l-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells

l-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 ( Btg2) regulates the G1/S transition phases of the cell cycle. N- myc downstream regulated gene 1 ( Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of l-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of l-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. l-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and l-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. l-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that l-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or l-mimosine in LNCaP cells. Our results indicated that hypoxia and l-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. l-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells.

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Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90929.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. E1344-E1353 ◽

Cited By ~ 23

Author(s):

Qing Huang ◽

Anthony P. Cheung ◽

Yu Zhang ◽

He-Feng Huang ◽

Nelly Auersperg ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Protein Expression ◽

Granulosa Cell ◽

Molecular Mechanisms ◽

Thymidine Incorporation ◽

Primary Cultures ◽

Cell Cycle Distribution ◽

Cyclin D ◽

Rb Protein

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [3H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [3H]thymidine incorporation, enhanced cell transition from G0/G1to S and G2/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D1and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15INK4Band p16INK4A. GDF-9 also activated Rb protein (a critical G1to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D1and E but not the decrease in p15INK4Band p16INK4Ainduced by GDF-9. In contrast, SIS3 reversed the decrease in p15INK4Band p16INK4Abut not the increase in cyclin D1and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D1and E and suppressing p15INK4Band p16INK4Avia both Smad-dependent and Smad-independent pathways.

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Hypoxia upregulates the gene expression of mitochondrial aconitase in prostate carcinoma cells

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0090 ◽

2013 ◽

Vol 51 (1) ◽

pp. 131-141 ◽

Cited By ~ 26

Author(s):

Ke-Hung Tsui ◽

Li-Chuan Chung ◽

Shyi-Wu Wang ◽

Tsui-Hsia Feng ◽

Phei-Lang Chang ◽

...

Keyword(s):

Gene Expression ◽

Prostate Carcinoma ◽

Fatty Acid Synthase ◽

Molecular Mechanisms ◽

Transient Gene Expression ◽

Carcinoma Cells ◽

Human Prostate ◽

Translation Initiation Site ◽

Transcriptional Level ◽

Mitochondrial Aconitase

Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1α was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with HIF-1α expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at −1013 to −842 upstream of the translation initiation site. l-mimosine, an iron chelator, stabilized HIF-1α but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells.

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The RNA-Binding Protein Musashi 2 Is Upregulated in the Proliferative Fraction of CLL Clones, Particularly in U-CLL Patients, and Its Silencing Induces Programmed Cell Death

Blood ◽

10.1182/blood.v128.22.3216.3216 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3216-3216

Author(s):

Florencia Palacios ◽

Xiao J. Yan ◽

Jacqueline C. Barrientos ◽

Jonathan E. Kolitz ◽

Steven L. Allen ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Poor Prognosis ◽

Protein Levels ◽

Poor Outcome ◽

Dividing Cells

CLL develops from a small fraction of dividing monoclonal CD5+ B cells. The size and rate of growth of this proliferative fraction (PF) correlates inversely with time-to-first-treatment and directly with poor outcome prognostic markers. Furthermore, since the dividing cells upregulate DNA mutators such as AID and APOBEC family members, the PF has a greater propensity for acquiring new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy for patients with worst outcome category. The PF (CXCR4DimCD5Bright) differs by more than 1000 genes from the resting fraction (RF, CXCR4Bright CD5Dim); these genes relate to replication, migration, and regulation of gene expression. Some of these genes are also preferentially expressed in the PF of U-CLL cases. One such gene is Musashi 2 (MSI2). MSI2 regulates gene expression by binding consensus sequences of mRNA and blocking protein translation. High MSI2 expression is involved in proliferation of normal and malignant stem cells, tumorigenesis, and poor outcome. In CLL, high MSI2 mRNA expression has been identified in patients with worse prognosis. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we report studies of the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression. First, we evaluated MSI2 protein levels by flow cytometry in CD19+CD5- and CD19+CD5+ cells from healthy donors (HDs; n=25) and in CD19+CD5+ from CLL patients (n=55). Higher MSI2 expression was observed in CLL than HD B cells, whereas no differences were found in CD19+CD5+ and CD19+CD5- cells from HDs. Also, MSI2 protein levels were higher in U-CLL than M-CLL, and M-CLL B cells express more MSI2 than HDs. Finally, MSI2 protein levels correlated with CD38, a CLL poor prognosis marker, suggesting MSI2 associates with poor prognosis in CLL. Within the leukemic clone, we observed 25% more MSI2 in the PF than the Int (defined as CXCR4intCD5int) and 15% more in the Int than the RF (PF>Int >RF). The PF contains 40% more MSI2 than the RF, suggesting the highest amounts of MSI2 protein are in dividing and recently-divided cells. Since CLL B cell proliferation occurs in the microenvironment of lymphoid organs, presumably delivered by external signals, we tested whether such signals could stimulate MSI2 expression. Results indicate that CD40L+IL4 and Toll-like 9 stimulation plus IL15 (TLR9+IL5) increase MSI2 synthesis in vitro 1.4 and 1.8 fold, respectively. The increases are associated with the appearance of phospho ERK and AKT. Also, inhibition of AKT signaling by a PI3K inhibitor decreases MSI2 levels, suggesting AKT is involved in MSI2 synthesis. In this regard, signals from the microenvironment inducing cell growth and proliferation promote MSI2 synthesis in B cells from CLL patients. In addition, cells entering the cell cycle (Ki-67+ cells, those incorporating the thymidine analogue EdU, and cells in S, G2 and M cell cycle phases) express higher MSI2 levels than quiescent cells. Furthermore, dividing cells contain higher MSI2 levels than non-dividing cells as determined by CFSE dilution. These results suggest that cells entering the cell cycle or recently dividing have greater MSI2 expression. Since high MSI2 levels associate with cell proliferation and its inhibition is said to promote apoptosis, we studied the effect of MSI2 downregulation in the CLL MEC1 cell line to determine if MSI2 is a potential therapeutic target for CLL. Our findings show that siRNAs decrease MSI2 mRNA (80%) and protein (40%) levels compared to negative controls. Downregulation of MSI2 in MEC1 led to cleaved caspase 3, TRAIL R1 and R2, FADD, TNFR1, P21, P27, phosho-p53, and decreased levels of inhibitors of apoptosis such as cIAP2 and survivin. Hence these data suggest downregulation of MSI2 in CLL cells could induce apoptosis. Thus, MSI2 levels are higher in B cells from poor outcome patients and also in the dividing/divided cells of the PF before and after stimulation. Also, MSI's downregulation induces apoptosis of CLL cell line. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention. Inhibiting its function and its role in cell proliferation will likely abort clonal evolution and disease progression, and make CLL an even more chronic and manageable condition. Disclosures No relevant conflicts of interest to declare.

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Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways

Scientific Reports ◽

10.1038/srep05511 ◽

2014 ◽

Vol 4 (1) ◽

Cited By ~ 11

Author(s):

Kun-Chun Chiang ◽

Ke-Hung Tsui ◽

Li-Chuan Chung ◽

Chun-Nan Yeh ◽

Tsui-Hsia Feng ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Prostate Carcinoma ◽

Carcinoma Cells

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Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180110141827 ◽

2018 ◽

Vol 18 (7) ◽

pp. 1025-1031

Author(s):

Cheng Luo ◽

Di Wu ◽

Meiling Chen ◽

Wenhua Miao ◽

Changfeng Xue ◽

...

Keyword(s):

Cell Proliferation ◽

Confocal Microscopy ◽

Protein Expression ◽

Mtt Assay ◽

Hepg2 Cells ◽

Molecular Mechanisms ◽

Migration And Invasion ◽

Rt Pcr ◽

Gene And Protein Expression ◽

Simulated Hypoxia

Background: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. Methods: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results： ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours’ relatively hypoxia incubation. Conclusion: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly

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LMNB2 promotes the progression of colorectal cancer by silencing p21 expression

Cell Death and Disease ◽

10.1038/s41419-021-03602-1 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Chen-Hua Dong ◽

Tao Jiang ◽

Hang Yin ◽

Hu Song ◽

Yi Zhang ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Gene Set Enrichment Analysis ◽

Molecular Therapy ◽

Cycle Progression ◽

Cancer Tissues

AbstractColorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

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miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

Cancer Cell International ◽

10.1186/s12935-020-01725-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jiong Ma ◽

Chunxia Zhou ◽

Xuejun Chen

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Protein Expression ◽

Signaling Pathway ◽

Luciferase Reporter ◽

Cell Proliferation And Migration ◽

Hh Signaling ◽

And Migration ◽

Proliferation And Migration

Abstract Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

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mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators

AJP Cell Physiology ◽

10.1152/ajpcell.00165.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. C1457-C1465 ◽

Cited By ~ 104

Author(s):

Gustavo A. Nader ◽

Thomas J. McLoughlin ◽

Karyn A. Esser

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cyclin D1 ◽

Ribosomal Rna ◽

Molecular Mechanisms ◽

D1 Protein ◽

Rna Content ◽

Cyclin D1 Protein ◽

Rb Phosphorylation ◽

Myotube Hypertrophy

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.

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BCL2 protein expression parallels its mRNA level in normal and malignant B cells

Blood ◽

10.1182/blood-2004-01-0243 ◽

2004 ◽

Vol 104 (9) ◽

pp. 2936-2939 ◽

Cited By ~ 17

Author(s):

Yulei Shen ◽

Javeed Iqbal ◽

James Z. Huang ◽

Guimei Zhou ◽

Wing C. Chan

Keyword(s):

B Cells ◽

Protein Expression ◽

B Cell ◽

Posttranscriptional Regulation ◽

Cell Lymphoma ◽

Dominant Role ◽

Mrna Level ◽

B Cell Lymphoma ◽

Transcriptional Level ◽

Bcl2 Protein

Abstract The regulation of B-cell lymphoma 2 (BCL2) protein expression in germinal center (GC) B cells has been controversial. Previous reports have indicated posttranscriptional regulation plays a dominant role. However, a number of recent studies contradicted these reports. Using real-time polymerase chain reaction (PCR) and Standardized Reverse Transcriptase-PCR (StaRT-PCR), we measured the level of mRNA expression in GC, mantle zone (MNZ), and marginal zone (MGZ) cells from laser capture microdissection. Both quantitative RT-PCR measurements of microdissected GC cells from tonsils showed that GC cells had low expression of BCL2 transcripts commensurate with the low protein expression level. These results are in agreement with microarray studies on fluorescence-activated cell sorter (FACS)-sorted cells and microdissected GC cells. We also examined BCL2 mRNA and protein expression on a series of 30 cases of diffuse large B-cell lymphoma (DLBCL) and found, in general, a good correlation. The results suggested that BCL2 protein expression is regulated at the transcriptional level in normal B cells and in the neoplastic cells in most B-cell lymphoproliferative disorders.

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