RESUMEN
We have previously demonstrated that uPA is overexpressed in pancreatic tumors. In an attempt to diagnose these tumors earlier, we sought to determine whether uPA could be identified in endoscopic retrograde cholangiopancreatography obtained brushings in patients with malignant pancreatic and biliary strictures. Secondarily, uPA was measured in the serum of this patient population. uPA overexpression was identified in the cytologic tissue in 8 of 11 patients (72.7%). Serum analysis demonstrated a 2-fold higher concentration of uPA in the pancreaticobiliary cancer patients (1.27 versus 0.56 ng/mL; P = .0182). Also, uPA overexpression correlated with serum levels (P < .0001). This study confirms that uPA can be detected in the ERCP cytologically obtained tissue and is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. A larger sample size will be required to address its value as a sensitive marker for the diagnosis of pancreatic or biliary cancers.
RESUMEN
BCL-2 is the prototypic anti-apoptotic protein involved in the regulation of apoptosis. Overexpression of BCL-2 is common in pancreatic cancer and confers resistance to the apoptotic effect of chemo- and radiotherapy. Although these cellular effects of BCL-2 are traditionally related to pathways involving the mitochondrial membrane, we sought to investigate whether BCL-2 is involved in other signaling pathways regulating cell survival and focused on AKT. We examined the effect of overexpression of BCL-2 in the MIA-PaCa-2 human pancreatic cancer cell line on the function and subcellular location of AKT. We observed that the stable subclones of MIA-PaCa-2 overexpressing BCL-2 demonstrated increased activity of AKT as well as IKK (a downstream target of AKT), increasing the transcriptional activity of NF-kappaB. Using immunoprecipitation techniques, we observed co-immunoprecipitation of AKT and BCL-2. Immunocytochemistry demonstrated co-localization of BCL-2 and AKT, which was abrogated by treatment with HA14-1, a small molecule inhibitor of BH-3-mediated protein interaction by BCL-2. Furthermore, treatment with HA14-1 decreased phosphorylation of AKT and increased sensitivity to the apoptotic effect of the chemotherapeutic agent, paclitaxel. These results demonstrate an additional mechanism of regulation of cell survival mediated by BCL-2, namely through AKT activation, in the MIA-PaCa-2 pancreatic cancer cell line. Therefore, directed inhibition of BCL-2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of pancreatic cancer.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Clonales , ADN Complementario , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Humanos , Inmunohistoquímica , Luciferasas de Renilla/metabolismo , FN-kappa B/fisiología , Nitrilos/farmacología , Paclitaxel/farmacología , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
Extremity soft tissue sarcomas (STS) represent a rare, heterogeneous malignancy. Surgery is the primary treatment for patients with no evidence of metastatic disease, and for small low-grade superficial tumors in which adequate margins can be obtained, it may be the only therapy indicated. For large, deep tumors or tumors that are close to important neurovascular structures or bone, the addition of radiotherapy to resection has improved local control and increased limb salvage without affecting overall survival. Adjuvant chemotherapy has been an issue of considerable debate. Because 50% of patients with high-risk tumors will develop metastatic disease, effective systemic treatment with chemotherapy is needed. Unfortunately, studies have shown minimal improvement in overall survival when chemotherapy is added to the local treatment of high-risk extremity STS. More recently, a few trials of neoadjuvant chemotherapy consisting of mesna, doxorubicin, ifosfamide, and dacarbazine and high-dose doxorubicin and ifosfamide have shown some early promising results, but at the price of increased toxicity. Targeted therapy has shown some of its best results with gastrointestinal stromal tumors, but so far there has been little success in treating extremity STS. At this time, high-dose adjuvant or neoadjuvant chemotherapy should be given in the setting of a clinical trial to patients with high-risk tumors who can tolerate a potentially toxic chemotherapeutic regimen. The goal of these trials should be to assess new combination therapies, possibly including targeted therapies, for the management of large high-grade, high-risk soft tissue sarcomas.
Asunto(s)
Sarcoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos Clínicos como Asunto , Terapia Combinada , Extremidades , Humanos , Terapia Neoadyuvante , Estadificación de Neoplasias , Radioterapia Adyuvante , Sarcoma/tratamiento farmacológico , Sarcoma/patología , Sarcoma/radioterapia , Sarcoma/cirugíaRESUMEN
Overexpression of the anti-apoptotic protein BCL-2 is frequently observed in small cell lung cancers (SCLC) and is associated with chemoresistance. We examined the signaling pathways involved in upregulation of BCL-2 in SCLC, and whether inhibition of NF-kappaB using the 26S proteasome inhibitor bortezomib had any effect on BCL-2 levels or apoptosis. Mutation of a NF-kappaB site in the BCL-2 promoter reduced promoter activity to less than 20% of the wild-type promoter. Treatment with bortezomib resulted in decreased transcription of the BCL-2 promoter, decreased BCL-2 levels, and induced apoptosis. These data provide the necessary laboratory background for further investigation of bortezomib in the treatment of SCLC.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Carcinoma de Células Pequeñas/patología , Neoplasias Pulmonares/patología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazinas/farmacología , Western Blotting , Bortezomib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Luciferasas/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/farmacología , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
BACKGROUND: BCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies. METHODS: Regulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-kappaB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-kappaB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis. RESULTS: BCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-kappaB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-kappaB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene. CONCLUSIONS: Diverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-kappaB activity and decrease BCL-2 levels.
Asunto(s)
Ácidos Borónicos/farmacología , Neoplasias de la Mama/patología , Ciclina D1/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , FN-kappa B/farmacología , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/patología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , Bortezomib , Femenino , Humanos , Masculino , FN-kappa B/antagonistas & inhibidores , Transducción de Señal , Transcripción Genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Non-small-cell lung cancer (NSCLC) has a poor prognosis. Despite advances in therapy, survival has improved only slightly. The 26S proteasome regulates multiple cellular processes through degradation of ubiquitin-tagged proteins. The proteasome inhibitor, bortezomib (Velcade, formerly PS-341), has been shown to be an active anticancer agent both in vitro and in vivo in multiple tumor types. PURPOSE: To determine the molecular and cellular effects of the proteasome inhibitor in NSCLC as well as to evaluate the effectiveness of sequential treatment with bortezomib and gemcitabine/carboplatin (G/C) chemotherapy both in vitro and in vivo. METHODS: All experiments were performed in the A549 NSCLC cell line. MTT assays were used to evaluate cytotoxicity. Western blotting evaluated protein levels. Measures of apoptosis included FACS analysis, DAPI staining and caspase-3 cleavage. Long-term cell viability was determined using an anchorage-dependent clonogenic assay. Sequential studies were performed in vitro and in vivo. RESULTS: Bortezomib increased p21(waf1/cip1), induced G(2)/M arrest, and triggered a small amount of apoptosis. The apoptotic effect of G/C chemotherapy was eliminated when bortezomib was administered prior to the chemotherapy; however, it was accentuated when the bortezomib was given simultaneously or after the chemotherapy. CONCLUSIONS: Bortezomib improves efficacy in combination with gemcitabine and carboplatin in NSCLC, but sequential effects are important and must be considered when developing therapeutic regimens.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ácidos Borónicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Pirazinas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácidos Borónicos/administración & dosificación , Ácidos Borónicos/farmacología , Bortezomib , Carboplatino/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Pirazinas/administración & dosificación , Pirazinas/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , GemcitabinaRESUMEN
BACKGROUND: When activated, the nuclear factor (NF)-kappaB pathway is a potent cellular signal that inhibits apoptotic cell death. Pancreatic cancer is resistant to the apoptotic effect of chemotherapy, though it is unclear whether this is an inherent feature or a survival signal engaged in response to chemotherapy. We investigated whether pancreatic cancer cells activate the NF-kappaB pathway in response to chemotherapy and whether inhibition of this response altered the apoptotic efficacy of chemotherapy. STUDY DESIGN: We determined NF-kappaB activity after chemotherapy treatment of the MIA-PaCa-2 human pancreatic cancer cell line using both physical (electrophoretic mobility shift assay) and functional (luciferase) techniques. The effect of chemotherapy on transcription of the antiapoptotic gene BCL-2, a target of NF-kappaB, was determined. We examined the effect of inhibition of Akt, an upstream activator of NF-kappaB, on the molecular (NF-kappaB function and BCL-2 transcription) and cellular (apoptosis) effect of chemotherapy. RESULTS: Both the chemotherapeutic agents gemcitabine and paclitaxel activated NF-kappaB and stimulated BCL-2 gene promoter activity. The stimulation of BCL-2 promoter function was directly regulated by NF-kappaB. These cellular responses were blocked by inhibition of Akt. The apoptotic effect of gemcitabine and paclitaxel also was enhanced after Akt inhibition. CONCLUSIONS: Part of the apoptotic resistance of pancreatic cancer may be mediated by activation of the NF-kappaB survival pathway in response to chemotherapy. Inhibition of this response may be an important adjunct to increase the efficacy of chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Desoxicitidina/análogos & derivados , FN-kappa B/fisiología , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Línea Celular Tumoral , Desoxicitidina/farmacología , Genes bcl-2/efectos de los fármacos , Humanos , Paclitaxel/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Éteres Fosfolípidos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , GemcitabinaRESUMEN
HYPOTHESIS: Despite advances in preoperative radiologic imaging, a significant fraction of potentially resectable pancreatic cancers are found to be unresectable at laparotomy. We tested the hypothesis that preoperative serum levels of CA19-9 (cancer antigen) and carcinoembryonic antigen will identify patients with unresectable pancreatic cancer despite radiologic staging demonstrating resectable disease. DESIGN AND SETTING: Academic tertiary care referral center. PATIENTS: From March 1, 1996, to July 31, 2002, 125 patients were identified who underwent surgical exploration for potentially resectable pancreatic cancer based on a preoperative computed tomographic scan; in 89 of them a preoperative tumor marker had been measured. MAIN OUTCOME MEASURES: Preoperative tumor markers (CA19-9 and carcinoembryonic antigen) were correlated with extent of disease at exploration. As CA19-9 is excreted in the biliary system, CA19-9 adjusted for the degree of hyperbilirubinemia was determined and analyzed. RESULTS: Of the 89 patients, 40 (45%) had localized disease and underwent resection, 25 (28%) had locally advanced (unresectable) disease, and 24 (27%) had metastatic disease. The mean adjusted CA19-9 level was significantly lower in those with localized disease than those with locally advanced (63 vs 592; P =.003) or metastatic (63 vs 1387; P<.001) disease. When a threshold adjusted CA19-9 level of 150 was used, the positive predictive value for determination of unresectable disease was 88%. Carcinoembryonic antigen level was not correlated with extent of disease. CONCLUSIONS: Among the patients with resectable pancreatic cancer based on preoperative imaging studies, those with abnormally high serum levels of CA19-9 may have unresectable disease. These patients may benefit from additional staging modalities such as diagnostic laparoscopy to avoid unnecessary laparotomy.
Asunto(s)
Adenocarcinoma/sangre , Antígeno CA-19-9/sangre , Antígeno Carcinoembrionario/sangre , Neoplasias Pancreáticas/sangre , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Valor Predictivo de las Pruebas , Cuidados Preoperatorios , Estudios Retrospectivos , Sensibilidad y Especificidad , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: 26S proteasome inhibitors are a novel class of compounds entering clinical trials as a method to increase tumor sensitivity to standard chemotherapy. We determined the effect of alternate sequencing regimens of a proteasome inhibitor and gemcitabine on molecular and cellular responses in pancreatic cancer cells. MATERIALS AND METHODS: MIA-PaCa-2 human pancreatic cancer cells were treated with the proteasome inhibitor bortezomib either before, simultaneously or following exposure to gemcitabine. Expression of the cell cycle proteins p21(WAF1/CIP1) and p27(KIP1), and the anti-apoptotic protein BCL-2 were determined by Western blotting. Cell cycle changes and immediate or delayed induction of apoptosis were quantitated. RESULTS: Gemcitabine followed by bortezomib induced the greatest induction of apoptosis and long-term inhibition of cell growth. Bortezomib treatment led to accumulation of p21(WAF1/CIP1) and p27(KIP1) and decreased BCL-2; gemcitabine decreased p27(KIP1), induced BCL-2 and had no effect on p21(WAF1/CIP1). When these agents were given in combination or sequence, intermediate changes in these proteins were observed, and the alterations did not correlate with immediate or delayed induction of apoptosis. CONCLUSIONS: Inhibition of the 26S proteasome following chemotherapy appears to be the most effective regimen, though changes in BCL-2, p21(WAF1/CIP1), p27(KIP1) do not necessarily correlate with the cellular effects when various sequences are examined. Therefore, these proteins may not be the most appropriate surrogate markers of efficacy of this regimen. These data provide the background for the development of the optimal regimen to be used in clinical trials.