| ObjectiveBufalin a cardiotonic steroid isolated from the Chinese toad venom preparation Chansu, a Chinese medicine prepared from the dried white secretion of auricular glands and skin glands of Chinese toad, has long been used as a therapeutic agent in China and shown to selectively induce leukemia cell differentiation and apoptosis under certain experimental conditions. Bufalin also has been shown to induce apoptosis in human leukemia K562,U937 and HL60 cells and human tumor cells lines such a Human Gastric Cancer Cell MGc - 803 and prostate cancer cell lines in vitro.Bufalin can inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. A variety of differentiation - inducing agents, such as camptothecin, VP16, geranylacetone and all - trans - retinoic acid, were shown to induce apoptosis in tumor and normal cells. Bufalin alone is only a modest inducer of differentiation of AML cells in primary culture. Bufalin restored RA sensitivity to previously resistant APL cells. Therefore, bufalin seems to act as a potent differentiation - and apoptosis - inducing agent in cancer cells. When U937 cells were treated with bufalin in the absence of serum, mitogen - activated protein ( MAP) kinase activity was markedly increased after the start of treatment and elevated. Prior to the activation of MAP kinase, increased activities of Ras, Raf - 1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf -1, and MAP kinase kinase to MAP kinase. That a pathway withthe persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apopto-sis. It has been reported that bufalin utilizes the extracellular signal - regulated kinase (ERK) cascade for signal transduction, leading not only to cell differentiation but also to apoptosis . It is thus postulated that the ERK cascade may control bufalin induced cell differentiation and apoptosis simultaneously.Caspases are cysteine proteases as the executioners of apoptosis. They activated , initiator Caspases activate downstream Caspases, resulting in an amplification of the Caspase cascade. Active Caspases cleave critical cellular protein substrates and systematically dismantle the cell. The availability and activation of Caspases are crucial steps in the commitment of a cell to die, are known to survive for the life of the organism, inhibition of apoptosis is a primary consideration in setting up cellular survival mechanisms. When in close proximity Caspases can autoactivate. Baculoviruses harbor yet another type of Caspase inhibitor , the inhibitor of apoptosis proteins (IAPs). These proteins are characterized by the presence of a homologous domain named the baculoviral IAP repeat (BIR) domain and IAPs . Survivin, a single BIR domain containing IAP, is upregulated in many common human tumors, implicating that the deregulation of IAPs contributes to human disease. Understanding of the mechanism by which IAPs inhibit apoptosis has come with the demonstration that XIAP can physically interact with and block Caspase -3, -7, and 9. Survivin, a novel inhibitor of apoptosis, expressing in a cell cycle -dependent manner,regulates the G(2)/M phase of the cell cycle by associating with mitotic spindle microtubules;it directly inhibits Caspase -3 and Caspase -7activity. During tumorigenesis, Survivin expression is inversely correlated with apoptosis and is positively correlated with proliferation and angiogenesis. Survivin expression up - regulation predicts short survival and poor prognosis in human cancers. Survivin targeting antisense nucleotide and Survivin mutants induce apoptosis, reduce tumor growth potential , and sensitize tumor cells to chemotherapeutic drugs and X - irradiation in vitro and in vivo. These results suggest that Survivin may has the potential function as a new target for the diagnosis and treatment of cancer.Mitogen - activated protein kinases ( MAPKs ) participate in signaling cas-cades from transmission of extracellular signals into their intracellular targets, which regulate important biologic activities. The MAP kinase family members have been implicated in events necessary for proliferation, differentiation, apop-tosis, and certain kinds of stress responses 0 Three major groups of Map kinases exist: the p38 Map kinase family, the extracellular signal - regulated kinase (ERk) family,and the c - Jun NH2 -terminal kinase (JNK) kinase. Map kinase signaling cascades are activated by a variety of different cellular stimuli ( stress, cytokine) and mediate diverse responses,The ERK pathway is activated in response to several cytokines and growth factors, and primarily mediates mito-genic and antiapoptotic signals. It has been suggested that the excessive activation of the ERK - kinase cascade, which is generally known to play a role in cell survival, is an event necessary for bufalin - mediated apoptosis. Such a hyper-excitation of the ERK - kinase cascade is due to the activation of an upper signal component Ras and the concomitant downregulation of the protein kinase A activity that may negatively regulate the Raf - 1 function. However, signal trans-duetion leading to bufalin mediated cell differentiation has not been explored.In order to characterize the mechanisms of apoptosis - inducing effect of by bufalin on NB4 cells, we examined the expression of Survivin and role of ERK pathway in bufalin - induced apoptosis, We also detected the effect of bufalin combined with ATRA on differentiation of APL cells in primary culture and NB4 cell, and characterize the related effects of Mitogen - activated Protein Kinases on the differention of APL cell by ATRA combined with bufalin.Methods1 . Assays for the proliferation of APL cellThe proliferation was determined by counting Btirker chamber and cell viability was determined by typan blue exclusion.2. Assay for apoptosisThe apoptosis of APL cell was assessed by morphological analysis. Cell cycle analysis using a Becton Dickinson FACScan flow cytometer ( Cambridge, MA) using a commercially available software program.3 . Assays for differentiation(1) The maturation was assessed by morphological analysis.(2) Differentiation of APL cell was assessed by the nitro blue tetrazolium (NBT) reduction test . The percentage of cells containing formazan depoisits, corresponding to the capacity to reduce NBT, was determined by counting 200 cells.(3) Expression of the granulocyte/macrophage - specific antigen CDllb was analyzed by monoclonal antibody labeling and flow cytometry using a FACScan.4. Western Blot analysisSurvivin , Caspase -3 and p -ERK were analyzed by Western Blot.5. RT - PCR Survivin mRNA ,PML/RARa mRNA expression was detected by semi - quantitative RT - PCR.6. Statistical Analysis. The significance of differences between experimental conditions was determined using the two - tailed Student t test.Results1. Bufalin induced apoptosis in a dose - and time - dependent fashion in NB4 cells. Proliferation of NB4 cells was inhibited by bufalin and the IC50 at 24h, 48h and 72h were 40 nmol/L, 27nmol/L and 17nmol/,respectively. Apoptosis of NB4 cells were induced when the cells were treated with bufalin at concentrations of 20nmol/L and higher, cell hypodiploid percentage at 24h, 48h and 72h was 13.7% ,20. 5%i￡ 37. 3% by flow cytometry analyses, respectively Compared with control,treatment with bufalin at 20nmol/L for 12 h,24 h and 48h resulted in decrease of Survivin protein expression . The cleaved active sub-units of Caspase - 3 were observed following bufalin treatment from 24h to 72h.2. We found that in NB4 acute promyelocytic cell line treatment with the MEK inhibitors PD98059 greatly enhances apoptotic cell death induced bybufalin alone. Combined treatment results in downregulation of the Survivin in NB4 cell line . The levels of p - ERK were detennined by Western Blot analysis at 5 minute after treatment with bufalin alone.3. Reinforcement by bufalin of RA - induced differentiation in APL cells.Bufalin alone did not induce NBT reduction of NB4 cells in 1 - lOnM concentration, if combined with 30 nM ATRA for 7 days, Bufalin did enhance differentia-teon induced by ATRA . Compared with ATRA, Bufalin combined with ATRA induces functional and morphologic differentiation of APL cells. The percentage of NBT reduction cells increased obviously, and expression of CDllb was also enhanced by combination treatment, although bufalin alone did not enhance CDllb expression. Combined effect of bufalin and RA on differentiation of APL cells in primary culture.We also examined the effect of treatment bufalin and RA on differentiation of APL cells in primary culture. APL cells from twelve patients were studied. Treatment with more than 50 nM bufalin induced apoptosis of APL cells in four of twelve cases. Our results suggest that bufalin combined with ATRA induces functional and morphologic differentiation of APL cells, A suboptimal concentration of RA ( 30nM ~ 50nM ) induced differentiation in six of twelve cases, these cases showed differentiation toward granulocytes after bufalin plus RA. Mature granulocytic cell differentiation was confirmed by expression of CDllb , Compared with ATRA ,expression of CDllb increases 16% ~69%of the combination. When concentration of ATRA alone is 30nM ~ 50nM, Differentiateon of APL cells were obviously observed in four of twelve cases. Compared with combination, there is no obvious difference. In one of twelve cases, APL cells exhibited spontaneous differentiation, and their differentiation was not enhanced by bufalin.DiscussionThough ATRA is efficiently differentiation inducer for APL, side effect, especially , Leukocytosis and RAS are serious problems in treatment, and there is no good solution to the problem about resistance to ATRA.Someone reports that bufalin induced apoptosis of HL60, ML1 and U937 cell in high concentration, and induced differentiation toward monocyte/macro-phage - like cells in low concentration. Our results showed that bufalin alone induced apoptosis of APL primary culture cells and NB4 in the concentration ofmore than 10 - 50nM, but there is no apoptosis in less than lOnM. Some observations were reported that MEKl/2 inhibitors potentiate the antitumor activity of various cytotoxic agents, including ara - C , cisplatin, and paclitaxel , suggests a possible role for MEKl/2 inhibitors in the treatment of human malignancies. At present, mechanism of bufalin is still unknown. It possibly actived APK , inhibit Topo II activity, and up - regulated Tiaml or down - regulated the express of c - myc and bcl - 2 mRNA to induce apoptosis;Someone reported distinct PKC isozymes regulate bufalin - induced differentiation and apoptosis in human monocytic cells.To explore details involved in the process , apoptosis - related protein Sur-vivin was determined by Western Blot and RT - PCR , Caspase - 3 also was tested by Western Blot, we found that exposure of NB4 cells to bufalin resulted in down - regulation of Survivin and Caspase - 3 protein , down - regulation of Survivin mRN A was also detected . Furthermore, Survivin downregulation and activation of Caspase - 3 became more marked in coadministration of bufalin with PD98059, the results indicates MEK/ERK signaling pathway mediates pro-gess of bufalin — induced apoptosis involved in Survivin downregulation in the post - transcription level.But there is enhancement by bufalin of RA - induced differentiation in NB4 cells . The synergistic effect is also showed in APL primary culture cells. In all cases we observed, the proportion of morphology changes of combination occupy 45% , the one of NBT reduction rate occupy 50% and the express of CDllb occupy 51 % Compared with the control , the concentration of bufalin that we used is no side effect on cell vitality , that is no cytotoxic effect . Effect concentration of combination decrease 30% of ATRA alone. The result suggests that bufain and ATRA are co - induction effect oh differentiateon of APL cells. The dosage of ATRA combined with bufalin is lower than ATRA alone . So decrease of dosage benefits to relieve side effect, especially Leukocytosis and RAS. We can detect the sensibitity of APL primary culture cells to inducers, and predict treatment effect of ATRA. Differentiation positive in vitro is easily obtained CR . The correlation is obvious.Conclusion1. Bufalin inhibited APL cell proliferation in a time - and dose - dependent manner and induced NB4 cell apoptosis accompanied by Survivin downregu-lation and activation of Caspase -3.2. MEK/ERK signaling pathway is negative regulation in bufalin - induced apoptosis in NB4 cell.3. Bufalin enhanced ATRA -induced differentiation in NB4 cell and primary culture in APL cells. |
