Dolastatin 15 from a Marine Cyanobacterium Suppresses HIF-1α Mediated Cancer Cell Viability and Vascularization

Abstract

Chemical investigation of a benthic marine cyanobacterium yielded the anticancer agent dolastatin 15, originally isolated from a mollusk. Dolastatin 15 is a microtubule-destabilizing agent with analogues undergoing clinical evaluation. Profiling against a panel of isogenic HCT116 colorectal cancer cells showed remarkable differential cytotoxicity against the parental cells over isogenic cells lacking HIF or other key players in the pathway, including oncogenic KRAS and VEGF. Dolastatin 15 displayed an antivascularization effect in human endothelial cells and in zebrafish vhl mutants with activated Hif, thus signifying its clinical potential as a treatment for solid tumors with an angiogenic component. Global transcriptome analysis with RNA sequencing suggested that dolastatin 15 could affect other major cancer pathways that might not directly involve tubulin or HIF. The identification of the true producer of a clinically relevant agent is important for sustainable supply, as is understanding the biosynthesis, and future genetic manipulation of the biosynthetic gene cluster for analogue production.

Keywords: HIF-1α; angiogenesis; cyanobacteria; dolastatin 15; zebrafish.

Figure 1:

Structure of natural dolastatin 15 (1) and variations designed to give different linkage sites at the N and C termini (2-5) to use as ADC warheads in clinical evaluations. Maleimido-caproyl (MC) modified N-terminus (2); Maleimido-caproyl-valine-citruline-p-aminobenzoyloxy-carbonyl linker (MC-VC-PABC) modified N-terminus (3); ester (4) and maleimide (5) modified C-termini conjugates used with trastuzumab.

Figure 2:

Cellular activity of dolastatin 15 (1) in parental HCT116 colon cells and their isogenic knockouts. A) and B) Parental HCT116, isogenic knockout and normal colon cells were treated with dolastatin 15, and cell viability was measured 48 h later in a MTT assay. (C) Western blot analysis of HCT116, HIF-1α isogenic knockout and normal epithelial colon cell lysates probed with HIF-1α and HIF-1β antibodies after 16 h culturing under normoxia. (D) HCT116 cells were pre incubated with CoCl₂ (100 μM, 4 h) to mimic hypoxia prior to treatment with increasing concentration of dolastatin 15 and solvent control (16 h). Cell lysates were analyzed by Western blotting to study the effect of dolastatin 15 on HIF-1α/β protein levels under hypoxic conditions. (E) HIF target gene expression after 24 h exposure of parental HCT116 cells to dolastatin 15. Error bars indicate mean ± SD of three replicates (*p = 0.05–0.01; **p = 0.01–0.001; ***p ≤ 0.001).

Figure 3:

Gene expression profiles of HCT116 cells treated with dolastatin 15 and overview of the differentially expressed genes for each comparison sample for 20 nM and 100 nM treatments at 16 h and 24 h (p < 0.05). (A) Heatmap representation of differentially expressed genes (>2-fold) based on log2-transformed values with dendrogram. (B) Volcano plots of differentially expressed genes with treated and untreated groups at 16 h and 24 h time points with 20 nM treatment. Red dots highlight the significantly upregulated genes and blue dots highlight the significantly downregulated genes. (C) Enriched Pathways in GO analysis for dolastatin 15 (20 nM, 24 h) treatment, showing major cancer related pathways highlighted in red. (D) IPA network analysis showing the highest number of up- (pink) and down- (green) regulated genes associated with cancer pathways at 100 nM (16 h) treatment of dolastatin 15.

Figure 4:

Effects of dolastatin 15 on HUVECs. (A) Dolastatin 15 shows anti-vascularization effect in vitro in a dose-dependent manner, determined by matrigel assay (scale bar 200 μm), 12 h. The known RTK inhibitor cabozantinib was tested in parallel. (B) Antiproliferative effect of dolastatin 15 and cabozantinib. Error bars indicate mean ± SD of three replicates. Both compounds do not significantly affect cell viability at this time point. (C) Branch point counting was used for quantification. Five random microscope view-fields were counted and the number of branch points were averaged. (D) Number of junctions analysed by the Angiogenesis Analyzer plug-in for ImageJ (n = 5 per group). Error bars in (C) and (D) indicate mean ± SEM of five fields.

Figure 5.

Dolastatin 15 ameliorates the phenotypes of VHL loss of function. (A-E): Lateral confocal images of the tail of 5dpf fli1a:egfp^y1 transgenic larvae, labelling blood vessels with eGFP. vhl^−/− mutant larvae display excessive vascularisation with extensive intersomitic vessel branching and a prominent dorsal plexus (bracket highlight; B), compared to wild-type siblings (A). Whilst treatment with 6 μM dolastatin15 had little effect on WT (C), it reduced the vhl^−/− vascular phenotype (D). The proportion of larvae displaying a vascular phenotype was significantly reduced upon dolastatin 15 treatment of offspring from a vhl ^+/− incross (E; Chi-squared test). (F-N): Lateral images of 5dpf larvae stained by in situ hybridisation for the Hif1α target genes, egln3 (F-I) and pfkfb3 (K-N). Whilst dolastatin 15 treated (H, L) or untreated (F, K) WT larvae show little egln3 and pfkfb3 RNA expression, vhl^−/− mutant larvae show strong staining due to higher Hif1α levels (G, M). Treatment with 6 μM dolastatin 15 significantly reduced the proportion of mutants in the strong RNA staining class for both probes (I, N, J; Chi-squared test for extent of egln3 staining, p<0.025).