Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a poor prognosis. Complex genetic alterations and the protective effect of the blood-brain barrier (BBB) have so far hampered effective treatment. Here, we investigated the cytotoxic effects of heat shock protein 90 (HSP90) inhibitors, geldanamycin (GDN) and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), in a panel of glioma tumor cell lines with various genetic alterations. We also assessed the ability of the main drug transporters, ABCB1 and ABCG2, to efflux GDN and 17-AAG. We found that GDN and 17-AAG induced extensive cell death with the morphological and biochemical hallmarks of apoptosis in all studied glioma cell lines at sub-micro-molar and nanomolar concentrations. Moderate efflux efficacy of GDN and 17-AAG mediated by ABCB1 was observed. There was an insignificant and low efflux efficacy of GDN and 17-AAG mediated by ABCG2. Conclusion: GDN and 17-AAG, in particular, exhibited strong proapoptotic effects in glioma tumor cell lines irrespective of genetic alterations. GDN and 17-AAG appeared to be weak substrates of ABCB1 and ABCG2. Therefore, the BBB would compromise their cytotoxic effects only partially. We hypothesize that GBM patients may benefit from 17-AAG either as a single agent or in combination with other drugs.

• Keywords
• ABC transporters, apoptosis, blood brain barrier, human glioma tumor cell panel, multidrug resistance, tanespimycin,
• Publication type
• Journal Article MeSH

Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 (LAMP1, LAMP2), vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca2+signaling. A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.

• Keywords
• Hl-60 cells, K562 cells, lysosomal fusion, lysosomal sequestration capacity, tyrosine kinase inhibitors,
• MeSH
• Organelle Biogenesis MeSH
• K562 Cells MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Gefitinib pharmacology   MeSH
• Imatinib Mesylate pharmacology   MeSH
• Humans MeSH
• Lysosomes drug effects   metabolism   MeSH
• Cell Line, Tumor MeSH
• Antineoplastic Agents pharmacology   MeSH
• Signal Transduction drug effects   MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors drug effects   metabolism   MeSH
• Protein-Tyrosine Kinases antagonists & inhibitors   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gefitinib MeSH
• Imatinib Mesylate MeSH
• Antineoplastic Agents MeSH
• TFEB protein, human MeSH Browser
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors MeSH
• Protein-Tyrosine Kinases MeSH

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.

• Keywords
• drug resistance, extracellular space, extralysosomal space, lysosomal sequestration, target sites, tyrosine kinase inhibitors,
• MeSH
• K562 Cells MeSH
• Drug Resistance, Neoplasm * MeSH
• Extracellular Space drug effects   metabolism   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Lysosomes drug effects   metabolism   MeSH
• Sunitinib pharmacology   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Kinase Inhibitors MeSH
• Sunitinib MeSH
• Protein-Tyrosine Kinases MeSH

The synthetic curcumin analogue, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), suppresses NF-κB activity and exhibits antiproliferative effects against a variety of cancer cells in vitro. Recently, it was reported that EF-24-induced apoptosis was mediated by a redox-dependent mechanism. Here, we studied the effects of N-acetylcysteine (NAC) on EF-24-induced cell death. We also addressed the question of whether the main drug transporters, ABCB1 and ABCG2, affect the cytotoxic of EF-24. We observed that EF-24 induced cell death with apoptotic hallmarks in human leukemia K562 cells. Importantly, the loss of cell viability was preceded by production of reactive oxygen species (ROS), and by a decrease of reduced glutathione (GSH). However, neither ROS production nor the decrease in GSH predominantly contributed to the EF-24-induced cell death. We found that EF-24 formed an adduct with GSH, which is likely the mechanism contributing to the decrease of GSH. Although NAC abrogated ROS production, decreased GSH and prevented cell death, its protective effect was mainly due to a rapid conversion of intra- and extra-cellular EF-24 into the EF-24-NAC adduct without cytotoxic effects. Furthermore, we found that neither overexpression of ABCB1 nor ABCG2 reduced the antiproliferative effects of EF-24. In conclusion, a redox-dependent-mediated mechanism only marginally contributes to the EF-24-induced apoptosis in K562 cells. The main mechanism of NAC protection against EF-24-induced apoptosis is conversion of cytotoxic EF-24 into the noncytotoxic EF-24-NAC adduct. Neither ABCB1 nor ABCG2 mediated resistance to EF-24.

• Keywords
• EF-24-GSH adduct, EF-24-NAC adduct, K562 cells, NF-κB, Nrf2,
• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 genetics   metabolism   MeSH
• Acetylcysteine metabolism   MeSH
• Apoptosis drug effects   MeSH
• Benzylidene Compounds pharmacology   MeSH
• Glutathione metabolism   MeSH
• Leukemia metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins genetics   metabolism   MeSH
• Oxidative Stress * MeSH
• ATP Binding Cassette Transporter, Subfamily B genetics   metabolism   MeSH
• Piperidones pharmacology   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,5-bis(2-fluorobenzylidene)piperidin-4-one MeSH Browser
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ABCB1 protein, human MeSH Browser
• ABCG2 protein, human MeSH Browser
• Acetylcysteine MeSH
• Benzylidene Compounds MeSH
• Glutathione MeSH
• Neoplasm Proteins MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Piperidones MeSH
• Antineoplastic Agents MeSH
• Reactive Oxygen Species MeSH