Inhibition of nuclear factor kappab activity by genistein is mediated via Notch-1 signaling pathway in pancreatic cancer cells

Pancreatic cancer remains the fourth most common cause of cancer related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of pancreatic cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on pancreatic cancer cells has not been fully elucidated. We have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway. Moreover, Notch signaling is known to play a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of pancreatic cancer. Hence, in our study, we investigated whether there is any cross talk between Notch and NF-kappaB during genistein-induced apoptosis in BxPC-3 pancreatic cancer cells. We used multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, Western blotting and EMSA for measuring DNA binding activity of NF-kappaB. We found that genistein inhibits cell growth and induces apoptotic processes in BxPC-3 pancreatic cancer cells. This was partly due to inhibition of Notch-1 activity. BxPC-3 cells transfected with Notch-1 cDNA showed induction of NF-kappaB activity, and this was inhibited by genistein treatment. From these results, we conclude that the inhibition of Notch-1 and NF-kappaB activity and their cross talk provides a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in pancreatic cancer cells.     

Down-Regulation of Notch1 and NF-κB by Curcumin in Breast Cancer Cells MDA-MB-231

Objective  To test whether the down-regulation of Notch1 gene expression by curcumin could inhibit cell growth and induce apoptosis, which may be associated mechanistically with the down-regulation of NF-κB in breast cancer cells. Methods  Breast cancer cell lines MDA-MB-231 were cultured in vitro and treated with different dosages of curcumin (0, 1.25, 5.0, 20.0μmol/L) for dose-dependent assay and different time (0, 24, 48, 72 h) at the dosage of 5.0μmol/L for time course assay. The changes of the mRNA and protein expression of Notch1 and NF-κB were measured by RT-PCR and Western Blot, and MTT assay was used to measure the change of proliferation. Results  The mRNA and protein levels of Notch1 and NF-κB were decreased significantly in human breast cancer cell line with the increase of dosage of curcumin(P<0.05), and with the extension of time course(P<0.05). These changes suggested a dose- and time-dependent manner. The proliferation rate of cells also was significantly inhibited(P<0.05). Conclusion  The current results show that the Notch-1 signaling pathway is associated mechanistically with NF-κB activity during curcumin-induced cell growth inhibition and apoptosis of breast cancer cells. These results suggest that the down-regulation of Notch signaling by curcumin may be a novel strategy for the treatment of patients with breast cancer.      

Down-regulation of notch-1 inhibits invasion by inactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrix metalloproteinase-9 in pancreatic cancer cells

Notch signaling plays a critical role in the pathogenesis and progression of human malignancies but the precise role and mechanism of Notch-1 for tumor invasion remains unclear. In our earlier report, we showed that down-regulation of Notch-1 reduced nuclear factor-kappaB (NF-kappaB) DNA-binding activity and matrix metalloproteinase-9 (MMP-9) expression. Because NF-kappaB, VEGF, and MMPs are critically involved in the processes of tumor cell invasion and metastasis, we investigated the role and mechanism(s) by which Notch-1 down-regulation (using molecular approaches) may lead to the down-regulation of NF-kappaB, vascular endothelial growth factor (VEGF), and MMP-9, thereby inhibiting invasion of pancreatic cancer cells through Matrigel. We found that the down-regulation of Notch-1 by small interfering RNA decreased cell invasion, whereas Notch-1 overexpression by cDNA transfection led to increased tumor cell invasion. Consistent with these results, we found that the down-regulation of Notch-1 reduced NF-kappaB DNA-binding activity and VEGF expression. Down-regulation of Notch-1 also decreased not only MMP-9 mRNA and its protein expression but also inactivated the pro-MMP-9 protein to its active form. Taken together, we conclude that the down-regulation of Notch-1 could be an effective approach for the down-regulation and inactivation of NF-kappaB and its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and metastasis.     

Notch-1信号通路在甲状腺癌中作用的研究进展

Notch信号通路参与调控机体细胞的增殖、分化、凋亡等，人类Notch信号通路包括了四种受体及五种配体，其中Notch-1受体在甲状腺癌中发挥重要作用。然而，Notch-1在甲状腺乳头状癌中的作用仍存在争议，大多数学者认为Notch-1信号通路能够促进乳头状癌细胞增殖、侵袭转移，且其表达水平与甲状腺乳头状癌侵袭性相关。而对于甲状腺滤泡状癌、髓样癌及未分化癌细胞，活化Notch-1信号通路可抑制他们的增殖。众多研究表明Notch-1信号通路有望成为甲状腺癌重要治疗靶点。本文就Notch-1在甲状腺癌发生、发展中的作用进行综述。     

Delta-tocotrienol augments cisplatin-induced suppression of non-small cell lung cancer cells via inhibition of the Notch-1 pathway

Non-small cell lung cancer (NSCLC), accounting for 80% of lung cancers, is the leading cause of all cancer deaths. Previously, we demonstrated that delta-tocotrienol inhibits NSCLC cell proliferation, invasion and induces apoptosis by down-regulation of the Notch-1 signaling pathway. The objective of this study was to investigate whether delta-tocotrienol, could enhance the anticancer effects of cisplatin. Treatment with a combination of delta-tocotrienol and cisplatin resulted in a dose-dependent, significant inhibition of cell growth, migration, invasiveness, and induction of apoptosis in NSCLC cells, as compared to the single agents. This was associated with a decrease in NF-κB DNA binding activity, decrease in Notch-1, Hes-1, Bcl-2 and increase in cleaved Caspase-3 and PARP expressions. These results suggest that down-regulation of Notch-1, via inhibition of NF-κB signaling pathways by delta-tocotrienol and cisplatin, in combination, could provide a potential novel approach for tumor arrest in NSCLC, while lowering the effective dose of cisplatin.     

Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 in T-ALL cell lines

Gain-of-function mutations in Notch-1 are common in T-cell lymphoblastic leukemia (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSIs). However, GSIs seem to be active in only a small fraction of T-ALL cell lines with constitutive Notch-1 activity and the downstream response of Notch signaling is only partially understood. To further investigate the molecular mechanisms underlying proliferation suppression and apoptosis and explore effective downstream target genes, we used RNA interference (RNAi) technology to down-regulate the expression of Notch-1 in GSIs-resistant T-ALL cell lines. Results showed that down-regulation of Notch-1 by transfection of a small interfering RNA (siRNA) could cause SupT1 cells proliferation inhibition by inducing G₀/G₁ cell cycle arrest and apoptosis. The proliferation inhibitory and apoptotic effects resulting from down-regulation of Notch-1 may be mediated through regulating the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 and the activity of Akt signaling. In addition, our results demonstrated that down-regulation of Notch-1 signaling could sensitize SupT1 cells to adriamycin. Taken together, cell cycle regulatory proteins and Akt signaling may be attractive targets in T-ALL.     

Potentiation of the effect of erlotinib by genistein in pancreatic cancer: the role of Akt and nuclear factor-kappaB

The epidermal growth factor receptor (EGFR) is a target of new therapies in most nonhematologic cancers. EGFR blockade alone may not be sufficient for the control of growth and invasion of human pancreas cancer because of the independent activation of Akt and nuclear factor-kappaB (NF-kappaB). The expression of EGFR, Akt, and NF-kappaB was determined in six human pancreatic cancer cell lines. Selected cells for specific expression were treated with erlotinib, genistein, gemcitabine, or the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. EGFR, phosphorylated EGFR, phosphorylated Akt, and survivin expressions were determined by immunoblotting. Electrophoretic mobility shift assay was used to evaluate the DNA binding activity of NF-kappaB. Genistein significantly increased (P < 0.05) erlotinib-induced growth inhibition and apoptosis in BxPC-3, CAPAN-2, and AsPC-1 cells. In the BxPC-3 cell line, significant down-regulation of EGFR, phosphorylated Akt, NF-kappaB activation, and survivin was observed in the cells treated with the combination compared with the erlotinib-treated cells. In the HPAC and MIAPaCa cell line, no potentiation of the effects of erlotinib by genistein on cell growth or inhibition of the EGFR/Akt/NF-kappaB was observed. Genistein potentiated growth inhibition and apoptosis of the gemcitabine and erlotinib combination in COLO-357 cell line. Genistein potentiates the growth inhibition and apoptosis induced by erlotinib and gemcitabine in certain pancreatic cancer cells. Akt and NF-kappaB inhibition represents one of the mechanisms for the potentiation of erlotinib- and gemcitabine-induced cell death by genistein.     

Notch信号阻断剂抑制鼻咽癌细胞增殖的作用及机制

目的探讨Notch信号阻断剂抑制鼻咽癌细胞增殖的作用及其机制。方法应用Notch信号特异性抑制剂GSI抑制Notch信号的表达,MTT法检测癌细胞的生长增殖变化;流式细胞及Hoechst 33258染色检测癌细胞凋亡;应用Western blot检测Notch信号受抑制后AKT、MEK信号通路的变化。结果应用Notch抑制剂GSI作用后,癌细胞Notch1、2、4表达明显下降,而Notch3无明显变化。GSI作用后癌细胞增殖明显受到抑制,凋亡增加;该作用有浓度及时间双重依赖性（P<0.05）。Notch信号阻断后磷酸化AKT、GSK3β和ERK1/2显著下降,而总AKT、总GSK3β及总ERK1/2无明显变化。结论阻断鼻咽癌细胞Notch信号可以显著抑制鼻咽癌细胞增殖及诱导癌细胞凋亡。该作用可能与下调AKT及MEK信号通路有关。     

姜黄素调控Notch信号通路对肺癌干细胞增殖及凋亡的影响

目的:研究姜黄素对肺癌干细胞增殖及凋亡的影响。方法:利用免疫磁珠标记肺癌表面标记物乙醛脱氢酶1(ALDH1)，从肺癌A549细胞系中分离肺癌干细胞；MTT实验检测姜黄素对肺癌干细胞增殖的影响；流式细胞仪检测姜黄素对肺癌干细胞凋亡的影响；Western blot检测Notch1和Hes1蛋白的表达情况；抑制剂γ-分泌酶抑制Notch信号通路研究对肺癌干细胞增殖及凋亡的影响。结果:利用免疫磁珠分选法分选出肺癌细胞系A549中ALDH1+肺癌干细胞。MTT检测结果显示，姜黄素能够呈浓度依赖性的抑制肺癌干细胞的增殖；经姜黄素处理后的肺癌干细胞，与对照组相比，凋亡率明显升高，Notch1和Hes1蛋白的表达明显下降。经γ-分泌酶抑制剂处理肺癌干细胞后，实验结果显示，γ-分泌酶抑制剂通过下调Notch1和Hes1蛋白的表达调控Notch信号通路抑制肺癌干细胞的增殖，诱导其凋亡。结论:姜黄素抑制肺癌干细胞的增殖，诱导其凋亡与Notch信号通路有关。     

Notch信号通路与消化系统肿瘤相关性的研究进展

Notch信号是一种遗传进化上高度保守,反映相邻细胞间通信作用的一种信号通路,其不仅在细胞正常发育、分化增殖和凋亡中起着重要的作用,而且与多种肿瘤的发生和发展具有相关性.食管鳞状细胞癌的发生常伴随Notch-1的低表达,但食管腺癌的发生却与Notch信号的高表达相关,且高表达的Notch信号对胃癌形成具有促进作用,其表达量提高的程度预示胃癌形成风险的高低.结肠癌中Notch-1表达的升高与病理分级、淋巴转移和病程相关,而Nctch配体Dll-4可促进结肠癌中新生血管的生成有助于癌细胞的转移和远端浸润,与之相反的是Notch-2却可能起到抑制结肠癌生长的作用.总之,目前Notch信号多被视为致癌因素,可促进肿瘤的生长,但在某些肿瘤中也能够诱导肿瘤细胞分化、抑制肿瘤细胞的增殖,表现为致癌与抑癌两种截然相反的作用.应用γ-分泌酶抑制剂(γ-secretase inhibitor,GSI)、小RNA干扰技术和单克隆抗体等方法阻断Notch信号通路,将成为肿瘤治疗的一个新方向.     

Down-regulation of platelet-derived growth factor-D inhibits cell growth and angiogenesis through inactivation of Notch-1 and nuclear factor-kappaB signaling

Platelet-derived growth factor-D (PDGF-D) signaling plays critical roles in the pathogenesis and progression of human malignancies; however, the precise mechanism by which PDGF-D causes tumor cell invasion and angiogenesis remain unclear. Because Notch-1, nuclear factor-kappaB (NF-kappaB), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether PDGF-D down-regulation could be mechanistically associated with the down-regulation of Notch-1, NF-kappaB, VEGF, and MMP-9, resulting in the inhibition of tumor cell invasion and angiogenesis. Our data showed that down-regulation of PDGF-D leads to the inactivation of Notch-1 and NF-kappaB DNA-binding activity and, in turn, down regulates the expression of its target genes, such as VEGF and MMP-9. We also found that the down-regulation of PDGF-D by small interfering RNA (siRNA) decreased tumor cell invasion, whereas PDGF-D overexpression by cDNA transfection led to increased cell invasion. Consistent with these results, we also found that the down-regulation of PDGF-D not only decreased MMP-9 mRNA and its protein expression but also inhibited the processing of pro-MMP-9 protein to its active form. Moreover, conditioned medium from PDGF-D siRNA-transfected cells showed reduced levels of VEGF and, in turn, inhibited the tube formation of human umbilical vascular endothelial cells, suggesting that down-regulation of PDGF-D leads to the inhibition of angiogenesis. Taken together, we conclude that the down-regulation of PDGF-D by novel approaches could lead to the down-regulation of Notch-1 and, in turn, inactivate NF-kappaB and its target genes (i.e., MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis.     

Numb蛋白及Numb/Notch信号通路对人胰腺癌细胞株放疗敏感性的影响

目的 探讨Numb蛋白及其Numb/Notch信号通路对人胰腺癌细胞株放疗敏感性的影响.方法 人胰腺癌细胞MIA PaCa-2经辐射处理.MTT法检测各组细胞生长活力;流式细胞术检测各组细胞总凋亡率.细胞划痕实验和Transwell实验分别检测细胞迁移和侵袭能力.Western blot方法检测Numb/Notch信号通路Numb蛋白及下游分子Notch1、Hes1和Hes5的mRNA和蛋白表达.结果 MTT实验显示随着辐射剂量的增加,MIA Pa-Ca-2细胞吸光度值均低于未受辐射细胞的吸光度值(P﹤0.05).细胞凋亡实验显示随着辐射剂量的增加,MIA PaCa-2细胞总凋亡率均高于未受辐射细胞的总凋亡率(P﹤0.05).细胞划痕实验和Transwell实验显示随着辐射剂量的增加,MIA PaCa-2细胞的迁移和侵袭能力减弱,其在1、3、5 Gy的放射剂量下迁移侵袭能力低于未受辐射细胞(P﹤0.05).qRT-PCR和Western blot方法检测显示,细胞随着辐射剂量的增加,Numb的mRNA和蛋白表达量增加,Notch1、Hes1和Hes5的mRNA和蛋白表达量较未受辐射的细胞的表达量降低(P﹤0.05).结论 Numb蛋白表达增高,Numb/Notch信号通路在胰腺癌细胞中呈激活状态,Numb/Notch信号通路在胰腺癌细胞中的强弱与X射线放射剂量的大小有关.     

Suppression of the notch signaling pathway by γ-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation

Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch-3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC.     

Notch-1 associates with IKKalpha and regulates IKK activity in cervical cancer cells

Notch-1 inhibits apoptosis in some transformed cells through incompletely understood mechanisms. Notch-1 can increase nuclear factor-kappa B (NF-kappaB) activity through a variety of mechanisms. Overexpression of cleaved Notch-1 in T-cell acute lymphoblastic leukemia cells activates NF-kappaB via interaction with the I kappa B kinase (IKK) signalosome. Concomitant activation of the Notch and NF-kappaB pathways has been described in a large series of cervical cancer specimens. Here, we show that wild-type, spontaneously expressed Notch-1 stimulates NF-kappaB activity in CaSki cervical cancer cells by associating with the IKK signalosome through IKKalpha. A significant fraction of tumor necrosis factor (TNF)-alpha-stimulated IkappaB kinase activity in CaSki cells is Notch-1-dependent. In addition, Notch-1 is found in the nucleus in association with IKKalpha at IKKalpha-stimulated promoters and is required for association of IKKalpha with these promoters under basal and TNF-alpha-stimulated conditions. Notch-1-IKKalpha complexes are found in normal human keratinocytes as well, suggesting that IKK regulation is a physiological function of Notch-1. Both Notch-1 and IKKalpha knockdown sensitize CaSki cells to cisplatin-induced apoptosis to equivalent extents. Our data indicate that Notch-1 regulates NF-kappaB in cervical cancer cells at least in part via cytoplasmic and nuclear IKK-mediated pathways.     

siRNA-mediated silencing of Notch-1 enhances docetaxel induced mitotic arrest and apoptosis in prostate cancer cells

Purpose: Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and theinitiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes totumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer isunclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancercells to docetaxel treatment. Methods: siRNA against Notch-1 was transfected into PC-3 prostate cancer cells.Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel byMTT and flow cytometry. Expression of p21waf1/cip1 and Akt as well as activation of Akt in PC-3 cells were detectedby Western blot and Real-time PCR. Results: Silencing of Notch-1 promoted docetaxel induced cell growthinhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increasedp21waf1/cip1 expression and decreased Akt expression and activation in PC-3 cells. Conclusion: Notch-1 promoteschemoresistance of prostate cancer and could be a potential therapeutic target.