Compensatory Mechanisms in Early Alzheimer's Disease and Clinical Setting: The Need for Novel Neuropsychological Strategies.

Despite advances in the detection of biomarkers and in the design of drugs that can slow the progression of Alzheimer's disease (AD), the underlying primary mechanisms have not been elucidated. The diagnosis of AD has notably improved with the development of neuroimaging techniques and cerebrospinal fluid biomarkers which have provided new information not available in the past. Although the diagnosis has advanced, there is a consensus among experts that, when making the diagnosis in a specific patient, many years have probably passed since the onset of the underlying processes, and it is very likely that the biomarkers in use and their cutoffs do not reflect the true critical points for establishing the precise stage of the ongoing disease. In this context, frequent disparities between current biomarkers and cognitive and functional performance in clinical practice constitute a major drawback in translational neurology. To our knowledge, the In-Out-test is the only neuropsychological test developed with the idea that compensatory brain mechanisms exist in the early stages of AD, and whose positive effects on conventional tests performance can be reduced in assessing episodic memory in the context of a dual-task, through which the executive auxiliary networks are 'distracted', thus uncover the real memory deficit. Furthermore, as additional traits, age and formal education have no impact on the performance of the In-Out-test.

New therapy for pancreatic cancer based on extracellular vesicles.

Pancreatic Ductal Adenocarcinoma (PDAC), is the most common aggressive cancer of the pancreas. The standard care of PDAC includes tumor resection and chemotherapy, but the lack of early diagnosis and the limited response to the treatment worsens the patient's condition. In order to improve the efficiency of chemotherapy, we look for more efficient systems of drug delivery. We isolated and fully characterized small Extracellular Vesicles (EVs) from the RWP-1 cell line. Our study indicates that the direct incubation method was the most efficient loading protocol and that a minimum total amount of drug triggers an effect on tumor cells. Therefore, we loaded the small EVs with two chemotherapeutic drugs (Temozolomide and EPZ015666) by direct incubation method and the amount of drug loaded was measured by high-performance liquid chromatography (HPLC). Finally, we tested their antiproliferative effect on different cancer cell lines. Moreover, the system is highly dependent on the drug structure and therefore RWP-1 small EVsTMZ were more efficient than RWP-1 small EVsEPZ015666. RWP-1 derived small EVs represent a promising drug delivery tool that can be further investigated in preclinical studies and its combination with PRMT5 inhibitor can be potentially developed in clinical trials for the treatment of PDAC.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

Glioblastoma-Derived Small Extracellular Vesicles: Nanoparticles for Glioma Treatment.

Glioblastoma (GBM), characterized by fast growth and invasion into adjacent tissue, is the most aggressive cancer of brain origin. Current protocols, which include cytotoxic chemotherapeutic agents, effectively treat localized disease; however, these aggressive therapies present side effects due to the high doses administered. Therefore, more efficient ways of drug delivery have been studied to reduce the therapeutic exposure of the patients. We have isolated and fully characterized small extracellular vesicles (EVs) from seven patient-derived GBM cell lines. After loading them with two different drugs, Temozolomide (TMZ) and EPZ015666, we observed a reduction in the total amount of drugs needed to trigger an effect on tumor cells. Moreover, we observed that GBM-derived small EVs, although with lower target specificity, can induce an effect on pancreatic cancer cell death. These results suggest that GBM-derived small EVs represent a promising drug delivery tool for further preclinical studies and potentially for the clinical development of GBM treatments.

CLytA-DAAO Chimeric Enzyme Bound to Magnetic Nanoparticles. A New Therapeutical Approach for Cancer Patients?

D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma.

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines.

We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.

Cell Death Mechanisms Induced by CLytA-DAAO Chimeric Enzyme in Human Tumor Cell Lines.

The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer.

CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, induces Cell Death in Human Cancer Cells.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.

Correction: Radiotherapy resistance acquisition in Glioblastoma. Role of SOCS1 and SOCS3.

[This corrects the article DOI: 10.1371/journal.pone.0212581.].

Radiotherapy resistance acquisition in Glioblastoma. Role of SOCS1 and SOCS3.

Glioblastoma multiforme (GBM) is a poor prognosis type of tumour due to its resistance to chemo and radiotherapy. SOCS1 and SOCS3 have been associated with tumour progression and response to treatments in different kinds of cancers, including GBM. In this study, cell lines of IDH-wildtype GBM from primary cultures were obtained, and the role of SOCS1 and SOCS3 in the radiotherapy response was analysed. Fifty-two brain aspirates from GBM patients were processed, and six new cell lines of IDH-wildtype GBM were established. These new cell lines were characterized according to the WHO classification of CNS tumours. SOCS1 and SOCS3 expression levels were determined, at mRNA level by Q-PCR, at protein level by immunocytochemistry, and Western blot analysis. The results showed that SOCS1 and SOCS3 are overexpressed in GBM, as compared to a non-tumoral brain RNA pool. SOCS1 and SOCS3 expression were reduced by siRNA, and it was found that SOCS3 inhibition increases radioresistance in GBM cell lines, suggesting a key role of SOCS3 in radioresistant acquisition. In addition, radioresistant clonal populations obtained by selective pressure on these cell cultures also showed a significant decrease in SOCS3 expression, while SOCS1 remained unchanged. Furthermore, the induction of SOCS3 expression, under a heterologous promoter, in a radiotherapy resistant GBM cell line increased its radiosensitivity, supporting an important implication of SOCS3 in radiotherapy resistance acquisition. Finally, the treatment with TSA in the most radioresistant established cell line produced an increase in the effect of radiotherapy, that correlated with an increase in the expression of SOCS3. These effects of TSA disappeared if the increase in the expression of SOCS3 prevented with an siRNA against SOCS3. Thus, SOCS3 signal transduction pathway (JAK/STAT) could be useful to unmask new putative targets to improve radiotherapy response in GBM.

In-Out-Test: A New Paradigm for Sorting the Wheat from the Chaff in Prodromal Alzheimer's Disease.

BACKGROUND: Assessment of hippocampal amnesia is helpful to distinguish between normal cognition and mild cognitive impairment (MCI), but not for identifying converters to dementia. Here biomarkers are useful but novel neuropsychological approaches are needed in their absence. The In-out-test assesses episodic memory using a new paradigm hypothesized to avoid reliance on executive function, which may compensate for damaged memory networks. OBJECTIVE: To assess the validity of the In-out-test in identifying prodromal Alzheimer's disease (PAD) in a clinical setting, by comparing this to the Free and Cued Selective Reminding Test (FCSRT) and cerebrospinal fluid biomarkers. METHODS: A cross-sectional study of 32 cognitively healthy, 32 MCI, and 30 progressive dementia subjects. All participants were given both the In-out-test and the FCSRT; 40 of them also received a lumbar puncture. RESULTS: Internal consistency was demonstrated using Cronbach Alpha (r = 0.81) and Inter-rater reliability with Kappa (k = 0.94). Intraclass correlation (ICC) for test-retest reliability: r = 0.57 (p = 0.57). ICC between the In-out-test and FCSRT r = 0.87 (p = 0.001). ICC between the In-out-test and Aß42 and P-tau/Aß42 for controls: 0.73 and 0.75, respectively; P-tau for MCI: 0.77 and total sample: 0.70; Aß42 for dementia: 0.71. All ICC measures between FCSRT and biomarkers were ≤0.264. AD diagnosis: In-out-test k = 0.71; FCSRT k = 0.49. PAD diagnosis (N = 35): In-out-test k = 0.69; FCSRT k = 0.44. CONCLUSIONS: The In-out-test detected prodromal AD with a higher degree of accuracy than a conventional hippocampal-based memory test. These results suggest that this new paradigm could be of value in clinical settings, predicting which patients with MCI will go on to develop AD.

PDGFR and IGF-1R Inhibitors Induce a G2/M Arrest and Subsequent Cell Death in Human Glioblastoma Cell Lines.

Glioblastomas are highly resistant to radiation and chemotherapy. Currently, there are no effective therapies for this type of tumor. Signaling mechanisms initiated by PDGFR and IGF-1R are important in glioblastoma, and inhibition of the signal transduction pathways initiated by these receptors could be a useful alternative strategy for glioblastoma treatment. We have studied the effects of the PDGFR inhibitor JNJ-10198409 (JNJ) and the IGF-1R inhibitor picropodophyllin (PPP) in glioblastoma cell lines as well as in primary cultures derived from patients affected by this type of tumor. JNJ and PPP treatment blocked PDGFR and IGF-1R signaling respectively and reduced Akt and Erk 1/2 phosphorylation. Both inhibitors diminished cell proliferation, inducing a G2/M block of the cell cycle. Cell death induced by JNJ was caspase-dependent, Annexin-V positive and caused PARP cleavage, especially in T98 cells, suggesting an apoptotic mechanism. However, cell death induced by PPP was not completely inhibited by caspase inhibitors in all cell lines apart from LN-229 cells, indicating a caspase-independent mechanism. Several inhibitors targeted against different cell death pathways could not block this caspase-independent component, which may be a non-programmed necrotic mechanism. Apoptotic arrays performed in T98 and LN-229 cells upon JNJ and PPP treatment revealed that procaspase 3 levels were augmented by both drugs in T98 cells and only by JNJ in LN229-cells. Furthermore, XIAP and survivin levels were much higher in LN-229 cells than in T98 cells, revealing that LN-229 cells are more susceptible to undergo caspase-independent cell death mechanisms. JNJ and PPP combination was more effective than each treatment alone.

HGUE-C-1 is an atypical and novel colon carcinoma cell line.

BACKGROUND: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab. METHODS: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry. RESULTS: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of ß-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms. CONCLUSIONS: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/ßcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol-based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922-sensitive cell lines lacked NAD(P)H: quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.

Resistance to Selumetinib (AZD6244) in colorectal cancer cell lines is mediated by p70S6K and RPS6 activation.

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Dual regulation of P-glycoprotein expression by trichostatin A in cancer cell lines.

BACKGROUND: It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. METHODS: A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study. RESULTS: The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. CONCLUSIONS: The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates differentially both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumour drugs that are substrates of Pgp. Finally, we also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines.

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G(1) arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G(1) arrest. This G(1) arrest was associated with up-regulation of p27(kip1), whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G(1) arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Protein kinase C-alpha antagonizes apoptosis induction by histone deacetylase inhibitors in multidrug resistant leukaemia cells.

Previous studies have documented that while several drug-resistant cells enter apoptosis upon treatment with histone deacetylase inhibitors (iHDACs), their drug-sensitive counterparts do not. In the present study, we have investigated at the molecular level why parental drug-sensitive tumor cells do not respond to Trichostatin A and suberoylanilide hydroxamic acid, two iHDACs that promote apoptosis in drug-resistant leukaemia cells. Taking murine leukaemia L1210 cells as a model, we have determined that: (i) PKC-alpha expression is more elevated in parental L1210 than in drug-resistant L1210/R cells, (ii) activation of PKC neutralizes iHDACs-mediated apoptosis in L1210/R cells, (iii) depletion of PKC in parental L1210 cells results in a positive response to iHDACs-mediated apoptosis, and (iv) transfection of a mutant constitutively active PKC-alpha form in L1210/R cells makes the cells refractory to apoptosis induction by iHDACs. These results allow us to conclude that activation/high expression of PKC-alpha protects parental drug-sensitive L1210 cells from iHDACs-mediated apoptosis. Thus, determination of PKC-alpha levels/activity in leukaemia seems to be relevant when choosing efficient chemotherapy protocols based on the use of apoptosis-inducing anticancer drugs.

Post-transcriptional regulation of P-glycoprotein expression in cancer cell lines.

The present study of inhibitors shows that the histone deacetylase-induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5' end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5' untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA.

Tumour cells resistance in cancer therapy.

Resistance to chemotherapeutic drugs presents a big caveat for cancer treatment. In this review we will describe the molecular mechanisms involved in chemoresistance, discussing the mechanisms of resistance related to tumour microenvironment, as well as their intracellular mechanisms. Chemoresistance can also appear as a consequence to treatments with new anticancer drugs. In this sense, we will exemplify this type of resistance discussing mechanisms of action of epidermal growth factor receptor (EGFR) inhibitors. We conclude that the main problem of chemoresistance is due to its pleiotropic and multifactorial nature.