RESUMO
PURPOSE: Cholangiocarcinoma is a fatal tumor with limited therapeutic options. We have reported that calmodulin antagonists tamoxifen and trifluoperazine induced apoptosis in cholangiocarcinoma cells. Here, we determined the effects of tamoxifen on tumorigenesis and the molecular mechanisms of tamoxifen-induced apoptosis. EXPERIMENTAL DESIGN: Nude mice xenograft model of cholangiocarcinoma was used and tamoxifen was given i.p. and intratumorally. Cholangiocarcinoma cells were used to characterize molecular mechanisms of tamoxifen-induced apoptosis in vitro. RESULTS: I.p. or intratumoral injection of tamoxifen decreased cholangiocarcinoma tumorigenesis by 40% to 80% in nude mice. In cells isolated from tumor xenografts, tamoxifen inhibited phosphorylation of AKT (pAKT) and cellular FLICE like inhibitory protein (c-FLIP). Immunohistochemical analysis further showed that pAKT was identified in all nontreated tumors but was absent in tamoxifen-treated tumors. In vitro, tamoxifen activated caspase-8 and caspase-10, and their respective inhibitors partially blocked tamoxifen-induced apoptosis. Overexpression of c-FLIP inhibited tamoxifen-induced apoptosis and enhanced tumorigenesis of cholangiocarcinoma cells in nude mice, whereas deletion of the calmodulin-binding domain on c-FLIP restored the sensitivity to tamoxifen and inhibited tumorigenesis. With two additional cholangiocarcinoma cell lines, we confirmed that the expression of FLIP is an important factor in mediating spontaneous and tamoxifen-induced apoptosis. CONCLUSIONS: Thus, tamoxifen inhibits cholangiocarcinoma tumorigenesis in nude mice. Tamoxifen-induced apoptosis is partially dependent on caspases, inhibition of pAKT, and FLIP expression. Further, calmodulin-FLIP binding seems to be important in FLIP-mediated resistance to tamoxifen. Therefore, the present studies support the concept that tamoxifen may be used as a therapy for cholangiocarcinoma and possibly other malignancies in which the calmodulin targets AKT and c-FLIP play important roles in the tumor pathogenesis.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Calmodulina/antagonistas & inibidores , Colangiocarcinoma/tratamento farmacológico , Tamoxifeno/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Calmodulina/fisiologia , Caspases/fisiologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tamoxifeno/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Calmodulina/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Apoptose , Sítios de Ligação , Cálcio/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
We have previously demonstrated that the antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells partially through Fas-mediated apoptosis pathways. Recently, CaM has been shown to bind to Fas, which is regulated during Fas or CaM antagonist-mediated apoptosis in Jurkat cells and osteoclasts. Accordingly, the present studies were designed to determine whether Fas interacts with CaM in cholangiocarcinoma cells and to elucidate its role in regulating Fas-mediated apoptosis. CaM bound to Fas in cholangiocarcinoma cells. CaM was identified in the Fas-mediated death inducing signaling complex (DISC). The amount of CaM recruited into the DISC was increased after Fas-stimulation, a finding confirmed by immunofluorescent analysis that demonstrated increased membrane co-localization of CaM and Fas upon Fas-stimulation. Consistently, increased Fas microaggregates in response to Fas-stimulation were found to bind to CaM. Fas-induced recruitment of CaM into the DISC was inhibited by the Ca(2+) chelator, EGTA, and the CaM antagonist, trifluoperazine (TFP). TFP decreased DISC-induced cleavage of caspase-8. Further, inhibition of actin polymerization, which has been demonstrated to abolish DISC formation, inhibited the recruitment of CaM into the DISC. These results suggest an important role of CaM in mediating DISC formation, thus regulating Fas-mediated apoptosis in cholangiocarcinoma cells. Characterization of the role of CaM in Fas-mediated DISC formation and apoptosis signaling may provide important insights in the development of novel therapeutic targets for cholangiocarcinoma.
Assuntos
Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Calmodulina/metabolismo , Colangiocarcinoma/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína Ligante Fas/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Caspase 8/metabolismo , Colangiocarcinoma/patologia , Humanos , Imunoprecipitação , Ligação Proteica , Transdução de Sinais , Trifluoperazina/farmacologia , Células Tumorais CultivadasRESUMO
A 63-year-old white female with a history of metastatic breast cancer to the liver developed acute-onset nausea, vomiting, mental status change, and generalized seizures following transarterial chemoembolization using doxorubicin. The patient was hospitalized with the above symptoms immediately following transarterial chemoembolization using drug eluting bead doxorubicin into the right hepatic artery. The patient developed intractable nausea, vomiting, and abdominal pain and had a generalized tonic-clonic seizure lasting for 40 seconds, approximately 24 hours after the procedure. The patient was confused and lethargic for 2 days with progressive improvement in her mental status. Her neurological examination showed encephalopathy with disorientation to time, place or person, and she also had a glassy look. Cranial nerves were normal other than lack of response to threat stimulus bilaterally; motor and sensory examination was unremarkable. Initial blood pressure was 130/90 mm Hg and routine chemistry and complete blood count on admission were within normal limits. The cerebrospinal fluid analysis showed clear and colorless fluid with glucose of 56 mg/dL, protein of 42 mg/dL, white blood cells of 2/µL, and red blood cells of 10/µL and did not show any evidence of infectious or toxic etiology on encephalopathy. Continuous electro encephalography showed diffuse slowing but no epileptiform discharges. The magnetic resonance imaging (MRI) revealed increased signal intensity in the bilateral parieto-occipital area, right more than the left, on fluid-attenuated inversion recovery, apparent diffusion coefficient, and T2-weighted imaging, with no increased signal on diffusion weighted image consistent with vasogenic edema. The patient's symptoms and MRI findings were consistent with diagnosis of posterior reversible encephalopathy syndrome. Resolution of the MRI changes is noted on the follow-up imaging 8 weeks later. Posterior reversible encephalopathy syndrome in this case is most likely related to intra-arterial doxorubicin infusion because of the temporal association between administration, symptom onset, and MRI changes.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Quimioembolização Terapêutica/efeitos adversos , Doxorrubicina/efeitos adversos , Síndrome da Leucoencefalopatia Posterior/induzido quimicamente , Antibióticos Antineoplásicos/administração & dosagem , Edema Encefálico/induzido quimicamente , Edema Encefálico/complicações , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/administração & dosagem , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Convulsões/induzido quimicamente , Convulsões/complicaçõesRESUMO
BACKGROUND: Amitriptyline is a tricyclic antidepressant useful for the treatment of depression. Amiodarone is a class III antiarrhythmic agent used for the treatment of cardiac dysrhythmias. OBJECTIVE: The objective of the current report is to describe the case of a previously asymptomatic patient receiving amitriptyline who developed extrapyramidal symptoms within 1 month of initiating concomitant treatment with amiodarone for atrial fibrillation. CASE SUMMARY: An 82-year-old, right-handed, white woman was brought to the medical center's emergency department with speech difficulty suggesting stroke. She was noted to have continuous orobuccal dyskinesias, upper and lower extremity shaking, and dry mouth. Once it was determined that no other focal neurologic findings indicated stroke, her medications were reviewed. The patient had been taking amitriptyline 50 mg/d for the past year for insomnia without any adverse events. However, 1 month before presentation, she also initiated treatment with amiodarone 200 mg/d for atrial fibrillation and had developed the symptoms of concern shortly thereafter. The patient's amitriptyline treatment was discontinued and she received benzotropine for extrapyramidal symptoms from amitriptyline toxicity. She experienced complete resolution of dysarthric speech and limb shaking within 2 days. A total score of 7 was achieved using Naranjo's adverse drug reaction causality algorithm, suggesting amitriptyline was a probable cause of these adverse events. CONCLUSION: This was a probable case of extrapyramidal symptoms in an elderly woman who began using amiodarone while also taking amitriptyline.
Assuntos
Amiodarona/efeitos adversos , Amitriptilina/efeitos adversos , Doenças dos Gânglios da Base/induzido quimicamente , Idoso de 80 Anos ou mais , Amiodarona/uso terapêutico , Amitriptilina/uso terapêutico , Antiarrítmicos/efeitos adversos , Antiarrítmicos/uso terapêutico , Antidepressivos Tricíclicos/efeitos adversos , Antidepressivos Tricíclicos/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Interações Medicamentosas , Feminino , HumanosRESUMO
Stimulation of Fas-mediated apoptosis has been promoted as a potential therapy for many cancers, including cholangiocarcinoma. We have previously reported that Fas-resistant, but not Fas-sensitive, cholangiocarcinoma cells are tumorigenic in nude mice. The present studies sought to identify molecular targets that promote Fas-mediated apoptosis in cholangiocarcinoma. We found that Fas-resistant cholangiocarcinoma cells exhibited increased constitutive phosphorylation of AKT compared with Fas-sensitive cells. Increased phosphorylation of AKT was also demonstrated in human cholangiocarcinoma tumors and was evident in a mouse xenograft cholangiocarcinoma model. Furthermore, we found that 3,3'-diindolylmethane (DIM), a vegetable autolysis product, promoted Fas-mediated apoptosis of cholangiocarcinoma cells. DIM inhibited phosphorylation of AKT and activation of FLICE-like-inhibitory-protein (FLIP). Inhibition of phosphatidylinositol 3-kinase/AKT decreased FLIP activation and promoted Fas-mediated apoptosis. By contrast, adenovirus-mediated constitutively activated AKT protected cholangiocarcinoma cells from Fas-mediated apoptosis. Decreased activation of extracellular signal-regulated kinase and nuclear factor-kappaB and increased activation of caspase-3, -8, and -9 were associated with inhibition of AKT and FLIP. These results support AKT and FLIP as potential molecular targets and DIM as a potent compound for cholangiocarcinoma intervention.