Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells

Abstract

Background: Translationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive.

Methods: Cell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP.

Results: In the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade.

Conclusions: TCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve as a therapeutic target for chemoresistance in cancer treatment.

Figure 1

TCTP inhibits anticancer drug-induced cell death and cleavage of EGFR and PLC-γ in HeLa cells. (A) TCTP-induced inhibition of cytotoxic drug-induced cell death. AdNull- and adTCTP-infected HeLa cells (MOI, 10) were incubated with 20 μM etoposide or 0.1 μM taxol and then DNA fragmentation was analyzed using PI staining and FACS as described in Materials and Methods. (B) TCTP-induced inhibition of cytotoxic drug-induced EGFR and PLC-γ fragmentation. After treatment of 20 μM etoposide or 0.1 μM taxol, adGFP (G)- and adTCTP-GFP (T)-infected HeLa cell (MOI, 10) extracts were blotted with anti-EGFR, -PLC- γ, and -GFP antibodies.

Figure 2

TCTP inhibits mitochondrial cytochrome c release and mitochondrial membrane depolarization in etoposide-induced cell death. (A) TCTP-induced inhibition of mitochondrial membrane perturbation. AdNull and adTCTP-infected cells (MOI, 10) were treated with 20 μM etoposide. Loss of mitochondrial membrane potential was measured using JC-1 dye by flow cytometry. The disrupter, CCCP was used as a positive control. JC-1 forms aggregates in the high mitochondrial membrane potential whereas the disrupted potential during apoptosis leads to form the JC-1 monomer. Loss of membrane potential was detected by measuring the shift of fluorescence from FL-2 (JC-1 aggregates, red fluorescence) to FL-1 (JC-1 monomers, green fluorescence) in FACS analysis. (B) TCTP-induced inhibition of mitochondrial cytochrome c release. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide. After fractionation of mitochondrial and cytosolic fractions, anti-cytochrome c antibodies were used for detecting its contents by western blot analysis. (C) Cytochrome c release was quantitated by expressing as RFU. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide. Following incubation with fluorescence-tagged cytochrome c-specific antibody, cells were subjected to FACS analysis. Data represent cytochrome c release relative to the control (mean ± S.D.) of two independent experiments.

Figure 3

TCTP inhibits caspase activation in etoposide-induced cell death. (A) TCTP-induced inhibition of caspase-9, -7, and -3 fragmentation. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide and the activation of caspases were determined by western blot assay using cleaved form-specific caspase-9, -7, and -3, and PARP antibodies (left panel). Cleavage of procaspase-9 was further assayed using specific antibodies detecting 35- and 37-kDa form of cleaved caspase-9 (right panel). (B) TCTP-induced inhibition of caspase-9 and -3 cleavages under in vitro assays of apoptosome reconstitution. S-100 extracts were incubated with dATP/cyto c to induce the apoptosome formation. Activation of caspase-9 and -3 was detected by western blotting in the presence or absence of TCTP in the reaction. (C) TCTP-induced inhibition of caspase-9 and -3 activities confirmed by inhibitor assay. The specific inhibitor of caspase-9 and -3, Ac-LEHD and Ac-DEVD, respectively, were used. Caspase activity was presented as RFU by using fluorogenic substrates for caspases. Error bar represent SD of two independent experiments. (D) Time- and dose-dependence of TCTP-induced inhibition of caspase-9 and -3 fragmentations. Following dose- and time-dependent incubation with His-tagged TCTP at a time of 30 min and at a dose of 1 μg/ml, respectively, western blot assay was performed to detect caspase activity using anti-Apaf-1, -cleaved caspase-9, -cleaved caspase-3, and -His-specific antibodies.

Figure 4Figure 4

TCTP interacts with CARD of Apaf-1 to form an apoptosome complex. (A) Binding of TCTP to Apaf-1 in apoptosome-forming condition. TCTP, procaspase-9, and cytochrome c binding to Apaf-1 was analyzed. Recombinant human TCTP, cytochrome c, dATP and S-100 extract were incubated, immunoprecipitated with anti-Apaf-1-specific antibody and then blotted with antibodies, as described in Methods. (B) Interaction of TCTP with CARD of Apaf-1. Schematic diagram of full-length Apaf-1 and its variants devoid of particular domain(s) was presented. The full-length Apaf-1 (residues 1-1194) contains CARD (residues 1-97), CED-4 (residues 98-412), and WD-40 repeats (residues 413-1194). Recombinant Full APAF-1, APAF-530 (residues 1-530), APAF-420 (residues 1-420), APAF-97 (residues 1-97), and TCTP-GST (GST-tagged TCTP) were constructed. Purified WDR (WD Repeat) protein lacking both CARD and CED-4 was obtained from Abnova Corporation. Binding of TCTP-GST and His-tagged Apaf-1 constructs was analyzed using pull-down assay. Protein binding complexes were separated on SDS-PAGE and stained with silver. Purified TCTP-GST protein was also resolved by SDS-PAGE for detection of TCTP (last lane). NC, negative control.

Figure 5

Cleaved TCTP specifically binds to Apaf-1 during apoptosis. (A) Full-length TCTP binding to Na,K-ATPase α1 subunit in TCTP-overexpressing HeLa cells in etoposide-induced cell death. Following treatment with 20 μM etoposide, Flag-tagged adNull- and adTCTP-infected cell extracts (MOI, 10) were immunoprecipitated with anti-Na,K-ATPase α1-specific antibodies and blotted with anti-Flag-specific antibody. (B) Fragmented TCTP binding to Apaf-1 in etoposide-induced cell death. Following treatment with 20 μM etoposide, Flag-tagged adNull- and adTCTP-infected cell (MOI, 10) extracts were immunoprecipitated with anti-Apaf-1-specific antibody and blotted with anti-Flag antibody.