In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells

Abstract

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100-200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Keywords: DNA damage; adhesion; cytotoxicity; flavonoids; irinotecan; polyphenols.

Figure 1

Cytotoxic properties of berry extracts of blackberry (BB), bilberry (Bil), black currant (BC) and elderberry (EB) (A,C) and single anthocyanins cyanidin-3-O-glucoside (Cy3glc), delphinidin-3-O-glucoside (Del3glc), cyanidin-3-O-rutinoside (Cy3rut), and cyanidin-3-O-sambubioside (Cy3sam) (B,D) assessed with the water-soluble tetrazolium (WST-1, top) and sulforhodamine B (SRB, bottom) assay. CT26 cells were incubated for 72 h with the respective substances. Results presented are the means + SD of at least three independent biological replicates measured in technical triplicates relative to the solvent control (Ctrl., 0.6% DMSO). Statistical differences to the control were calculated with one-sample Student’s t-tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and among the concentrations with one-way ANOVA via post hoc Bonferroni test (p < 0.05, a–e; e.g., the letter a represents significance compared to the lowest concentration of 6.25 µg/mL or µM).

Figure 2

Combined cytotoxic effects of anthocyanin-rich extracts (A,C) or single anthocyanins (B,D) and SN-38. CT26 cells were pre-incubated for 30 min with anthocyanin and subsequently additionally challenged with 10 nM SN-38 for 72 h. Metabolic cell activity and cell protein content were measured with WST-1 (A,B) and SRB (C,D) assays. Data presented are the means + SD of at least three independent replicates measured in technical triplicates and expressed as test over control (T/C) in percent relative to 10 nM SN-38. Statistical significances compared to the SN-38 incubation were calculated with one-sample Student’s t-tests (* p < 0.05, ** p < 0.01, *** p < 0.001). Significances among the tested concentrations were calculated with one-way ANOVA via post hoc Bonferroni test (p < 0.05, a–e; e.g., the letter a represents significance compared to the lowest concentration of 6.25 µg/mL or µM).

Figure 3

DNA-damaging properties of anthocyanin-rich (A) BB, (B) Bil, (C) BC, and (D) EB extracts alone and in combination with 10 µM SN-38. CT26 cells were pre-incubated for 30 min with the extract and then additionally challenged with SN-38 for 1 h; 1 min UV irradiation was used as a positive control. Clear and striped bars indicate an additional treatment without or with formamidopyrimidine-DNA glycosylase (FPG), respectively. Results presented are the means + SD of at least three different replicates expressed as T/C in percent relative to SN-38. Statistical significances to the respective solvent control were calculated with one- and two-sample Student’s t-tests and marked with $ (without FPG, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001) and § (with FPG, § p < 0.05, §§§ p < 0.001). Statistical differences to SN-38 were calculated with one-sample Student’s t-test (without FPG, * p < 0.05, ** p < 0.01) or two-sample Student’s t-test (with FPG, ^## p < 0.01). One-way ANOVA with post hoc Bonferroni test was used to calculate statistical differences of the applied concentrations (p < 0.05, a, b; e.g., the letter a represents significance compared to the lowest concentration of 0.1 µg/mL).

Figure 4

Influence of (A) Cy3glc, (B) Del3glc, (C) Cy3rut, and (D) Cy3sam on SN-38-induced genotoxicity. For the comet assay, cells were pre-incubated with the respective anthocyanin for 30 min and subsequently additionally challenged with 10 µM SN-38 for 1 h. Irradiation with UV light for 1 min served as positive control. Striped bars represent samples additionally treated with FPG. Data presented are the means + SD of at least three independent replicates shown as T/C in percent relative to SN-38. Statistical differences to the respective solvent control were calculated with one- or two-sample Student’s t-tests and indicated with $ (without FPG, $ p < 0.05, $$$ p < 0.001) or § (with FPG, §§ p < 0.01, §§§ p < 0.001). Statistical differences compared to SN-38 treated without (* p < 0.05) or with FPG (# p < 0.05, ## p < 0.01) were calculated with one- or two-sample Student’s t-tests, respectively. Significances among the tested concentrations were calculated with one-way ANOVA via a post hoc Bonferroni test (p < 0.05) and indicated with a–c (without FPG; e.g., the letter a represents significance compared to the lowest concentration of 1 µM) or x–z (with FPG).

Figure 5

Effects of anthocyanin-rich extracts (A) and single anthocyanins (B) on cell adhesion in CT26 cells measured with the crystal violet assay. Cells were incubated with the test substances for 2 h, and adhered cells were fixed and stained according to the assay protocol. Results presented are the means + SD of at least three independent biological replicates measured in technical triplicates relative to the solvent control (Ctrl., 0.6% DMSO). Statistical differences were calculated with one-sample Student’s t-tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and one-way ANOVA via a posthoc Bonferroni test (p < 0.05, a–e; e.g., the letter a represents significance compared to the lowest concentration of 6.25 µg/mL or µM).