The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8

Abstract

Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficiently partitioned into liposomes while retaining full antiproliferative activity. Consistent with the induction of apoptosis via the lipid compartment, we noted accumulation and aggregation of ceramide in treated cells and subsequent colocalization with caspase 8. Angiogenic endothelial cells were extremely sensitive to ScA. Picomolar concentrations of ScA disrupted proliferation and endothelial tubule formation in vitro. Systemic treatment of zebrafish or local treatment of the chick chorioallantoic membrane with ScA resulted in dose-dependent inhibition of angiogenesis, whereas topical treatment blocked tumor growth among caspase-8-expressing tumors. Together, the results reveal an unexpected mechanism of action for this unique lipopeptide and suggest future development of this and similar agents as antiangiogenesis and anticancer drugs.

Fig. 1.

ScA induces apoptosis selectively via caspase-8-dependent mechanisms. (A) Treatment of A549 cells with 100 nM ScA induces a blebbing morphology, as observed via bright-field microscopy. (B) A549 cells were fixed and costained with antibodies directed against ceramide (red channel). (Scale bars in A and B: 25 μM.) Arrows indicate regions of blebbing. The nuclear compartment was visualized by using DAPI to stain DNA (blue channel). The assessments were performed 2 h after treatment with 300 nM ScA. (C) Immunoblot analysis of Jurkat cells was performed after treatment with 100 nM ScA. Then, 25 μg of cell lysates was probed for caspases 8, 3, and 9, for PARP, and for actin (loading control) as indicated. (D) Caspase 8-deficient (Casp 8−), FADD-deficient (FADD−), or parental Jurkat cells (Jurkat) were incubated with 50 or 300 nM for 6 h and analyzed for the presence of apoptotic cells via FACS analysis of DNA content. Results shown are the mean and standard error of triplicate determinations. (E) A549 cells subjected to lentivirus-delivered shRNA-mediated knockdown of caspase 8 or treated with a scrambled shRNA lentivirus. Cells then were cultured in the presence of increasing doses of ScA, as indicated. Viability was measured by XTT assay after 72 h and normalized to controls not treated with drug. (F) Similarly, the viability of neuroblastoma cultures deficient in caspase expression, or reconstituted for caspase 8 expression, was determined as for the A549 cells above. Both experiments are representative, with each point shown the mean ± SE of triplicate wells.

Fig. 2.

ScA partitions into the lipid compartment. (A) ScA was mixed with DOPE:cholesterol:DSPC:DSPE-mPEG to form liposomes, as described in Materials and Methods. ScA completely partitioned into the lipid nanosomes, which had an effective diameter of 109 nm and a polydispersity of 0.205. A549 cells were cultured with free ScA added in DMSO diluent or with ScA incorporated into nanosomes, and cell viability was assessed by XTT assay as described above. (B and C) A549 cells were stained with anti-ceramide (red channel), DAPI (blue channel), and anti-caspase 8 (green channel) 30 min after treatment with 300 nM arachidonic acid, a control lipid (B), or 300 nM ScA (C). Colocalization of the green and red channels is shown by the merge (yellow signal). (D) A limited structure–function analysis of the required elements for ScA (shown at left) activity was performed. Derivatives of ScA synthesized or derived included a “monomeric” form (with <0.1% activity) and forms in which the disulfide was replaced with alternative linkers, which showed no activity below 50 μM. A “truncated” lipopeptide that maintained the disulfide linkage also lacked activity below 50 μM.

Fig. 3.

Endothelial cells are highly sensitive to ScA. (A) Human endothelial cells were incubated with ScA at decreasing concentrations, as shown, and viability was assessed by XTT assay after 72 h. Data shown are the mean ± SE of triplicate wells from a representative experiment. (B) Human endothelial cells were plated on Matrigel-coated surfaces and allowed to form tubules for 48 h in the presence of DMSO diluent (Top). When ScA was added (Middle and Bottom) cell viability was compromised, and endothelial cell tube formation was disrupted in dose-dependent manner.

Fig. 4.

Antiangiogenic and antitumor activity of ScA. (A–F) Transgenic Tg(fli1:EGFP) zebrafish embryos in which GFP is expressed in endothelial cells were incubated without (A) or with increasing concentrations of ScA: 80 nM (B), 160 nM (C), 300 nM (D) 1.6 μM (E), or 3 μM (F). Blood vessel morphology was recorded by fluorescence microscopy. (G) Filter disks impregnated with 100 ng of basic FGF were placed on the chorioallantoic membrane of 11-day-old chicks to induce angiogenesis in the absence or presence of ScA, as shown. After 72 h, disks were removed and the vascularity of the underlying chorioallantoic membrane determined by direct counting of branch points using a dissecting microscope. (H) NB7 neuroblastoma cells lacking caspase 8 (filled bar) or NB7C8 cells reconstituted for caspase 8 expression (open bars) were seeded into 10-day-old chick chorioallantoic membranes to form tumors. After 72 h, ScA was added topically to each growing tumor mass. Tumors were harvested and resected on day 8, and mass (wet weight) was determined. Data shown is the mean ± SD (n = 8–12). The mass of the NB7C8 is significantly decreased (P < 0.002).