189 research outputs found
PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in HT29 human colorectal cancer cell lines
Ajuts: Beca bianual de la FundaciĂł Olga Torres 2008-2009Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells
Estudi del paper de la Isoforma M2 de Piruvat Quinasa (PKM2) en la resposta i resistĂšncia a oxaliplatĂ en lĂnies celâlulars i en pacients amb cĂ ncer colorectal avançat
El cĂ ncer colorectal Ă©s la segona causa de mort per cĂ ncer al mĂłn i el tumor mĂ©s freqĂŒent a lâEstat Espanyol englobant ambdĂłs sexes. El tractament de la malaltia avançada es basa en la combinaciĂł de fĂ rmacs citotĂČxics, citostĂ tics i noves molĂšcules anti-diana, assolint-se medianes de supervivĂšncia al voltant dels 2 anys. Encara que les darreres tendĂšncies de tractament del cĂ ncer estan enfocades principalment cap al desenvolupament de molĂšcules anti-diana, lâadministraciĂł dâoxaliplatĂ continua essent clau i essencial en el tractament en el CCR avançat. Malgrat els grans avenços que sâhan donat en el tractament del cĂ ncer de cĂČlon metastĂ tic, tots ells estan al voltant de la introducciĂł de nous medicaments i seguim indefensos pel que fa a lâapariciĂł de resistĂšncies, que sĂłn la causa fonamental del fracĂ s del tractament. Per tant, lâobjectiu principal dâaquesta tesi doctoral Ă©s conĂšixer millor els mecanismes de resistĂšncia adquirida a oxaliplatĂ mitjançant lâestudi de perfils dâexpressiĂł proteica en models cel·lulars in vitro per desprĂ©s traslladar els resultats a la selecciĂł del tractament de primera lĂnia en pacients amb cĂ ncer colorectal avançat.
AixĂ, a partir dâuna aproximaciĂł de proteĂČmica comparativa, sâha identificat la proteĂŻna piruvat quinasa M2 (PKM2) en relaciĂł amb el procĂ©s dâadquisiciĂł de resistĂšncia a oxaliplatĂ en el nostre model cel·lular. PKM2 catalitza lâĂșltim pas de la glicĂČlisi i regula de manera clau la glicĂČlisi aerĂČbica o efecte Warburg en les cĂšl·lules canceroses. Recentment, se li han atribuĂŻt altres funcions no-glicolĂtiques; aixĂ, pot translocar al nucli i activar la transcripciĂł gĂšnica interaccionant i modificant proteĂŻnes nuclears participant en funcions tant dispars com la supervivĂšncia o la mort cel·lular.
Els nostres resultats demostren que PKM2 es troba infraexpressada en les lĂnies resistents, tambĂ© a nivell dâARNm i que desprĂ©s del tractament amb oxaliplatĂ, els nivells dâexpressiĂł disminueixen. Mitjançant tĂšcniques dâARN dâinterferĂšncia hem demostrat que la inhibiciĂł de lâexpressiĂł de PKM2 augmenta la viabilitat de les cĂšl·lules sensibles, amb p53 mutada, quan es tracten amb el fĂ rmac en un 50% mentre que lâefecte Ă©s oposat en un model amb p53 salvatge. Tanmateix no hem pogut demostrar que aquest augment en la viabilitat sigui conseqĂŒĂšncia dâuna disminuciĂł en la mort cel·lular.
Dâaltra banda, hem pogut observar un canvi en la compartimentaciĂł de la proteĂŻna que nomĂ©s es dona en les cĂšl·lules sensibles a oxaliplatĂ, donat que PKM2 Ă©s capaç de translocar al nucli de les cĂšl·lules sensibles al llarg de 72 hores de tractament continu perĂČ no en les cĂšl·lules derivades resistents. En tercer lloc, a partir dâuna aproximaciĂł de silenciament gĂšnic i proteĂČmica comparativa en 2 dimensions, sâhan pogut establir els perfils proteics associats a la presĂšncia/absĂšncia de PKM2 i determinar possibles proteĂŻnes vinculades a lâexpressiĂł dâaquesta com son, FKBP4 i HSC70. Finalment, pel que fa a lâestudi en pacients, hem demostrat que el grup de pacients amb els nivells mĂ©s baixos de PKM2 presenten pitjors rĂ tios de resposta a la quimioterĂ pia basada en oxaliplatĂ i que aquest fet tambĂ© pot tenir relaciĂł amb lâestat mutacional de p53 en aquests pacients, correlacionant aquests resultats amb els obtinguts a lâestudi in vitro. AixĂ doncs, en aquesta tesi doctoral es descriu un nou mecanisme dâadquisiciĂł de resistĂšncia a oxaliplatĂ i se li atribueix un nou paper a la proteĂŻna PKM2. Tanmateix, sâobren noves vies dâinvestigaciĂł, tant a nivell de funcionalitat de dita proteĂŻna com tambĂ© de noves vies dâintervenir en la resistĂšncia adquirida a aquest fĂ rmac platinat.Colorectal cancer is the second most common cause of cancer death in the world and the most frequent tumor in Spain including both genders. Survival of the advanced disease has increased from 12 months with monotherapy to roughly 2 years with the addition of cytotoxic and cytostatic drugs combined with targeted drugs. Despite the state of the art in cancer treatment is mainly focused on the development of target molecules, oxaliplatin administration remains essential in the treatment of advanced CRC. Regardless of the great improvements achieved in the treatment of metastatic CRC, all of them are focused in the introduction of new drugs and we are still defenseless in front of resistance appearance, which is the main cause of treatment failure. According to this, the main objective of this doctoral thesis is to better know and understand oxaliplatin acquired resistance mechanisms by studying protein expression profiles in in vitro models in order to translate these results in a better selection of first line treatment in advanced CRC patients.
Thus, from a comparative proteomic study, we have identified the protein M2 pyruvate kinase (PKM2) related to the oxaliplatin resistance acquisition process in our cellular model. PKM2 catalyzes the last step of glycolysis and is a key enzyme in the regulation of aerobic glycolysis (known as a Warburg effect) in cancer cells. Recently, other non-glycolytic functions have been assigned to PKM2. It has been shown this protein can translocate to the nucleus and, consequently, it activates transcription of several genes. PKM2 can also interact and modify nuclear proteins taking part in different and opposed cellular processes such as proliferation or death.
Our results have demonstrated that PKM2 expression is down-regulated in oxaliplatin resistant cells as compared to sensitive cells, also at mRNA level. Moreover, we have shown that oxaliplatin treatment decreases PKM2 levels. By using RNA interference techniques, we have shown that PKM2 gene silencing increases cell proliferation of sensitive cells harboring p53 mutation in about 50% when those cells are treated with the drug, while PKM2 silencing in sensitive and wild type p53 cells has the opposite result. The effect in increased cell proliferation was not related to a cell death decrease, though.
On the other hand, we could observe a change in the subcellular localization of PKM2 only in treated sensitive cells, done by the fact that this protein is able to translocate to the nucleus of these cells in response to the drug and this is not observed in oxaliplatin resistant cells. Moreover, by using gene silencing and a 2-D DIGE approach, we obtained protein profiles associated to PKM2 presence/absence and we have determined possible proteins whose expression depends on that of PKM2 such as FKBP4 and HSC70. Finally, we have demonstrated that patients with the lowest PKM2 mRNA levels showed the worst response ratios to oxaliplatin-based first line treatment. Tumors from these patients presented more frequently p53 mutations linking these results with our in vitro study.
To conclude, in this doctoral thesis we describe a new mechanism of oxaliplatin resistance acquisition in which PKM2 is an important player. Further investigations are guaranteed to elucidate molecular mechanisms underlying PKM2 functionality as well as to discover new pathways to interfere in acquired resistance to this platinum agent
Estudi del paper de la isoforma M2 de Piruvat Quinasa (PKM2) en la resposta i resistĂšncia a oxaliplatĂ en lĂnies cel·lulars i en pacients amb cĂ ncer colorectal avançat
Colorectal cancer is the second most common cause of cancer death in the world and the most frequent tumor in Spain including both genders. Survival of the advanced disease has increased from 12 months with monotherapy to roughly 2 years with the addition of cytotoxic and cytostatic drugs combined with targeted drugs. Despite the state of the art in cancer treatment is mainly focused on the development of target molecules, oxaliplatin administration remains essential in the treatment of advanced CRC. Regardless of the great improvements achieved in the treatment of metastatic CRC, all of them are focused in the introduction of new drugs and we are still defenseless in front of resistance appearance, which is the main cause of treatment failure. According to this, the main objective of this doctoral thesis is to better know and understand oxaliplatin acquired resistance mechanisms by studying protein expression profiles in in vitro models in order to translate these results in a better selection of first line treatment in advanced CRC patients. Thus, from a comparative proteomic study, we have identified the protein M2 pyruvate kinase (PKM2) related to the oxaliplatin resistance acquisition process in our cellular model. PKM2 catalyzes the last step of glycolysis and is a key enzyme in the regulation of aerobic glycolysis (known as a Warburg effect) in cancer cells. Recently, other non-glycolytic functions have been assigned to PKM2. It has been shown this protein can translocate to the nucleus and, consequently, it activates transcription of several genes. PKM2 can also interact and modify nuclear proteins taking part in different and opposed cellular processes such as proliferation or death. Our results have demonstrated that PKM2 expression is down-regulated in oxaliplatin resistant cells as compared to sensitive cells, also at mRNA level. Moreover, we have shown that oxaliplatin treatment decreases PKM2 levels. By using RNA interference techniques, we have shown that PKM2 gene silencing increases cell proliferation of sensitive cells harboring p53 mutation in about 50% when those cells are treated with the drug, while PKM2 silencing in sensitive and wild type p53 cells has the opposite result. The effect in increased cell proliferation was not related to a cell death decrease, though. On the other hand, we could observe a change in the subcellular localization of PKM2 only in treated sensitive cells, done by the fact that this protein is able to translocate to the nucleus of these cells in response to the drug and this is not observed in oxaliplatin resistant cells. Moreover, by using gene silencing and a 2-D DIGE approach, we obtained protein profiles associated to PKM2 presence/absence and we have determined possible proteins whose expression depends on that of PKM2 such as FKBP4 and HSC70. Finally, we have demonstrated that patients with the lowest PKM2 mRNA levels showed the worst response ratios to oxaliplatin-based first line treatment. Tumors from these patients presented more frequently p53 mutations linking these results with our in vitro study. To conclude, in this doctoral thesis we describe a new mechanism of oxaliplatin resistance acquisition in which PKM2 is an important player. Further investigations are guaranteed to elucidate molecular mechanisms underlying PKM2 functionality as well as to discover new pathways to interfere in acquired resistance to this platinum agent.El cĂ ncer colorectal Ă©s la segona causa de mort per cĂ ncer al mĂłn i el tumor mĂ©s freqĂŒent a l'Estat Espanyol englobant ambdĂłs sexes. El tractament de la malaltia avançada es basa en la combinaciĂł de fĂ rmacs citotĂČxics, citostĂ tics i noves molĂšcules anti-diana, assolint-se medianes de supervivĂšncia al voltant dels 2 anys. Encara que les darreres tendĂšncies de tractament del cĂ ncer estan enfocades principalment cap al desenvolupament de molĂšcules anti-diana, l'administraciĂł d'oxaliplatĂ continua essent clau i essencial en el tractament en el CCR avançat. Malgrat els grans avenços que s'han donat en el tractament del cĂ ncer de cĂČlon metastĂ tic, tots ells estan al voltant de la introducciĂł de nous medicaments i seguim indefensos pel que fa a l'apariciĂł de resistĂšncies, que sĂłn la causa fonamental del fracĂ s del tractament. Per tant, l'objectiu principal d'aquesta tesi doctoral Ă©s conĂšixer millor els mecanismes de resistĂšncia adquirida a oxaliplatĂ mitjançant l'estudi de perfils d'expressiĂł proteica en models cel·lulars in vitro per desprĂ©s traslladar els resultats a la selecciĂł del tractament de primera lĂnia en pacients amb cĂ ncer colorectal avançat. AixĂ, a partir d'una aproximaciĂł de proteĂČmica comparativa, s'ha identificat la proteĂŻna piruvat quinasa M2 (PKM2) en relaciĂł amb el procĂ©s d'adquisiciĂł de resistĂšncia a oxaliplatĂ en el nostre model cel·lular. PKM2 catalitza l'Ășltim pas de la glicĂČlisi i regula de manera clau la glicĂČlisi aerĂČbica o efecte Warburg en les cĂšl·lules canceroses. Recentment, se li han atribuĂŻt altres funcions no-glicolĂtiques; aixĂ, pot translocar al nucli i activar la transcripciĂł gĂšnica interaccionant i modificant proteĂŻnes nuclears participant en funcions tant dispars com la supervivĂšncia o la mort cel·lular. Els nostres resultats demostren que PKM2 es troba infraexpressada en les lĂnies resistents, tambĂ© a nivell d'ARNm i que desprĂ©s del tractament amb oxaliplatĂ, els nivells d'expressiĂł disminueixen. Mitjançant tĂšcniques d'ARN d'interferĂšncia hem demostrat que la inhibiciĂł de l'expressiĂł de PKM2 augmenta la viabilitat de les cĂšl·lules sensibles, amb p53 mutada, quan es tracten amb el fĂ rmac en un 50% mentre que l'efecte Ă©s oposat en un model amb p53 salvatge. Tanmateix no hem pogut demostrar que aquest augment en la viabilitat sigui conseqĂŒĂšncia d'una disminuciĂł en la mort cel·lular. D'altra banda, hem pogut observar un canvi en la compartimentaciĂł de la proteĂŻna que nomĂ©s es dona en les cĂšl·lules sensibles a oxaliplatĂ, donat que PKM2 Ă©s capaç de translocar al nucli de les cĂšl·lules sensibles al llarg de 72 hores de tractament continu perĂČ no en les cĂšl·lules derivades resistents. En tercer lloc, a partir d'una aproximaciĂł de silenciament gĂšnic i proteĂČmica comparativa en 2 dimensions, s'han pogut establir els perfils proteics associats a la presĂšncia/absĂšncia de PKM2 i determinar possibles proteĂŻnes vinculades a l'expressiĂł d'aquesta com son, FKBP4 i HSC70. Finalment, pel que fa a l'estudi en pacients, hem demostrat que el grup de pacients amb els nivells mĂ©s baixos de PKM2 presenten pitjors rĂ tios de resposta a la quimioterĂ pia basada en oxaliplatĂ i que aquest fet tambĂ© pot tenir relaciĂł amb l'estat mutacional de p53 en aquests pacients, correlacionant aquests resultats amb els obtinguts a l'estudi in vitro. AixĂ doncs, en aquesta tesi doctoral es descriu un nou mecanisme d'adquisiciĂł de resistĂšncia a oxaliplatĂ i se li atribueix un nou paper a la proteĂŻna PKM2. Tanmateix, s'obren noves vies d'investigaciĂł, tant a nivell de funcionalitat de dita proteĂŻna com tambĂ© de noves vies d'intervenir en la resistĂšncia adquirida a aquest fĂ rmac platinat
PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in HT29 human colorectal cancer cell lines
Ajuts: Beca bianual de la FundaciĂł Olga Torres 2008-2009Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells
Perfil clĂnico, tratamiento y pronĂłstico de los pacientes con infarto agudo de miocardio no ingresados en una unidad coronaria: utilidad de una unidad de cuidados intermedios como lugar de ingreso inicial
The Role of Regulations in the Spanish Housing Dispossession Crisis: Towards Dispossession by Regulations?
Measurement of electrons from semileptonic heavy-flavour hadron decays at midrapidity in pp and PbâPb collisions at âsNN = 5.02 TeV
The differential invariant yield as a function of transverse momentum (pT) of electrons from semileptonic heavy-flavour hadron decays was measured at midrapidity in central (0â10%), semi-central (30â50%) and peripheral (60â80%) leadâlead (PbâPb) collisions at âsNN = 5.02 TeV in the pT intervals 0.5â26 GeV/c (0â10% and 30â50%) and 0.5â10 GeV/c (60â80%). The production cross section in protonâproton (pp) collisions at âs = 5.02 TeV was measured as well in 0.5 < pT < 10 GeV/c and it lies close to the upper band of perturbative QCD calculation uncertainties up to pT = 5 GeV/c and close to the mean value for larger pT. The modification of the electron yield with respect to what is expected for an incoherent superposition of nucleonânucleon collisions is evaluated by measuring the nuclear modification factor RAA. The measurement of the RAA in different centrality classes allows in-medium energy loss of charm and beauty quarks to be investigated. The RAA shows a suppression with respect to unity at intermediate pT, which increases while moving towards more central collisions. Moreover, the measured RAA is sensitive to the modification of the parton distribution functions (PDF) in nuclei, like nuclear shadowing, which causes a suppression of the heavy-quark production at low pT in heavy-ion collisions at LHC
Charged-particle multiplicity fluctuations in PbâPb collisions at âsNN = 2.76 TeV
Measurements of event-by-event fluctuations of charged-particle multiplicities in PbâPb collisions at sNNââââ = 2.76 TeV using the ALICE detector at the CERN Large Hadron Collider (LHC) are presented in the pseudorapidity range |η|<0.8 and transverse momentum 0.2<pT<2.0 GeV/c. The amplitude of the fluctuations is expressed in terms of the variance normalized by the mean of the multiplicity distribution. The η and pT dependences of the fluctuations and their evolution with respect to collision centrality are investigated. The multiplicity fluctuations tend to decrease from peripheral to central collisions. The results are compared to those obtained from HIJING and AMPT Monte Carlo event generators as well as to experimental data at lower collision energies. Additionally, the measured multiplicity fluctuations are discussed in the context of the isothermal compressibility of the high-density strongly-interacting system formed in central PbâPb collisions
Enhanced deuteron coalescence probability in jets
The transverse-momentum (pT) spectra and coalescence parameters B2 of (anti)deuterons are measured in pp collisions at sâ=13 TeV for the first time in and out of jets. In this measurement, the direction of the leading particle with the highest pT in the event (pleadT>5 GeV/c) is used as an approximation for the jet axis. The event is consequently divided into three azimuthal regions and the jet signal is obtained as the difference between the Toward region, that contains jet fragmentation products in addition to the underlying event (UE), and the Transverse region, which is dominated by the UE. The coalescence parameter in the jet is found to be approximately a factor of 10 larger than that in the underlying event. This experimental observation is consistent with the coalescence picture and can be attributed to the smaller average phase-space distance between nucleons inside the jet cone as compared to the underlying event. The results presented in this Letter are compared to predictions from a simple nucleon coalescence model, where the phase space distributions of nucleons are generated using PYTHIA 8 with the Monash 2013 tuning, and to predictions from a deuteron production model based on ordinary nuclear reactions with parametrized energy-dependent cross sections tuned on data. The latter model is implemented in PYTHIA 8.3. Both models reproduce the observed large difference between in-jet and out-of-jet coalescence parameters, although the almost flat trend of the BJet2 is not reproduced by the models, which instead give a decreasing trend
Measurement of the production cross section of prompt Î0c baryons in pâPb collisions at âsNN = 5.02 TeV
The transverse momentum (pT) differential production cross section of the promptly-produced charm-strange baryon Î0c (and its charge conjugate Î0cÂŻÂŻÂŻÂŻÂŻÂŻ) is measured at midrapidity via its hadronic decay into Ï+Îâ in pâPb collisions at a centre-of-mass energy per nucleonânucleon collision sNNââââ = 5.02 TeV with the ALICE detector at the LHC. The Î0c nuclear modification factor (RpPb), calculated from the cross sections in pp and pâPb collisions, is presented and compared with the RpPb of Î+c baryons. The ratios between the pT-differential production cross section of Î0c baryons and those of D0 mesons and Î+c baryons are also reported and compared with results at forward and backward rapidity from the LHCb Collaboration. The measurements of the production cross section of prompt Î0c baryons are compared with a model based on perturbative QCD calculations of charm-quark production cross sections, which includes only cold nuclear matter effects in pâPb collisions, and underestimates the measurement by a factor of about 50. This discrepancy is reduced when the data is compared with a model in which hadronisation is implemented via quark coalescence. The pT-integrated cross section of prompt Î0c-baryon production at midrapidity extrapolated down to pT = 0 is also reported. These measurements offer insights and constraints for theoretical calculations of the hadronisation process. Additionally, they provide inputs for the calculation of the charm production cross section in pâPb collisions at midrapidity
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