salinomycin and Brain-Neoplasms

Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair.. In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships.. Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell-like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action.. Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

Topics: Animals; Autophagy; Brain Neoplasms; DNA Replication; Drug Discovery; Glioblastoma; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Pyrans; Recombinational DNA Repair; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is the deadliest and most common form of primary brain tumor. Conventional treatments are ineffective at treating GBM due to the heterogeneous cellular makeup of the tumors as well as the existence of drug‑resistant cells known as cancer stem cells (CSCs). CSCs have the ability to initiate tumorigenesis and self‑renew, which can lead to recurrence. Salinomycin, an antibiotic commonly used in agricultural feed, has been revealed to target CSCs in other cancer types. A few studies have suggested salinomycin can be effective at treating glioblastoma stem cells (GSCs); however, no study has examined the effect of salinomycin treatment on GSC markers. In the present study, flow cytometry, RT‑qPCR, and limiting dilution assays were used to further analyze the effects of salinomycin on GSCs. It was revealed that salinomycin decreased the expression of the GSC marker SOX2 at both the transcriptional and translational level. However, the effect of salinomycin on the GSC markers Nestin and CD133 was inconsistent between GBM subtypes. Additionally, the present findings provide initial evidence of caspase‑3‑dependent and independent apoptosis as the method by which salinomycin induces cell death in GBM. The present results indicated that salinomycin is an effective candidate as a chemotherapeutic agent that can treat GBM by targeting both bulk tumor cells as well as CSCs.

Topics: Apoptosis; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Neoplastic Stem Cells; Pyrans; SOXB1 Transcription Factors; Spheroids, Cellular

Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O(6)-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide.. MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided.. Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models.. These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Dacarbazine; DNA Damage; DNA Glycosylases; DNA Modification Methylases; DNA Repair Enzymes; Down-Regulation; Endoplasmic Reticulum Stress; Glioblastoma; Humans; Mice; Pyrans; Rad51 Recombinase; Survival Analysis; Temozolomide; Tumor Suppressor Proteins

Glioblastoma is the most frequent malignant brain tumor. Even with aggressive treatment, prognosis for patients is poor. One characteristic of glioblastoma cells is its intrinsic resistance to apoptosis. Therefore, drugs that induce alternative cell deaths could be interesting to evaluate as alternative therapeutic candidates for glioblastoma. Salinomycin (SLM) was identified through a chemical screening as a promising anticancer drug, but its mechanism of cell death remains unclear. In the present work we set out to elucidate how SLM causes cell death in glioblastoma cell lines (both established cell lines and brain tumor stem cell lines), aiming to find a potential antitumor candidate. In addition, we sought to determine the mechanism of action of SLM so that this mechanism can be can be exploited in the fight against cancer. Our data showed that SLM induces a potent endoplasmic reticulum (ER) stress followed by the trigger of the unfolded protein response (UPR) and an aberrant autophagic flux that culminated in necrosis due to mitochondria and lysosomal alterations. Of importance, the aberrant autophagic flux was orchestrated by the production of Reactive Oxygen Species (ROS). Alleviation of ROS production restored the autophagic flux. Altogether our data suggest that in our system the oxidative stress blocks the autophagic flux through lipid oxidation. Importantly, oxidative stress could be instructing the type of cell death in SLM-treated cells, suggesting that cell death modality is a dynamic concept which depends on the cellular stresses and the cellular mechanism activated.

Topics: Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Self Renewal; Endoplasmic Reticulum Stress; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Necrosis; Neoplastic Stem Cells; Pyrans; Reactive Oxygen Species; Unfolded Protein Response

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment.

Topics: Animals; Antineoplastic Agents; Benzeneacetamides; beta Catenin; Brain Neoplasms; Camptothecin; Celecoxib; Cisplatin; Colorectal Neoplasms; Dacarbazine; DNA Modification Methylases; DNA Repair Enzymes; Doxorubicin; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Glucose-6-Phosphate Isomerase; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Immunohistochemistry; Irinotecan; Medulloblastoma; Mice; Neoplasm Transplantation; Neoplasms; Neuroblastoma; Pyrans; Pyrazines; Pyridines; Real-Time Polymerase Chain Reaction; Sulfones; Temozolomide; Triazoles; Tumor Suppressor Proteins; Vincristine; Wnt Proteins; Wnt Signaling Pathway