Cucurbitacin-I inhibits Aurora kinase A, Aurora kinase B and survivin, induces defects in cell cycle progression and promotes ABT-737-induced cell death in a caspase-independent manner in malignant human glioma cells

Abstract

Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; MTS,[3][4]]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H, tetrazolium; FITC, fluorescein isothiocyanate; NF-kB, nuclear factor kB; PI3K, Phosphatidylinositol 3-Kinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PDGFR, platelet derived growth factor receptor; TBS, Tris-buffered saline; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; PI, propidium iodide.Because STAT signaling is commonly activated in malignant gliomas as a result of constitutive EGFR activation, strategies for inhibiting the EGFR/JAK/STAT cascade are of significant interest. We, therefore, treated a panel of established glioma cell lines, including EGFR overexpressors, and primary cultures derived from patients diagnosed with glioblastoma with the JAK/STAT inhibitor cucurbitacin-I. Treatment with cucurbitacin-I depleted p-STAT3, p-STAT5, p-JAK1 and p-JAK2 levels, inhibited cell proliferation, and induced G2/M accumulation, DNA endoreduplication, and multipolar mitotic spindles. Longer exposure to cucurbitacin-I significantly reduced the number of viable cells and this decrease in viability was associated with cell death, as confirmed by an increase in the subG1 fraction. Our data also demonstrated that cucurbitacin-I strikingly downregulated Aurora kinase A, Aurora kinase B and survivin. We then searched for agents that exhibited a synergistic effect on cell death in combination with cucurbitacin-I. We found that cotreatment with cucurbitacin-I significantly increased Bcl -2/Bcl -xL family member antagonist ABT-737-induced cell death regardless of EGFR/PTEN/p53 status of malignant human glioma cell lines. Although >50% of the cucurbitacin-I plus ABT-737 treated cells were annexin V and propidium iodide positive, PARP cleavage or caspase activation was not observed. Pretreatment of z-VAD-fmk, a pan caspase inhibitor did not inhibit cell death, suggesting a caspaseindependent mechanism of cell death. Genetic inhibition of Aurora kinase A or Aurora kinase B or survivin by RNA interference also sensitized glioma cells to ABT-737, suggesting a link between STAT activation and Aurora kinases in malignant gliomas.

STAT3 inhibition suppresses hepatic stellate cell fibrogenesis: HJC0123, a potential therapeutic agent for liver fibrosis

“…Previous studies examining apoptosis after STAT3 inhibition in malignant glioma cells found neither PARP cleavage nor caspase activation while caspase inhibitor treatment did not affect cell death index, suggesting mechanisms of caspase-independent apoptosis. 41 Chen et al recently described caspase-independent apoptosis in HSCs while studying the antifibrogenic properties of Saikosaponin d. 42 Although we did not fully explore the involved apoptotic mechanisms, taking together with previous literature, our data suggest that HJC0123 treatment and subsequent STAT3 inhibition might lead to caspase-independent apoptosis.…”

Cell-based phenotypic screening of mast cell degranulation unveils kinetic perturbations of agents targeting phosphorylation

“…A previous study shows that 150 nM of JSI124 can effectively block Stat3 signaling and initiate apoptosis in CD133-positive NSCLC cells 35 . JSI124 induces suppression of serine 727 phosphorylation of Stat3, leading to cell-cycle arrest through alterations in gene transcription and promoting cell death in a caspase-independent manner in many normal and cancer cells 36 37 38 . Of note, different levels of p-Stat3 in cells may have an influential role in dissecting two different cell deaths when treating them with JSI124.…”

Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737-Mediated Cell Death in Malignant Human Glioma Cell Lines

“…Genetic features (Table 1) of these glioma cell lines have been characterized elsewhere (Furnari et al, 1997;Weller et al, 1998). Cell culture conditions of these cell lines were as previously described (Nagane et al, 1996;Premkumar et al, 2015). Cell lines used in this study were authenticated using short tandem repeat (STR) analysis by ATCC cell line authentication service.…”