Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation

Mammalian target of rapamycin, also known as mechanistic target of rapamycin (mTOR) is a protein kinase that belongs to the PI3K/AKT/mTOR signaling pathway, which is involved in several fundamental cellular functions such as cell growth, proliferation, and survival. This protein and its associated pathway have been implicated in cancer development and the regulation of immune responses, including the rejection response generated following allograft transplantation. Inhibitors of mTOR (mTORi) such as rapamycin and its derivative everolimus are potent immunosuppressive drugs that both maintain similar rates of efficacy and could optimize the renal function and diminish the side effects compared with calcineurin inhibitors. These drugs are used in solid-organ transplantationtoinduceimmunosuppression while also promoting the expansion of CD4+CD25+FOXP3+ regulatory T-cells that could favor a scenery of immunological tolerance. In this review, we describe the mechanisms by which inhibitors of mTOR induce suppression by regulation of these pathways at different levels of the immune response. In addition, we particularly emphasize about the main methods that are used to assess the potency of immunosuppressive drugs, highlighting the studies carried out about immunosuppressive potency of inhibitors of mTOR.     

雷帕霉素抑制门静脉高压症大鼠肠系膜血管新生

目的:研究雷帕霉素对门静脉高压症大鼠肠系膜血管新生的抑制作用,并探讨其机制。方法 :采用部分静脉结扎制备门静脉高压症模型。将雄性SD大鼠随机分为假手术对照组、假手术干预组、手术对照组和手术干预组。术后1周起,假手术干预组和手术干预组给予2 mg/(kg·d)雷帕霉素腹腔注射,持续2周,而假手术对照组和手术对照组给予同等剂量二甲基亚砜腹腔注射。病理学和血液动力学分别评估肠系膜血管新生和门静脉压力。Western印迹法测定血管内皮生长因子(vascular endothelial growth factor,VEGF)和m TOR信号通路标志物P-核糖体蛋白S6激酶(P-P70S6K)和P-真核细胞翻译启始因子4E结合蛋白1(P-4E-BP1)的表达。结果:术后3周,部分门静脉结扎大鼠脾脏肿大,肠系膜血管新生和门静脉压力显著增高(P<0.01);VEGF、P-P70S6K和P-4E-BP1的相对表达量也明显增高(P<0.01)。雷帕霉素干预2周后,有效改善脾脏肿大,抑制肠系膜血管新生和降低门静脉压力(P<0.01),下调VEGF蛋白表达量、P-P70S6K和P-4E-BP1的相对表达量(P<0.01)。结论:哺乳动物雷帕霉素靶蛋白信号通路活化促进门静脉高压症大鼠肠系膜血管新生,雷帕霉素阻断该通路,可抑制肠系膜血管新生,降低内脏血流高动力循环。     

雷帕霉素对人宫颈癌HeLa细胞增殖和HIF-1α及VEGF表达的影响

目的 探讨不同浓度雷帕霉素对宫颈癌HeLa细胞生长的抑制作用及对HIF-1α、VEGF表达的影响.方法 采用四甲基偶氮唑盐(MTT)比色法检测分别以不同浓度雷帕霉素处理的体外培养人宫颈癌HeLa细胞的抑制率,采用反转录聚合酶链反应(RT-PCR)、Western blot法检测各组细胞HIF-1 α、VEGF mRNA和蛋白的表达.结果 不同浓度雷帕霉素对宫颈癌HeLa细胞的生长有明显的抑制作用,并呈剂量依赖性;随雷帕霉素浓度增加细胞内HIF-1α、VEGF mRNA和蛋白的表达均呈下调趋势.结论 雷帕霉素对宫颈癌HeLa细胞的生长有抑制作用并呈剂量依赖性,雷帕霉素具有明显下调HIF-1α、VEGF mRNA和蛋白表达的作用.     

腺苷酸环化激酶-雷帕霉素靶蛋白信号通路参与小鼠急性肾损伤的机制研究

目的 探讨腺苷酸环化激酶(AMPK) 雷帕霉素靶蛋白(mTOR)信号通路参与小鼠急性肾损伤的机制。方法 将健康雄性C57BL/6小鼠随机分为对照组、模型组及SB2111组,每组各10只。模型组与SB2111组小鼠采用脂多糖(LPS)建立急性肾损伤模型,对照组以等剂量的生理盐水代替。SB2111组另外给予经腹腔右侧注射2 mL AMPK阻断剂SB2111治疗。观察与记录三组小鼠的一般状况、病理特征并检测肾功能情况。结果 模型组与SB2111组的体重都显著少于对照组,差异有统计学意义(P<0.05);模型组的肾脏重量大于对照组(P<0.05),而SB2111组的肾脏重量与对照组比较,差异无统计学意义(P>0.05)。模型组与SB2111组的24 h尿蛋白、血肌酐、血尿素氮均高于对照组,差异有统计学意义(P<0.05),而SB2111组的24 h尿蛋白、血肌酐、血尿素氮均低于模型组,差异有统计学意义(P<0.05)。对照组肾小管上皮细胞胞质丰富,肾组织的肾小球和肾小管结构正常;模型组肾小球充血肿胀、肾小管腔有管型形成;SB2111组部分肾小球充血,近端肾小管上皮细胞轻度肿胀,出现少量炎症细胞浸润。模型组与SB2111组的肾组织TNF-α、TGF-β1水平与AMPK、mTOR相对表达水平都显著高于对照组,而SB2111组的肾组织TNF-α、TGF-β1水平与AMPK、mTOR相对表达水平均低于模型组,差异均有统计学意义(P<0.05)。结论 AMPK-mTOR信号通路在小鼠急性肾损伤中呈现激活状况,抑制AMPK-mTOR信号通路的表达能抑制TNF-α和TGF-β1的表达水平,促进小鼠肾功能的恢复。     

低氧通过HIF-1α和HIF-2α上调SKOV3细胞Syndecan-1的表达

目的:探讨缺氧环境下,卵巢癌细胞株SKOV3中HIF-1α、HIF-2α和Synde-can-1在转录及蛋白水平的表达及相互作用关系。方法:利用MatInspector在线分析软件,分析Syndecan-1的转录因子结合位点。在CoCl2诱导缺氧环境以及低氧诱导因子(HIF)抑制剂雷帕霉素(Rapamycin)的干预下,实时荧光定量RT-PCR检测HIF-1α、HIF-2α及Syndecan-1 mRNA水平的表达;Western blot检测HIF-1α、HIF-2α蛋白水平的表达。结果:Syndecan-1的转录因子结合位点中有3处HIFF(低氧诱导因子家族)/HIF保守序列。缺氧时,HIF-1αmRNA降低(P<0.05),HIF-1α蛋白、HIF-2αmRNA和蛋白、Syndecan-1mRNA水平均升高(P<0.05)。Rapamycin不能抑制CoCl2模拟缺氧条件下HIF-1α的蛋白表达,可抑制HIF-2α的表达。Syndecan-1 mRNA动态变化曲线和HIF-1α及HIF-2α蛋白水平的动态变化曲线一致。结论:低氧可诱导HIF-1α和HIF-2α的表达,并通过HIF-1α、HIF-2α途径增强Syndecan-1的表达。     

Everolimus immunosuppression reduces the serum expression of fibrosis markers in liver transplant recipients

AIM: To evaluate the expression of serum fibrosis markers in liver transplantation (LT) recipients on everolimus monotherapy compared to patients on an anti-calcineurin regimen.METHODS: This cross-sectional case-control study included LT patients on everolimus monotherapy (cases) (E) (n = 30) and matched controls on an anti-calcineurin regimen (calcineurin inhibitors, CNI), paired by etiology of liver disease and time since LT (n = 30). Clinical characteristics, blood tests and elastography were collected. Serum levels of transforming growth factor-β (TGF-β), angiopoietin-1, tumor necrosis factor (TNF), platelet derived growth factor, amino-terminal propeptide of type III procollagen (PIIINP), hyaluronic acid (HA), VCM-1 (ng/mL), interleukin (IL)-10, interferon-inducible protein 10 (IP-10), vascular endothelial growth factor and hepatocyte growth factor (HGF) (pg/mL) were determined by enzyme-linked immunosorbent assay. Expression of these markers between E and CNI was compared. Stratified analysis was done according to factors that may influence liver fibrosis. Variables are described with medians (interquartillic range) or percentages.RESULTS: A total of 60 patients [age: 59 (49-64), hepatitis C virus (HCV): n = 21 (35%), time from LT: 73 mo (16-105)] were included. Patients had been on everolimus for a median of 15 mo. No differences in inflammatory activity, APRI test or liver elastography were found between the groups. No significant differences were observed between the groups in serum levels of PIIINP, metalloproteinase type = 1, angiopoietin, HGF, IP-10, TNF-α, IL-10 and vascular cell adhesion molecule. Patients on E had a lower expression of TGF-β [E: 12.7 (3.7-133.6), CNI: 152.5 (14.4-333.2), P = 0.009] and HA [E: 702.89 (329.4-838.2), CNI: 1513.6 (691.9-1951.4), P = 0.001] than those on CNI. This difference was maintained in the stratified analysis when recipient age is more than 50 years (TFG-β1: P = 0.06; HA: P = 0.005), in patients without active neoplasia (TFG-β1, P = 0.009; HA: P = 0.01), according to time since LT (> than 5 years, TFG-β1: P = 0.001; HA: P = 0.002), related to previous history of biliary complications (HA: P = 0.01) and HCV recurrence (HA: P = 0.004). Liver transplant recipients with everolimus monotherapy had less serum expression of TGF-β y HA than matched patients with anti-calcineurins. This difference remains when classifying patients according to donor age and time since LT. Due to the small sample size, when examining patients with a prior history of biliary complications or recurrent HCV, the difference was non-significant but trends towards the lower expression of TFG-β1 in the everolimus group. Mammalian target of rapamycin (mTOR) plays a role in the transformation of quiescent hepatocellular stellate cell to their active profibrotic state, and experimental models have demonstrated the potential activity of mTOR inhibition in attenuating fibrogenesis.CONCLUSION: This study supports a possible role of everolimus in liver fibrosis modulation after LT in a clinical setting and suggests that tailoring immunosuppression could avoid fibrosis progression in the allograft.     

mTOR inhibitors and the anti-diabetic biguanide metformin: new insights into the molecular management of breast cancer resistance to the HER2 tyrosine kinase inhibitor lapatinib (Tykerb®)

The small molecule HER2 tyrosine kinase inhibitor (TKI) lapatinib (Tykerb®) is approved for the therapy of patients with HER2-positive breast carcinomas who have progressed on trastuzumab (Herceptin®). Unfortunately, the efficacy of this HER2 TKI is limited by both primary (inherent) and acquired resistance, the latter typically occurring within 12 months of starting therapy. One of the key factors limiting our understanding of the mechanisms involved in lapatinib resistance is the lack of published preclinical models. We herein review lapatinib-refractory models recently developed at the bench and the survival pathways discovered. As hyperactivation of the pharmacologically targetable PI3K/mTOR/p70S6K1 axis appears to be central to the occurrence of lapatinib resistance, preclinical data showing enhanced antitumour effects when combining lapatinib with mTOR inhibitors (e.g., rapamycin analogues and NVP-BEZ235) highlight the importance of translational work to yield clinically useful regimens capable of delaying or treating lapatinib resistance. The unexpected ability of the anti-type II diabetes drug metformin to inactivate mTOR and decrease p70S6K1 activity further reveals that this biguanide, generally considered non-toxic and remarkably inexpensive, might be considered for new combinatorial lapatinib-based protocols in HER2-overexpressing breast cancer patients.     

Chemopreventive efficacy of rapamycin on Peutz–Jeghers syndrome in a mouse model

Germline mutations in LKB1 cause Peutz–Jeghers syndrome (PJS), an autosomal dominant disorder with a predisposition to gastrointestinal polyposis and cancer. Hyperactivation of mTOR-signaling has been associated with PJS. We previously reported that rapamycin treatment of Lkb1^+/− mice after the onset of polyposis reduced the polyp burden. Here we evaluated the preventive efficacy of rapamycin on Peutz–Jeghers polyposis. We found that rapamycin treatment of Lkb1^+/− mice initiated before the onset of polyposis in Lkb1^+/− mice led to a dramatic reduction in both polyp burden and polyp size and this reduction was associated with decreased phosphorylation levels of S6 and 4EBP1. Together, these findings support the use of rapamycin as an option for chemoprevention and treatment of PJS.     

Blocking on the CXCR4/mTOR signalling pathway induces the anti-metastatic properties and autophagic cell death in peritoneal disseminated gastric cancer cells

Patients with advanced gastric carcinoma, especially peritoneal dissemination, have a poor prognosis even after any treatment. Chemokines are now known to play an important role in cancer growth and metastasis. We recently reported that the chemokine CXCL12 plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. In this study, we investigated signalling pathway involved in the peritoneal carcinomatosis induced by chemokine CXCL12.Akt was rapidly and strongly phosphorylated by chemokine CXCL12. CXCL12 also induced the activation of p70S6K (S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) included in mammalian target of rapamycin (mTOR) pathways which are located downstream of Akt, resulting in enhancements of metastatic properties such as MMP production, cell migration and cell growth in peritoneal disseminated gastric cancer, NUGC4 cells. Furthermore, mTOR inhibitor rapamycin not only drastically inhibited migration and MMP production, but also induced type II programmed cell death, autophagic cell death.In the present study, we have shown for the first time that the mTOR pathway plays a central role in the development of peritoneal carcinomatosis, and blocking this pathway induces autophagic cell death in disseminated gastric cancer.Therefore, blocking on the CXCR4/mTOR signalling pathway may be useful for the future development of a more effective therapeutic strategy for gastric cancer involved in peritoneal dissemination.     

Awareness among patient at risk of developing Medication Related Osteonecrosis of the Jaw (MRONJ) – A primary prevention strategy

The quality of life, in itself, in cancer patients or in osteoporotic individuals, without even considering the side effects of the medication in the first place, has a considerable negative impact on the clinical outcome. The Medication Related Osteonecrosis of the Jaw (MRONJ), in the maxillofacial region, although rare, needs to be addressed with the prime importance. One of the key components of any given preventive treatment strategy is to, create awareness about the medication related unwanted effects, among health care professionals and patients.ObjectiveThis study is aimed to explore and assess the awareness level among dental patients about MRONJ, the risk factors, and the high-risk category (who are prone to develop MRONJ).Material and methodsThis is a prospective interviewer administered research electronic data capture (REDCap) survey. The sample included 68 patients, who are currently taking or will be taking Bisphosphonate (BP), and/or Denosumab, and anti-Angiogenic agent. Data have been analyzed using IBM SPSS software,ResultsSixty-eight patients (18 males and 50 females), participated in this study. Only 23 subjects (33.82%) were aware about the MRONJ. Females were more aware about the complications than males. The awareness among the subjects with education at college level appears to be higher than the subjects having education less than high school level. Even though, a dental check- up, is mandatory, prior to starting these medications, to see if any dental treatment is required, only slightly more than half of the patients (54.72%) had a dental checkup.ConclusionThis is a novel study in the Middle- East, used to assess awareness about the MRONJ including three type of related medications. Low awareness of MRONJ is alarming. The results of the study will help to initiate the process of providing the education materials, about the side effects and importance of oral hygiene maintenance, giving priority to improve the quality of life in such patients. Awareness of patients regarding the complications must be an important part of health care practice guidelines.