Antiproliferative Effects of the Natural Oxadiazine Nocuolin A Are Associated With Impairment of Mitochondrial Oxidative Phosphorylation

Abstract

Natural products are interesting sources for drug discovery. The natural product oxadiazine Nocuolin A (NocA) was previously isolated from the cyanobacterial strain Nodularia sp. LEGE 06071 and here we examined its cytotoxic effects against different strains of the colon cancer cell line HCT116 and the immortalized epithelial cell line hTERT RPE-1. NocA was cytotoxic against colon cancer cells and immortalized cells under conditions of exponential growth but was only weakly active against non-proliferating immortalized cells. NocA induced apoptosis by mechanism(s) resistant to overexpression of BCL family members. Interestingly, NocA affected viability and induced apoptosis of HCT116 cells grown as multicellular spheroids. Analysis of transcriptome profiles did not match signatures to any known compounds in CMap but indicated stress responses and induction of cell starvation. Evidence for autophagy was observed, and a decrease in various mitochondrial respiration parameter within 1 h of treatment. These results are consistent with previous findings showing that nutritionally compromised cells in spheroids are sensitive to impairment of mitochondrial energy production due to limited metabolic plasticity. We conclude that the antiproliferative effects of NocA are associated with effects on mitochondrial oxidative phosphorylation.

Keywords: anti-cancer drugs; autophagy; colon cancer; cyanobacteria; mitochondria; natural products; spheroids.

Figure 1

Representative IncuCyte images of HCT116^wt spheroids. Top image represents a spheroid in the beginning (0 h) and at the end of the experiment (72 h), without exposure to NocA while the bottom images represent a spheroid exposed to 20 μM of NocA. Quantitative analysis is shown on Supplementary Figure 4 derived from 3 independent experiments in triplicates.

Figure 2

Analysis of apoptosis on monolayer culture and multicellular spheroids (MTS). (A) Apoptosis analysis of monolayer culture HCT116 cells using flow cytometry with Annexin V and Propidium iodide staining. (B) Quantifications of flow cytometry results. Apoptosis is observed after 48 h at 2.5 μM. ^***p < 0.001; ^**p < 0.01. Ten thousand cells per gated replicate were counted, n = 3. (C) M30 CytoDeathTM ELISA absorbance readings on HCT116 MTS exposed to NocA up to 20 μM. High absorbance values represent higher levels of caspase-cleaved K18. Two independent assays were performed, n = 3. (D) Bright field images of MTS after 48 h of exposure, being the top image a control and the bottom image an MTS exposed to Noc A at 10 μM; ^***p < 0.001.

Figure 3

Gene Set Enrichment Analysis mapped clustered with Cytoscape. Clusters represent different responses of exposed cells to NocA. Node sizes represent the number of genes and darken of the color increases with significance (FDR Q-value Cut-off = 0.1; P-value Cut-off = 0.005).

Figure 4

Evidence of autophagy induction on monolayer cell culture of HT116 and multicellular systems (MTS). (A) Western blot of HCT116 cells exposed to NocA at 2.5 and 1.25 μM for 6 and 18 h with detection of LC3-B (B). (C) MDC staining of autophagic compartments on HCT116^wt monolayer cultured cells exposed to NocA 7 μM over 18 h. (D) Western blot of MTS of HCT116 cells exposed to NocA at 10 μM for 24 and 48 h with detection of LC3B-II. (E) LC3B-II quantification. (F) MDC staining of MTS exposed to NocA 7 μM over 48 h. Three independent assays, ^***p < 0.001; ^**p < 0.01.

Figure 5

Seahorse analysis of cellular respiration. (A) Oxygen consumption rates by HCT116^wt cells over 6 h in response to exposure to NocA at 1.25, 2.5, and 7 μM. (B) Mitochondrial key parameters—Basal respiration, ATP production, Maximal Respiration—calculated after addition of sequential kit reagents (Oligomycin, FCCP, Antimycin A, and Rotenone) from Seahorse XF Cell Mito Stress Test, Kit ^***p < 0.001. (C) Dose-response for glucose-free (galactose supplemented) HCT116^wt cells treated with NocA and CCCP and respective cell viability after 2 h of exposure (according to Mitochondrial ToxGlo™ Assay kit). 1 = Log (10 μM) to −0.81 = Log (0.16 μM). (D) Comparison of Oxygen consumption rates between HCT116^wt and RPE-1 hTERT cells in response to 6 h exposure to NocA at 7 μM. (E) Mitochondrial key parameters—Basal respiration, ATP production, Maximal Respiration- calculated after using Seahorse XF Cell Mito Stress Test Kit on RPE-1 hTERT and HCT116^wt exposed to NocA 7 μM for 6 h. ^***p < 0.001; ^**p < 0.01, n > 5, 3 independent assays.