DDEL-11. SELECTIVE TARGETING OF GLIOBLASTOMA USING FOLATE-DECORATED NANO-PARTICULATE CYTOCHROME C DRUG CONSTRUCTS Josue Davila1, Kimberleve Rolon Reyes1, Moraima Morales Cruz2, Michael Inyushin1, Yuriy Kucheryavykh1, Kai Griebenow2, and Lilia Kucheryavykh1; UniversidadCentraldel Caribe,Bayamon,Puerto Rico; University of Puerto Rico, Rio Piedras, Puerto Rico BACKGROUND: Glioblastoma (GBM) is one of the most malignant forms of brain cancers due to resistance to chemoand radiotherapy, mostly caused by enhanced resistance to apoptosis. We propose the development of targeted apoptosis-inducing drugs for glioblastomatreatment andapprobationof these drugs in glioblastoma cell lines and animal model. We developed nano-sized protein particles containing cytochrome C (NPs). For targeted delivery of the drug specifically to GBM cells we decorated the drug nano-particles with the ligand folic acid (FA). Many GBMs express Proton Coupled Folate Transporters that provide selective internalization of the designed NPs. Protein drug stability problems were countered by covalently decorating the constructs with glycans. RESULTS: Confocal imaging revealed the specific up-take of FITC tagged FA-NPs by GL261 cells, but not by primary cultured astrocytes. Examination of live brain slices encompassing glioblastoma tumor showed some non-specific accumulation of NPs in healthy tissue in two hours after application of FA-NPs, but 20-times less compared to the tumorarea.NPs, used invitro inconcentration of100 mg/ml,havenocytotoxic effect in astrocytes but cause 40% death in tumor cells. The use of NPs in combination with LY294002 (PI3K/AKT blocker), 50 mM, caused 90% death in tumor cells. In vivo TUNEL investigation of tumors generated in C57Bl/6 mice by implantation of GL261 cells revealed the strong signs of apoptosis after three days of in-site administration of NPs through the microosmotic pumps without the evident signs of apoptosis in healthy tissue. In CONCLUSION: We can state that the designed NPs demonstrate good specificity for GL261 cells, effectively cause the apoptosis in these cells when used in combination with LY294002, and give a good baseline for the development of efficient methods for treating GBM. Acknowledgement: NIH Grant Numbers 1SC2GM102040-01A1, SC2GM095410-01A1, 8G12MD007583-27, R25GM110513, Title V grant number P031S130068. Neuro-Oncology 17:v73–v77, 2015. doi:10.1093/neuonc/nov212.11 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.
Abstract In this study we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to glioma cells. Using the techniques of confocal imaging, western blotting, and siRNA knockdown against the PCFT (encoded by the SLC46A1 gene), we demonstrated that Gl261 glioma cells, but not primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by glioma cell death. FA-Cyt c NPs, used at a concentration of 100 µg/ml, had no cytotoxic effects in astrocytes but caused 40% death in GL261 cells. Whole-cell patch clamp recording revealed FA-induced membrane currents, with maximum activity at pH 6.0, which is specific for the PCFT. Currents were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a major carrier of FA and a route for internalization of FA-conjugated NPs in GL261 cells. We conclude that the PCFT provides a mechanism for targeted delivery of FA-conjugated nanodrugs to glioma cells with high specificity for GL261 cells, effectively causing apoptosis in these cells, and provides a starting point for the development of efficient methods for treating gliomas. The research was supported by NIH grants SC2GM102040, SC2GM095410, G12MD007583, R25GM110513, and US Department of Education Grant P031S130068. Citation Format: Yuriy Kucheryavykh, Jescelica Ortiz-Rivera, Michael Inyushin, Luis Cubano, Moraima Morales-Cruz, Alejandra Cruz-Montañez, Kai Griebenow, Lilia Kucheryavykh. Targeted delivery of nanoparticulate cytochrome c into GL261 glioma cells through the proton-coupled folate transporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2179. doi:10.1158/1538-7445.AM2017-2179
The extent of an ischemic insult is less in brain regions enriched in astrocytes suggesting that astrocytes maintain function and buffer glutamate during ischemia.Astrocytes express a wide variety of potassium channels to support their functions including TREK-2 channels which are regulated by polyunsaturated fatty acids, intracellular acidosis and swelling; conditions that pertain to ischemia.The present study investigated the possible involvement of TREK-2 channels in cultured cortical astrocytes during experimental ischemia (anoxia/hypoglycemia) by examining TREK-2 protein levels, channel activity and ability to clear glutamate.We found that TREK-2 protein levels were increased rapidly within 2 hrs of the onset of simulated ischemia.This increase corresponded to an increase in temperature-sensitive TREK-2-like channel conductance and the ability of astrocytes to buffer extracellular glutamate even during ischemia.Together, these data suggest that up-regulation of TREK-2 channels may help rescue astrocyte function and lower extracellular glutamate during ischemia.
Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203
It is known that secondary transporters, which utilize transmembrane ionic gradients to drive their substrates up a concentration gradient, can reverse the uptake and instead release their substrates. Unfortunately, the Michaelis-Menten kinetic scheme, which is popular in transporter studies, does not include transporter reversal, and it completely neglects the possibility of equilibrium between the substrate concentrations on both sides of the membrane. We have developed a complex two-substrate kinetic model that includes transport reversal. This model allows us to construct analytical formulas allowing the calculation of a "heteroexchange" and "transacceleration" using standard Michaelis coefficients for respective substrates. This approach can help to understand how glial and other cells accumulate substrates without synthesis and are able to release such substrates and gliotransmitters.
