Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Inhibitory effect of curcumin on oral carcinoma CAL-27 cells via suppression of Notch-1 and NF-κB signaling pathways

Curcumin has been reported to inhibit cell growth and induce apoptosis in oral cancer cells. Although many studies have been done to uncover the mechanisms by which curcumin exerts its antitumor activity, the precise molecular mechanisms remain to be unclear. In the present study, we assessed the effects of curcumin on cell viability and apoptosis in oral cancer. For mechanistic studies, we used multiple cellular and molecular approaches such as gene transfection, real-time RT-PCR, Western blotting, invasion assay, and ELISA. For the first time, we found a significant reduction in cell viability in curcumin-treated cells, which was consistent with induction of apoptosis and also associated with down-regulation of Notch-1 and nuclear factor-κB (NF-κB). Taken together, we conclude that the down-regulation of Notch-1 by curcumin could be an effective approach, which will cause down-regulation of NF-κB, resulting in the inhibition of cell growth and invasion. These results suggest that antitumor activity of curcumin is mediated through a novel mechanism involving inactivation of Notch-1 and NF-κB signaling pathways.     

Curcumin induces cell death in esophageal cancer cells through modulating Notch signaling

Background

Curcumin inhibits the growth of esophageal cancer cell lines; however, the mechanism of action is not well understood. It is becoming increasingly clear that aberrant activation of Notch signaling has been associated with the development of esophageal cancer. Here, we have determined that curcumin inhibits esophageal cancer growth via a mechanism mediated through the Notch signaling pathway.

Methodology/Principal Findings

In this study, we show that curcumin treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in esophageal cancer cell lines. Furthermore, curcumin treatment induced apoptosis through caspase 3 activation, confirmed by an increase in the ratio of Bax to Bcl2. Cell cycle analysis demonstrated that curcumin treatment induced cell death and down regulated cyclin D1 levels. Curcumin treatment also resulted in reduced number and size of esophagospheres. Furthermore, curcumin treatment led to reduced Notch-1 activation, expression of Jagged-1 and its downstream target Hes-1. This reduction in Notch-1 activation was determined to be due to the down-regulation of critical components of the γ-secretase complex proteins such as Presenilin 1 and Nicastrin. The combination of a known γ-secretase inhibitor DAPT and curcumin further decreased proliferation and induced apoptosis in esophageal cancer cells. Finally, curcumin treatment down-regulate the expressions of Notch-1 specific microRNAs miR-21 and miR-34a, and upregulated tumor suppressor let-7a miRNA.

Conclusion/Significance

Curcumin is a potent inhibitor of esophageal cancer growth that targets the Notch-1 activating γ-secretase complex proteins. These data suggest that Notch signaling inhibition is a novel mechanism of action for curcumin during therapeutic intervention in esophageal cancers.     

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.     

Activation of Notch pathway is linked with epithelial-mesenchymal transition in prostate cancer cells

Notch signaling has been reported to play an essential role in tumorigenesis. Several studies have suggested that Notch receptors could be oncoproteins or tumor suppressors in different types of human cancers. Emerging evidence has suggested that Notch pathway regulates cell growth, apoptosis, cell cycle, and metastasis. In the current study, we explore whether Notch-1 could regulate the cell invasion and migration as well as EMT (epithelial-mesenchymal transition) in prostate cancer cells. We found that overexpression of Notch-1 enhanced cell migration and invasion in PC-3 cells. However, downregulation of Notch-1 retarded cell migration and invasion in prostate cancer cells. Importantly, we observed that overexpression of Notch-1 led to EMT in PC-3 cells. Notably, we found that EMT-type cells are associated with EMT markers change and cancer stem cell phenotype. Taken together, we concluded that downregulation of Notch-1 could be a promising approach for inhibition of invasion in prostate cancer cells, which could be useful for the treatment of metastatic prostate cancer.     

非小细胞肺癌靶向治疗的临床前研究进展

针对表皮生长因子受体（EGFR）和血管生成（angiogenesis）信号通路的靶向治疗已经在晚期非小细胞肺癌的治疗上取得成功,但由于抗药性的存在,大多数晚期患者的生存时间仍然提高有限。继发性的EGFR T790M突变和原癌基因肝细胞生长因子受体（MET）的扩增被鉴定为两种主要的抗药机制。最近转化生长因子-β（TGF-β）/白介素-6信号通路被报道能介导选择性和适应性地对erlotinib的抗药。另一方面,Kras突变所致肺癌的靶向治疗方面也取得了一些进展。双重抑制磷脂酰肌醇3-激酶（PI3K）和促分裂素原活化蛋白激酶激酶（MEK）信号通路可导致Kras突变肿瘤的显著消退,联合抑制SRC、PI3K和MEK可使丝氨酸/苏氨酸蛋白激酶11（Lkb1）缺失,Kras突变的肺癌小鼠的肿瘤明显消退,抑制核因子-κB（NF-κB）信号通路导致p53缺失,Kras突变的肿瘤发展显著减慢。这些发现都为发展非小细胞肺癌患者的靶向治疗提供了有力的支持。     

Curcumin inhibits proliferation and invasion of osteosarcoma cells through inactivation of Notch-1 signaling

The Notch signaling pathway plays critical roles in human cancers, including osteosarcoma, suggesting that the discovery of specific agents targeting Notch would be extremely valuable for osteosarcoma. Curcumin, a naturally occurring phenolic compound found in curcuma longa, has been shown to inhibit proliferation and induce apoptosis of osteosarcoma cells in vitro and tumor growth in xenotransplant or orthotransplant models. However, the precise molecular mechanisms by which curcumin exerts its antitumor activity remain unclear. Here we used multiple molecular approaches, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the invasion assay, gene transfection, real-time RT-PCR, western blot and gelatin zymography, to investigate whether the downregulation of Notch-1 contributes to curcumin-induced inhibition of proliferation and invasion in osteosarcoma cells. The results showed that curcumin caused marked inhibition of osteosarcoma cell growth and G2/M phase cell cycle arrest. This was associated with concomitant attenuation of Notch-1 and downregulation of its downstream genes, such as matrix metalloproteinases, resulting in the inhibition of osteosarcoma cell invasion through Matrigel. We also found that specific downregulation of Notch-1 via small-interfering RNA prior to curcumin treatment resulted in enhanced inhibition of cell growth and invasion. These results suggest that antitumor activity of curcumin is mediated through a novel mechanism involving inactivation of the Notch-1 signaling pathway. Our data provide the first evidence that the downregulation of Notch-1 by curcumin may be an effective approach for the treatment of osteosarcoma.     

Notch-1 regulates cell death independently of differentiation in murine erythroleukemia cells through multiple apoptosis and cell cycle pathways

Notch signaling is a potential therapeutic target for various solid and hematopoietic malignancies. We have recently shown that downregulation of Notch-1 expression has significant anti-neoplastic activity in pre-clinical models. However, the mechanisms through which Notch modulation may affect cell fate in cancer remain poorly understood. We had previously shown that Notch-1 prevents apoptosis and is necessary for pharmacologically induced differentiation in murine erythroleukemia (MEL) cells. We investigated the mechanisms of these effects using three experimental strategies: (1) MEL cells stably transfected with antisense Notch-1 or constitutively active Notch-1, (2) activation of Notch-1 by a cell-associated ligand, and (d3) activation of Notch-1 by a soluble peptide ligand. We show that: (1) downregulation of Notch-1 sensitizes MEL cells to apoptosis induced by a Ca(2+) influx or anti-neoplastic drugs; (2) Notch-1 downregulation induces phosphorylation of c-Jun N-terminal kinase (JNK) while constitutive activation of Notch-1 or prolonged exposure to a soluble Notch ligand abolishes it; (3) Notch-1 has dose- and time-dependent effects on the levels of apoptotic inhibitor Bcl-x(L) and cell cycle regulators p21(cip1/waf1), p27(kip1), and Rb; and (4) Notch-1 activation by a cell-associated ligand is accompanied by rapid and transient induction of NF-kappaB DNA-binding activity. The relative effects of Notch-1 signaling on these pathways depend on the levels of Notch-1 expression, the mechanism of activation, and the timing of activation. The relevance of these findings to the role of Notch signaling in differentiation and cancer are discussed.     

NF-κB targeting for overcoming tumor resistance and normal tissues toxicity

Radiotherapy and chemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.     

Down-regulation of epidermal growth factor receptor by curcumin-induced UBE1L in human bronchial epithelial cells

UBE1L, ubiquitin-activating enzyme E1-like, is the activating enzyme of ISG15ylation (ISG15, interferon stimulated gene 15). Loss of UBE1L and activation of epidermal growth factor receptor (EGFR) signaling are common events in lung carcinogenesis. Curcumin, a well-studied chemopreventive agent, is known to down-regulate EGFR. The present study demonstrated that curcumin decreased EGFR expression in human bronchial epithelial (HBE) Beas-2B cells and lung cancer A549 cells. For the first time, UBE1L was found to be induced by curcumin in HBE cells. Interestingly, overexpression of UBE1L reduced EGFR at posttranslational level in HBE cells. UBE1L triggered EGFR membrane internalization and promoted complex formation between ISG15 and EGFR. Curcumin decreased EGFR downstream signaling pAKT and nuclear factor κB (NF-κB). Overexpression or knockdown of UBE1L also resulted in down-regulation or up-regulation of phosphoinositide 3-kinase/AKT/NF-κB correspondently. In human samples, there was an inverse relationship between UBE1L and EGFR/AKT/NF-κB in non-small cell lung cancer tissues and adjacent tissues. These results uncover a novel chemopreventive mechanism of curcumin in inducing UBE1L and down-regulating EGFR signaling in HBE cells.     

Notch signaling protects retina from nuclear factor-κB- and poly-ADP-ribose-polymerase-mediated apoptosis under high-glucose stimulation

Proliferative diabetic retinopathy, the primary cause of vision loss in adults, is one of serious microvascular complications caused by diabetes. Both poly-ADP-ribose-polymerase (PARP) and nuclear factor (NF)-κB signaling are involved in the injury process. Injury activates PARP, which in turn potentiates NF-κB activation and causes cell apoptosis. Like the NF-κB pathway, Notch1 signaling plays a key role in the regulation of cell proliferation, differentiation, and apoptosis. However, the connections between these signaling pathways are not well understood. In this study, we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships. We found that apoptosis was increased in both STZ-induced diabetic mice and high-glucose-treated HRVECs, which was due to increased activation of PARP, cleaved caspase3, and reduced expression of Notch1 and p-Akt. The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50, and inhibited PARP- and p50-mediated apoptosis directly. These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels. Thus, our study demonstrated that Notch1 signaling protects cells from PARP- and NF-κB-induced apoptosis under high glucose through the activation of Akt.     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of NF-κB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner

A skin-specific block in NF-κB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-κB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IκBα protein (IκBαDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IκBαDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IκBαDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IκBαDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IκBαDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-κB inhibition.     

Down‐regulation of Notch‐1 is associated with Akt and FoxM1 in inducing cell growth inhibition and apoptosis in prostate cancer cells

Although many studies have been done to uncover the mechanisms by which down‐regulation of Notch‐1 exerts its anti‐tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch‐1 down‐regulation and also assessed the molecular consequence of Notch‐1‐mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down‐regulation of Notch‐1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down‐regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch‐1 signaling. Moreover, we found that a “natural agent” (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down‐regulation of Notch‐1, Akt, and FoxM1. These results suggest that down‐regulation of Notch‐1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa. J. Cell. Biochem. 112: 78–88, 2011. © 2010 Wiley‐Liss, Inc.     

Modulation of apoptosis-related cell signalling pathways by curcumin as a strategy to inhibit tumor progression

A hallmark of cancer is resistance to apoptosis, with both the loss of proapoptotic signals and the gain of anti-apoptotic mechanisms contributing to tumorigenesis. As inducing apoptosis in malignant cells is one of the most challenging tasks regarding cancer, researchers increasingly focus on natural products to regulate apoptotic signaling pathways. Curcumin, a polyphenolic derivative of turmeric, is a natural compound derived from Curcuma longa, has attracted great interest in the research of cancer during the last half century. Extensive studies revealed that curcumin has chemopreventive properties, which are mainly due to its ability to arrest cell cycle and to induce apoptosis in cancer cells either alone or in combination with chemotherapeutic agents or radiation. The underlying action mechanisms of curcumin are diverse and has not been elucidated so far. By regulating multiple important cellular signalling pathways including NF-κB, TRAIL, PI3 K/Akt, JAK/STAT, Notch-1, JNK, etc., curcumin are known to activate cell death signals and induce apoptosis in pre-cancerous or cancer cells without affecting normal cells, thereby inhibiting tumor progression. Several phase I and phase II clinical trials indicate that curcumin is quite safe and may exhibit therapeutic efficacy. This article reviews the main effects of curcumin on the different apoptotic signaling pathways involved in curcumin induced apoptosis in cancer cells via cellular transduction pathways and provides an in depth assessment of its pharmacological activity in the management of tumor progression.     

The tyrosine kinase c-Src directly mediates growth factor-induced Notch-1 and Furin interaction and Notch-1 activation in pancreatic cancer cells

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth.