Project description:Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.
Project description:Recently, we presented evidence that high mitochondrial ATP production is a new therapeutic target for cancer treatment. Using ATP as a biomarker, we isolated the "metabolically fittest" cancer cells from the total cell population. Importantly, ATP-high cancer cells were phenotypically the most aggressive, with enhanced stem-like properties, showing multi-drug resistance and an increased capacity for cell migration, invasion and spontaneous metastasis. In support of these observations, ATP-high cells demonstrated the up-regulation of both mitochondrial proteins and other protein biomarkers, specifically associated with stemness and metastasis. Therefore, we propose that the "energetically fittest" cancer cells would be better able to resist the selection pressure provided by i) a hostile micro-environment and/or ii) conventional chemotherapy, allowing them to be naturally-selected for survival, based on their high ATP content, ultimately driving tumor recurrence and distant metastasis. In accordance with this energetic hypothesis, ATP-high MDA-MB-231 breast cancer cells showed a dramatic increase in their ability to metastasize in a pre-clinical model in vivo. Conversely, metastasis was largely prevented by treatment with an FDA-approved drug (Bedaquiline), which binds to and inhibits the mitochondrial ATP-synthase, leading to ATP depletion. Clinically, these new therapeutic approaches could have important implications for preventing treatment failure and avoiding cancer cell dormancy, by employing ATP-depletion therapy, to target even the fittest cancer cells.
Project description:PURPOSE:In acute lymphoblastic leukemia (ALL), multidrug resistance is often mediated by ATPase Binding Cassette (ABC) proteins, which principally involve ABCB1 (multidrug resistance 1, MDR1) and ABCC1 (multidrug resistance protein 1, MRP1). However, direct comparisons between the differential effects of ABCB1 and ABCC1 have been difficult, since identical cell lines with differential expression of these transporters have not been developed. EXPERIMENTAL DESIGN:In this study, we developed and compared the biological profiles of Jurkat cell lines that selectively over-expressed ABCB1 and ABCC1. Vincristine (VCR) plays an important role in the treatment of T-lineage ALL (T-ALL), and is often the first drug given to newly-diagnosed patients. Because of its importance in treatment, we provided escalating, sub-lethal doses of VCR to Jurkat cells, and extended our observations to expression profiling of newly diagnosed patients with T-ALL. RESULTS:We found that VCR-resistant cells over-expressed ABCC1 nearly 30-fold. The calcein AM assay confirmed that VCR-resistant cells actively extruded VCR, and that ABCC1-mediated drug resistance conferred a different spectrum of multidrug resistance than other T-ALL induction agents. siRNA experiments that blocked ABCC1 export confirmed that VCR resistance could be reversed in vitro. Analyses of T-lymphoblasts obtained from 92 newly diagnosed T-ALL patients treated on Children's Oncology Group Phase III studies 8704/9404 showed that induction failure could be explained in all but one case by the over-expression of ABCB1 or ABCC1. CONCLUSIONS:Taken together, these results suggest that over-expression of ABC transporters plays a contributing role in mediating treatment failure in T-ALL, and underscore the need to employ alternate treatment approaches in patients for whom induction failed or for those with relapsed disease.
Project description:Adenylate kinase 4 (AK4) is localized in the mitochondrial matrix, and is believed to be involved in stress, drug resistance, the malignant transformation of cancer, and ATP regulation. We produced AK4 knockdown HeLa cells by using shRNA and used these to analyze resistance, and found that sensitivity to hypoxia and drugs increased. 3 samples of HeLa cells haboring AK4 shRNA plasmids and 4 samples of HeLa cells haboring control shRNA plasmid.
Project description:The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc(2)155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic beta-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain beta-lactams in M. smegmatis.
Project description:The vinorelbine (VRB) plus cisplatin regimen is widely used to treat non-small cell lung cancer (NSCLC), but its cure rate is poor. Drug resistance is the primary driver of chemotherapeutic failure, and the causes of resistance remain unclear. By focusing on the focal adhesion (FA) pathway, we have highlighted a signaling pathway that promotes VRB resistance in lung cancer cells. First, we established VRB-resistant (VR) lung cancer cells (NCI-H1299 and A549) and examined its transcriptional changes, protein expressions, and activations. We treated VR cells by Src Family Kinase (SFK) inhibitors or gene silencing and examined cell viabilities. ATP-binding Cassette Sub-family B Member 1 (ABCB1) was highly expressed in VR cells. A pathway analysis and western blot analysis revealed the high expression of integrins β1 and β3 and the activation of FA pathway components, including Src family kinase (SFK) and AKT, in VR cells. SFK involvement in VRB resistance was confirmed by the recovery of VRB sensitivity in FYN knockdown A549 VR cells. Saracatinib, a dual inhibitor of SFK and ABCB1, had a synergistic effect with VRB in VR cells. In conclusion, ABCB1 is the primary cause of VRB resistance. Additionally, the FA pathway, particularly integrin, and SFK, are promising targets for VRB-resistant lung cancer. Further studies are needed to identify clinically applicable target drugs and biomarkers that will improve disease prognoses and predict therapeutic efficacies.
Project description:Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1—an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis—also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity.
Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.