Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells

Abstract

Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

Figure 1. (A, B, C) Effect of the diatom PUA 2-trans,4-trans-decadienal (DD) on the human lung adenocarcinoma cell lines A549 and COLO 205, and the lung/brunch normal epithelial BEAS-2B cell line.

(D) Effect of DD on A549 cell line in the presence of caspase-3 inhibitor (9.7 µM). Percentage of viable cells for A549 and COLO 205 calculated with the Trypan blue viability assay and for BEAS-2B with the MTT viability assay. Values are reported as mean ±S.D compared to controls (100% viability); ▴ 2 µM; ▪ 5 µM, ♦ 10 µM.

Figure 2. (A, B, C) Effect of the diatom PUA 2-trans,4-trans-octadienal (OD) on the human lung adenocarcinoma cell lines A549 and COLO 205, and the lung/brunch normal epithelial BEAS-2B cell line.

(D) Effect of OD on A549 cell line in the presence of caspase-3 inhibitor (9.7 µM). Percentage of viable cells for A549 and COLO 205 calculated with the Trypan blue viability assay and for BEAS-2B with the MTT viability assay. Values are reported as mean ±S.D compared to controls (100% viability); ▴ 2 µM; ▪ 5 µM, ♦ 10 µM.

Figure 3. (A, B, C) Effect of the diatom PUA 2-trans,4-trans-heptadienal (HD) on the human lung adenocarcinoma cell lines A549 and COLO 205, and the lung/brunch normal epithelial BEAS-2B cell line.

(D) Effect of HD on A549 cell line in the presence of caspase-3 inhibitor (9.7 µM). Percentage of viable cells for A549 and COLO 205 calculated with the Trypan blue viability assay and for BEAS-2B with the MTT viability assay. Values are reported as mean ±S.D compared to controls (100% viability); ▴ 2 µM; ▪ 5 µM, ♦ 10 µM.

Figure 4. Effect of the diatom PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the human lung adenocarcinoma cell line A549.

Control and treated cells double stained with acridine orange and ethidium bromide after 48 µM DD, OD and HD observed at the confocal microscope. Numbers indicate (1) normal cells; (2) early apoptotic cells; (3) late apoptotic cells; (4) necrotic cells (see material and methods for details). Arrows indicate cells with fragmented nuclei.

Figure 5. The histograms show the effects of 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the expression levels of target proteins (TNFR2, TNFR1, Fas, FADD, RIP, caspase-3) and control (actin) in lung adenocarcinoma A549 cells.

Immunoblot analysis shows that PUAs induce TNF signaling after 24(DD and HD) and 48 h (OD) of treatment with actin. Asterisk denotes significant increase in protein levels measured. **p≤0.05 versus control; error bars represent ±SD.

Figure 6. Histograms show the effects of 2-trans,4-trans-decadienal (DD) and 2-trans,4-trans-heptadienal (HD) on the expression levels of target genes in lung adenocarcinoma A549 cells.

Gene expression analysis was conducted after 2 µM DD and HD; error bars represent ±SD.

Figure 7. (A) Flow cytometric analysis of DNA content.

Cells were exposed to 2-trans,4-trans-heptadienal (HD) 5 µM for 24 h. (B), The corresponding percentage of cells in each phase, obtained by ModFit LT Software.

Figure 8. Schematic representation of pathways induced by 2-trans,4-trans-decadienal (DD), 2-trans,4-trans–octadienal (OD) and 2-trans,4-trans-heptadienal (HD) in A549 cell line.

Black arrows indicate possible correlated pathways not involved in the response of cells to aldehyde treatment.