Expression of microRNA-15b and the glycosyltransferase GCNT3 correlates with antitumor efficacy of Rosemary diterpenes in colon and pancreatic cancer

Abstract

Colorectal and pancreatic cancers remain important contributors to cancer mortality burden and, therefore, new therapeutic approaches are urgently needed. Rosemary (Rosmarinus officinalis L.) extracts and its components have been reported as natural potent antiproliferative agents against cancer cells. However, to potentially apply rosemary as a complementary approach for cancer therapy, additional information regarding the most effective composition, its antitumor effect in vivo and its main molecular mediators is still needed. In this work, five carnosic acid-rich supercritical rosemary extracts with different chemical compositions have been assayed for their antitumor activity both in vivo (in nude mice) and in vitro against colon and pancreatic cancer cells. We found that the antitumor effect of carnosic acid together with carnosol was higher than the sum of their effects separately, which supports the use of the rosemary extract as a whole. In addition, gene and microRNA expression analyses have been performed to ascertain its antitumor mechanism, revealing that up-regulation of the metabolic-related gene GCNT3 and down-regulation of its potential epigenetic modulator miR-15b correlate with the antitumor effect of rosemary. Moreover, plasmatic miR-15b down-regulation was detected after in vivo treatment with rosemary. Our results support the use of carnosic acid-rich rosemary extract as a complementary approach in colon and pancreatic cancer and indicate that GCNT3 expression may be involved in its antitumor mechanism and that miR-15b might be used as a non-invasive biomarker to monitor rosemary anticancer effect.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Viability inhibition and death induction of colon and pancreatic cancer cells by five different RE's.

Colon (SW620 and DLD-1) and pancreatic (MIA-PaCa-2 and PANC-1) cancer cells were treated with increasing concentrations of five different RE's during 48 h and cell viability was determined by MTT assay. (A) Bars represent the IC50 values (50% cell viability inhibition) of the five different RE's in each cancer cell line. (B-C) IC50s of the five RE's in the four cancer cell lines plotted against the carnosic acid (B) or carnosol (C) content of the RE's. IC50 values are expressed as the mean ± SEM of at least three independent experiments, each performed in quadruplicate. (D-E) PARP1 cleavage induced by different RE's in colon and pancreatic cancer cells after 48 h treatment. (D) Colon cancer cells (DLD-1) were treated with 70 µg/mL of five different RE's (RE-1 to RE-5) or vehicle (Cntrl). (E) Two colon (DLD-1 and SW620) and two pancreatic (MIA-PaCa-2 and PANC-1) cancer cell lines were treated with 110 µg/mL of RE-3, 90 µg/mL of RE-4 or vehicle (Cntrl). Western blot figures show representative results of three independent experiments. DLD: DLD-1, M: marker of molecular weight, MIA: MIA-PaCa-2, P: PANC-1, RE: supercritical rosemary extract, SW: SW620.

Figure 2. Inhibition of cancer cell viability by different RE's in comparison with their major active components.

Cell viability inhibition of cancer cells by different RE's and their equivalent concentrations of carnosic acid alone, carnosol alone, or both compounds in combination. SW620 colon and PANC-1 pancreatic cancer cells were treated with increasing concentrations of RE-2, RE-4 and RE-5, and the same concentrations of carnosic acid, carnosol, and the combination of both present in the RE's at each assayed condition. Extract ID and tumor cell line used in the assay are indicated in each graph. Results are shown as the mean ± SEM of three independent experiments, each performed in quadruplicate. Asterisks indicate statistically significant differences of carnosic acid or extract in comparison with the combination of carnosic acid and carnosol (U de Mann-Whitney; *p≤0.05; **p≤0.01). RE: supercritical rosemary extract.

Figure 3. Antitumorigenic activity of different RE's in human colon cancer xenografts.

RE (test groups) or vehicle (control groups) was administered to nude mice bearing SW620 colon cancer xenografts in the drinking water (1 mg RE and 20 μL ethanol as vehicle per mL of drinking water). Three RE's with different composition (RE-3, RE-4 and RE-5, indicated in each graph) were assayed. Tumor volumes were monitored twice a week during 32–35 days. Results are shown as mean ± SEM (n = 16–20) and repeated measures ANOVA with Bonferroni's test was used to determine the statistically significant differences between treated and control groups (*p≤0.05; **p≤0.01; ***p≤0.001). RE: supercritical rosemary extract.

Figure 4. Modulation of gene and miRNA expression by RE.

(A) Venn diagram representing overlap of the genes significantly modulated (p<0.05 and fold change (FC) >±50%) by RE-2 at 30, 60 and 100 µg/mL after 48 h treatment in SW620 cells according to microarray data. (B) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by five RE's with different composition (RE-1, RE-2, RE-3, RE-4 and RE-5) at the same concentration (90 µg/mL) during 48 h. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. ANOVA with Bonferroni's post hoc test was applied to test statistically significant differences among RE's. (C) Up-regulation of GCNT3 gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Bars represent the mean ± SEM of two independent experiments, each performed with biological triplicates and technical duplicates. Student's t test was applied to determine the statistically significant differences between treated and control cells. (D) Down-regulation of miR-15b gene expression in SW620 colon cancer cells by 48 h treatment with RE-4 at 60 µg/mL and carnosic acid, carnosol, and the combination of both compounds at the concentrations present in this RE. Student's t test was used to test statistically significant differences between treated and control cells. (E) Modulation of miR-15b and miR-939 (as a negative control) in plasma of colon cancer xenograft nude mice after orally intake of RE-5 (1 mg/mL in drinking water), in comparison with the control group of mice. Bars represent mean ± SEM (n = 8). Student's t test was used to assess statistically significant differences between treated and control groups. * p≤0.05, ** p≤0.01, *** p≤0.001. RE: supercritical rosemary extract, Cc+Cl: Carnosic acid and carnosol.