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The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

ABSTRACT: Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ?70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

SUBMITTER: Barbone D

PROVIDER: S-EPMC3169000 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Barbone D D Ryan J A JA Kolhatkar N N Chacko A D AD Jablons D M DM Sugarbaker D J DJ Bueno R R Letai A G AG Coussens L M LM Fennell D A DA Broaddus V C VC

Cell death & disease 20110623

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surp ...[more]

PMID: 21697949

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Project description:Purpose:Peritoneal metastasis is the most common pathway for the spread of ovarian cancer. Ovarian cancer cells in ascites prefer to aggregate into the more chemoresistant multicellular spheroids (MCSs), leading to treatment failure and disease recurrence. We previously established a suspension MCS model of ovarian cancer cells in vitro and found that the MCS cells acquired drug resistance to cisplatin. In the present study, we aimed to uncover the underlying mechanism of the platinum resistance of MCS and the potential targets to reverse the drug resistance. Materials and methods:MCS models were established for the phenotypic studies, including proliferation, invasion, migration, drug resistance, apoptosis assays, and signaling pathway analysis. The key molecule, Bcl-2, was screened by profile analysis and validated by Western blotting. siRNA was used to verify the anti-cisplatin-induced apoptosis effect of Bcl-2. The Bcl-2 inhibitor, ABT-737, was used for improving the sensitivity of MCS to cisplatin. The 50% inhibitory concentrations (IC50) were measured by viability assays treated with different concentrations of cisplatin. Flow cytometry and Western blotting were used for quantification of drug-induced apoptosis. Results:The ovarian cancer MCS showed a proliferation-stagnant but invasive phenotype when resuspended. When treated with cisplatin, MCS cells showed much higher viability, with significantly fewer apoptotic cells than the adherent cells. Levels of Bcl-2 were upregulated in ovarian cancer ascitic cells and MCS cells. Bcl-2 knockdown by siRNA or blockage by ABT-737 enhanced the cisplatin-induced apoptosis and reduced the 50% inhibitory concentrations of cisplatin for MCS by 58.5% and 88.2%, respectively. Conclusion:The upregulated Bcl-2 contributes to cisplatin resistance in our MCS model and targeting it sensitizes the MCS to cisplatin treatment. This provides us a preliminary treatment method for ovarian cancer peritoneal metastasis.

Project description:Uveal melanoma (UM) has a high propensity for metastatic spread, and approximately 40-50% of patients die of metastatic disease. Metastases can be found at the time of diagnosis but also several years after the tumor has been removed. The survival of disseminated cancer cells is known to be linked to anchorage independence, anoikis resistance, and an adaptive cellular metabolism. The cultivation of cancer cells as multicellular tumor spheroids (MCTS) by anchorage-independent growth enriches for a more aggressive phenotype. The present study examines the differential gene expression of adherent cell cultures, non-adherent MCTS cultures, and uncultured tumor biopsies from three patients with UM. We elucidate the biochemical differences between the culture conditions to find whether the culture of UM as non-adherent MCTS could be linked to an anchorage-independent and more aggressive phenotype, thus unravelling potential targets for treatment of UM dissemination.The various culture conditions were evaluated with microarray analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNAscope, immunohistochemistry (IHC), and transmission electron microscopy (TEM) followed by gene expression bioinformatics.The MCTS cultures displayed traits associated with anoikis resistance demonstrated by ANGPTL4 upregulation, and a shift toward a lipogenic profile by upregulation of ACOT1 (lipid metabolism), FADS1 (biosynthesis of unsaturated fatty acids), SC4MOL, DHCR7, LSS (cholesterol biosynthesis), OSBPL9 (intracellular lipid receptor), and PLIN2 (lipid storage). Additionally, the present study shows marked upregulation of synovial sarcoma X breakpoint proteins (SSXs), transcriptional repressors related to the Polycomb group (PcG) proteins that modulate epigenetic silencing of genes.The MCTS cultures displayed traits associated with anoikis resistance, a metabolic shift toward a lipogenic profile, and upregulation of SSXs, related to the PcG proteins.

Dataset Information

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Publications

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

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