The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-KB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI doublelabeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blotting.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

Musca domestica pupae lectin induces apoptosis in HepG2 cells through a NF-κB/p65-mediated caspase pathway

A new lectin (42 kDa) from Musca domestica pupae (MPL) has been known to inhibit proliferation in tumor cells. In this study, flow cytometry analysis showed that MPL induced HepG2 cells apoptosis significantly and the cells were arrested at S phase. MPL inhibited IκB-α degradation and NF-κB/p65 translocation from cytoplasm into nucleus. Simultaneously, the expressions of FLIP, which is a target gene of NF-κB/p65 were down-regulated and the caspase-8 and caspase-3 were then activated to induce apoptosis. Taken together, these results showed that MPL induced a caspase-dependent apoptosis via NF-κB/p65 pathway in HepG2 cells.     

三氧化二砷诱导A375细胞凋亡过程中的信号传导途径研究

背景与目的： 探讨三氧化二砷(arsenic trioxide, As2O3)在诱导人黑素瘤A375细胞凋亡中,对NF-κB、C-IAP2以及Caspase-3信号传导途径的影响。 材料与方法： 将As2O3和Caspase-3 抑制剂Ac-devd-cho单独或联合作用A375细胞后,采用Western blot 法检测NF-κB p65和Caspase-3表达水平,免疫组织化学法检测Caspase-3蛋白的表达,RT-PCR法检测C-IAP2 mRNA表达。 结果： 5.0 μmol/L As2O3单独处理A375细胞12 h和24 h以及As2O3联合Ac-devd-cho处理24 h组,随着As2O3作用时间的延长,Caspase-3激活蛋白增多、NF-κB p65核蛋白和C-IAP2 mRNA表达下降,Ac-devd-cho能够阻断Caspase-3蛋白的活化,而对NF-κBp 65核蛋白和C-IAP2 mRNA表达无明显影响;免疫组织化学法也显示,Caspase-3激活型蛋白随着As2O3作用时间的延长而增高,但能被Ac-devd-cho阻断。 结论： As2O3可诱导A375细胞凋亡,能抑制A375细胞NF-κB、C-IAP2基因的表达,且不被Ac-devd-cho所阻断; As2O3能活化Caspase-3蛋白表达,而此效应可被Ac-devd-cho阻断。推测NF-κB-C-IAP2-Caspase-3通路可能是As2O3诱导A375细胞凋亡的途径之一。     

Phase I dose-escalation study of F60008, a novel apoptosis inducer,in patients with advanced solid tumours

Resistance of cancer cells to cytotoxic therapy can be caused by the activation of strong anti-apoptotic effectors, for example NF-κB. Therefore, compounds that inhibit NF-κB stimulation might overcome chemotherapy resistance. F60008, a semi-synthetic derivate of triptolide, is converted to triptolide in vivo and activates apoptosis in human tumour cells. We performed a phase I and pharmacological study of F60008 given intravenously as a weekly infusion for 2 weeks every 3 weeks in patients with advanced solid tumours. Twenty patients were enrolled, and a total of 35 cycles were administered. The most frequent haematological side-effect was mild grade 1–2 anaemia. Non-haematological toxicities included fatigue, nausea, vomiting, diarrhoea and constipation, all grade 1–2. Two lethal events were observed in which an increase in caspase-3 activity and overt apoptosis in monocytes and neutrophils could be seen. Pharmacokinetic studies showed high inter-individual variability and rendered F60008 a far from optimal derivate of triptolide.     

Anti-neoplastic effects of fucoxanthin and its deacetylated product, fucoxanthinol, on Burkitt's and Hodgkin's lymphoma cells

Fucoxanthin (FX) is a natural carotenoid with reported antitumorigenic activity. This study explored the effects of FX and its deacetylated product, fucoxanthinol (FXOH), on B-cell malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma and Epstein-Barr virus-immortalized B cells. Both FX and FXOH reduced the viability of these malignant B cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during G1 phase and caspase-dependent apoptosis. FXOH was approximately twice more potent than FX in these activities. In contrast, normal peripheral blood mononuclear cells were resistant to FX and FXOH. Strong and constitutive activation of nuclear factor-κB (NF-κB) is a common characteristic of many B-cell malignancies, and FXOH suppressed constitutive NF-κB activity. NF-κB inhibition was accompanied by downregulation of NF-κB-dependent anti-apoptotic and cell cycle regulator gene products, including Bcl-2, cIAP-2, XIAP, cyclin D1 and cyclin D2. The results indicated that FX and FXOH are potentially useful therapeutic agents in B-cell malignancies characterized by aberrant regulation of NF-κB.     

NEMO-binding domain peptide inhibits proliferation of human melanoma cells

Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-κB (NF-κB) pathway. In vitro studies have shown that NF-κB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-β NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFα-induced NF-κB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-κB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-κB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-κB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process.     

Inhibitory effect and mechanism of mesenchymal stem cells on melanoma cells

Purpose

To explore the inhibitory effect and mechanism of MSCs on melanoma proliferation.

Methods

The inhibitory effect of MSCs on melanoma A375 cells was detected by co-culture and conditioned medium (CM) experiments using MTT method. The cell cycle was analyzed by flow cytometry. Then, Western Blot experiment detected the expression of proteins related to NF-κB signaling in A375 cells. The expression of IL-1Ra in MSCs was proved by RT-PCR. The over-expression and silencing vector pcDNA3.1-EGFP-IL-1Ra and pGPH1-IL-1R were constructed and transfected into MSCs cells. After that, the changes of inhibitory effect and cell cycle from MSCs-S and MSCs-O CM on A375 cells were explored. The expression of proteins related to NF-κB signaling in A375 cells after MSCs-S or MSCs-O CM treatment was detected by Western Blot. MSCs, MSCs-S, or MSCs-O and A375 cells were co-injected into nude mice under the arms, the growth of tumor was observed, the frozen sections were made, and H&E staining of tumor tissue was performed.

Results

The proliferation of A375 cells was inhibited and the cell cycle of A375 was arrested by MSCs. The expressions of cytokines related to NF-κB signaling were down-regulated. Over-expression and silence of Interleukin 1 receptor antagonist (IL-1Ra), specifically blocking activation of NF-κB signaling, indicated that inhibitory effect from MSCs was enhanced or weakened respectively, which suggested that IL-1Ra was involved in the inhibitory effect. In vivo, tumor initiation and growth were significantly inhibited when A375 cells were co-injected with MSCs into nude mice, which were related to the expression level of IL-1Ra.

Conclusion

MSCs could inhibit the proliferation and tumor initiation of melanoma A375 cells through NF-κB signaling. MSCs could secret IL-1Ra and inhibit expressions of NF-κB signaling-related factors of tumor cells, and cause cell cycle arrest in G1 phase.

     

Antitumor effect of berberine against primary effusion lymphoma via inhibition of NF-κB pathway

Primary effusion lymphoma (PEL) is an infrequent and distinct entity among the aggressive non-Hodgkin B cell lymphomas that occurs predominantly in patients with advanced AIDS. It shows serous lymphomatous effusion in body cavities, and is resistant to conventional chemotherapy with a poor prognosis. Thus, the optimal treatment for PEL is not well defined and there is a need for novel agents. PEL has been recognized as the tumor caused by Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), and nuclear factor (NF)-κB activation plays a critical role in the survival and growth of PEL cells. In this study, we assessed the antitumor effect of berberine, a naturally occurring isoquinoline alkaloid, on this pathway. The methylthiotetrazole assay showed that cell proliferation in the PEL cell lines was inhibited by berberine. Berberine also induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IκB kinase (IKK) phosphorylation, IκB phosphorylation and IκB degradation, upstream targets of the NF-κB pathway, in PEL cells. In a xenograft mouse model that showed ascites and diffuse organ invasion of PEL cells, treatment with berberine inhibited the growth and invasion of PEL cells significantly compared with untreated mice. These results show that the suppression of NF-κB is a molecular target for treating PEL, and berberine is a potential antitumor agent for PEL.     

Minnelide reduces tumor burden in preclinical models of osteosarcoma

Osteosarcoma is the most common bone cancer in children and adolescents with a 5-year survival rate of about 70%. In this study, we have evaluated the preclinical therapeutic efficacy of the novel synthetic drug, Minnelide, a prodrug of triptolide on osteosarcoma. Triptolide was effective in significantly inducing apoptosis in all osteosarcoma cell lines tested but had no significant effect on the human osteoblast cells. Notably, Minnelide treatment significantly reduced tumor burden and lung metastasis in the orthotopic and lung colonization models. Triptolide/Minnelide effectively downregulated the levels of pro-survival proteins such as heat shock proteins, cMYC, survivin and targets the NF-κB pathway.     

Insulin-like growth factor-binding protein-3 suppresses tumor growth via activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling

Nuclear factor-kappaB (NF-κB) is constitutively activated in a variety of human cancers including prostate cancer and involved in tumorigenesis, tumor progression and chemo-resistance. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a potent tumor suppressor and is significantly suppressed in a variety of cancers. Diverse biological effects of IGFBP-3 have been reported to be both dependent and independent of the IGF/IGF-I receptor (IGF-IR) axis. The precise underlying mechanisms of IGF/IGF-IR-independent, antiproliferative actions of IGFBP-3 are yet to be elucidated. We found an inverse correlation between NF-κB activity and IGFBP-3 expression during prostate cancer progression using an in vitro prostate cancer progression model. Restoration of IGFBP-3 resulted in significant inhibition of constitutively elevated NF-κB activity in prostate cancer cells. IGFBP-3 further inhibited the expression of NF-κB-regulated angiogenic factors such as VEGF and IL-8, and cell adhesion molecules, ICAM-1 and VCAM-1. This inhibitory action of IGFBP-3 was IGF/IGF-IR-independent since IGFBP-3 mutant devoid of IGF binding affinity had a similar inhibitory effect. We identified that IGFBP-3 degrades the key NF-κB regulatory molecules-IκBα and p65-NF-κB proteins through activation of caspase-8 and -3/-7, thereby inhibiting elevated NF-κB activity in prostate cancer. Finally intratumoral administration of IGFBP-3 resulted in significant tumor suppression as well as sensitization of antitumor effect of doxorubicin. Our findings indicate that IGFBP-3 exerts antitumor effects via IGF-independent mechanisms which involve activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling. The use of IGFBP-3 as a cancer therapeutic with this distinctive suppression mechanism may offer alternate means to treat chemotherapy resistant tumors.     

HA14-1 sensitizes TNF-α-induced apoptosis via inhibition of the NF-κB signaling pathway: Involvement of reactive oxygen species and JNK

Nuclear factor-kappa B (NF-κB) activation by tumor necrosis factor-alpha (TNF-α) attenuates the TNF-α-induced apoptosis pathway. Thus, blockage of NF-κB activity may improve the anti-cancer activity of TNF-α. HA14-1 induces apoptosis in various human cancer cells, and the molecular mechanisms of this action remain to be fully characterized. The present study evaluated the involvement of NF-κB, reactive oxygen species (ROS), and c-Jun N-terminal kinase (JNK) in the effects of HA14-1 by examining the sensitization effect on TNF-α-induced apoptosis in human leukemia cells. Such sensitization is closely associated with the inhibitory effect of HA14-1 on TNF-α-mediated NF-κB activation. HA14-1 suppressed NF-κB activation through inhibition of phosphorylation and degradation of IκBα. This inhibition was correlated with suppression of NF-κB-dependent gene products (c-myc, cyclin D1, cox-2, and IAP-1). Additionally, the present findings provide evidence of a critical role of ROS accumulation induced by HA14-1 in TNF-α-induced apoptosis. Moreover, HA14-1 also markedly sustained TNF-α-mediated JNK activation. A specific JNK inhibitor abolished the sensitization effect of HA14-1 on TNF-α-induced apoptosis. Taken together, these results indicate that ROS and JNK represent important signals in HA14-1 sensitization in TNF-α-induced apoptosis.     

5-Fu诱导结肠癌细胞凋亡与NF-κB活化及uPA表达的关系

 [摘要] 目的 研究5-氟尿嘧啶（5-Fu）诱导结肠癌细胞HCT116凋亡与核转录因子-κB (NF-κB) 活化以及uPA表达的关系，并观察吡咯烷二硫氨基甲酸盐（PDTC）抑制NF-κB活化对5-Fu诱导结肠癌细胞凋亡及uPA表达的影响。方法 采用流式细胞仪Annexin Ⅴ-FITC法检测结肠癌细胞HCT116凋亡;采用免疫组织化学方法半定量分析HCT116细胞NF-κB及uPA表达的动态变化。结果 5-Fu在诱导HCT116细胞凋亡的同时有NF-κB活化，1umol/LPDTC能显著增加5-Fu诱导HCT116细胞凋亡及抑制5-Fu诱导的HCT116细胞NF-κB活化作用(P<0.05), uPA变化与NF-κB一致。结论5-Fu在诱导HCT116细胞凋亡的同时活化了NF-κB，uPA表达亦随之增强；PDTC在抑制5-Fu诱导的HCT116细胞NF-κB活化的同时增强了5-Fu诱导细胞凋亡的作用，uPA表达也相应下降     

Biologic activity of triptolide in t(8;21) acute myeloid leukemia cells

Triptolide is a compound isolated from the traditional Chinese medicinal herb Tripterygium wilfordii that shows potent anti-tumor activities, but its effects on acute myeloid leukemia with t(8;21) remain unclear. Here we report that triptolide inhibits cell proliferation and induces apoptosis in a dose- and time-dependent manner of t(8;21)-bearing Kasumi-1, SKNO-1 and CD34+ cells harvested from bone marrow samples of patients with t(8;21) leukemia. We show that triptolide triggers cleavage of the resultant AML1-ETO fusion protein of t(8;21), and causes downregulation of C-KIT followed by inhibition of JAK-STAT signaling. Triptolide downregulates p65 and inhibits the DNA-binding activity of NF-κB. Our data indicate that triptolide might be an effective agent for t(8;21) leukemia.     

The anti-CD20 mAb LFB-R603 interrupts the dysregulated NF-κB/Snail/RKIP/PTEN resistance loop in B-NHL cells: role in sensitization to TRAIL apoptosis

LFB-R603 is a chimeric anti-CD20 mouse/human monoclonal antibody with a human IgG1 constant (Fc) region. In comparison to rituximab, LFB-R603 exhibits a high affinity to the Fcγ RIII (CD16) and a potent in vitro antibody-dependent cellular cytotoxicity (ADCC). We examined several experimental designs for the biological anti-tumor activity of LFB-R603 as well as its sensitizing activity to apoptosis in resistant non-Hodgin's B-cell lymphoma (B-NHL) in comparison to rituximab. Treatment of the B-NHL cell line Ramos with LFB-R603 was not toxic at the concentrations used and induced cell aggregation following culture at 24 and 48 h similarly to rituximab. Hence, we hypothesized that LFB-R603 may signal the tumor cells and modify intracellular survival/anti-apoptotic pathways. Treatment of Ramos cells with LFB-R603 inhibited the constitutively active NF-κB survival pathway, followed by significant potentiation of TRAIL-mediated apoptosis. We examined the underlying molecular mechanism by which the LFB-R603-mediated NF-κB inhibition results in the reversal of tumor cell resistance to TRAIL. We hypothesized that downstream gene products regulated by NF-κB that are involved in the resistance may be involved in LFB-R603-mediated sensitization. We found that LFB-R603 inhibited NF-κB activation and the anti-apoptotic factor Snail and induced the pro-apoptotic factor RKIP. The direct roles of Snail and RKIP modulation by LFB-R603 in the reversal of B-NHL resistance to TRAIL were examined by knocking down Snail and overexpressing RKIP in Ramos cells, respectively. Both approaches increased significantly the cell sensitivity to TRAIL apoptosis. In addition to changes observed in the expression levels of Snail and RKIP, Ramos cells treated with LFB-R603 induced the expression of PTEN along with inhibition of the PI3K-AKT activated pathway. PTEN silencing in Ramos cells pretreated with LFB-R603 and TRAIL inhibited TRAIL apoptosis; thus, demonstrating that PTEN induction by LFB-R603 has a direct role in tumor cell sensitization to TRAIL apoptosis. Several of the findings obtained in the experimental designs with LFB-R603 were superior to those obtained with rituximab. Overall, the findings demonstrate that LFB-R603 interferes with the dysregulated NF-κB/Snail/RKIP/PTEN/AKT resistance circuitry in B-NHL cells. Further, the findings suggest that LFB-R603 may sensitize tumor cells to various apoptotic stimuli including cytotoxic ligands such as TRAIL and chemotherapeutic drugs.     

Inhibition of proliferation and induction of apoptosis by trimethoxyl stilbene (TMS) in a lung cancer cell line

Trimethoxyl stilbene (TMS) is a derivative of resveratrol, a compound shown to inhibit development of a variety of tumor types. We aimed to evaluate the effect of TMS on cell proliferation and apoptosis in the A549 non-small cell lung cancer cell line. Growth inhibition rate and colony formation was measured and apoptosis was determined with Hoechst 33258 staining. Protein expression levels of caspase-3, STAT3, STAT5b, JAK2, NF-κB, and IκB were examined by Western blotting. Furthermore, localization of NF-κB protein was also explored. TMS inhibited proliferation (IC50 8.6 μmol/L) and induced apoptosis of the cells in a concentration-dependent manner., also inducing apoptosis accompanied by up-regulated expression and cleavage activation of caspase-3, up-regulation of IκB and down-regulation of NFκB, STAT3, STAT5b, and JAK2 signal transduction. TMS has potential as a new drug for treatment of non-small cell lung cancer patients with anti-proliferation and apoptosis inducing effect of TMS to A549 cells apparently related to its inhibitory effect on STATs and NF-κB signal transduction. Up-regulation of caspase-3 further supports the potential clinical use of TMS for the treatment of non-small cell lung adenocarcinoma.     

p38 MAPK and NF-κB pathways are involved in naphtho[1,2-b] furan-4,5-dione induced anti-proliferation and apoptosis of human hepatoma cells

Naphtho[1,2-b] furan-4,5-dione (NFD) was investigated for its anti-proliferation effect on human hepatocellular carcinoma (HCC), Hep3B, HepG₂, and Huh-7 cells. The effect of NFD on inhibiting proliferation and apoptosis was correlated with up-regulation of pro-apoptotic protein and down-regulation of pro-survival proteins. Remarkably, we found that NFD inhibited the nuclear translocation of NF-κB, likely accounting for the down-regulation of pro-survival Bcl-2 family. Furthermore, suppression of p38 MAPK activity by a specific inhibitor significantly rescued the cell proliferation inhibited by NFD. These findings suggest that signaling imbalance between p38 MAPK and NF-κB by NFD results in the proliferative inhibition and apoptosis of HCC tumor cells.