Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2-null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2−/− and TSC2-/meth α-actin smooth muscle (ASM) cells have been investigated. The TSC2−/− and TSC2-/meth ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2-/meth ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2-/meth ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2−/− ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2−/− and TSC2-/meth ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.
Tuberous sclerosis complex (TSC) is caused by germline mutations in the TSC1 or TSC2 genes and is associated with aberrant up-regulation of the mammalian target of rapamycin (mTOR) signaling pathway, resulting in growth of tumors, such as renal angiomyolipomas (AMLs), cutaneous angiofibromas, cardiac rhabdomyomas, and subependymal giant cell astrocytomas (Young and Povey, 1998). Tuberin and hamartin, the proteins encoded by TSC2 and TSC1 gene, respectively, form a complex that regulates signaling through the Rheb/mTOR/p70S6K pathway, which controls processes, such as growth, cell cycle progression, and apoptosis (Inoki et al., 2005). Tuberin acts as a GTPase-activating protein to regulate Ras homolog enriched in brain (Rheb) function (Garami et al., 2003). Active Rheb activates mTOR, and the up-regulation of the TSC/mTOR signaling pathway leads to increased protein synthesis, cell proliferation, and ultimately, tumorigenesis (Inoki et al., 2005).
From an AML of a patient with TSC2, we isolated α-actin smooth muscle (ASM) cells displaying loss of heterozygosity with no expression of tuberin (TSC2−/− ASM cells) (Lesma et al., 2005). More recently, we isolated another type of α actin-positive TSC2 cell population from the AML of a male patient. These latter cells do not express tuberin for the methylation of the TSC2 promoter (TSC2-/meth ASM cells); thus, epigenetic defects might also originate AML pathogenesis (Lesma et al., 2009). Aberrant DNA methylation of CpG islands in promoter regions of many genes has been observed in several types of cancer and is associated with tumor suppressor gene silencing (Jones and Baylin, 2002). In spite of the different type of genetic mutation, TSC2−/− and TSC2-/meth ASM cells share most proliferative and biochemical characteristics. Growth and proliferation require specifically the presence of epidermal growth factor (EGF) in the growth medium. EGF cannot be replaced by insulin-like growth factor-1 (Lesma et al., 2005, 2009; Carelli et al., 2007). The EGF dependency ceases when these TSC2-deficient cells are made able to produce tuberin either by transfection of TSC2 gene in TSC2−/− ASM cells or treatment with chromatin remodeling agents in the case of TSC2-/meth ASM cells. In both cases, the restored function of the TSC2 gene normalizes rate of cell proliferation and extent of S6 phosphorylation (Lesma et al., 2008, 2009). The blockade of EGF or insulin-like growth factor-1 receptors by antibodies results in cell death. In contrast, the inhibition of mTOR with rapamycin does not affect TSC2−/− ASM cell survival, and the cytostatic action is restricted when the drug is added at plating time. Later addition of the drug is rather ineffective (Lesma et al., 2008). TSC2-/meth ASM cells are perhaps more responsive to the cytostatic action of rapamycin (Lesma et al., 2009). These data are consistent with recent clinical studies that showed the effectiveness of rapamycin and its derivate everolimus in promoting the regression of TSC AMLs and their tendency to recover to the original size when therapy has been stopped (Bissler et al., 2008, Pollizzi et al., 2009).
Statins are a class of drugs that inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of mevalonate pathway of lipid synthesis. The effects of statins are mediated by blockade of mevalonate formation, which results in reduction of cholesterol synthesis (Bulhak et al., 2007). The inhibition of mevalonate formation also greatly affects the formation of isoprenoid derivatives, such as geranylgeranyl pyrophosphate, which is involved in the isoprenylation of small GTPases of the Rho/Rac/Cdc42 family (Takai et al., 2001). Rosuvastatin, a hydrophilic member of the statin family, is among the most effective statins in decreasing low-density lipoprotein cholesterol levels (Ai et al., 2008). Recently, it has been reported that atorvastatin inhibited the growth of tuberin-null cells and failed to affect the tumor size in TSC2+/− mice (Finlay et al., 2007, 2009).
On the basis of these observations that loss of tuberin leads to constitutively activated Rheb and tuberin-null cells present elevated levels of GTP-RhoA (Goncharova et al., 2004), we investigated rosuvastatin as possible active pharmacological agent in purified human TSC2-deficient cells. Our results show that TSC2−/− ASM cell proliferation is unaffected by rosuvastatin, whereas TSC2-/meth ASM cells are sensitive; this is secondary to the restoration of tuberin expression through the demethylating action of rosuvastatin on TSC2 promoter. RhoA activation was prevented very effectively in TSC2-/meth ASM cells and partially in TSC2−/− ASM cells by rosuvastatin. The addition of rapamycin extended the action of rosuvastatin also to TSC2−/− ASM cells. Together, these results suggest that the action of mTOR inhibitors on human TSC2-deficient cells may be reinforced by the addition of statins acting as demethylating agents.
Materials and Methods
TSC2−/− and TSC2-/meth ASM cells were isolated, characterized, and grown as previously described (Arbiser et al., 2001, Lesma et al., 2005, 2009). TSC2−/− and TSC2-/meth ASM cells were obtained from renal angiomyolipomas during total nephrectomy from a 42-year-old female and from a 36-year-old man, respectively. Both patients had a history of TSC2 and had given written informed consent according to the Declaration of Helsinki. The study was approved by the Institutional Review Board of Milan’s San Paolo Hospital. The culture medium of both TSC2−/− and TSC2-/meth ASM cells contained a 50/50 mixture of Dulbecco’s modified Eagle’s medium/Ham F12 (EuroClone, Paignton, UK) supplemented with hydrocortisone (2 × 10−7 mol/l; Sigma-Aldrich, St. Louis, MO), EGF (10 ng/ml; Sigma-Aldrich), sodium selenite (5 × 10−8 mol/l; Sigma-Aldrich), insulin (25 μg/ml; Sigma-Aldrich), transferrin (10 μg/ml; Sigma-Aldrich), ferrous sulfate (1.6 × 10−6 mol/l; Sigma-Aldrich), and 15% fetal bovine serum (EuroClone), as described by Arbiser et al. (2001). Cells were maintained in a humidified incubator under 5% carbon dioxide. For all experiments, cells were plated 3 hours before treatments or immediately before drug treatments.
Evaluation of Cell Proliferation.
After incubation with rosuvastatin (AstraZeneca, Basiglio, Italy; 10 nM, 100 nM, and 1 µM) and rapamycin (Rapamune-Sirulimus; Wyeth Europe, Maidenhead, UK; 1 ng/ml), cell proliferation was evaluated by counting at least 400–500 cells in a Neubauer chamber. Each data point was the mean of three independent experiments.
Western Blot Analysis.
TSC2−/− and TSC2-/meth ASM cells were incubated with rosuvastatin (100 nM and 1 µM), mevalonate (Sigma-Aldrich; 100 µM), and rapamycin (1 ng/ml) for the indicated time. Cells were lysed in lysis buffer (5 mM EDTA, 100 mM deoxycholic acid, 3% sodium dodecyl sulfate). Samples (25 µg per lane) were boiled for 5 minutes and analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes (Amersham, Arlington Height, IL) and blocking at room temperature for 3 hours with 5% dry milk (Merck, Darmstadt, Germany), membranes were incubated overnight at 4°C with antibodies against phospho-Akt (Ser473; 1:1000; Cell Signaling Technology, Beverly, MA), Akt (1:1000; Cell Signaling Technology), phospho-S6 (Ser 235/236; 1:1000; Cell Signaling Technology), S6 (1:1000; Cell Signaling Technology), phospho-extracellular signal-regulated kinase 1 and 2 (ERK1/2; Thr 202/Tyr 204; 1:1000; Cell Signaling Technology), ERK1/2 (1:1000; Cell Signaling Technology), RhoA (1:500; Cytoskeleton Inc., Denver, CO), or β-actin (1:1000; Sigma-Aldrich). Membranes were washed and incubated for 1 hour with the appropriate secondary antibodies (1:10000; EMD Millipore, Billerica, MA). The reaction was quantified using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). Images were purchased using Kodak 1D 3.6 Software after acquisition on a Kodak image station 440 CF (Kodak, Milano, Italy).
Rho Activation Assay.
TSC2−/− and TSC2-/meth ASM cells were treated with rosuvastatin (100 nM and 1 µM) and rapamycin (1 ng/ml) for 48 hours. RhoA activity was evaluated using Rho activation pulldown assay (Cytoskeleton Inc.). Cellular GTP-Rho-A was affinity purified using glutathione S-transferase-rhotekin Rho binding domain agarose beads according to the manufacturer’s recommendation. In brief, after treatment, cells were washed with ice-cold phosphate-buffered saline and lysed in ice-cold lysis buffer with 1× protease inhibitor cocktail supplemented in Rho activation pulldown assay. Protein concentrations were assayed, and 300 µg total cell protein was added to 50 µg rhotekin-Rho binding domain beads. Then samples were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis.
TSC2−/− and TSC2-/meth ASM cells were cultured on glass slides and were incubated with rosuvastatin (100 nM and 1 µM), rapamycin (1 ng/ml), or 5-azacytidine (1 µM). Cells were fixed with cytoskelfix (Cytoskeleton Inc.) for 4 minutes at −20°C and dried in air. The primary antibody directed against RhoA (1:500; Cytoskeleton Inc.) or against tuberin C-terminal (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) was applied overnight at 4°C. The samples were incubated for 3 hours at room temperature with Alexa Fluor 555 goat anti-rabbit or with Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA). Nuclei were stained with 4′,6′-diamino-2-phenylindole (2 µg/ml; Sigma-Aldrich). After washing, the slides were mounted with FluorSave reagent (Calbiochem, Darmstadt, Germany). RhoA and tuberin labeling were achieved with a confocal microscope (Leica TCS-SP2; Leica Microsystems GmbH, Wetzlar, Germany). Quantification of RhoA expression was calculated as percentage of positive and negative cells on total cell number. The level of statistical significance was determined using Student’s t test.
Flow Cytometric Analysis.
TSC2-/meth ASM cells were treated for 5 days with rosuvastatin (1 µM) and for 96 hours with 5-azacytidine (1 µM). Cells were collected by centrifugation and washed in phosphate-buffered saline. Cells were fixed with fixation buffer [Becton Dickinson (BD) Italia, Buccinasco, Milan] for 1 hour at 4°C. Then, cells were washed and permeabilized with Perm/Wash buffer I (BD) for 1 hour at room temperature. Permeabilized samples were incubated with anti-tuberin C-terminal primary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature and then with Alexa fluor 488 goat anti-rabbit secondary antibody (Invitrogen). After washing twice with Perm/Wash buffer, samples were analyzed with the flow cytometer Cytomics FC500 (Beckman Coulter SpA, Milan, Italy). Fluorescence signal was collected using amplifier that reported on a logarithmic scale (FL1); forward and side scatter signals were recorded on a linear scale. Data acquisition and analysis were done using CXP 2.2 software (Beckman Coulter).
Enzyme-Linked Immunosorbent Assay.
TSC2−/− and TSC2-/meth ASM cells were treated overnight with cycloheximide (10 μg/ml; Sigma-Aldrich), methylprednisolone (10 nM; Sigma-Aldrich), and reparixin (1 nM; Dompè Pharma SpA, L’Aquila, Italy). Rosuvastatin (1 μM) and rapamycin (1 ng/ml) were added for 48 hours. After treatments, media were collected and interleukin (IL) 6 and 8 production was measured by specific enzyme-linked immunosorbent assay (Invitrogen) according to the manufacturer’s instruction.
All data are expressed as mean values ± S.E.M. and were statistically analyzed using Student’s t test; significance is indicated for P values of <0.05; <0.01; and <0.001.
Effect of Rosuvastatin on TSC2−/− and TSC2-/meth ASM Cell Proliferation.
The effect of rosuvastatin on growth of TSC2−/− and TSC2-/meth ASM cells was evaluated after the addition to the growth medium of the statin at different concentrations. At 100 µM, rosuvastatin was cytotoxic for both cell types (Fig. 1, A and C). TSC2−/− ASM cells were quite resistant to rosuvastatin, and no effects on cell growth were observed at any applied concentration (Fig. 1A). Their proliferation was markedly reduced by the statin, applied at the higher dose of 1 μM, when coadministered with rapamycin (Fig. 1B). We previously reported that rapamycin alone could affect proliferation of both TSC2−/− and TSC2-/meth ASM cells when added at plating time with the cells still in suspension and was only slightly effective on attached TSC2-/meth ASM cells (Lesma et al., 2005, 2009). The proliferation of these latter cells is rather sensitive to rosuvastatin alone, and the combined application with rapamycin (1 ng/ml) inhibits proliferation at any tested concentration and leads to cell death within 11 days (Fig. 1, C and D).
Evaluation Phosphatidyl-Inositol 3 Kinase (PI3K) and Mitogen-Activated Protein Kinases (MAPKs) Pathways after Rosuvastatin Incubation.
The involvement of PI3K and MAPKs pathway in rosuvastatin (1 μM) action was tested after 2 and 10 days of incubation. Neither activation nor expression of Akt, ERK1/2, and S6 were modified by the statin, even at dosages that effectively reduced TSC2 cell proliferation (Fig. 2, A and B). β-actin was used as loading control.
Evaluation of Mevalonate Pathway.
The observed antiproliferative effects of rosuvastatin on TSC2-/meth ASM cells may be attributable to the blockade of HMG-CoA reductase activity with the inhibition of the formation of products, such as mevalonate (Grundy, 1988). To this end, we evaluated cell growth in the presence of rosuvastatin and mevalonate. Statin inhibition of TSC2-/meth ASM cell proliferation was reverted by the coaddition of mevalonate at the concentration of 100 μM (Fig. 3B), whereas mevalonate alone was ineffective (Fig. 3A). PI3K and MAPKs pathways were also examined after exposure to mevalonate. As expected, the phosphorylation of S6 was increased in TSC2−/− and TSC2-/meth ASM cells after 48 hours of incubation. Such an activation was inhibited by rapamycin (1 ng/ml), whereas rosuvastatin (1 or 5 μM) did not have any effect at any used concentration (Fig. 3). Rosuvastatin slightly decreased ERK1/2 phosphorylation at the higher concentration of 5 μM when added with rapamycin (Fig. 3, C and D). Expression of ERK1/2 was not altered in any experimental condition. Activation and expression of Akt were not modified by any treatment. β-actin was used as loading control.
Effect of Rosuvastatin on RhoA Activity.
On the basis of the observations that loss of tuberin leads to constitutively activated Rheb and that tuberin-null cells additionally have elevated levels of GTP-RhoA (Goncharova et al., 2004), we investigated whether rosuvastatin exposure might regulate RhoA activity in TSC2−/− and TSC2-/meth ASM cells. The levels of active RhoA (bound to GTP) were determined by pull-down assay. The basal activity of RhoA was higher in TSC2−/− ASM cells than in TSC2-/meth ASM cells. Rosuvastatin (1 μM and 100 nM) affected RhoA activation in TSC2−/− ASM cells, but the complete effectiveness was achieved only with the coaddition of rapamycin, which is only slightly effective when alone (Fig. 4A). In contrast, rosuvastatin (100 nM and 1 μM) strongly inhibited activation of RhoA in TSC2-/meth ASM cells, even in the absence of rapamycin (Fig. 4B). Rapamycin alone slightly reduced RhoA activity. The expression of RhoA was unchanged (Fig. 4, A and B).
The percentage of TSC2−/− and TSC2-/meth ASM cells expressing RhoA was evaluated by immunofluorescence. After 48 hours incubation with rosuvastatin (1 μM), in presence or absence of rapamycin (1 ng/ml), cells were assessed for positive or negative labeling to RhoA. TSC2−/− and TSC2-/meth ASM cells were 80 and 67.3% positive, respectively (Fig. 4, C and D). Rosuvastatin alone did not affect the percentage of RhoA-expressing cells in both cell populations. The coadministration of rosuvastatin and rapamycin significantly reduced the extent of positively labeled TSC2-/meth ASM cells, whereas it was only slightly effective in TSC2−/− ASM cells. In both cell populations, rapamycin alone did not significantly affect RhoA labeling (Fig. 4, C and D).
Effect of Rosuvastatin on Tuberin Expression in TSC2-/meth ASM Cells.
We previously described the presence of an epigenetic modification in the TSC2 promoter of TSC2-/meth ASM cells and the induction of tuberin expression after exposure to the chromatin-remodeling agents 5-azacytidine and trichostatin-A (Lesma et al., 2009). Because statins may act as demethylating agents (Kodach et al., 2011), tuberin expression in TSC2-/meth ASM cells was quantitatively evaluated by immunofluorescence and flow cytometric analysis after incubation with rosuvastatin. The addition of rosuvastatin at the concentration of 1 µM for 5 days resulted in the induction of tuberin expression in about 47% of TSC2-/meth ASM cells, and in the control cells, the percentage was 2.5% (Fig. 5). The addition of 5-azacydine to rosuvastatin further enhanced the expression of tuberin in 90% of the cells (Fig. 5B). Rosuvastatin did not affect the expression of tuberin in TSC2−/− ASM cells (Fig. 5A).
Anti-Inflammatory Effects of Rosuvastatin.
Statins are also known for their anti-inflammatory properties; thus, the effects of rosuvastatin on the release of IL-6 and IL-8 by TSC2−/− and TSC2-/meth ASM cells was evaluated. TSC2−/− and TSC2-/meth ASM cells synthesize and secrete IL-6 and IL-8, because the release is CHX dependent (Fig. 6). This process was not inhibited by rosuvastatin (1 μM) but, as expected, was markedly inhibited by anti-inflammatory agents, such as methylprednisolone (10 nM) and reparixin (1 nM). Reparixin is a noncompetitive allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2 (Gorio et al., 2007).
Statins, as pharmacologic inhibitors of HMG-CoA reductase, have a wide array of biologic effects. Statins are synthetic agents that inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A reductase with potent effects on cholesterol biosynthesis in vivo and in vitro (Istvan and Deisenhofer, 2001). In addition to lipid-lowering effects, statins have anti-inflammatory and antineoplastic activities, possibly regulated through the mevalonate pathway (Chan et al., 2003; Demierre et al., 2005; Greenwood and Mason, 2007). By inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase and controlling mevalonate synthesis, statins not only affect cholesterol biosynthesis but also control the production of several other major cell products, such as dolichol, geranylpyrophosphate, and farnesylpyrophosphate (Goldstein and Brown, 1990). Atorvastatin addition to the culture medium of tuberin-null cells attenuates the increased levels of activated RhoA and blocks isoprenylation of Rheb and phosphorylation of mTOR-S6K-S6, thus affecting growth ability of these cells (Finlay et al., 2007). It has also been reported that statins, such as simvastatin alone or in combination with rapamycin, counteract both TSC-null cell abnormal proliferation and TSC-null tumor growth (Goncharova et al., 2011). On the other hand, treatment with atorvastatin failed to reduce size and number of cystadenomas, spontaneous liver hemangiomas, and subcutaneous tumors when applied as a single agent or in combination with rapamycin in Tsc+/− mice (Finlay et al., 2009; Lee et al., 2009). These contrasting data on TSC-null cells and the need of novel and improved approaches for the treatment of TSC and correlated diseases prompted us to further investigate the possible actions of statins on human TSC2 cells purified from renal angiomyolipomas surgically derived (Lesma et al., 2005, 2009).
Human TSC2−/− and TSC2-/meth ASM cells have been isolated and then well characterized in our laboratory (Lesma et al., 2005, 2009). Rapamycin slightly inhibits TSC2-/meth ASM cell proliferation and is ineffective with TSC2−/− ASM cells. In this study, we observed that rosuvastatin did not affect TSC2−/− cell growth, but reduced TSC2-/meth cell proliferation without causing cell death. However, addition of rapamycin to rosuvastatin promoted TSC2-/meth cell growth blockade and cell death. In contrast, TSC2−/− cell proliferation was affected only when higher doses of rosuvastatin were added to rapamycin. Both TSC2−/− and TSC2-/meth ASM cells share molecular mechanisms underlying their proliferation, such as the EGF growth dependency, the specific efficacy of EGF receptor antibodies, and the germline TSC2 gene mutations. The loss of heterozigosity is caused by a second-hit mutation in TSC2−/− ASM cells and by the methylation of the TSC2 promoter in TSC2-/meth ASM cells (Lesma et al., 2005, 2009). Promoter hypermethylation is an important and potentially reversible mechanism of tumor suppressor gene silencing, and the aberrant methylation of CpG islands in the promoter regions of many genes has been observed in many types of cancer (Toyota et al., 1999; Jiang et al., 2005; Sirchia et al., 2005). Exposure to rosuvastatin resulted in tuberin expression in TSC2-/meth ASM cells, whereas this was not observed in TSC2−/− ASM cells. This effect of rosuvastatin was further reinforced when the drug and 5-azacytidine were added together. These data suggest that rosuvastatin inhibitory effects on TSC2-/meth ASM cell proliferation might be dependent on its demethylating properties on TSC2 gene that promote the expression of tuberin, thus normalizing the rate of cell proliferation. Several evidences show that statins may act as epigenetic modulators in different cell types (Kim et al., 2010; Kodach et al., 2011). The effects of simvastatin on immunosuppression are associated with demethylation of the Foxp3 promoter, because it exerts a potent synergistic effect on Foxp3 induction when combined with a low concentration of TGF-β in T cells (Kim et al., 2010). Lovastatin inhibits the DNA methyltransferase activity and promotes the demethylation of the promoters of bone morphogenetic protein 2 in colorectal cancer cells with the expression of protein 2, thereby leading to differentiation and reduced proliferation of colorectal cancer cells (Kodach et al., 2011). In the same way, the action of rosuvastatin described in this study appears to be related to the induction of tuberin expression in TSC2-/meth ASM cells, where loss of heterozygosity is caused by an epigenetic event.
Rosuvastatin inhibited RhoA activity in TSC2-/meth ASM cells, and the same effect in TSC2−/− ASM cells was achieved when cells were cotreated also with rapamycin. RhoA belongs to the small GTPase family and is constitutively activated in TSC2-null cells (Castro et al., 2003; Goncharova et al., 2004). This mTORC2-dependent RhoA GTPase activation is necessary for cell proliferation and survival in TSC2-null cells (Goncharova et al., 2011). RhoA and Rac GTPases regulate actin cytoskeleton through mTORC2 promoting formation of stress fiber and focal adhesion processes (Lamb et al., 2000; Sarbassov et al., 2004). The inhibition of cytoskeleton reorganization and cell motility by rapamycin is also mediated by the suppression of mTORC1 pathway through the down-regulation of expression and activity of Rho-family small GTPases, such as Cdc42, Rac, and RhoA (Liu et al., 2006, 2010). Thus, it is conceivable that the efficacy of the cotreatment in blocking RhoA activity in TSC2−/− and TSC2-/meth ASM cells may be caused by the suppression of HMG-CoA reductase by rosuvastatin and the inhibition of mTOR by rapamycin. These effects appear to be additive to the demethylating action of rosuvastatin in TSC2-/meth cells.
Statins have been reported to exert pleiotropic effects on cellular signaling and cellular functions involved in inflammation, and release of inflammatory cytokines, such as IL-6 and IL-8, are lowered by statin treatment (Rosenson et al., 1999; Bessler et al., 2005; Ma et al., 2011). The statin anti-inflammatory actions appear to be regulated through inhibition of the mevalonic cascade (Iwata et al., 2012). In our experimental conditions, TSC2−/− and TSC2-/meth ASM cells secrete IL-6 and IL-8, but such a processs is not sensitive to rosuvastatin. Methylprednisolone and repertaxin (IL-8 receptor antagonist), used as positive control active agents, greatly reduce secretion of both interleukins, indicating that the event is typically glucocorticoid sensitive and is regulated by a positive feedback mechanism, because a IL-8 receptor antagonist, such as repertaxin, blocks the release of both. The failure of rosuvastatin to impair IL-6 and IL-8 release in TSC2−/− and TSC2-/meth ASM cells might be attributable to altered regulatory pathways in TSC. Thus, the mechanisms of beneficial effects of statins are not totally clear yet and appear to be dependent on the kind of cells and statins used (Jain and Ridker, 2005).
In conclusion, our study suggests that rosuvastatin may be useful as an adjunct to mTOR-inhibitors in TSC, particularly when the genetic alteration is attributable to an epigenetic modification, as in the case of the methylation of the TSC2 gene promoter.
Participated in research design: Lesma, Grande, Gorio.
Conducted experiments: Lesma, Ancona, Orpianesi, Grande.
Performed data analysis: Lesma, Ancona, Grande, Gorio.
Wrote or contributed to the writing of the manuscript: Lesma, Di Giulio, Gorio.
- Received January 9, 2013.
- Accepted February 19, 2013.
This work was supported by Associazione Italiana Linfangioleiomiomatosi AILAM (to A.G. and E.L.); Associazione Sclerosi Tuberosa (to E.L.); and Accordo Quadro di Collaborazione tra Università Lombarde from Lombardy Region (Research Collaborative Program between Lombard Universities and Region; to A.G.).
- α-actin smooth muscle
- epidermal growth factor
- extracellular signal-regulated protein kinase
- 3-hydroxy-3-methylglutaryl coenzyme A
- mitogen-activated protein kinases
- mammalian target of rapamycin
- phosphatidyl-inositol 3 kinase
- Ras homolog enriched in brain
- tuberous sclerosis complex
- TSC2-/meth ASM cells
- ASM cells bearing a TSC2 mutation and a TSC2 promoter methylation
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics