The traditional Chinese medical compound Rocaglamide protects nonmalignant primary cells from DNA damage-induced toxicity by inhibition of p53 expression

Abstract

One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors.

Figure 1

Roc-A protects nonmalignant cells from DNA damage-induced cytotoxicity. (a) A cell viability screen reveals Roc-A to be a potential DNA damage-protective compound. Peripheral blood T cells from healthy donors were treated with solvent (DMSO) or 50 μM Etoposide in the presence of different TCM compounds or solvent as indicated. Cell viability was determined after 24 h of treatment by Cell-Titer Glo viability assay. Data are an average of three independent experiments. (b) Roc-A protects T cells from Etoposide-induced cell death in a dose- and time-dependent manner. Left panel: T cells were treated with solvent (DMSO) or increasing amounts of Etoposide in the presence of different concentrations of Roc-A or solvent (DMSO) for 24 h. Cell death was determined by DNA fragmentation. Data are an average of three independent experiments. Error bars (S.D.) are shown. Middle panel: T cells were treated with 50 μM Etoposide in the presence of different concentrations of Roc-A or solvent (DMSO) for the indicated time periods. Cell death was determined by DNA fragmentation. Data are an average of three independent experiments. Error bars (S.E.M.) are shown. Right panel: Roc-A was added 2 h before, in parallel or 2 and 4.5 h after Etoposide (50 μM) treatment. Data are presented as percent of protection of T cells from Etoposide-induced cell death. Results are an average of three independent experiments. Error bars (S.E.M.) are shown. (c) Roc-A reduces Teniposide-, Doxorubicin- and Bleomycin-induced cell death in T cells. Peripheral blood T cells were treated with Teniposide (left panel), Doxorubicin (middle panel), Bleomycin (right panel) or solvent (DMSO) in the presence of Roc-A (75 nM) or solvent (DMSO) as indicated. Cell death was determined by DNA fragmentation for Teniposide and Bleomycin treatment or by FSC/SSC profile for Doxorubicin treatment. Data are an average of three independent experiments. Error bars (S.D.) are shown. (d) Roc-A protects a panel of nontransformed primary cells from Etoposide-induced cell death. Primary human B cells, NK cells, neutrophils, HSPCs and cardiomyocytes were treated with solvent (DMSO) or Etoposide in the presence of different concentrations of Roc-A or solvent (DMSO) for different times as indicated. Cell death was determined by DNA fragmentation. Results are an average of three to four independent experiments. Error bars (S.D.) are shown

Figure 2

Roc-A does not protect T cells from genotoxin-induced DNA damage. (a) Roc-A does not prevent Etoposide-induced increase in γ-H2AX. T cells were treated with solvent (DMSO) or different concentrations of Etoposide in the presence of Roc-A (75 nM) or solvent (DMSO) for 4 h. DSB induction was assessed by determination of the mean fluorescence intensity (MFI) of γ-H2AX-stained cells. Data are an average of three independent experiments. Error bars (S.D.) are shown. (b) Kinetic analysis of the effect of Roc-A on Etoposide-induced DSBs. T cells were treated with 50 μM Etoposide in the presence of Roc-A (75 nM) or solvent (DMSO) for different times and DSB induction was determined as described in (a). Data are an average of three independent experiments. Error bars (S.D.) are shown. (c) Confocal microscopy analysis of Etoposide-induced γ-H2AX-foci in the absence or presence of Roc-A. Nuclei of cells, obtained from (a) and (b), were stained with DAPI (blue) and imaged by confocal microscopy. γ-H2AX staining is shown in green. Three representative nuclei are shown for each treatment. (d) Comet assay to monitor the effect of Roc-A on Etoposide-induced DNA damage in T cells. Peripheral blood T cells were treated with solvent (DMSO) or Etoposide (50 μM) in the presence of Roc-A (75 nM) or solvent (DMSO) for different time periods as indicated. A comet assay was carried out subsequently. The distribution of olive tail moments (OTM) among treated cells is shown. Data are representative of three different healthy donors. (e) Summary of the data obtained from (d). Results are an average of the mean OTM of three different healthy donors. Error bars (S.D.) are shown. *P<0.05, calculated by the unpaired Student's t-test with Welch's correction

Figure 3

Roc-A blocks genotoxin-induced upregulation of p53. (a and b) Roc-A inhibits Etoposide- (a), Bleomycin-, Teniposide- and Doxorubicin (b)-induced p53 upregulation in T cells. T cells were treated with solvent or different anticancer drugs in the presence of different concentrations of Roc-A or solvent (DMSO) as indicated. Cell lysates were subjected to immunoblot analysis with antibodies against p53. Actin or tubulin were used as loading controls. Data are representative of three independent experiments. (c) Kinetic analysis of the effect of Roc-A on Etoposide-induced p53 upregulation. T cells were treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for different time periods as indicated. Cell lysates were subjected to immunoblot analysis with antibodies against p53 and tubulin. Data are representative of two independent experiments. (d and e) Roc-A inhibits Etoposide-induced p53 upregulation in B cells (d) and NK cells (e). Cells were treated with solvent (DMSO) or Etoposide in the presence of Roc-A or solvent (DMSO) as indicated and cell lysates were subjected to immunoblot analysis. Data are representative of two independent experiments. In (a, b, d), the double band observed for p53 is likely to occur because of a heterozygous p53 R72P polymorphism

Figure 4

Roc-A-mediated chemoprotection depends on p53. (a) Roc-A inhibits Nutlin-3-induced p53 upregulation and protects T cells from Nutlin-3-induced cell death. T cells were treated with solvent (DMSO) or Nutlin-3 in the presence of Roc-A or solvent (DMSO) as indicated. After 24 h of treatment, the expression levels of p53 were analyzed by immunoblot (upper panel). Cell death was determined after 48 h of treatment by FSC/SSC profile (lower panel). Data are representative of three independent experiments. Error bars (S.D.) are shown. (b) Roc-A reduces Etoposide-induced mRNA expression of p53 target genes. T cells were treated with solvent (DMSO) or Etoposide (50 μM) in the presence of Roc-A (75 nM) or solvent (DMSO) for different time periods as indicated. Total RNA was isolated and subjected to quantitative real-time PCR for BAX, MDM2, BBC3 (PUMA), FAS and BCL2L11 (BIM). Data are presented as fold change in mRNA levels and normalized to control treatment with solvent (DMSO). Data are representative of three independent experiments. Error bars (minimum and maximum) are shown. (c) siRNA-mediated knockdown of p53 mimics the protective effect of Roc-A. T cells were transfected with scrambled (si-Ctrl.) or specific siRNA targeting p53 (si-p53). At 24 h after transfection, T cells were treated with solvent (DMSO) or Etoposide (50 μM) in the presence of Roc-A (75 nM) or solvent (DMSO) as indicated for 24 h. The expression levels of p53 were analyzed by immunoblot and cell death was determined by FSC/SSC profile. Data are representative of three independent experiments. Error bars (S.D.) are shown. (d) Roc-A-mediated protection is abolished in p53^−/− splenocytes. Splenocytes from p53^−/− or p53^+/+ mice were treated with 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for indicated time periods. Cell death was determined by DNA fragmentation. Data are an average of four independent experiments. Error bars (S.D.) are shown

Figure 5

Roc-A does not protect cancer cell lines with nonfunctional p53. p53-mutated or -deficient cancer cell lines (a) and p53 WT cell lines (b) were treated with solvent (DMSO) or different concentrations of Etoposide in the presence of increasing amounts of Roc-A or solvent (DMSO) as indicated. Cell death was determined by DNA fragmentation after 24 or 48 h of treatment as indicated. Results are averages of three independent experiments. Error bars (S.D.) are shown

Figure 6

Roc-A inhibits upregulation of p53 via inhibition of protein synthesis. (a) Inhibition of proteasome-mediated degradation does not influence Roc-A-mediated chemoprotection. T cells were treated with solvent (DMSO) or 100 nM Bortezomib to block proteasome-mediated protein degradation and treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for 4 h. Cell lysates were subjected to immunoblot analysis with antibodies against p53 and Actin. Data are representative of three independent experiments. (b) Roc-A does not influence ubiquitination of p53. T cells were treated with Bortezomib, Etoposide and Roc-A as in (a). After 4 h of treatment, cell lysates were used for immunoprecipitation against p53. Isotype-matched antibody-coupled beads and uncoupled beads were used as controls. Ubiquitinated p53 ((UB)_n-p53) is indicated. Data are representative of two independent experiments. (c) Inhibition of protein translation by Roc-A or its derivatives correlates with their chemoprotective effects. Effects of Roc-A and its derivatives (-AB, -J, -AR, -Q, -I, -AF and -AA) on protein synthesis in T cells was determined by measuring the amounts of incorporation of [³⁵S]-labeled methionine. Cell death was determined by DNA fragmentation of T cells treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM of different Roc-derivatives or solvent (DMSO) for 24 h. The percentage of chemoprotection was determined by calculating the percentage of protection against Etoposide-induced cell death. The data are shown by plotting the percentage of translation inhibition against the percentage of chemoprotection. Data are an average of three independent experiments. Error bars (S.E.M.) are shown. An allosteric sigmoidal regression curve was plotted against the experimental data. R²=0.96. (d) Roc-A inhibits p53 protein translation. T cells were treated according to (a), followed by metabolic pulse-labeling for indicated time periods and immunoprecipitation. Data are representative of three independent experiments. (e) Roc-A does not reduce the mRNA levels of p53. T cells were treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for indicated time periods. Total RNA was isolated and was subjected to quantitative real-time PCR for TP53. Data are presented as fold increase in mRNA levels determined by comparison of HPRT1 gene expression levels with TP53 and normalized to control treatment with solvent (DMSO). Data are an average of three independent experiments. Error bars (S.D.) are shown