The PI3K/Akt/FOXO3a/p27 signaling contributes to anti-inflammatory drug-suppressed proliferation of human osteoblasts

Akt has been reported to suppress p27^Kip1 promoter activity through Forkhead box O (FOXO) in different kinds of cells. Previous studies indicated that anti-inflammatory drugs up-regulated p27^Kip1, and this effect might play an important role in anti-inflammatory drug-induced cell cycle arrest of human osteoblasts (hOBs). In this study, we hypothesized that these drugs might increase p27^Kip1 expression in hOBs by altering the Akt/FOXO signaling. We tested this hypothesis by examining the influences of three anti-inflammatory drugs on the levels and/or activities of Akt, FOXO and p27^Kip1 as well as the relationship between these factors and proliferation of hOBs. We tested the effects of indomethacin (10⁻⁵ and 10⁻⁴ M), celecoxib (10⁻⁶ and 10⁻⁵ M), and dexamethasone (10⁻⁷ and 10⁻⁶ M) using PI3K inhibitor, LY294002 (10⁻⁵ M) as the basis of comparison. The three drugs suppressed the canonical level of phosphorylated Akt in hOBs. This was accompanied by elevated FOXO3a level and increased promoter activity, mRNA expression and protein level of p27^Kip1. Furthermore, the anti-inflammatory drugs suppressed the EGF-induced increases in proliferation, phosphorylation, and nucleus translocation of Akt. Simultaneously, they suppressed EGF-induced decreases of FOXO3a nucleus accumulation and p27^Kip1 mRNA expression. On the other hand, FOXO silencing significantly attenuated the drug-induced up-regulation of p27^Kip1 and suppression of proliferation in hOBs. To the best of our knowledge, this study represents the first to demonstrate that Akt/FOXO3a/p27^Kip1 pathway contributes to suppression of hOB proliferation by anti-inflammatory drugs. We suggest that anti-inflammatory drugs suppress hOB proliferation, at least partly, through inactivating Akt, activating FOXO3a, and eventually up-regulating p27^Kip1 expression.     

辛基酚对MCF-7乳腺癌细胞增殖及凋亡作用研究

目的探讨辛基酚（octylphenols,OP）诱导乳腺癌细胞凋亡及抑制乳腺癌细胞增殖的可能机制。方法应用MTT法及流式细胞仪检测OP对乳腺癌细胞MCF-7的增殖抑制及细胞周期的变化;Real Time PCR和Western blot法检测OP对MCF-7乳腺癌细胞中细胞周期相关蛋白p27Kip1和CyclinE的mRNA及蛋白表达的影响及对凋亡抑制蛋白PAK4表达的影响。结果 OP对MCF-7细胞增殖有明显的抑制作用,MCF-7细胞阻滞在G0/G1期,使S期的细胞减少;Real Time PCR和Western Blot检测发现培养的MCF-7细胞,随着OP的处理浓度增加,CyclinE蛋白在分子水平和蛋白水平均降低,p27Kip1的表达量升高。结论 OP可能通过调控细胞周期相关蛋白的表达,抑制MCF-7乳腺癌细胞增殖。     

A novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells

A key asset of cytotoxic drugs in cancer therapeutics is their ability to discriminate between proliferating and mitotically inert cells and eliminate preferentially neoplastic ones. We have designed a high throughput-compatible mammalian cell-based assay for the discovery of cytotoxic drugs, which selectively kill proliferation-competent target cells. This cytotoxic drug discovery assay is based on a transgenic CHO-K1-derived cell line engineered for a conditional G1-specific growth arrest following tetracycline-responsive overexpression of the human cyclin-dependent kinase inhibitor p27(Kip1). The CHO-derived cell line CHO-p27(Kip1) shows wild type proliferation rates and can be expanded in the presence of tetracycline antibiotics when p27(Kip1) expression is repressed. Upon withdrawal of regulating antibiotics CHO-p27(Kip1) differentiates into a 1:1 mixed population consisting of two different proliferation phenotypes: (i) a G1-arrested cell population induced by heterologous expression of p27(Kip1) which mimics mitotically inactive terminally differentiated cells and (ii) a proliferation-competent cell population which eliminated the p27(Kip1) expression unit and imitates neoplastic cell characteristics. Addition of chemical or metabolic libraries to CHO-p27(Kip1) populations cultivated in tetracycline-free medium followed by scoring for cell viability will reveal cytotoxic drug candidates associated with a high viability ratio of proliferation-competent/arrested populations.We have validated the cell-based cytotoxic drug discovery assay using the clinically licensed cancer drugs mitomycin C, doxorubicin, etoposide and 5-fluorouracil. Comparative proof-of-concept studies showed that these top-prescribed cancer therapeutics preferentially eliminate proliferating cells while showing less interference with the viability of G1-arrested cell populations. These results demonstrate the CHO-p27(Kip1)-based cytotoxic drug finder technology is ready-to-apply for high throughput screenings of chemical as well as metabolic libraries to discover novel cancer therapeutics which show reduced cytotoxicity on terminally differentiated cells.     

UBE2M-mediated p27(Kip1) degradation in gemcitabine cytotoxicity

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.     

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation

Aim:

Probucol, an anti-hyperlipidemic drug, has been reported to exert antitumor activities at various stages of tumor initiation, promotion and progression. In this study we examined whether the drug affected glioma cell growth in vitro and the underlying mechanisms.

Methods:

Human glioma U87 and glioblastoma SF295 cell lines were used. Cell proliferation was accessed using the cell proliferation assay and BrdU incorporation. The phosphorylation of AMPK, liver kinase B1 (LKB1) and p27^Kip1 was detected by Western blot. The activity of 26S proteasome was assessed with an in situ fluorescent substrate. siRNAs were used to suppress the expression of the relevant signaling proteins.

Results:

Treatment of U87 glioma cells with probucol (10–100 μmol/L) suppressed the cell proliferation in dose- and time dependent manners. Meanwhile, probucol markedly increased the ROS production, phosphorylation of AMPK at Thr172 and LKB1 at Ser428 in the cells. Furthermore, probucol significantly decreased 26S proteasome activity and increased p27^Kip1 protein level in the cells in an AMPK-dependent manner. Probucol-induced suppression of U87 cell proliferation could be reversed by pretreatment with tempol (a superoxide dismutase mimetic), MG132 (proteasome inhibitor) or compound C (AMPK inhibitor), or by gene silencing of LKB1, AMPK or p27^Kip1. Similar results were observed in probucol-treated SF295 cells.

Conclusion:

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation, which reduces 26S proteasome-dependent degradation of p27^Kip1.     

Nebivolol inhibits human aortic smooth muscle cell growth: effects on cell cycle regulatory proteins

An enhanced vasoconstriction and vascular smooth muscle cell proliferation are involved in pathogenesis of hypertension. Beta3-blockers are effective for treatment of hypertensive patients. Recently the new beta1-receptor blocker nebivolol showed a different hemodynamic profile from those of other classic beta-blockers. In this study we hypothesized that nebivolol may also have different effects on smooth muscle cell proliferation compared with other beta-blockers such as atenolol. Human aortic smooth muscle cells (SMCs) were cultured, and cell growth was determined by increase in cell number. Growth-signaling molecules such as mitogen-activated protein kinase (p42mapk) and S6-kinase (p70S6K) and cell-cycle regulatory proteins (i.e., Cdk2, p27Kip1, and pRb) were analyzed by immunoblotting. In cultured human aortic SMCs, cell number was markedly increased in response to 5% fetal calf serum (FCS) over 6 days (87 +/- 11 x 10(3)/well), which was inhibited by nebivolol (10(-8)-10(-5) M; 25 +/- 2 x 10(3)/well; n = 6; p < 0.05), but not by atenolol. 5% FCS activated p42mapk, S6K, and Cdk2, but downregulated p27Kip1 and hyperphosphorylated pRb. Nebivolol prevented Cdk2 activation without influencing p42mapk, S6K, pRB, and p27Kip1. Thus, the new beta1-blocker nebivolol exhibits antiproliferative effect on human SMC through inactivation of Cdk2. This effect of nebivolol may have advantages over other beta-blockers in treatment of patients with cardiovascular disease.     

UBE2M-mediated p27 degradation in gemcitabine cytotoxicity

Gemcitabine (2′-deoxy-2′, 2′-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27^Kip1 protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27^Kip1 protein degradation. The induction of UBE2M and reduction of p27^Kip1 by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27^Kip1. Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27^Kip1. Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27^Kip1 expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27^Kip1 in drug resistance.     

Helioxanthin suppresses the cross talk of COX‐2/PGE2 and EGFR/ERK pathway to inhibit Arecoline‐induced Oral Cancer Cell (T28) proliferation and blocks tumor growth in xenografted nude mice

Helioxanthin, an active compound from Taiwania cryptomerioides Hayata, has been shown to have various biological activities. However, their anticancer effect in oral squamous cell carcinoma has not been well established yet. Helioxanthin inhibited the proliferation of oral squamous cell carcinoma cells in a dose‐dependent manner by inducing G2/M phase arrest. Similarly, helioxanthin inhibited cyclooxygenase‐2, (COX‐2), phosphorylated EGFR, and extracellular‐signal‐regulated kinases (ERK) protein level and further reduced the nuclear accumulation of phosphorylated epidermal growth factor receptor (pEGFR) and activator protein‐1(AP‐1) family protein, c‐fos. Moreover, helioxanthin at the dose of 20 and 30 mg kg^?1 for 15 days reduced the tumor growth in animal model. This study demonstrated that Helioxanthin exerts its anticancer activity against oral cancer cells by downregulating EGFR/ERK/c‐fos signaling pathway to inhibit COX‐2 level and by activating cyclin‐dependent kinase inhibitor (p27) to further induce G2/M cell cycle arrest. This helioxanthin may serve as a novel candidate for oral cancer prevention. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2045–2056, 2016.     

Physalis angulata induced G2/M phase arrest in human breast cancer cells.

Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.     

Molecular mechanism of cell cycle blockage of hepatoma SK-Hep-1 cells by Epimedin C through suppression of mitogen-activated protein kinase activation and increased expression of CDK inhibitors p21(Cip1) and p27(Kip1).

Reports elsewhere demonstrated that Epimedin C, a constituent isolated from the leaves of Epimedium sagittatum, possessed anti-tumor activity. However, its mechanism of action remains unresolved. Using SK-Hep-1 cells, a poorly-differentiated hepatoma subline, as an experimental model, we present evidence here that the anti-tumor activity of Epimedin C may involve cell cycle blockage. Immunoblotting analyses demonstrated that Epimedin C caused a decreased expression of hyperphosphorylated retinoblastoma (Rb) protein, cyclin D1, c-Myc, and c-Fos. In parallel, we measured the kinase activities and found that CDK2 and CDK4 were suppressed with commensurate increased levels of CDK inhibitors, p21(Cip1) and p27(Kip1). These data suggested that Epimedin C arrested the proliferation of these cells at G0/G1 phase through inhibition of CDK2 and CDK4 activities via an increased induction of p21(Cip1) and p27(Kip1). Alternatively, we investigated whether the anti-proliferative effect of Epimedin C on these cells might involve MAP kinase cascade. Using western blotting technique, we demonstrated that Epimedin C also selectively decreased ERK1/2 phosphorylation. Among the downstream effectors of ERK examined, we found that Epimedin C selectively decreased the expression of c-Fos, but not c-Jun. By EMSA assay, we further demonstrated that decreased c-Fos resulted in the downregulation of AP-1/DNA binding activity. Taken together, the molecular mechanisms of anti-tumor activity of Epimedin C may be proceeded by the combined effects of the cell cycle blockage via either the inhibition of CDK2 and CDK4 activities, with commensurate increase in their inhibitors, p21(Cip1) and p27(Kip1) or negatively modulates the ERK/c-Fos/AP-1 signaling pathway.     

人神经前体细胞分化过程中p27~(Kip1)的作用及其调节机制研究

目的研究视黄酸(RA)诱导人神经前体细胞(hNPCs)分化的过程中,细胞周期调节蛋白p27Kip1的变化及其调节机制,以掌握hNPCs的增殖分化特性。方法取原代hNPCs体外培养,在细胞进入对数生长期时给予RA诱导,在诱导d3、5、7收集细胞,用流式细胞分析术观察细胞周期的变化、用Westernblot法观察p27Kip1及其相关的细胞周期调节蛋白p21Cip1、cdk2的变化,以及参与p27Kip1泛素-蛋白酶降解途径的重要因子skp2的变化。结果RA诱导d3,hNPCs细胞G0/G1期的比例由诱导前的65.20%上升至80.72%;而S期的比例由诱导前的28.39%下降到8.62%;p27Kip1蛋白表达在RA诱导d3增加,并在d5达到高峰;p21Cip1和cdk2的表达在RA诱导前后变化不明显,但反映cdk2活性的p-cdk2蛋白的表达在RA诱导后明显下降;skp2的表达在RA诱导后明显下降。结论在RA诱导hNPCs分化的过程中,p27Kip1蛋白泛素降解途径受阻,致使其含量增加,并通过抑制cdk2的活性而发挥了促进hNPCs分化的作用。     

白血病患者P27(Kip1)表达的初步研究

目的：研究白血病患P27（Kipl)的表达及其与白血病预后的关系。方法：用蛋白印迹法（Western blot)加化学发光法研究23例急性白血病患骨髓或外周血中白血病细胞的P27（Kipl)蛋白表达。结果：P27（Kipl)总阳性率为78．26％（18／23），但表达强度不一。急性非淋巴细胞白血病（ANLL）阳性率82．35％（14／17），4例急性淋巴细胞白血病（ALL）中2例阳性一，1例慢性粒细胞白血病急变期（CML－BC）为阳性，1例淋巴肉瘤白血病为阳性。P27（Kipl)低表达组与高表达组生存率无显差异。13例已死亡的ANLL中P27（Kipl)高表达组总生存期长于低表达组（秩和检验P＝0．05）。所有进行化疗的18例患达到完全缓解（CR）P27（Kipl)平均表达强度高于未完全缓解（NR），但无统计学意义（秩和检验P＞0．05）。诊断时外周血白细胞数、血乳酸脱氢酶水平与P27（Kipl)无相关性。结论：P27（Kipl)的表达与ANLL的预后可能相关，高P27（Kipl)预后好。     

Role of connexin 43 in cadmium‐induced proliferation of human prostate epithelial cells

Connexins (Cxs), the subunits of gap junction channels, are involved in many physiological processes. Aberrant control of Cxs and gap junction intercellular communication may contribute to many diseases, including the promotion of cancer. Cd exposure is associated with increased risk of human prostate cancer and benign prostatic hyperplasia. The roles of Cxs in the effects of Cd on the prostate have, however, not been reported previously. In this study, the human prostate epithelial cell line RWPE‐1 was exposed to Cd. A low dose of Cd stimulated cell proliferation along with a lower degree of gap junction intercellular communication and an elevated level of the protein Cx43. Cd exposure increased the levels of intracellular Ca²⁺ and phosphorylated Cx43 at the Ser368 site. Knockdown of Cx43 using siRNA blocked Cd‐induced proliferation and interfered with the Cd‐induced changes in the protein levels of cyclin D1, cyclin B1, p27^Kip1 (p27) and p21^Waf1/Cip1 (p21). The increase in Cx43 expression induced by Cd was presumably mediated by the androgen receptor, because it was abolished upon treatment with the androgen receptor antagonist, flutamide. Thus, a low dose of Cd promotes cell proliferation in RWPE‐1, possibly mediated by Cx43 expression through an effect on cell cycle‐associated proteins. Cx43 might be a target for prostatic diseases associated with Cd exposure. Copyright © 2017 John Wiley & Sons, Ltd.     

p27(kip1) functional regulation in human cancer: a potential target for therapeutic designs

The mitotic cell cycle is a tightly regulated process that ensures the correct division of one cell into two daughter cells. Progress along the different phases of the cell cycle is positively regulated by the sequential activation of a family of serine-threonine kinases called CDKs (Cyclin Dependent Kinases). Their activity is counteracted by small proteins known as CDK inhibitors (CKI) that ensure the correct timing of CDK activation in the different phases of the cell cycle. The present review will deal with the role of one of this CKI, p27(kip1), in human cancer, focusing in particular on the mechanisms underlying its functional inactivation in tumor cells. p27(kip1) protein downregulation is usually achieved by proteasomal degradation and is often correlated to a worse prognosis in several types of human cancers, resulting in the reduction of disease free and overall survival. More recently, it has been proposed that p27(kip1) protein, rather than degraded, can be functionally inactivated. The mechanisms and the implications of these two types of p27(kip1) deregulation will be discussed and some potential therapeutic approaches targeting p27(kip1) functions will be proposed.     

Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies

In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular.     

Resveratrol increases serine15-phosphorylated but transcriptionally impaired p53 and induces a reversible DNA replication block in serum-activated vascular smooth muscle cells

Resveratrol (RV), a polyphenolic stilbene derivative, has been proposed to exert a plethora of beneficial cardiovascular effects. Of these, in particular, inhibition of vascular smooth muscle cell (VSMC) proliferation shows great promise for preventing cardiovascular disease. In the present study, we show that RV leads to a reversible arrest in early S phase of the VSMC cycle, accompanied by an accumulation of hyperphosphorylated retinoblastoma protein. In contrast to studies with other cell systems, RV decreases cellular levels of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). This is of particular interest because phosphorylated p53 protein (serine(15)) is strongly enhanced by this substance. We further found that RV only slightly inhibits phosphorylation of Erk 1/2, protein kinase B/Akt, and p70(S6) kinase upon serum stimulation. Thus, inhibition of these kinases is not likely to contribute to the cell cycle effect of RV. Importantly, the observed S phase arrest is not linked to an increase in apoptotic cell death: there was no detectable increase in apoptotic nuclei and in levels of the proapoptotic protein Bax. This is the first study elucidating the molecular pathways mediating the antiproliferative properties of RV in VSMCs.