ABSTRACT
Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.
Subject(s)
Antineoplastic Agents/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Rhabdomyosarcoma/pathology , src-Family Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Blotting, Western , Cell Differentiation/physiology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Fluorescent Antibody Technique , Humans , Mice , Polymerase Chain Reaction , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The activity of the Retinoblastoma protein, the master regulator of the cell cycle, is finely regulated by phosphorylation. CDKs and cyclins are major players in phosphorylation and it has been recently discovered that the prolyl isomerase Pin1 is an essential protein that orchestrates this process. In this article, we report new findings regarding the role of Pin1 in the pRb pathway. Our data suggest that PI3K, CDKs, and the Pin1 axis have a critical role in sustaining the complete phosphorylation of pRb. Furthermore, we analyze the correlation between Pin1 and pRb phosphorylation in vivo. We show that, in human malignant glioma tissue microarrays (TMA) and in Pin1 knockout (KO) mice, there is a positive correlation between Pin1 and pRb phosphorylation. Prospectively, our findings suggest that the synergism between CDKs, Pin1, and PI3K inhibitors hold great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.
Subject(s)
Gene Expression Regulation/physiology , Peptidylprolyl Isomerase/metabolism , Retinoblastoma Protein/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Central Nervous System Neoplasms/metabolism , Glioma/metabolism , Humans , Immunohistochemistry , Mice , Mice, Knockout , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/genetics , Phosphorylation , Retinoblastoma Protein/genetics , Signal TransductionABSTRACT
Imatinib (IM) is considered the gold standard for chronic myeloid leukemia (CML) treatment, although resistance is emerging as a significant problem. The proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) play an important role in cell proliferation, survival, and resistance to glucocorticoid-mediated cell death. Several transcription factors such as NF-KB and AP-1 are activated in response to physiopathological increases and modulation of intracellular calcium levels. Our previous study demonstrated that lymphocytes from CML patients showed dysregulated calcium homeostasis and oxidative stress. Alteration in ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation. In this study, we hypothesized that IL-6, IL-8, NF-kB, AP-1, and intracellular calcium may be used as selective and prognostic factors to address the follow-up in CML patients treated with imatinib. Our results demonstrated a significant down-regulation in IL-6 and IL-8 release as well as NF-kB and AP-1 activation in lymphomonocytes from Imatinib-treated patients, compared to samples from untreated patients. In parallel, IM treatment, in vivo and in vitro, were able to modulate the intracellular calcium concentration of peripheral blood mononuclear cells of CML patients by acting at the level of InsP(3) receptor in the endoplasmic reticulum and at the level of the purinergic receptors on plasma membrane. The results of this study show that measurements of NF-kB, AP-1, IL-6, IL-8, and intracellular calcium in CML patients treated with Imatinib may give important information to the hematologist on diagnostic criteria and are highly predictive in patients with newly diagnosed CML.
Subject(s)
Antineoplastic Agents/therapeutic use , Calcium/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukocytes, Mononuclear/drug effects , NF-kappa B/metabolism , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Transcription Factor AP-1/metabolism , Adult , Benzamides , Case-Control Studies , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Down-Regulation , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Female , Humans , Imatinib Mesylate , Inositol 1,4,5-Trisphosphate Receptors/drug effects , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Italy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Receptors, Purinergic/drug effects , Receptors, Purinergic/metabolism , Treatment OutcomeABSTRACT
BACKGROUND: The steroid hormone testosterone has been found to be greatly reduced by opioids in different experimental and clinical conditions. The purpose of this study on male rats was to determine the effects of a single injection of morphine (5 mg/Kg) on persistent pain (formalin test) and the single or combined effects on p450-aromatase and 5-alpha reductase type 1 mRNA expression in the brain, liver and testis. Testosterone was determined in the plasma and in the brain, morphine was assayed in the plasma. RESULTS: In the morphine-treated rats, there were increases of 5-alpha reductase mRNA expression in the liver and aromatase mRNA expression in the brain and gonads. Morphine was detected in the blood of all morphine-treated rats even though there were no clear analgesic affects in the formalin-treated animals three hours after treatment. Testosterone was greatly reduced in the plasma and brain in morphine-treated subjects. CONCLUSIONS: It appears that morphine administration can induce long-lasting genomic effects in different body areas which contribute to the strong central and peripheral testosterone levels. These changes were not always accompanied by behavioral modifications.
Subject(s)
Aromatase/genetics , Cholestenone 5 alpha-Reductase/genetics , Gene Expression Regulation, Enzymologic/drug effects , Morphine/pharmacology , Pain/enzymology , Pain/genetics , Animals , Aromatase/metabolism , Behavior, Animal/drug effects , Brain/drug effects , Brain/metabolism , Cholestenone 5 alpha-Reductase/metabolism , Formaldehyde , Male , Morphine/administration & dosage , Morphine/therapeutic use , Organ Specificity/drug effects , Organ Specificity/genetics , Pain/drug therapy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Testosterone/bloodABSTRACT
RB loss has long been recognized as the causative genetic alteration underlying retinoblastoma but it is increasingly evident that other alterations are required for the tumor to develop. Therefore, we set out to identify additional inheritable susceptibility markers and new potential preventive and therapeutic targets for retinoblastoma. We focused on the p16INK4A tumor suppressor gene because of its possible role in retinoblastoma pathogenesis and its involvement in predisposition to familial cancer. p16INK4A expression was analyzed in tumor samples from retinoblastoma patients by immunohistochemistry and in peripheral blood cells from both patients and their parents by real-time quantitative reverse transcription-PCR (qRT-PCR). Since promoter methylation is a common mechanism regulating p16INK4A expression, the methylation status of its promoter was also analyzed in blood samples from patients and their parents by methylation-specific PCR. A downregulation of p16INK4A was observed in 55% of retinoblastoma patients. Interestingly, in 56% of the cases showing p16INK4A downregulation at least one of the patients' parents bore the same alteration in blood cells. Analysis of p16INK4A promoter methylation showed hypermethylation in most patients with p16INK4A downregulation and in the parents with the same alteration in p16INK4A expression. The finding that p16INK4A was downregulated both in patients and their parents suggests that this alteration could be a novel inheritable susceptibility marker to retinoblastoma. The observation that p16INK4A downregulation seems to be due to its promoter hypermethylation opens the way for the development of new preventive and therapeutic strategies using demethylating agents.
