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Signal Transduction and Functional Imaging

Reactive Oxygen Species (ROS)-Inducing Triterpenoid Inhibits Rhabdomyosarcoma Cell and Tumor Growth through Targeting Sp Transcription Factors

Ravi Kasiappan, Indira Jutooru, Kumaravel Mohankumar, Keshav Karki, Alexandra Lacey and Stephen Safe
DOI: 10.1158/1541-7786.MCR-18-1071 Published March 2019

Abstract

Methyl 2-trifluoromethyl-3,11-dioxo-18β-olean-1,12-dien-3-oate (CF3DODA-Me) is derived synthetically from glycyrrhetinic acid, a major component of licorice, and this compound induced reactive oxygen species (ROS) in RD and Rh30 rhabdomyosarcoma (RMS) cells. CF3DODA-Me also inhibited growth and invasion and induced apoptosis in RMS cells, and these responses were attenuated after cotreatment with the antioxidant glutathione, demonstrating the effective anticancer activity of ROS in RMS. CF3DODA-Me also downregulated expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and prooncogenic Sp-regulated genes including PAX3-FOXO1 (in Rh30 cells). The mechanism of CF3DODA-Me–induced Sp-downregulation involved ROS-dependent repression of c-Myc and cMyc-regulated miR-27a and miR-17/20a, and this resulted in induction of the miRNA-regulated Sp repressors ZBTB4, ZBTB10, and ZBTB34. The cell and tumor growth effects of CF3DODA-Me further emphasize the sensitivity of RMS cells to ROS inducers and their potential clinical applications for treating this deadly disease.

Implications: CF3DODA-Me and HDAC inhibitors that induce ROS-dependent Sp downregulation could be developed for clinical applications in treating rhabdomyosarcoma.

Introduction

Rhabdomyosarcoma (RMS) is primarily a disease of children and adolescents, and 50% of all pediatric soft-tissue sarcomas are RMS (1–4). RMS is primarily a muscle-derived tumor that forms in the head and neck and genitourinary regions and also in the extremities, and current treatments include radiation, surgery, and chemotherapy with cytotoxic drugs. Two of the major forms of RMS, namely embryonal (ERMS) and alveolar (ARMS), are characterized by their unique pathologies, and in ARMS, chromosomal rearrangements result in formation of the chimeric fusion genes PAX3-FOXO1 and PAX7-FOXO1 in 55% and 22% of patients with ARMS, respectively (5, 6). Patients with ERMS respond favorably to the cytotoxic treatments, whereas children with ARMS respond poorly to current treatments, and the overall survival of metastatic patients with ARMS is less than 10% (7, 8). The highly cytotoxic drug treatment of childhood cancers results in development of chronic diseases in 95.5% of these children as adults (9), suggesting an urgent need for developing new and less toxic therapeutic regimens.

Examination of gene expression data in patients with RMS indicated elevated expression of genes associated with reactive oxygen species (ROS), and anticancer agents such as histone deacetylase (HDAC) inhibitors that induce ROS were highly effective inhibitors of ERMS tumor growth using patient-derived xenografts (10). Moreover, mouse models of ARMS that express PAX3-FOXO1 showed that the HDAC inhibitor entinostat was highly effective for inhibiting tumor growth and increasing survival; however, the precise role of induced ROS in mediating the antitumor activity was not determined (11). We reported that induction of ROS by HDAC inhibitors was a critical element for their anticancer activity, and this was due, in part, to downregulation of the specificity protein (Sp) transcription factors (TF) Sp1, Sp3, and Sp4 (12) that are overexpressed in RMS, other sarcomas, and other cancer cell lines (13–19). Moreover, individual or combined knockdown of Sp1, Sp3, and Sp4 decreases cancer cell (including RMS) growth, survival, and migration, and this is due to downregulation of prooncogenic Sp-regulated genes (12, 15, 20, 21).

Pentacyclic triterpenoids such as methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me; bardoxolone Me) are potent anticancer agents (22), and in pancreatic cancer cells, CDDO-Me inhibits growth and survival through ROS-dependent downregulation of Sp TFs (23). In this laboratory, we have also been developing synthetic triterpenoids derived from glycyrrhetinic acid (GA), a major phytochemical in licorice (23–26). Results of cancer cell growth–inhibitory effects identified methyl 2-trifluoromethyl-3,11-dioxo-18β-olean-1,12-dien-30-oate (CF3DODA-Me) as the most potent analogue, (24) and the compound induces ROS in bladder cancer cells (27). In this study, we show that CF3DODA-Me induces ROS in RMS cells, and this is accompanied by ROS-dependent downregulation of Sp TFs in RD (ERMS) and Rh30 (ARMS) cells (24) and demonstrates not only the efficacy of this compound but also potential clinical applications for treating patients with RMS.

Materials and Methods

Ethics statement

This study was approved by the Texas A&M University Institution Animal Care and Use Committee (#2018-084).

Cell lines

C2C12 cells were purchased from the ATCC and received during May 2016. The human RD (embryonal rhabdomyosarcoma) and Rh30 (alveolar rhabdomyosarcoma) cells were purchased from the ATCC and were authenticated in 2014 (Promega Powerplex 18D) at the Duke University DNA Analysis Laboratory (Durham, NC). RPMI1640 medium containing 10% FBS and 1× antibiotic/antimycotic solution (Sigma-Aldrich) was used for maintaining the cells that were incubated at 37°C in a humidified atmosphere composed of 5% CO2.

Cell proliferation and viability assays

Cells (5 × 104/well) were plated in 12-well plates with RPMI1640 medium containing 2.5% charcoal-stripped FBS, and after 24 hours, cells were treated with different concentrations of CF3DODA-Me or vehicle (DMSO) for 48 hours. Experiments that were designed to determine the effects of drug-induced ROS used cells pretreated with 5 mmol/L glutathione (GSH) for 30 minutes prior to addition of CF3DODA-Me. Cells were counted with a Coulter Z1 cell counter and mean cell numbers were determined.

Measurement of ROS

The cell-permeable probe CM-H2DCFDA (5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester) was used to measure ROS using the protocol outlined by the manufacturer (Life Technologies). The RMS cells (1.5 × 105/well) were seeded in 6-well plates using RPMI1640 medium and FBS that had been previously stripped with 2.5% charcoal. GSH (5 mmol/L) was added to the cells and after 30 minutes, cells were treated with 2.5 μmol/L CF3DODA-Me alone, or in combination with GSH for 1 hour, and control cells were treated with the solvent vehicle DMSO. Flow cytometry was used to determine ROS levels (12).

Measurement of apoptosis and invasion

Lab-Tek II Chamber Slide (Thermo Fisher Scientific) containing two wells were seeded with RMS cells (5 × 104/well) treated with 2.5 μmol/L CF3DODA-Me alone or in combination with GSH (preincubation for 30 minutes) for 24 hours. The effects of GSH were determined by preincubating cells with GSH for 3 hours. Apoptosis was detected using FITC-Annexin V and Hoechst 33342 Apoptosis Assay Kit (Biotium). The effect of various treatments on cell invasion was determined using Corning BioCoat Matrigel Invasion Chamber (Corning) as described previously (12). Six randomly selected fields were examined by fluorescence microscopy to quantify the number of apoptotic or invasive cells, and our protocols for using human cells were previously approved by the Texas A&M University Institutional Review Board (IRB).

Western blot analysis

After various treatments, proteins were extracted from the RMS cells using RIPA lysis buffer containing 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100 (w/v), 0.5% sodium deoxycholate, and 0.1% SDS with 10 μL/mL protease and phosphatase inhibitor cocktail (GeneDepot/Thermo Fisher Scientific). SDS-PAGE (10%) was used for separation of protein lysates, which were then transferred to nitrocellulose membranes and incubated with primary antibodies for 12 hours at 4°C. Details on primary antibodies are summarized in Supplementary Table S1. The corresponding horseradish peroxidase–conjugated secondary IgG antibodies were used, and immunoreactive proteins were detected with chemiluminescence reagent. Protein band densities were normalized to β-actin densities and quantitated, and relative expression of each band from 3 gels is presented as a fraction (%) of the DMSO control (100%).

Chromatin immunoprecipitation (ChIP) assay

The ChIP-IT Express magnetic chromatin immunoprecipitation kit (Active Motif) was used for ChIP analysis using the manufacturer's protocol. Cells (5 × 106) were treated with 2.5 μmol/L of CF3DODA-Me and after 3 hours, cells were cross-linked with 1% formaldehyde and then the reaction was stopped by addition of 0.125 mol/L glycine. Cells were removed in PBS and lysed with buffer containing 10 μL/mL protease inhibitor cocktail and PMSF; nuclei were recovered by centrifugation and sheared by sonication (10 pulses for 1 second). Selected antibodies (Supplementary Table S1) and protein A–conjugated magnetic beads were used for immunoprecipitation of sonicated chromatin at 4°C for overnight. Proteinase K digestion was used to extract DNA, and selected primers (Supplementary Table S2) were used for PCR amplification. PCR products were separated on a 2% agarose gel and detected by Green Glo DNA dye (Denville Scientific Inc.).

RT-PCR

Cells (4 × 105) were plated in 60-mm dish, and then cells were allowed to attach for 24 hours. RMS cells were treated with 2.5 μmol/L of CF3DODA-Me alone or in combination with GSH (preincubation for 30 minutes) for 6 hours. Total RNA was extracted using the mirVana miRNA isolation kit (Ambion), and RNA levels were measured using TaqMan microRNA assays (Life Technologies). RNU6B was used as a control to determine relative miRNA levels (23, 28).

siRNA transfection

siRNAs for c-Myc (siMyc) and GL12 as control (siCtl) were purchased from Santa Cruz Biotechnology, and knockdown studies were carried out in cells (6 × 104/well in 6-well plates) grown in RPMI1640 medium with 2.5% charcoal-stripped FBS without antibiotic. Cells were allowed to attach for 24 hours and specific siRNAs were transfected with Lipofectamine 2000 as outlined by the manufacturer, and after 72 hours, cells were harvested and used for subsequent analysis of functional activity and protein expression.

Xenograft study

The in vivo studies used female athymic nude mice (4–6 weeks old) obtained from Harlan Laboratories. RD cells (1 × 106) suspended in 100 μL of RPMI1640 medium with ice-cold Matrigel (1:1 ratio) were used for the xenograft study and were injected (100 μL) subcutaneously into either side of the flank area of nude mice. Mice were selected randomly for the control and treated groups, and 7 days after tumor cell injection mice (7/treatment group) were treated every 2 days with either vehicle (corn oil) or CF3DODA-Me (20 mg/kg body weight), which was administered intraperitoneally in a volume of 100 μL. Body weight changes were determined weekly, and after 30 days mice were sacrificed and weights of all tumors were determined individually; some of the mice exhibited more than one tumor. The Texas A&M University Institutional Animal Care and Use Committee reviewed and approved our animal treatment and use protocol.

Statistical analysis

Statistical significance of differences between the groups was determined by Student t test. The in vitro results are presented with three independent experiments as mean with SE at 95% confidence intervals. Results were considered statistically significant at a P value of less than 0.05.

Results

The synthetic CF3DODA-Me drug (Fig. 1A) was synthesized from GA, the major triterpenoid in licorice (24), and treatment of RD and Rh30 cells with CF3DODA-Me significantly inhibited cell proliferation with IC50 values of 0.50 and 0.83 μmol/L, respectively (Fig. 1B). Treatment of the RMS cell lines with 2.5 μmol/L CF3DODA-Me for 1 hour induced ROS as determined by FACS analysis using cell-permeant CM-H2DCFDA, and cotreatment with the antioxidant GSH significantly attenuated this response (Fig. 1C). In contrast, CF3DODA-Me did not induce ROS in C2C12 muscle cells and had minimal effects on cell growth (Supplementary Fig. S1A). The importance of CF3DODA-Me–induced ROS on cell proliferation was confirmed (Fig. 1D) because the growth-inhibitory effects of CF3DODA-Me were also inhibited after cotreatment with GSH.

Figure 1.
Figure 1.

CF3DODA-Me inhibits RMS cell growth through the induction of ROS. A, Chemical structure of CF3DODA-Me. B, RD and Rh30 cells were treated with 0, 1, 2.5, and 5 μmol/L concentrations of CF3DODA-Me for 48 hours. Cell numbers were counted by using a Coulter Z1 cell counter. C, RD and Rh30 cells were pretreated with 5 mmol/L GSH for 30 minutes and then treated with vehicle (DMSO, Control), 2.5 μmol/L of CF3DODA-Me alone, or in combination with GSH for 1 hour. ROS levels were measured by FACS using cell-permeant CM-H2DCFDA dye. D, RD and Rh30 cells were pretreated with 5 mmol/L GSH for 30 minutes and then treated with 2.5 μmol/L of CF3DODA-Me alone, or in combination with 5 mmol/L GSH for 48 hours. Cell numbers were determined by using a Coulter Z1 cell counter. Data represent three independent experiments and expressed as mean ± SE, and significant (P < 0.05) growth inhibition (*) or induction of ROS or reversal by GSH (#) is indicated.

We also examined the effects of CF3-DODA-Me on apoptosis and observed that this compound induced Annexin V staining (Fig. 2A) and PARP cleavage (cPARP; Fig. 2B) in RD and Rh30 cells, and these responses were significantly attenuated in cells cotreated with GSH. CF3DODA-Me also decreased RMS cell invasion in a Boyden chamber assay, and the response was attenuated after cotreatment with GSH (Fig. 2C), demonstrating that the inhibitory effects of CF3DODA-Me on RMS cell growth, survival, and invasion were ROS-dependent. CF3DODA-Me exhibited minimal effects on growth inhibition after treatment for 24 hours; however, this may contribute to the observed CF3DODA-Me–induced inhibition of RMS cell invasion.

Figure 2.
Figure 2.

ROS-dependent induction of apoptosis and inhibition of invasion by CF3DODA-Me. A, RD and Rh30 cells were pretreated with 5 mmol/L GSH for 3 hours and then treated with vehicle, 2.5 μmol/L of CF3DODA-Me alone, or in combination with 5 mmol/L GSH for 24 hours, and Annexin V staining was determined and quantitated by fluorescence microscopy. B, RD and Rh30 cells were treated as indicated in A, and whole lysates were analyzed for cleaved PARP by Western blots, and β-actin was used a loading control. The signals were quantitated by ImageJ software. C, RD and Rh30 cells were treated as indicated in A, and cell invasion was analyzed and quantitated by using Corning BioCoat Matrigel Invasion Chamber. Data represent three independent experiments and expressed as mean ± SE, and significant (P < 0.05) induction of apoptosis or inhibition of invasion (*) or reversal by GSH (#) is indicated.

Previous studies with ROS inducers in RMS and other cancer cell lines show that these compounds downregulate Sp proteins (12, 28–34). Figure 3A shows that CF3DODA-Me decreased expression of Sp1, Sp3 (high and low molecular weight forms), and Sp4 in RD and Rh30 cells, and a time-dependent decrease in Sp proteins was also observed (Supplementary Fig. S1B). In cells cotreated with GSH, the effects of CF3DODA-Me on Sp TFs was reversed (Fig. 3B). Treatment with CF3DODA-Me also decreased expression of the Sp-regulated genes survivin and cyclin D1 and induced PARP cleavage in RD and Rh30 cells (Fig. 3C). Previous studies showed that Sp TFs regulate expression of the PAX3-FOXO1 gene in Rh30 cells (13, 35). In this study, CF3DODA-Me decreased expression of PAX3-FOXO1 in this cell line and this was accompanied by downregulation of PAX3-FOXO1–regulated gene products including NMyc, RASSF4, MyoD1, Gremlin, and DAPK1 (Fig. 3D; refs. 36–39).

Figure 3.
Figure 3.

CF3DODA-Me downregulates Sp1, Sp3, Sp4, and Sp- and PAX3-FOXO1–regulated proteins. A, RD and Rh30 cells were treated with 0, 1, and 2.5 μmol/L CF3DODA-Me for 24 hours, and whole lysates were analyzed in Western blots for Sp1, Sp3, and Sp4 proteins. B, RD and Rh30 cells were pretreated with 5 mmol/L of GSH for 3 hours and then treated with 2.5 μmol/L of CF3DODA-Me alone, or in combination with GSH for 24 hours. The whole lysates were analyzed for Sp1, Sp3, and Sp4 proteins. C and D, RD and Rh30 cells were treated as indicated in A and analyzed for Sp-regulated prosurvival and growth-promoting proteins (C) and PAX3-FOXO1–regulated proteins in Western blots (D). β-Actin was used as a loading control. The signals were quantitated by ImageJ software. Data represent three independent experiments and expressed as mean ± SE, and significant (P < 0.05) induction or suppression of target genes (*) or reversal by GSH (#) is indicated. High and low molecular weight forms of Sp3 are designated “h” and “l,” respectively.

A model for ROS-dependent downregulation of Sp TFs (Fig. 4A) has been reported (12, 28). ROS induces epigenetic downregulation of Myc through genome-wide migration of chromatin-modifying complexes (12, 28, 40), resulting in decreased expression of Myc-regulated miR-27a and miR-20a/miR-17 (miR-17-92 complex) and induction of the miRNA-repressed transcriptional repressors (Sp repressors) ZBTB10, ZBTB4, and ZBTB34. Results illustrated in Fig. 4B and C show that after treatment of RD and Rh30 cells with CF3DODA-Me, there was a decrease in expression of Myc protein, and this response was attenuated in cells cotreated with GSH. Knockdown of Myc by RNA interference (siMyc) also decreased expression of Sp1, Sp3, and Sp4 in RD and Rh30 cells (Fig. 4D), confirming that Myc is upstream from Sp TFs as reported previously in pancreatic cancer cells (28). After treatment of the RMS cells with CF3DODA-Me for 3 hours, a ChIP assay showed a decreased association of pol II with the proximal region of the Myc promoter consistent with observed decreased expression of Myc protein (Fig. 5A). In RD cells, there was an increase in the gene-repressing H3K27me3 mark with minimal changes in H3K4me3 or H4K16Ac; whereas in Rh30 cells, changes in the methylation marks and H4K16Ac (increased) were not consistent with the observed change in Myc repression, suggesting that other epigenetic effects are induced by ROS and these are currently being investigated. siMyc also downregulated PAX3-FOXO1 and downstream genes in Rh30 cells (Fig. 5B), and the loss of Myc also resulted in decreased proliferation of RD and Rh30 cells (Fig. 5C).

Figure 4.
Figure 4.

CF3DODA-Me downregulates Myc expression. A, Proposed mechanism of CF3DODA-Me–induced ROS-dependent downregulation of Sp transcription factors. B, RD and Rh30 cells were treated with 0, 1, and 2.5 μmol/L CF3DODA-Me for 24 hours. C, RD and Rh30 cells were pretreated with 5 mmol/L GSH for 3 hours and then treated with 2.5 μmol/L of CF3DODA-Me alone, or in combination with 5 mmol/L GSH for 24 hours. The whole-cell lysates from experiments illustrated in B and C were analyzed by Western blots for expression of Myc protein. D, RD and Rh30 cells were transfected with siRNAs for control (siCtl) or Myc (siMyc) for 72 hours, and whole lysates were analyzed in Western blots for expression of Myc protein. Significantly (P < 0.05) decreased expression of proteins after Myc knockdown is indicated (*), and results are expressed as means ± SE for three replicate experiments. The high and low molecular weight forms of Sp3 are designated “h” and “l,” respectively.

Figure 5.
Figure 5.

CF3DODA-Me epigenetically regulates Myc levels. A, RD and Rh30 cells were treated with 2.5 μmol/L CF3DODA-Me for 3 hours and ChIP assays were performed with control (IgG), polymerase II, H3K27me3, H3K4me3 and H4K16Ac antibodies used to detect their interactions on the Myc promoter region. B, Rh30 cells were transfected as in Fig. 4D, and whole lysates were analyzed in Western blots for Myc-regulated proteins. β-Actin was used as a loading control. C, RD and Rh30 cells were transfected with siMyc for 72 hours, and cell survival was determined by cell counting. The signals were quantitated by ImageJ software. Data represent three independent experiments and expressed as mean ± SE, and significant (P < 0.05) induction or suppression of target genes and cell growth (*) or reversal by GSH (#) is indicated.

Treatment of RD and Rh30 cells with CF3DODA-Me also decreased expression of miR-27a and miR-20a/miR-17 (Fig. 6A), and in cells cotreated with GSH, the decreased miR expression was significantly reversed (Fig. 6B). The CF3DODA-Me–dependent decreased expression of the miRNAs was accompanied by the time-dependent induction of the miR-27a–regulated ZBTB10/ZBTB34 and miR-20a/miR-17–regulated ZBTB4 transcriptional repressor (Sp repressor) proteins in RD and Rh30 cells (Fig. 6C), and these responses were also blocked after cotreatment with GSH (Fig. 6D). In a ChIP assay, we also observed that treatment with CF3DODA-Me decreased association of cMyc from the miR-27a and miR-17-92 promoter and also pol II from the miR-17-92 (both cell lines) and miR-23-27a (RD cells only) promoters (Fig. 6E).

Figure 6.
Figure 6.

CF3DODA-Me modifies miRNA–ZBTB interactions. A, RD and Rh30 cells were treated with 0, 1 and 2.5 μmol/L of CF3DODA-Me for 6 hours. B, RD and Rh30 cells were pretreated with 5 mmol/L of GSH for 3 hours and then treated with 2.5 μmol/L CF3DODA-Me alone, or in combination with GSH for 6 hours, and total RNA was extracted and expression of miR-17, miR-20a, and miR-27a was determined by real-time PCR. RNU6 was used as endogenous control. C, RD and Rh30 cells were treated with 2.5 μmol/L of CF3DODA-Me for the indicated times, and ZBTB4, ZBTB10, and ZBTB34 proteins were analyzed by Western blots. D, Cells were treated with DMSO (control), CF3DODA-Me and 5 mmol/L GSH alone and in combination for 12 hours, and whole-cell lysates were analyzed by Western blots. β-Actin was used as a loading control. E, RD and Rh30 cells were treated with 2.5 μmol/L of CF3DODA-Me for 3 hours, and a ChIP assay was performed with control (IgG), polymerase II, and Myc antibodies to determine their interactions on the miR-23a/27a and miR-17/92 cluster promoter regions. The signals were quantitated by ImageJ software. Results shown are expressed as mean ± SE for replicate determinants, and significant (P < 0.05) changes by CF3DODA-Me (*) or reversal by GSH (#) is indicated.

The effect of CF3DODA-Me on the growth of RMS tumors was investigated in athymic nude mice bearing RD cells as xenografts. Treatment with CF3DODA-Me (20 mg/kg/day) resulted in the loss of 1 tumor, and tumors from the remaining 5 mice are shown. In contrast, larger tumors developed in untreated mice and in some animals, multiple tumors developed and are illustrated in Fig. 7A. In addition, at the 20 mg/kg/day dose, we did not observe any weight loss or organ toxicity compared with control mice (Fig. 7C). Western blot analysis of tumor lysates showed that CF3DODA-Me decreased expression of Sp1, Sp3, and Sp4, and these results complemented the in vitro studies where CF3DODA-Me decreased cell growth and downregulated Sp1, Sp3, and Sp4 (Figs. 1 and 3).

Figure 7.
Figure 7.

CF3DODA-Me suppresses in vivo ERMS tumor growth. A–C, RD cells were injected into athymic nu/nu mice, and representative tumor images (A), relative tumor weights (B), and body and organ weight changes over the course of 30 days treatment with corn oil (control) or CF3DODA-Me (20 mg/kg body weight; C) are shown. D, Protein lysates from control and CF3DODA-Me–treated xenograft tumor tissues were analyzed by Western blots for expression of Sp1, Sp3, and Sp4 proteins. The signals were quantitated by ImageJ software. Results are as mean ± SE for 7 animals in each group, and significant (P < 0.05) changes by CF3DODA-Me (*) are indicated. The high and low molecular weight forms of Sp3 and designated “h” and “l,” respectively. Visual inspection of the organs did not detect any lesions associated with toxicity.

Discussion

The Sp1 and/or Sp3 transcription factors are negative prognostic factors for patients with colon, gastric, head and neck, lung, pancreatic, prostate, and breast cancer (16, 19, 41–46), and Sp1 is also highly expressed in tumors from RMS patients (13). The importance of Sp1, Sp3, and Sp4 overexpression in cancer cells is associated with their regulation of prooncogenic factors/genes associated with cell proliferation (cyclin D1 and multiple receptor tyrosine kinases), survival (survivin and bcl-2), angiogenesis/migration/invasion (MMP-9, VEGF, and its receptors), and inflammation (p65-NFκB; ref. 15). Not surprisingly, knockdown of Sp TFs individually or combined decreases growth, survival, and migration/invasion of kidney, breast, pancreatic, lung, and colon cancer cells (20), and similar results were observed in RD and Rh30 cells (13). Previous studies with HDAC inhibitors and tolfenamic acid showed that both compounds were highly effective inhibitors of RMS tumor growth, and they decreased expression of Sp1, Sp3, Sp4, and prooncogenic Sp-regulated genes through ROS-independent and -dependent pathways, respectively (12, 13).

Bardoxolone-methyl shows promising anticancer activity (47) and has been in clinical trials for treating kidney disease (48). Studies in this laboratory have developed a series of GA derivatives (24–26), which differ structurally from bardoxolone-methyl only by the en-one position in the C-ring and the location of the carboxymethyl substituent in the E-ring (C-28 for bardoxolone-methyl and C-30 for CF3DODA-Me). Among the GA derivatives, CF3DODA-Me was the most potent anticancer drug and in cancer cell proliferation assays, bardoxolone-methyl ≥ CF3DODA-Me in terms of potency; however, these differences were cell context–dependent (24, 49). The pharmacokinetics of bardoxolone-methyl have been extensively investigated in both animal and human models (22, 47, 50) and show bardoxolone-methyl to be readily bioavailable. Because of the similarities in structure between CF3DODA-Me and bardoxolone-methyl, we expect to observe comparable pharmacokinetics for both compounds, and these studies on CF3-DODA-Me are currently being investigated in rodent models with an emphasis on bioavailability in the brain. The advantages of CF3DODA-Me versus bardoxolone-methyl include ease of synthesis, availability and relative cost of starting materials, and indications of potentially lower toxic side effects for the former compound because, unlike bardoxolone methyl, CF3DODA-Me does not alkylate thiol groups via a Michael addition (49). Results of this study in RD and Rh30 cells showed that CF3DODA-Me induced ROS-dependent inhibition of growth, survival, and invasion and also downregulated Sp1, Sp3, Sp4, and some Sp-regulated genes (Figs. 1-3). The CF3DODA-Me–induced functional effects and Sp downregulation were significantly attenuated in cells cotreated with the antioxidant GSH, indicating that ROS plays an important role in the anticancer activity of CF3DODA-Me.

ROS-inducing anticancer agents are being used clinically for cancer chemotherapy and there is evidence that this could be particularly effective for treating cancers such as ERMS where endogenous ROS levels are high (10), and therefore the threshold for drug-induced cytotoxicity is relatively low compared with other cancer cells and nontumor tissue. Activation of ROS via disabling extramitochondrial genes or by directly targeting mitochondria results in activation of apoptosis. Results of this study confirm that ROS-mediated downregulating of Sp TFs via disruption of the Myc–miR-27a–ZBTB10/ZBTB34 and Myc–miR-27-91–miR–ZBTB4 pathways (Fig. 4A) also contributes to the anticancer activity of ROS-inducing agents, and this is primarily due to the targeting of prooncogenic Sp-regulated genes. For example, in Rh30 cells, the PAX3-FOXO1 fusion gene is also regulated by a nuclear receptor 4A1 (NR4A1)/Sp4 complex and is decreased by compounds that inactivate NR4A1 or drugs such as tolfenamic acid and CF3DODA-Me (13), and this is accompanied by downregulation of several PAX3-FOXO1–regulated prooncogenic genes including NMyc, RASSF4, MyoD1, Gremlin, and DAPK1 (Fig. 3D). The results of this study coupled with previous reports showing the high expression and important role of Sp TFs in maintaining the RMS phenotype (12, 13) suggest that agents such as CF3DODA-Me and HDAC inhibitors that induce ROS-dependent Sp downregulation should be further developed for clinical applications in treating this deadly disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: R. Kasiappan, I. Jutooru, S. Safe

Development of methodology: R. Kasiappan, K. Mohankumar, S. Safe

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Kasiappan, I. Jutooru, K. Mohankumar, K. Karki, A. Lacey, S. Safe

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Kasiappan, I. Jutooru, K. Mohankumar, K. Karki

Writing, review, and/or revision of the manuscript: R. Kasiappan, K. Mohankumar, S. Safe

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Safe

Study supervision: S. Safe

Others (performed experiments): K. Mohankumar

Acknowledgments

This work was supported by grants from the NIH (grant nos. P30-ES023512, to S. Safe and T32-ES026568 to, K. Karki), the Kleberg Foundation (to S. Safe), the College of Veterinary Science and Biomedical Sciences postdoctoral research grant and the DBT-Ramalingaswami Fellowship (to R. Kasiappan), Texas AgriLife Research (to S. Safe), and the Sid Kyle endowment (to S. Safe).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received October 3, 2018.
  • Revision received November 13, 2018.
  • Accepted December 17, 2018.
  • Published first January 4, 2019.

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Reactive Oxygen Species (ROS)-Inducing Triterpenoid Inhibits Rhabdomyosarcoma Cell and Tumor Growth through Targeting Sp Transcription Factors
Ravi Kasiappan, Indira Jutooru, Kumaravel Mohankumar, Keshav Karki, Alexandra Lacey and Stephen Safe
Mol Cancer Res March 1 2019 (17) (3) 794-805; DOI: 10.1158/1541-7786.MCR-18-1071
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