JQ1 is a bromo- and extraterminal (BET) domain inhibitor that downregulates MYC expression and impairs the DNA damage response. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent DNA damage sensing and repair. We hypothesized that JQ1 would promote a DNA repair-deficient phenotype that sensitizes neuroblastoma cells to PARP inhibition.
Four human neuroblastoma cell lines were examined: two MYCN-amplified (BE(2)-C and IMR-32), and two non-MYCN-amplified (SK-N-SH and SH-SY5Y). Cells were treated with JQ1 (BET inhibitor), Olaparib (PARP inhibitor), or in combination to assess for therapeutic synergy of JQ1 and Olaparib. Treated cells were harvested and analyzed. Quantitative assessment of combination treatment synergy was performed using the median effect principle of Chou and Talalay.
Combination treatment with Olaparib decreased the IC50 of JQ1 by 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C, IMR-32, SK-N-SH, and SH-SY5Y cell lines, respectively. In the MYCN-amplified cell lines, BE(2)-C and IMR-32, combination treatment decreased gene expression of MYCN relative to single-drug treatment alone or control. Combination treatment decreased protein expression of DNA repair proteins Ku80 and RAD51, led to accumulation of DNA damage marker phospho-histone H2A.X, and increased caspase activity. In the non-MYCN-amplified cell lines, SK-N-SH and SH-SY5Y, combination treatment induced G0/G1 cell cycle arrest.
Combination BET and PARP inhibition synergistically inhibited neuroblastoma tumorigenesis in vitro. In MYCN-amplified neuroblastoma cells, this effect may be induced by downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest.
© 2022. The Author(s).

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is frequently associated to high treatment resistance. Gemcitabine (GEM) alone or in combination is the most used chemotherapy for unresecable PDACs. Here we studied whether modulated electro-hyperthermia (mEHT), a non-invasive complementary treatment, can support the effect of GEM on PDAC cells in vitro. The LD20 for the GEM-resistant Panc1 cells proved to be 200× higher than for the drug-sensitive Capan1. The mEHT alone caused significant apoptosis in Capan1 cultures as confirmed by the elevated SubG1 phase cell fraction and increased number of cleaved Caspase-3 positive cells 48 h after treatment, with an additive effect when GEM was used after hyperthermia. These were accompanied by reduced number of G1, S, and G2/M phase cells and elevated expression of the cyclin-dependent kinase inhibitor p21waf1 protein. In GEM-resistant Panc1 cells, an initial apoptosis was detected by flow cytometry 24 h after mEHT ± GEM treatment, which however diminished by 48 h at persistent number of cleaved Caspase-3 positive tumor cells. Though GEM monotherapy reduced the number of tumor progenitor colonies in Capan1 cell line, an additive colony inhibitory effect of mEHT was observed after mEHT + GEM treatment. The heat shock induced Hsp27 and Hsp70 proteins, which are known to sensitize PDAC cells to GEM were upregulated in both Capan1 and Panc1 cells 24 h after mEHT treatment. The level of E-Cadherin, a cell adhesion molecule, increased in Capan1 cells after mEHT + GEM treatment. In conclusion, in GEM-sensitive PDAC cells mEHT treatment alone induced cell death and cell cycle inhibition and improved GEM efficiency in combination, which effects were milder and short-term up to 24 h in the GEM-resistant Panc1 cells. Our data further support the inclusion of hyperthermia, in particular of mEHT, into the traditional oncotherapy regimens of PDAC.
Copyright © 2021 Forika, Kiss, Petovari, Danko, Gellert and Krenacs.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.
Copyright © 2017 Elsevier Inc. All rights reserved.

Autophagy and senescence are both well-established responses to chemotherapy and radiation that often occur in parallel, contributing to growth arrest in tumor cells. However, it has not been established whether this growth arrest is reversible. This question was addressed using non-small cell lung cancer models exposed to the cancer chemotherapeutic drug, etoposide. Senescent cells that were sorted, identified by β-galactosidase staining and alterations in morphology, isolated by flow cytometric cell sorting based on C 12 FDG staining, and real-time live microscopy were found to be capable of recovering proliferative capacity. Autophagy, monitored by vacuole formation, SQSTM1/p62 degradation, and LC3BII generation did not interfere with either the senescence arrest or proliferative recovery and was nonprotective in function (i.e. autophagy inhibition via both pharmacological and genetic strategies had negligible impact on the response to etoposide). These observations argue against the premise that (chemotherapy-induced) senescence is irreversible and indicate that therapy-induced senescence may ultimately be a transient process in that at least a subpopulation of tumor cells can and will remain metabolically active and recover proliferative capacity independently of autophagic turnover. We therefore propose that dormant tumor cells may be capable of prolonged survival in a state of autophagy/senescence and that disease recurrence may reflect escape from this senescence-arrested state.