Identificación de biomarcadores de quimiorresistencia e invasividad para la mejora del tratamiento del cáncer colorrectal

El cáncer colorrectal es un tipo de cáncer muy prevalente en el mundo. La cirugía junto con la quimioterapia como adyuvante han resultado efectivas en fases de I a la III. El uso de quimioterápicos puede fracasar debido a fenómenos de quimiorresistencia. Uno de los objetivos de este trabajo fue investigar la respuesta frente al fármaco quimioterapéutico Oxaliplatino. Para ello se utilizó un panel de líneas celulares de cáncer de colon y tumores de pacientes con cáncer colorrectal extirpados en el Hospital Clínico Universitario Virgen de la Arrixaca, a partir de los cuales se realizaron cultivos primarios. Como resultado, dichas líneas celulares y cultivos primarios se clasificaron en sensibles y resistentes. Al estudiar la señalización del daño en el ADN, se obtuvo que Chk1 y Chk2, proteínas control de dicha señalización, conseguían activarse por una vía distinta de ATR y ATM, respectivamente. Una de las posibles quinasas activadora de Chk1 podría ser VRK1 y además, la proteína Rad51, implicada en la reparación del daño, se activaría en ausencia de la proteína ATR. En el análisis de mutaciones no se pudo demostrar una asociación entre la presencia de mutaciones en KRAS, BRAF y PIK3CA y la respuesta a Oxaliplatino. La mutación p.R273H en el gen TP53 podría impedir la correcta reparación del daño en el ADN causado por Oxaliplatino, a diferencia de la mutación p.S241F

Modifications in Gene Expression in the Process of Osteoblastic Differentiation of Multipotent Bone Marrow-Derived Human Mesenchymal Stem Cells Induced by a Novel Osteoinductive Porous Medical-Grade 3D-Printed Poly(ε-caprolactone)/β-tricalcium Phosphate Composite

In this work, we evaluated the influence of a novel hybrid 3D-printed porous composite scaffold based on poly(ε-caprolactone) (PCL) and β-tricalcium phosphate (β-TCP) microparticles in the process of adhesion, proliferation, and osteoblastic differentiation of multipotent adult human bone marrow mesenchymal stem cells (ah-BM-MSCs) cultured under basal and osteogenic conditions. The in vitro biological response of ah-BM-MSCs seeded on the scaffolds was evaluated in terms of cytotoxicity, adhesion, and proliferation (AlamarBlue Assay®) after 1, 3, 7, and 14 days of culture. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization (Alizarin Red Solution, ARS), expression of surface markers (CD73, CD90, and CD105), and reverse transcription–quantitative polymerase chain reaction (qRT-PCR) after 7 and 14 days of culture. The scaffolds tested were found to be bioactive and biocompatible, as demonstrated by their effects on cytotoxicity (viability) and extracellular matrix production. The mineralization and ALP assays revealed that osteogenic differentiation increased in the presence of PCL/β-TCP scaffolds. The latter was also confirmed by the gene expression levels of the proteins involved in the ossification process. Our results suggest that similar bio-inspired hybrid composite materials would be excellent candidates for osteoinductive and osteogenic medical-grade scaffolds to support cell proliferation and differentiation for tissue engineering, which warrants future in vivo research

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

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