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. 2023 Feb 1;15(3):740.
doi: 10.3390/nu15030740.

The Cytotoxic Effect of Curcumin in Rhabdomyosarcoma Is Associated with the Modulation of AMPK, AKT/mTOR, STAT, and p53 Signaling

Sara Salucci  1 , Alberto Bavelloni  2 , Anna Bartoletti Stella  3 , Francesco Fabbri  4 , Ivan Vannini  4 , Manuela Piazzi  5   6 , Karyna Volkava  7 , Katia Scotlandi  2 , Giovanni Martinelli  4 , Irene Faenza  1 , William Blalock  5   6
Affiliations

The Cytotoxic Effect of Curcumin in Rhabdomyosarcoma Is Associated with the Modulation of AMPK, AKT/mTOR, STAT, and p53 Signaling

Sara Salucci et al. Nutrients. .

Abstract

Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin's anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors.

Keywords: cancer therapy; drug design and targeting; muscle tissue; nutraceuticals; sarcoma; signal transduction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Curcumin treatment leads to decreased cell proliferation/viability and a G2/M cell cycle arrest in RMS cell lines. (A) The effects of curcumin on the proliferation and viability of A204, RD, and SJRH30 (RH30) cells was assayed using a resazurin-based cell proliferation/viability assay at 24, 48, and 72 h in the presence of various concentrations of curcumin (0.8–50 µM). IC50 values were obtained after 48 and 72 h treatment. Three replicates were tested per concentration and at least three independent experiments were performed (bars, ±s.d.). (B) Rhabdomyosarcoma cells (A204, RD, and RH30) were treated with 20 µM curcumin for 24 h and then analyzed by flow cytometry to determine the cell cycle distribution of the cells. The graph shows the percent distribution of cells in each of the cell cycle phases and is representative of three independent experiments. (C) The ability of curcumin to induce cell killing in RMS was measured by Trypan blue viability assay. A204, RD, and RH30 cells were treated with curcumin (10 or 20 µM) or left untreated (control) for 48 and 72 h. Both detached and attached cells were collected and stained with 0.4% Trypan blue solution and counted on a hemocytometer. Results are the mean of three different experiments ± s.d. Ctrl, Untreated cells. ** p < 0.01, **** p < 0.0001.
Figure 2
Figure 2
Curcumin treatment induces apoptotic cell death. Rhabdomyosarcoma cell lines (A204, RD, and RH30) were treated for 24 h with 20 μM of curcumin. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. The dot plots are representative of three independent experiments.
Figure 3
Figure 3
Curcumin influences rhabdomyosarcoma cell migration and colony forming ability. (A) Rhabdomyosarcoma cells (A204, RD, RH30) 90% confluence were “wounded” with a 200 μL pipet tip. Three “wounds” were formed on each culture dish in duplicate per experiment. Loose cells were removed with washing and fresh complete medium containing vehicle (DMSO) or curcumin (10 or 20 µM) was added to each cell monolayer. The “wounds” were photographed under light microscopy at 6 and 24 h after the initial “wounding”, and the diameters measured using the AxioVision software. Data were plotted as percent healing of the original “wound” and graphed using GraphPad Prism software. ** p < 0.01, **** p < 0.0001. (B) Rhabdomyosarcoma cells (A204, RD, and RH30) were treated with either vehicle control (DMSO) or curcumin (10 or 20 µM) for 24 h, then plated (500 viable cells/well in 6-well culture plates) and cultured in normal growth media for 10 days. Cells were fixed and stained with crystal violet. Colony number and size were determined using ImageJ software. Results are the mean of three different experiments ±s.d. **** p < 0.0001.
Figure 4
Figure 4
Curcumin influences signaling through AKT/mTOR, STAT, AMPK, and p53 associated pathways. (A,C,E) Whole cell lysates prepared from A204 (A,B), RH30 (C,D), and RD (E,F) cell lines that were treated with DMSO (control) or 20 µM curcumin for 24 h were analyzed using Proteome Profiler Phospho-Kinase arrays. Exemplary array blots are shown. (B,D,F) Percent changes in spot density of Proteome Profiler array proteins/phospho-proteins of interest that were quantified using ImageJ software and normalized to positive controls on the same membrane. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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