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Rational Cisplatin based cancer therapy is an affordable and effective standard therapy for several solid cancers, including lung, ovarian and head and neck cancers. However, the clinical use of cisplatin is routinely limited by the development of drug resistance and subsequent therapeutic failure. Therefore, methods of circumventing cisplatin resistance have the potential to increase therapeutic efficiency and dramatically increase overall survival. Cisplatin resistance can be mediated by alterations to the DNA damage response, where multiple components of the repair machinery have been described to be client proteins of HSP90. In the present study, we have investigated whether therapy with the novel HSP90 inhibitor onalespib can potentiate the efficacy of cisplatin and potentially reverse cisplatin resistance in ovarian and head and neck cancer cells. Methods Cell viability, cancer cell proliferation and migration capacity were evaluatedin vitroon models of ovarian and head and neck cancer cells. Western blotting was used to assess the downregulation of HSP90 client proteins and alterations in downstream signaling proteins after exposure to cisplatin and/or onalespib. Induction of apoptosis and DNA damage response were evaluated in both monotherapy and combination therapy groups. Results Results demonstrate that onalespib enhances the efficiency of cisplatin in a dose-dependent manner. Tumor cells treated with both drugs displayed lower viability and a decreased migration rate compared to vehicle-control cells and cells treated with individual compounds. An increase of DNA double strand breaks was observed in both cisplatin and onalespib treated cells. The damage was highest and most persistent in the combination group, delaying the DNA repair machinery. Further, the cisplatin and onalespib co-treated cells had greater apoptotic activity compared to controls. Conclusion The results of this study demonstrate that the reduced therapeutic efficacy of cisplatin due to drug-resistance could be overcome by combination treatment with onalespib. We speculate that the increased apoptotic signaling, DNA damage as well as the downregulation of HSP90 client proteins are important mechanisms promoting increased sensitivity to cisplatin treatment.

Background: Glioblastoma (GBM) is the deadliest form of brain cancer, impacting both adults and children, marked by exceptionally high morbidity and mortality rates, even with current standard treatments such as surgery, radiation therapy, and chemotherapy. Therefore, there is a pressing need for new therapeutic strategies to improve survival and reduce treatment side effects. In this study, we investigated the effect of HSP90 inhibition in combination with radiotherapy in established and patient-derived glioblastoma cell lines.  

Methods: Potential radiosensitizing effects of the HSP90 inhibitor Onalespib were studied in XTT and clonogenic survival assays as well as in tumor-mimicking multicellular spheroid models. Further, migration capacity and effects on protein expression were studied after exposure to Onalespib and radiation using Proximity Extension Assay analysis.  

Results: HSP90 inhibition with Onalespib synergistically enhanced the radiosensitivity of glioblastoma cells grown in 2D and 3D models, resulting in increased cell death, reduced migration capacity and activation of the apoptotic signaling pathway. The proteomic analysis of glioblastoma cells treated with Onalespib, radiation, and their combination revealed significant alterations in protein expression profiles, involved in growth signaling, immune modulation pathways and angiogenesis. Moreover, the combination treatment indicated potential for enhancing cell cycle arrest and apoptosis, suggesting promising antitumor effects.  

Conclusion: These findings demonstrate that HSP90 inhibition may be a promising strategy to enhance the efficacy of radiotherapy in the treatment of GBM, potent

The bone-seeking radiopharmaceutical Xofigo (Radium-223 dichloride) has demonstrated both extended sur-vival and palliative effects in treatment of bone metastases in prostate cancer. The alpha-particle emitter Ra-223, targets regions undergoing active bone remodeling and strongly binds to bone hydroxyapatite (HAp). However, the toxicity mechanism and properties of Ra-223 binding to hydroxyapatite are not fully understood. By exposing 2D and 3D (spheroid) prostate cancer cell models to free and HAp-bound Ra-223 we here studied cell toxicity, apoptosis and formation and repair of DNA double-strand breaks (DSBs). The rapid binding with a high affinity of Ra-223 to bone-like HAp structures was evident (KD= 19.2 x 10-18 M) and almost no dissociation was detected within 24 h. Importantly, there was no significant uptake of Ra-223 in cells. The Ra-223 alpha-particle decay produced track-like distributions of the DNA damage response proteins 53BP1 and gamma H2AX induced high amounts of clustered DSBs in prostate cancer cells and activated DSB repair through non-homologous end-joining (NHEJ). Ra-223 inhibited growth of prostate cancer cells, independent of cell type, and induced high levels of apoptosis. In summary, we suggest the high cell killing efficacy of the Ra-223 was attributed to the clustered DNA damaged sites induced by alpha-particles.

Background: Ascorbate is used for combination therapy with promising results in clinical trials. The proposed main ascorbate cytotoxic effect is DNA damage induction through excessive oxidative stress. However, the involvement of DNA double-strand breakage (DSB) formation and repair in ascorbate-induced DNA damage remains to be revealed.  

Methods: We here used wild-type cells and cells with knock-outs (KO) of the key non-homologous end-joining (NHEJ) repair proteins DNA-PKcs and XRCC4 and tested 2D and 3D cell viability, growth, DSB (DNA fragmentation and DSB surrogate markers 53BP1 and γH2AX), apoptosis, necrosis, and cell cycle in response to ascorbate.  

Results: Ascorbate reduced cell survival and viability in a concentration-dependent manner, with a tendency toward XRCC4 KO cells more sensitivity. Unlike radiation, ascorbate did not produce prompt DSB. However, 24 h after exposure, there was a clear increase in 53BP1 foci in both NHEJproficient and KO cells and evident pan-nuclear γH2AX response, especially in XRCC4 KO cells, which may indicate nuclear degradation leading to DSB formation over time. Further, ascorbate induced G2/M arrest with a more pronounced effect in XRCC4 KO cells. For higher doses (>1 mM) we observed a rapid (24 h) necrotic response without activation of apoptosis. The cell line's different response seems to be related to their cell cycle regulation rather than the NHEJ status. These findings provide novel mechanistic insights into DNA damage formation and cellular response to ascorbate.  

Conclusion: Our data suggest that ascorbate does not generate prompt or direct DSBs. Instead, it induces delayed DSBs ste

Anticancer agents kill cancer cells by inducing DNA double-strand breaks (DSBs). DSBs vary in type and complexity, posing different challenges to repair mechanisms. The primary DSB repair pathway is non-homologous end-joining (NHEJ). However, NHEJ response to different types and complexity of DSB remains elusive. Here we examined agents which produce DSB and varying ratios of single-strand breaks (SSB) and base damage, calicheamicin, X-rays, phleomycin, etoposide, and temozolomide in wild-type cells and cells with knock-outs (KO) of DNA-PKcs or XRCC4 and analyzed clonogenicity, induction of prompt DSB, non-DSB clusters and DSB repair kinetics. In wild-type cells, DSB were repaired by both fast and slow repair kinetics. However, in the absence of NHEJ there was essentially only a fast repair phase (30-60 min), whereafter there was no repair the following 1-24 hours. Treatment with X-rays and calicheamicin resulted in only 20% repair in the NHEJ defective cells, whereas 40-50%, and 10-20% of DSB were repaired after treatment with phleomycin and etoposide, respectively. Ttemozolomide did not induce prompt DSB. The anticancer agent temozolomide, which do not induce prompt DSB, reduced cell survival in a dose-dependent manner but independent of NHEJ status. Non-DSB clusters, e.g., combination of two or more oxidized bases, single-strand breaks or other DNA lesion that do not form a prompt DSB, increased as the DSB:SSB ratio decreased from calicheamicin, to X-rays, phleomycin, etoposide, and temozolomide, respectively. Notably, removal of non-DSB clusters occurred rapidly, independent of NHEJ. Although the NHEJ defective cells were hypersensitive to all agents, except temozolomide, the cell survival did not directly correlate to the capacity of NHEJ defective cell to repair DSB, suggesting that the role of NHEJ-independent repair pathways vary for DSB of different types or complexity. Overall, DSB type/complexity clearly affects the repair efficiency. These insights could be vital for understanding the choice of DSB repair pathway and optimization of DNA repair modulation.