Flow Cytometry Analysis Of Cell Cycle Research Articles

Abstract 5836: SMC4 promotes prostate cancer organ-specific metastasis by modifying cancer cell metabolism

Abstract Structural maintenance of chromosomes 4 (SMC4), one subunit of condensin complexes, is a key structural component of mitotic chromosomes. Accumulating evidence has suggested that SMC4 regulates prostate carcinogenesis. However, its roles on regulation of prostate cancer development remain unclear. Our previous studies showed that the expression of SMC4 is increased in highly lung-metastatic prostate cancer cells, RM1-LM, an organ-specific metastasis cell line that we developed. To investigate the function of SMC4, SMC4 gene was knockdown in RM1-LM cells by CRISPR/Cas9 system. Knockdown of SMC4 significantly diminished the cancer cell growth in RM1-LM. In addition, transwell assay results showed that SMC4 knockdown in RM1-LM suppressed the cell migration and invasion. Furthermore, we observed that SMC4 protein is important in the non-dividing phase of the cell cycle. In particular, cell cycle flow cytometry analysis indicated that SMC4 knockdown leaded to cell arrest in S phase. Finally, RM1-LM cells showed increased lung metastases compared with RM1-SMC4+/- cells in a murine model. The H&E and IHC results confirmed that RM1-LM cells induced greater number of lung metastases and colonization than RM1-SMC4+/-cells, suggesting the loss of SMC4 suppressed prostate cancer metastasis. The interactome of SMC4 was uncovered through IP-MS and revealed that SMC4 may interacted with GLUT1, a key protein in glycolysis pathway. The ATP rate assay and Glycolysis rate assay illustrated a significantly metabolic switch existing in RM1-SMC4+/-cells compared with their parental cells. Collectively, our study suggested that the interaction between SMC4 and GLUT1, confirmed by Co-IP, promotes prostate cancer cells metastasis via modifying the cell metabolism. This work was supported by NSFC projects 81802949, 81773146, 81972766, 81972420, 82173336; Shenzhen Science and Technology Commission Project JCYJ20190809161811237, JCYJ2021032410424040; Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research (2017B030301018);and College students' innovative entrepreneurial training program (S202114325028) Citation Format: Wei Zhang, Xin Huang, yueming Li, yingxuan Gong, xiuzhi Li. SMC4 promotes prostate cancer organ-specific metastasis by modifying cancer cell metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5836.

Long Noncoding RNA RAET1K Enhances CCNE1 Expression and Cell Cycle Arrest of Lung Adenocarcinoma Cell by Sponging miRNA-135a-5p.

Molecular dysregulation is believed to participate in the onset and progression of lung adenocarcinoma (LUAD). This study aimed to identify and evaluate the potential key long noncoding RNAs (lncRNAs) involved in the significant dysfunctional process of LUAD. We found that lncRNA retinoic acid early transcript 1K (RAET1K) was upregulated in tumor tissues and were correlated with a poor prognosis of patients with LUAD; further, for the first time, we detected the biological roles of RAET1K. Weighted gene correlation network and gene set enrichment analysis revealed that high RAET1K expression is related to cell cycle dysfunction through upregulated cyclin E1 (CCNE1) by targeting miR-135. The dual-luciferase reporter gene assay was performed to clarify the binding relationship between RAET1K and miR-135a-5p in transgenic A549 and H1299 cells. Real-time PCR and Western blot analyses showed that RAET1K overexpression and miR-135a-5p inhibition exerted a strong synergistic effect on CCNE1 expression, and cell cycle flow cytometry analysis was used to confirm the arrest of A549 and H1299 cells at the G1/S phase. The lncRNA RAET1K/miR-135a-5p axis might participate in the regulation of LUAD progression by influencing CCNE1 expression and the accumulation of cells arrested at the G1/S phase boundary.

Arsenic Trioxide and Thalidomide Combination Induces Autophagy Along with Apoptosis in Acute Myeloid Cell Lines.

Objective Autophagy and apoptosis play key roles in cancer survival and pathogenesis and are governed by specific genes which have a dual role in both cell death and survival. Arsenic trioxide (ATO) and thalidomide (THAL) are used for treatment of many types of hematologic malignancies. ATO prevents the proliferation of cells and induces apoptosis in some cancer cells. Moreover, THAL has immunomodulatory and antiangiogenic effects in malignant cells. The aim of present study was to examine the effects of ATO and THAL on U937 and KG-1 cells, and evaluation of mRNA expression level of VEGFs genes, PI3K genes and some of autophagy genes. Materials and Methods In this in vitro experimental study, U937 and KG-1 cells were treated by ATO (0.4-5 µM) and THAL (5-100 µM) for 24, 48 and 72 hours. Cell viability was measured by MTT assay. The apoptosis rate and cell cycle arrest were evaluated by flow cytometry (Annexin/PI) and cell cycle flow cytometry analysis, respectively. The effect of ATO/THAL on mRNAs expression was evaluated by real-time polymerase chain reaction (PCR). Results ATO/THAL combination enhanced cell apoptosis in a dose-dependent manner. Also, ATO/THAL induced SubG1/ G1 phase arrest. mRNA expression levels of VEGFC (contrary to other VEGFs isoform), PI3K, AKT, mTOR, MEK1, PTEN, IL6, LC3 and P62 genes were upregulated in acute myeloid leukemia (AML) cells following treatment with ATO/THAL. ConclusionCombined treatment with ATO and THAL can inhibit proliferation and invasion of AML cells by down-regulating ULK1 and BECLIN1 and up-regulating PTEN and IL6, and this effect was more marked than the effects of ATO and THAL alone.

Effect of shRNA Mediated Silencing of YB-1 Protein on the Expression of Matrix Collagenases in Malignant Melanoma Cell In Vitro.

Background and Objective: YB-1 is a transcription and oncogenic factor capable of binding to DNA and RNA performing versatile functions within normal and cancer cells. Some studies reported the binding of YB-1 with a collagenases gene promoter and influencing their expression. In addition, the role of YB-1 in malignant melanoma was not elucidated. Thus, in this study, the aim was to knock down the expression of YB-1 in A375 malignant melanoma cancer cell using the shRNA approach and study its effect on cancer cell proliferation, migration, and expression of collagenases. Methods: A375 malignant melanoma cell lines were grown in standard conditions and were transfected with three plasmids containing a retroviral pGFP-V-RS vector, two of them containing targeting sequences for YB-1 mRNA. The third plasmid contained a scrambled mRNA sequence as a negative control. Expression of YB-1 was validated using immune-fluorescence staining, RT-PCR and western blotting. The cancer cell proliferation was determined using MTT assay, serial trypan blue cell counting and cell cycle flow-cytometry analysis. Expression of collagenases (MMP1, MMP8, and MMP13) was evaluated using RT-PCR and western blotting analysis. In addition, a wound-healing assay was used to assess cell migration potential. Statistical analysis was performed using one-way ANOVA test with Bonferroni post hoc analysis to compare the quantitative results among samples. Results: The established silenced cell strains (P1 and P2) had nearly 70% knockdown in the expression of YB-1. These YB-1 silenced strains had a significant cell cycle-specific reduction in cell proliferation (p < 0.05 in serial cell counting and cell cycle flow cytometry analysis, p < 0.001 in MTT assay). In addition, YB-1 silenced strains had a remarkable reduction in cell migration potential. Expression of MMP13 was significantly reduced in YB-1 silenced strains. Conclusion: YB-1 oncoprotein is a promising target in the treatment of malignant melanoma. Silencing of this protein is associated with significant anti-proliferative, anti-invasive and MMP13 insulating properties in A375 malignant melanoma cancer cell lines.

Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513.

BackgroundEvaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c.MethodsThe activity of 12 ozonides, artemisinin, and two semisynthetic artemisinins were tested for activity against two neuroblastoma cell-lines (BE (2)-c and IMR-32) and the Ewing’s Sarcoma cell line A673 in an MTT viability assay. Time course data indicated that peak effect was seen 18 h after the start of treatment thus 18 h pre-treatment was used for all subsequent experiments. The most active ozonide (OZ513) was assessed in a propidium iodide cell cycle flow cytometry analysis which measured cell cycle transit and apoptosis. Metabolic effects of OZ513 in BE (2)-c cells was evaluated. Western blots for the apoptotic proteins cleaved capase-3 and cleaved PARP, the highly amplified oncogene MYCN, and the cell cycle regulator CyclinD1, were performed. These in-vitro experiments were followed by an in-vivo experiment in which NOD-scid gamma immunodeficient mice were injected subcutaneously with 1 × 106 BE (2)-c cells followed by immediate treatment with 50–100 mg/kg/day doses of OZ513 administered IP three times per week out to 23 days after injection of tumor. Incidence of tumor development, time to tumor development, and rate of tumor growth were assessed in DMSO treated controls (N = 6), and OZ513 treated mice (N = 5).ResultsIt was confirmed that five commonly used chemotherapy drugs had no cytotoxic activity in BE (2)-c cells. Six of 12 ozonides tested were active in-vitro at concentrations achievable in vivo with OZ513 being most active (IC50 = 0.5 mcg/ml). OZ513 activity was confirmed in IMR-32 and A673 cells. The Ao peak on cell-cycle analysis was increased after treatment with OZ513 in a concentration dependent fashion which when coupled with results from western blot analysis which showed an increase in cleaved capase-3 and cleaved PARP supported an increase in apoptosis. There was a concentration dependent decline in the MYCN and a cyclinD1 protein indicative of anti-proliferative activity and cell cycle disruption. OXPHOS metabolism was unaffected by OZ513 treatment while glycolysis was increased. There was a significant delay in time to tumor development in mice treated with OZ513 and a decline in the rate of tumor growth.ConclusionsThe antimalarial ozonide OZ513 has effective in-vitro and in-vivo activity against a pleiotropic drug resistant neuroblastoma cell-line. Treatment with OZ513 increased apoptotic markers and glycolysis with a decline in the MYCN oncogene and the cell cycle regulator cyclinD1. These effects suggest adaptation to cellular stress by mechanism which remain unclear.

Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells

AimsCell cycle dysregulation is important in tumorigenesis. Transcriptional silencing of cell cycle regulatory genes, due to DNA methylation, is a common epigenetic event in malignancies. As2O3 has been shown to induce cell cycle arrest and also to be a potential hypomethylating agent. Our study aimed to investigate DNA methylation patterns of cell cycle regulatory genes promoters, the effects of Arsenic trioxide (As2O3) on the methylated genes and cell cycle distribution in colorectal cancer (CRC) cell lines. Main methodsThe methylation-specific PCR (MSP) and/or restriction enzyme-based methods were used to study the promoter methylation patterns of 24 cell cycle regulatory genes in CRC cell lines. Gene expression level and cell cycle distribution were determined by Real-time PCR and flow cytometric analyses, respectively. Key findingsOur methylation analysis indicated that only promoters of RBL1 (p107), CHFR and p16 genes were aberrantly methylated in three cell lines. As2O3 significantly decreased DNA methylation in promoter regions of these genes and restored their expression. We found that As2O3 significantly reduced the expression of DNA methyltransferase 1 (DNMT1) and increased arsenic methyltransferase (AS3MT). Furthermore, As2O3 altered transcriptional activity of several unmethylated cell cycle regulatory genes including cyclin B1, E1, D1, GADD45A and p21. Cell cycle flow cytometry analysis showed As2O3 induced G2/M arrest in all three cell lines. SignificanceThese data suggest that demethylation and alteration in the expression level of the cell cycle-related genes may be possible mechanisms in As2O3-induced cell cycle arrest in colorectal cancer cells.

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.

Cytotoxic Effect of Ethanol Extract of Microalga,Chaetoceros calcitrans, and Its Mechanisms in Inducing Apoptosis in Human Breast Cancer Cell Line

Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 μg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug.