Daniele Tibullo

Nuclear translocation of heme oxygenase-1 confers resistance to imatinib in chronic myeloid leukemia cells.

Identification of imatinib mesylate as a potent inhibitor of the Abl kinase and the subsequent findings that this compound displays growth inhibitory and pro-apoptotic effects in Bcr-Abl+ cells, has deeply conditioned CML treatment. Unfortunately the initial striking efficacy of this drug has been overshadowed by the development of clinical resistance. A wide variety of molecular mechanisms can underlie such resistance mechanisms. In the recent years, heme oxygenase-1 (HO-1) expression has been reported as an important protective endogenous mechanism against physical, chemical and biological stress and this cytoprotective role has already been demonstrated for several solid tumors and acute leukemias. The aim of the present study was to investigate the effect of HO-1 expression on cell proliferation and apoptosis in chronic myeloid leukemia cells, K562 and LAMA-84 cell lines following imatinib treatment. Cells were incubated for 24h with Imatinib (1 μM) alone or in combination with Hemin (10μM), an inducer of HO-1. In addition, cells were also treated with HO byproducts, bilirubin and carbon monoxide (CO), or with a protease inhibitor (Ed64) to inhibit HO-1 nuclear translocation. Pharmacological induction of HO-1 was able to overcome the effect of imatinib. The cytoprotective effect of HO-1 was further confirmed after silencing HO-1 by siRNA. Interestingly, neither bilirubin nor CO was able to protect cells from Imatinib-induced toxicity. By contrast, the protective effect of HO-1 was mitigated by the addition of E64d, preventing HO-1 nuclear translocation. Finally, imatinib was able to increase the formation of cellular reactive oxygen species (ROS) and this effect was reversed by HO-1 induction or the addition of N-acetylcisteine (NAC). In conclusion, the protective effect of HO-1 on imatinib-induced cytotoxicity does not involve its enzymatic byproducts, but rather the nuclear translocation of HO-1 following proteolytic cleavage.

Silibinin improves hepatic and myocardial injury in mice with nonalcoholic steatohepatitis

BACKGROUND Nonalcoholic fatty liver disease is a chronic metabolic disorder with significant impact on cardiovascular and liver mortality. AIMS In this study, we examined the effects of silibinin on liver and myocardium injury in an experimental model of nonalcoholic fatty liver disease. METHODS A four-week daily dose of silibinin (20 mg/kg i.p.) was administrated to db/db mice fed a methionine-choline deficient diet. Hepatic and myocardial histology, oxidative stress and inflammatory cytokines were evaluated. RESULTS Silibinin administration decreased HOMA-IR, serum ALT and markedly improved hepatic and myocardial damage. Silibinin reduced isoprostanes, 8-deoxyguanosine and nitrites/nitrates in the liver and in the heart of db/db fed the methionine-choline deficient diet, whereas glutathione levels were restored to lean mice levels in both tissues. Consistently, liver mitochondrial respiratory chain activity was significantly impaired in untreated mice and was completely restored in silibinin-treated animals. TNF-α was increased whereas IL-6 was decreased both in the liver and heart of db/db fed methionine-choline deficient diet. Silibinin reversed heart TNF-α and IL-6 expression to control mice levels. Indeed, liver JNK phosphorylation was reduced to control levels in treated animals. CONCLUSIONS This study demonstrates a combined effectiveness of silibinin on improving liver and myocardial injury in experimental nonalcoholic fatty liver disease.

Effects of imatinib mesylate in osteoblastogenesis.

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Evaluation of novel aryloxyalkyl derivatives of imidazole and 1,2,4-triazole as heme oxygenase-1 (HO-1) inhibitors and their antitumor properties.

A novel series of aryloxyalkyl derivatives of imidazole and 1,2,4-triazole, 17-31, was designed and synthesized as inhibitors of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). Some of these compounds were found to be good inhibitors of HO-1, in particular those carrying an imidazole moiety as azolyl group and a 3-bromo or 4-iodophenyl as aryl moiety. The most potent compounds 6 and 30 were selected and studied for their antitumor properties in a model of LAMA-84 R cell line overexpressing HO-1 and resistant to imatinib mesylate (IM), a tyrosine-kinase inhibitor used in the treatment of multiple types of cancer, most notably Philadelphia Chromosome positive (Ph(+)) Chronic Myelogenous Leukemia (CML). Results show that both 6 and 30 sensitized LAMA-84 R cell line to antitumor properties of IM.

Consequences of metaphase II oocyte cryopreservation on mRNA content

INTRODUCTION We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). MATERIAL AND METHODS Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. RESULTS There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. CONCLUSIONS Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte.

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity Together with Polymorphonuclear Leukocytes (PMNs) in Chronic Myeloid Leukemia Patients

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.

Determination of chitinases family during osteoclastogenesis

Mammalian chitinases consisting of CHIA, CHIT1, CHI3L1, CHI3L2 and CHID1 exert important biological roles in the monocyte lineage and chronic inflammatory diseases. Pathological bone resorption is a cause of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, osteoporosis, periodontitis and cancer metastasis. The biologic role of chitinases in bone resorption is poorly understood. In this study, we evaluated the expression of the chitinases family during osteoclast differentiation. The expression of CHIA, CHI3L2 and CHID1 resulted unchanged during osteoclast differentiation, whereas CHIT1 and CHI3L1 increased significantly. We also observed that CHIT1 and CHI3L1 are involved in osteoclast function. Indeed, silencing CHIT1 and CHI3L1 with siRNA resulted in a significant decrease in bone resorption activity. In addition, transfection with CHIT1 or CHI3L1 siRNA and co-transfection with both decreased the levels of the pro-differentiative marker MMP9. Overall, these discoveries reveal a novel and crucial role for both CHIT1 and CHI3L1 in promoting bone resorption and identifying new potential candidate markers for therapeutic targeting.

Effects of second-generation tyrosine kinase inhibitors towards osteogenic differentiation of human mesenchymal cells of healthy donors

The BCR‐ABL inhibitor imatinib is a standard first‐line therapy for patients with chronic myeloid leukemia. However, it has been demonstrated that this long‐term treatment is associated with altered bone metabolism. The mechanisms of this effect are not fully understood, but an inhibition of the platelet‐derived growth factor receptor (PDGF‐R) β axis has been suspected on the basis of some in vitro findings. We evaluated the osteoblastic differentiation of mesenchymal stem cells derived from bone marrow (hBM‐MSCs) after in vitro treatment with dasatinib, nilotinib or bosutinib. Human bone marrow mesenchymal stem cells were induced to differentiate in osteoblastic cells by treatment with osteogenic medium with or without dasatinib, nilotinib or bosutinib. We found that the addition of dasatinib, and to a greater extend nilotinib, induced expression of osteogenic mRNA markers as compared with cultures with standard medium or osteogenic medium only. However, treatment with bosutinib did not induce an increase of osteogenic markers. In conclusion, we show that besides imatinib, other tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib, but not bosutinib, increase osteogenic markers in hBM‐MSCs. Because bosutinib differs from the other TKIs because of its low affinity to other kinases such as PDGF‐R, these experiments suggest that inhibition of PDGF‐R may be involved in the induction of osteoblastogenesis by TKIs. Copyright

A cytoprotective role for the heme oxygenase-1/CO pathway during neural differentiation of human mesenchymal stem cells

The inducible protein heme oxygenase‐1 (HO‐1) catalyzes the oxidation of heme to carbon monoxide (CO) and biliverdin, which play a concerted action in cytoprotection against oxidative stress and in the modulation of cell proliferation and differentiation. Here we report that both HO‐1 expression and activity can be highly increased in undifferentiated human mesenchymal stem cells (MSCs) treated with hemin, a known HO‐1 inducer. However, HO‐1 mRNA and protein expression gradually decrease when MSCs undergo neural differentiation in vitro, making them extremely susceptible to glutamate‐mediated cytotoxicity. A time course for HO‐1 revealed that this protein is markedly down‐regulated after 2 days and returns to control levels 6 days after differentiation. Treatment with glutamate (250 μM) after 2 days of neural differentiation resulted in a more pronounced lactate dehydrogenase release, a marker of cell injury, compared with undifferentiated cells. Notably, cells pretreated with hemin (50 μM) or compounds that release small amounts of CO (10 μM CORM‐3 and CORM‐A1) rendered cells more resistant to glutamate‐induced toxicity; this effect was evident in both undifferentiated and differentiated MSCs. Our findings indicate that MSCs become more vulnerable to oxidative injury during the early stages of differentiation via mechanisms that involve a temporary inhibition of HO‐1 expression. Thus, overexpression of HO‐1 and CO‐releasing molecules could provide a possible therapeutic strategy to improve cell viability during neural differentiation in applications that use stem cell technology.

Expression of CHI3L1 and CHIT1 in osteoarthritic rat cartilage model. A morphological study

Osteoarthritis is a degenerative joint disease, which affects millions of people around the world. It occurs when the protective cartilage at the end of bones wears over time, leading to loss of flexibility of the joint, pain and stiffness. The cause of osteoarthritis is unknown, but its development is associated with different factors, such as metabolic, genetic, mechanical and inflammatory ones. In recent years the biological role of chitinases has been studied in relation to different inflammatory diseases and more in particular the elevated levels of human cartilage glycoprotein 39 (CHI3L1) and chitotriosidase (CHIT1) have been reported in a variety of diseases including chronic inflammation and degenerative disorders. The aim of this study was to investigate, by immunohistochemistry, the distribution of CHI3L1 and CHIT1 in osteoarthritic and normal rat articular cartilage, to discover their potential role in the development of this disease. The hypothesis was that the expression of chitinases could increase in OA disease. Immunohistochemical analysis showed that CHI3L1 and CHIT1 staining was very strong in osteoarthritic cartilage, especially in the superficial areas of the cartilage most exposed to mechanical load, while it was weak or absent in normal cartilage. These findings suggest that these two chitinases could be functionally associated with the development of osteoarthritis and could be used as markers, so in the future they could have a role in the daily clinical practice to stage the severity of the disease. However, the longer-term in vivoand in vitro studies are needed to understand the exact mechanism of these molecules, their receptors and activities on cartilage tissue.