The palladacycle complex AJ-5 induces apoptotic cell death while reducing autophagic flux in rhabdomyosarcoma cells

Abstract

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anti-cancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.

Galenia africana plant extract exhibits cytotoxicity in breast cancer cells by inducing multiple programmed cell death pathways

“…Cells were seeded at 2.5–3.0 × 10 5 cells per 6 cm dish and treated with vehicle or KB2 for 24 h. Cells were then trypsinized, washed with PBS and re-plated at low densities (800–2000 cells per 35 mm dishes) and allowed to form colonies for 7–21 days depending on the cell line. The clonogenic assays were performed as described by Bleloch et al (2019) .…”

“…Cells were seeded in 12 well plates and allowed to reach 100% confluency and a scratch motility assay was performed as described by Bleloch et al (2019) . On the day that the scratch was made, the cells were treated with vehicle or KB2 and the wound was monitored and photographed using the Evos® XL Core microscope (Thermo Fisher Scientific, Massachusetts, USA) at 3 h intervals for up to 24 h. The areas of the wounds were measured using the ImageJ software.…”

“…The cells were treated with vehicle or KB2 for 48 h and processed as previously described ( Bleloch et al, 2019 ). The cell cycle profiling was done using the BD FACS Calibur flow cytometer (Becton Dickinson, New Jersey, USA) with a 488 nm Coherent laser (Santa Clara, California, USA).…”

“…The cells were treated with vehicle or KB2 for 24 h and 48 h. The cells were lysed in whole-cell lysis buffer and western blotting was carried out as previously described ( Bleloch et al, 2019 ). The primary antibodies used in this study were, rabbit polyclonal antibodies to phospho-histone H2A.X (Ser139) (#2577), cleaved caspase-3 (Asp175) (#9661), PARP (#9542), caspase-9 (#9502), LC3II (#2775), rabbit monoclonal antibody to β-Catenin (D10A8) (#8480), cleaved caspase-7 (Asp198) (D6H1) (#8438), mouse monoclonal antibodies to E-cadherin (4A2) (#14472), vimentin (R28) (#3932), Cyclin B1 (V152) (#4135), Caspase-8 (1C12) (#9746) from Cell Signaling Technology (Massachusetts, USA); mouse monoclonal antibody to p53 (DO-1) (sc-126), rabbit polyclonal antibodies to p21 (C-19) (sc-397), Cyclin A (H-432) (sc-751) from Santa Cruz Biotechnology (Texas, USA); rabbit polyclonal antibody to p38 MAP kinase (M0800) from Sigma Aldrich.…”

“…Cells were treated with vehicle or KB2 for 24 h or 48 h and immunocytochemistry and analyses thereof carried out as previously described ( Bleloch et al, 2019 ). Briefly, before treatment, cells were seeded on glass coverslips in 35 mm dishes.…”