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Abstract：
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with a poor prognosis. Doxorubicin is part of standard curative therapy for TNBC, but chemotherapy resistance remains an important clinical challenge. Bocodepsin (OKI-179) is a small molecule class I histone deacetylase (HDAC) inhibitor that promotes apoptosis in TNBC preclinical models. The purpose of this study was to investigate the combination of bocodepsin and doxorubicin in preclinical TNBC models and evaluate the impact on terminal cell fate, including apoptosis and senescence.
TNBC cell lines were treated with doxorubicin and CellTiter-Glo was used to assess proliferation and determine doxorubicin sensitivity. Select cell lines were treated with OKI-005 (in vitro version of bocodepsin) and doxorubicin and assessed for proliferation, apoptosis as measured by Annexin V/PI, and cell cycle by flow cytometry. Immunoblotting was used to assess changes in mediators of apoptosis, cell cycle arrest, and senescence. Senescence was measured by the senescence-associated β-galactosidase assay. An MDA-MB-231 xenograft in vivo model was treated with bocodepsin, doxorubicin, or the combination and assessed for inhibition of tumor growth. shRNA knockdown of p53 was performed in the CAL-51 cell line and proliferation, apoptosis and senescence were assessed in response to combination treatment.
OKI-005 and doxorubicin resulted in synergistic antiproliferative activity in TNBC cells lines regardless of p53 mutation status. The combination led to increased apoptosis and decreased senescence. In vivo, the combination resulted in increased tumor growth inhibition compared to either single agent. shRNA knock-down of p53 led to increased doxorubicin-induced senescence that was decreased with the addition of OKI-005 in vitro.
The addition of bocodepsin to doxorubicin resulted in synergistic antiproliferative activity in vitro, improved tumor growth inhibition in vivo, and promotion of apoptosis which makes this a promising combination to overcome doxorubicin resistance in TNBC. Bocodepsin is currently in clinical development and has a favorable toxicity profile compared to other HDAC inhibitors supporting the feasibility of evaluating this combination in patients with TNBC.
TNBC cell lines were treated with doxorubicin and CellTiter-Glo was used to assess proliferation and determine doxorubicin sensitivity. Select cell lines were treated with OKI-005 (in vitro version of bocodepsin) and doxorubicin and assessed for proliferation, apoptosis as measured by Annexin V/PI, and cell cycle by flow cytometry. Immunoblotting was used to assess changes in mediators of apoptosis, cell cycle arrest, and senescence. Senescence was measured by the senescence-associated β-galactosidase assay. An MDA-MB-231 xenograft in vivo model was treated with bocodepsin, doxorubicin, or the combination and assessed for inhibition of tumor growth. shRNA knockdown of p53 was performed in the CAL-51 cell line and proliferation, apoptosis and senescence were assessed in response to combination treatment.
OKI-005 and doxorubicin resulted in synergistic antiproliferative activity in TNBC cells lines regardless of p53 mutation status. The combination led to increased apoptosis and decreased senescence. In vivo, the combination resulted in increased tumor growth inhibition compared to either single agent. shRNA knock-down of p53 led to increased doxorubicin-induced senescence that was decreased with the addition of OKI-005 in vitro.
The addition of bocodepsin to doxorubicin resulted in synergistic antiproliferative activity in vitro, improved tumor growth inhibition in vivo, and promotion of apoptosis which makes this a promising combination to overcome doxorubicin resistance in TNBC. Bocodepsin is currently in clinical development and has a favorable toxicity profile compared to other HDAC inhibitors supporting the feasibility of evaluating this combination in patients with TNBC.
摘要：
背景：三阴性乳腺癌（TNBC）是一种侵袭性乳腺癌亚型，预后不良。阿霉素是TNBC标准治疗的一部分，但化疗耐药仍然是一个重要的临床挑战。Bocodepsin（OKI-179）是一种小分子I类组蛋白脱乙酰酶（HDAC）抑制剂，可促进TNBC临床前模型中的细胞凋亡。本研究的目的是研究临床前TNBC模型中博考地辛和阿霉素的组合，并评估其对终末细胞命运的影响。包括细胞凋亡和衰老。
方法：TNBC细胞系用阿霉素处理，CellTiter-Glo用于评估增殖并确定阿霉素敏感性。用OKI-005（体外版本的博科地辛）和阿霉素处理选择的细胞系，并评估其增殖情况。通过膜联蛋白V/PI测量的细胞凋亡，和细胞周期通过流式细胞术。免疫印迹用于评估细胞凋亡介质的变化，细胞周期停滞,和衰老。通过衰老相关的β-半乳糖苷酶测定来测量衰老。MDA-MB-231异种移植体内模型用bocodepsin处理，阿霉素,或组合并评估肿瘤生长的抑制作用。在CAL-51细胞系中进行p53的shRNA敲低，评估细胞凋亡和衰老对联合治疗的反应。
结果：OKI-005和多柔比星在TNBC细胞系中产生协同抗增殖活性，无论p53突变状态如何。该组合导致细胞凋亡增加和衰老减少。在体内,与任一单一药物相比,该组合导致肿瘤生长抑制增加.p53的shRNA敲低导致阿霉素诱导的衰老增加,其在体外添加OKI-005时降低。
结论：在多柔比星中添加博可地辛导致体外协同抗增殖活性，改善体内肿瘤生长抑制，和促进细胞凋亡，使其成为克服TNBC中阿霉素抗性的有希望的组合。Bocodepsin目前处于临床开发中，与其他HDAC抑制剂相比，具有良好的毒性特征，支持在TNBC患者中评估这种组合的可行性。
方法：TNBC细胞系用阿霉素处理，CellTiter-Glo用于评估增殖并确定阿霉素敏感性。用OKI-005（体外版本的博科地辛）和阿霉素处理选择的细胞系，并评估其增殖情况。通过膜联蛋白V/PI测量的细胞凋亡，和细胞周期通过流式细胞术。免疫印迹用于评估细胞凋亡介质的变化，细胞周期停滞,和衰老。通过衰老相关的β-半乳糖苷酶测定来测量衰老。MDA-MB-231异种移植体内模型用bocodepsin处理，阿霉素,或组合并评估肿瘤生长的抑制作用。在CAL-51细胞系中进行p53的shRNA敲低，评估细胞凋亡和衰老对联合治疗的反应。
结果：OKI-005和多柔比星在TNBC细胞系中产生协同抗增殖活性，无论p53突变状态如何。该组合导致细胞凋亡增加和衰老减少。在体内,与任一单一药物相比,该组合导致肿瘤生长抑制增加.p53的shRNA敲低导致阿霉素诱导的衰老增加,其在体外添加OKI-005时降低。
结论：在多柔比星中添加博可地辛导致体外协同抗增殖活性，改善体内肿瘤生长抑制，和促进细胞凋亡，使其成为克服TNBC中阿霉素抗性的有希望的组合。Bocodepsin目前处于临床开发中，与其他HDAC抑制剂相比，具有良好的毒性特征，支持在TNBC患者中评估这种组合的可行性。
