Control of advanced and refractory acute myelogenous leukaemia with sirolimus-based non-myeloablative allogeneic stem cell transplantation

Non-myeloablative conditioning has extended the use of allogeneic haematopoietic transplant to many previously ineligible patients. We added the immunosuppressive and antitumour agent sirolimus (rapamycin) to an established transplant regimen of fludarabine 25 mg/m(2) days -7 through -3 and cyclophosphamide 1000 mg/m(2) days -7 and -6, with tacrolimus and methotrexate immunoprophyllaxis. A total of 23 patients with acute myelogenous leukaemia (AML) were treated, with a median age of 59 years (range: 28-72) at transplant. Only seven patients in total were in complete remission prior to transplantation. Nine patients were in chemotherapy-refractory progression and seven were primarily refractory to induction therapy. Six patients received matched sibling, 11 unrelated donor, 1-5/6 matched and five haploidentical (haplo - three of six or four of six matched) transplants. The haplo-recipients also received antithymocyte globulin, all patients engrafted. Only two, both recipients of haploidentical cells, have died from transplant-related causes. Twelve of 23 patients survived at 198-1162-d post-transplant (median 578). Four of 12 survivors relapsed at 83, 88, 243 and 508 d and three were in remission after chemotherapy and donor lymphocyte infusion. Although follow up is short, this data suggests that non-myeloablative haematopoietic cell transplantation with sirolimus (rapamycin)-based immunosuppression may provide disease control over several years in some patients with advanced and poor prognosis AML.     

Ribosomal protein S6 kinase 1 signaling in prefrontal cortex controls depressive behavior

Current treatments for major depressive disorder (MDD) have a time lag and are ineffective for a large number of patients. Development of novel pharmacological therapies requires a comprehensive understanding of the molecular events that contribute to MDD pathophysiology. Recent evidence points toward aberrant activity of synaptic proteins as a critical contributing factor. In the present studies, we used viral-mediated gene transfer to target a key mediator of activity-dependent synaptic protein synthesis downstream of mechanistic target of rapamycin complex 1 (mTORC1) known as p70 S6 kinase 1 (S6K1). Targeted delivery of two mutants of S6K1, constitutively active or dominant-negative, to the medial prefrontal cortex (mPFC) of rats allowed control of the mTORC1/S6K1 translational pathway. Our results demonstrate that increased expression of S6K1 in the mPFC produces antidepressant effects in the forced swim test without altering locomotor activity. Moreover, expression of active S6K1 in the mPFC blocked the anhedonia caused by chronic stress, resulting in a state of stress resilience. This antidepressant response was associated with increased neuronal complexity caused by enhanced S6K1 activity. Conversely, expression of dominant-negative S6K1 in the mPFC resulted in prodepressive behavior in the forced swim test and was sufficient to cause anhedonia in the absence of chronic stress exposure. Together, these data demonstrate a critical role for S6K1 activity in depressive behaviors, and suggest that pathways downstream of mTORC1 may underlie the pathophysiology and treatment of MDD.Major depressive disorder (MDD) affects nearly one-fifth of the US population (1) and represents the second-leading cause of disability worldwide. Despite its prevalence, successful treatment of MDD is hindered by a lack of understanding of the molecular mechanisms underlying the disorder. Recent evidence points to a critical role for the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, which controls synaptic protein synthesis in mediating antidepressant behavior.Drugs that demonstrate rapid antidepressant activity in preclinical models of depression, such as the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine, the NR2B-selective antagonist Ro 25-6981, the muscarinic antagonist scopolamine, and the metabotropic glutamate receptor 2/3 (mGluR2/3) antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495, all share a common signaling profile through cellular protein synthesis cascades. All of these drugs increase activity of mTORC1 and its downstream substrates p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (2–4). Pharmacological inhibition of mTORC1 in the medial prefrontal cortex (mPFC) of rats completely blocks the behavioral effects of these drugs (2–4). These data suggest that enhanced signaling through the mTORC1 pathway is required for rapid antidepressant responses. It remains unknown, however, whether direct modulation of this translational pathway is sufficient to control depressive behavior, and which downstream targets of mTORC1 play a critical role.To address this question, we used a viral-mediated approach to target S6K1 in discrete brain regions to assess depressive behavior. We demonstrate that targeted activation or suppression of S6K1 activity in the mPFC is sufficient to produce antidepressant or depressive behavior, respectively. These data demonstrate that manipulations of S6K1 in the mPFC can bidirectionally control depressive behavior, and suggest that aberrant activity of synaptic protein synthesis pathways may underlie the pathophysiology of MDD.     

Angiographic and clinical outcome for the treatment of in-stent restenosis with sirolimus-eluting stent compared to vascular brachytherapy

Summary Background With the use of coronary stents for the treatment of coronary artery disease, in-stent restenosis became a major clinical problem. In this non-randomized study, we examined the use of stent-based delivery of sirolimus (rapamycin) for the treatment of in-stent restenosis in comparison to intracoronary -brachytherapy, regarding the clinical effectiveness and the angiographic results for the treatment of in-stent restenosis after 6–9 months. Methods and results Between July 2001 and May 2002, 28 patients (65±11 years) with instent restenosis were treated with intracoronary brachytherapy. Consecutively, between May 2002 and April 2003, 28 patients (65±10 years) with in-stent restenosis were treated with the implantation of a sirolimus-eluting stent (SES). Patients with in-stent restenosis treated by implantation of a SES had significantly lower incidence of in-stent restenosis (1/28 (3.6%) vs 10/28 (36%); p=0.007) and insegment restenosis (4/28 (14%) vs 14/28 (50%); p=0.013) compared to patients treated with brachytherapy. Target lesion and target vessel revascularization rate tended to be lower in the SES group (14 vs 25%) but did not yet reach statistical significance. One patient died in the group treated by implantation of a SES eight months after stenting, one patient suffered from myocardial infarction due to a subtotal in-stent restenosis after brachytherapy. Two patients after brachytherapy underwent surgical revascularization due to recurrent in-stent restenosis similar to the patient with in-stent restenosis after SES implantation. Conclusion In this study we show the feasibility and safety of the treatment of in-stent restenosis by implantation of sirolimus-eluting stents and demonstrate a lower incidence of recurrent in-stent restenosis as well as lower late luminal loss compared to treatment by intravascular brachytherapy.

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Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells

The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.     

Phosphatidylethanolamine-binding protein 4 promotes lung cancer cells proliferation and invasion via PI3K/Akt/mTOR axis

Background

While phosphatidylethanolamine-binding protein 4 (PEBP4) is a key factor in the malignant proliferation and metastasis of tumor cells, the exact regulatory network governing its roles remains unclear. This study was designed to investigate the effect of PEBP4 on PI3K/Akt/mTOR pathway and explore its molecular network that governs the proliferation and metastasis of tumor cells.

Methods

After the recombinant plasmid pcDNA3.1-PEBP4 was constructed, the recombinant plasmid pcDNA3.1-PEBP4 and PEBP4-targeting siRNA were transfected into lung cancer HCC827 cell line. The expressions of PI3K/Akt/mTOR pathway components in HCC827 cells in each group were determined using Western blotting. In the HCC827 cells, the effect of PI3K pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Furthermore, the effect of mTOR inhibitor rapamycin (RAPA) on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of RAPA on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay.

Results

As shown by Western blotting, the protein expressions of p-Akt and phosphorylated mTOR (p-mTOR) were significantly higher in the pcDNA3.1-PEBP4-transfected group than in the normal control group and PEBP4 siRNA group (P<0.05); furthermore, the protein expressions of p-Akt and p-mTOR significantly decreased in the PEBP4 targeting siRNA-transfected group (P<0.05). Treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly inhibited the protein expressions of p-Akt and p-mTOR in HCC827 cells (P<0.05). In contrast, treatment with RAPA only significantly inhibited the protein expression of p-mTOR (P<0.05). As shown by MTT, flow cytometry, and Transwell migration assay, both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and RAPA could significantly lower the viability of HCC827 cells and inhibit their proliferation and invasion (P<0.05); meanwhile, they could reverse the effect of PEBP4 in promoting the proliferation and migration of HCC827 cells (P<0.05).

Conclusions

The overexpression of PEBP4 increases the phosphorylation levels of Akt and mTOR in lung cancer cells. The PI3K/Akt/mTOR signaling axis may be a key molecular pathway via which PEBP4 promotes the proliferation and invasion of non-small cell lung cancer (NSCLC) cells; also, it may serve as a potential therapeutic target.     

Low-Dose Rapamycin (Sirolimus) Effects in Autosomal Dominant Polycystic Kidney Disease: An Open-Label Randomized Controlled Pilot Study

Background and objectives

The two largest studies of mammalian target of rapamycin inhibitor treatment of autosomal dominant polycystic kidney disease (ADPKD) demonstrated no clear benefit on the primary endpoint of total kidney volume (TKV) or on eGFR. The present study evaluated two levels of rapamycin on the 12-month change in ¹²⁵I-iothalamate GFR (iGFR) as the primary endpoint and TKV secondarily.

Design, setting, participants, & measurements

In a 12-month open-label pilot study, 30 adult patients with ADPKD were randomly assigned to low-dose (LD) rapamycin (rapamycin trough blood level, 2–5 ng/ml) (LD group, n=10), standard-dose (STD) rapamycin trough level (>5–8 ng/ml) (STD group, n=10), or standard care (SC group, n=10). They were evaluated with iGFR and noncontrast computed tomography.

Results

Change in iGFR at 12 months was significantly higher in the LD group (7.7±12.5 ml/min per 1.73 m²; n=9) than in the SC group (−11.2±9.1 ml/min per 1.73 m²; n=9) (LD versus SC: P<0.01). Change in iGFR at 12 months in the STD group (1.6±12.1 ml/min per 1.73 m²; n=8) was not significantly greater than that in the SC group (P=0.07), but it was in the combined treatment groups (LD+STD versus SC: P<0.01). Neither eGFR calculated by the CKD-Epidemiology Collaboration equation nor TKV (secondary endpoint) changed significantly from baseline to 12 months in any of the groups. On the basis of results of the mixed model, during the study, patients in the LD group had significantly lower trough blood levels of rapamycin (mean range±SD, 2.40±0.64 to 2.90±1.20 ng/ml) compared with those in the STD group (3.93±2.27 to 5.77±1.06 ng/ml) (P<0.01).

Conclusion

Patients with ADPKD receiving LD rapamycin demonstrated a significant increase in iGFR compared with those receiving standard care, without a significant effect on TKV after 12 months.     

雷帕霉素洗脱支架系统中雷帕霉素的含量测定

目的：建立高效液相色谱法测定雷帕霉素洗脱支架中雷帕霉素含量测定的方法并对样品制备方法进行了验证.方法：用waters 1525高效液相色谱仪进行检测,色谱柱为C18柱（250mm ×4.6mm,5μm）,流动相为甲醇∶乙腈∶水（67∶ 15∶ 18）,流速1mL/min,紫外检测器,检测波长为280nm,外标法定量.结果：雷帕霉素的线性范围为19.2～77.6μg/mL（r=0.9993）,检出限为0.388μg/mL,平均加样回收率为98.86％.结论：该方法简便、快速、准确,可用于雷帕霉素洗脱支架系统中雷帕霉素的含量测定.