Tumor-targeted delivery of a C-terminally truncated FADD (N-FADD) significantly suppresses the B16F10 melanoma via enhancing apoptosis

Abstract

Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182-205 aa) named N-FADD (m-FADD, 1-181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma.

Figure 1. Alignment of FADD proteins from different species and diagram of N-FADD.

(A) Alignment of FADD proteins from several different species. Only the C-terminal regions are shown to highlight the conservation of the known phosphorylation sites, ser 191 of mouse FADD (equivalent to Ser194 of human FADD), and the other two putative ones, Ser 187, Ser 202, are indicated by asterisks to highlight their conservation in mammals. The residue numbers are based on the corresponding amino acid residue position in the mouse FADD protein. All of the FADD protein sequences were obtained from NCBI and aligned by Clustalw. (B) FADD and N-FADD of mouse. The numbers indicate the amino acid residue position in the mouse FADD protein.

Figure 2. Overexpression of FADD or N-FADD induced apoptosis of B16F10 melanoma cells in vitro.

(A–C) Representative FACS analysis of Annexin V and propidium iodide (PI) staining after transfection of FADD or N-FADD expressing or empty vectors for 16 h. (D–F) Representative FACS analysis of Annexin V and PI staining after transfection of FADD or N-FADD expressing or empty vectors for 24 h. (G) Overexpression of FADD or N-FADD induced apoptosis of B16F10 melanoma cells (Mean ± SD, n = 3 independent experiments); **P < 0.01. (H) Western blots analysis of expression of FADD after transfection of FADD or N-FADD expressing or empty plasmids in B16F10 cells. β-actin was served as loading control. (I) Statistical analysis of western blot by Image J; **P < 0.01, compared with pcDNA3.1. One representative of three independent experiments is displayed.

Figure 3. Overexpression of FADD or N-FADD in B16F10 melanoma cells activation of caspase-3.

(A) Representative immunofluorescence staining for cleaved caspase-3 (green) after transfection of FADD or N-FADD expressing or empty vectors for 16 h. (B) Representative immunofluorescence staining for cleaved caspase-3 (green) after transfection of FADD or N-FADD expressing or empty vectors for 24 h. (C) Quantification analysis of relative cleaved caspase3 expression level (fluorescence intensity of cleaved caspase3/DAPI) by Image J software after transfecting FADD or N-FADD expressing or empty vectors, *P < 0.05, **P < 0.01. Data are expressed as mean ± SD of three independent experiments.

Figure 4. FADD and N-FADD delivered by S. typhimurium strain VNP20009 expressed in vitro and in vivo under the control of NirB promoter.

(A) pQE30-NirB-FADD and pQE30-NirB-N-FADD plasmids were constructed and transformed into VNP (VNP-pN-FADD and VNP-pN-N-FADD). VNP-pN-FADD and VNP-pN-N-FADD were grown for 72 h under anaerobic or aerobic conditions. Bacterial lysates were subjected to immunoblotting assays using anti-FADD (N term) antibodies (B). (C) The relative tissue distribution of indicated stable strains was detected by colony formation assay. *p < 0.001, tumor compared with the other tissues. Data are expressed as mean ± SD of five animals. (D) Detection of the expression of FADD or N-FADD in tumor tissues of the mice bearing melanoma by western blotting. β-actin was served as loading control. (E) Statistical analysis the results of western blot by Image J; **P < 0.01, compared with PBS group. One representative of three independent experiments is displayed.

Figure 5. VNP-pN-N-FADD delayed tumor growth and enhanced survival time in B16F10 mice model.

(A) Tumor growth curves of groups as indicated. B16F10 tumor mice per group (n = 10 mice) were injected i.p. with 2 × 10⁵ cfu of VNP, VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD or with PBS at day 7. Tumor volumes among different groups were compared. Data are presented as mean ± SD. *P < 0.05 for VNP-pN-FADD versus VNP or VNP-pN; **P < 0.01 for VNP-pN-N-FADD versus VNP-pN-FADD; ***P < 0.001 for VNP-pN-N-FADD versus VNP or VNP-pN. (B) Kaplan-Meier survival curves of mice bearing B16F10 melanomas. Data were analyzed by the log-rank test. *P < 0.05 for VNP-pN-N-FADD versus PBS, VNP, VNP-pN and VNP-pN-FADD. (C,D) Representative therapeutic efficacy of the recombinant VNP strains for melanoma therapy. (E,F) Tumor doubling time and growth delay. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, (n = 10 mice). (G) Immunofluorescence staining of FADD (stained red), the representative images (100×) revealing FADD or N-FADD expression in B16F10 tumor tissue treated with PBS, VNP, VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD.

Figure 6. Salmonella typhimurium strain VNP20009 carrying FADD or N-FADD induced apoptosis of melanoma cells.

(A) H&E staining of the tumor sections. The representative images (100×) revealing necrotic areas of B16F10 tumor tissue treated with PBS, VNP, VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD; N, necrotic tumor regions; V, vital tumor regions. (B) TUNEL assay of melanoma. The representative images (100×) revealing apoptosis of tumor cells treated with appropriate VNP strains; bright red, apoptotic cells. (C) Immunofluorescence staining of cleaved caspase-3 (stained green and indicated with blank arrows), the representative images (200×) revealing cleaved caspase-3 in B16F10 tumor tissue treated with PBS, VNP, VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD. (D) Quantification analysis of relative cleaved caspase-3 expression level (fluorescence intensity of cleaved caspase-3 DAPI) by Image J software, *P < 0.05, **P < 0.01, bar represents the mean ± SD of five optical fields. Each experiment was carried out in triplicate.