Avian sperm biology has demonstrated specific features in preparation for fertilization. For example, capacitationlike processes and motility hyperactivation do not exist in the form described in ...mammals. The present study investigated the potential involvement of several signaling pathways, including protein kinase A (PKA), phosphatidylinositol 3 kinase (PIK3), mitogen-activated protein kinase 3/1 (MAPK3/1), and MAPK14 in the chicken acrosome reaction (AR). The presence in chicken spermatozoa of key proteins involved in these signaling pathways (i.e., cAMP-responsive element-binding protein CREB, AKT, MAPK1, and MAPK14 and their respective phosphorylated forms) was detected using immunoblotting and localized by immunocytochemistry, mainly in the heads. The potential involvement of these pathways in the AR induced by inner perivitelline layer (IPVL) and Ca²⁺ was then examined using specific inhibitors and phosphorylation status measurements. The effects of the specific inhibitors on motility were also measured. Phosphorylations of AKT, CREB, and MAPK1, but not MAPK14, were increased at the time of AR. Phosphorylation of AKT was increased in the presence of IPVL alone, whereas both IPVL and Ca²⁺ were needed to increase CREB and MAPK1 phosphorylations. Inhibition of the three corresponding pathways blocked the increase in phosphorylation and significantly decreased AR. Inhibitions of the PKA and MAPK1 pathways also significantly decreased motility, whereas MAPK14 and PIK3 inhibition had no effect on motility. Our results suggest that the AR could be mediated by activation of the PKA, PIK3, and MAPK1 pathways through a sequential action involving, successively, PIK3 and then PKA and MAPK1 activations.
Histone deacetylases (HDACs) play an important role in the regulation of gene expression. In addition to histones, HDACs can modulate the function of many other proteins involved in the regulation of ...cell survival and proliferation, angiogenesis, inflammation, and immunity. Deregulated HDACs have been shown to be commonly associated with many types of cancer, and are considered promising targets for cancer therapy. Several HDAC inhibitors are in clinical trials as monotherapies or in combination with other anticancer agents, but only two such inhibitors -- vorinostat (suberoylanilide hydroxamic acid) and romidepsin (depsipeptide) -- have been approved by the US Food and Drug Administration for treating relapsed cutaneous T-cell lymphoma. Other HDAC inhibitors, such as belinostat (PXD101), mocetinostat (MGCD0103), entinostat (SNDX-275), and panobinostat (LBH589), are currently in clinical development. This review focuses on the use of HDAC inhibitors in the treatment of relapsed lymphoma.
The pan-deacetylase inhibitor panobinostat (LBH589) recently has been shown to have significant clinical activity in patients with relapsed Hodgkin lymphoma, but its mechanism of action in Hodgkin ...lymphoma remains unknown. In this study, we demonstrate that panobinostat has potent antiproliferative activity against Hodgkin lymphoma–derived cell lines. At the molecular level, panobinostat activated the caspase pathway, inhibited STAT5 and STAT6 phosphorylation, and down-regulated hypoxia-inducible factor 1 α and its downstream targets, glucose transporter 1 (GLUT1) and vascular endothelial growth factor. Paradoxically, panobinostat inhibited LKB1 and AMP-activated protein kinase, leading to activation of mammalian target of rapamycin (mTOR) that promotes survival. Combining panobinostat with the mTOR inhibitor everolimus (RAD001) inhibited panobinostat-induced mTOR activation and enhanced panobinostat antiproliferative effects. Collectively, our data demonstrate that panobinostat is a potent deacetylase inhibitor against Hodgkin lymphoma–derived cell lines, and provide a mechanistic rationale for combining panobinostat with mTOR inhibitors for treating Hodgkin lymphoma patients. Furthermore, the effect of panobinostat on GLUT1 expression suggests that panobinostat may modulate the results of clinical diagnostic imaging tests that depend of functional GLUT1, such as fluorodeoxyglucose positron emission tomography.
Chicken spermatozoa may remain in the female oviduct for a prolonged period before induction of the acrosome reaction on contact with the inner perivitelline layer (IPVL). By contrast, the acrosome ...reaction may be induced very rapidly in vitro in the presence of IPVL and Ca²⁺. In the present study, we examined the extent to which the chicken acrosome reaction can be induced in media of various compositions in the presence or absence of IPVL and/or Ca²⁺ and other factors known to be efficient in mammals. We also compared the efficacy of perivitelline layer (PL) taken at various states of oocyte maturation in initiating the reaction. The acrosome reaction was induced in less than 5 min in the presence of Ca²⁺ and IPVL. Incubation of spermatozoa in different saline media (Beltsville poultry semen extender (BPSE); Dulbecco's modified eagle medium; NaCl-TES buffer) without IPVL showed a significant induction of acrosome reaction in BPSE supplemented with 5 mM Ca²⁺ and in the three media after supplementation with Ca²⁺ and Ca²⁺ ionophore A23187. By contrast, the acrosome reaction was never induced without Ca²⁺. BSA, NaHCO₃, and progesterone did not stimulate the acrosome reaction. Ca²⁺ plus PL taken at various physiological states (follicle IPVL, ovulated IPVL, oviposited IPVL, and/or outer perivitelline layer) strongly stimulated the acrosome reaction, the latest states being the most efficient. Although PL induced the acrosome reaction in the presence of extracellular Ca²⁺, it was not possible to induce hyperactivation in chicken spermatozoa. Taken together, these results emphasize the central role of Ca²⁺ in the in vitro initiation of the acrosome reaction in chickens and show specific features of this induction in birds.
The effects of
in vitro storage on the sperm's ability to undergo the acrosome reaction (AR) have never been studied in avian species despite its major importance for reproduction management.
The ...ability of chicken sperm to undergo the AR was measured after liquid storage at 4 °C and after cryopreservation, and its relationship with other semen quality parameters, including viability, mass motility and objective motility parameters measured by computer semen analyser (CASA) was analysed in two different flocks. The percentage of intact acrosome-reacting spermatozoa (IAR) was dramatically decreased by 48 h liquid storage (loss of 2/3 among the spermatozoa initially able to undergo the AR) whereas motility, viability and morphological integrity were reduced by 10–15%. By contrast, cryopreservation did not affect the induction of AR in flock 1 (29% IAR) whereas it was strongly affected in flock 2 (7% IAR). Motility parameters, viability and morphology were considerably altered by freezing in every case (more that 50% loss). Positive correlations were found between the percentage of intact acrosome-reacting spermatozoa and viability, mass motility and many objective motility parameters.
Our results showed that the sperm's ability to undergo the AR was much more affected than other sperm functions after storage at 4 °C, while cryopreservation only had an effect in semen with the lowest initial quality. These results raise questions regarding the specific features of chicken sperm biology that must be taken into account in the treatment of semen.
Abstract 4965
The Phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway is frequently deregulated in Hodgkin (HL) and non-Hodgkin lymphoma (NHL), and has been linked with tumor cell growth and ...survival. Although several proteins/enzymes in this pathway can be targeted by a variety of small molecules in vitro and in vivo, it remains unclear which protein target is the ideal for clinical testing. Previous studies demonstrated that the clinical activity of mTOR inhibitors may be attenuated by a negative feedback loop that involves activation of AKT, suggesting that a dual inhibition of AKT and mTOR activation may produce a better therapeutic outcome. To test this hypothesis, we evaluated the in vitro activity of NVP-BEZ235, a dual inhibitor of PI3K and mTOR, in a panel of 13 HL and NHL cell lines. NVP-BEZ235 inhibited cell growth and induced apoptosis in lymphoma cell lines in a time and dose dependent manner. After 48 hours of incubation, the IC50 ranged between 50 and 100 nM, and it was equally effective in ABC and GCB-derived DLBCL cell lines. NVP-BEZ235 induced cell death was primarily due to induction of apoptosis, as evident by the annexin-V and PI dual staining method, and the induction of caspase 3 and PARP cleavage. NVP-BEZ235 effectively inhibited the activation of the PI3K pathway at several steps, including decreasing the phosphorylation level of p-Akt (Ser473), p-Akt (Thr308), p-mTOR, p-4-EBPI and pP70S6K. Because lymphoma cells frequently depend on multiple activated signaling pathways to promote their survival, including the JAK/STAT pathway, we investigated the potential synergy between PI3K and JAK/STAT pathway inhibitors. Lymphoma cells were variably sensitive to the JAK1/2 inhibitor INCB16562 in vitro. Submaximal concentrations of NVP-BEZ235 demonstrated a synergistic activity with INCB16562. Collectively, our data show that the PI3K/mTOR inhibitor NVP-BEZ235 is highly effective against a wide range of lymphoma cell lines, and warrants evaluating it alone and in combination with JAK/STAT inhibitors in phase I/II clinical trials in patients with relapsed lymphoma.
No relevant conflicts of interest to declare.
Abstract 2731
Constitutive activation of Janus Kinases has been shown to be an important mechanism supporting human lymphomagenesis. Aberrantly activated JAK/STAT signaling has been described in ...Hodgkin lymphoma (HL), Anaplastic Large T-cell lymphoma and Diffuse large B cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, making it an attractive target for therapy. In this study we report the activity profile of the JAK2 inhibitor INCB16562 in a panel of lymphoma cell lines (n=17) including Hodgkin (HL), Mantle Cell (MCL), Anaplastic Large T-cell (ALCL), and DLBCL. JAK-STAT pathway was found to be constitutively active (baseline p-STAT3 Tyr 705 detected by western immunoblotting) in the HL (HDLM-2, L.-428 and L-540), ALCL (SUP-M2, KARPAS-299, SUDHL-1) and the ABC derived DLBCL cell lines (HBL-1, U2932, TMD8, OCI-LY-3). In contrast, no activation of JAK-STAT was observed in the HL cell line KM-H2, in the MCL cell lines (Mino, Jeko-1, SP-53) and in the GCB-derived cell lines (SUDHL-4, SUDHL-6, BJAB). To assess the effect of INCB16562 on cell proliferation, cells were first incubated with increasing concentrations of INC16562 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs) and cell viability was evaluated by MTS assay. The IC50 values at 48 hrs ranged from 1 to 9 μM. The ABC derived DLBCL cell line TMD8 showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with INCB16562 1μM for 24 hours. Of note, INCB16562 demostrated activity also in primary DLBCL cells. pSTAT3 negative cell lines were resistant to treatment (IC50 from 4.8 to 6.5 μM). However pretreatment level of pJAKs and pSTATs did not predict sensitivity to INCB16562. In fact inhibition of STAT3 phosphorylation alone was not sufficient to predict sensitivity to the drug in pSTAT3 expressing cells. Simultaneous inhibition of STAT3 and ERK phosphorylation was observed by western immunoblotting in the sensitive cell lines (TMD8, HBL-1, SUP-M2, KARPAS 299, SUDHL-1) (IC50 from 1 to 3 μM), whereas in the resistant cell lines (U2932, OCI-LY3, HD-LM2, L-540, L-428) a paradoxical hyperactivation of pERK was observed after incubation with INCB16562 (IC50 from 5–9 μM). Since it has been reported that sensitivity to JAK inhibition is associated to downregulation of bcl-2 family genes such as Bcl-xL and MCL-1, we investigated the baseline expression of these bcl-2 family members in our cell lines by western blot. Bcl-xL and MCL-1 were expressed at various levels, but there was no correlation between levels of baseline expression and outcome. Both Bcl-xL and MCL-1 were dowregulated in the sensitive cell lines following treatment with INCB16562. Furthermore The bcl-2 inhibitor ABT-737 synergistically enhanced the effect of INCB16562 particularly in HL cell lines (HDLM-2 and L-540) and DLBCL cells lines (HBL-1, TMD8, SUDHL-4). These data demonstrate that INCB16562 has a promising therapeutic value in lymphoma, and provide a rationale for combining INCB16562 with bcl-2 family inhibitors.
No relevant conflicts of interest to declare.
Avian sperm biology has demonstrated specific features in preparation for fertilization. For example, capacitationlike processes and motility hyperactivation do not exist in the form described in ...mammals. The present study investigated the potential involvement of several signaling pathways, including protein kinase A (PKA), phosphatidylinositol 3 kinase (PIK3), mitogen-activated protein kinase 3/1 (MAPK3/1), and MAPK14 in the chicken acrosome reaction (AR). The presence in chicken spermatozoa of key proteins involved in these signaling pathways (i.e., cAMP-responsive element-binding protein CREB, AKT, MAPK1, and MAPK14 and their respective phosphorylated forms) was detected using immunoblotting and localized by immunocytochemistry, mainly in the heads. The potential involvement of these pathways in the AR induced by inner perivitelline layer (IPVL) and Ca2+ was then examined using specific inhibitors and phosphorylation status measurements. The effects of the specific inhibitors on motility were also measured. Phosphorylations of AKT, CREB, and MAPK1, but not MAPK14, were increased at the time of AR. Phosphorylation of AKT was increased in the presence of IPVL alone, whereas both IPVL and Ca2+ were needed to increase CREB and MAPK1 phosphorylations. Inhibition of the three corresponding pathways blocked the increase in phosphorylation and significantly decreased AR. Inhibitions of the PKA and MAPK1 pathways also significantly decreased motility, whereas MAPK14 and PIK3 inhibition had no effect on motility. Our results suggest that the AR could be mediated by activation of the PKA, PIK3, and MAPK1 pathways through a sequential action involving, successively, PIK3 and then PKA and MAPK1 activations.
Abstract 2851
Deacetylases (DAC) inhibitors are promising new class of anticancer agents. Panobinostat (LBH589) is a pan-DAC inhibitor with evidence of clinical activity in patients with relapsed ...classical Hodgkin Lymphoma (HL). However the mechanisms of action of this new drug are not completely understood. The aim of this study was to investigate the mechanisms of antiproliferative effect induced by LBH589 in HL-derived cell lines. Experiments were performed in HL derived-cell lines (HDLM-2, KM-H2 and L-428). Cells were treated with 0.01–1μM of LBH589 alone or in combination with 0.01–1μM of everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR), for 24–72 hours. Growth inhibition and apoptosis were analyzed in response to treatment using MTS assay, Western blotting and flow cytometric analyses. Effect of panobinostat on a panel of 30 cytokines and chemokines was assayed on cells after incubation of 24 hours using a multiplex assay. Panobinostat demonstrated antiproliferative activity in HL cell lines in a dose-and time-dependant manner with an IC50 ranging between 20 and 40nM at 72 hours. The antiproliferative activity was associated with downregulation of the X-linked inhibitor of apoptosis protein (XIAP), activation of caspase 9 and caspase 3, cleavage of Poly ADP Ribosome polymerase (PARP) and induction of apoptosis. In addition, panobinostat downregulated the level of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) a major regulator of tumor cell adaptation to hypoxic stress. Furthermore, panobinostat decreased the level of the vascular endothelial growth factor (VEGF), an HIF-1α target gene, in the cell culture supernatants.The combination of panobinostat and everolimus had synergistic antiproliferative activity in the cells lines. This synergy was due to reciprocal inhibition of negative feedback loops induced by mTOR inhibitor and panobinostat therapy. Collectively, our data demonstrate that panobinostat induces apoptosis in HL cell lines by modulating several survival pathways. Furthermore, the observed synergy between panobinostat and everolimus provides rationale for combining these two active agents in a phase I/II clinical trial in patients with relapsed HL.
No relevant conflicts of interest to declare.
Abstract 3729
The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. ...Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma.
No relevant conflicts of interest to declare.
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