Rational development of a cytotoxic peptide to trigger cell death

Abstract

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

Figure 1. CT20p, based on the C-terminus of Bax, Associates with Mitochondrial Membranes

(A) Mitochondrial translocation of HA-tagged wild type Bax (Bax-KK) and K189/K190 mutants, expressed in 293 cells using the Flp-In T-Rex system, was examined by immunoblot. p38 MAPK and Prohibitin were blotted for cytosolic and mitochondrial content, respectively. Data are representative of five independent assays. Images from full-length blots were cropped for concise presentation. (B) The mitochondrial translocation of DD-tagged Bax full-length (FL-Bax) and DD-tagged CT20 peptides, wild-type and EE, LL and RR mutants, was examined in Bax^+/+ HCT-116 cells by immunoblot. Endogenous Bax was probed with anti-Bax antibody. p38 MAPK and prohibitin indicated cytosolic and mitochondrial content, respectively. DD-fusions were detected with an anti-DD antibody. Controls are cells transfected with empty vector or untransfected. Data are representative of three independent assays. Images from full-length blots were cropped for concise presentation.

Figure 2. CT20p causes the Release of Sequestered Contents from Mitochondrial-like Lipid Vesicles without Loss of Membrane Integrity

(A) CT20p was commercially synthesized and calcein-loaded mitochondrial-like LUVs prepared. Calcein release from CT20p-treated LUVs was measured as described in Experimental Procedures. In the first Control figure, dotted lines indicate maximal release of calcein with Triton X-100. In samples treated with CT20p or variants, dotted lines indicate addition of peptide to LUVs and solid lines are LUVs alone in buffer. CT20 is the wild-type peptide and CT20-LL and CT20-EE are mutations of the double lysines (K189/K190 in the full-length Bax protein). (B) Light scatter analysis of LUVs from (A). Data are representative of three independent assays.

Figure 3. CT20p can be Encapsulated in NPs for Delivery to Cells

(A) Schematic representation of the three dimensional structure of aliphatic hyperbranched NPs. (B) HCT-116 cells were treated with NPs loaded with DiI or DiI + CT20p (0.07 nM) for 24 hours. Time-lapse movies were acquired as described in the Experimental Section using a 10× air objective. For each sample, three different fields of view were acquired. (C) Bax^+/+ and Bax^−/− HCT-116 cells were treated with NPs loaded with DiI at amounts of 5, 10 and 15 µg for 24 hours and cell death was measured using Sytox AAD as described in the Experimental Section. Results or images are representative “snapshots” of three independent experiments.

Figure 4. CT20p-NPs Kill Bax-containing or Bax-deficient HCT116 cells

(A–B) Bax^+/+ (A) and Bax^−/− (B) HCT-116 cells were treated with AM- or COOH-NPs loaded with CT20p (350 pM) for 24 hours. To visualize mitochondria, cells were treated with MitoTracker Red 580 and time-lapse movies were acquired as described in the Experimental Section using a 63× Oil objective. For each sample, three different fields of view were acquired. Images are representative “snapshots” of three independent experiments. Insets are digitally enlarged 4-fold. (C) HCT-116 cells were treated with CT20p-NPs (350 pM) and, after three hours, cell death was measured using Sytox AAD as described in Experimental Procedures.

Figure 5. CT20p-NPs Kill Breast Cancer Cells

(A,C) MCF-7 (A) or MDA-MB-231 (C) cells were treated with AM- or COOH-NPs loaded with CT20p (350 pM) for 24 hours. To visualize mitochondria, cells were treated with MitoTracker Red 580 and time-lapse movies were acquired as described in the Experimental Section using a 40× Oil objective. For each sample, three different fields of view were acquired. Images are representative “snapshots” of three independent experiments. (B, D) MCF-7 (B) or MDA-BB-231 (D) cells were treated CT20p-NPs (350 pM) and, after three hours, cell death was measured using Sytox AAD as described in Experimental Procedures.

Figure 5. CT20p-NPs Kill Breast Cancer Cells

(A,C) MCF-7 (A) or MDA-MB-231 (C) cells were treated with AM- or COOH-NPs loaded with CT20p (350 pM) for 24 hours. To visualize mitochondria, cells were treated with MitoTracker Red 580 and time-lapse movies were acquired as described in the Experimental Section using a 40× Oil objective. For each sample, three different fields of view were acquired. Images are representative “snapshots” of three independent experiments. (B, D) MCF-7 (B) or MDA-BB-231 (D) cells were treated CT20p-NPs (350 pM) and, after three hours, cell death was measured using Sytox AAD as described in Experimental Procedures.

Figure 6. Mechanism of Cancer Cell Death Mediated by CT20p is Non-apoptotic

(A) MDA-MB-231 cells were cultured with COOH-NPs, encapsulated with CT20p (350 pM), and treated with ZVAD-Fmk and/or CDDP as described in the Experimental Section. Cells were also transiently transfected with Bcl-2. Cell death was assayed with Sytox AAD. Table shows median peak values for each sample displayed in histograms. (B) MDA-MB-231 cells were treated as described in (A) and membrane asymmetry measured with a violet radiometric probe. Dot blots show a combination of results from Sytox (cell death) and changes in membrane symmetry. (C) Cells treated as described above were stained with JC-1 as described in Experimental Procedures to measure mitochondrial membrane potential changes. Dependent on the mitochondrial membrane potential, JC-1 aggregates accumulate in the mitochondrial matrix, while JC-1 monomers are cytosolic. A measurement of JC-1 aggregates to monomers indicates mitochondrial with high (H), intermediate (I) or low (L) membrane potential. Data from a 6 hour treatment time point is shown. Results shown are representative of at least three independent experiments.

Figure 7. CT20p Causes Regression of Tumors when Delivered Intratumorally or Systemically

(A) MDA-MB-231 cells were implanted in nude mice and changes in tumor area (length × width) assessed by two-dimensional ultrasound as described in the Experimental Section. Mice were treated with either two intratumoral (IT) injections or one intravenous (IV) injection of unloaded NPs (control) or CT20p-NPs. Graph is representative of at least 4 mice per group. (B) Figures are representative ultrasound images of tumor regression induced by treatment with unloaded or CT20p loaded NPs.