Lovastatin-induced Apoptosis Research Articles

Lovastatin-Induced Mitochondrial Oxidative Stress Leads to the Release of mtDNA to Promote Apoptosis by Activating cGAS-STING Pathway in Human Colorectal Cancer Cells.

Statins are 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors widely used in the treatment of hyperlipidemia. The inhibition of HMG-CoA reductase in the mevalonate pathway leads to the suppression of cell proliferation and induction of apoptosis. The cyclic GMP-AMP synthase (cGAS) stimulator of the interferon genes (STING) signaling pathway has been suggested to not only facilitate inflammatory responses and the production of type I interferons (IFN), but also activate other cellular processes, such as apoptosis. It has not been studied, however, whether cGAS-STING activation is involved in the apoptosis induced by statin treatment in human colorectal cancer cells. In this study, we reported that lovastatin impaired mitochondrial function, including the depolarization of mitochondrial membrane potential, reduction of oxygen consumption, mitochondrial DNA (mtDNA) integrity, and mtDNA abundance in human colorectal cancer HCT116 cells. The mitochondrial dysfunction markedly induced ROS production in mitochondria, whereas the defect in mitochondria respiration or depletion of mitochondria eliminated reactive oxygen species (ROS) production. The ROS-induced oxidative DNA damage by lovastatin treatment was attenuated by mitochondrial-targeted antioxidant mitoquinone (mitoQ). Upon DNA damage, mtDNA was released into the cytosol and bound to DNA sensor cGAS, thus activating the cGAS-STING signaling pathway to trigger a type I interferon response. This effect was not activated by nuclear DNA (nuDNA) or mitochondrial RNA, as the depletion of mitochondria compromised this effect, but not the knockdown of retinoic acid-inducible gene-1/melanoma differentiation-associated protein 5 (RIG-I/MDA5) adaptor or mitochondrial antiviral signaling protein (MAVS). Moreover, lovastatin-induced apoptosis was partly dependent on the cGAS-STING signaling pathway in HCT116 cells as the knockdown of cGAS or STING expression rescued cell viability and mitigated apoptosis. Similarly, the knockdown of cGAS or STING also attenuated the antitumor effect of lovastatin in the HCT116 xenograft model in vivo. Our findings suggest that lovastatin-induced apoptosis is at least partly mediated through the cGAS-STING signaling pathway by triggering mtDNA accumulation in the cytosol in human colorectal cancer HCT116 cells.

Lovastatin Induces Apoptosis through Deprivation of Intracellular Geranylgeranyl Pyrophosphate and Induction of Puma Expression in C6 Glioma Cells

Lovastatin is widely used to decrease serum cholesterol levels and reduce the risk of cardiovascular diseases. Lovastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that converts HMG-CoA to mevalonate. Mevalonate is a precursor of various metabolic intermediates such as isopentenyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and cholesterol. It has been reported that statins can induce apoptosis in various cell lines. In this study, we examined the mechanism of lovastatin-induced apoptosis in C6 and HCT116 cell lines. C6 is a rat glioma cell line and HCT116 is a human colon cancer cell line. Cells were maintained in Dulbecco's minimal essential medium containing 10% (v/v) fetal bovine serum. After treatment, the number of viable cells was estimated using a colorimetric assay based on tetrazolium salt. The percentage of dead cells was estimated using trypan blue cell viability assay or lactate dehydrogenase assay. The levels of Puma mRNA were estimated by reverse transcription quantitative polymerase chain reaction assay. The levels of PUMA were estimated by Western blot assay. Our data showed that lovastatin increased both mRNA and protein levels of Puma in C6 cells, which could be prevented in the presence of 100 μM geranylgeranyl pyrophosphate. Treatment with geranylgeranyl pyrophosphate, cycloheximide, and siPuma (siRNA against Puma) prevented the death of lovastatin-treated cells. Pifithrin-a, an inhibitor of tumor protein p53, neither prevented cell death nor decreased PUMA levels in lovastatin-treated cells. Pifithrin-a attenuated cell death and reduced PUMA levels in etoposide-treated C6 cells. Etoposide, an anticancer drug, has been known to induce p53-dependent apoptosis. To evaluate the role of p53 on the induction of Puma expression in lovastatin-treated cells, cytotoxic effects of lovastatin were examined in HCTm and HCTw cells. HCTm is a HCT116 cell line expressing inactive mutant p53 and HCTw is the wild-type. When cells were cultured in the presence of 25 μM lovastatin for 60 hours, the values of the percentage of cell death were 46% and 43% in HCTm and HCTw groups respectively: there was little difference in lovastatin-induced cell death between two groups. Etoposide-induced cell death was exacerbated in HCTw cells as compared to HCTm cells. When cells were cultured with 100 μM etoposide for 72 hours, the values of the percentage of cell death were 35% and 45% in HCTm and HCTw groups respectively. Pifithrin-a reduced etoposide-induced cell death in HCTw cells from 45% to 25%. Pifithrin-a did not attenuate etoposide-induced cell death in HCTm cells. Our data show that lovastatin depletes intracellular geranylgeranyl pyrophosphate to increase Puma expression in a p53-independent manner, resulting in apoptosis.

Lovastatin induces platelet apoptosis.

Statins are widely used in the prevention of atherosclerosis and treatment of coronary artery disease because of pleiotropic effects on thrombosis. Thrombocytopenia and hemorrhage occurred in some statin-treated patients, but the reason remains unclear. In the current study, we show that lovastatin dose-dependently induces depolarization of mitochondrial inner transmembrane potential, leading to up-regulation of Bak, down-regulation of Bcl-XL, and activation of caspase-3/8/9. Lovastatin treatment did not increase the surface expression of P-selectin or PAC-1 binding but led to strongly reduced collagen- and thrombin-induced platelet aggregation. The integrin αIIbβ3 antagonist, RGDS, inhibited lovastatin-induced apoptosis in both human platelets and Chinese hamster ovary (CHO) cells stably expressing integrin αIIbβ3. The number of circulating platelets in mice was significantly reduced after intraperitoneal injections with lovastatin. Taken together, these data indicate that lovastatin induced caspase-dependent platelet apoptosis. Lovastatin does not incur platelet activation, whereas impairs platelet function and reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia and hemorrhage in patients treated with statins.

Lovastatin‐induced apoptosis is mediated by activating transcription factor 3 and enhanced in combination with salubrinal

We have previously demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to induce tumor-specific apoptosis. The apoptotic effects of lovastatin were regulated in part by the integrated stress response (ISR) that regulates cellular responses to a wide variety of stress inducers. A key regulator of the ISR apoptotic response is activating transcription factor 3 (ATF3) and its target gene CHOP/GADD153. In our study, we demonstrate that in multiple lovastatin-resistant clones of the squamous cell carcinoma (SCC) cell line SCC9, lovastatin treatment (1-25 μM, 24 hr) in contrast to the parental line failed to significantly induce ATF3 expression. Furthermore, the SCC-derived cell lines SCC25 and HeLa that are sensitive to lovastatin-induced apoptosis also preferentially induce ATF3 expression compared to resistant breast (MCF-7) and prostate carcinoma (PC3)-derived cell lines. In HeLa cells shRNA targeting ATF3 expression as well as in ATF3-deficient murine embryonic fibroblasts, lovastatin-induced cytotoxicity and apoptosis were attenuated. In ex vivo HNSCC tumors, lovastatin also induced ATF3 mRNA expression in two of four tumors evaluated. Salubrinal, an agent that can sustain the activity of a key regulator of the ISR eIF2α, further increased the expression of ATF3 and demonstrated synergistic cytotoxicity in combination with lovastatin in SCC cells. Taken together, our results demonstrate preferential induction of ATF3 in lovastatin-sensitive tumor-derived cell lines that regulate lovastatin-induced apoptosis. Importantly, combining lovastatin with salubrinal enhanced ATF3 expression and induced synergistic cytotoxicity in SCC cells.

Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line

Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD.We demonstrate that apoptosis in neuronal lovastatin treated-cells is induced by the mitochondrial pathway, with caspase-9 as the initiator and caspase-3 as the effector caspase. The presence of geranylgeraniol modulates both the caspase-9 and caspase-3 activity in a dose-dependent way, confirming that this isoprenoid enters the mevalonate pathway, is metabolized and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. According to our findings, it should not be the time course adopted that modulates the apoptotic response but rather the isoprenoid itself.Being aware that our results have been obtained using a biochemical model of MKD, and not cells from patients with the disease, we believe our findings increase the knowledge of MA pathogenesis, and may possibly contribute to the development of novel therapeutic strategies.

Synergistic anti-tumor efficacy of lovastatin and protein kinase C-beta inhibitor in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is characterized by hypervascularity and chemoresistance. Protein kinase C (PKC) participates in cancer progression by enhancing anti-apoptotic signals, angiogenesis, and chemoresistance. Statins have a selective anti-cancer effect due to over-expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) in cancer cells, but statins may also activate PKC. Thus, we hypothesized that simultaneous treatment with statin and PKC inhibitor might synergistically enhance their anti-tumor efficacies against HCCs. Hepatocellular carcinoma cell growth was assessed using MTS assays, apoptotic cell death by DAPI staining, and apoptotic signaling cascades were explored by immunoblotting. An in vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells. Lovastatin and/or a PKC inhibitor (enzastaurin) was subsequently administered. Anti-tumor efficacies were evaluated by measuring tumor volumes and quantifying apoptotic cells and microvessel densities (MVD). Co-treatment with lovastatin and enzastaurin was found to synergistically suppress HCC cell growth in vitro. Lovastatin induced HCC cellular apoptosis by activating mitochondrial apoptotic signals, and although enzastaurin alone did not induce apoptosis, its addition significantly enhanced lovastatin-induced apoptosis. This enhanced apoptosis was attributed to increased caspase-9 activation in these cells. Moreover, tumor growth was significantly suppressed in mice co-treated with lovastatin and enzastaurin, and percentages of TUNEL-positive cells were significantly increased and MVDs were significantly decreased in those mice. Combinatorial treatment with statin and PKC inhibitor was found to enhance anti-tumor efficacy in vivo and in vitro. Further studies are warranted to prove anti-tumor efficacy of this potential therapy in human HCCs.

Lovastatin induces apoptosis of k-ras-transformed thyroid cells via inhibition of ras farnesylation and by modulating redox state

Transformation of thyroid cells with either K-ras or H-ras viral oncogenes produces cell types with different phenotype and different response to the inhibition of the prenylation pathway by 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyltransferase inhibitors. These inhibitors induce apoptosis in K-ras-transformed FRTL-5 cells (FRTL-5-K-Ras) whereas cell cycle arrest is induced in H-ras-transformed FRTL-5 (FRTL-5-H-Ras). In FRTL-5-K-Ras cells, the product of K-ras gene is implicated in the scavenging of reactive oxygen species (ROS) through the activation of extracellular-signal-regulated kinase (ERK)1/2 kinases. We observed that lovastatin blocked ras activation through inhibition of farnesylation and induced apoptosis, increasing ROS levels through inhibition of ERK1/2 signaling and Mn-SOD expression. Lovastatin-induced apoptosis was due to intracellular ROS increase since both, the antioxidant compound pyrrolidinedithiocarbamate or the SOD-mimetic compound, antagonized apoptosis. Moreover, both p38 mitogen-activated protein kinase and nuclear factor kappaB pathways, activated as a consequence of high ROS levels, are involved in the apoptotic effect, indicating that cell death induced by lovastatin was dependent on oxidative stress. Lovastatin antitumor efficacy in K-ras-dependent thyroid tumors was further confirmed in vivo, proposing a new therapeutic strategy for those tumor diseases that are sustained by an inappropriate K-ras expression.

Activation of the Integrated Stress Response Regulates Lovastatin-induced Apoptosis

Lovastatin, a potent inhibitor of mevalonate synthesis, can readily induce apoptosis in a subset of human tumor types including head and neck squamous cell carcinomas (HNSCC). We recently identified activation of transcription factor (ATF) 4 as a lovastatin induced gene in HNSCC cells. ATF4 plays a significant role in regulating cellular responses to a wide variety of stress inducers known as the integrated stress response (ISR). These cell stresses lead to the phosphorylation of eukaryotic initiation factor (eIF) 2alpha shutting down global protein translation. However, the translation of ATF4 is enhanced. In this study, lovastatin treatment induced eIF2alpha phosphorylation and inhibited global protein translation. ATF4 expression was induced followed by increased ATF3 and CHOP expression, targets of ATF4 activity, in SCC25 HNSCC cells. In CHOP(-/-) murine embryonic fibroblasts (MEFs), lovastatin-induced apoptosis was attenuated indicating a role for CHOP in this response. Furthermore, the eIF2alpha kinase GCN2 mediates lovastatin induction of ATF4 and lovastatin-induced apoptosis was also attenuated in GCN2(-/-) MEFs. The pro-drug version of lovastatin has potential proteasome inhibitory activity and recently a variety of well established proteasome inhibitors were shown to activate the ISR. In this study, neither the pro-drug nor the active forms of lovastatin had any significant effect on proteasome activity. Therefore, lovastatin, by targeting mevalonate synthesis, is a potent inducer of the ISR through a novel and as yet unrecognized mechanism.

Lovastatin Induces Apoptotic Cell Death by Activation of Intracellular Ca2+Signal in HepG2 Human Hepatoma Cells

Although lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, has been shown to have anti-cancer actions, the effect on human hepatoma cells was not investigated. Moreover, the exact mechanism of this action is not fully understood. In this study we investigated the mechanism by which lovastatin induces apoptosis using HepG2 human hepatoblastoma cells. Lovastatin induced apoptotic cell death in a dose-dependent manner in the cells, assessed by the flow cytometric analysis. Treatment with mevalonic acid, a precursor of cholesterol, did not significantly suppress the lovastatin-induced apoptosis. Lovastatin induced a rapid and sustained increase in intracellular <TEX>$Ca^{2+}$</TEX> concentration. Treatment with EGTA, an extracellular <TEX>$Ca^{2+}$</TEX> chelator did not significantly alter the lovastatin-induced intracellular <TEX>$Ca^{2+}$</TEX> increase and apoptosis, whereas intracellular <TEX>$Ca^{2+}$</TEX> reduction with BAPTA/AM and intracellular <TEX>$Ca^{2+}$</TEX> release blockers (dantrolene and TMB-8) completely blocked these actions of lovastatin. In addition, the lovastatin-induced apoptosis was significantly reduced by a calpain inhibitor, a broad spectrum caspase inhibitor z-VAD-fmk and inhibitors specific for caspase-9 and caspase-3 (z-LEHD-fmk and z-DEVD-fmk, respectively), but not by an inhibitor specific for caspase-8 (z-IETD-fmk). Collectively, these results suggest that lovastatin induced apoptosis of HepG2 hepatoma cells through intracellular <TEX>$Ca^{2+}$</TEX> release and calpain activation, leading to triggering mitochondrial apoptotic pathway. These results further suggest that lovastatin may be valuable for the therapeutic management of human hepatoma.

The Effect of Statins in Colorectal Cancer Is Mediated Through the Bone Morphogenetic Protein Pathway

Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs. We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model. CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell lines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. In a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed. Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Survivin Down-regulation Plays a Crucial Role in 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitor-induced Apoptosis in Cancer

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-X(L), revealed that only survivin expression was decreased by lovastatin. Survivin down-regulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin down-regulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.

Determinants of sensitivity to lovastatin-induced apoptosis in multiple myeloma

Statins, commonly used to treat hypercholesterolemia, have been shown to trigger tumor-specific apoptosis in certain cancers, including multiple myeloma (MM), a plasma cell malignancy with poor prognosis. In this article, we show that of a panel of 17 genetically distinct MM cell lines, half were sensitive to statin-induced apoptosis and, despite pharmacodynamic evidence of drug uptake and activity, the remainder were insensitive. Sensitive cells were rescued from lovastatin-induced apoptosis by mevalonate, geranylgeranyl PPi, and partially by farnesyl PPi, highlighting the importance of isoprenylation. Expression profiling revealed that Rho GTPase mRNAs were differentially expressed upon lovastatin exposure in sensitive cells, yet ectopic expression of constitutively active Rho or Ras proteins was insufficient to alter sensitivity to lovastatin-induced apoptosis. This suggests that sensitivity involves more than one isoprenylated protein and that statins trigger apoptosis by blocking many signaling cascades, directly or indirectly deregulated by the oncogenic lesions of the tumor cell. Indeed, clustering on the basis of genetic abnormalities was shown to be significantly associated with sensitivity (P = 0.003). These results suggest that statins may be a useful molecular targeted therapy in the treatment of a subset of MM.

Lovastatin-Induced Apoptosis in Macrophages through the Rac1/Cdc42/JNK Pathway

Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been used successfully in the treatment of hypercholesterolemia for more than a decade. Statins also exhibit overall clinical benefits on cardiovascular diseases independent of their effects on lowering serum cholesterol levels. These beneficial effects of statin therapy are believed to be due, at least in part, to the anti-inflammatory and immunomodulatory roles of statins. Statin treatment reduces the levels of inflammatory markers, decreases the activation and recruitment of immune cells, and delays the progression of atherosclerosis, a chronic inflammatory disease. However, little is known about the direct impact of statins on immune cells, particularly on macrophages. We report that lovastatin, a member of the statin family, effectively induces apoptosis in macrophages. Further investigation of the molecular mechanism has revealed that Rac1 and Cdc42, the small GTPase family members, may play an important role in lovastatin-induced macrophage apoptosis. Moreover, the activation of the JNK pathway may contribute to this event. Our findings provide a better understanding of the molecular basis underlying the anti-inflammatory clinical benefits of statin therapy in cardiovascular diseases.

Rho/ROCK pathway as a target of tumor therapy

This study emphasizes the importance of Rho/ROCK pathway in lovastatin-induced apoptosis as replenishment with exogenous isoprenoid, geranylgeranylpyrophosphate (GGPP), resulted in inhibition of apoptosis in cultured tumor cells. Treatment of C6 glioma cells with Toxin B and exoenzyme C3 resulted in cell death suggesting the role of geranylgeranylated protein(s) in the survival of glioma cells. Relative apoptotic death observed in cells transfected with dominant negative constructs of RhoA, Rac, and cdc42 imply Rho A as playing the major role in cell survival. Furthermore, the inhibition of Rho A kinase (ROCK), a direct downstream effector of Rho A, by Y-27632 or dominant negative of ROCK, induced apoptosis in glioma cells. These findings indicate that RhoA/ROCK pathway is involved negatively in the regulation of glioma cell death pathway. Moreover, in vivo studies of lovastatin treatment in animals implanted with C6 glioma cell tumors also resulted in smaller tumor size and induced apoptosis in the tumor tissue. The implantation of stably transfected C6 glioma cells with expression vector of C3 exoenzyme, dominant negative of RhoA and ROCK, resulted in significant smaller tumor mass, further establishing the importance of geranylgeranylated proteins, specifically RhoA and its downstream effecter ROCK, in cell survival and tumor genesis.

The association between RhoB and caspase-2: changes with lovastatin-induced apoptosis

Because cytoskeletal actin is regulated, in part, by Rho, and because Rho and caspases are involved in apoptosis, we sought to determine whether there was an association between RhoB and caspase-2. A RhoB-caspase-2 association was consistently demonstrated in neonatal mouse cardiomyocytes with Western Blotting, either after immunoprecipitation with RhoB followed by immunoblotting with caspase-2, or in reciprocal experiments after immuno precipitation with caspase-2 and immunoblotting with RhoB (n = 14). Although the RhoB-caspase-2 complex was constitutively present, the link between RhoB and caspase-2 may be operative in apoptosis because the HMG-CoA reductase inhibitor lovastatin increased the RhoB-caspase complex, especially in the nuclear fraction of the cell, with a peak occurrence 2 h after treatment. This association was unaffected by the caspase-2 inhibitor zVDVAD. Lovastatin produced apoptosis that was accompanied by an activation of caspase-2, as demonstrated by its immunohistochemistry and by the fact that the caspase-2 inhibitor zVDVAD reduced lovastatin-induced apoptosis. Lovastatin induced dramatic changes in cell morphology and a reduction in F-actin. Immunoblotting for actin suggests that lovastatin does not induce a degradation of the actin molecule, but rather affects filamentous F-actin. Caspase-2 inhibition with zVDVAD reduced lovastatin-induced alteration in cytoskeletal F-actin. The Rho inhibitor, Clostridium difficile toxin B, blunted the ability of lovastatin to induce apoptosis. In summary, these data show a previously unrecognized association between RhoB and caspase-2 in the cytosolic and nuclear fractions, which has ramifications for processes regulated by RhoB and caspase-2, including apoptosis.

Lovastatin-induced apoptosis in human melanoma cell lines

The cholesterol-lowering medications, statins, inhibit cellular proliferation and induce apoptosis in an array of cancer cell lines, including melanoma. We investigated the apoptotic mechanism of lovastatin on human melanoma cell lines in vitro. The cytotoxicity of statins on multiple cell lines was examined by Cell Titer 96 Aqueous One solution cell proliferation assay (MTS assay). Apoptosis was assayed by ethidium bromide and acridine orange morphologic assays, an Annexin V apoptosis detection kit and active caspase 3 assays. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate add-back experiments were performed to better define the molecular mechanisms mediating lovastatin cytotoxicity. Lovastatin caused cytotoxicity in human and murine melanoma cells, but did not induce toxicity in an epidermoid carcinoma cell line A431. For human melanoma cells, lovastatin precipitated cell rounding, increased the percentage of apoptotic cells detected by ethidium bromide and acridine orange staining and by the Annexin V apoptosis detection kit, and resulted in a 50-fold increase in active caspase 3, corroborating that lovastatin induced apoptosis. Adding back geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, reversed the effects of lovastatin in A375 cells. Of the five statins tested, pravastatin was least effective in killing melanoma cells. Lovastatin induced caspase-dependent apoptosis in multiple melanoma cell lines via a geranylation-specific mechanism. This study supports a possible role of lovastatin as a therapeutic, adjuvant or chemopreventive agent for melanoma.

Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis.

The statin family of drugs are well-established inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and are used clinically in the control of hypercholesterolemia. Recent evidence, from ourselves and others, shows that statins can also trigger tumor-specific apoptosis by blocking protein geranylgeranylation. We and others have proposed that statins disrupt localization and function of geranylgeranylated proteins responsible for activating signal transduction pathways essential for the growth and/or survival of transformed cells. To explore this further, we have investigated whether the mitogen-activated protein kinase (MAPK) signaling cascades play a role in regulating statin-induced apoptosis. Cells derived from acute myelogenous leukemia (AML) are used as our model system. We show that p38 and c-Jun NH2-terminal kinase/stress-activated kinase MAPK pathways are not altered during lovastatin-induced apoptosis. By contrast, exposure of primary and established AML cells to statins results in significant disruption of basal extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Addition of geranylgeranyl PPi reverses statin-induced loss of ERK1/2 phosphorylation and apoptosis. By establishing and evaluating the inducible Raf-1:ER system in AML cells, we show that constitutive activation of the Raf/MAPK kinase (MEK)/ERK pathway significantly represses but does not completely block lovastatin-induced apoptosis. Our results strongly suggest statins trigger apoptosis by regulating several signaling pathways, including the Raf/MEK/ERK pathway. Indeed, down-regulation of the Raf/MEK/ERK pathway potentiates statin-induced apoptosis because exposure to the MEK1 inhibitor PD98059 sensitizes AML cells to low, physiologically achievable concentrations of lovastatin. Our study suggests that lovastatin, alone or in combination with a MEK1 inhibitor, may represent a new and immediately available therapeutic approach to combat tumors with activated ERK1/2, such as AML.

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.

Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a p53-independent mitochondrial-mediated apoptotic mechanism.

The effects of lovastatin, a potent inhibitor of hydroxymethylglutaryl-coenzyme A reductase, were studied in a mouse model of metastatic mammary cancer carrying a p53 mutation. Mice bearing mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879 cells, were treated with lovastatin at 0, 25 and 50 mg/kg three times a week. Tumor volumes were significantly reduced in a dose-dependent manner throughout the 6 week study and were associated with both a decrease in DNA synthesis and an increase in apoptosis. The high dose of lovastatin also inhibited lung metastasis. In a corollary in vitro study, flow cytometric analyses of lovastatin-treated mammary cancer cells additionally showed cell cycle arrest at G1 phase and decreases in S and G2/M phases. Laser scanning cytometric analyses further demonstrated that cancer cells in S and G2/M were particularly susceptible to the effects of lovastatin. Transmission electron microscopic evaluation of TUNEL-confirmed apoptotic bodies in lovastatin-treated mammary carcinoma cells revealed many free 3'-OH ends of DNA in condensed chromatin within fragmented nuclei that occasionally assumed a characteristic half-moon shape. Consistent with initiation of apoptosis, cellular caspase-8, caspase-9 and caspase-3 activities were elevated in lovastatin-treated cells. The mitochondrial membrane potential was also decreased, with subsequent release of cytochrome c. However, lovastatin-induced cell death was significantly reduced by the broad spectrum caspase inhibitor z-VAD-fmk, as well as the caspase-9 inhibitor z-LEHD-fmk and the caspase-3 inhibitor z-DEVD-fmk, but not by the specific caspase-8 inhibitor z-IETD-fmk. Since immunoelectron microscopy showed translocation of Bax to the mitochondria in lovastatin-treated cells, lovastatin-induced apoptosis may, therefore, be ultimately dependent on Bax induction of cytochrome c release. These results suggest that lovastatin may be useful as an adjuvant therapy in breast cancers containing p53 mutations due to its ability to both suppress DNA synthesis and induce p53-independent mitochondria-mediated apoptosis.

Lovastatin-induced cardiac toxicity involves both oncotic and apoptotic cell death with the apoptotic component blunted by both caspase-2 and caspase-3 inhibitors

The objective of this study was to evaluate the cardiac toxicity of the HMG–CoA reductase inhibitors by testing the hypothesis that lovastatin induces apoptotic and/or oncotic cell death in the myocyte element of the heart and further that cell death is mediated through interruption of the mevalonate pathway and that apoptosis is induced through activation of caspase-2 and caspase-3. Cardiomyocytes were cultured from embryonic chick heart. Lovastatin-induced apoptosis in these cells was demonstrated by three independent techniques, namely (1) FACS analysis of low DNA content by propidium iodide (PI); (2) microscopic assessment for cellular changes of apoptosis; and (3) FACS analysis of cells stained with PI and fluoroscein diacetate. Lovastatin produced a concentration-dependent increase in apoptotic cell death and 100 μM lovastatin showed over a 4-fold increase in apoptosis compared to control. Lovastatin also induced oncotic cell death, as there was a 2.5-fold increase in the amount of oncotic cell death compared to control. Lovastatin-induced apoptosis operated, in part, through the mevalonate pathway. The caspase-2 inhibitor z-VDVAD-fmk and the caspase-3 inhibitor Ac-DEVD-CHO reduced the extent of lovastatin-induced cardiac apoptosis. In contrast, lovastatin-induced oncosis was not only insensitive to these caspase-2 or -3 inhibitors but occurred through a mevalonate-independent mechanism of action. In summary, lovastatin-induced cardiotoxicty is complex and represents the sum of two distinct modes of cell death operating in part through the mevalonate pathway with the apoptotic component subject to modification by inhibitors of the initiator caspase, caspase-2, as well as the effector caspase, caspase-3.