Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

MON [6], 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.

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 [11].

Propidium iodide staining and FACS analysis were performed as described previously [9]. 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 [9]. 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.

To determine the effect of combining flavopiridol with 4OH-Tam, we treated MON, G401, and A204 RT cells with increasing concentrations of flavopiridol (0 to 200 nM) in combination with 2.5, 5.0, or 10.0 μM 4OH-Tam for two days. Flavopiridol inhibited RT cell growth with IC₅₀ values of ~50-100 nM, with MON cells showing an intermediate level of sensitivity compared to G401 and A204 cells (Figure 1A-D). Exposure of all cells to flavopiridol and 4OH-Tam in combination resulted in a dramatic reduction in IC₅₀ values, with 10 μM 4OH-Tam reducing the IC₅₀s to ~0.12-0.2 nM (Figure 1A-D). Furthermore, increasing the time of exposure to the combination of drugs enhanced the inhibitory effects in both MON and G401 cells (compare Figures 1A and 1B with Figure 2A, B, E, and 2F). Bar graphs derived from the data in Figure 2A and 2E are given to show the decreases in cell survival induced by the specific concentrations of the drugs after three days of exposure (Figure 2D and 2H). We also tested the effect of 4OH-Tam alone and found that 4OH-Tam decreased cell survival significantly (Figure 2C and 2G). However, increasing time of exposure to 4OH-Tam alone from one to three or five days, did not result in further decreases of cell survival (Figure 2C and 2G). Thus, while the flavopiridol and 4OH-Tam combination resulted in a time dependent decrease of survival, the effect of 4OH-Tam alone was independent of time of exposure. Notably, by day five of treatment, combinations of very low concentrations of flavopiridol (~ 0.2 nM) and 10 μM 4OH-Tam decreased cell survival by 80% in MON and 99% in G401 cells (Figure 2B and 2F). These results indicated that treatment with low concentrations of flavopiridol in combination with 4OH-Tam for prolonged periods potently inhibits RT cell growth.

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To determine the mechanism of growth inhibition by these drugs, we studied MON cells which showed intermediate responses to the combination of drugs with an intermediate level of 4OH-Tam (5 μM) and duration of treatment. MON cells were exposed to combinations of flavopiridol and 5 μM 4OH-Tam for two days and subjected to cell cycle analysis. Treatment of cells with 5 μM 4OH-Tam alone resulted in G₁ arrest (p = 0.0117, Figure 3A). Our previous results indicated that flavopiridol at 200-400 nM induces G₁ arrest [13]. However, treatment with 100 nM flavopiridol induced G₂ arrest in the presence or absence of 4OH-Tam (p = 0.0039 and p = 0.0003, respectively, Figure 3A) indicating that the stage of cell cycle arrest induced is dose-dependent. These results also indicate that G₂ arrest induced by flavopiridol is dominant over 4OH-Tam-induced G₁ arrest. Analysis of the sub-G₁ fraction of cells after single or combination treatment indicated that the percentage of sub-G₁ cells increased in a dose-dependent manner upon treatment with flavopiridol, which was further enhanced by combination with 4OH-Tam (Figure 3B). Very high levels of cell death are induced by these treatments and this likely plays the major role, compared to induction of cell cycle arrest, in inducing cytotoxicity in response to treatment with these drugs. To determine if cell death was due to apoptosis, the cells were stained for annexin (indicative of apoptosis) and propidium iodide (indicative of necrosis) and the percentage of cells positive for either or both stains was determined by FACS (Figure 3C). While the percentage of necrotic or dead cells remained largely unchanged, there was a dose-dependent increase in annexin-positive cells demonstrating that combination treatment with flavopiridol and 4OH-Tam induces significant levels of apoptosis.

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Since it is known that flavopiridol can induce apoptosis by caspase-dependent or caspase-independent mechanisms, we investigated the effect of a caspase inhibitor on drug-induced cytotoxicity. MON cells were exposed for two days to flavopiridol and 4OH-Tam in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK, and FACS analysis was carried out to assess the effects on cell cycle and apoptosis. The results indicated that drug-induced cell cycle arrest was unaffected (p = 0.0458 and p = 0.0025 for G1-arrest induced by 100 nM flavopiridol with and without 4OH-Tam respectively) (Figure 4A), but induction of apoptosis by flavopiridol with or without 4OH-Tam was significantly inhibited by Z-VAD-FMK (p < 0.0001, Figure 4B). This established that cell death induced by flavopiridol, 4OH-Tam, or their combination is mediated by caspase-dependent apoptosis in RT cells. We further investigated the kinetics of induction of caspase 3 and 7 activities upon drug treatment. The results indicated that, while flavopiridol induced peak caspase 3 and 7 activities at six hours, 4OH-Tam exhibited delayed kinetics with peak induction at 12 hours. Interestingly, combination of 4OH-Tam and flavopiridol elevated caspase 3 and 7 activities at both 6 and 12 hours (Figure 4C). These results suggest that combination of flavopiridol and 4OH-Tam could increase apoptosis by inducing and prolonging caspase 3/7 activities.

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To determine the mechanism of cell cycle arrest induced by flavopiridol and 4OH-Tam, we examined the expression levels of several cell cycle regulatory and tumor suppressor proteins in the presence and absence of drugs. 100 nM flavopiridol induced p53 and p21^Waf-1 and repressed cyclin B1, consistent with the observed G₂ arrest (Figure 5A). We found that the majority of cyclins and cdks, including cyclins D1, A and E, and cdks 2, 4 and 6 were unaffected by concentrations of flavopiridol ≤ 100 nM (Figure 5A). Interestingly, the presence of 4OH-Tam did not significantly alter the effects of flavopiridol on cyclin and cdk expression, but further decreased the level of pRb protein and phosphorylation (Figure 5A compare lanes 4 and 8). We also observed differential effects on pRb phosphorylation. Concentrations of flavopiridol ≤ 25 nM increased the level of phosphorylated pRb (Figure 5A lanes 2 and 3), but 100 nM dramatically decreased the phosphorylated form of pRb (Figure 5A lane 4). Addition of 4OH-Tam eliminated the flavopiridiol-induced phosphorylation of pRb at the 25 nM concentration (Figure 5A lane 7). However, 4OH-Tam had no effect on 100 nM flavopiridol-induced inhibition of pRB phosphorylation, consistent with the observed dominant effect of 100 nM flavopiridol in inducing G₂ arrest.

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Our previous analysis indicated that flavopiridol efficacy in mouse xenografts correlated to both down-modulation of cyclin D1 and up-regulation of p21^Waf-1 [13]. Therefore, we examined p21^Waf-1 levels upon flavopiridol treatment with and without 4OH-Tam. We found that p21^Waf-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 p21^Waf-1(Figure 5A, compare lanes 1 and 5). We also investigated the accumulation of nuclear p21^Waf-1 upon drug treatment by immunocytochemical analysis. The results indicated that flavopiridol treatment led to increased nuclear p21^Waf-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 p21^Waf-1 (Figure 5C) and, taken together, indicate that combination treatment results in both increased protein levels and increased nuclear localization of p21^Waf-1.

The above studies pointed to the involvement of p21^Waf-1 in flavopiridol or flavopiridol plus 4OH-Tam-mediated cytotoxicity. It has been established that p21^Waf-1 is a downstream effector of p53 [38]. Furthermore, tumorigenesis in Ini1 heterozygous mice is enhanced upon abrogation of p53 [39]. Since p53 is up regulated upon flavopiridol treatment, we further investigated its role. To determine whether p53 is necessary for flavopiridol-induced cell cycle arrest and/or apoptosis, RNA interference was used to down-modulate p53 expression. Transfection of RT cells with p53 siRNA slightly enhanced cell proliferation (data not shown). As expected, flavopiridol markedly increased p53 expression in cells transfected with control siRNA (Cy3), but not in cells transfected with p53 siRNA (Figure 6A, compare lanes 2 and 6). FACS analysis of cells transfected with control or p53 siRNA and treated with single or combination drugs for two days indicated that, while control siRNA did not affect drug-induced cell cycle arrest (p = 0.0221 and p = 0.0035 for G₁-arrest induced by 100 nM flavopiridol with and without 4-OH-Tam respectively, Figure 6B), knock-down of p53 resulted in abrogation of flavopiridol-mediated G₂ arrest both in the presence and absence of 4OH-Tam (p = 1.000 (not significant), Figure 6C). These results indicated that p53 is necessary for flavopiridol-mediated G₂ arrest in RT cells.

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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.

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.

Analysis of the activation kinetics of caspases 2, 3, 8 and 9 indicated that neither flavopiridol nor 4OH-Tam induced caspase 8 and 9 activities in RT cells (Figure 7A and 7B and Additional File 1A-C). Induction of caspases 2 and 3 was affected by treatment with flavopiridol and flavopiridol with 4OH-Tam (p = 0.0022 for caspase 2 and p.0001 for caspase 3) and was correlated to p53 levels (p = 0.0074 for caspse 2 and p < 0.0001 for caspase 3) (Figure 7C and 7D). Flavopiridol increased caspase 2 and 3 activity two-fold within 6 hours and abrogation of p53 allowed flavopiridol to increase caspase 2 and 3 activity four-fold in the same time (Additional File 1D and 1G). Exposure of cells to flavopiridol with 4OH-Tam caused an overall increase in caspase 2 and 3 activity, with maximum induction observed at 12 hours (Figure 7C and Additional File 1F and 1I). Abrogation of p53 led to a further increase in caspase 2 and 3 activity after combination treatment, similar to that caused by flavopiridol alone (Figure 7C and Additional File 1F and 1I). 4OH-Tam alone caused only slight increases in caspase 2 and 3 activities in the presence or absence of p53 (Figure 7C and Additional File 1E and 1H).

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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).