RESUMEN
BACKGROUND: Resistance to 5-fluorouracil (5-FU) in patients with colorectal cancer prevents effective treatment and leads to unnecessary and burdensome chemotherapy. Therefore, prediction of 5-FU resistance is imperative. METHODS: To identify the proteins linked to 5-FU resistance, two-dimensional gel electrophoresis-based proteomics was performed using the human colon cancer cell line SNU-C4R with induced 5-FU resistance. Proteins showing altered expression in SNU-C4R were identified by matrix-associated laser desorption/ionization-time-of-flight analysis, and their roles in susceptibility to 5-FU or radiation were evaluated in various cell lines by transfection of specific siRNA or creation of overexpression constructs. Changes in cellular signaling and expression of mitochondrial apoptotic factors were investigated by Western Blot analysis. A mitochondrial membrane potential probe (JC-1 dye) and a flow cytometry system were employed to determine the mitochondrial membrane potential. Finally, protein levels were determined by Western Blot analysis in tissues from 122 patients with rectal cancer to clarify whether each identified protein is a useful predictor of a chemoradiation response. RESULTS: We identified mitochondrial phosphoenolpyruvate carboxykinase (mPEPCK) as a candidate predictor of 5-FU resistance. PEPCK was downregulated in SNU-C4R compared with its parent cell line SNU-C4. Overexpression of mPEPCK did not significantly alter the susceptibility to either 5-FU or radiation. Suppression of mPEPCK led to a decrease in both the cellular level of phosphoenolpyruvate and the susceptibility to 5-FU and radiation. Furthermore, the cellular levels of phosphoenolpyruvate (an end product of PEPCK and a substrate of pyruvate kinase), phosphorylated AKT, and phosphorylated 4EBP1 were decreased significantly secondary to the mPEPCK suppression in SNU-C4. However, mPEPCK siRNA transfection induced changes in neither the mitochondrial membrane potential nor the expression levels of mitochondrial apoptotic factors such as Bax, Bcl-2, and Bad. Downregulation of total PEPCK was observed in tissues from patients with rectal cancer who displayed poor responses to preoperative 5-FU-based radiation therapy. CONCLUSION: Our overall results demonstrate that mPEPCK is a useful predictor of a response to chemoradiotherapy in patients with rectal cancer.
Asunto(s)
Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Quimioradioterapia , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Femenino , Fluorouracilo/farmacología , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Mitocondrias/metabolismo , Clasificación del Tumor , Estadificación de Neoplasias , Fosfoenolpiruvato/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Tolerancia a Radiación/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Cancer stem cells (CSCs) are known to be resistant to conventional chemotherapy and radiotherapy. Specific CSC targeting and eradication is therefore a therapeutically important challenge. CD133 is a colorectal CSC marker with unknown function(s). Assessing proteomic changes induced by CD133 may provide clues not only to new CD133 functions but also to the chemotherapy and radiation susceptibility of colon cancer cells. To identify the proteins affected by CD133, CD133-positive (CD133+), and CD133-negative (CD133-) human colon cancer cells were obtained by cell sorting. Whole proteomes were profiled from SW620/CD133+ and SW620/CD133- cells and analyzed by 2D-based proteome analysis. Nucleophosmin (NPM1) was identified as a protein regulated by CD133. CD133 protein level was not affected by NPM1, and an interaction between the two proteins was not observed. CD133 and NPM1 protein levels were positively correlated in 11 human colon cancer cell lines. The CD133+ subpopulation percentage or its value normalized against CD133 protein level was only linked to intrinsic susceptibility of human colon cancer cells to 5-fluorouracil (5-FU). However, either suppression of CD133 or NPM1 significantly increased 5-FU susceptibility of SW620. The present study suggests that CD133-regulated NPM1 protein level may provide a clue to novel CD133 function(s) linked to human colon cancer cell susceptibility to chemotherapy.
Asunto(s)
Antígenos CD/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Fluorouracilo/farmacología , Glicoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Antígeno AC133 , Antígenos CD/análisis , Antígenos CD/química , Línea Celular Tumoral , Neoplasias del Colon/química , Resistencia a Antineoplásicos/efectos de los fármacos , Electroforesis en Gel Bidimensional , Citometría de Flujo , Glicoproteínas/análisis , Glicoproteínas/química , Humanos , Células Madre Neoplásicas , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Nucleofosmina , Péptidos/análisis , Péptidos/químicaRESUMEN
BACKGROUND: Caldesmon (CaD), a major actin-associated protein, is found in smooth muscle and non-muscle cells. Smooth muscle caldesmon, h-CaD, is a multifunctional protein, and non-muscle cell caldesmon, l-CaD, plays a role in cytoskeletal architecture and dynamics. h-CaD is thought to be an useful marker for smooth muscle tumors, but the role(s) of l-CaD has not been examined in tumors. METHODS: Primary colon cancer and liver metastasis tissues were obtained from colon cancer patients. Prior to chemoradiotherapy (CRT), normal and cancerous tissues were obtained from rectal cancer patients. Whole-tissue protein extracts were analyzed by 2-DE-based proteomics. Expression and phosphorylation level of main cellular signaling proteins were determined by western blot analysis. Cell proliferation after CaD siRNA transfection was monitored by MTT assay. RESULTS: The expression level of l-CaD was significantly increased in primary colon cancer and liver metastasis tissues compared to the level in the corresponding normal tissues. In cancerous tissues obtained from the patients showing poor response to CRT (Dworak grade 4), the expression of l-CaD was increased compared to that of good response group (Dworak grade 1). In line with, l-CaD positive human colon cancer cell lines were more resistant to 5-fluorouracil (5-FU) and radiation treatment compared to l-CaD negative cell lines. Artificial suppression of l-CaD increased susceptibility of colon cancer cells to 5-FU, and caused an increase of p21 and c-PARP, and a decrease of NF-kB and p-mTOR expression. CONCLUSION: Up-regulated expression of l-CaD may have a role for increasing metastatic property and decreasing CRT susceptibility in colorectal cancer cells.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Anciano , Antimetabolitos Antineoplásicos/farmacología , Biomarcadores de Tumor/fisiología , Western Blotting , Proteínas de Unión a Calmodulina/fisiología , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Femenino , Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Fosforilación , ARN Interferente Pequeño , Regulación hacia ArribaRESUMEN
Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca(2+) biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca(2+) oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca(2+) oscillation, which determines the physiological functions of HMSCs.