Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K_isom) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K_isom values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway.As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K_isom calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.     

Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.Bisphosphonates (BPs), synthetic analogues of pyrophosphates, are clinically in use for diseases with excessive bone absorption such as osteoporosis and malignancy-associated hypercalcemia. BPs administered in vivo are accumulated in the bone matrix and inhibit activities of osteoclasts.¹ The first generation of BPs, without nitrogen in the structure, is converted into cytotoxic non-hydrolyzable ATP analogues and achieves cytotoxic effects thorough decreased mitochondrial membrane potentials.^2,3 The second and the third generations, containing nitrogen, inhibit farnesyl pyrophosphate synthetase, a key enzyme in the mevalonate pathways, and deplete isoprenoid pools, which subsequently results in decreased prenylation of small guanine-nucleotide-binding regulatory proteins (small G proteins) (Supplementary Figure S1).⁴Isoprenoid lipids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are substrates for prenylation processes that mediate farnesylation and geranylgeranylation of small G proteins, respectively.^5,6 Ras family proteins are either farnesylated by farnsyl transferase or geranylgeranylated by geranylgeranyl transferase I. In contrast, the majority of Rho family proteins and Rab family proteins are geranylgeranylated by geranylgeranyl transferase I and II, respectively. These lipid modifications are essential for most of small G proteins to bind to cytoplasmic and organelle membranes where prenylated small G proteins become functional, whereas unprenylated small G proteins remain in the cytoplasm and non-functional.⁵The nitrogen-containing BPs (N-BPs) also induce cytotoxicity to osteoclasts, which is favorable for enhanced bone mineralization, and recent studies also showed that N-BPs had cytotoxic activities on tumors such as breast and prostate cancer.^7,8 These cytotoxic actions are attributable to a number of mechanisms including apoptosis induction and anti-angiogenesis,^9,10 but it is not well investigated as to which small G proteins produce the cytotoxic effects.We recently showed that zoledronic acid (ZOL), which is one of the N-BPs to inhibit farnesyl pyrophosphate synthetase, produced cytotoxic activities to human mesothelioma.¹¹ ZOL treatments induced apoptotic cell death or S-phase arrest in cell cycle, and moreover caused morphological changes from fibroblast-like to spherical shapes. In the present study, we examined what kinds of small G proteins are responsible to these ZOL-mediated effects using inhibitors or small interfering RNA (siRNA) for the respective small G proteins and for prenylating enzymes.     

Bisphosphonate mechanism of action

Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption widely used in the treatment of osteoporosis and other bone degrading disorders. At the tissue level, N-BPs reduce bone turnover, increase bone mass and mineralization, measured clinically as a rise in bone mineral density, increase bone strength and reduce fracture risk. At the cellular level, N-BPs, localize preferentially at sites of bone resorption, where mineral is exposed, are taken up by ostoclasts and inhibit osteoclast activity. The bone formation that follows incroporates the N-BP in the matrix, where it becomes pharmacologically inactive until released at a future time during bone remodeling. At the molecular level, N-BPs inhibit an enzyme in the cholesterol synthesis pathway, farnesyl diphosphate synthase. As a result, there is a reduction in the lipid geranylgeranyl diphosphate, which prenylates GTPases required for cytoskeletal organization and vesicular traffic in the osteoclast, leading to osteoclast inactivation.     

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

Zoledronic acid-induced IPP/ApppI production in vivo

Bisphosphonates are currently the most important class of anti-resorptive drugs used for the treatment of diseases involving excess bone resorption. Recently we discovered a new mechanism of action for bisphosphonates. Previously it has been shown that nitrogen-containing bisphosphonates (N-BPs) are not metabolized. However, our studies revealed that N-BPs induce formation of a novel pro-apoptotic ATP analog (ApppI), as a consequence of the inhibition of FPP synthase in the mevalonate pathway, and the subsequent accumulation of isopentenyl pyrophosphate (IPP) in vitro. The primary aim of the current study was to determine whether zoledronic acid (a N-BP) induces IPP/ApppI formation in vivo. Mass spectrometry was used to identify whether in vivo administration of zoledronic acid-induced IPP/ApppI production by mouse peritoneal macrophages or bone marrow cells. IPP/ApppI could be detected in extracts from peritoneal macrophages isolated from zoledronic acid-treated animals. Increasing IPP/ApppI accumulation was determined up to 7 days after drug injection, indicating prolonged FPP synthase inhibition by zoledronic acid. Importantly, this is the first report of in vivo production of ApppI, supporting the biological significance of this molecule.     

Farnesyl diphosphate synthase localizes to the cytoplasm of Trypanosoma cruzi and T. brucei

The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes.     

Bisphosphonates, specific inhibitors of osteoclast function and a class of drugs for osteoporosis therapy

Osteoporosis is a result of the disruption of bone homeostasis that is carried out by bone-forming osteoblasts and bone-degrading osteoclasts. The most common treatment of osteoporosis is N-containing bisphosphonates, a class of non-hydrolyzable pyrophosphate analogs. They have strong affinity to Ca(2+) of hydroxyapatite with high specificity and can only be liberated from the bone in an acidic environment. These properties bestow them unique pharmacokinetic features including specific and strong retention at bone resorption surface, uptaken specifically by osteoclasts, quick excretion of non-retained free bisphosphonates, long half-life, and recyclability. Such properties underlie the drugs' high efficacy, minor side effects, and intermittent dosing regimens. Further studies show that bisphosphonates inhibit farnesyl pyrophosphate synthase, a critical enzyme required for synthesis of isoprenyl and geranylgeranyl, and inhibit prenylation and geranylgeranylation of small G-proteins such as Rac and Rho. This leads to defective actin ring formation at the sealed zone, a subcellular structure essential for bone resorption, and a decrease in bone resorption. Bisphosphonates are also used to treat Paget's disease of bone, osteolytic bone metastases, and hypercalcemia. Moreover, these properties also make N-BPs a good candidate as a bone-seeking agent. Here we update our understanding of this remarkable class of anti-resorption drugs.     

Shotgun proteomics analysis reveals new unsuspected molecular effectors of nitrogen-containing bisphosphonates in osteocytes

Nitrogen-containing bisphosphonates (N-BPs) are therapeutic agents used to treat osteoporosis and promote osteoblast and osteocyte survival. The molecular mechanisms underlying this effect have been extensively studied, but the global changes induced by N-BPs at the protein level are not known. In this context, we investigated the effect of 10(-7)M Risedronate for 1h and 48h on MLO-Y4 osteocytic cells, through a quantitative, label free shotgun proteomic analysis. We described herein a preliminary proteome map of untreated MLO-Y4 cells, composed of 353 protein species. Moreover, we identified 10 and 15 differentially expressed proteins after 1h and 48h of Risedronate treatment, respectively. Among these, PARK7/DJ-1 protein levels were induced up to 3 times and this event was associated with the activation of the pro-survival Akt pathway that we propose as a novel player in the effect of N-BPs on osteocytes. Risedronate was also able to induce the expression and the secretion of the growth factor pro-granulin. In addition, protein prenylation inhibition appeared to be involved in the modulation of MLO-Y4 proteome by RIS in a protein-specific manner. In conclusion, these findings unveil novel functions targeted by N-BPs in osteocytes and could be useful to design novel pharmaceutical compounds.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

Inhibition of zoledronic acid derivatives with extended methylene linkers on osteoclastogenesis involve downregulation of JNK and Akt pathways

Bisphosphonates (BPs), especially zoledronic acid (ZOL), are clinically used to treat osteolytic bone lesions. However, serious side-effects may be also induced during the therapeutic process. To improve the BPs drugs, here, we investigated the effects of a series of ZOL derivatives with increasing number of methylene linker between the imidazole ring and the P–C–P backbone named IPrDP, IBDP, IPeDP, and IHDP on cell viability and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, function and apoptosis induction in mouse bone marrow-derived macrophages (BMMs). Our results suggested that IPeDP and IHDP, which contains 4 and 5 methylene linkers, respectively, exerted lower toxicity on BMMs compared with ZOL, IPrDP, and IBDP, which contains 1, 2, and 3 methylene linkers respectively. At concentrations below cytotoxicity threshold, IPeDP and IHDP possessed strong abilities of antiosteoclast formation, antibone absorption, and inducing osteoclast apoptosis, which were similar to ZOL and more powerful than IPrDP and IBDP. The mechanism behind these effects of IPeDP and IHDP might involve the interference of small GTPases prenylation through suppression of mevalonate pathway. The downregulation of JNK and Akt phosphorylation and subsequent inhibition of the expression of c-Fos and NFATc1 might also be involved. Our results supported the potential usage of IPeDP and IHDP to treat bone-related disorders involving increased osteoclastogenesis. Our attempt to extend the methylene linker between the imidazole ring and the P–C–P backbone of ZOL also reveals some regularities between the structure and properties of the BPs drugs.     

Osteonecrosis of the Jaw Developed in Mice: DISEASE VARIANTS REGULATED BY γδ T CELLS IN ORAL MUCOSAL BARRIER IMMUNITY*

Osteonecrosis of the jaw (ONJ), an uncommon co-morbidity in patients treated with bisphosphonates (BP), occurs in the segment of jawbone interfacing oral mucosa. This study aimed to investigate a role of oral mucosal barrier γδ T cells in the pathogenesis of ONJ. Female C57Bl/6J (B6) mice received a bolus zoledronate intravenous injection (ZOL, 540 μg/kg), and their maxillary left first molars were extracted 1 week later. ZOL-treated mice (WT ZOL) delayed oral wound healing with patent open wounds 4 weeks after tooth extraction with characteristic oral epithelial hyperplasia. γδ T cells appeared within the tooth extraction site and hyperplastic epithelium in WT ZOL mice. In ZOL-treated γδ T cell null (Tcrd^−/− ZOL) mice, the tooth extraction open wound progressively closed; however, histological ONJ-like lesions were identified in 75 and 60% of WT ZOL and Tcrd^−/− ZOL mice, respectively. Although the bone exposure phenotype of ONJ was predominantly observed in WT ZOL mice, Tcrd^−/− ZOL mice developed the pustule/fistula disease phenotype. We further addressed the role of γδ T cells from human peripheral blood (h-γδ T cells). When co-cultured with ZOL-pretreated human osteoclasts in vitro, h-γδ T cells exhibited rapid expansion and robust IFN-γ secretion. When h-γδ T cells were injected into ZOL-treated immunodeficient (Rag2^−/− ZOL) mice, the oral epithelial hyperplasia developed. However, Rag2^−/− ZOL mice did not develop osteonecrosis. The results indicate that γδ T cells are unlikely to influence the core osteonecrosis mechanism; however, they may serve as a critical modifier contributing to the different oral mucosal disease variations of ONJ.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells

Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.     

R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Akt inactivation and reduced Bcl-2 and bad phosphorylation

Zoledronic acid (ZOL) has proved activity in bone metastases from prostate cancer through inhibition of mevalonate pathway and of prenylation of intracellular proteins. We have reported that ZOL synergizes with R115777 farnesyltransferase inhibitor (FTI, Zarnestra) in inducing apoptosis and growth inhibition on epidermoid cancer cells. Here, we have studied the effects of the combination of these agents in prostate adenocarcinoma models and, specifically, on androgen-independent (PC3 and DU145) and -dependent (LNCaP) prostate cancer cell lines. We have found that ZOL and R115777 were synergistic in inducing both growth inhibition and apoptosis in prostate adenocarcinoma cells. These effects were paralleled by disruption of Ras-->Erk and Akt survival pathways, consequent decreased phosphorylation of both mitochondrial bcl-2 and bad proteins, and caspase activation. Finally, ZOL/R115777 combination induced cooperative effects also in vivo on tumor growth inhibition of prostate cancer xenografts in nude mice with a significant survival increase. These effects were paralleled by enhanced apoptosis and inactivation of both Erk and Akt. In conclusions, the combination between ZOL and FTI leads to enhanced anti-tumor activity in human prostate adenocarcinoma cells likely through a more efficacious inhibition of ras-dependent survival pathways and consequent bcl-related proteins-dependent apoptosis.     

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.     

Site specific effects of zoledronic acid during tibial and mandibular fracture repair

Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones.     

New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: a comparative study

Zoledronic acid (ZOL) is a drug whose potent anti-cancer activity is limited by its short plasma half-life and rapid uptake and accumulation within bone. We have recently proposed new delivery systems to avoid ZOL accumulation into the bone, thus improving extra-skeletal bioavailability. In this work, we have compared the technological and anti-cancer features of either ZOL-containing self-assembly PEGylated nanoparticles (NPs) or ZOL-encapsulating PEGylated liposomes (LIPO-ZOL). ZOL-containing NPs showed superior technological characteristics in terms of mean diameter, size distribution, and ZOL encapsulation efficiency, compared to LIPO-ZOL. Moreover, the anti-cancer activity of NPs in nude mice xenografted with prostate cancer PC3 cells was higher than that one induced by LIPO-ZOL. In addition, NPs induced the complete remission of tumour xenografts and an increase of survival time higher than that one observed with LIPO-ZOL. It has also to be considered that PC3 tumour xenografts were almost completely resistant to the anti-cancer effects induced by free ZOL. Both nanotechnological products did not induce toxic effects not affecting the mice weight nor inducing deaths. Moreover, the histological examination of some vital organs such as liver, kidney and spleen did not find any changes in terms of necrotic effects or modifications in the inflammatory infiltrate. On the other hand, NPs but not LIPO-ZOL caused a statistically significant reduction of the tumour associated macrophages (TAM) in tumour xenografts. This effect was paralleled by a significant increase of both necrotic and apoptotic indexes. The effects of the NPs were also higher in terms of neo-angiogenesis inhibition. These results suggest the future preclinical development of ZOL-encapsulating NPs in the treatment of human cancer.     

Syntheses and characterization of non-bisphosphonate quinoline derivatives as new FPPS inhibitors

Background

Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in the biosynthesis of cholesterol and in the post-translational modification of signaling proteins. It has been reported that non-bisphosphonate FPPS inhibitors targeting its allosteric binding pocket are potentially important for the development of promising anti-cancer drugs.

Methods

The following methods were used: organic syntheses of non-bisphosphonate quinoline derivatives, enzyme inhibition studies, fluorescence titration assays, synergistic effect studies of quinoline derivatives with zoledronate, ITC studies for the binding of FPPS with quinoline derivatives, NMR-based HAP binding assays, molecular modeling studies, fluorescence imaging assay and MTT assays.

Results

We report our syntheses of a series of quinoline derivatives as new FPPS inhibitors possibly targeting the allosteric site of the enzyme. Compound 6b showed potent inhibition to FPPS without significant hydroxyapatite binding affinity. The compound showed synergistic inhibitory effect with active-site inhibitor zoledronate. ITC experiment confirmed the good binding effect of compound 6b to FPPS, and further indicated the binding ratio of 1:1. Molecular modeling studies showed that 6b could possibly bind to the allosteric binding pocket of the enzyme. The fluorescence microscopy indicated that these compounds could get into cancer cells.

Conclusions

Our results showed that quinoline derivative 6b could become a new lead compound for further optimization for cancer treatment.

General significance

The traditional FPPS active-site inhibitors bisphosphonates show poor membrane permeability to tumor cells, due to their strong polarity. The development of new non-bisphosphonate FPPS inhibitors with good cell membrane permeability is potentially important.     

Anti-proliferative action of endogenous dehydroepiandrosterone metabolites on human cancer cell lines

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.