Cyanophora paradoxa

Herbert, Helen, Parkes, Rachel, Barone, Maria Elena, Picciotto, Sabrina, Adamo, Giorgia, Paterna, Angela, Manno, Mauro, Bongiovanni, Antonella, Campion, Eva & Touzet, Nicolas, 2024, Effects of Cultivation Stress on the Glaucophyte Cyanophora paradoxa and Bioactive Potential in Human Cancer Cell Lines, Phycologia 63 (3), pp. 278-289 : 281-282

The growth of Cyanophora paradoxa

under various cultivation conditions including control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM sodium chloride (NaCl) was monitored ( Fig. 1 ). The BLU (0.03 d −1 ± 0.03) and NaCl (0.08 d −1 ± 0.03) treatments returned significantly lower specific growth rates as compared to the CONT (0.16 d −1 ± 0.01) and N- (0.14 d −1 ± 0.02) treatments (ANOVA, p <0.001, n = 12) ( Table S1 ). Varying amounts of dried biomass were then recovered, following centrifugation, from the cultures (CONT 724 mg, BLU 57 mg, N- 100 mg and NaCl 304 mg).

Effect of Cultivation Conditions on Pigment and Phycobiliprotein Composition

Alterations to the culture medium of C. paradoxa influenced pigment abundance as shown in Table 1 . Chlorophyll a, β-carotene and zeaxanthin were detected in all extracts ( Figs 2, 3 ). A significant 3-fold decrease in the yield of zeaxanthin was observed following BLU treatment (2.2 mg g −1) compared to the CONT treatment (6.8 mg g −1) (ANOVA, p <0.001, n = 12). This pattern was also observed for β-carotene, with BLU treatment having significantly less pigment (2.1 mg g −1) as compared to the CONT (7.5 mg g −1), N- (4.6 mg g −1) and NaCl (8.7 mg g −1) treatments (ANOVA, p <0.001, n = 12). There were also significant differences in chlorophyll a yields, with BLU and N- treatments having significantly less (~ 7.5 mg g −1) than the CONT and NaCl treatments (~19.0 mg g −1) (ANOVA, p <0.001, n = 12).

Both the phycocyanin and allophycocyanin contents were significantly higher in the CONT as compared to the three treatments (ANOVA, p <0.001, n = 12) ( Fig. 4 ). The phycocyanin content ranged from 113.9 mg g − 1 in the CONT treatment to 19.4 mg g − 1 in the BLU treatment. The allophycocyanin yields were 71.1 and 12.6 mg g −1 for the CONT and BLU treatments, respectively.

Effect of Cultivation Conditions on Fatty Acid Composition and EPA Content

Significant changes in the relative proportions of some FAMEs were observed in response to stress conditions ( Figs 5, 6 ). There was a significant decrease in the proportions of C20:4 following the BLU treatment (7.1%) compared to the CONT (22.7%), N- (24.5%) and NaCl (22.3%) treatments (ANOVA, p <0.001, n = 12) ( Table S2). There was also a significant increase in C16:1 following the BLU treatment (11.2%) as compared to the CONT (1.3%), N- (1.4%) and NaCl (1.8%) treatments (ANOVA, p <0.001, n = 12). In addition, the BLU treatment enhanced the accumulation of unsaturated forms of C18 (34.3%) compared to the CONT (6.5%), N- (6.6%) and NaCl (19.4%) treatments (ANOVA, p <0.001, n = 12). As shown in Table 2, the BLU treatment also led to a significant reduction in polyunsaturated fatty acids (PUFA) proportions, while increasing those of monounsaturated fatty acids (MUFA) (ANOVA, p <0.001, n = 12). The EPA content was negatively affected by all the treatments compared to the CONT (ANOVA, p <0.001, n = 12) ( Fig. 7 ). The highest EPA yield obtained for the CONT (1.1 mg g −1) was ~4-fold greater than that of the BLU treatment (0.3 mg g −1).

Antiproliferative Effect of Crude Extracts on Cancer Cell Lines - MTS Assay

Extracts of Et 2 O, EA and MeOH (100 µg ml −1) obtained from each treatment were used to monitor their effect on the cell viability of human lung (A549) and breast (MCF-7) cancer cell lines. There was no significant difference in A549 cell viability with any extract ( Fig. 8 ). The MCF-7 cell line appeared more sensitive to extracts, in particular to the EA extract obtained following the N- treatment, for which a significant reduction in viability was observed (~70%) (ANOVA, p <0.001, n = 12) as compared to the Et 2 O extracts (CONT and N-) ( Fig. 9 ).

Genotoxicity Assay - Acridine Orange Staining

A549 and MCF-7 human cancer exposed to C. paradoxa extracts were stained with acridine orange and examined microscopically to assess the presence of genotoxic effects after 72 h. The nuclei of treated cells appeared uniformly bright green, indicating that DNA within the nucleus remained undamaged, with a cellular structure comparable to that of the untreated control cells ( Fig. 10 ). Only a minor population of MCF-7 cells (exposed to NaCl derived extract) exhibited red and orange fluorescence outside the nucleus, presumably associated with lysosomes.

Principal Component Analysis (PCA)

A PCA examined patterns among the treatments and parameters measured. The PCA revealed two components, accounting for 88% of the variance ( Fig. 11 ). Component 1 (CP1) was positively related to variables such as phycobiliproteins and Et 2 O extract bioactivity on A549 cells and negatively associated with the Et 2 O and EA extract bioactivities on A549 and MCF-7 cells. Component 2 (CP2) was more closely aligned with the fatty acid content. In addition, both the zeaxanthin content and MeOH extract bioactivity on A549 cells were strongly associated with the positive domain on CP2.

The four treatments applied in this study were projected in different locations of the PCA plane. In particular, the CONT and N- treatments were firmly associated to the positive axes of CP1 and CP2, respectively, while the BLU treatment was instead located along the negative domain of CP2.

Figures

Figs 1–4. Growth (cells ml−1) and pigment production of Cyanophora paradoxa in various cultivation conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM of NaCl. Fig. 1. Growth (cells ml−1) of Cyanophora paradoxa for ~27 days. All data are mean ± SD (n = 3). Fig. 2. HPLC-UV chromatogram representation of identified pigments. Fig. 3. Pigment content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p <0.001, n = 12). Fig. 4. Phycocyanin, allophycocyanin and phycoerythrin content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p <0.001, n = 12).

Figs 5–7. Fatty acid analysis of Cyanophora paradoxa cultivated under various conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM NaCl. Fig. 5. GC-MS chromatogram representation of identified FAMEs. Fig. 6. FAME proportions (%). Fig. 7. EPA (mg g−1) content. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p <0.001, n = 12).

Figs 8, 9. Relative cell viability of human cancer cell lines exposed to diethyl ether (Et2O), ethyl acetate (EA) and methanol (MeOH) extracts derived from Cyanophora paradoxa at a concentration of 100 µg ml−1 over 72 h. Values are expressed as mean ± SD of three separate experiments, each assay was performed in triplicate. Lettering indicates homogenous subsets (ANOVA, p <0.001, n = 12). Fig. 8. A549 human cancer cell line. Fig. 9. MCF-7 human cancer cell line.

Fig. 10. Epi-fluorescence microscopy images of acridine orange stained A549 (A-E) and MCF-7 (F-J) cells following treatment (72 h) with 100 µg ml−1 of Cyanophora paradoxa derived extracts. Untreated cells (A and F) are also included. Scale bar = 100 µm.

Fig. 11. Principal Component Analysis (PCA) projections indicating the relationships between the treatment conditions applied to C. paradoxa and the variables measured (compounds including carotenoids, fatty acids and phycobiliproteins , bioactivity, biomass production).