Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen
- Velasco Cimica†1,
- Melissa E Smith†1,
- Zhikai Zhang1,
- Deepti Mathur1,
- Sridhar Mani1, 2, 3 and
- Ganjam V Kalpana1, 3Email author
© Cimica et al; licensee BioMed Central Ltd. 2010
Received: 11 November 2009
Accepted: 19 November 2010
Published: 19 November 2010
Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs.
The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined.
We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G2 arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G2 arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G2 arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities.
Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.
RTs, including Malignant Rhabdoid Tumors (MRT), Atypical Teratoid and Rhabdoid Tumors (AT/RT), and extra renal rhabdoid tumors (ERRT) are rare, but highly aggressive pediatric solid tumors with poor prognosis . Current therapy for RTs includes surgical resection, radiation therapy, and/or chemotherapy with empirically selected and highly toxic chemotherapeutics, which are largely ineffective [2, 3]. Despite aggressive treatment, mean survival with surgical intervention alone is only 3 months and with adjuvant chemotherapy and radiotherapy is only 8 months . Therefore, strategies based on understanding the genesis of RTs will aid in the development of novel therapies. RTs are characterized by biallelic deletions and/or mutations in INI1/hSNF5, a tumor suppressor and component of the chromatin remodeling SWI/SNF complex [5, 6]. Reintroduction of INI1/hSNF5 into RT cells induces G1 cell cycle arrest and senescence. INI1/hSNF5 mediates these effects by directly activating p16Ink4a by recruiting the SWI/SNF complex and by directly repressing cyclin D1 by recruiting the HDAC1 complex [7–10]. We have found that cyclin D1 is de-repressed in human and mouse RTs and is required for rhabdoid tumorigenesis in mouse models [9, 11, 12]. Such studies indicated that therapeutic targeting of cyclin D1 and its pathway could be an effective and novel therapeutic strategy for RTs.
We previously reported that down-modulating cyclin D1 and inhibiting cyclin dependent kinases (cdks) using either flavopiridol or a combination of N-(4-hydroxyphenyl)retinamide (4-HPR) with 4OH-Tam is effective in inhibiting RTs in vitro and in xenograft tumor models in vivo [11, 13]. The effectiveness of 4-HPR and flavopiridol was correlated with down-modulation of cyclin D1 in xenograft tumors .
Flavopiridol is one of the first cdk inhibitors to enter clinical trials. Although early clinical trials were unsuccessful, design of a novel schedule of administration based on the in vitro and in vivo pharmacokinetic modeling of flavopiridol's effect has shown promising efficacy in refractory chronic lymphocytic leukemia . Phase I trials of flavopiridol in children have revealed that its toxicity profile, pharmacokinetics, and maximum tolerable dose were similar to that in adults, indicating that using flavopiridol in RT patients, a largely pediatric population, is feasible .
The effects of flavopiridol on cancer cells are varied and cell type dependent. In many cell lines flavopiridol leads to G 1 arrest due to down-modulation of cyclin D1 and inhibition of its pathway by various mechanisms [16–24]. In other cells, flavopiridol induces G 2 arrest, in part due to its potent ability to inhibit cdk 7, 8 and 9 activities . Flavopiridol also inhibits transcription of Mdm-2 resulting in the accumulation of its proteolytic target, p53, which triggers p21Waf1 up-regulation, cyclin B1 down-regulation, and ultimately G2 arrest . Flavopiridol can induce apoptosis at nanomolar concentrations and its pro-apoptotic action is either caspase-dependent or -independent . Flavopiridol can trigger apoptosis by activation of caspases 2, 3 and 8  or by activation of apoptosis inducing factor (AIF) via its release from the mitochondria . At this point, the mechanism of action of flavopiridol in RT cells is not completely understood.
Since flavopiridol can be toxic at high doses, recent studies have focused on combining low concentrations of flavopiridol with other anti-neoplastic agents [29, 30]. In this report, we tested the combination of flavopiridol with 4OH-Tam to determine its ability to increase therapeutic efficacy against RT cells. 4OH-Tam inhibits tumor cell growth in part by deregulating cyclins and cdks. Breast cancer cells over-expressing cyclin D1 are resistant to 4OH-Tam and the level of cyclin D1 is negatively correlated to responsiveness to 4OH-Tam [31–34]. 4OH-Tam suppresses the growth of estrogen-receptor positive tumors by down-modulating cyclin D1 . Furthermore, treatment of tumor cell lines with 10μM 4OH-Tam induces the expression of p21Waf1 and p27Kip1, which are known to block the effects of cyclin D1 . Previous studies have demonstrated that 4OH-Tam is effective in inducing cytotoxic effects in RT cells . In this study it was demonstrated that the expression of ERα receptor in RT cells is variable and that the cytotoxic effects of 4OH-Tam are independent of ERα expression . Since the efficacy of flavopiridol in xenograft RTs was correlated with down-modulation of cyclin D1 and up-regulation of p21Waf1, we considered combining 4OH-Tam with flavopiridol to enhance its therapeutic efficacy in RT cells.
We report here that the combination of flavopiridol with 4OH-Tam potently inhibited the survival of RT cells. A low concentration of flavopiridol (100 nM) induced G2 arrest in RT cells in a p53-dependent manner and resulted in a moderate amount of apoptosis. Addition of 4OH-Tam significantly increased flavopiridol-mediated apoptosis. Down-modulation of p53 did not affect 4OH-Tam-induced cytotoxicity, but significantly enhanced flavopiridol-mediated apoptosis. Furthermore, we found that the increased cytotoxic effects of flavopiridol and 4OH-Tam correlated with augmentation of caspase 2 and 3 activities and that these effects were independent of p53. These studies indicate that the combination of flavopiridol and 4OH-Tam can be effective in potently inhibiting RT cell growth and is potentially a novel therapeutic strategy against RTs.
Cell culture and drug treatment
MON , G401 (American Type Culture Collection), and A204 cells (American Type Culture Collection) were maintained in RPMI supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine. For drug studies, the cells were transferred one day before drug treatment to RPMI containing 10% charcoal and dextran treated fetal bovine serum (HyClone, Cat. SH30068.03). Flavopiridol was obtained from the CTEP program at NCI (courtesy of Dr. Colevas). 4OH-Tam and pan-caspase inhibitor z-VAD-FMK were purchased from SIGMA (Catalogue #H7904) and Promega (Catalogue #G7232) respectively.
Cell Survival analysis
8,000 cells/well (MON and A204) or 6,500 cells/well (G401) were plated in 96-well plates and treated with different concentrations/combinations of drugs, using the epMotion 5070 automated liquid handler system (Eppendorf). The CellTiter 96 AQueous One Solution Cell Proliferation Assay was used to determine cell survival (Promega, Catalogue #G3580). Data elaboration of cell survival, IC50, and drug combination effects were performed according to previously described methods .
Propidium iodide staining and FACS analysis were performed as described previously . Annexin staining was performed using Annexin V-FITCH Apoptosis Detection Kit I (BD Pharmingen Catalogue #556547) according to the manufacturer's instructions. Data was elaborated using CellQuest Pro program (BD Pharmingen).
Data was analyzed using GraphPad Prism by applying the ANOVA test or t-test.
Immunoblot analysis was carried out as described previously with minor modifications . Dry milk was used as a blocking agent for the following antibodies: p53 (Santa Cruz Catalogue #sc-126), cyclin A (Santa Cruz Catalogue #sc-239), cyclin B1 (Santa Cruz Catalogue #sc-752), cyclin E (LabVision Catalogue #MS-870-P), cyclin D1 (Lab Vision Catalogue #RB-010-P), E2F-1 (Santa Cruz Catalogue #sc-251), GAPDH (Chemicon Catalogue #MAB374) and Rb (Santa Cruz Catalogue #sc-102); and bovine serum albumin for the following antibodies: cdk 2 (Santa Cruz Catalogue #sc-163), cdk 4 (Santa Cruz Catalogue #sc-260), cdk 6 (Santa Cruz Catalogue #sc-7961) and p21 (Calbiochem Catalogue #OP64). Chemiluminescence detection was achieved using SuperSignal West Pico Chemiluminescence Substrate (Pierce Catalogue #34080).
MON cells were plated at 300,000 cells/well in 6-well plates one day before transfection. Cells were transfected using DharmaFECT siRNA transfection reagent (Dharmacon Catalogue #T-200(01-07)-01) according to the manufacturer's instruction and using previously published p53 siRNA (Qiagen Catalogue #024849) and control (Cy3-Luciferase GL2 Duplex, Dharmacon Catalogue #D-001110-01-05). One day post-transfection, cells were split into 50,000 cells/well using medium containing 10% charcoal and dextran treated fetal bovine serum, for drug treatment the following day.
MON cells were grown on glass coverslips and treated with drugs. Cells were fixed with 4% paraformaldehyde-PBS solution for 10 minutes, and permeabilized with 0.1% triton-PBS solution for 10 minutes. Cover-slips were treated for 1 hour at room temperature with 1:250 diluted αp21 antibody (Calbiochem Catalogue #OP64). Staining was detected using the Vectastain ABC kit (Vector Laboratories Catalogue #PK-6102) according to manufacturer's instruction. Peroxidase staining was developed using the DAB enhanced liquid substrate system (SIGMA Catalogue #D3939-1SET). The percentage of cells with nuclear expression of p21 was quantified by counting 250 to 300 individual cells, noting whether or not their nuclei showed positive staining above the background. The background staining was defined as an intensity of staining at or below the intensity of the negative control (i.e. any background staining that occurred in the absence of a p21-specific antibody).
Caspase 3/7 Assay was performed using the Caspase-Glo 3/7 Assay kit (Promega Catalogue #G8093). Caspase activity profiling assays were performed using ApoAlert Caspase Profiling Plate (Clontech Catalogue #630225), according to the manufactures' instruction with minor modification, using 50 μg of protein for each well of the assay.
Results and Discussion
Combination treatment with flavopiridol and 4OH-Tam inhibits the growth of RT cells
Induction of apoptosis by flavopiridol and 4OH-Tam
Induction of apoptosis by flavopiridol and 4OH-Tam is caspase-dependent
Mechanism of G2 arrest mediated by flavopiridol and 4OH-Tam
Flavopiridol induced p21Waf-1 expression in RT cells
Our previous analysis indicated that flavopiridol efficacy in mouse xenografts correlated to both down-modulation of cyclin D1 and up-regulation of p21Waf-1 . Therefore, we examined p21Waf-1 levels upon flavopiridol treatment with and without 4OH-Tam. We found that p21Waf-1 was up-regulated in a dose-dependent manner upon treatment with flavopiridol (Figure 5A, lanes 1-4), but 4OH-Tam alone only modestly up-regulated p21Waf-1(Figure 5A, compare lanes 1 and 5). We also investigated the accumulation of nuclear p21Waf-1 upon drug treatment by immunocytochemical analysis. The results indicated that flavopiridol treatment led to increased nuclear p21Waf-1, which was further increased upon addition of 4OH-Tam (Figure 5B and 5C). These results were confirmed by quantifying the percentage of cells with nuclear p21Waf-1 (Figure 5C) and, taken together, indicate that combination treatment results in both increased protein levels and increased nuclear localization of p21Waf-1.
Abrogation of p53 inhibited cell cycle arrest but enhanced apoptosis induced by flavopiridol
Interestingly, knock-down of p53 demonstrated differential effects on flavopiridol and 4OH-Tam-mediated apoptosis in RT cells. Knock-down of p53 had no effect on 4OH-Tam-induced apoptosis indicating that 4OH-Tam-induced apoptosis is independent of p53 (Figure 6D). However, p53-knockdown enhanced flavopiridol-mediated apoptosis in the presence or absence of 4OH-Tam (Figure 6D). These results indicated that p53 is detrimental to flavopiridol-induced apoptosis in MON cells. Additionally, these results indicated that flavopiridol and 4OH-Tam induce apoptosis in RT cells by two different mechanisms; flavopiridol-mediated apoptosis being inhibited by p53 and 4OH-Tam-induced apoptosis being independent of p53.
Role of caspases in flavopiridol and 4OH-Tam induced apoptosis
Expression of p53 has been associated with resistance to radiation-induced apoptosis in some cancers including gliomas and keratinomas [40, 41]. Many RTs are resistant to chemotherapy and radiotherapy; however the mechanistic basis for this resistance is not clearly understood [42, 43]. Based on our results we surmised that induction of p53 by flavopiridol is counter-productive to induction of apoptosis in RT cells. It has been reported that p53 is expressed in a majority of RTs and sequence analysis of mRNA does not show any abnormality in the p53 coding region [44, 45]. Therefore, understanding the role of p53 in inhibiting drug-induced apoptosis might shed light on the mechanism of drug resistance exhibited by these tumors. Since flavopiridol increased p53 levels in RT cells, which was inversely correlated to induction of apoptosis, we explored the possibility that flavopiridol induced apoptosis through specific caspases. We profiled the kinetics of induction of caspase 2, 3, 8 and 9 activities in RT cells in the presence or absence of p53 and upon treatment with flavopiridol, 4OH-Tam, or their combination.
To demonstrate that induction of caspase 2 and 3 activities were specific, we treated MON cells in the presence and absence of p53 and also in the presence and absence of specific caspase 2 or 3 inhibitors. Caspase 2 and 3 activities were determined at the 24 hr. time point in all of the treatment conditions. We found that treatment of cells with caspase 2 or caspase 3 inhibitors significantly reduced the induction of caspase 2 or 3 activities upon drug treatment respectively. These results indicated that flavopiridol and 4OH-Tam drug treatment selectively induces caspases 2 and 3 in MON cells, as inhibitors to these caspases eliminated these increases (Additional File 1J and 1K).
Our report demonstrates, for the first time, that RT cell growth and survival is potently inhibited by combination treatment with clinically achievable concentrations of 4OH-Tam (2.5 or 5 μM) and flavopiridol (< 200 nM). Since high concentrations of flavopiridol may cause significant toxicities, and since only low concentrations may be achievable in areas such as the brain due to the blood brain barrier, our results provide a method to increase the efficacy of low concentrations of flavopiridol.
Flavopiridol and 4OH-Tam together induce a significant increase in RT cell death. Induction of cell death by flavopiridol and the combination of flavopiridol with 4OH-Tam is due to caspase-dependent apoptosis and caspase-profiling assays indicate that these treatments can potently induce caspases 2 and 3. In addition to inducing cell death, these treatments also induce cell cycle arrest. While our previous report indicated that 400 nM flavopiridol induces G1 arrest , this current report indicates that 100 nM flavopiridol is sufficient to cause G2 arrest indicating that flavopiridol potently induces cell cycle arrest in a dose-dependent manner. Flavopiridol-induced G2 arrest was correlated to down-regulation of cyclin B1 and up-regulation of p53 and p21Waf-1. On the contrary, 4OH-Tam inhibited p53 expression; however, this effect was nullified by addition of 100 nM flavopiridol, explaining the dominant effect of flavopiridol in mediating G2 arrest. These results suggest that the effect of flavopiridol in inducing p53 is upstream of the mechanism by which 4OH-Tam inhibits p53.
Interestingly, we found that p53 differentially regulated flavopiridol-mediated cell cycle arrest and apoptosis. RNA interference analysis of p53 indicated that while flavopiridol-mediated G2 arrest was dependent on p53, flavopiridol-mediated apoptosis (but not that mediated by 4OH-Tam) was countered by p53. This is an intriguing observation since there was a clear enhancement of apoptosis induced by flavopiridol when p53 was abrogated by RNA interference (Figure 6D). Previously it has been reported that lack of p53 enhances radio-sensitivity via activation of E2F-1 and induction of caspase 8 activity in glioma cells . Furthermore, radio-sensitivity of keratinocytes was enhanced by abrogation of p53 and was mediated by down-regulation of anti-apoptotic proteins Mcl-1 and Bcl-XL . To our knowledge, this is the first report which indicates that down-modulation of p53 also enhances drug-induced apoptosis. Our results indicate that the potency of flavopiridol can be enhanced if p53 can be inhibited by some means.
Most RTs express p53, though a percentage of RTs do show mutations within the p53 gene [44–46]. Some RT cell lines express p53 at high levels or with increased nuclear distribution, however, the p53 pathway has been tested and considered to be functionally intact [42, 44]. The mutation status of p53 in MON RT cells has not been determined but the proper expression of p53 and responsiveness of p21Waf1 to p53 levels leads us to believe that the p53 pathway is intact in these cells. However, other studies indicated that the pro-apoptotic pathway downstream of p53 may be dysfunctional in MON cells but the exact nature of this defect is unknown . Although this defect in the pro-apoptotic pathway downstream of p53 could account for the observed effects reported here, our observation indicates that flavopiridol-induced apoptosis is inhibited by p53. More experiments are needed to delineate the exact role of the p53 pathway in flavopiridol-induced cytotoxicity in MON and other RT cells.
The relationship of flavopiridol and p53 in inducing apoptosis seems to be paradoxical in different cell lines and in different treatment approaches. Perhaps this is related to the paradoxical anti-apoptotic activities of p53 itself in various cancer cells . The activities of p53 are cell type dependent and can be either pro-apoptotic and/or pro-survival. For example, in RT cells p53 is deleterious to flavopiridol-mediated apoptosis, but in other cancer cell lines flavopiridol-induced apoptosis is actually dependent on p53. An example is provided by Ambrosini et al who demonstrated that enhanced apoptosis induced by a combination therapy of flavopiridol with a G1-arrest-inducing agent (namely SN-38) was dependent on p53 . In this study the combination of flavopiridol and SN-38 was tested on isogenic pairs of cells differing only in p53. They found that the enhanced apoptosis by combination of flavopiridol and SN-38 was observed only in p53+/+ cells. Similar to SN-38, 4OH-Tam also induces G1 arrest in our system, however; the effects of combining flavopiridol with 4OH-Tam obtained in RT cells are different in terms of inducing apoptosis. While flavopiridol-mediated G2 arrest was dependent on p53, flavopiridol-induced apoptosis was abrogated by p53 (Figure 6C and 6D). On the contrary, p53 had no effect on 4OH-Tam-mediated apoptosis. Because of these results, we believe that p53 has compromising effects on flavopiridol-induced apoptosis in RT cells, similar to the effect of p53 in protecting cells from radiation induced apoptosis as discussed above.
Our studies involving p53 knock-down indicate that stimulation of p53 by flavopiridol limits its ability to induce apoptosis in RT cells. Thus, it is possible that 4OH-Tam increases the effects of flavopiridol because it down-modulates p53 in addition to inducing apoptosis by p53-independent mechanisms. This new understanding of p53's role in drug-induced apoptosis in RT cells might shed light on the mechanism of resistance to therapies exhibited by these tumors. Also, evaluation of p53 levels induced by flavopiridol and other treatments may be necessary to implement effective treatment strategies for RTs.
Flavopiridol induces apoptosis by additional p53-independent mechanisms. It is able to block RNA polymerase II phosphorylation by inhibiting cdk 9, thereby blocking transcriptional elongation. This activity, as well as flavopiridol's ability to reduce antiapoptotic protein MCL-1, has been implicated in the induction of apoptosis in multiple myeloma cell lines . Additionally, induction of the mitochondrial permeability transition by flavopiridol has been correlated with induction of apoptosis in chronic lymphocytic leukemia cells . These functions of flavopiridol may also contribute to the apoptosis occurring in RT cells in a p53-independent manner.
RTs are notoriously resistant to therapeutic interventions . Potent chemotherapy in combination with surgery and radiotherapy have proven futile in increasing survival rates and only a handful of RT survivors have been reported . Therefore, efforts to develop molecularly targeted therapies are needed. Based on the molecular understanding of RTs, it is known that INI1/hSNF5 mediates tumor suppression in part by targeting cyclins and cdks [7–9]. Furthermore, cyclin D1 is up-regulated in, and necessary for, rhabdoid tumorigenesis [9, 12]. Thus, it appears that therapeutically inhibiting the cyclin/cdk pathway is a novel, targeted treatment strategy for RTs. Our report suggests that combination of flavopiridol and 4OH-Tam could be used as a novel combination therapy for RTs. Furthermore, this combination could be effective in inducing apoptosis in other tumor models, especially those lacking p53.
At this point, the concentrations of 4OH-Tam required to increase the effects of flavopiridol appear to be high. Nevertheless, in the pediatric population, where RTs most often occur, it has been reported that high doses of tamoxifen (100 mg/m2 twice a day) can be administered with minimal toxicity . Furthermore, in cases where sustained high concentrations (≥ 10 uM) of 4OH-Tam may not be attained, alternative formulations, such as a liposomal formulation, of both tamoxifen and 4OH-Tam have been used that would result in the delivery of high concentrations directly to the tumor . Therefore, further preclinical and clinical studies to test the efficacy of these drugs in children may lead to the development of definitive therapeutic strategies against RTs that may improve prognosis.
The authors thank Dr. Prasad at AECOM for critically reading the manuscript and Dr. Kitsis at AECOM for useful discussions, Dr. Colevas at CTEP, NCI for providing flavopiridol. This work was supported by grants from ACS (#CCG-10493) and Children's Brain Tumor Foundation (CBTF, NY) to G.V.K, who is a Mark Trauner faculty scholar and a recipient of the Irma T. Hirschl Career Scientist Award. M.E.S. is supported by Institutional training grant, NIGMS (T32 GM 07491).
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