Marine guanidine alkaloids crambescidins inhibit tumor growth and activate intrinsic apoptotic signaling inducing tumor regression in a colorectal carcinoma zebrafish xenograft model

Abstract

The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo.Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.

Keywords: apoptosis; cancer treatment; cell cycle inhibition; crambescidins; zebrafish xenograft model.

Figure 1. Crambescidins decrease viability of a wide range of tumor cells

A. Chemical structures of crambescidin 816, crambescidin 830 and crambescidin 800. B. Viability of HOP-92, OVCAR-3, HT-29, SK-MEL-2, OU-31 and MCF-7 cells after C830 and C800 treatments for 24 and 48 h, determined by the MTT method. *Significant difference with respect to controls (p <0.01). C. Dose-dependent decrease of cell viability in response to C816, C830 and C800 determined by the alamarBlue^® assay. HepG2 cells were treated with different concentrations of C816, C830 and C800 ranging from 0.1 to 15 μM for 48 h to determine the IC₅₀ dose. D. Dose-dependent decrease of HepG2 cells viability in response to 0.1-15 μM C816, C830 and C800 and IC₅₀ determination at 72 h.

Figure 2. Crambescidins alter cell adherence and cytoskeletal integrity of tumor cells

A. OCLN (green), VCL (red), F-ACTA (green) and β-TUBB (red) detection by confocal microscopy in control and 2.5 μM C816, C830 and C800-treated cells after 24 h. Colocalization of F-ACTA and VCL is shown in yellow and representative images of control and treated cells are shown. Hoechst 33258 was included for nuclei counterstaining (blue). B. Left: Determination of soluble CLDN2, OCLN, TUBB, VLC and GADPH levels. HepG2 cells were treated with 2.5 μM C816, C830 and C800 for 24 h and then soluble protein fractions obtained from cell lysates of treated and untreated cells were analyzed by western blot. Right: Quantification of the differences in protein levels among control and 2.5 μM C816, C830 and C800 treated cells.* Significant differences respect to controls (p <0.05).

Figure 3. Crambescidins arrest cell cycle progression in the G0/G1 phase

A. Representative histograms of the cell cycle obtained after flow cytometry analysis of 2.5 μM C816, C830 and C800-treated HepG2 cells for 24 h. B. Quantification of the cell population percentages in each phase of the cell cycle in control and HepG2 cells treated with 2.5 μM C816, C830, C800 and 1 μM C830 and C800 for 24 h (p <0.05, n =2). C. Down-regulation of CDK1, CDK2, CCND2 and CCNA2 mRNA and up-regulation of CDKN2A and CDKN2D mRNA in 2.5 μM C816, C830 and C800-treated cells after 24 h as determined by qRT-PCR. D. Quantification of the cell population percentages in each phase of the cell cycle in control and HepG2 cells treated with 0.5, 1 and 2.5 μM C800 for 48 h (p <0.05, n =2). E. Representative histograms of the cell cycle obtained after flow cytometry analysis of 0.5, 1 and 2.5 μM C800-treated cells for 48 h.

Figure 4. Crambescidins induce intrinsic apoptotic death of tumor cells

A. Annexin V and PI staining of HepG2 cells after 0.5, 1 and 2.5 μM C816, C830 and C800 treatments for 24 and 48 h analyzed by confocal microscopy. B. Annexin V apoptosis determination of HepG2 cells treated with 1 μM C816, 2.5 μM C830 and 2.5 μM C800 for 24 h and 2.5 μM C800 for 48 h. Representative plots. C. Quantification of the different subpopulations as determined by flow cytometry.

Figure 5. Crambescidins' effect on mitochondrial function

A. Effects of 0.5, 1 and 2.5 μM C816, C830 and 0.5, 1 and 2.5 μM C800 treatments on mitochondrial membrane potential after 24 and 48 h, respectively as determined using MitoTracker^® Red CM-H2XRos staining (p<0.05). B. Representative overlay histograms of mitochondrial viability of control (red line), 0.5 (green line), 1 (blue line) and 2.5 μM (black line) treated cells as determined by flow cytometry using Mito Tracker^® Deep Red-FM. C. Population percentages of HepG2 cells (mean ± SEM) in the R regions (for R1, 2, 3 and 4 regions determination see Supplementary Figure S1) after 24 h treatments with C816, C830 and 48 h with C800 (p <0.05).

Figure 6. Death signaling and effector pathways activated by crambescidins

A. Caspase-3 activity in HepG2 cells treated with C816 and C830 for 24 h and with C800 for 48 h. *Significant differences with respect to controls (p <0.05, n = 3). B. Up-regulation of BBC3, CDKN1A, GADD45, IGFBP3 and 14-3-3σ genes mRNA in C816-treated cells for 24 h as determined by qRT-PCR. C. Up-regulation of BBC3, CDKN1A, GADD45, IGFBP3 and 14-3-3σ genes mRNA in 2.5 μM C816 and C830-treated cells for 24 h as determined by qRT-PCR. D. Schematic illustration of the molecular downstream targets of the cellular response elicited by crambescidins that mediate their different biological outcomes. E. Western blot analysis of proteins involved in crambescidin-mediated cell cycle arrest (p21, Cdk2 and cyclin D) and apoptosis (PUMA) induction. * Significant differences respect to controls (p <0.05).

Figure 7. p-53 implication in crambescidin-induced apoptosis

A. Caspase-3 activity determination for C816, C830 and C800-treated cells after p-53 inhibition. *Significant differences with respect to controls and between treatments groups (p <0.01). B. Viability of p53-null PC3 cells after C830 and C800 treatments during 24 and 48 h as determined by the MTT method. *Significant difference with respect to controls (p <0.01).

Figure 8. Crambescidin 816 induces tumor regression in an in vivo colorectal carcinoma model

A. In vivo anticancer activity of C816 in a human tumor xenograft model. Representative Z-projection images of the tumor development in control and treated embryos after 48 h. B. Three-dimensional reconstruction of tumor development in control and treated embryos after 48 h. C. Tumor fluorescence intensities (mean ± SEM) and percentages of decrease in tumor volumes of treated embryos after 48 h. D. Survival rates of control and treated embryos (median and Q₁, Q₃). *Significant difference with respect to controls (p <0.05).