearly identify an exogenous requirement of ser and gly for hMPC expansion and challenges the notion that ser and gly are strictly NEAA, particularly in states of tissue regeneration.Support or Funding InformationPresident's Council of Cornell Women and Cornell Division of Nutritional SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A16: The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB)

Abstract Introduction: We previously reported a series of novel marinopyrroles as Mcl-1/Bcl-2 inhibitors for potential anticancer activity. The marinopyrrole agents have not been studied in pediatric tumors including neuroblastoma (NB). Temsirolimus (TEM) is an mTOR inhibitor that has been used clinically in renal cell carcinoma and breast cancer and more recently has been used in relapse/refractory pediatric tumors, including NB. In the following study we describe the in vitro and in vivo activity of the most active of the marinopyrroles (RL71) alone or with the mTOR inhibitor TEM in a chemoresistant NB cell line. Methods: RL71, which was determined to be the most active of 39 pyrrolomycin derivatives, was tested in a MYCN amplified BE-2c NB cell line using an MTT assay and was selected for study in experiments to determine potential mechanism of action and for evaluation of in vivo activity. RL71 was tested in a series of in vitro assays to evaluate effects on apoptosis, metabolism, and cell ultrastructure. Apoptosis was evaluated using propidium iodide cell cycle analysis by flow cytometry and measurement of the sub-G1 fraction. Apoptosis was also evaluated by the measurement of cleaved PARP by Western blot. The effect of RL71 alone and combined with TEM on metabolism was determined using Seahorse Xp Metabolic Analyzer. The effect of agent(s) on OXPHOS metabolism was measured by oxygen consumption rate and effect on glycolysis by extracellular acidification rate (ECAR). Electron microscopy (EM) was used to determine changes in cell ultrastructure, particularly of mitochondria, in treated and nontreated cells. Using a mouse xenograft model, the antitumor effect for RL71 and the combination of RL71 plus TEM was tested. RL71 concentrations were measured in blood, tumor, and brain. Results: RL71 at low concentrations (500 nM) caused disruption of ATP production and increased proton leak in mitochondria after injection. This effect was associated with sharp drop in ATP cellular levels. There was also a modest decline in glycolytic capacity and a lack of compensation for loss of mitochondrial ATP production. These effects were not seen with TEM alone, nor did TEM combined with RL71 show potentiation. Measures of apoptosis, which included cleaved PARP and sub-G1 peaks on flow cytometry, were not consistently elevated in either RL71 or TEM alone or when the two were combined. EM showed mitochondrial and cell membrane disruption with RL71 treatment alone but not with TEM alone; drugs combined appeared to produce greater disruption of cell membranes. There was no clear effect on nuclear morphology on EM, further supporting that apoptosis was unlikely to be the mechanism of cell death. The cell membrane disruption was consistent with necrosis, perhaps as a result of sharp decline in ATP. Three of 5 mice dosed with the combination of RL71 plus TEM had complete responses while none of the mice in the control group, TEM-alone group, or PL71-alone group responded (p = 0.009). Concentrations of RL71 in blood, tumor, and brain generally achieved concentrations exceeding the IC50 when it was combined with TEM (IC50=25 nM). Conclusion: The combination of RL71 and TEM may be active in chemoresistant, MYCN-amplified NB. The mechanism of action of the combination continues to be evaluated; it is unlikely to be completely explained by disrupted metabolism or induction of apoptosis but likely results from nonapoptotic mechanisms of cell death such as necrosis or autophagy. Citation Format: Timothy R. McGuire, Donald W. Coulter, Yan Liu, Yashpal S. Chhonker, Daryl J. Murry, Gracey R. Alexander, Erin M. McIntyre, Jason A. Sughroue, Graham J. Sharp, Rongshi Li. The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A16.

Abstract B07: Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials

Abstract Objective: To evaluate the anti-tumor activity of a class of experimental antimalarial drugs in an MYCN amplified chemoresistant neuroblastoma cell line (BE-2c) intended to model poor prognostic neuroblastoma. Methods: Etoposide cytotoxicity was studied at concentrations (250 ng/ml,500 ng/ml, 1 mcg/ml, 2 mcg/ml, and 3 mcg/ml) to confirm chemoresistance to a commonly used cytotoxic agent in the treatment of neuroblastoma. Concentrations chosen for the ozonide antimalarials were those that have been achieved in-vivo when studied in a malarial model in rodents. Ozonide antimalarial drugs were tested in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which measures cell viability colorimetrically. A series of 13 ozonide compounds were evaluated for activity using a concentration response scheme (0, 250 ng/ml, 500 ng/ml, 1 mcg/ml, 5 mcg/ml, and 10 mcg/ml) that allowed the generation of IC50 for each agent based on mean MTT absorption values from 10 replicates at each concentration. The most active ozonide (OZ513) identified was then studied in a cell cycle flow cytometry analysis using propidium idodide with measurement of the A0 peak which estimates apoptosis. A neurosphere assay was also used which is considered enriched for “cancer stem cells” and is able to model movement of drug into a small avascular tumor was used to further evaluate the activity of OZ513. Spheres were counted and characterized after treatment and compared to non-treatment controls. Because it has been postulated that the anti-cancer mechanism of action of the ozonide antimalarials is the modulation of cancer cell metabolism (autophagy, disruption of oxidative metabolism) the effect of OZ513 on mitochondrial oxidative metabolism and glycolysis was evaluated in BE-2c cells using Seahorse metabolic analysis. Results: Etoposide had no cytotoxic activity at any of the concentrations studied. OZ513 antimalarials were consistently active in BE-2c cell culture with the MTT assay generating IC50s on two separate experiments of 0.03 and 0.05 mcg/ml. The MTT results were confirmed using trypan blue staining to directly measure cell death. The Ao peak on PI flow cytometry increased after treatment with OZ513 indicating an increase in apoptosis. In addition, 5 mcg/ml of OZ513 increased G1, S, and G2 aneuploid fraction on propidium iodide cell cycle analysis. OZ513 disrupted BE-2c neurospheres confirming their activity in these multicellular tumorspheres. Metabolic studies demonstrated little effect on mitochondria based oxidative metabolism or glycolysis. Structure activity relationship suggests that the alcohol of the ozonide structure is required for optimal activity. Conclusion: The antimalarial agent OZ513 has promising activity against the chemoresistant neuroblastoma cell-line BE-2c by increasing apoptosis by mechanisms that remain unclear, but increased cell cycle aneuploidy with treatment of cells with 5 mcg/ml of OZ513 indicates defects in mitosis and cytokinesis. Citation Format: Don W. Coulter, Timothy R. McGuire, John G. Sharp, Yuxiang Dong, Xiaofang Wang, Erin McIntyre, Xiaoyu Chen, Jon Vennerstrom. Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B07.

Abstract A047: Targeting androgen receptor decreases proliferation of triple-negative breast cancer

Abstract Recent studies demonstrate that the androgen receptor (AR) is expressed in up to a third of triple negative breast cancer (TNBC) and define a subtype of TNBC with a “luminal AR” gene signature. We hypothesized that AR+TNBC are dependent on AR for growth and that inhibiting AR activity by preventing nuclear localization and DNA binding will decrease proliferation and tumor burden in preclinical models of breast cancer. Indeed, knock down of AR expression using a lentiviral shRNA significantly reduced proliferation in cell line models of TNBC compared to cells transduced with a non-targeting control (2-fold reduction, p<0.001). Propidium iodide cell cycle analysis demonstrated that TNBC cells with reduced AR expression had a 22% increase in the percentage of cells in S-phase (p<0.001). In TNBC cells that exhibit androgen-stimulated growth, reduced AR expression inhibited response to ligand. Enzalutamide, an AR inhibitor, inhibited ligand mediated nuclear translocation of AR as assessed by nuclear cytosolic fractionation and significantly decreased baseline proliferation of TNBC in vitro (p<0.001). In MDA453 xenografts, enzalutamide significantly decreased tumor volume and weight by up to 85% and increased apoptosis as measured by cleaved caspase-3 by 60% compared to mice that received DHT alone. In addition, a 50% reduction in nuclear AR was observed in the tumors of mice treated with enzalutamide. Thus AR may play an important role in a subset of TNBC and presents an opportunity for targeted therapy. Citation Format: Valerie Barton, Nicholas D'Amato, Jennifer Richer. Targeting androgen receptor decreases proliferation of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A047.

Abstract IA18: Targeting the androgen receptor in multiple breast cancer subtypes

Abstract The androgen receptor (AR) is even more widely expressed in breast cancer (BC) than estrogen receptor alpha (ER) or progesterone receptor (PR), and recently AR has emerged as a useful marker to refine classification of breast cancer (BC). However, we still understand relatively little about the specific effects of AR or its potential as a therapeutic target in the different subtypes of BC. Our data from clinical samples suggests that some ER+ tumors can switch from growth driven by estrogens to growth driven by adrenal androgens, particularly when the estradiol (E2)/ER pathway is inhibited by standard ER-directed endocrine treatments. Indeed, we have evidence showing that a high ratio of AR to ER (≥2.0) leads to a > 4 fold increased risk for relapse while on tamoxifen (HR=4.43, P<0.0001) and is a strong independent predictor of disease-free survival (HR=4.04, P=0.002). Since approximately 30% of metastatic ER+ tumors exhibit de novo resistance to standard endocrine therapy and all patients with metastatic disease ultimately progress with acquired resistance, targeting AR in ER+ BC may be clinically useful. Thus, clinical trials evaluating the role of androgen receptor inhibition in BC are underway. Importantly, we find that new generation anti-androgens that block AR DNA binding affect BC differently than older generation anti-androgens (which allow DNA binding), leading to new insights into the role of AR. When AR is excluded from the nucleus, both androgen- and estradiol (E2)-stimulated proliferation is inhibited in ER+ breast cancers, suggesting that AR plays a previously-unknown role in E2-mediated ER activity. Both enzalutamide (ENZ) and bicalutamide inhibited androgen-mediated proliferation of breast cancer lines in vitro. Interestingly, ENZ uniquely inhibited E2-mediated proliferation of ER.+/AR+ breast cancer cells in vitro, and also inhibited E2-driven tumor growth as effectively as tamoxifen in vivo. When opposing androgen-stimulated tumor growth in ER+ BC in vivo models, ENZ inhibited proliferation and increased apoptosis, while in ER- models it increased apoptosis but did not alter proliferation. When opposing E2-stimulated tumor growth in vivo ENZ decreased proliferation but did not increase apoptosis. In Her2+ but non-amplified, BC models the AR inhibitor ENZ, inhibited proliferation as well or better than trastuzumab (TRAS), whereas TRAS showed a greater inhibitory effect than ENZ in Her2 amplified lines. In all models tested, the combination of ENZ and TRAS inhibited proliferation more effectively than either agent alone. In TRAS resistant lines, addition of ENZ to TRAS significantly inhibited proliferation. Liganded AR upregulates Her3 in some Her2+ BC lines; however, in other Her2+ lines, Her3 is not increased by androgen stimulation, yet anti-androgens still inhibit proliferation. Our data suggest that the AR and Her2 pathways are linked in ways not previously understood and that there are novel Her3-independent mechanisms whereby AR impacts Her2+BC. A subset of TNBC express AR and knock down of AR using lentiviral shRNAs significantly reduced proliferation in multiple TNBC lines. Propidium iodide cell cycle analysis demonstrated that knockdown of AR in TNBC decreased the percentage of cells in G2/M. Lastly, inhibition of AR with ENZ, blocked ligand-mediated nuclear translocation of AR as assessed by nuclear and cytosolic fractionation and significantly decreased proliferation of TNBC in vitro. In summary, our studies demonstrate that AR plays varying roles in the different BC subtypes and will serve to guide appropriate combinations with AR inhibitor and existing therapeutic strategies. Further, we find that assessment of the amount of AR relative to ER may be informative. Citation Format: Nicholas C. D'Amato, Dawn R. Cochrane, Valerie N. Barton, Liu Bolin, Susan M. Edgerton, Sebastian Bernales, Haihua Gu, Ann D. Thor, Jennifer K. Richer. Targeting the androgen receptor in multiple breast cancer subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA18.

Abstract 1085: RbBP6 isoform 3/DWNN is essential for arsenic trioxide-induced G2/M arrest in cancer cell lines

Abstract Retinoblastoma binding protein 6 (RbBP6) is a gene that encodes RbBP6 protein products (isoforms 1,2 and 3) that has previously been documented to bind retinoblastoma protein (pRb) and p53, which are key components of cell cycle regulation and localises on chromosome 16p12.2 in humans. These isoforms are derived from two major mRNA transcripts, a 1.1 kb (Isoform 3) and 6.1 kb (isoform 1and 2). These two tumour suppressors are important to the understanding the function of the RbBP6. Arsenic trioxide has been used to treat APLS and has been shown to arrest cells at G1 and G2. In this study, the involvement of the DWNN in cell cycle arrest caused by As2O3 was investigated. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was used to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. RNA interference was employed to investigate the involvement of the DWNN in cell cycle regulation. Immunocytochemistry was employed to determine the localization of the DWNN-containing proteins in human cancer cells as well as non-cancerous cells. Propidium iodide cell cycle analysis was utilised to quantify cell cycle arrest induced by As2O3. Western blotting and real-time PCR were used to determine the expression of the DWNN in both control and treated cells. DWNN knockdown increased cell proliferation and did not have an effect on apoptosis. Treatment of human cells with low concentrations of As2O3 induced G2 cell cycle arrest and resulted in induced expression of DWNN. This induced cell cycle arrest was also associated with deglycosylation of the DWNN domain. This finding is in line with the localization of the DWNN proteins in human cancer cells, which showed high expression of the DWNN proteins in G2/M cell cycle phase. Expression analysis showed that the up-regulation of the DWNN domain resulted in p53 up-regulation. These results strongly suggest that the DWNN may be either required for G2 cell cycle arrest or may be required for p53 stabilization during this phase or may be needed for signalling for progression to the next phase of cell cycle. DWNN involvement in cell cycle may be in a p53-dependent manner. Staurosporine which induces G1 cell cycle arrest did not result in DWNN up-regulation and that suggest that DWNN may not be required for G1 cell cycle arrest or regulation. The effect of DWNN in pRb phosphorylation and G1 cell cycle arrest still needs more attention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1085.