Therapeutic Potential of 5'-Methylschweinfurthin G in Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5'-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.

Cytotoxic Effects of the Schweinfurthin Analog 5'-Methylschweinfurthin G in Malignant Plasma Cells.

INTRODUCTION: Multiple myeloma (MM) is a B plasma cell malignancy currently incurable, and novel therapeutics are needed. Evidences regarding the effect of natural compound schweinfurthins suggest that hematological cancers showed growth inhibitory effects to this family of compounds at single nanomolar concentrations. In this study, we evaluated the cytotoxicity of the schweinfurthin synthetic analog 5'-methylschweinfurthin G (MeSG) in MM cell lines, to better understand the validity of this compound as a therapeutic candidate for further studies in MM. METHODS: MeSG toxicity against MM cell lines RPMI-8226, MM.1S, and H-929 was evaluated. Trypan blue exclusion and MTT assays measured cell viability and mitochondrial activity, respectively. Flow cytometry was performed to detect apoptotic mitochondria. Flow cytometry and Western blotting techniques were used to investigate apoptosis and to examine the cell cycle. Western blotting was used to determine AKT activation upon MeSG treatment. RESULTS: We provide evidence that in all MM cells analyzed, MeSG exerts diverse cytotoxic effects. MeSG treatment of MM.1S and H-929, but not in RPMI-8226, causes a loss of mitochondria membrane potential. MeSG causes an arrest in G2/M, especially in RPMI-8226, supported by decreased levels of cyclin-B1 and early increased levels of p21. Finally, there is a diverse response to the MeSG treatment for AKT phosphorylation. MM.1S and H-929 showed a marked decrease in AKT phosphorylation at earlier time points compared to the RPMI-8226 line. CONCLUSIONS: MeSG cytotoxicity has been confirmed in all of 3 cell lines studied. Results suggest an early event of increased reactive oxygen species, and/or involvement of cholesterol homeostasis via decreased AKT activation, both of which are currently under investigation.

Bisphosphonates: The role of chemistry in understanding their biological actions and structure-activity relationships, and new directions for their therapeutic use.

The bisphosphonates ((HO)2P(O)CR1R2P(O)(OH)2, BPs) were first shown to inhibit bone resorption in the 1960s, but it was not until 30 years later that a detailed molecular understanding of the relationship between their varied chemical structures and biological activity was elucidated. In the 1990s and 2000s, several potent bisphosphonates containing nitrogen in their R2 side chains (N-BPs) were approved for clinical use including alendronate, risedronate, ibandronate, and zoledronate. These are now mostly generic drugs and remain the leading therapies for several major bone-related diseases, including osteoporosis and skeletal-related events associated with bone metastases. The early development of chemistry in this area was largely empirical and only a few common structural features related to strong binding to calcium phosphate were clear. Attempts to further develop structure-activity relationships to explain more dramatic pharmacological differences in vivo at first appeared inconclusive, and evidence for mechanisms underlying cellular effects on osteoclasts and macrophages only emerged after many years of research. The breakthrough came when the intracellular actions on the osteoclast were first shown for the simpler bisphosphonates, via the in vivo formation of P-C-P derivatives of ATP. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates in the 1980s and 1990s led to the key discovery that the antiresorptive effects of these more complex analogs on osteoclasts result mostly from their potency as inhibitors of the enzyme farnesyl diphosphate synthase (FDPS/FPPS). This key branch-point enzyme in the mevalonate pathway of cholesterol biosynthesis is important for the generation of isoprenoid lipids that are utilized for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Since then, it has become even more clear that the overall pharmacological effects of individual bisphosphonates on bone depend upon two key properties: the affinity for bone mineral and inhibitory effects on biochemical targets within bone cells, in particular FDPS. Detailed enzyme-ligand crystal structure analysis began in the early 2000s and advances in our understanding of the structure-activity relationships, based on interactions with this target within the mevalonate pathway and related enzymes in osteoclasts and other cells have continued to be the focus of research efforts to this day. In addition, while many members of the bisphosphonate drug class share common properties, now it is more clear that chemical modifications to create variations in these properties may allow customization of BPs for different uses. Thus, as the appreciation for new potential opportunities with this drug class grows, new chemistry to allow ready access to an ever-widening variety of bisphosphonates continues to be developed. Potential new uses of the calcium phosphate binding mechanism of bisphosphonates for the targeting of other drugs to the skeleton, and effects discovered on other cellular targets, even at non-skeletal sites, continue to intrigue scientists in this research field.

Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium-dependent, non-D2 receptor-dependent, manner.

Dopamine D2 -like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood-brain barrier. Despite a plethora of data reporting anticancer effects for D2 R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D2 receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D2 antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D2 receptors (D2 R) on these cells. Although several chemotypes of D2 R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D2 R antagonist-induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D2 R. Instead, cytotoxicity was associated with a rapid, high-magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D2 R antagonists involves calcium signaling mechanisms, not D2 R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Bisphosphonates in dentistry: Historical perspectives, adverse effects, and novel applications.

Studies of the potential role of bisphosphonates in dentistry date back to physical chemical research in the 1960s, and the genesis of the discovery of bisphosphonate pharmacology in part can be linked to some of this work. Since that time, parallel research on the effects of bisphosphonates on bone metabolism continued, while efforts in the dental field included studies of bisphosphonate effects on dental calculus, caries, and alveolar bone loss. While some utility of this drug class in the dental field was identified, leading to their experimental use in various dentrifice formulations and in some dental applications clinically, adverse effects of bisphosphonates in the jaws have also received attention. Most recently, certain bisphosphonates, particularly those with strong bone targeting properties, but limited biochemical effects (low potency bisphosphonates), are being studied as a local remedy for the concerns of adverse effects associated with other more potent members of this drug class. Additionally, low potency bisphosphonate analogs are under study as vectors to target active drugs to the mineral surfaces of the jawbones. These latter efforts have been devised for the prevention and treatment of oral problems, such as infections associated with oral surgery and implants. Advances in the utility and mechanistic understanding of the bisphosphonate class may enable additional oral therapeutic options for the management of multiple aspects of dental health.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

The Dopamine D2 Receptor Contributes to the Spheroid Formation Behavior of U87 Glioblastoma Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is a common and lethal cancer of the central nervous system. This cancer is difficult to treat because most anticancer therapeutics do not readily penetrate into the brain due to the tight control at the cerebrovascular barrier. Numerous studies have suggested that dopamine D2 receptor (D2R) antagonists, such as first generation antipsychotics, may have anticancer efficacy in vivo and in vitro. The role of the D2R itself in the anticancer effects is unclear, but there is evidence suggesting that D2R activation promotes stem-like and spheroid forming behaviors in GBM. OBJECTIVES: We aimed to observe the role of the dopamine D2R and its modulators (at selective concentrations) in spheroid formation and stemness of GBM cell line, U87MG, to clarify the validity of the D2R as a therapeutic target for cancer therapy. METHODS: Spheroid formation assays and Western blotting of the glioblastoma cell line, U87MG, were used to observe responses to treatment with the D2R agonists sumanirole, ropinirole, and 4-propyl-9-hydroxynaphthoxazine (PHNO); and the D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride. Extreme limiting dilution analysis was done to determine the impact of sumanirole and remoxipride treatment on sphere-forming cell frequency. Proliferation was also measured by crystal violet staining. Stable lentiviral transduction of DRD2 or shDRD2 was used to validate the role of the D2R in assay behaviors. RESULTS: D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride decrease spheroid formation behaviors at a selective 100 nmol/L concentration, while D2R agonists PHNO, sumanirole, and ropinirole increase the formation of spheroids. Similarly, 100 nmol/L remoxipride decreased sphere-forming cell frequency. These results were recapitulated with genetic overexpression and knockdown of the D2R, and combination experiments indicate that the D2R is required for the effects of the pharmacological modulators. Furthermore, spheroid proliferation and invasive capacity increased under treatment with 100 nmol/L sumanirole and decreased under treatment with 100 nmol/L thioridazine. Expression levels of the stemness markers Nestin and Sox2, as well as those of differentiation marker glial fibrillary acidic protein, were not altered by 100 nmol/L thioridazine or sumanirole for 72 h or continuous treatment with these compounds for 7 days during a spheroid formation assay. CONCLUSIONS: Signaling activity of the dopamine D2R may be involved in the spheroid formation phenotype in the context of the U87MG cell line. However, this modulation may not be due to alterations in stemness marker expression, but due to other factors that may contribute to spheroid formation, such as cell-cell adhesion or EGFR signaling.

Increased flux through the mevalonate pathway mediates fibrotic repair without injury.

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to ß oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

Cancer and the Dopamine D2 Receptor: A Pharmacological Perspective.

The dopamine D2 receptor (D2R) family is upregulated in many cancers and tied to stemness. Reduced cancer risk has been correlated with disorders such as schizophrenia and Parkinson's disease, in which dopaminergic drugs are used. D2R antagonists are reported to have anticancer efficacy in cell culture and animal models where they have reduced tumor growth, induced autophagy, affected lipid metabolism, and caused apoptosis, among other effects. This has led to several hypotheses, the most prevalent being that D2R ligands may be a novel approach to cancer chemotherapy. This hypothesis is appealing because of the large number of approved and experimental drugs of this class that could be repurposed. We review the current state of the literature and the evidence for and against this hypothesis. When the existing literature is evaluated from a pharmacological context, one of the striking findings is that the concentrations needed for cytotoxic effects of D2R antagonists are orders of magnitude higher than their affinity for this receptor. Although additional definitive studies will provide further clarity, our hypothesis is that targeting D2-like dopamine receptors may only yield useful ligands for cancer chemotherapy in rare cases.

Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity.

Metastatic melanoma is a significant clinical problem with a 5-year survival rate of only 15-20%. Recent approval of new immunotherapies and targeted inhibitors have provided much needed options for these patients, in some cases promoting dramatic disease regressions. In particular, antibody-based therapies that block the PD-1/PD-L1 checkpoint inhibitory pathway have achieved an increased overall response rate in metastatic melanoma, yet durable response rates are reported only around 15%. To improve the overall and durable response rates for advanced-stage melanoma, combined targeted and immune-based therapies are under investigation. Here, we investigated how the natural products called schweinfurthins, which have selective anti-proliferative activity against many cancer types, impact anti-(α)PD-1-mediated immunotherapy of murine melanomas. Two different compounds efficiently reduced the growth of human and murine melanoma cells in vitro and induced plasma membrane surface localization of the ER-resident protein calreticulin in B16.F10 melanoma cells, an indicator of immunogenic cell death. In addition, both compounds improved αPD-1-mediated immunotherapy of established tumors in immunocompetent C57BL/6 mice either by delaying tumor progression or resulting in complete tumor regression. Improved immunotherapy was accomplished following only a 5-day course of schweinfurthin, which was associated with initial tumor regression even in the absence of αPD-1. Schweinfurthin-induced tumor regression required an intact immune system as tumors were unaffected in NOD scid gamma (NSG) mice. These results indicate that schweinfurthins improve αPD-1 therapy, leading to enhanced and durable anti-tumor immunity and support the translation of this novel approach to further improve response rates for metastatic melanoma.

Inhibiting geranylgeranyl diphosphate synthesis reduces nuclear androgen receptor signaling and neuroendocrine differentiation in prostate cancer cell models.

BACKGROUND: Following androgen deprivation for the treatment of advanced adenocarcinoma of the prostate, tumors can progress to neuroendocrine prostate cancer (NEPC). This transdifferentiation process is poorly understood, but trafficking of transcriptional factors and/or cytoskeletal rearrangements may be involved. We observed the role of geranylgeranylation in this process by treatment with digeranyl bisphosphonate (DGBP), a selective inhibitor of geranylgeranyl pyrophosphate synthase which blocks the prenylation of small GTPases such as Rho and Rab family proteins, including Cdc42 and Rac1. METHODS: We examined the therapeutic potential of DGBP in LNCaP, C4-2B4, and 22Rv1 cell culture models. Cell morphology and protein expression were quantified to observe the development of the neuroendocrine phenotype in androgen-deprivation and abiraterone-treated LNCaP models of NEPC development. Luciferase reporter assays were utilized to examine AR activity, and immunofluorescence visualized the localization of AR within the cell. RESULTS: Essential genes in the isoprenoid pathway, such as HMGCR, MVK, GGPS1, and GGT1, were highly expressed in a subset of castration resistant prostate cancers reported by Beltran et al. Under treatment with DGBP, nuclear localization of AR decreased in LNCaP, 22Rv1, and C4-2B4 cell lines, luciferase reporter activity was reduced in LNCaP and 22Rv1, and AR target gene transcription also decreased in LNCaP. Conversely, nuclear localization of AR was enhanced by the addition of GGOH. Finally, induction of the NEPC structural and molecular phenotype via androgen deprivation in LNCaP cells was inhibited by DGBP in a GGOH-dependent manner. CONCLUSIONS: DGBP is a novel compound with the potential to reduce AR transcriptional activity and inhibit PCa progression to NEPC phenotype. These results suggest that DGBP may be used to block cell growth and metastasis in both hormone therapy sensitive and resistant paradigms.

Schweinfurthins: Lipid Modulators with Promising Anticancer Activity.

The schweinfurthin family of compounds displays exciting potent and differential cytotoxicity against human cancer cell lines. Currently, the effect of schweinfurthins on tumor development and progression is being explored in animal models of cancer with promising results. The first schweinfurthin family member, vedelianin, was isolated in 1992, followed by other schweinfurthins in 1998. This opened up the door for the synthesis of additional analogs. At present, the focus of research lies on delineating the mechanism of schweinfurthin action and identifying the nature of sensitivity. It appears that many of the intracellular effects of schweinfurthins are due to, or impacted by, the effect of schweinfurthins on lipid metabolism, synthesis, and homeostasis. These effects include impaired trafficking from the trans-golgi network, disruption of lipid rafts, changes in oxysterol-binding protein activity, and interference with the isoprenoid biosynthesis pathway (IBP). Cancer cells are known to rely heavily on fatty acid, lipid, and sterol synthesis for growth and proliferation. Therefore, compounds that target these needs, such as schweinfurthins, display promise as novel therapeutics. This timely review will take an in-depth look at the history of schweinfurthins, their synthesis, where the research presently stands, and the questions that remain.

Selective opioid growth factor receptor antagonists based on a stilbene isostere.

As part of an ongoing drug development effort aimed at selective opioid receptor ligands based on the pawhuskin natural products we have synthesized a small set of amide isosteres. These amides were centered on lead compounds which are selective antagonists for the delta and kappa opioid receptors. The amide isomers revealed here show dramatically different activity from the parent stilbene compounds. Three of the isomers synthesized showed antagonist activity for the opioid growth factor (OGF)/opioid growth factor receptor (OGFR) axis which is involved in cellular and organ growth control. This cellular signaling mechanism is targeted by "low-dose" naltrexone therapy which is being tested clinically for multiple sclerosis, Crohn's disease, cancer, and wound healing disorders. The compounds described here are the first selective small molecule ligands for the OGF/OGFR system and will serve as important leads and probes for further study.

Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms.

Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a "target and release" chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.

Targeting protein geranylgeranylation slows tumor development in a murine model of prostate cancer metastasis.

The isoprenoid biosynthetic pathway (IBP) plays a critical role in providing substrates and enzymes necessary for the post-translational modification and thus activation of a number of proteins involved in prostate cancer metastasis. Previous work by our lab found novel compound disodium [(6Z,11E,15E)-9-[bis(sodiooxy)phosphoryl]-17-hydroxy-2,6,12,16-tetramethyheptadeca-2,6,11,15-tetraen-9-yl]phosphonate (GGOHBP), which inhibits the IBP enzyme geranylgeranyl diphosphate synthase (GGDPS), reduced protein geranylgeranylation without altering protein farnesylation. This activity significantly reduced adrenal gland tumor burden in a murine model of human prostate cancer metastasis which relied on treatment of established disease. The present study determined the ability of GGDPS inhibition to slow the development of prostate cancer metastasis in a preventative murine model. Using tail vein injection of human derived PC-3 prostate cancer cells 4 d after initiating daily GGOHBP or vehicle treatments, we found GGOHBP significantly reduced whole body tumor burden, significantly slowed the development of tumors, and prolonged overall survival as compared to vehicle treated animals. The observed reduction in soft tissue tumor burden corresponded to a biochemical reduction in Rap1A geranylgeranylation, which for prostate cancer is important in its own merit and which serves as a surrogate marker for Rho family, i.e. Rac, protein modification. This effect was present in all treated mice pointing to strong target engagement, which was not observed in non-tumor burdened tissues or control mice. Our findings reiterate a role for protein geranylgeranylation in the development of prostate cancer metastasis in vivo.

A selective delta opioid receptor antagonist based on a stilbene core.

Studies of directed ortho metalation reactions on an aromatic substrate with multiple potential directing groups have identified conditions that favor either of two regioisomers. One of these regioisomers has been converted to an analogue of the stilbene pawhuskin A, and been shown to have high selectivity as an antagonist of the delta opioid receptor. Docking studies have suggested that this compound can adopt a conformation similar to naltrindole, a known delta antagonist.

3-Deoxyschweinfurthin B Lowers Cholesterol Levels by Decreasing Synthesis and Increasing Export in Cultured Cancer Cell Lines.

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or ß. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

Targeting geranylgeranylation reduces adrenal gland tumor burden in a murine model of prostate cancer metastasis.

The isoprenoid biosynthetic pathway (IBP) is critical for providing substrates for the post-translational modification of proteins key in regulating malignant cell properties, including proliferation, invasion, and migration. Inhibitors of the IBP, including statins and nitrogenous bisphosphonates, are used clinically for the treatment of hypercholesterolemia and bone disease respectively. The statins work predominantly in the liver, while the nitrogenous bisphosphonates are highly sequestered to bone. Inhibition of the entire IBP is limited by organ specificity and side effects resulting from depletion of all isoprenoids. We have developed a novel compound, disodium [(6Z,11E,15E)-9-[bis(sodiooxy)phosphoryl]-17-hydroxy-2,6,12,16-tetramethyheptadeca-2,6,11,15-tetraen-9-yl]phosphonate (GGOHBP), which selectively targets geranylgeranyl diphosphate synthase, reducing post-translational protein geranylgeranylation. Intracardiac injection of luciferase-expressing human-derived 22Rv1 PCa cells into SCID mice resulted in tumor development in bone (100 %), adrenal glands (72 %), mesentery (22 %), liver (17 %), and the thoracic cavity (6 %). Three weeks after tumor inoculation, daily subcutaneous (SQ) injections of 1.5 mg/kg GGOHBP or the vehicle were given for one month. Dissected tumors revealed a reduction in adrenal gland tumors corresponding to a 54 % (P < 0.005) reduction in total adrenal gland tumor weight of the treated mice as compared to vehicle-treated controls. Western blot analysis of the harvested tissues showed a reduction in Rap1A geranylgeranylation in adrenal glands and mesenteric tumors of the treated mice while non-tumorous tissues and control mice showed no Rap1A alteration. Our findings detail a novel bisphosphonate compound capable of preferentially altering the IBP in tumor-burdened adrenal glands of a murine model of PCa metastasis.

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis.

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H2O2) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H2O2 attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H2O2 generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys(189)) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.