Oxaliplatin was the first platinum drug with proven activity against colorectal tumors, becoming a standard in the management of this malignancy. It is also considered for the treatment of pancreatic ...and gastric cancers. However, a major reason for treatment failure still is the existence of tumor intrinsic or acquired resistance. Consequently, it is important to understand the molecular mechanisms underlying the appearance of this phenomenon to find ways of circumventing it and to improve and optimize treatments. This review will be focused on recent discoveries about oxaliplatin tumor-related resistance mechanisms, including alterations in transport, detoxification, DNA damage response and repair, cell death (apoptotic and nonapoptotic), and epigenetic mechanisms.
Oxaliplatin is a third-generation platinum agent used in colorectal cancer treatment. Oxaliplatin resistance acquisition is a complex process mainly based on alteration of genes and pathways involved ...in its mechanism of action. Therefore, our purpose was to perform a gene expression screening in an in vitro model to identify genes that could play a role in oxaliplatin resistance acquisition processes. Four colorectal cancer cell lines and their oxaliplatin-resistant derived sublines were compared. Microarray analysis was done using Human 19K Oligo Array Slides. RNA from cells were hybridized with a commercial RNA reference sample and labeled with both fluorochromes Cy3 and Cy5. Data were analyzed by hierarchical clustering method. Subsequently, quantitative real-time PCR (qRT-PCR) was used to corroborate microarray data, considering as positively validated those genes that showed significant differences in expression levels between groups and a correlation between microarray and qRT-PCR data. By microarray analysis, 32 candidate genes were identified. After validation process by qRT-PCR, the genes AKT1, CDK5, TRIP, GARP, RGS11, and UGCGL1 were positively validated. The 3 first genes proved to be involved in regulation of nuclear factor-kappabeta antiapoptotic transcription factor previously related to drug resistance, and the other 3 genes are novel finds. We have identified 6 genes related to oxaliplatin resistance acquisition. These findings are of paramount importance to understand these processes better and open new lines of study to elucidate the relevance of this pharmacogenomic approach into the clinic.
Chemoresistance 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.
We aimed to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer by using a proteomic approach. A 5-fold oxaliplatin-resistant cell line, ...HTOXAR3, was compared with its parental cell line, HT29, using two-dimensional PAGE. Mass spectrometry, Western blot, and real-time quantitative PCR confirmed the down-regulation of pyruvate kinase M2 (PK-M2) in HTOXAR3 cells. In a panel of eight colorectal cancer cell lines, we found a negative correlation between oxaliplatin resistance and PK-M2 mRNA levels (Spearman r=-0.846, P=0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PK-M2 mRNA up-regulation. PK-M2 mRNA levels were measured by real-time quantitative PCR in 41 tumors treated with oxaliplatin/5-fluorouracil. Tumors with the lowest PK-M2 levels attained the lowest response rates (20% versus 64.5%, P=0.026). High PK-M2 levels were associated with high p53 levels (P=0.032). In conclusion, the data provided clearly link PK-M2 expression and oxaliplatin resistance mechanisms and further implicate PK-M2 as a predictive marker of response in patients with oxaliplatin-treated colorectal cancer.
Abstract We studied the role of TS ( 5’VNTR, 5’SNP and 3’UTR ), XRCC1-399, XPD-751, ERCC1-118 and XRCC3-241 genetic polymorphisms in tailoring fluroropyrimidine/oxaliplatin treatment. For this ...purpose, 110 XELOX (capecitabine/oxaliplatin)- or FUOX (fluorouracil/oxaliplatin)-treated metastatic colorectal cancer patients were selected prospectively for genotyping. In the FUOX group, TS-3’UTR +6 bp/+6 bp (hazards ratio, HR = 2.62, p = 0.007) and ERCC1- 118 C/T or C/C (HR = 1.96, p = 0.050) genotypes correlated with a shorter progression free survival (PFS). When analysed jointly, the higher the number of favourable genotypes (FG) the longer the PFS (6.8 m, 9.6 m and 25.8 m for 0, 1 or 2 FG; p = 0.005). Disease-control rate was 100% in patients with 2 FG (87% and 38.5% for 1 or 0 FG; p = 0.001). In the multivariate analysis, ERCC1-118 (HR = 2.12, p = 0.0037) and TS-3’UTR (HR = 2.68, p = 0.006) were strong independent prognostic factors. According to this, patients harbouring TS-3’UTR +6 bp/+6 bp and ERCC1- 118 C/T or C/C genotypes may better receive capecitabine instead of 5FU in an oxaliplatin-based first-line treatment.
El càncer colorectal és la segona causa de mort per càncer al món i el tumor més freqüent a l’Estat Espanyol englobant ambdós sexes. El tractament de la malaltia avançada es basa en la combinació de ...fàrmacs citotòxics, citostàtics i noves molècules anti-diana, assolint-se medianes de supervivència al voltant dels 2 anys. Encara que les darreres tendències de tractament del càncer estan enfocades principalment cap al desenvolupament de molècules anti-diana, l’administració d’oxaliplatí continua essent clau i essencial en el tractament en el CCR avançat. Malgrat els grans avenços que s’han donat en el tractament del càncer de còlon metastàtic, tots ells estan al voltant de la introducció de nous medicaments i seguim indefensos pel que fa a l’aparició de resistències, que són la causa fonamental del fracàs del tractament. Per tant, l’objectiu principal d’aquesta tesi doctoral és conèixer millor els mecanismes de resistència adquirida a oxaliplatí mitjançant l’estudi de perfils d’expressió proteica en models cel·lulars in vitro per després traslladar els resultats a la selecció del tractament de primera línia en pacients amb càncer colorectal avançat.
Així, a partir d’una aproximació de proteòmica comparativa, s’ha identificat la proteïna piruvat quinasa M2 (PKM2) en relació amb el procés d’adquisició de resistència a oxaliplatí en el nostre model cel·lular. PKM2 catalitza l’últim pas de la glicòlisi i regula de manera clau la glicòlisi aeròbica o efecte Warburg en les cèl·lules canceroses. Recentment, se li han atribuït altres funcions no-glicolítiques; així, pot translocar al nucli i activar la transcripció gènica interaccionant i modificant proteïnes nuclears participant en funcions tant dispars com la supervivència o la mort cel·lular.
Els nostres resultats demostren que PKM2 es troba infraexpressada en les línies resistents, també a nivell d’ARNm i que després del tractament amb oxaliplatí, els nivells d’expressió disminueixen. Mitjançant tècniques d’ARN d’interferència hem demostrat que la inhibició de l’expressió de PKM2 augmenta la viabilitat de les cèl·lules sensibles, amb p53 mutada, quan es tracten amb el fàrmac en un 50% mentre que l’efecte és oposat en un model amb p53 salvatge. Tanmateix no hem pogut demostrar que aquest augment en la viabilitat sigui conseqüència d’una disminució en la mort cel·lular.
D’altra banda, hem pogut observar un canvi en la compartimentació de la proteïna que només es dona en les cèl·lules sensibles a oxaliplatí, donat que PKM2 és capaç de translocar al nucli de les cèl·lules sensibles al llarg de 72 hores de tractament continu però no en les cèl·lules derivades resistents. En tercer lloc, a partir d’una aproximació de silenciament gènic i proteòmica comparativa en 2 dimensions, s’han pogut establir els perfils proteics associats a la presència/absència de PKM2 i determinar possibles proteïnes vinculades a l’expressió 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 més baixos de PKM2 presenten pitjors ràtios de resposta a la quimioteràpia basada en oxaliplatí i que aquest fet també pot tenir relació amb l’estat mutacional de p53 en aquests pacients, correlacionant aquests resultats amb els obtinguts a l’estudi in vitro. Així doncs, en aquesta tesi doctoral es descriu un nou mecanisme d’adquisició de resistència a oxaliplatí i se li atribueix un nou paper a la proteïna PKM2. Tanmateix, s’obren noves vies d’investigació, tant a nivell de funcionalitat de dita proteïna com també de noves vies d’intervenir en la resistència adquirida a aquest fàrmac 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.
Non-alcoholic fatty liver disease (NAFLD) is rapidly increasing alongside overweight and obesity, not only in adults but also in children and adolescents. It is unknown what impact the development of ...NAFLD in childhood may have in later life. The importance of early detection and treatment lies in its potential for progression to cirrhosis, liver cancer and liver-related death, as well as its associated extrahepatic comorbidities. Vibration-Controlled Transient Elastography (VCTE) with Controlled Attenuation Parameter (CAP) is an effective, non-invasive and safe diagnostic method to estimate the degree of fibrosis and steatosis in the liver, but little is known about its applicability in the paediatric population. 1) To assess the prevalence of significant liver fibrosis (Liver Stiffness Measurement (LSM) greater than or equal to6.5 kPa) using VCTE, and that of non-alcoholic fatty liver disease (greater than or equal to225 dB/m) using CAP in children and adolescents. 2) To determine the optimal cut-off points of the CAP to achieve maximum concordance with the Magnetic Resonance Imaging (MRI) findings in the diagnosis of mild, moderate and severe NAFLD in children and adolescents. Cross-sectional population-based study which will include 2,866 subjects aged between 9 and 16 years. Participants will undergo: anamnesis, physical examination, blood extraction, VCTE, MRI and questionnaires on socio-demographic data, personal and family medical history and lifestyle assessment.
To demonstrate the efficacy of the anti-interleukin-6 receptor monoclonal antibody tocilizumab in patients with moderate-to-severe corticosteroid-resistant Graves orbitopathy (GO).
Double-masked ...randomized clinical trial.
Setting and Participants: Thirty-two adults with moderate-to-severe corticosteroid-resistant GO from 10 medical centers in Spain were randomized (1:1). Intervention: Randomization to either 8 mg/kg body weight tocilizumab or placebo administered intravenously at weeks 0, 4, 8, and 12, and follow-up for an additional 28 weeks. Main Outcomes and Measures: The primary outcome was the proportion of patients with a change from baseline to week 16 of at least 2 in the clinical activity score (CAS).
The primary outcome was met by 93.3% (95% confidence interval CI 70.1%-98.8%) of the patients receiving tocilizumab and 58.8% (36%-78.3%) receiving placebo (P = .04; odds ratio, 9.8 CI 1.3-73.2). A significant difference was also observed in the proportion of patients achieving a CAS < 3 (86.7% CI 62.1%-96.2% vs 35.2% CI 17.3%-58.7%, P = .005; OR 11.9 CI 2.1-63.1) at week 16. Additionally, a larger proportion of patients with improvement in the European Group on GO–proposed composite ophthalmic score at week 16 (73.3% CI 48%-89.1% vs 29.4% CI 13.2%-53.1%; P = .03), and exophthalmos size change from baseline to week 16 (-1.5 -2.0 to 0.5 mm vs 0.0 -1.0 to 0.5 mm; P = .01) were seen with tocilizumab. One patient experienced a moderate increase in transaminases at week 8; another had an acute pyelonephritis at week 32 in the tocilizumab-treated group.
Tocilizumab offers a meaningful improvement in activity and severity in corticosteroid-resistant GO. This trial justifies further studies to characterize the role of tocilizumab in GO.
Liver fibrosis is the main determinant of long-term outcome in chronic liver diseases. Little is known about the prevalence of liver fibrosis in the general population. The aim of the study was to ...investigate the prevalence of liver fibrosis in the general adult population with unknown liver disease.
This was a population-based, cross-sectional study performed in the Barcelona metropolitan area. Subjects aged 18 to 75 years old were identified randomly from citizens included in the primary health care registry. Of 4866 subjects invited, 3076 participated (63.2%). Liver fibrosis was estimated by measuring liver stiffness (LS) with transient elastography (TE). Liver histology was assessed in 92 subjects with increased LS.
Prevalence estimates of increased LS (≥6.8, ≥8.0, and ≥9.0 kPa) were 9.0%, 5.8%, and 3.6%, respectively. The etiology of liver disease was mainly nonalcoholic fatty liver disease (NAFLD), followed by alcohol risk consumption (consumption of ≥21 standard drinking units/wk in men and ≥14 standard drinking units/wk in women). Factors independently associated with increased LS were male sex, abdominal obesity, type 2 diabetes, serum glucose, high-density lipoprotein, and triglyceride levels. Subjects without risk factors for NAFLD or without alcohol risk consumption had a very low prevalence of increased LS. The best cut-off value of LS for significant liver fibrosis (F2–F4) was 9.2 kPa, with high sensitivity and specificity. TE was more accurate than alanine aminotransferase, NAFLD fibrosis score, or Fibrosis 4. An algorithm for screening for liver fibrosis using TE in the community setting is proposed.
These findings show a high prevalence of silent liver disease with advanced fibrosis mainly related to NAFLD in adult European subjects without known liver disease. An LS value less than 9.2 kPa predicts the absence of significant liver fibrosis with high accuracy and could be used for screening purposes.
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