Toxoplasma gondii GRA8-derived peptide immunotherapy improves tumor targeting of colorectal cancer

Abstract

Targeted tumor and efficient, specific biological drug delivery in vivo has been one of the main challenges in protein-based cancer-targeted therapies. Mitochondria are potential therapeutic targets for various anti-cancer drugs. We have previously reported that protein kinase Cα-mediated phosphorylation of Toxoplasma gondii GRA8 is required for mitochondrial trafficking and regulating the interaction of the C-terminal of GRA8 with ATP5A1/SIRT3 in mitochondria. Furthermore, SIRT3 facilitates ATP5A1 deacetylation, mitochondrial activation, and subsequent antiseptic activity in vivo. Herein we developed a recombinant acidity-triggered rational membrane (ATRAM)-conjugated multifunctional GRA8 peptide (rATRAM-G8-M/AS) comprising ATRAM as the cancer-targeting cell-penetrating peptide, and essential/minimal residues for mitochondrial targeting or ATP5A1/SIRT3 binding. This peptide construct showed considerably improved potency about cancer cell death via mitochondria activity and biogenesis compared with rGRA8 alone in HCT116 human carcinoma cells, reaching an IC₅₀ value of up to 200-fold lower in vitro and 500-fold lower in vivo. Notably, rATRAM-G8-M/AS treatment showed significant therapeutic effects in a mouse xenograft model through mitochondrial metabolic resuscitation, and it produced negligible immunogenicity and immune responses in vivo. Thus, these results demonstrate that rATRAM-G8-M/AS represents a useful therapeutic strategy against tumors, particularly colon cancer. This strategy represents an urgently needed paradigm shift for therapeutic intervention.

Keywords: Toxoplasma gondii GRA8 peptide; metabolism; mitochondria; tumor-targeting.

Figure 1. The rGRA8 treatment increases the HCT116 cell death by mitochondria pathways.

(A and B) The various cancer cells were incubated with WT rGRA8 (20 μg/ml) and its mutants for the indicated times and then cell viability measured with MTT assay (upper) and subjected to IB with αATP5A1, αSIRT3, or αActin (lower). (C) HCT116 cells were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-PKCα or SIRT3 (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days. The cells were incubated with WT rGRA8 for the indicated times and subjected to IP with αLys-AC or αHis and IB with αATP5A1, αATP5C1, αSIRT3, αPGC-1, αPKCα, or αActin. The data are representative of five independent experiments with similar results. Statistical significance was determined by Student’s t-test with Bonferroni adjustment; ^** P < 0.01, ^*** P < 0.001 compared with rVector-treated.

Figure 2. Design and expression of ATRAM-conjugated multifunctional GRA8 peptide-based protein.

(A) Subcellular fractionation of 293T cells are expressing GST-GRA8 WT or incubation with WT rGRA8 (1 μg/ml) BMDMs and treated with several Tat-GRA8 peptide (10 µM) for 6 h. Mitochondrial and cytosolic fractions were fractionated and analyzed for expression of GST by IB. Purity of the fractions was assessed by blotting for VDAC (mitochondrial protein) and Actin (cytosolic protein). (B) At 12 hr post-transfection with mammalian GST-GRA8 constructs together with V5-ATP5A1, or Flag-SIRT3 and 293T cells treated with several Tat-GRA8 peptide (10 µM) for 6 h. 293T cells were used for GST pulldown, followed by IB with αV5 or αFlag. WCLs were used for IB with αGST, αV5, αFlag or αActin. (C) Schematic in design of ATRAM-GRA8-M/AS and its mutants. (D) Bacterially purified 6xHis-rATRAM-GRA8-M/AS and its mutants were analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right). (E) BMDMs were incubated with rATRAM-GRA8-M/AS and its mutants (20 μg/ml) for the indicated times and then cell viability measured with MTT assay. The data are representative of four independent experiments with similar results (A, B, D, and E).

Figure 3. The rATRAM-GRA8-M/AS-induced the HCT116 cell death by mitochondria activation.

(A and B) HCT116 cells were incubated with WT rGRA8 or rATRAM-GRA8-M/AS and its mutants for the indicated times and then cell viability measured with MTT assay. (C–E) HCT116 cells were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-PKCα or SIRT3 (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days. The cells were stimulated with rATRAM-GRA8-M/AS for 1 day and subjected to enzymatic activity of OXPHOS V (C), Mitotracker fluorescence signals assessed by a flow cytometric analysis. Bar graph indicates the mitochondrial mass MFIs (D), or mtDNA content in BMDMs measured by quantitative real-time PCR. The mtDNA content was normalized to nuclear DNA (E). Statistical significance was determined by Student’s t-test with Bonferroni adjustment; ^** P < 0.01, ^*** P < 0.001 compared with control-treated. The data are representative of five independent experiments with similar results.

Figure 4. The rATRAM-GRA8-M/AS showed an antitumor activity in HCT116 xenografts.

(A and B) Schematic of the xenograft model treated with or rATRAM-GRA8-M/AS or its mutants (upper). HCT116 cells were subcutaneously injected into the flanks of nude mice. The length and width of the tumors were measured using calipers, and the tumor volume was calculated every third day for 30 days. At day 15 after administration, the mice were euthanatized, and the interaction with GRA8, levels of acetylation of ATP5A1, or expression of OXPHOS were analyzed (A, lower). Representative images of tumors from mice treated with rATRAM-GRA8-M/AS or mutants on day 15 (right). (C–F) HCT116 cells were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-PKCα, SIRT3, or ATP5A1 with polybrene (C–E) or HCT116-expressed ATP5A1 WT and mutant cells were subcutaneously injected into the flanks of nude mice. Individual tumor volumes from each mouse from each group were averaged and plotted against the number days postinoculation. Statistical significance was determined by two-way analysis of variance (ANOVA) with Tukey’s posttest; ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 compared with rVector. Each group contained ten mice. The data are representative of two independent experiments with similar results.