Resveratrol induces apoptosis by modulating the reciprocal crosstalk between p53 and Sirt-1 in the CRC tumor microenvironment

Abstract

Introduction: P53 represents a key player in apoptosis-induction in cancers including colorectal cancer (CRC) that ranks third worldwide in cancer prevalence as well as mortality statistics. Although a pro-apoptotic effect of resveratrol has been repeatedly proven in CRC cells, its pathway mechanisms are not completely understood, as there are controversial statements in the literature regarding its activation or inhibition of the counteracting proteins Sirt-1 and p53.

Methods: CRC cells as wild-type (HCT-116 WT) or p53-deficient (HCT-116 p53^-/-) were cultured using multicellular tumor microenvironment (TME) cultures containing T-lymphocytes and fibroblasts to elucidate the role of p53/Sirt-1 modulation in resveratrol's concentration-dependent, pro-apoptotic, and thus anti-cancer effects.

Results: Resveratrol dose-dependently inhibited viability, proliferation, plasticity as well as migration, and induced apoptosis in HCT-116 WT more effectively than in HCT-116 p53^-/- cells. Moreover, resveratrol stimulated Sirt-1 expression when administered at low concentrations (<5µM) but suppressed it when added at high concentrations (>10µM) to CRC-TME. In parallel, similar to the knockdown of Sirt-1 at the mRNA level, treatment with high-concentration resveratrol boosted the acetylation of p53, the expression of p21, Bax, cytochrome C, caspase-3, and ultimately induced apoptosis in CRC WT but not in CRC p53^-/- cells. Notably, increasing concentrations of resveratrol were found to promote hyperacetylation of p53 and FOXO3a as post-translational substrates of Sirt-1, indicating a negative regulatory loop between Sirt-1 and p53.

Discussion: These results demonstrate for the first time, a negative reciprocal crosstalk between the regulatory circuits of p53 and Sirt-1, consequently, apoptosis induction by higher resveratrol concentrations in CRC-TME.

Keywords: Sirt-1 modulation; apoptosis; caspase-3; colorectal cancer; p53; reciprocal crosstalk; resveratrol; tumor microenvironment.

Figure 1

Resveratrol’s impact on CRC cell viability. (A) Chemical structure of trans-resveratrol. (B) HCT-116 WT or HCT-116 p53^-/- cells were cultivated in alginate beads, then isolated and their viability was measured by MTT assay. X-axis shows the treatments: basal control (Ba.Co., without TME, without resveratrol), TME control (with MRC-5, Jurkat, without resveratrol), or TME with resveratrol (1, 5, 10, 20, or 40µM Res). Y-axis shows the number of viable CRC cells, measured at 550nm. Grey bars represent HCT-116 WT cells, while black bars represent HCT-116 p53^-/- cells. Values related to TME control: *p<0.05, **p<0.01.

Figure 2

Impact of resveratrol and role of Sirt-1 on CRC cell proliferation. HCT-116 WT (A) or HCT-116 p53^-/- (B) cells were grown on small cover glasses in a 3D culture environment, treated differently, and photographed (phase contrast, x400 magnification). Scale bar corresponds to 30nm. (C) illustrates statistic evaluation. X-axis shows the treatments: TME control or TME with 1, 5, 10, 20, 40µM resveratrol. Y-axis shows cell numbers of adhered CRC cells. They were calculated by counting 5 microscopic fields per culture. Grey bars represent HCT-116 WT cells, and black bars represent HCT-116 p53^-/- cells. Compared to TME control: *p<0.05, **p<0.01.

Figure 3

Resveratrol’s impact on nuclear Sirt-1 or p53 expression in CRC cells. HCT-116 WT cells were grown on glass coverslips, treated differently (TME control (Co) or TME with 5, 10, 20, 40µM resveratrol), and afterward immunolabeled against Sirt-1 (red, row 1) or p53 (red, row 3) and stained with DAPI (blue, row 2 and row 4). White arrows mark immunolabelling, and white arrowheads mark apoptosis. Additionally, statistics were applied by counting 5 microscopic fields each. X-axis: treatments. Y-axis: apoptotic CRC cells (white bars) or positive immunolabeled CRC cells (black bars), stated in %. Values: *p<0.05, **p<0.01, in reference to TME control.

Figure 4

Resveratrol’s impact on CRC cell plasticity. (A) HCT-116 WT (a-e) or HCT-116 p53^-/- (f-j) cells were grown on small cover-glasses in 3D TME as treatment-free control or enriched with 5, 10, 20, or 40µM resveratrol and then evaluated by transmission electron microscopy (TEM). Scale bars: 1µm, black arrowheads: active pseudopodia, black arrows: epithelial cell surface, black stars: apoptotic bodies. (B) X-axis: treatments, black bars: HCT-116 WT cells, white bars: HCT-116 p53^-/- cells. Y-axis: mitochondrial changes (MC) and apoptosis in % by counting 5 microscopic fields. *p<0.05, **p<0.01, relative to TME control.

Figure 5

Resveratrol’s impact on CRC cell migration. (A) HCT-116 WT (upper row) or HCT-116 p53^-/- (lower row) were cultivated in 3D TME and their migration on a wound incision after 5 days was compared. The first column shows a fresh wound on day 1. Further columns show selected treatments: TME control and TME with resveratrol addition (1, 5, 10, 20µM) or Sirt-1-SO/ASO addition (0.5µM). (B) Statistic evaluation compares the non-migrated glass area (in %) after 5 days, measured related to fresh incision on day 1. The non-migrated area is marked by yellow dashed lines. In comparison with TME control: *p<0.05, **p<0.01.

Figure 6

Resveratrol’s impact on apoptosis-related protein expression in CRC cells. HCT-116 WT or HCT-116 p53^-/- cells were detached from alginate beads and the expression of apoptosis-associated proteins was investigated per Western Blot. (A) HCT-116 WT cells grown as TME control or TME with supplementation of resveratrol (5, 10, 20, 30, 40, 50, 60µM Res). The concentration-dependent effect of resveratrol on Sirt-1, acetyl-p53/Lysin382, p53, cleaved-caspase-3, or β-actin (loading control) was investigated. Furthermore, a control peptide was used that verified Sirt-1. (B) HCT-116 WT or HCT-116 p53^-/- cells were processed as TME control or enriched with resveratrol (5, 10, 20µM Res), and its concentration-dependent impact on p53, p21, cyclin D1, cleaved-caspase-3 or β-actin (housekeeping protein control) expression was tested. (C) HCT-116 WT cells were left treatment-free or transfected with 0.5µM Sirt-1-SO (control substance) or 0.5µM Sirt-1-ASO (knockdown substance). Effects of Sirt-1 knockdown on the expression of Sirt-1, acetyl-p53/Lysin382, p21, cyclin D1, Bax, cytochrome C, cleaved-caspase-3, and β-actin (loading control) were investigated. *p<0.05, **p<0.01.

Figure 7

Resveratrol’s impact on p53/FOXO3a acetylation and p53/Sirt-1 negative functional interaction. HCT-116 WT cells were isolated from alginate beads, which were left untreated in TME or treated with resveratrol (5, 10, 20µM Res). Western Blot samples were generated from it and immunoprecipitated (IP) with anti-acetyl-lysin (A), anti-acetyl-p53 or anti-Sirt-1 (B). Then, immunoblotting against acetylated p53 and FOXO3a (A) and Sirt-1 or acetylated p53 (B) was performed to elucidate their functional connections. IgH means immunoglobulin heavy chain. Non-acetylated p53 verified the CRC samples.