The Protective Anticancer Effect of Natural Lycopene Supercritical CO2 Watermelon Extracts in Adenocarcinoma Lung Cancer Cells

Abstract

Carotenoids may have different effects on cancer and its progression. The safety of carotenoid supplements was evaluated in vitro on human non-small cell lung cancer (NSCLC) adenocarcinoma A549 cells by the administration of three different oleoresins containing lycopene and other lipophilic phytochemicals, such as tocochromanols. The oleoresins, obtained by the supercritical CO₂ green extraction technology from watermelon (Lyc W), gấc(Lyc G) and tomato (Lyc T) and chlatrated in α-cyclodextrins, were tested in comparison to synthetic lycopene (Lyc S), by cell cycle, Annexin V-FITC/PI, clonogenic test, Mytosox, intracellular ROS, Western Blot for NF-kB and RT-PCR and ELISA for IL-8. The extracts administered at the same lycopene concentration (10 µM) showed conflicting behaviors: Lyc W, with the highest lycopene/tocochromanols ratio, significantly increased cell apoptosis, mitochondrial stress, intracellular ROS, NF-kB and IL-8 expression and significantly decreased cell proliferation, whereas Lyc G and Lyc T significantly increased only cell proliferation. Lyc S treatment was ineffective. The highest amount of lycopene in Lyc W was able to counteract and revert the cell survival effect of tocochromanols supporting the importance of evaluating the lycopene bio-availability and the real effect of antioxidant tocochromanols' supplementation which may not only have no anticancer benefits but may even increase cancer aggressivity.

Keywords: antioxidants; carotenoids; inflammation; lung cancer; lycopene; lycopene from gấc; lycopene from tomato; lycopene from watermelon; supercritical fluid extraction; synthetic lycopene; tocochromanols.

Figure 1

Flow cytometry analysis on A549 cells for cell cycle. (A) Lyc W significantly increased the apoptotic cells in comparison to medium, Lyc G, Lyc T, Lyc S and to its carrier α-CDs. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = four experiments; *, p-value < 0.05 (B) Representative graph bar with means for M1, Apoptosis (Sub G1), M2, G0/G1, M3, S, M4, G2/M; (C) Representative examples of flow cytometric analysis. The numbers indicate the percentage of positive cells.

Figure 2

Flow cytometry analysis on A549 cells for Annexin V test, redistribution of phosphatidylserine (PS). (A) Lyc W significantly increased total apoptosis in A549 cells in comparison to medium, Lyc G, Lyc T, Lyc S, its carrier α-CDs and to ACTD. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = four experiments; *, p-value < 0.05, **, p-value < 0.01 (B) Representative examples of flow cytometric analysis for percentage of positive cells: the numbers indicate the percentage of positive cells for PS, both early and late apoptosis (LR + UR quadrants). FL1-heights, Annexin V; FL2-heights, PI.

Figure 3

Cell colony assay. Effect of all of the three α-CDs oleoresin extracts on colony formation ability in A549 cell line. (A) Lyc W significantly reduced colony numbers whereas Lyc G and Lyc T significantly increased colony numbers when compared to untreated cells. Lyc S treatment was ineffective. The results are shown as box plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = six experiments; *, p-value < 0.05. (B) Bright-field pictures of A549 cell colony are reported.

Figure 4

Lyc W effect on mitochondrial stress. (A) Lyc W significantly increased mitochondrial stress when compared to cells grown in medium or in Lyc G, Lyc T and Lyc S or in its carrier α-CDs. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = five experiments; *, p-value < 0.05. (B) representative examples of flow cytometric analysis for percentage of positive cells. The numbers indicate the percentage of positive cells for MITOSOX.

Figure 5

Lyc W affect intracellular ROS generation. (A) Lyc W significantly increased intracellular ROS when compared to cells grown in medium or in Lyc G, Lyc T and Lyc S or in its carrier α-CDs. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = five experiments; *, p-value < 0.05. (B) representative examples of flow cytometric analysis for percentage of positive cells. The numbers indicate the percentage of positive cells for DCF.

Figure 6

Lyc W affects inflammation markers in A549 cell line. (A,B) Lyc W significantly increased nuclear translocation of NF-kB expression in comparison to cells treated with medium and in comparison, to cells treated with Lyc G, Lyc T and Lyc S; (A) Three Western blots were semi-quantified by densitometric scanning, normalized and expressed after correction with the density of the bands obtained for housekeeping proteins for nuclear proteins. Data are expressed as arbitrary units. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD, n = three experiments; *, p-value < 0.05. (B) representative Western blot for NF-kB and for the nuclear housekeeping gene Lamin B1; (C,D) Lyc W significantly increased both m-RNA (C) and IL-8 protein expression (D) in comparison to cells treated with medium and in comparison, to cells treated with Lyc G, Lyc T and Lyc S. The results are shown as box-plots with medians (lines inside the boxes). Analysis of variance (ANOVA), Fisher’s PLSD; *, p-value < 0.05. (C) n = three experiments; quantitative real-time PCR of IL-8; (D) n = six experiments, ELISA for IL-8 release.