Histone deacetylase inhibitor ITF2357 leads to apoptosis and enhances doxorubicin cytotoxicity in preclinical models of human sarcoma

Abstract

Sarcomas are rare tumors with generally poor prognosis, for which current therapies have shown limited efficacy. Histone deacetylase inhibitors (HDACi) are emerging anti-tumor agents; however, little is known about their effect in sarcomas. By using established and patient-derived sarcoma cells with different subtypes, we showed that the pan-HDACi, ITF2357, potently inhibited in vitro survival in a p53-independent manner. ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by induction of pro-apoptotic BH3-only proteins and a caspases-dependent mechanism. ITF2357 also induced autophagy, which protected sarcoma cells from apoptotic cell death. ITF2357 activated forkhead box (FOXO) 1 and 3a transcription factors and their downstream target genes, however, silencing of both FOXO1 and 3a did not protect sarcoma cells against ITF2357-induced apoptosis and upregulated FOXO4 and 6. Notably, ITF2357 synergized with Doxorubicin to induce cell death of established and patient-derived sarcoma cells. Furthermore, combination treatment strongly impaired xenograft tumor growth in vivo, when compared to single treatments, suggesting that combination of ITF2357 with Doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma. Overall, our study highlights the therapeutic potential of ITF2357, alone or in rational combination therapies, for bone and soft tissue sarcomas management.

Fig. 1. ITF2357 affects survival of human sarcoma cells.

a Analysis of cell viability by MTT assay in the indicated sarcoma cell lines treated for 72 h with increasing concentrations of ITF2357. The results are reported as “viability of drug-treated cells/viability of control cells” × 100 and represent the mean ± SD of three independent experiments performed in triplicate. b Colony formation of HT1080 cells treated for 24 h with increasing concentrations of ITF2357. Representative images are shown. c Western blot analysis of acetylated histone H3 (Ac-H3), acetylated α-Tubulin (Ac-Tubulin) in total cell lysates from the indicated cell lines treated with 0.1 and 0.5 μM ITF2357 for 24 h. d Representative experiment of apoptotic cells evaluation by AnnexinV/PI staining in the indicated cell lines exposed to increasing concentration of ITF2357 for 72 h. e Quantification of apoptosis by AnnexinV/PI staining in the indicated cell lines treated for 72 h with increasing concentrations of ITF2357. The results represent the mean ± SD of three independent experiments. f Western blot analysis of cleaved form of PARP (PARP cl.) in total cell lysates from HT1080 cells treated with 1 and 5 μM ITF2357 for 72 h. c, f HSP72/73 expression was used as loading and transferring control. Western blots representative of two independent experiments with similar results are shown. g Flow cytometric analysis of active caspase-3-PE staining in HT1080 cells untreated or treated for 72 h with 1 μM ITF2357 alone or in combination with 50 μM pan-caspase inhibitor zVAD-fmk (zVAD). A representative experiment out of two is shown. a, e p values were calculated between untreated and treated cells, *p < 0.01

Fig. 2. ITF2357 activates mitochondrial apoptosis.

a Western blot analysis of the indicated BH3-only pro-apoptotic proteins in total cell lysates from the HT1080 cell line treated with 1 and 5 μM ITF2357 for 48 h. b–d Analysis of the indicated mRNA expression evaluated by qRT-PCR in HT1080 b, SW872 c, and SaOS2 d cell lines exposed to 1 and 5 μM ITF2357 for 48 h. Results are presented as the mean ± SEM of at least two independent experiments. p values were calculated between untreated and treated cells, *p < 0.05. e Western blot analysis of p53 protein expression in total cell lysates from the indicated cell lines treated for 48 h with ITF2357 (1 μM). As positive control of p53 induction, cells were exposed to Doxorubicin (Doxo, 0.1 μM, 24 h). a, e HSP72/73 expression was used as loading and transferring control. Western blots representative of two independent experiments with similar results are shown

Fig. 3. ITF2357 induces a canonical autophagic process.

a Representative images of autophagosomal structures by fluorescence microscopy in HT1080 cells stably transfected with EGFP-LC3B vector (HT1080/EGFP-LC3) and treated with 1 and 5 μM ITF2357 for the indicated times. b Quantification of cells positive for punctate autophagosomal structures treated as indicated in a. The results represent the mean ± SEM of three independent experiments. c Western blot analysis of microtubule-associated protein 1 (LC3B-I/II) conversion and of p62/SQSTM1 protein expression in total cell lysates from HT1080 cell line treated with increasing concentration of ITF2357 for 24 h. Western blots representative of two independent experiments with similar results are shown. d Analysis of the indicated mRNA expression evaluated by qRT-PCR in HT1080 exposed to ITF2357 (1 and 5 μM for 24 h). Results are presented as the mean ± SEM of at least two independent experiments. p values were calculated between untreated and treated cells, (*p < 0.05). e Analysis of cell death evaluated by PI staining in HT1080 cells exposed to ITF2357 for 48 h in absence or presence of Chloroquine (CQ), a late stage autophagy inhibitor, *p < 0.05. The results represent the mean ± SD of three independent experiments

Fig. 4. ITF2357 induces FOXOs protein expression and transcriptional activity.

a Analysis of FOXO1 and FOXO3 mRNA expression evaluated by qRT-PCR in the indicated cell lines treated for 24 h with 1 and 5 μM ITF2357. b Representative images of immunofluorescence for FOXO1 and FOXO3 proteins in HT1080 cells after treatment with 1 μM ITF2357. c qRT-PCR analysis of the indicated mRNA expression in HT1080 cells treated for 24 h with 1 and 5 μM ITF2357. a, c Results are presented as the mean ± SEM of two independent experiments. p values were calculated between untreated and treated cells, *p < 0.05

Fig. 5. Critical role of FOXOs protein in ITF2357-induced apoptosis.

a Western blot analysis of FOXO1 and FOXO 3a protein expression in total cell lysates from HT1080 cells after 48 h from trasfection with siRNA-mediated knockdown of FOXO1 and FOXO3a, alone (siFOXO1 and siFOXO3) and in combination (siFOXO1/3) or with a control sequence (siScr). β-actin expression was used as loading and transferring control. Western blots representative of two independent experiments with similar results are shown. b Flow cytometric analysis (left panel) and quantification (right panel) of apoptotic cells by AnnexinV/PI staining HT1080 transfected with the indicated siRNAs (siScr, siFOXO1, siFOXO3a, and siFOXO1/3) and exposed to 1 µM ITF2357 for 48 h. The results represent the mean ± SD of three independent experiments. c qRT-PCR analysis of the indicated mRNA expression in HT1080 transfected with the indicated siRNAs (siScr, siFOXO1, siFOXO3a, and siFOXO1/3) and exposed to 1 µM ITF2357 for 48 h. p values were calculated between untreated and treated cells, *p < 0.05

Fig. 6. ITF2357 sensitizes human sarcoma cells to Doxorubicin treatment.

a, b Analysis of cell viability by MTT assay in the indicated established a and patient-derived b cell lines treated with ITF2357 and Doxorubicin (Doxo, drug ratio 5:1) alone or in combination. The results are reported as “viability of drug-treated cells/viability of control cells” × 100 and represent the mean ± SD of three independent experiments performed in triplicate. c Cytofluorimetric analysis of apoptotic cells in the indicated cell lines treated with ITF2357 (0.5 μM) and Doxo (0.1 μM) alone or in combination for 24, 48, and 72 h. The results are reported as mean ± SD of three independent experiments

Fig. 7. ITF2357 reduces tumor growth and potentiates Doxorubicin effect in vivo.

a In vivo response of SW982 xenograft to vehicle (daily), Doxorubicin (Doxo, 2.2 mg/kg per week) or ITF2357 (100 mg/kg per day for 4 days) alone, or in combination (weekly Doxo followed by daily ITF2357 for 4 days). Treatments lasted 4 weeks. b Representative images of immunohistochemical detection of apoptosis by TUNEL assay of tumor xenografts treated as reported in a