Rapamycin and its analogs rapalogs , those are the mTOR inhibitor, exhibit immunosuppressant and antitumor activity, and extend survival of patients with metastatic renal-cell carcinoma mRCC , advanced breast cancer and Pancreatic Neuroendocrine Tumour PNET. Drug Instructions of Everolimus and Temsirolimus mention that patients taking any medicines for heart conditions or high blood pressure might need to be prescribed a different medicine or the dose of rapalogs may need to be changed. Similar to their human counterparts, these Nf1 mouse gliomas have low proliferative indices, and exhibit microglial infiltration and increased vascularity 9 , Based on their similarity to NF1-associated optic glioma, Nf1 GEM have been successfully employed for proof-of-principle preclinical studies using conventionally-used chemotherapy temozolomide to demonstrate tumor shrinkage, reduced glioma proliferation, and increased tumor apoptosis Analysis of Nf1 -deficient astrocytes as well as other NF1 -deficient cell types previously revealed that the NF1 protein, neurofibromin, functions to negatively regulate cell growth by inactivating the Ras proto-oncogene 12 , Neurofibromin contains a amino acid residue domain with sequence similarity to members of the GTPase activating protein GAP family of molecules that serve to accelerate the conversion of Ras from its active GTP-bound to its inactive GDP-bound form 14 — Subsequent studies further showed that neurofibromin Ras-mediated growth regulation operates through the mammalian target of rapamycin mTOR pathway 17 , In this regard, NF1 -deficient human and mouse cells exhibit increased mTOR pathway activation as evidenced by high levels of activity of the mTOR effector, ribosomal S6.
Using activation-specific S6 phospho-antibodies, several groups showed increased mTOR activation in human and mouse NF1-associated tumors, including human and mouse optic gliomas. This exciting finding prompted investigators to perform preclinical studies using the rapamycin macrolide to inhibit mTOR activation and NF1 -deficient tumor growth in vivo 11 , 17 , In these studies, we previously showed that Nf1 mouse optic glioma proliferation was reduced following rapamycin treatment.
These interesting results prompted us to define the molecular basis for this treatment effect. However, neither Ki67 nor phospho-S6 activity were robust biomarkers for the in vivo response to rapamycin.
Instead, phospho-histone-H3 most strongly correlated with combined inhibition of both S6 and AKT phosphorylation. We recapitulated these in vivo results using Nf1 -deficient mouse low-grade glioma cells in vitro to demonstrate that combined treatment with rapamycin and the LY PI3-Kinase inhibitor suppressed cell growth to levels seen with higher doses of rapamycin alone. Collectively, these data suggest that additional biomarkers will be required to adequately assess mTOR target inhibition and tumor proliferative responses to rapamycin treatment in vivo.
All mice were used in accordance with established and approved animal studies protocols at the Washington University School of Medicine. Vehicle-treated mice received daily injections of an identical solution lacking rapamycin. At least five mice were included in each treatment group. After the final injection, mice were euthanized. Blood samples were collected for rapamycin concentration determination, and then the mice were perfused with ice-cold normal saline. The remaining brain was divided into two parts for Western blotting and vibratome sectioning for Ki67 determinations.
Calibrators and quality controls were prepared by spiking known amounts of Sirolimus into blank EDTA mouse blood or homogenized mouse brain tissue Bioreclamation, Hicksville, NY. Samples were vortexed for 2. For on-line sample clean-up, an extraction column 4. The analytes were then back-flushed onto a C8 analytical column 4. The mass spectrometer was run in the positive MRM multiple reaction monitoring mode.
In vitro treatments were for 16—18h unless otherwise indicated. Experiments were performed at least three times with identical results. K mouse glioma cells were plated 10, cells per well in well dishes and allowed to adhere for 24 h followed by treatment with rapamycin, NVP-BEZ, or LY at the indicated concentrations. All assays were performed thrice with identical results 18 , The number of Ki—immunoreactive cells in the dentate gyrus was quantified by direct counting on three consecutive sections from each mouse.
Brain tissues were harvested in NP lysis buffer with protease and phosphatase inhibitors and homogenized by mechanical disruption. K cells were lysed in standard NP lysis buffer with protease and phosphatase inhibitors. Western blotting was performed as previously described All antibodies were purchased from Cell Signaling Technology Beverly, MD and used at a , dilution unless otherwise stated. Primary phospho-histone-H3 Ser 10 antibody was purchased from Abcam, Inc. Following horseradish peroxidase—conjugated secondary antibody Cell Signaling Technology incubation, detection was accomplished by enhanced chemiluminescence Amersham Biosciences, Pittsburgh, PA.
Densitometry analysis was performed with Gel-Pro Analyzer 4. The measures of all biomarkers were summarized using the mean and standard error of mean SEM. The raw data were checked graphically and data transformation was performed as necessary. The relationship between blood and brain rapamycin concentrations was described by linear regression analysis. For each biomarker, the differences of means among rapamycin dose levels were compared using 1-way ANOVA, followed by post-hoc tests to compare individual treatment conditions to the wild-type or untreated group.
To control the family-wise false-positive rate at the designed 0. One of the major obstacles in translating preclinical drug studies to human clinical trials is a relative paucity of reported blood and target tissue drug levels. In these studies, we sought to determine whether rapamycin crosses the blood-brain barrier and how brain tissue levels correlated with rapamycin dose and circulating levels. First, we measured rapamycin concentrations in the blood and brain tissue of 6—8 week-old Nf1 GFAP CKO mice either treated with rapamycin or vehicle daily for 2 consecutive weeks.
These observations demonstrated that intraperitoneal rapamycin administration increases both blood and brain levels.
However, brain rapamycin levels were exponentially correlated with blood rapamycin levels Fig. Error bars represent the SEM. B, Rapamycin concentrations in brain tissue were increased as a function of dose top.
Data from five mice per treatment group were included and all the R 2 and P values were obtained after logarithm data transformation. PLoS Med. Article Google Scholar. Download references. Reprints and Permissions. Rapamycin hits the target. Nat Rev Cancer 8, Download citation. Issue Date : March Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.
Provided by the Springer Nature SharedIt content-sharing initiative. Advanced search. For example, in , McCormack et al reported a double-blind, randomized, placebo controlled trial in which 89 patients with LAM who had moderate lung impairment were assigned to either sirolimus also known as rapamycin or placebo for 1 year, followed by 1-year observation period. Their study demonstrated that treatment with sirolimus for 1-year stabilized lung function and improved quality of life in patients with LAM.
However, once patients stopped taking sirolimus, their lung function deteriorated again. The authors concluded that additional trials are required to investigate the duration of treatment and long-term safety of sirolimus McCormack et al. Their results showed that sirolimus or everolimus decreased AML volume Bissler et al. However, the tumors returned to their original states when sirolimus was discontinued, underscoring the cytostatic and not cytotoxic effects of rapamycin and rapalogs.
Since rapamycin-based therapy suppresses immune function and may cause serious side effects such as thrombocytopenia and hyperlipidemia, impaired wound healing, nephrotoxicity, and altered insulin sensitivity, the safety of long-term use still remains uncertain. A growing body of evidence suggests that the mTORC1 pathway may also be involved in a number of neurological disorders. For instance, patients with TSC are prone to a wide range of neurological diseases, such as epilepsy, neurocognitive dysfunction and autism.
Therefore, inhibition of mTORC1 is a potential therapeutic option. In a mouse model of TSC, rapamycin treatment was shown to suppress epilepsy, and shortly after, the study by Muncy et al demonstrated that rapamycin reduced seizure frequency in a year-old girl with TSC Muncy et al.
A recent study by Sato et al reported that rapamycin reverses impaired social interactions associated with autism spectrum disorder in a mouse model of TSC. Their data suggested that the abnormal behaviors displayed by TSC -deficient mice were correlated with altered gene and protein expressions involved in the mTOR signaling Sato et al.
Future therapies involving the use of ATP-competitive mTOR inhibitors or in combinations with other drugs, such as estrogen antagonists for the treatment of LAM are being explored. Mounting evidence suggested that accumulation of misfolded and aggregated proteins is a common feature of these diseases, possibly caused by mTORC1-driven protein synthesis and defective autophagic degradation. Thus, suppression of protein synthesis and induction of autophagy by rapamycin may prevent or diminish protein aggregation.
For more detailed descriptions of the molecular mechanisms underlying how rapamycin exerts its neuroprotective effect, we refer the readers to a comprehensive review Bove et al. Genetic or pharmacological inhibition of mTOR signaling has been found to extend lifespan of invertebrates including yeast, nematodes and fruit flies Lamming et al. In , Harrison et al showed that rapamycin extends both median and maximal lifespan of male and female genetically heterogeneous mice when beginning treatment at 9 or months of age 1.
Subsequent work by other groups confirmed the positive effect of rapamycin on lifespan in mice with different genetic backgrounds and other model organisms Lamming et al. Two classes of explanations may account for these observations: 1. To test the hypothesis that rapamycin might retard aging in mice, Wilkinson et al used a genetically heterogeneous mouse model and analyzed a number of age-related pathologies as well as age-dependent spontaneous activity of mice upon rapamycin treatment beginning at 9-months of age 1 year duration.
Their results suggested that age-dependent changes occur more slowly in rapamycin-treated mice, including alterations in heart, liver, endometrium, adrenal gland, and tendon elasticity. Rapamycin was also shown to diminish age-related decline in spontaneous activity of mice Wilkinson et al.
As rapamycin is known to have modest anti-proliferative properties in many forms of cancer, lifespan extension by rapamycin could also be caused by suppression of specific life-limiting pathologies e. In a recent article by Neff et al , several concerns were raised with regard to the previous reports on the effect of rapamycin in slowing aging.
For example, cancer is the main cause of death in mice including the mice strain used in the study by Wilkinson et al. In addition, aging-independent effects by rapamycin were not examined previously. After completing the treatment, they performed a large assessment of diverse structural and functional aging phenotypes in a variety of cell types, tissues and organ systems.
Intriguingly, while rapamycin did extend lifespan in mice, age-related traits were largely unaffected. Although rapamycin was able to rescue a subset of aging-dependent phenotypes, such as spatial learning and memory impairments, as well as declined exploratory activity, similar positive effects on many of these attributes were also observed in young mice, indicating an age-independent effect.
They reasoned that the discrepancy in findings could be due to different mouse models genetic backgrounds used in the studies or technical variations in the analysis Neff et al. Clearly, future studies with other mouse strains and gender are warranted Figure 2. In response to nutrients and growth factors, mTOR signaling serves as a key regulator of cell metabolism to coordinate the balance between anabolic and catabolic processes.
When fasting, muscle and liver produce glucose via glycogenolysis glycogen breakdown and gluconeogenesis glucose synthesis , and adipose tissue generates fatty acids through lipolysis, whereas, upon feeding, glycogenesis glycogen synthesis is favored in muscle and liver, and lipid uptake is favored in adipose tissue.
Dysregulation of mTOR signaling has been linked to the development of a few metabolic diseases, such as diabetes and obesity Laplante and Sabatini, Inhibition of the mTOR pathway by rapamycin has been demonstrated to have both beneficial and detrimental effects on metabolism. For example, acute rapamycin treatment improves insulin sensitivity in vitro and in vivo by disrupting a S6K-mediated feedback loop described below Krebs et al.
Moreover, rapamycin treatment inhibits human adipocyte differentiation in vitro Bell et al. As discussed above, rapamycin appears capable of extending the lifespan of mice and preventing the onset of many age-related diseases, however, deleterious metabolic effects associated with rapamycin were also reported in other studies.
For instance, Fradenkel et al found that rapamycin treatment 2 weeks worsened hyperglycemia in a nutrition-dependent type 2 diabetes mouse model Fraenkel et al. Moreover, the study by Houde et al reported that rapamycin treatment 2 weeks promoted insulin resistance and hyperlipidemia in rats Houde et al. It is currently unclear how rapamycin can have both positive and negative effects. In , Lamming et al showed that rapamycin exerts different effects via separate mechanisms.
They found that while reduced mTORC1 increases longevity and maintains normal glucose homeostasis, disruption of mTORC2 contributes to insulin resistance in vivo Lamming et al.
Additionally, Fang and colleagues suggested that these seemingly controversial findings might be explained by the duration of rapamycin treatment Fang et al. Their study compared various metabolic effects in male mice after 2, 6, or 20 weeks of rapamycin treatment. After 2 weeks of rapamycin treatment, the mice displayed smaller pancreas and enlarged liver. However, with prolonged treatment, these features returned to normal levels while adiposity, body weight BW , and food consumption were significantly reduced.
More strikingly, insulin sensitivity was altered with respect to different lengths of rapamycin treatment. Under normal physiological conditions, insulin suppresses hepatic gluconeogenesis while increasing lipogenesis, but in individuals with impaired insulin sensitivity, excess insulin is secreted to compensate for the insulin resistance.
In agreement with previous findings by Houde et al Houde et al.
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