Celastrol and Resveratrol Modulate SIRT Genes Expression and Exert Anticancer Activity in Colon Cancer Cells and Cancer Stem-like Cells

Abstract

Metastatic colorectal cancer (CRC) remains a hard-to-cure neoplasm worldwide. Its curability declines with successive lines of treatment due to the development of various cancer resistance mechanisms and the presence of colorectal cancer stem cells (CSCs). Celastrol and resveratrol are very promising phytochemicals for colon cancer therapy, owing to their pleiotropic activity that enables them to interact with various biological targets. In the present study, the anticancer activities of both compounds were investigated in metastatic colon cancer cells (LoVo cells) and cancer stem-like cells (LoVo/DX). We showed that celastrol is a very potent anti-tumor compound against metastatic colon cancer, capable of attenuating CSC-like cells at the molecular and cellular levels. In contrast, resveratrol has a much greater effect on colon cancer cells that are expressing standard sensitivity to anticancer drugs, than on CSC-like cells. In addition, both polyphenols have different influences on the expression of SIRT genes, which seems to be at least partly related to their anti-tumor activity.

Keywords: cancer stem cells; celastrol; colon cancer; resveratrol; sirtuins.

Figure 1

Molecular targets modulated by celastrol and resveratrol (Adapted from [10,11]).

Figure 2

LoVo/DX cells display cancer stem cell–like properties compared to LoVo cells. (A) Intracellular accumulation of Rhodamine 123 (Rh123) and Hoechst 33342 (Ho342) fluorescent dyes. The results are expressed as E/E₀ × 100%, where the MFI (mean fluorescent intensity) of Rh123 or Ho342 in LoVo/DX cells (E) was compared to the MFI in LoVo cells (E₀); (B) Frequency of Side Population cells in LoVo and LoVo/DX cell-lines. Representative cytograms of flow cytometric analysis of Side Population cells. Side Population is defined as a subpopulation of cells that shows the lowest Ho342 content (a low-Ho342 fluorescence “tail” on dual wavelength of fluorescence emission: Hoechst red [630 nm] and Hoechst blue [455 nm]); (C) CD44 expression after immunofluorescence staining using FITC mouse anti-human CD44; (D) Intracellular ROS level measured by the means of DCF-DA assay; (E,F) mRNA expression level of BRCA1, PARP1, SIRT1, SIRT2, SIRT3, and SIRT6 genes obtained from real-time RT-PCR analysis. The results are expressed as E/E₀, where the mRNA expression levels of the tested genes in LoVo/DX (E) cells were compared with the mRNA expression levels of these genes in LoVo (E₀) cells. All data are the mean ± SD from at least three independent experiments. The significance of the differences was determined by Student’s t-test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

Effect of celastrol (CEL) (A) and resveratrol (RSV) (B) on the frequency of apoptotic and necrotic cells in LoVo and LoVo/DX cell cultures after 24 h of incubation. The cells were double-stained with Annexin V Alexa Fluo^®r 488 and PI fluorescent dyes, and analyzed by flow cytometry. Control: cells incubated with the solvent (DMSO). The results are presented as a percentage of early apoptotic cells (Annexin V Alexa Fluor^® 488+ and PI−) and late apoptotic cells (Annexin V Alexa Fluor^® 488+ and PI+) and necrotic (Annexin V Alexa Fluor^® 488+ and PI+). The results are the mean ± SD of at least three independent experiments. The significance of the differences was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Effect of celastrol (CEL) (A); and resveratrol (RSV) (B) on cell cycle distribution in LoVo and LoVo/DX cell cultures after 24 h of incubation. The bar graphs show the percentage of cells in G1, S, and G2/M phases. Control comprises of cells incubated with solvent (DMSO). The results are the mean ± SD of at least three independent experiments. The significance of the differences was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Impact of celastrol (CEL) (A) and resveratrol (RSV) (B) on the frequency of DNA double-strand breaks (γ-H2AX+ cells) in LoVo and LoVo/DX cell cultures after 24 h of incubation. γ-H2AX+ cells were detected using phospho-histone H2A.X (Ser139) monoclonal antibody (CR55T33) Alexa Fluor^® 488. Control constitutes cells incubated with solvent (DMSO). The results are the mean ± SD of at least three independent experiments. The significance of the differences was determined by Student’s t-test. ** p < 0.01, *** p < 0.001.

Figure 6

Impact of celastrol (CEL) and resveratrol (RSV) on intracellular ROS level (A); and mRNA expression of BRCA1 and PARP1genes (B) in LoVo and LoVo/DX cell cultures after 24 h of incubation. Intracellular ROS level measured by the means of DCF-DA assay. The MFI (mean fluorescent intensity) of DCF obtained in the presence of celastrol or resveratrol (E) was compared to the relevant control (E₀), i.e., cells incubated in the presence of the solvent (DMSO). The mRNA expression level of BRCA1 and PARP1 genes was obtained from real-time RT-PCR analysis. The results are the mean ± SD of at least three independent experiments. The significance of the differences was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

Impact of celastrol (CEL) (A) and resveratrol (RSV) (B) on mRNA expression of sirtuins (SIRT) genes in LoVo and LoVo/DX cell cultures after 24 h of incubation. The mRNA expression level was obtained from real-time RT-PCR analysis. The results are the mean ± SD of at least three independent experiments. The significance of the differences was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Possible mechanism of anticancer activity of celastrol on colon cancer cells.

Figure 9

Possible mechanism of anticancer activity of resveratrol on LoVo colon cancer cells.

Figure 10

The interplay between SIRT3, ROS, and celastrol/resveratrol effects on colon cancer cells.