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Electrophoresis Aug 2017Inherent electrical properties of cells can be beneficial to characterize different cell lines and their response to experimental drugs. This paper presents a novel...
Inherent electrical properties of cells can be beneficial to characterize different cell lines and their response to experimental drugs. This paper presents a novel method to characterize the response of breast cancer cells to drug stimuli through use of off-chip passivated-electrode insulator-based dielectrophoresis (OπDEP) and the application of AC electric fields. This work is the first to demonstrate the ability of OπDEP to differentiate between two closely related breast cancer cell lines, LCC1 and LCC9 while assessing their drug sensitivity to an experimental anti-cancer agent, Obatoclax. Although both cell lines are derivatives of estrogen-responsive MCF-7 breast cancer cells, growth of LCC1 is estrogen independent and anti-estrogen responsive, while LCC9 is both estrogen-independent and anti-estrogen resistant. Under the same operating conditions, LCC1 and LCC9 had different DEP profiles. LCC1 cells had a trapping onset (crossover) frequency of 700 kHz and trapping efficiencies between 30-40%, while LCC9 cells had a lower crossover frequency (100 kHz) and showed higher trapping efficiencies of 40-60%. When exposed to the Obatoclax, both cell lines exhibited dose-dependent shifts in DEP crossover frequency and trapping efficiency. Here, DEP results supplemented with cell morphology and proliferation assays help us to understand the response of these breast cancer cells to Obatoclax.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Cell Size; Drug Resistance, Neoplasm; Electrodes; Electrophoresis; Estrogen Antagonists; Female; Humans; Indoles; MCF-7 Cells; Microfluidic Analytical Techniques; Pyrroles
PubMed: 28608427
DOI: 10.1002/elps.201600447 -
The FEBS Journal Dec 2017The ERK1/2 signalling pathway is best known for its role in connecting activated growth factor receptors to changes in gene expression due to activated ERK1/2 entering... (Review)
Review
The ERK1/2 signalling pathway is best known for its role in connecting activated growth factor receptors to changes in gene expression due to activated ERK1/2 entering the nucleus and phosphorylating transcription factors. However, active ERK1/2 also translocate to a variety of other organelles including the endoplasmic reticulum, endosomes, golgi and mitochondria to access specific substrates and influence cell physiology. In this article, we review two aspects of ERK1/2 signalling at the mitochondria that are involved in regulating cell fate decisions. First, we describe the prominent role of ERK1/2 in controlling the BCL2-regulated, cell-intrinsic apoptotic pathway. In most cases ERK1/2 signalling promotes cell survival by activating prosurvival BCL2 proteins (BCL2, BCL-x and MCL1) and repressing prodeath proteins (BAD, BIM, BMF and PUMA). This prosurvival signalling is co-opted by oncogenes to confer cancer cell-specific survival advantages and we describe how this information has been used to develop new drug combinations. However, ERK1/2 can also drive the expression of the prodeath protein NOXA to control 'autophagy or apoptosis' decisions during nutrient starvation. We also describe recent studies demonstrating a link between ERK1/2 signalling, DRP1 and the mitochondrial fission machinery and how this may influence metabolic reprogramming during tumorigenesis and stem cell reprogramming. With advances in subcellular proteomics it is likely that new roles for ERK1/2, and new substrates, remain to be discovered at the mitochondria and other organelles.
Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bridged Bicyclo Compounds, Heterocyclic; Drug Screening Assays, Antitumor; Humans; Indoles; MAP Kinase Signaling System; Mitochondrial Dynamics; Molecular Targeted Therapy; Neoplasm Proteins; Neoplasms; Oncogene Addiction; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides
PubMed: 28548464
DOI: 10.1111/febs.14122 -
Cancer Research Jul 2017Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof...
Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors and in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. .
Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Biomimetic Materials; Cell Line, Tumor; Cell Proliferation; Female; HSP90 Heat-Shock Proteins; Humans; Indoles; Male; Mice; Mice, SCID; Mitochondria; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Xenograft Model Antitumor Assays
PubMed: 28522750
DOI: 10.1158/0008-5472.CAN-16-3424 -
Oncotarget May 2017Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL...
Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL and c-myc inhibition accomplished by bromodomain protein inhibitors. Established, patient-derived xenograft and stem cell-like glioma cells were treated with the novel bromodomain protein inhibitors, JQ1 and OTX015, along with BH3-mimetics, ABT263 or Obatoclax. Synergy was assessed by calculation of CI values. Small interfering RNAs (siRNAs) were used for gene silencing and mechanistic studies. In vivo experiments were performed in a glioblastoma xenograft model. Single treatments with JQ1 and OTX015 had only moderate effects on the reduction of cellular viability. However, the combination treatment of BH3-mimetics along with JQ1 or OTX015 resulted in a highly synergistic reduction of cellular viability in a broad range of different model systems of malignant glioma. Similarly, knockdown of c-myc sensitized glioma cells for ABT263 mediated cell death. The enhanced loss of cellular viability in the combination treatment was mediated by activation of apoptosis with dissipation of mitochondrial membrane potential and caspase cleavage. The combination treatment led to a modulation of anti- and pro-apoptotic Bcl-2 family members with an increase in pro-apoptotic Noxa mediated by ATF4. Small interfering RNA mediated knockdown of Bak and Noxa protected glioma cells from ABT263/JQ1 mediated apoptosis. Finally, the combination treatment of ABT263 and OTX015 resulted in a regression of tumors and a significantly smaller tumor size as compared to single or vehicle treated tumors. Thus, these results warrant clinical testing for the drug combination of BH3-mimetics along with bromodain protein inhibitors.
Topics: Acetanilides; Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Azepines; Bcl-2-Like Protein 11; Caspases; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Synergism; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Heterocyclic Compounds, 3-Ring; Humans; Membrane Potential, Mitochondrial; Mice; Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Sulfonamides; Triazoles; Xenograft Model Antitumor Assays
PubMed: 28418907
DOI: 10.18632/oncotarget.16365 -
Journal of Experimental & Clinical... Apr 2017Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with...
BACKGROUND
Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance.
METHODS
Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays.
RESULTS
KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46.
CONCLUSION
Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma.
Topics: Antineoplastic Combined Chemotherapy Protocols; Autophagy; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Indoles; Molecular Targeted Therapy; Organometallic Compounds; Osteosarcoma; Oxyquinoline; Pyrroles
PubMed: 28403890
DOI: 10.1186/s13046-017-0527-z -
Theranostics 2017Although the prognosis of differentiated thyroid cancer (DTC) is relatively good, 30-40% of patients with distant metastases develop resistance to radioactive iodine...
Although the prognosis of differentiated thyroid cancer (DTC) is relatively good, 30-40% of patients with distant metastases develop resistance to radioactive iodine therapy due to tumor dedifferentiation. For DTC patients harboring BRAF mutation, Vemurafenib, a BRAF kinase inhibitor, has dramatically changed the therapeutic landscape, but side effects and drug resistance often lead to termination of the single agent treatment. In the present study, we showed that either LY3009120 or Obatoclax (GX15-070) efficiently inhibited cell cycle progression and induced massive death of DTC cells. We established that BRAF/CRAF dimerization was an underlying mechanism for Vemurafenib resistance. LY3009120, the newly discovered pan-RAF inhibitor, successfully overcame Vemurafenib resistance and suppressed the growth of DTC cells in vitro and in vivo. We also observed that expression of anti-apoptotic Bcl-2 increased substantially following BRAF inhibitor treatment in Vemurafenib-resistant K1 cells, and both Obatoclax and LY3009120 efficiently induced apoptosis of these resistant cells. Specifically, Obatoclax exerted its anti-cancer activity by inducing loss of mitochondrial membrane potential (ΔΨm), dysfunction of mitochondrial respiration, reduction of cellular glycolysis, autophagy, neutralization of lysosomes, and caspase-related apoptosis. Furthermore, the cancer killing effects of LY3009120 and Obatoclax extended to two more Vemurafenib-resistant DTC cell lines, KTC-1 and BCPAP. Taken together, our results highlighted the potential value of LY3009120 for both Vemurafenib-sensitive and -resistant DTC and provided evidence for the combination therapy using Vemurafenib and Obatoclax for radioiodine-refractory DTC.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Drug Resistance; Enzyme Inhibitors; Heterografts; Humans; Indoles; Mice, Nude; Phenylurea Compounds; Protein Multimerization; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Pyrimidines; Pyrroles; Sulfonamides; Thyroid Neoplasms; Treatment Outcome; Vemurafenib
PubMed: 28382170
DOI: 10.7150/thno.17322 -
Tumour Biology : the Journal of the... Mar 2017Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the...
Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the plasticity of cancer cells have been identified as important mechanisms of resistance; therefore, treatments targeting cancer cells independent of stemness phenotype would be much more effective in treating lung cancer. In this article, we have characterized the anticancer effects of the antibiotic Nigericin in cells displaying varying degrees of stemness and resistance to anticancer drugs, arising from (1) routine culture conditions, (2) prolonged periods of serum starvation. These cells are highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea, Colchicine, Obatoclax, Wortmannin, and LY294002, and the multidrug-resistant phenotype of cells growing under prolonged periods of serum starvation is likely the result of extensive rewiring of signaling pathways, and (3) lung tumorspheres that are enriched for cancer stem-like cells. We found that Nigericin potently inhibited the viability of cells growing under routine culture conditions, prolonged periods of serum starvation, and lung tumorspheres. In addition, we found that Nigericin downregulated the expression of key proteins in the Wnt canonical signaling pathway such as LRP6, Wnt5a/b, and β-catenin, but promotes β-catenin translocation into the nucleus. The antitumor effects of Nigericin were potentiated by the Wnt activator HLY78 and by therapeutic levels of the US Food and Drug Administration-approved drug Digitoxin and its novel synthetic analog MonoD. We believe that Nigericin may be used in a co-therapy model in combination with other novel chemotherapeutic agents in order to achieve potent inhibition of cancers that display varying degrees of stemness, potentially leading to sustained anticancer effects.
Topics: Antineoplastic Agents; Benzodioxoles; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Neoplastic Stem Cells; Nigericin; Phenanthridines; Wnt Signaling Pathway
PubMed: 28351327
DOI: 10.1177/1010428317694310 -
Nature Communications Jan 2017Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL...
Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Survival; Chromatin; Cyclin-Dependent Kinases; Female; Gain of Function Mutation; Gene Expression Regulation, Neoplastic; Humans; Indoles; Lymphoma, T-Cell; Male; Mice; Mice, Inbred NOD; Mice, SCID; Phenylenediamines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Pyrroles; STAT3 Transcription Factor; Signal Transduction; Transcription, Genetic; Treatment Outcome; Xenograft Model Antitumor Assays; Cyclin-Dependent Kinase-Activating Kinase
PubMed: 28134252
DOI: 10.1038/ncomms14290 -
Current Hematologic Malignancy Reports Feb 2017The BCL-2 family of proteins integrates pro- and anti-apoptotic signals within the cell and is responsible for initiation of caspase-dependent apoptosis. Chronic... (Review)
Review
The BCL-2 family of proteins integrates pro- and anti-apoptotic signals within the cell and is responsible for initiation of caspase-dependent apoptosis. Chronic lymphocytic leukemia (CLL) cells are particularly dependent on the anti-apoptotic protein BCL-2 for their survival, making this an attractive therapeutic target in CLL. Several early efforts to create inhibitors of the anti-apoptotic family members faced significant challenges, but eventually, the BCL-2 specific inhibitor venetoclax moved forward in CLL. Overall and complete response rates to venetoclax monotherapy in relapsed, refractory CLL are approximately 80 and 20%, respectively, even in patients with high-risk 17p deletion. Toxicities have been manageable and include neutropenia, diarrhea, and nausea. The risk of tumor lysis syndrome (TLS), seen in early experience with the drug, has been mitigated by the use of appropriate TLS risk assessment, prophylaxis, and management. Future studies of venetoclax will focus on combination approaches, predictive biomarker discovery, and mechanisms of resistance.
Topics: Aniline Compounds; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Gossypol; Humans; Indoles; Leukemia, Lymphocytic, Chronic, B-Cell; Neutropenia; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Thionucleotides
PubMed: 28116634
DOI: 10.1007/s11899-017-0359-0 -
Oncotarget Jan 2017Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Schwann cell-derived neoplasms of the peripheral nervous system that have recently been shown to...
Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Schwann cell-derived neoplasms of the peripheral nervous system that have recently been shown to possess an autocrine CXCL12/CXCR4 signaling loop that promotes tumor cell proliferation and survival. Importantly, the CXCL12/CXCR4 signaling axis is driven by availability of the CXCL12 ligand rather than CXCR4 receptor levels alone. Therefore, pharmacological reduction of CXCL12 expression could be a potential chemotherapeutic target for patients with MPNSTs or other pathologies wherein the CXCL12/CXCR4 signaling axis is active. AT101 is a well-established BCL-2 homology domain 3 (BH3) mimetic that we recently demonstrated functions as an iron chelator and thus acts as a hypoxia mimetic. In this study, we found that AT101 significantly reduces CXCL12 mRNA and secreted protein in established human MPNST cell lines in vitro. This effect was recapitulated by other BH3 mimetics [ABT-737 (ABT), obatoclax (OBX) and sabutoclax (SBX)] but not by desferrioxamine (DFO), an iron chelator and known hypoxia mimetic. These data suggest that CXCL12 reduction is a function of AT101's BH3 mimetic property rather than its iron chelation ability. Additionally, this study investigates a potential mechanism of BH3 mimetic-mediated CXCL12 suppression: liberation of a negative CXCL12 transcriptional regulator, poly (ADP-Ribose) polymerase I (PARP1) from its physical interaction with BCL-2. These data suggest that clinically available BH3 mimetics might prove therapeutically useful at least in part by virtue of their ability to suppress CXCL12 expression.
Topics: Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Chemokine CXCL12; Down-Regulation; Gene Expression Regulation, Neoplastic; Gossypol; Humans; Indoles; Molecular Mimicry; Neurilemmoma; Nitrophenols; Piperazines; Poly (ADP-Ribose) Polymerase-1; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides
PubMed: 28055968
DOI: 10.18632/oncotarget.14398