Decreased Cyclin B1 Research Articles

Intermittent Fasting Attenuates Obesity-Induced Triple-Negative Breast Cancer Progression by Disrupting Cell Cycle, Epithelial-Mesenchymal Transition, Immune Contexture, and Proinflammatory Signature.

Obesity is associated with one-fifth of cancer deaths, and breast cancer is one of the obesity-related cancers. Triple-negative breast cancer (TNBC) lacks estrogen and progesterone receptors and human epidermal growth factor receptor 2, leading to the absence of these therapeutic targets, followed by poor overall survival. We investigated if obesity could hasten TNBC progression and intermittent fasting (IF) could attenuate the progression of obesity-related TNBC. Our meta-analysis of the TNBC outcomes literature showed that obesity led to poorer overall survival in TNBC patients. Fasting-mimicking media reduced cell proliferation disrupted the cell cycle, and decreased cell migration and invasion. IF decreased body weight in obese mice but no change in normal mice. Obese mice exhibited elevated plasma glucose and cholesterol levels, increased tumor volume and weight, and enhanced macrophage accumulation in tumors. The obesity-exacerbated TNBC progression was attenuated after IF, which decreased cyclin B1 and vimentin levels and reduced the proinflammatory signature in the obesity-associated tumor microenvironment. IF attenuated obesity-induced TNBC progression through reduced obesity and tumor burdens in cell and animal experiments, supporting the potential of a cost-effective adjuvant IF therapy for TNBC through lifestyle change. Further evidence is needed of these IF benefits in TNBC, including from human clinical trials.

Abstract 406: Novel therapeutic combination targets the growth of letrozole resistant breast cancer through decreased cyclin B1

Abstract As breast cancer cells transition from letrozole-sensitive to letrozole resistance they over-express EGFR, MAPK, and HER2 and acquire enhanced motility and EMT-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Since previous reports from our lab demonstrate that the combination of lapatinib, a dual EGFR and HER2 inhibitor, and glyceollin, a novel phytochemical, induce apoptosis in hormone-dependent aromatase inhibitor (AI)-resistant breast cancer cells, we hypothesized that combination therapy could reverse motility in hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by altering proteins involved in motility and cell cycle progression. We compared the effects of 10 μM glyceollin ± 5 μM lapatinib treatment on both cell lines and evaluated cell viability, cell migration, cell cycle analysis, and protein expression of cell cycle regulators. Glyceollin ± lapatinib treatment caused a time-dependent decrease in cell viability which was comparable between each cell line. When wound healing assays were conducted, glyceollin prevented the migration in the AC-1 and LTLT-Ca cell lines by approximately 55% and 51% respectively, while combination treatment exhibited synergistic inhibitory properties by preventing 88% and 93% wound closure in the AC-1 cells and LTLT-Ca cells, respectively. To determine if the glyceollin + lapatinib-induced decrease in cell viability was a consequence of cell cycle dysregulation, flow cytometric analyses were performed and drug treatment had no effect on the AC-1 cell cycle distribution however, combination therapy reduced the number of LTLT-Ca cells in the G1/G0 phase, while causing accumulation of the cells in both the S phase and G2/M phase. Since combination therapy altered the cell cycle distribution of the LTLT-Ca, we measured the effect of glyceollin ± lapatinib treatment on the expression of key cell cycle regulators such as p21, p27, cyclin B1, cdk1, and cdk2. Interestingly, while there there was no effect on protein expression in the AC-1 cells, glyceollin ± lapatinib treatment selectively decreased the expression of cyclin B1 in the LTLT-Ca cells, without significantly altering the expression of the other proteins. These results suggest that while both AI-sensitive and AI-resistant cells respond favorably to the growth inhibitory and anti-migratory properties of glyceollin + lapatinib, the LTLT-Ca cells exhibit increased sensitivity to cell cycle progression, which may represent an opportunity to exploit and refine second-line therapies for AI-resistant cancers. This work was supported by NIH grant #1SC1GM126617 and U54MD007582. Citation Format: Bipika Banjara, Jankiben R. Patel, A Michael Davidson, Syreeta L. Tilghman. Novel therapeutic combination targets the growth of letrozole resistant breast cancer through decreased cyclin B1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 406.

Novel Therapeutic Combination Targets the Growth of Letrozole-Resistant Breast Cancer through Decreased Cyclin B1.

As breast cancer cells transition from letrozole-sensitive to letrozole-resistant, they over-express epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and human epidermal growth factor receptor 2 (HER2) while acquiring enhanced motility and epithelial-to-mesenchymal transition (EMT)-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Interestingly, glyceollin inhibits the proliferation and tumor progression of triple-negative breast cancer (TNBC) and estrogen-independent breast cancer cells; however, it is unlikely that a single phytochemical would effectively target aromatase-inhibitor (AI)-resistant metastatic breast cancer in the clinical setting. Since our previous report indicated that the combination of lapatinib and glyceollin induced apoptosis in hormone-dependent AI-resistant breast cancer cells, we hypothesized that combination therapy would also be beneficial for hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by decreasing the expression of proteins associated with proliferation and cell cycle progression. While glyceollin + lapatinib treatment caused comparable inhibitory effects on the proliferation and migration in both cell lines, combination treatment selectively induced S and G2/M phase cell cycle arrest of the LTLT-Ca cells, which was mediated by decreased cyclin B1. This phenomenon may represent a unique opportunity to design novel combinatorial therapeutic approaches to target hormone-refractory breast tumors.

MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.

Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

N6 -methyladenosine (m6 A) RNA modification of G protein-coupled receptor 133 increases proliferation of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) accounts for almost 40% of lung cancers, leading to significant associated morbidity and mortality rates. However, the mechanism of LUAD tumorigenesis remains far from clear. Here, we scanned down‐regulated genes involved in LUAD sourced from The Cancer Genome Atlas and Gene Expression Omnibus data and focused on G protein‐coupled receptor 133 (GPR133). We offer compelling evidence that GPR133 was expressed at low levels in the setting of LUAD, and higher expression was positively related to a better prognosis among patients with LUAD. Functionally, GPR133 inhibited cell proliferation and tumor growth in vitro and in vivo. Regarding the mechanism, flow cytometry assays and western blot assays showed that GPR133 enhanced p21 and decreased cyclin B1 expression, thus triggering LUAD cells at G2/M‐phase arrest. Consistent with this, we evaluated the expression levels of cell‐cycle biomarkers and found that bioinformatics analysis combined with N6‐methyladenosine (methylation at the N6 position in adenosine) RNA immunoprecipitation‐qPCR assay indicated that GPR133 expression was down‐regulated by this modification. Moreover, we observed that methyltransferase‐like 3 was impaired in LUAD, and that it is able to significantly increase levels of GPR133 by enhancing its RNA stability. In conclusion, we found that GPR133 expression was down‐regulated in LUAD via N6‐methyladenosine modification. Increasing GPR133 levels could suppress LUAD cell proliferation and tumor growth.

Cucurbitacin B regulates lung cancer cell proliferation and apoptosis via inhibiting the IL-6/STAT3 pathway through the lncRNA XIST/miR-let-7c axis

Context Lung cancer, the most common type of cancer, has a high mortality rate. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae plants, has antitumor effects. Objective We investigated the role of CuB on lung cancer and its potential mechanisms. Materials and methods A549 cells were treated with 0.1, 0.3, 0.6, and 0.9 μM CuB for 12, 24, and 48 h or untreated. Gene and protein levels were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Enzyme-linked immunosorbent assay (ELISA) detected inflammatory factors levels (TNF-α and IL-10). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and colony formation assays measured cell viability, apoptosis, and proliferation. The interaction between miR-let-7c and long non-coding RNA X inactive-specific transcript (XIST) or interleukin-6 (IL-6) was verified by dual-luciferase reporter assays. Results CuB treatment inhibited the proliferation of lung cancer cells and promoted cell apoptosis, and increased the expression of Bax and cleaved caspase3, decreased cyclin B1 and Bcl-2 expression. CuB suppressed XIST and IL-6 expression, and enhanced miR-let-7c expression. XIST silencing enhanced the inhibitory effect of CuB on cell proliferation and the promotion effect on apoptosis via upregulating miR-let-7c. Moreover, XIST targeted miR-let-7c to activate the IL-6/STAT axis. MiR-let-7c overexpression enhanced the regulatory effect of CuB on proliferation and apoptosis via suppressing the IL-6/STAT3 pathway. Discussion and conclusion CuB regulated cell proliferation and apoptosis by inhibiting the XIST/miR-let-7c/IL-6/STAT3 axis in lung cancer. These findings indicate CuB may have the possibility of clinical application in lung cancer treatment.

LncRNA MALAT1 participates in metformin inhibiting the proliferation of breast cancer cell.

In recent years, the repurposing of conventional and chemotherapeutic drugs is recognized as an alternative strategy for health care. The main purpose of this study is to strengthen the application of non‐oncological drug metformin on breast cancer treatment in the perspective of epigenetics. In the present study, metformin was found to inhibit cell proliferation, promote apoptosis and induce cell cycle arrest in breast cancer cells at a dose‐dependent manner. In addition, metformin treatment elevated acH3K9 abundance and decreased acH3K18 level. The expression of lncRNA MALAT1, HOTAIR, DICER1‐AS1, LINC01121 and TUG1 was up‐regulated by metformin treatment. In metformin‐treated cells, MALAT1 knock‐down increased the Bax/Bcl2 ratio and enhanced p21 but decreased cyclin B1 expression. The expression of Beclin1, VDAC1, LC3‐II, CHOP and Bip was promoted in the cells received combinatorial treatment of metformin and MALAT1 knock‐down. The reduced phosphorylation of c‐Myc was further decreased in the metformin‐treated cells in combination with MALAT1 knock‐down than metformin treatment alone. Taken together, these results provide a promising repurposed strategy for metformin on cancer treatment by modulating epigenetic modifiers.

Deciphering the Molecular Mechanism of Spontaneous Senescence in Primary Epithelial Ovarian Cancer Cells.

Spontaneous senescence of cancer cells remains a puzzling and poorly understood phenomenon. Here we comprehensively characterize this process in primary epithelial ovarian cancer cells (pEOCs). Analysis of tumors from ovarian cancer patients showed an abundance of senescent cells in vivo. Further, serially passaged pEOCs become senescent after a few divisions. These senescent cultures display trace proliferation, high expression of senescence biomarkers (SA-β-Gal, γ-H2A.X), growth-arrest in the G1 phase, increased level of cyclins D1, D2, decreased cyclin B1, up-regulated p16, p21, and p53 proteins, eroded telomeres, reduced activity of telomerase, predominantly non-telomeric DNA damage, activated AKT, AP-1, and ERK1/2 signaling, diminished JNK, NF-κB, and STAT3 pathways, increased formation of reactive oxygen species, unchanged activity of antioxidants, increased oxidative damage to DNA and proteins, and dysfunctional mitochondria. Moreover, pEOC senescence is inducible by normal peritoneal mesothelium, fibroblasts, and malignant ascites via the paracrine activity of GRO-1, HGF, and TGF-β1. Collectively, pEOCs undergo spontaneous senescence in a mosaic, telomere-dependent and telomere-independent manner, plausibly in an oxidative stress-dependent mechanism. The process may also be activated by extracellular stimuli. The biological and clinical significance of pEOC senescence remains to be explored.

MiR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase.

Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.

Antitumor properties of Salvianolic acid B against triple-negative and hormone receptor-positive breast cancer cells via ceramide-mediated apoptosis.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options. It is urgent to develop new therapeutics against this disease. Salvinolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bunge, a well-known Chinese medicine for treating various diseases without appreciable adverse effects. To understand the antitumor properties of Sal-B against TNBC, we analyzed its effects on the cell viability, cell cycle and apoptosis of triple-negative MDA-MB-231 cells with the hormone receptor-positive MCF-7 cells as the control. The in vitro analysis showed that Sal-B could significantly reduce the cell viability and suppress the proliferation of both MDA-MB-231 and MCF-7 cells with decreased cyclin B1 expression, but with no noticeable cell cycle phase change. In mouse models, Sal-B markedly inhibited the growth, decreased the PCNA expression, and increased the cell apoptosis of MDA-MB-231 tumor xenografts. To understand the antitumor mechanisms, we analyzed the expression levels of ceramides, and anti-apoptotic (Bcl-xL and survivin) and pro-apoptotic (caspase-3 and caspase-8) proteins. We found that Sal-B enhanced the ceramide accumulation and inhibited the anti-apoptotic protein expression. Interestingly, the ceramide accumulation was accompanied by decreased expression of glucosylceramide and GM3 synthases, two key enzymes regulating ceramide metabolism. These findings indicate that Sal-B exerts its antitumor effects at least partially by inducing the ceramide accumulation and ceramide-mediated apoptosis via inhibiting the expression of glucosylceramide and GM3 synthases, which was independent of estrogen receptor α. Sal-B appears to be a promising therapeutic agent against TNBC.

KT2 and RT2 modified antimicrobial peptides derived from Crocodylus siamensis Leucrocin I show activity against human colon cancer HCT-116 cells

Conventional colon cancer treatments have been associated with side effects. Consequently, the discovery of novel effective and safe therapies is urgently needed. Hence, cationic antimicrobial peptides KT2 and RT2 were evaluated towards human colon cancer HCT-116 cells. The MTT assay indicated that both KT2 and RT2 exhibited anticancer activity with good therapeutic indices, and were found to be non-toxic to non-cancerous Vero cells. The IC50 values of KT2 were determined as 111.96 and 90.25 μg/mL while RT2 showed IC50 as 104.07 and 87.84 μg/mL after 12 and 24 h treatments, respectively. Moreover, KT2 and RT2 treatment caused a significant reduction in PI3K, AKT1 and mTOR mRNA expression levels, which resulted in suppression either of HCT-116 proliferation or migration. The mechanism involved in apoptosis induction were due to decreased Bcl-2 and XIAP and increased p53, cytochrome c, caspase-2, caspase-3, caspase-8, and caspase-9 mRNA expression levels. These effects increased the level of cell cycle associated gene p21 and decreased cyclin B1 and cyclin D1 expression.

MiR-519d-3p Inhibits Cell Proliferation and Invasion of Gastric Cancer by Downregulating B-Cell Lymphoma 6

miR-519d inhibits cell growth, migration, and invasion, but its role in gastric cancer (GC) cells is obscure. We showed that miR-519d-3p was lowly expressed in GC tissues and was associated with the clinical stage and lymph node metastasis of GC tissues. We found that miR-519d-3p repressed cell proliferation and invasion of MGC803 cells and delayed the G1/S phase transition, resulting in decreased cyclin B1 and MMP2 and increased E-cadherin levels. Furthermore, miR-519d-3p targeted and downregulated B-cell lymphoma 6 (BCL6) expression. BCL6 overexpression partially abrogated the suppressive function of miR-519d in MGC803 cells. In conclusion, our study demonstrated that miR-519d-3p functions as a tumor suppressor by targeting and downregulating the expression of BCL6 in GC cells.

Abstract 2739: Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo

Abstract Carnosic acid (CA), one of the purified components of rosemary, has shown anti-inflammatory, chemo-preventive, and anticancer activities. In this study, we treated melanoma cell line B16F10 with CA and detected the anticancer effects. After 24-hour treatment, CA decreased cell proliferation rate, wound healing activity, and inhibited colony formation of B16F10. Additionally, CA induced G0/G1 cell cycle arrest, up-regulated p21 expression, and down-regulate cyclin E, cyclin D and CDK4 expression. Carmustine (BCNU) and Lomustine (CCNU) are used in clinical treatment of melanoma. To assay the combinational effects of CA and BCNU or CCNU, B16F10 cells were treated with CA, BCNU, CCNU alone, or CA combined with BCNU or CCNU. Our results showed, BCNU and CCNU arrested cell cycle at G2/M phase after 24-hour treatment. Dramatically, CA enhenced BCNU- and CCNU-induced cell cycle arrest at G2/M phase, and decreased cyclin B1 and p-CDK1 expresion. In the in vivo xenograft model, B16F10 cells (5×105) were inoculated subcutaneously in C57BL/6 mice, and the tumor grew for one week. The mice were divided into six groups and provided different treated strategies: control, CA (50 mg/kg) alone, BCNU (50 mg/kg) alone, CCNU (25 mg/kg) alone, CA combined with BCNU, and CA combined with CCNU. After two-week treatment, compared with control group, CA had better treated effects than BCNU or CCNU. Compared with CA, BCNU or CCNU treatment alone, the combined strategies inhibited the tumor growth in melanoma mice model more effectively. Furthermore, BCNU and CCNU treatment in melanoma mice caused high values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). However, CA significantly decreased AST and ALT values caused by BCNU and CCNU in combined trestment group. Therefore, CA is more safe and effective than BCNU and CCNU, and the combined treatment strategies of CA and BCNU or CCNU result in an increased antitumor efficacy. CA may be suitable for translation to future clinic. Citation Format: Kun-I Lin, Chih-Chien Lin, Ming-Feng Chen, Chang-Han Chen, Li-Yen Shiu. Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2739.

Tellimagrandin I Decreases Cyclin B1 Protein Levels through SP1 in MDA‐MB‐231 Cells

Consumption of walnuts has been shown to inhibit MDA‐MB‐231 breast cancer xenograft growth in nude mice. However, identification of compounds and mechanism of action have not been identified. We recently reported that a walnut extract treatment of the triple‐negative breast cancer cell type, MDA‐MB‐231 cells, could a) arrest the cells in G2/M and decreased cyclin B1 levels, b) decrease mitochondrial membrane potential and release cytochrome c from the mitochondria, and c) increase phosphotidyl serine flipping in the cell membrane, and that these events were associated with cell growth inhibition and apoptotic death. Over‐expression of Cyclin B1 has been shown in breast cancers that lack estrogen receptor, progesterone receptor, and HER2 (i.e. the triple‐negative breast cancer) and is a potential emerging biomarker as well as therapeutic target. In an attempt to conduct detailed mechanistic studies with active compounds from walnut extracts, we were able to identify numerous ellagitannins which induced cell death in breast cancer cells. Of the ellagitannins isolated, tellimagrandin I (TMI) showed the strongest cell death. We have gone on further to characterize the mechanism of action of TMI. We have performed time and dose response analysis of TMI on MDA‐MB‐231 cells for cell death and show that TMI has an IC50 of 60 μM. TMI is also able to reduce Cyclin B1 mRNA and protein levels similar to a walnut extract. We see that mRNA levels are reduced relatively quickly (within 1 hour) while protein levels begin to be reduced at a much later time suggesting that the effects are transcriptional. We have identified other cell cycle specific mRNAs that are also reduced by TMI. A careful examination of all mRNAs that are reduced show that they all have SP1 binding sites. Interestingly, we show that SP1 mRNA levels are also reduced upon TMI treatment in a similar fashion as Cyclin B1. Taken together our results suggest that TMI is able to reduce Cyclin B1 through inhibition of SP1 in MDA‐MB‐231 cells.Support or Funding InformationThis work was supported by a grant from the California Walnut Commission to BW.

Maturation promoting factor destabilization facilitates postovulatory aging-mediated abortive spontaneous egg activation in rat.

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging-mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee-1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging-mediated abortive SEA in rats eggs aged invivo. For this purpose, sexually immature female rats were given a single injection (20IU IM) of pregnant mare serum gonadotropin for 48h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21h post-hCG surge to induce postovulatory aging invivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr-14/Tyr-15 and dephosphorylation of Thr-161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging-mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging-mediated abortive SEA in rat eggs.

CenpH regulates meiotic G2/M transition by modulating the APC/CCdh1-cyclin B1 pathway in oocytes

ABSTRACTMeiotic resumption (G2/M transition) and progression through meiosis I (MI) are two key stages for producing fertilization-competent eggs. Here, we report that CenpH, a component of the kinetochore inner plate, is responsible for G2/M transition in meiotic mouse oocytes. Depletion of CenpH by morpholino injection decreased cyclin B1 levels, resulting in attenuation of maturation-promoting factor (MPF) activation, and severely compromised meiotic resumption. CenpH protects cyclin B1 from destruction by competing with the action of APC/CCdh1. Impaired G2/M transition after CenpH depletion could be rescued by expression of exogenous cyclin B1. Unexpectedly, blocking CenpH did not affect spindle organization and meiotic cell cycle progression after germinal vesicle breakdown. Our findings reveal a novel role of CenpH in regulating meiotic G2/M transition by acting via the APC/CCdh1-cyclin B1 pathway.

The Role of the Y Box Binding Protein 1 C-Terminal Domain in Vascular Endothelial Cell Proliferation, Apoptosis, and Angiogenesis

Different domains of the multifunctional transcription factor Y-box binding protein 1 (YB1) regulate proliferation, differentiation, and apoptosis by transactivating or repressing the promoters of various genes. Here we report that the C-terminal domain of YB1 (YB1 CTD) is involved in endothelial cell proliferation, apoptosis, and tube formation. The oligo pull-down assays demonstrated that YB1 directly binds double-stranded GC box sequences in endothelial cells through the 125-220 amino acids. Adenovirus expression vectors harboring green fluorescent protein (GFP) or GFP-tagged YB1 CTD were constructed and used to infect EA.hy926 endothelial cells. Overexpression of the YB1 CTD significantly increased p21 expression, decreased cyclin B1 expression, and inhibited the proliferation of EA.hy926 cells. YB1 CTD overexpression also increased Bax and active caspase 3 expression, decreased Bcl-2 expression, and induced apoptosis in EA.hy926 cells. Furthermore, overexpression of the YB1 CTD significantly suppressed migration and tube formation in EA.hy926 cells. Finally, YB1 CTD decreased ERK1/2 phosphorylation in EA.hy926 cells. These findings demonstrated vital roles for YB1 in endothelial cell proliferation, apoptosis, and tube formation through transcriptional regulation of GC box-related genes.

Carvacrol inhibits proliferation and induces apoptosis in human colon cancer cells.

Colon cancer is one of the most common malignancies worldwide and has a high mortality rate. Carvacrol is a major component of oregano and thyme essential oils and shows antitumor properties. Here, we investigated the effects of carvacrol on the proliferation and apoptosis of two human colon cancer cell lines, HCT116 and LoVo, and studied the molecular mechanisms of its antitumor properties. We found that carvacrol inhibited the proliferation and migration of the two colon cancer cell lines in a concentration-dependent manner. Cell invasion was suppressed after carvacrol treatment by decreasing the expression of matrix metalloprotease-2 (MMP-2) and MMP-9. Carvacrol treatment also caused cell cycle arrest in the G2/M phase and decreased cyclin B1 expression. Finally, carvacrol induced cell apoptosis in a dose-dependent manner. At the molecular level, carvacrol downregulated the expression of Bcl-2 and induced the phosphorylation of the extracellular-regulated protein kinase and protein kinase B (p-Akt). In parallel, carvacrol upregulated the expression of Bax and c-Jun N-terminal kinase. These results indicate that carvacrol might induce apoptosis in colon cancer cells through the mitochondrial apoptotic pathway and the MAPK and PI3K/Akt signaling pathways. Together, our results suggest that carvacrol may have therapeutic potential for the prevention and treatment of colon cancer.

Cellular and Molecular Effects of NVP-BKM120 in Lymphoblastic Leukemia Cells

Background: PI3K/AKT/mTOR signaling controls most hallmarks of cancer. Constitutive activation of PIK3 pathway in T cells acute lymphoblastic leukemia (T-ALL) has been reported; in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. NVP-BKM120 is an orally bioavailable 2,6-dimorpholine pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. In preclinical studies, it has shown efficacy in a variety of malignancies and is currently being investigated in phase I/II/III clinical testing, mainly for advanced solid tumors (clinicaltrials.gov).Aims: Here, we described the effects of the pan-PI3K/AKT/mTOR inhibitor NVP-BKM120 on T-ALL and BL cell lines.Methods: T-ALLcell lines, Jurkat and MOLT-4, and BL cell lines, NAMALWA and Daudi, were obtained from ATCC. NVP-BKM120 was kindly provided by Novartis, and was prepared as a 10mM stock solution in DMSO. Different concentrations of the drug were used as indicated, where cells treated only with DMSO served as control. Cell viability was measured by MTT. Colony formation was carried out in semisolid methyl cellulose medium. The induction of apoptosis was assessed by annexin-V-APC/PI and by caspase cleavage. The cell cycle was analyzed by a PI-staining method. Western blot analysis was performed by standard methods. Vital staining and flow cytometry analysis with acridine orange was performed for the detection and quantification of acidic vesicular organelles (AVOs). Comparisons between the two groups were performed by the t test. Pvalue <0.05 was considered statistically significant.Results: Cell viability decreased in a concentration-dependent manner, with an IC50 range of 7-8 μM and the clonogenic growth was significantly decreased at the concentration of 1μM and at 10µM the colony formation was completely inhibited in all cells tested. After 6 hours of NVP-BKM120 treatment, we observed an increase in apoptotic cells, as well as an increase in the cleavage of procaspase 3, 8 and 9 in Jurkat, MOLT-4 and NAMALWA cells. Compared with DMSO control, NVP-BKM120 does not have any effect during apoptosis induction in the Daudi cell line. NVP-BKM120 treatment also resulted in G2/M arrest, associated with a decrease in the G1population and a decrease in Cyclin B1 protein levels. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, P70SK6 and 4EBP1, with stable total proteins levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, followed by an increase in BAX:BCL2 ratio. Quantification of AVOs showed a dose-dependent increase of AVOs in all cells tested, after NVP-BKM120 treatment.Conclusions: NVP-BKM120 induced apoptosis in a dose-dependent manner in Jurkat, MOLT-4 and NAMALWA cells, while effects of the drug in the Daudi cell line were mainly cytostatic. In those cells, the induction of apoptosis suggested that the death receptor and mitochondrial pathways were activated after drug treatment. In our study, we found that NVP-BKM120 decreased the phosphorylation levels of BAD, which is linked to a pro-apoptotic activity, and up-regulated the BAX:BCL2 ratio. These results are consistent with the activation of caspase-9 and 3, related to the mitochondrial apoptosis. The accumulation of leukemia cells in the G2/M phase of the cell cycle has been associated with enhanced apoptosis. Our results suggest that decreased Cyclin B1 protein expression might be the molecular mechanism through which NVP-BKM120 induces G2/M arrest. The effects of NVP-BKM120 on the PI3K pathway indicate that NVP-BKM120 treatment may overcome rapamycin-induced AKT activation. P70SK6 and 4EBP1 are the two best-characterized substrates of mTOR1. Hence, the decrease in the phosphorylation levels of P70SK6 and 4EBP1 results in impaired oncogenic protein synthesis. Moreover, P70SK6 is one of the kinases whose phosphorylation by mTOR1 results in opposing autophagy. Accordingly, NVP-BKM120 resulted in increased AVOs, which is a characteristic feature of cells engaged in autophagy. In summary, our present study establishes that NVP-BKM120 effectively presents an antitumor activity against T-ALL and BL cell lines. The reduction of proliferation is possibly by down-regulation of Cyclin B1 and the increased BAX:BCL2 ratio is one of the mechanisms involved in the induction of apoptosis. DisclosuresOff Label Use: NVP-BKM120 is an orally bioavailable 2,6-dimorpholino pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. .

Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway.

Telekin, isolated from the Chinese herb Carpesium divaricatum, has shown anti-proliferation effects against various cancer cells, including hepatocellular carcinoma cells. In this study, we investigated the anti-proliferation mechanisms of telekin in human hepatocellular carcinoma HepG2 cells in vitro. HepG2 cells were treated with telekin. Cell viability was evaluated using MTT assay. Flow cytometry was used to measure cell cycle profiles, ROS level and apoptosis. The protein expression levels were analyzed with Western blotting. Telekin (3.75-30 μmol/L) dose-dependently inhibited the viability of HepG2 cells and induced l apoptosis. Furthermore, the treatment induced cell cycle arrest at G2/M phase, accompanied by significantly increased the phosphorylation of Cdc25A and Cdc2, and decreased Cyclin B1 level. Moreover, the treatment significantly stimulated ROS production, and increased the phosphorylation of p38 and MAPKAPK-2 in the cells. Pretreatment with the antioxidant NAC (2.5, 5, and 10 mmol/L), or the p38 MAPK inhibitor SB203580 (2.5 and 5 μmol/L) dose-dependently attenuated these telekin-induced effects in the cells. Telekin suppresses hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via activating the p38 MAPK pathway.