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BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies targeting glioblastoma (GBM)-associated antigens such as interleukin-13 receptor subunit alpha-2 (IL-13Rα2) have achieved limited clinical efficacy to date, in part due to an immunosuppressive tumor microenvironment (TME) characterized by inhibitory molecules such as transforming growth factor-beta (TGF-ß). The aim of this study was to engineer more potent GBM-targeting CAR-T cells by countering TGF-ß-mediated immune suppression in the TME. METHODS: We engineered a single-chain, bispecific CAR targeting IL-13Rα2 and TGF-ß, which programs tumor-specific T cells to convert TGF-ß from an immunosuppressant to an immunostimulant. Bispecific IL-13Rα2/TGF-ß CAR-T cells were evaluated for efficacy and safety against both patient-derived GBM xenografts and syngeneic models of murine glioma. RESULTS: Treatment with IL-13Rα2/TGF-ß CAR-T cells leads to greater T-cell infiltration and reduced suppressive myeloid cell presence in the tumor-bearing brain compared to treatment with conventional IL-13Rα2 CAR-T cells, resulting in improved survival in both patient-derived GBM xenografts and syngeneic models of murine glioma. CONCLUSION: Our findings demonstrate that by reprogramming tumor-specific T-cell responses to TGF-ß, bispecific IL-13Rα2/TGF-ß CAR-T cells resist and remodel the immunosuppressive TME to drive potent anti-tumor responses in GBM.
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Glioblastoma (GBM) is the most common primary malignant brain tumor, with a median overall survival of less than 2 years and a nearly 100% mortality rate under standard therapy that consists of surgery followed by combined radiochemotherapy. Therefore, new therapeutic strategies are urgently needed. The success of chimeric antigen receptor (CAR) T cells in hematological cancers has prompted preclinical and clinical investigations into CAR-T-cell treatment for GBM. However, recent trials have not demonstrated any major success. Here, we delineate existing challenges impeding the effectiveness of CAR-T-cell therapy for GBM, encompassing the cold (immunosuppressive) microenvironment, tumor heterogeneity, T-cell exhaustion, local and systemic immunosuppression, and the immune privilege inherent to the central nervous system (CNS) parenchyma. Additionally, we deliberate on the progress made in developing next-generation CAR-T cells and novel innovative approaches, such as low-intensity pulsed focused ultrasound, aimed at surmounting current roadblocks in GBM CAR-T-cell therapy.
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Glioblastoma , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Glioblastoma/terapia , Glioblastoma/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Microambiente Tumoral/imunologia , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/imunologia , Linfócitos T/imunologia , AnimaisRESUMO
Cancers and neurological disorders are two major types of diseases in humans. We developed the concept called the "Aberrant Cell Cycle Disease (ACCD)" due to the accumulating evidence that shows that two different diseases share the common mechanism of aberrant cell cycle re-entry. The aberrant cell cycle re-entry is manifested as kinase/oncoprotein activation and tumor suppressor (TS) inactivation, which are associated with both tumor growth in cancers and neuronal death in neurological disorders. Therefore, some cancer therapies (e.g., kinase/oncogene inhibition and TS elevation) can be leveraged for neurological treatments. MicroRNA (miR/miRNA) provides a new style of drug-target binding. For example, a single tumor suppressor miRNA (TS-miR/miRNA) can bind to and decrease tens of target kinases/oncogenes, producing much more robust efficacy to block cell cycle re-entry than inhibiting a single kinase/oncogene. In this review, we summarize the miRNAs that are altered in both cancers and neurological disorders, with an emphasis on miRNA drugs that have entered into clinical trials for neurological treatment.
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The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.
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Glioma , Microambiente Tumoral , Camundongos , Humanos , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Encéfalo/metabolismo , Linfócitos T , Serina-Treonina Quinases TOR/metabolismoRESUMO
The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.
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Chromosomal instability (CIN) is a driver of cancer metastasis1-4, yet the extent to which this effect depends on the immune system remains unknown. Using ContactTracing-a newly developed, validated and benchmarked tool to infer the nature and conditional dependence of cell-cell interactions from single-cell transcriptomic data-we show that CIN-induced chronic activation of the cGAS-STING pathway promotes downstream signal re-wiring in cancer cells, leading to a pro-metastatic tumour microenvironment. This re-wiring is manifested by type I interferon tachyphylaxis selectively downstream of STING and a corresponding increase in cancer cell-derived endoplasmic reticulum (ER) stress response. Reversal of CIN, depletion of cancer cell STING or inhibition of ER stress response signalling abrogates CIN-dependent effects on the tumour microenvironment and suppresses metastasis in immune competent, but not severely immune compromised, settings. Treatment with STING inhibitors reduces CIN-driven metastasis in melanoma, breast and colorectal cancers in a manner dependent on tumour cell-intrinsic STING. Finally, we show that CIN and pervasive cGAS activation in micronuclei are associated with ER stress signalling, immune suppression and metastasis in human triple-negative breast cancer, highlighting a viable strategy to identify and therapeutically intervene in tumours spurred by CIN-induced inflammation.
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Instabilidade Cromossômica , Progressão da Doença , Neoplasias , Humanos , Benchmarking , Comunicação Celular , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Microambiente Tumoral , Interferon Tipo I/imunologia , Metástase Neoplásica , Estresse do Retículo Endoplasmático , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
Neuronal activity-driven mechanisms impact glioblastoma cell proliferation and invasion 1-7 , and glioblastoma remodels neuronal circuits 8,9 . Distinct intratumoral regions maintain functional connectivity via a subpopulation of malignant cells that mediate tumor-intrinsic neuronal connectivity and synaptogenesis through their transcriptional programs 8 . However, the effects of tumor-intrinsic neuronal activity on other cells, such as immune cells, remain unknown. Here we show that regions within glioblastomas with elevated connectivity are characterized by regional immunosuppression. This was accompanied by different cell compositions and inflammatory status of tumor-associated macrophages (TAMs) in the tumor microenvironment. In preclinical intracerebral syngeneic glioblastoma models, CRISPR/Cas9 gene knockout of Thrombospondin-1 (TSP-1/ Thbs1 ), a synaptogenic factor critical for glioma-induced neuronal circuit remodeling, in glioblastoma cells suppressed synaptogenesis and glutamatergic neuronal hyperexcitability, while simultaneously restoring antigen-presentation and pro-inflammatory responses. Moreover, TSP-1 knockout prolonged survival of immunocompetent mice harboring intracerebral syngeneic glioblastoma, but not of immunocompromised mice, and promoted infiltrations of pro-inflammatory TAMs and CD8+ T-cells in the tumor microenvironment. Notably, pharmacological inhibition of glutamatergic excitatory signals redirected tumor-associated macrophages toward a less immunosuppressive phenotype, resulting in prolonged survival. Altogether, our results demonstrate previously unrecognized immunosuppression mechanisms resulting from glioma-neuronal circuit remodeling and suggest future strategies targeting glioma-neuron-immune crosstalk may open up new avenues for immunotherapy.
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STING signaling in cancer is a crucial component of response to immunotherapy and other anti-cancer treatments. Currently, there is no robust method of measuring STING activation in cancer. Here, we describe an immunohistochemistry-based assay with digital pathology assessment of STING in tumor cells. Using this novel approach in estrogen receptor-positive (ER+) and ER- breast cancer, we identify perinuclear-localized expression of STING (pnSTING) in ER+ cases as an independent predictor of good prognosis, associated with immune cell infiltration and upregulation of immune checkpoints. Tumors with low pnSTING are immunosuppressed with increased infiltration of "M2"-polarized macrophages. In ER- disease, pnSTING does not appear to have a significant prognostic role with STING uncoupled from interferon responses. Importantly, a gene signature defining low pnSTING expression is predictive of poor prognosis in independent ER+ datasets. Low pnSTING is associated with chromosomal instability, MYC amplification and mTOR signaling, suggesting novel therapeutic approaches for this subgroup.
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BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
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Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias da Próstata/terapia , Hipofracionamento da Dose de Radiação , Microambiente Tumoral , Animais , Antígeno B7-H1/análise , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologiaRESUMO
PURPOSE: Glycogen synthase kinase 3 (GSK3) is a key controlling element of many cellular processes including cell-cycle progression and recent studies suggest that GSK3 is a potential anticancer target. Changes in glucose metabolism associated with GSK3 inhibition may impact on lipid synthesis, whilst lipid metabolites can act as molecular response markers. METHODS: Here, SKBr3 breast and HCT8 colorectal cancer cells were treated with the GSK3 inhibitor SB216763, and [14C (U)] glucose and [3H] choline incorporation into lipids was determined. Cell extracts from treated cells were subject to 31P NMR spectroscopy. RESULTS: SB216763 treatment decreased choline incorporation into lipids and caused an accumulation of CDP-choline which was accompanied by decreased conversion of glucose into lipid components. CONCLUSION: SB216763 profoundly inhibits phospholipid synthesis in cancer cells which demonstrate accumulation of CDP-choline detectable by 31P NMR spectroscopy. Metabolic changes in lipid metabolism present potential response markers to drugs targeting GSK3.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Citidina Difosfato Colina/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Indóis/farmacologia , Maleimidas/farmacologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Colina/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Glucose/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Fosfatidilcolinas/metabolismoRESUMO
Signal transduction pathways, which regulate cell growth and survival, are up-regulated in many cancers and there is considerable interest in their pharmaceutical modulation for cancer treatment. However inhibitors of single pathway components induce feedback mechanisms that overcome the growth moderating effect of the inhibitor. Combination treatments have been proposed to provide a more complete pathway inhibition. Here the effect of dual treatment of cancer cells with a pan-Akt and a pan-mTOR inhibitor was explored. Breast (SKBr3 and MDA-MB-468) and colorectal (HCT8) cancer cells were treated with the pan-Akt inhibitor MK2206 and pan-mTOR inhibitor AZD8055. Cytotoxic effect of the two drugs were determined using the MTT assay and the Combination Index and isobolomic analysis used to determine the nature of the interaction of the two drugs. Flow cytometry and western blot were employed to demonstrate drug effects on cell cycle distribution and phosph-Aktser473 expression. Radiolabelled ([methyl-3H]) Choline uptake was measured in control and drug-treated cells to determine the modulatory effects of the drugs on choline incorporation. The two drugs acted synergistically to inhibit the growth rate of each cancer cell line. Flow cytometry demonstrated G0/G1 blockade with MK2206 and AZD8055 which was greater when cells were treated with both drugs. The incorporation of [methyl-3H] choline was found be decreased to a greater extent in cells treated with both drugs compared with cells treated with either drug alone. Conclusions Pan-mTOR and pan-Akt inhibition may be highly effective in cancer treatment and measuring changes in choline uptake could be useful in detecting efficacious drug combinations.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Colina/metabolismo , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Feminino , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Humanos , Morfolinas/administração & dosagem , Fosforilação , Células Tumorais CultivadasRESUMO
Androgen receptor (AR) activation is the primary driving factor in prostate cancer which is initially responsive to castration but then becomes resistant (castration-resistant prostate cancer (CRPC)). CRPC cells still retain the functioning AR which can be targeted by other therapies. A recent promising development is the use of inhibitors (Epi-1) of protein-protein interaction to inhibit AR-activated signalling. Translating novel therapies into the clinic requires sensitive early response indicators. Here potential response markers are explored. Growth inhibition of prostate cancer cells with flutamide, paclitaxel, and Epi-1 was measured using the MTT assay. To simulate choline-PET scans, pulse-chase experiments were carried out with [3H-methyl]choline and proportion of phosphorylated activity was determined after treatment with growth inhibitory concentrations of each drug. Extracts from treated cells were also subject to 31P-NMR spectroscopy. Cells treated with flutamide demonstrated decreased [3H-methyl]choline phosphorylation, whilst the proportion of phosphorylated [3H-methyl]choline that was present in the lipid fraction was increased in Epi-1-treated cells. Phospholipid breakdown products, glycerophosphorylcholine and glycerophosphoethanolamine levels, were shown by 31P-NMR spectroscopy to be decreased to undetectable levels in cells treated with Epi-1. LNCaP cells responding to treatment with novel protein-protein interaction inhibitors suggest that 31P-NMR spectroscopy may be useful in detecting response to this promising therapy.
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Fosfolipídeos/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colina/metabolismo , Flutamida/farmacologia , Humanos , Masculino , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Propano/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Espectroscopia de Prótons por Ressonância Magnética , Resultado do Tratamento , Trítio/metabolismoRESUMO
Akt is an intracellular signalling pathway that serves as an essential link between cell surface receptors and cellular processes including proliferation, development and survival. The pathway has many downstream targets including glycogen synthase kinase3 which is a major regulatory kinase for cell cycle transit as well as controlling glycogen synthase activity. The Akt pathway is frequently up-regulated in cancer due to overexpression of receptors such as the epidermal growth factor receptor, or mutation of signalling pathway kinases resulting in inappropriate survival and proliferation. Consequently anticancer drugs have been developed that target this pathway. MDA-MB-468 breast and HCT8 colorectal cancer cells were treated with inhibitors including LY294002, MK2206, rapamycin, AZD8055 targeting key kinases in/associated with Akt pathway and the consistency of changes in 31P-NMR-detecatable metabolite content of tumour cells was examined. Treatment with the Akt inhibitor MK2206 reduced phosphocholine levels in MDA-MB-468 cells. Treatment with either the phosphoinositide-3-kinase inhibitor, LY294002 and pan-mTOR inhibitor, AZD8055 but not pan-Akt inhibitor MK2206 increased uridine-5'-diphosphate-hexose cell content which was suppressed by co-treatment with glycogen synthase kinase 3 inhibitor SB216763. This suggests that there is an Akt-independent link between phosphoinositol-3-kinase and glycogen synthase kinase3 and demonstrates the potential of 31P-NMR to probe intracellular signalling pathways.
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Quinase 3 da Glicogênio Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Receptores ErbB/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Espectroscopia de Ressonância Magnética/métodos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
INTRODUCTION: The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined. METHODS: MDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [3H] Radiolabelled metabolites were determined using thin layer chromatography. RESULTS: Metformin-treated cells exhibited decreased formation of [3H]phosphocholine but increased accumulation of [3H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [14C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [14C(U)]glucose. CONCLUSION: This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.
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Neoplasias da Mama/metabolismo , Metformina/farmacologia , Fosfolipídeos/metabolismo , Linhagem Celular Tumoral , Colina/metabolismo , Feminino , Humanos , Fosfatidilcolinas/metabolismoRESUMO
BACKGROUND: Anticancer drug treatment, particularly with anthracyclines, is frequently associated with cardiotoxicity, an effect exacerbated by trastuzumab. Several compounds are in use clinically to attenuate the cardiac-damaging effects of chemotherapy drugs, including angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, the anti-diabetic drug metformin, and dexrazoxane. However, there is concern that the cardiac-preserving mechanisms of these drugs may also limit the anticancer efficacy of the chemotherapeutic agents. MATERIALS AND METHODS: Herein two breast cancer cell lines, SKBr3 and BT474, overexpressing human epithelial receptor 2 (HER2), the target of the humanised antibody trastuzumab, were treated with a range of concentrations (20-2000 nM) of doxorubicin with and without trastuzumab in the presence of clinically relevant doses of the ACE inhibitor enalapril, the beta-blocker carvedilol, metformin or dexrazoxane, and cell survival determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: None of the drugs reduced the anticancer effect of doxorubicin or trastuzumab (nor of the two drugs combined). Using Chou and Talalay's combination index, dexrazoxane and doxorubicin were found to act synergistically on the SKBr3 cells. (18)F-Fluoro-2-deoxy-D-glucose ((18)F-FDG) incorporation was reduced by treatment of SKBr3 cells with doxorubicin and this was shown to be due to reduced phosphorylation of (18)F-FDG in doxorubicin-treated cells. Treatment of SKBr3 cells with doxorubicin and dexrazoxane further reduced (18)F-FDG incorporation, indicating that the synergy in the cytotoxicity of these two drugs was reflected in their combined effect on (18)F-FDG incorporation. CONCLUSION: Commonly administered cardioprotective drugs do not interfere with anticancer activity of doxorubicin or tratsuzumab. Further studies to establish the effect of cardioprotective drugs on anticancer drug efficacy would be beneficial.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cardiomiopatias/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carbazóis/farmacologia , Cardiomiopatias/induzido quimicamente , Cardiotônicos/farmacologia , Fármacos Cardiovasculares/toxicidade , Carvedilol , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dexrazoxano/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Interações Medicamentosas , Enalapril/farmacologia , Feminino , Fluordesoxiglucose F18 , Humanos , Metformina/farmacologia , Propanolaminas/farmacologia , Cintilografia , Compostos Radiofarmacêuticos , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologiaRESUMO
PURPOSES: Metformin, currently undergoing clinical trials as an adjuvant for the treatment of breast cancer, modulates the activity of key intracellular signalling molecules which affect 2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) incorporation. Here, we investigate the effect of drugs used in the treatment of breast cancer combined with metformin on [(18)F]FDG incorporation in HER2- or HER1-overexpressing breast cancer cells to determine whether or not metformin may obscure changes in [(18)F]FDG incorporation induced by clinically utilised anticancer drugs in the treatment of breast cancer. METHODS: Three breast cancer cell lines expressing HER2 and one HER2 negative but HER1 positive were exposed to metformin, doxorubicin and trastuzumab or cetuximab. Cytotoxicity was measured by the MTT assay. Expression of active (phospho-) AMPK, PKB (Akt) and ERK was determined by Western blotting. [(18)F]FDG incorporation by cells exposed to drug combinations with metformin was determined. Glucose transport was assessed by measuring the initial rate of uptake of [(3)H]O-methyl-D-glucose ([(3)H]OMG). Phosphorylation of [(18)F]FDG was determined in intact cells after exposure to [(18)F]FDG. RESULTS: Phospho-AMPK was increased by metformin in all cell lines whilst phospho-Akt and phospho-ERK expressions were decreased in two. Metformin treatment increased [(18)F]FDG incorporation in all cell lines, and treatment with anti-HER antibodies or doxorubicin only produced minor modulations in the increase induced by metformin alone. Glucose transport was increased in BT474 cells and decreased in SKBr3 and MDA-MB-468 cells after treatment with metformin. The fraction of phosphorylated [(18)F]FDG was increased in metformin-treated cells compared with controls, suggesting that hexokinase efficiency was increased by metformin. CONCLUSION: This is the first study to show that increased [(18)F]FDG incorporation by breast cancer cells induced by metformin overwhelms the effect of doxorubicin and anti-HER treatments on [(18)F]FDG incorporation. Metformin-induced increased [(18)F]FDG incorporation was consistently associated with enhanced [(18)F]FDG phosphorylation.