ABSTRACT
BACKGROUND: Less than 20 % of patients with resectable oesophageal adenocarcinoma obtain a pathological response following neoadjuvant chemotherapy. Studies using oesophageal cancer cell lines have shown that drug sensitive tumour cells undergo apoptosis in response to drug treatment, whereas resistant cells induce autophagy and can recover following withdrawal of drug. In this study, we evaluated markers of apoptosis (active/cleaved caspase-3) and autophagy (LC3B) to establish whether these markers are useful prognostic indicators following neoadjuvant therapy. METHODS: Oesophageal adenocarcinoma tumour tissue from the Northern Ireland Biobank at Queens University Belfast was examined retrospectively. Tumours from 144 patients treated with platinum-based neoadjuvant chemotherapy followed by surgical resection were assembled into tissue microarrays prior to immunohistochemical analysis. Kaplan-Meier survival curves and log-rank tests were used to assess the impact of cleaved caspase-3 and LC3B expression on survival. Cox regression was used to examine association with clinical risk factors. RESULTS: High levels of cleaved caspase-3 were found in 14.6 % of patients and this correlated with a significantly better overall survival (p = 0.03). 38.9 % of patients had high cytoplasmic LC3B expression, which correlated with poor overall survival (p = 0.041). In addition, a distinct globular pattern of LC3B expression was identified in 40.3 % of patients and was also predictive of overall survival (p < 0.001). LC3B globular structures are also associated with tumour recurrence (p = 0.014). When these markers were assessed in combination, it was found that patients who showed low/negative cleaved caspase-3 staining and high/positive staining for both patterns of LC3B had the worst overall survival (p < 0.001). Multi-variate analysis also indicated that this marker combination was an independent predictor of poor prognosis (p = 0.008; HR = 0.046, 95% CI = (0.005-0.443). CONCLUSIONS: The expression of cleaved caspase-3 and specific LC3B staining patterns are associated with overall survival following neoadjuvant treatment. The combination of these markers is an independent indicator of outcome in neoadjuvant chemotherapy treated oesophageal adenocarcinoma.
Subject(s)
Adenocarcinoma , Esophageal Neoplasms , Adenocarcinoma/pathology , Apoptosis , Autophagy , Biomarkers, Tumor/metabolism , Caspase 3 , Humans , Neoadjuvant Therapy , Prognosis , Retrospective StudiesABSTRACT
OBJECTIVE: In acute promyelocytic leukemia (APL), normal retinoid signaling is disrupted by an abnormal PML-RARα fusion oncoprotein, leading to a block in cell differentiation. Therapeutic concentrations of all-trans-retinoic acid (ATRA) can restore retinoid-induced transcription and promote degradation of the PML-RARα protein. Autophagy is a catabolic pathway that utilizes lysosomal machinery to degrade intracellular material and facilitate cellular re-modeling. Recent studies have identified autophagy as an integral component of ATRA-induced myeloid differentiation. METHODS: As the molecular communication between retinoid signaling and the autophagy pathway is not defined, we performed RNA sequencing of NB4 APL cells treated with ATRA and examined autophagy-related transcripts. RESULTS: ATRA altered the expression of >80 known autophagy-related transcripts, including the key transcriptional regulator of autophagy and lysosomal biogenesis, TFEB (11.5-fold increase). Induction of TFEB and its transcriptional target, sequestosome 1 (SQSTM1, p62), is reduced in ATRA-resistant NB4R cells compared to NB4 cells. TFEB knockdown in NB4 cells alters the expression of transcriptional targets of TFEB and reduces CD11b transcript levels in response to ATRA. CONCLUSIONS: We show for the first time that TFEB plays an important role in ATRA-induced autophagy during myeloid differentiation and that autophagy induction potentiates leukemic cell differentiation (Note: this study includes data obtained from NCT00195156, https://clinicaltrials.gov/show/NCT00195156).
Subject(s)
Antineoplastic Agents/pharmacology , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Differentiation/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/pathology , Tretinoin/pharmacology , Antineoplastic Agents/therapeutic use , Autophagy/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Biopsy , Bone Marrow/pathology , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Tretinoin/therapeutic useABSTRACT
Following publication of the original article [1], the authors reported an omission in the affiliations.
ABSTRACT
Increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for ATP generation. Altered energy metabolism is a biochemical fingerprint of cancer cells that represents one of the "hallmarks of cancer". The immune system can prevent tumour growth by eliminating cancer cells but this editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour growth. In this review we look at the glycolysis pathway as a target for cancer treatments. We also examine the interplay between the glycolysis modulation and the immune response as an anti-cancer therapy.
Subject(s)
Glycolysis/drug effects , Immunity, Cellular/drug effects , Neoplasms/drug therapy , Adenosine Triphosphate/metabolism , Energy Metabolism/drug effects , Humans , Metabolic Networks and Pathways/drug effects , Molecular Targeted Therapy , Neoplasms/metabolism , Neoplasms/pathologyABSTRACT
BACKGROUND: Successful treatment of oesophageal cancer is hampered by recurrent drug resistant disease. We have previously demonstrated the importance of apoptosis and autophagy for the recovery of oesophageal cancer cells following drug treatment. When apoptosis (with autophagy) is induced, these cells are chemosensitive and will not recover following chemotherapy treatment. In contrast, when cancer cells exhibit only autophagy and limited Type II cell death, they are chemoresistant and recover following drug withdrawal. METHODS: MicroRNA (miRNA) expression profiling of an oesophageal cancer cell line panel was used to identify miRNAs that were important in the regulation of apoptosis and autophagy. The effects of miRNA overexpression on cell death mechanisms and recovery were assessed in the chemoresistant (autophagy inducing) KYSE450 oesophageal cancer cells. RESULTS: MiR-193b was the most differentially expressed miRNA between the chemosensitive and chemoresistant cell lines with higher expression in chemosensitive apoptosis inducing cell lines. Colony formation assays showed that overexpression of miR-193b significantly impedes the ability of KYSE450 cells to recover following 5-fluorouracil (5-FU) treatment. The critical mRNA targets of miR-193b are unknown but target prediction and siRNA data analysis suggest that it may mediate some of its effects through stathmin 1 regulation. Apoptosis was not involved in the enhanced cytotoxicity. Overexpression of miR-193b in these cells induced autophagic flux and non-apoptotic cell death. CONCLUSION: These results highlight the importance of miR-193b in determining oesophageal cancer cell viability and demonstrate an enhancement of chemotoxicity that is independent of apoptosis induction.
Subject(s)
Apoptosis/genetics , Autophagy/genetics , Drug Resistance, Neoplasm/genetics , Esophageal Neoplasms/metabolism , MicroRNAs/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Esophageal Neoplasms/genetics , Fluorouracil/pharmacology , Humans , MicroRNAs/geneticsABSTRACT
BACKGROUND: Esophageal adenocarcinoma has the fastest growing incidence of any solid tumor in the Western world. Prognosis remains poor with overall five-year survival rates under 25 %. Only a limited number of patients benefit from chemotherapy and there are no biomarkers that can predict outcome. Previous studies have indicated that induction of autophagy can influence various aspects of tumor cell biology, including chemosensitivity. The objective of this study was to assess whether expression of the autophagy marker (LC3B) correlated with patient outcome. METHODS: Esophageal adenocarcinoma tumor tissue from two independent sites, was examined retrospectively. Tumors from 104 neoadjuvant naïve patients and 48 patients post neoadjuvant therapy were assembled into tissue microarrays prior to immunohistochemical analysis. Kaplan-Meier survival curves and log-rank tests were used to assess impact of LC3B expression on survival. Cox regression was used to examine association with clinical risk factors. RESULTS: A distinct globular pattern of LC3B expression was found to be predictive of outcome in both patient groups, irrespective of treatment (p < 0.001). Multivariate analysis found that this was a strong independent predictor of poor prognosis (p < 0.001). CONCLUSIONS: This distinctive staining pattern of LC3B represents a novel prognostic marker for resectable esophageal adenocarcinoma.
Subject(s)
Biomarkers, Tumor/metabolism , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Microtubule-Associated Proteins/metabolism , Autophagy , Cell Line, Tumor , Esophageal Neoplasms/drug therapy , Humans , Prognosis , Retrospective Studies , Survival AnalysisABSTRACT
Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.
Subject(s)
Autophagy/drug effects , Benzamides/pharmacology , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Cell Line, Tumor , Down-Regulation/drug effects , Fusion Proteins, bcr-abl/biosynthesis , Fusion Proteins, bcr-abl/genetics , Gene Knockdown Techniques , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phagosomes/drug effects , Phagosomes/enzymology , Proteasome Endopeptidase Complex/metabolism , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Signal Transduction , TransfectionABSTRACT
Acute myeloid leukemia (AML) is an aggressive blood cancer with an overall survival of 30%. One form of AML, acute promyelocytic leukemia (APL) has become more than 90% curable with differentiation therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Application of differentiation therapy to other AML subtypes would be a major treatment advance. Recent studies have indicated that autophagy plays a key role in the differentiation of ATRA-responsive APL cells. In this study, we have investigated whether differentiation could be enhanced in ATRA resistant cells by promoting autophagy induction with valproic acid (VPA). ATRA sensitive (NB4) and resistant leukemia cells (NB4R and THP-1) were co-treated with ATRA and valproic acid, followed by assessment of autophagy and differentiation. The combination of VPA and ATRA induced autophagic flux and promoted differentiation in ATRA-sensitive and -resistant cell lines. shRNA knockdown of ATG7 and TFEB autophagy regulators impaired both autophagy and differentiation, demonstrating the importance of autophagy in the combination treatment. These data suggest that ATRA combined with valproic acid can promote differentiation in myeloid leukemia cells by mechanism involving autophagy.
ABSTRACT
Aim: To investigate alterations in transcription of genes, encoding Ca2+ toolkit proteins, in oesophageal adenocarcinoma (OAC) and to assess associations between gene expression, tumor grade, nodal-metastatic stage, and patient survival. Methods: The expression of 275 transcripts, encoding components of the Ca2+ toolkit, was analyzed in two OAC datasets: the Cancer Genome Atlas [via the University of Alabama Cancer (UALCAN) portal] and the oesophageal-cancer, clinical, and molecular stratification [Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS)] dataset. Effects of differential expression of these genes on patient survival were determined using Kaplan-Meier log-rank tests. OAC grade- and metastatic-stage status was investigated for a subset of genes. Adjustment for the multiplicity of testing was made throughout. Results: Of the 275 Ca2+-toolkit genes analyzed, 75 displayed consistent changes in expression between OAC and normal tissue in both datasets. The channel-encoding genes, N-methyl-D-aspartate receptor 2D (GRIN2D), transient receptor potential (TRP) ion channel classical or canonical 4 (TRPC4), and TRP ion channel melastatin 2 (TRPM2) demonstrated the greatest increase in expression in OAC in both datasets. Nine genes were consistently upregulated in both datasets and were also associated with improved survival outcomes. The 6 top-ranking genes for the weighted significance of altered expression and survival outcomes were selected for further analysis: voltage-gated Ca2+ channel subunit α 1D (CACNA1D), voltage-gated Ca2+ channel auxiliary subunit α2 δ4 (CACNA2D4), junctophilin 1 (JPH1), acid-sensing ion channel 4 (ACCN4), TRPM5, and secretory pathway Ca2+ ATPase 2 (ATP2C2). CACNA1D, JPH1, and ATP2C2 were also upregulated in advanced OAC tumor grades and nodal-metastatic stages in both datasets. Conclusions: This study has unveiled alterations of the Ca2+ toolkit in OAC, compared to normal tissue. Such Ca2+ signalling findings are consistent with those from studies on other cancers. Genes that were consistently upregulated in both datasets might represent useful markers for patient diagnosis. Genes that were consistently upregulated, and which were associated with improved survival, might be useful markers for patient outcome. These survival-associated genes may also represent targets for the development of novel chemotherapeutic agents.
ABSTRACT
[This corrects the article DOI: 10.18632/oncotarget.15182.].
ABSTRACT
Ubiquitin/ISG15-conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post-translational protein modification that conjugates the ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all-trans-retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200-fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation-two cell line models were employed: the human APL cell line NB4 and its ATRA-resistant NB4R counterpart, as well as the ATRA-sensitive human AML HL60 cells along with their ATRA-resistant subclone-HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA-sensitive HL60 AML cells, but not in the ATRA-resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA-mediated UBE2L6 depletion in NB4 cells impeded ATRA-mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA-induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA-induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA-induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.
Subject(s)
Cell Differentiation , Leukemia, Promyelocytic, Acute/pathology , Protein Processing, Post-Translational , Tretinoin/pharmacology , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Cell Differentiation/drug effects , Cell Line, Tumor , Gene Expression Regulation, Leukemic , Gene Knockdown Techniques , Humans , Leukemia, Promyelocytic, Acute/genetics , Neutrophils/metabolism , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Pancreatic cancer represents a major challenge in oncology. Poor permeability of the pancreas and resistance to currently available therapies are impediments to improved patient survival. By transiently increasing cell membrane porosity and increasing drug uptake, Electrochemotherapy (ECT) has the potential to overcome these issues. In this study, we have evaluated the response of human and murine pancreatic cancer cells, in vitro, to electroporation in combination with Bleomycin, Cisplatin, or Oxaliplatin (ECT). The cytotoxic actions of all three drugs are potentiated when combined with electroporation in these cells. The biochemical and morphological changes post ECT are associated with immunogenic cell death that occurs with necroptosis rather than apoptosis. Moreover, ECT-induced cell death is rescued by Nec-1 suggesting that necroptosis may play a role in cell death mediated by cancer therapies.
ABSTRACT
Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a "differentiation block." Pharmacological override of this block is one attractive avenue of therapy, termed "differentiation therapy." The most successful example of this therapeutic strategy is the use of the physiologic retinoid all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). In this chapter, we will outline the methods used to characterize the mechanisms mobilized by retinoid signaling and will use the activation of a key regulator of autophagy, ATG7, as an example of the functional characterization of a retinoid regulated gene during differentiation. We will discuss how lentiviral delivery of shRNA constructs into cultured APL cells, such as NB4, can be used to functionally deplete key proteins. We will also describe how the effect of protein knockdown on ATRA-induced differentiation and autophagy can be assessed using quantitative PCR, Western blotting, and flow cytometry.
Subject(s)
Autophagy-Related Protein 7/genetics , Leukemia, Myeloid, Acute/genetics , RNA, Small Interfering/pharmacology , Tretinoin/pharmacology , Autophagy , Cell Differentiation , Flow Cytometry , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Leukemia, Myeloid, Acute/metabolism , Proteomics , Signal Transduction , Tumor Cells, CulturedABSTRACT
Esophageal cancer remains a poor prognosis cancer due to advanced stage of presentation and drug resistant disease. To understand the molecular mechanisms influencing response to chemotherapy, we examined genes that are differentially expressed between drug sensitive, apoptosis competent esophageal cancer cells (OE21, OE33, FLO-1) and those which are more resistant and do not exhibit apoptosis (KYSE450 and OE19). Members of the ISG15 (ubiquitin-like) protein modification pathway, including UBE2L6 and ISG15, were found to be more highly expressed in the drug sensitive cell lines. In this study, we evaluated the contribution of these proteins to the response of drug sensitive cells. Depletion of UBE2L6 or ISG15 with siRNA did not influence caspase-3 activation or nuclear fragmentation following treatment with 5-fluorouracil (5-FU). We assessed autophagy by analysis of LC3II expression and Cyto-ID staining. Depletion of either ISG15 or UBE2L6 resulted in enhanced endogenous autophagic flux. An increase in autophagic flux was also observed following treatment with cytotoxic drugs (5-FU, rapamycin). In ISG15 depleted cells, this increase in autophagy was associated with improved recovery of drug treated cells. In contrast, UBE2L6 depleted cells, did not show enhanced recovery. UBE2L6 may therefore influence additional targets that limit the pro-survival effect of ISG15 depletion. These data identify UBE2L6 and ISG15 as novel inhibitors of autophagy, with the potential to influence chemosensitivity in esophageal cancer cells.
Subject(s)
Autophagy/genetics , Cytokines/genetics , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitins/genetics , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Autophagy/drug effects , Blotting, Western , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cytokines/metabolism , Drug Resistance, Neoplasm/drug effects , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Fluorouracil/pharmacology , Gene Expression Profiling/methods , Humans , Microtubule-Associated Proteins/metabolism , RNA Interference , Signal Transduction/drug effects , Signal Transduction/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitins/metabolismABSTRACT
This article discusses the main principles of infection prevention and control in non-acute healthcare settings. It explores the use of a set of ten tools developed by the Infection Control Nurses Association (ICNA) to audit infection prevention and control, using the standard statements and criteria within the tools as a checklist. The results of the audit of facilities, commodities and practice using the ICNA audit tools will help staff to identify areas of best practice and areas where improvements are needed to enhance patient care.
Subject(s)
Chronic Disease/nursing , Cross Infection/prevention & control , Infection Control/methods , Long-Term Care/methods , Skilled Nursing Facilities/organization & administration , Benchmarking , Cross Infection/etiology , Cross Infection/transmission , Evidence-Based Medicine , Guideline Adherence/standards , Hand Disinfection , Housekeeping, Hospital , Humans , Infection Control/standards , Long-Term Care/standards , Medical Waste Disposal , Nursing Audit , Nursing Evaluation Research , Practice Guidelines as Topic , Protective Clothing , Specimen Handling , Sterilization , Universal PrecautionsABSTRACT
Many epithelial cancers, particularly gastrointestinal tract cancers, remain poor prognosis diseases, due to resistance to cytotoxic therapy and local or metastatic recurrence. We have previously shown that apoptosis incompetent esophageal cancer cells induce autophagy in response to chemotherapeutic agents and this can facilitate their recovery. However, known pharmacological inhibitors of autophagy could not enhance cytotoxicity. In this study, we have examined two well known, clinically approved autophagy inducers, rapamycin and lithium, for their effects on chemosensitivity in apoptosis incompetent cancer cells. Both lithium and rapamycin were shown to induce autophagosomes in esophageal and colorectal cancer cells by western blot analysis of LC3 isoforms, morphology and FACS quantitation of Cyto-ID or mCherry-GFP-LC3. Analysis of autophagic flux indicates inefficient autophagosome processing in lithium treated cells, whereas rapamycin treated cells showed efficient flux. Viability and recovery was assessed by clonogenic assays. When combined with the chemotherapeutic agent 5-fluorouracil, rapamycin was protective. In contrast, lithium showed strong enhancement of non-apoptotic cell death. The combination of lithium with 5-fluorouracil or oxaliplatin was then tested in the syngenic mouse (balb/c) colorectal cancer model--CT26. When either chemotherapeutic agent was combined with lithium a significant reduction in tumor volume was achieved. In addition, survival was dramatically increased in the combination group (p < 0.0001), with > 50% of animals achieving long term cure without re-occurrence (> 1 year tumor free). Thus, combination treatment with lithium can substantially improve the efficacy of chemotherapeutic agents in apoptosis deficient cancer cells. Induction of compromised autophagy may contribute to this cytotoxicity.
Subject(s)
Autophagy/drug effects , Colorectal Neoplasms/pathology , Esophageal Neoplasms/pathology , Lithium Chloride/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Drug Synergism , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/metabolism , Female , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Genes, Reporter , Humans , Lithium Chloride/therapeutic use , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/therapeutic use , Oxaliplatin , Sirolimus/pharmacology , Sirolimus/therapeutic use , Transplantation, HeterologousABSTRACT
Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has achieved success only in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In the study described here, we investigated the functional importance of autophagy in APL cell differentiation. We found that autophagy is increased during all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of the APL cell line NB4 and that this is associated with increased expression of LC3II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we found that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Cell Differentiation/drug effects , Leukemia, Promyelocytic, Acute/drug therapy , Tretinoin/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Antirheumatic Agents/pharmacology , Autophagy-Related Protein 7 , Autophagy-Related Protein 8 Family , Chloroquine/pharmacology , Granulocytes/metabolism , Granulocytes/pathology , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolismABSTRACT
Resistance to tyrosine kinase inhibitors (TKIs) remains a limitation to the treatment of chronic myeloid leukaemia (CML), due in part, to the induction of autophagy. We examined whether disruption of autophagy with the pharmacological agents, brefeldin A, vincristine and chloroquine, improves the cytotoxicity of imatinib. In K562 CML cells, all drugs tested, in combination with imatinib impaired the expression or cellular distribution of LC3 and Beclin 1 (autophagy markers) and reduced the recovery of cells following drug withdrawal. The combination of imatinib and an agent that impedes autophagy demonstrates impressive potential as a more curative regime for CML.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Benzamides/pharmacology , Brefeldin A/pharmacology , Chloroquine/pharmacology , Piperazines/pharmacology , Pyrimidines/pharmacology , Vincristine/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Drug Synergism , Humans , Imatinib Mesylate , K562 CellsABSTRACT
We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A 1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Autophagy/physiology , Carcinoma, Squamous Cell/drug therapy , Drug Resistance, Neoplasm , Esophageal Neoplasms/drug therapy , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/rehabilitation , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Autophagy/drug effects , Autophagy/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/rehabilitation , Caspase 3/metabolism , Cell Survival/genetics , Cisplatin/pharmacology , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Esophageal Neoplasms/rehabilitation , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic/physiology , Humans , Recovery of Function/genetics , Tumor Cells, Cultured , Up-Regulation/genetics , Up-Regulation/physiologyABSTRACT
Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.