Pepita Giménez-Bonafé

Ki-67 proliferative index predicts clinical outcome in patients with atypical or anaplastic meningioma

Meningiomas represent the second most common central nervous system neoplasms in adults and account for 26% of all primary brain tumors. Although most are benign, between 5% and 15% of meningiomas are atypical (grade II) whereas 1–2% are anaplastic meningiomas (grade III). Although histological grade is the most relevant prognostic factor, there are some unusual cases in which establishing a diagnosis of high‐grade meningioma following 2000 World Health Organization (WHO) histological criteria is extremely difficult. Therefore, the aim of the present study was to evaluate the predictive value of Ki‐67 labeling index and its contribution to current WHO classification in predicting tumor recurrence and overall survival in patients with high‐grade meningiomas. A total of 28 patients (with 16 atypical meningiomas and 12 anaplastic meningiomas) were evaluated for demographic, clinical, radiological and therapeutic variables, and for Ki‐67 immunohistochemistry. Median Ki‐67 labeling index in the whole series was 7.0 (0.5–31.5) with no differences with respect to the histological grade (P = 0.87). In the univariate analysis, Ki‐67 labeling index and postoperative Karnofsky performance status were identified as significant prognostic factors of tumor recurrence and overall survival. The multivariate analysis demonstrated that Ki‐67 labeling index is the only independent predictor of both tumor recurrence and overall survival. More importantly, this predictive value was maintained in both patients with atypical and patients with anaplastic meningioma.

Overcoming Drug Resistance by Enhancing Apoptosis of Tumor Cells

Drug resistance remains a major clinical challenge for cancer treatment. One mechanism by which tumor cells develop resistance to cytotoxic agents and radiation is related to resistance to apoptosis. Apoptosis is a well-organised process of cell death pre-programmed inside the cell. Apoptosis can be initiated either by activation of death receptors on the cell surface membranes (extrinsic pathway) or through a series of cellular events primarily processed at mitochondria (intrinsic pathway). Apoptosis has been shown to be important for tumorigenesis and cancer treatment. Defects in apoptosis can result in the expansion of a population of neoplastic cells. However, because the death of tumor cells induced by chemotherapy and radiotherapy is largely mediated by activation of apoptosis, inhibition of apoptosis will make tumor cells resistant to anti-tumor treatment. Herein, we will review the molecular changes that have the potential to cause apoptotic dysregulation, including activation of antiapoptotic factors (Bcl-2, BCLX(L), Bfl1/A1 etc.), inactivation of pro-apoptotic effectors (p53, p53 pathway), and /or reinforcement of survival signals (Survivin, FLIP, NF-kappaB etc). Furthermore, we will discuss therapeutic intervention and/or strategies that can lower the threshold for apoptosis of tumor cells that could became useful approaches to treat cancer with special emphasis placed on the important priority to develop new cancer therapeutics toward tumor stem cells.

Activation of p53 by Nutlin-3a Induces Apoptosis and Cellular Senescence in Human Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells

BACKGROUND AND PURPOSE The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P(2)) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines. METHODS/RESULTS After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P(2), lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in γ-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects. CONCLUSION TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses.

Different renal toxicity profiles in the association of cyclosporine and tacrolimus with sirolimus in rats

BACKGROUND The association of calcineurin inhibitors (CNIs) with mTOR inhibitors (mTORi) is still a problem in clinical practice and there is substantial interest in better understanding the impact of these associations on kidney toxicity. We aimed to analyse the functional and histological profiles of damage and to define the contribution of inflammatory and pro-fibrotic mediators in the association of cyclosporine (CsA) and/or tacrolimus (Tac) with sirolimus (SRL). METHODS A well-defined model of nephrotoxicity in salt-depleted male rats was used. Monotherapy groups were distributed as a non-treated control group with saline solution (n = 12), the Tac group (n = 16) (tacrolimus 6 mg/kg/day) and the CsA group (n = 13) (CsA 15 mg/kg/day). The groups with different associations were scattered as the Tac + SRL group (n = 14) (tacrolimus 6 mg/kg/day and rapamycin 3 mg/kg/day) and the CsA + SRL group (n = 7) (CsA 15 mg/kg/day and rapamycin 3 mg/kg/day). Groups were divided into 30 and 70 days of follow-up, but the CsA + SRL group was only studied for 30 days because animals became sick. RESULTS Rats with the CsA + SRL association were the only ones which showed a significant reduction in body weight, impairment of renal function and severe and diffuse tubular vacuolization and tubular atrophy following a striped distribution, and scarce areas of the kidney were still preserved. The Tac + SRL association did not produce renal function impairment, and mild histological damage including enhanced periglomerular tubular atrophy was observed. This local damage affected the distal convoluted tubule involving macula densa and juxtaglomerular apparatus. Pro-inflammatory mediators paralleled functional and structural data. ED-1 and TNF-alpha were noticeably higher in the CsA + SRL than in the Tac + SRL association. Only in the CsA + SRL association an important increase in alpha-SMA+ cells was seen, mainly found in the areas with tubular atrophy. TGF-beta1 was also markedly enhanced in the CsA + SRL association whilst monotherapy or Tac + SRL groups at 30 days TGF-beta1 did not show any changes. However, at 70 days of treatment TGF-beta1 was significantly increased in the Tac + SRL group. Animals receiving SRL showed a decrease in renal vascular endothelial growth factor (VEGF) expression. This growth factor was significantly down-regulated in both CNI associations than in SRL monotherapy. P-glycoprotein (Pgp) was overexpressed in CsA and CsA + SRL therapy whilst Tac and TAC + SRL showed a middle increase Pgp expression but higher than the control and SRL group. CONCLUSION We conclude that the association of SRL with high doses of CsA or Tac produces a different functional, histological, inflammatory and pro-fibrogenic pattern. Thus, the addition of SRL to high doses of CsA leads to severe renal injury. Combination with high doses of Tac is clearly less deleterious in the short term. However, there is a low grade of pro-fibrotic inflammatory expression when this association is prolonged.

Mdm2 antagonists induce apoptosis and synergize with cisplatin overcoming chemoresistance in TP53 wild-type ovarian cancer cells

Ovarian cancer (OVCa) is the leading cause of death from gynecological malignancies. Although treatment for advanced OVCa has improved with the introduction of taxane–platinum chemotherapy, the majority of patients will develop resistance to the treatment, leading to poor prognosis. One of the causes of chemoresistance is the reduced ability to undergo apoptosis. Cisplatin is a genotoxic drug that leads cells to apoptosis through the activation of the p53 pathway. Defective signaling in this pathway compromises p53 function, and thus cisplatin does not induce apoptosis. A new group of nongenotoxic small molecules called Nutlins have been developed to inhibit p53‐Mdm2 binding, inducing apoptosis in chemoresistant tumors through the activation of the p53 pathway. The wild‐type p53 cisplatin‐resistant ovarian cancer cell‐line A2780cis was used to test the effect of Nutlin‐3a (Nut3a) on apoptosis response. The results showed that Nut3a synergized with cisplatin, inducing cell‐cycle arrest in G2/M and potentiating apoptotic cell death. Increased apoptosis was also induced in wild‐type TP53 primary OVCa cultures by double cisplatin–Nut3a treatment. In conclusion, Nut3a appears to sensitize chemoresistant OVCa cells to cisplatin, inducing apoptosis. As increased response was generalized in primary tumors, this cisplatin–Nut3a combination could be useful for the treatment of patients harboring wild‐type TP53 who do not respond to standard chemotherapy.

YM155 sensitizes ovarian cancer cells to cisplatin inducing apoptosis and tumor regression.

OBJECTIVE The objective of this study is to chemosensitize ovarian cancer (OVCa) cells to cisplatin (CDDP) using an inhibitor of Survivin, YM155. The efficacy of YM155 in combination with CDDP was determined in vitro, ex vivo and in vivo. METHODS Human OVCa cell lines A2780p and their cisplatin-resistant derivative A2780cis, were treated with CDDP, YM155, and the combined treatment (YM155+CDDP), and cell viability, mRNA and protein expression levels, cell-cycle distribution, and DNA damage were then evaluated. Furthermore, the efficacy of YM155 combined with CDDP was further examined in established primary cell cultures and xenograft models. RESULTS The combination of YM155 with CDDP induced G2/M cell cycle arrest and apoptosis, increased DNA damage, and decreased Survivin levels, especially in A2780cis CDDP-resistant cells. Additionally, YM155 in combination with CDDP sensitized primary cell cultures to CDDP. Studies in vivo showed how this combination significantly decreased the tumor size of OVCa xenografts. CONCLUSIONS Our results demonstrate that in OVCa cells the expression of Survivin did not affect their sensitivity to YM155, suggesting that Survivin was not the only target of YM155. The combination of YM155 with CDDP could be a good option for therapy of CDDP-resistant OVCa, independently of p53 status.

Do drug transporter (ABCB1) SNPs and P‐glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study

The function of the efflux pump P‐glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp‐low pumpers) showed lower Pgp activity than noncarriers. Pgp‐high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3+ peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

Delayed mTOR Inhibition with Low Dose of Everolimus Reduces TGFβ Expression, Attenuates Proteinuria and Renal Damage in the Renal Mass Reduction Model

Background The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival. The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFβ expression in the 5/6 nephrectomy model in Wistar rats. Methods This study evaluated the effects of everolimus (0.3 mg/k/day) introduced 15 days after surgical procedure on renal function, proteinuria, renal histology and mechanisms of fibrosis and proliferation. Results Everolimus treated group (EveG) showed significantly less proteinuria and albuminuria, less glomerular and tubulointerstitial damage and fibrosis, fibroblast activation cell proliferation, when compared with control group (CG), even though the EveG remained with high blood pressure. Treatment with everolimus also diminished glomerular hypertrophy. Everolimus effectively inhibited the increase of mTOR developed in 5/6 nephrectomy animals, without changes in AKT mRNA or protein abundance, but with an increase in the pAKT/AKT ratio. Associated with this inhibition, everolimus blunted the increased expression of TGFβ observed in the remnant kidney model. Conclusion Delayed mTOR inhibition with low dose of everolimus significantly prevented progressive renal damage and protected the remnant kidney. mTOR and TGFβ mRNA reduction can partially explain this anti fibrotic effect. mTOR can be a new target to attenuate the progression of chronic kidney disease even in those nephropathies of non-immunologic origin.

Radioresistance of mesenchymal glioblastoma initiating cells correlates with patient outcome and is associated with activation of inflammatory program

Glioblastoma (GBM) still remains an incurable disease being radiotherapy (RT) the mainstay treatment. Glioblastoma intra-tumoral heterogeneity and Glioblastoma-Initiating Cells (GICs) challenge the design of effective therapies. We investigated GICs and non-GICs response to RT in a paired in-vitro model and addressed molecular programs activated in GICs after RT. Established GICs heterogeneously expressed several GICs markers and displayed a mesenchymal signature. Upon fractionated RT, GICs reported higher radioresistance compared to non-GICs and showed lower α- and β-values, according to the Linear Quadratic Model interpretation of the survival curves. Moreover, a significant correlation was observed between GICs radiosensitivity and patient disease-free survival. Transcriptome analysis of GICs after acquisition of a radioresistant phenotype reported significant activation of Proneural-to-Mesenchymal transition (PMT) and pro-inflammatory pathways, being STAT3 and IL6 the major players. Our findings support a leading role of mesenchymal GICs in defining patient response to RT and provide the grounds for targeted therapies based on the blockade of inflammatory pathways to overcome GBM radioresistance.Glioblastoma (GBM) still remains an incurable disease being radiotherapy (RT) the mainstay treatment. Glioblastoma intra-tumoral heterogeneity and Glioblastoma-Initiating Cells (GICs) challenge the design of effective therapies. We investigated GICs and non-GICs response to RT in a paired in-vitro model and addressed molecular programs activated in GICs after RT. Established GICs heterogeneously expressed several GICs markers and displayed a mesenchymal signature. Upon fractionated RT, GICs reported higher radioresistance compared to non-GICs and showed lower α- and β-values, according to the Linear Quadratic Model interpretation of the survival curves. Moreover, a significant correlation was observed between GICs radiosensitivity and patient disease-free survival. Transcriptome analysis of GICs after acquisition of a radioresistant phenotype reported significant activation of Proneural-to-Mesenchymal transition (PMT) and pro-inflammatory pathways, being STAT3 and IL6 the major players. Our findings support a leading role of mesenchymal GICs in defining patient response to RT and provide the grounds for targeted therapies based on the blockade of inflammatory pathways to overcome GBM radioresistance.