Tumor suppression by cell competition through regulation of the Hippo pathway

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. Here we investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib(-) cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib(-) cells are prevented from overproliferating. We report that cell competition between scrib(-) and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib(-) cells. Suppressing Yki activation is critical for scrib(-) clone elimination by cell competition, and experimental elevation of Yki activity in scrib(-) cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib(-) cells.     

曲格列酮抑制宫颈癌SiHa细胞增殖中的作用

目的观察曲格列酮对宫颈癌SiHa细胞增殖的影响,并探讨S期激酶相关蛋白2（s-phase kinase associated protein 2,skp2）、p27在此过程中的作用。方法曲格列酮（0、100、200、400μg/ml）处理人宫颈癌SiHa细胞,采用四甲基偶氮唑蓝（MTT）法测定细胞增殖活性,膜联蛋白-V（Annexin V）-FITC法检测细胞凋亡;流式细胞术检测细胞周期;倒置显微镜观察SiHa细胞形态学变化;构建skp2表达质粒并转染SiHa细胞,Western blot检测蛋白的表达。结果曲格列酮明显抑制宫颈癌SiHa细胞增殖,且呈明显时间及浓度依赖性（P〈0.05）;曲格列酮增加SiHa细胞周期G1/S期阻滞,细胞凋亡率未见显著性变化（P〉0.05）;曲格列酮上调SiHa细胞p27表达,下调skp2表达;过表达skp2可明显抑制曲格列酮对p27表达的升高作用,同时抵消曲格列酮对细胞周期抑制的作用。结论曲格列酮通过调节细胞周期而非凋亡途径,抑制宫颈癌SiHa细胞增殖,其机制可能与调节skp2、p27蛋白表达有关。     

Phorbol 12-myristate 13-acetate inhibits FRO anaplastic human thyroid cancer cell proliferation by inducing cell cycle arrest in G1/S phase: evidence for an effect mediated by PKCdelta

Phorbol 12-myristate 13-acetate (PMA) is known to affect a variety of cellular processes, including cell proliferation, differentiation, and migration. PMA has been shown to promote antiproliferative and antimigratory effects in many types of cancer cells. Our findings show that PMA induced a strong antiproliferative effect in two anaplastic (FRO and ARO) and one follicular (ML-1) thyroid cancer cell lines, and increased the fraction of FRO cells in G1 phase of the cell cycle. The fractions in the S and G2 phases were decreased. Moreover, PMA evoked a significant increase in the levels of the cell cycle regulators p21(Waf1/Cip1) and p27(Kip1). The levels of cyclin D3 and the cyclin-dependent kinases cdk4 and cdk6 decreased, as did the phosphorylation of the Rb-protein. PMA did not induce apoptosis. PMA stimulated the translocation of protein kinase C (PKC) alpha, betaI and delta isoforms to the cell membrane. PKCdelta small interfering RNA attenuated the PMA-induced antiproliferative effect and prevented the upregulation of p21(Waf1/Cip1) and p27(Kip1). Prolonged stimulation with PMA decreased the phosphorylation of mitogen-activated protein (MAP) kinase. PMA also decreased the phosphorylation of Akt and evoked a biphasic change in the phosphorylation of the forkhead box class-O protein (FOXO): an increase in phosphorylation, followed by a dephosphorylation. In addition, PMA inhibited FRO, ARO and ML-1 cell migration toward serum. The inactive phorbol ester analog 4alpha-phorbol and the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol were without an effect on proliferation and migration. The results indicate that PMA is an effective inhibitor of thyroid cancer cell proliferation and migration by a mechanism involving PKC-MAP kinase/Akt and FOXO signaling.     

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.     

曲古菌素A对食管癌EC1细胞增殖和细胞周期的影响及其分子机制

目的:探讨不同浓度曲古菌素A对食管癌细胞系EC1 细胞增殖、细胞周期的影响及其对细胞周期调控基因p21 WAF1/CIP1 表达的影响. 方法:用0.3,0.5,1.0 μmol/L 的TSA 处理EC1 细胞,MTT 检测TSA 作用24 、48 h 对EC1 细胞的抑制作用,流式细胞仪检测0.3,0.5,1.0 μmol/L 的TSA 作用24 h 后EC1 细胞周期的改变,Western blot 法检测p21 WAF1/CIP1 变化. 结果:TSA 在0.5 μmol/L 以上时对EC1 细胞有抑制作用;0.3 μmol/L TSA 处理细胞后细胞周期与对照组相比,无明显变化;0.5 μmol/L TSA 处理EC1 细胞后,G0/G1期细胞较对照组明显增加,S 期细胞较对照组明显减少(74.56% ±1.34% vs 62.12%±0.52%;14.52%±1.81% vs 27.50%±0.66%,均P <0.05);0.5,1.0 μmol/L TSA 处理细胞后p21 WAF1/CIP1 表达明显增加(均P<0.05). 结论:一定浓度的TSA 对人食管癌细胞EC 具有的增殖抑制作用,引起EC1 细胞发生G0/G1 期阻滞,其部分机制与p21 WAF1/CIP1 上调有关.     

Nimesulide inhibits proliferation via induction of apoptosis and cell cycle arrest in human gastric adenocarcinoma cell line

AIM: To evaluate the potential role of Nimesulide, a selective COX-2 inhibitor, in proliferation and apoptosis of gastric adenocarcinoma cells SGC7901. METHODS: Cell counts and MTT assay were used to quantify the influence of Nimesulide in the proliferation of SGC7901 cells. Transmission electron microscopy and flow cytometry were used to observe the induction of Nimesulide the apoptosis of SGC7901 cells and influence in the distribution of cell cycle. The expression of P27(kip1) protein was observed by immunocytochemical staining. RESULTS: SGC-7901 Cells treated with Nimesulide at various concentrations exhibited a profound dose- and time-dependent reduction in the proliferation rate over the 72 h test period. The highest survival rate of the cells was 78.7 %, but the lowest being 22.7 %. Nimesulide induced apoptosis of the cells in a dose-dependent and non-linear manner and increased the proportion of cells in the G(0)/G(1) phase and decreased the proportion in the S and G(2)/M phase of the cell cycle. Meanwhile, Nimesulide could up-regulate the expression of P27(kip1) protein. CONCLUSION: The induction of apoptosis and cell cycle arrest are both anti-proliferative responses that likely contribute to the antineoplastic action of nimesulide on SGC-7901 cells. The up-regulation of P27(kip1) gene may contribute to the accumulation of these cells in the G(0)/G(1) phase following treatment with Nimesulide. Selective COX-2 inhibitor may be a new channel of the chemoprevention and chemotherapy for gastric carcinoma.     

Dihydrotestosterone inhibits granulosa cell proliferation by decreasing the cyclin D2 mRNA expression and cell cycle arrest at G1 phase

Hyperandrogenism is known to perturb ovarian physiology resulting in anovulatory conditions. In the ovary, androgens produced by theca-interstitial cells are converted to estrogens in granulosa cells under the influence of FSH and LH. In some of the target organs, including the ovary, androgens are also converted into their 5alpha reduced metabolites. In the present study, we examined the molecular mechanism by which dihydrotestosterone (DHT), a 5alpha reduced metabolite of testosterone, mediates the inhibition of granulosa cell proliferation, using a rat model. Immature female rats were primed with estradiol, followed by DHT administration for 2 d and granulosa cells were cultured in the presence or absence of forskolin. Granulosa cells from the DHT-treated rats showed reduced [(3)H]thymidine incorporation into DNA and reduced cell number in response to forskolin stimulation, compared with control. The decreased responsiveness of DHT-treated granulosa cells to forskolin was not due to increased apoptosis because the expression of cleaved caspase 3 remained the same in both control and DHT-exposed granulosa cells stimulated with forskolin. Forskolin treatment stimulated the expression of cyclin D2 mRNA in control granulosa cells, whereas DHT treatment abolished this response. In vitro DHT treatment of granulosa cells for 48 h resulted in a cell cycle arrest with 70% of cells at G1 phase and 26% at S phase, and control cells exhibited a distribution of 42% and 55% at G1 and S phase, respectively. In conclusion, the present study shows that DHT inhibits the granulosa cell proliferation through a decrease in cyclin D2 mRNA expression, which leads to cell cycle arrest at the G1 phase.     

Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways

Intestinal stem cells (ISCs) in the Drosophila adult midgut are essential for maintaining tissue homeostasis and replenishing lost cells in response to tissue damage. Here we demonstrate that the Hippo (Hpo) signaling pathway, an evolutionarily conserved pathway implicated in organ size control and tumorigenesis, plays an essential role in regulating ISC proliferation. Loss of Hpo signaling in either midgut precursor cells or epithelial cells stimulates ISC proliferation. We provide evidence that loss of Hpo signaling in epithelial cells increases the production of cytokines of the Upd family and multiple EGFR ligands that activate JAK-STAT and EGFR signaling pathways in ISCs to stimulate their proliferation, thus revealing a unique non-cell-autonomous role of Hpo signaling in blocking ISC proliferation. Finally, we show that the Hpo pathway mediator Yorkie (Yki) is also required in precursor cells for injury-induced ISC proliferation in response to tissue-damaging reagent DSS.     

The antiprogestin Lonaprisan inhibits breast cancer cell proliferation by inducing p21 expression

The ovarian steroid hormone progesterone is essential for normal mammary gland physiology but may also play a role in breast cancer. Highly potent and selective antiprogestins may therefore represent a new treatment option for this disease. Here we studied the effects of the new antiprogestin Lonaprisan on the T47D breast cancer cell line. Strong inhibition of cell proliferation and arrest in the G0/G1 phase were observed, as well as induction of a senescence-like phenotype. This was accompanied by p21 induction through direct binding of Lonaprisan-bound progesterone receptor (PR) to the promoter. Reduction of p21 levels blunted the antiproliferative effects of Lonaprisan. Mutation analysis showed that intact PR DNA-binding properties were needed for p21 induction. Phosphorylation of PR Ser345 was stimulated by Lonaprisan, but this post-translational modification was not required for p21 promoter activation, nor was the interaction with c-Src needed. These results support the rationale for using antiprogestins in breast cancer treatment and warrant further studies to better understand their mode of action.     

miR-21在大鼠肝卵圆细胞活化和增殖过程中作用研究

目的 探讨miR-21调控肝卵圆细胞活化和增殖过程的可能机制.方法 采用2-乙酰氨基芴(2-AAF)/部分肝切除法诱导SD大鼠肝卵圆细胞活化模型,分别于0、6、12、24、72、168 h处死动物,同时以单纯肝切除的相应时间点作为对照,分别提取各标本RNA逆转录后行SYBR Green实时荧光定量PCR,检测各组miR-21的表达,行两样本t检验,并用生物信息学方法分析其调控的miRNA转录分子和靶基因.实时荧光定量PCR检测其靶基因表达,Westem blot法检测其靶基因蛋白表达,进行两样本均值的t检验,以P＜ 0.05为差异有统计学意义. 结果 成功建立大鼠肝卵圆细胞活化模型,检测miR-21的扩增信号,miR-21在实验组表达12h开始升高,24 h达到峰值,随后开始降低,168 h再次升高;而对照组6h开始升高,24h后开始下降后基本恢复原水平.实验组与对照组表达比较,实验组在6 h miR-21的表达低于对照组,t=3.029,P=0.039,差异有统计学意义;而在24 h和168 h的表达高于对照组,t值分别为-3.433、-5.105,P值均＜0.05,差异有统计学意义.根据三个数据库的综合评分,结合相关文献,我们选取Smad7为研究的靶基因.检测两组Smad7mRNA表达,对照组中在6h略有下降,后开始升高,在24 h达到峰值后开始下降恢复原水平;在实验组中,6h升高后至24 h达到峰值,随后开始降低,168 h又略有升高.对照组Smad7蛋白表达从6h开始降低,到24 h达到最低后开始升高;实验组6h升高,随后下降,168 h达到最低.Smad7 mRNA表达量变化趋势与miR-21表达变化趋势基本一致,Smad7蛋白表达和miR-21表达变化趋势呈负相关. 结论 成功建立SYBR Green实时荧光定量PCR检测大鼠miR-21的方法,证实miR-21在肝卵圆细胞活化与增殖中起重要作用,Smad7作为miR-21的靶基因可能参与这一过程.     

Kangxian ruangan keli inhibits hepatic stellate cell proliferation mediated by PDGF

AIM: To investigate the effect of Kangxian ruangan keli (KXR)on hepatic stellate cell (HSC) proliferation mediated by platelet-derived growth factor (PDGF) and the underlying mechanism.METHODS: In a serum-free culture system, HSCs were treated with a KXR preparation for 24 hours, followed by stimulation with PDGF-BB for 24 hours. Then the cells were incubated again in the medium containing KXR for 3 hours stimulated with PDGF-BB for 5 minutes, and collected. The proliferation of HSC was examined using an MTT assay and flow cytometry. Tyrosine phosphorylation was detected with Western blotting and visualized by the enhenced chemiluminescent (ECL) method.RESULTS: The OD values for the HSCs growing in the media without and with addition of PDGF were 0.17±0.06 and 0.82±0.05, respectively. The PDGF-induced increase was hindered remarkably by KXR preparation in a dosedependent manner. The reaction values for the systems with 5 mg/mL, 2.5 rng/mL and 1.25 mg/mL of KXR were 0.28±0.03,0.37±0.02 and 0.43±0.04, respectively. Mloreover, the percentages of S-phase cells in these KXR-containing culture systems were 10.95±1.35, 32.76±1.07 and 43.19±1.09,respectively, all of which were significantly lower than that in the culture free of KXR (68.24±2.72). In addition, the values for tyrosine-phosphorylated protein in HSCs treated with 5 mg/mL and 1.25 mg/mL of KXR were 0.1349±0.0072and 0.1658±0.0025, respectively, which were smaller than that in the cells treated only with PDGF-BB (0.1813±0.0117).CONCLUSION: Within the dose range used in the present study, KXR preparation shows an inhibitory effect on HSC proliferation induced by PDGF. The mechanism of this process may involve interference with tyrosine phosphorylation mediated by PDGF.     

Kangxian ruangan keli inhibits hepatic stellate cell proliferation mediated by PDGF

AIM: To investigate the effect of Kangxian ruangan keli (KXR)on hepatic stellate cell (HSC) proliferation mediated by platelet-derived growth factor (PDGF) and the underlying mechanism.METHODS: In a serum-free culture system, HSCs were treated with a KXR preparation for 24 hours, followed by stimulation with PDGF-BB for 24 hours. Then the cells were incubated again in the medium containing KXR for 3 hours stimulated with PDGF-BB for 5 minutes, and collected. The proliferation of HSC was examined using an MTT assay and flow cytometry. Tyrosine phosphorylation was detected with Western blotting and visualized by the enhenced chemiluminescent (ECL) method.RESULTS: The OD values for the HSCs growing in the media without and with addition of PDGF were 0.17±0.06 and 0.82±0.05, respectively. The PDGF-induced increase was hindered remarkably by KXR preparation in a dosedependent manner. The reaction values for the systems with 5 mg/mL, 2.5 rng/mL and 1.25 mg/mL of KXR were 0.28±0.03,0.37±0.02 and 0.43±0.04, respectively. Mloreover, the percentages of S-phase cells in these KXR-containing culture systems were 10.95±1.35, 32.76±1.07 and 43.19±1.09,respectively, all of which were significantly lower than that in the culture free of KXR (68.24±2.72). In addition, the values for tyrosine-phosphorylated protein in HSCs treated with 5 mg/mL and 1.25 mg/mL of KXR were 0.1349±0.0072and 0.1658±0.0025, respectively, which were smaller than that in the cells treated only with PDGF-BB (0.1813±0.0117).CONCLUSION: Within the dose range used in the present study, KXR preparation shows an inhibitory effect on HSC proliferation induced by PDGF. The mechanism of this process may involve interference with tyrosine phosphorylation mediated by PDGF.