Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 23
Filter
Add more filters










Publication year range
1.
Biomolecules ; 14(6)2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38927098

ABSTRACT

Cytochrome c (Cytc) is important for both mitochondrial respiration and apoptosis, both of which are altered in cancer cells that switch to Warburg metabolism and manage to evade apoptosis. We earlier reported that lysine 53 (K53) of Cytc is acetylated in prostate cancer. K53 is conserved in mammals that is known to be essential for binding to cytochrome c oxidase and apoptosis protease activating factor-1 (Apaf-1). Here we report the effects of this acetylation on the main functions of cytochrome c by expressing acetylmimetic K53Q in cytochrome c double knockout cells. Other cytochrome c variants analyzed were wild-type, K53R as a control that maintains the positive charge, and K53I, which is present in some non-mammalian species. Intact cells expressing K53Q cytochrome c showed 49% decreased mitochondrial respiration and a concomitant increase in glycolytic activity (Warburg effect). Furthermore, mitochondrial membrane potential was decreased, correlating with notably reduced basal mitochondrial superoxide levels and decreased cell death upon challenge with H2O2 or staurosporine. To test for markers of cancer aggressiveness and invasiveness, cells were grown in 3D spheroid culture. K53Q cytochrome c-expressing cells showed profoundly increased protrusions compared to WT, suggesting increased invasiveness. We propose that K53 acetylation of cytochrome c is an adaptive response that mediates prostate cancer metabolic reprogramming and evasion of apoptosis, which are two hallmarks of cancer, to better promote tumor survival and metastasis.


Subject(s)
Apoptosis , Cytochromes c , Lysine , Prostatic Neoplasms , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Humans , Cytochromes c/metabolism , Male , Acetylation , Lysine/metabolism , Cell Line, Tumor , Mitochondria/metabolism , Membrane Potential, Mitochondrial , Metabolic Reprogramming
2.
bioRxiv ; 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38586033

ABSTRACT

Monounsaturated fatty acids (MUFAs) play a pivotal role in maintaining endoplasmic reticulum (ER) homeostasis, an emerging hallmark of cancer. However, the role of polyunsaturated fatty acid (PUFAs) desaturation in persistent ER stress driven by oncogenic abnormalities remains elusive. Fatty Acid Desaturase 1 (FADS1) is a rate-limiting enzyme controlling the bioproduction of long-chain PUFAs. Our previous research has demonstrated the significant role of FADS1 in cancer survival, especially in kidney cancers. We explored the underlying mechanism in this study. We found that pharmacological inhibition or knockdown of the expression of FADS1 effectively inhibits renal cancer cell proliferation and induces cell cycle arrest. The stable knockdown of FADS1 also significantly inhibits tumor formation in vivo. Mechanistically, we show that while FADS1 inhibition induces ER stress, its expression is also augmented by ER-stress inducers. Notably, FADS1-inhibition sensitized cellular response to ER stress inducers, providing evidence of FADS1's role in modulating the ER stress response in cancer cells. We show that, while FADS1 inhibition-induced ER stress leads to activation of ATF3, ATF3-knockdown rescues the FADS1 inhibition-induced ER stress and cell growth suppression. In addition, FADS1 inhibition results in the impaired biosynthesis of nucleotides and decreases the level of UPD-N-Acetylglucosamine, a critical mediator of the unfolded protein response. Our findings suggest that PUFA desaturation is crucial for rescuing cancer cells from persistent ER stress, supporting FADS1 as a new therapeutic target.

3.
Chemistry ; 28(24): e202104430, 2022 Apr 27.
Article in English | MEDLINE | ID: mdl-35235227

ABSTRACT

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.


Subject(s)
Rhodium , Ruthenium , Triple Negative Breast Neoplasms , Cell Line, Tumor , Humans , Immunotherapy , Rhodium/pharmacology , Ruthenium/pharmacology , Tumor-Associated Macrophages
4.
Photochem Photobiol ; 98(2): 378-388, 2022 03.
Article in English | MEDLINE | ID: mdl-34866185

ABSTRACT

We report the synthesis, photochemical and biological characterization of two new Ru(II) photoactivated complexes based on [Ru(tpy)(Me2 bpy)(L)]2+ (tpy = 2,2':6',2''-terpyridine, Me2 bpy = 6,6'-dimethyl-2,2'-bipyridine), where L = pyridyl-BODIPY (pyBOD). Two pyBOD ligands were prepared bearing flanking hydrogen or iodine atoms. Ru(II)-bound BODIPY dyes show a red-shift of absorption maxima relative to the free dyes and undergo photodissociation of BODIPY ligands with green light irradiation. Addition of iodine into the BODIPY ligand facilitates intersystem crossing, which leads to efficient singlet oxygen production in the free dye, but also enhances quantum yield of release of the BODIPY ligand from Ru(II). This represents the first report of a strategy to enhance photodissociation quantum yields through the heavy-atom effect in Ru(II) complexes. Furthermore, Ru(II)-bound BODIPY dyes display fluorescence turn-on once released, with a lead analog showing nanomolar EC50 values against triple negative breast cancer cells, >100-fold phototherapeutic indexes under green light irradiation, and higher selectivity toward cancer cells as compared to normal cells than the corresponding free BODIPY photosensitizer. Conventional Ru(II) photoactivated complexes require nonbiorthogonal blue light for activation and rarely show submicromolar potency to achieve cell death. Our study represents an avenue for the improved photochemistry and potency of future Ru(II) complexes.


Subject(s)
Iodine , Ruthenium , Coloring Agents , Ligands , Photosensitizing Agents/chemistry , Ruthenium/chemistry
5.
J Bone Oncol ; 30: 100386, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34466376

ABSTRACT

Prostate cancer (PCa) metastasis research has been hamstrung by lack of animal models that closely resemble the disease present in most patients - that metastasize to bone, are dependent on the androgen receptor (AR), and grow in an immune competent host. Here, we adapt the Myc-CaP cell line for use as a PCa androgen dependent, immune competent bone metastases model and characterize the metastases. After injection into the left cardiac ventricle of syngeneic FVB/NJ mice, these cells formed bone metastases in the majority of animals; easily visible on H&E sections and confirmed by immunohistochemistry for Ar and epithelial cell adhesion molecule. Mediastinal tumors were also observed. We also labeled Myc-CaP cells with tdTomato, and confirmed the presence of cancer cells in bone by flow cytometry. To adapt the model to a bone predominant metastasis pattern and further examine the bone phenotype, we labeled the cells with luciferase, injected in the tibia and observed tumor formation only in tibia with a mixed osteolytic/osteoblastic phenotype. The presence of Myc-CaP tumors significantly increased tibia bone volume as compared to sham injected controls. The osteoclast marker, TRAcP-5b was not significantly changed in plasma from tibial tumor bearing animals vs. sham animals. However, conditioned media from Myc-CaP cells stimulated osteoclast formation in vitro from FVB/NJ mouse bone marrow. Overall, Myc-CaP cells injected in the left ventricle or tibia of syngeneic mice recapitulate key aspects of human metastatic PCa.

6.
Biochim Biophys Acta Mol Cell Res ; 1868(11): 119101, 2021 10.
Article in English | MEDLINE | ID: mdl-34280426

ABSTRACT

A critical unknown in the field of skeletal metastases is how cancer cells find a way to thrive under harsh conditions, as exemplified by metastatic colonization of adipocyte-rich bone marrow by prostate carcinoma cells. To begin understanding molecular processes that enable tumor cells to survive and progress in difficult microenvironments such as bone, we performed unbiased examination of the transcriptome of two different prostate cancer cell lines in the absence or presence of bone marrow adipocytes. Our RNAseq analyses and subsequent quantitative PCR and protein-based assays reveal that upregulation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) genes is a shared signature between metastatic prostate carcinoma cell lines of different origin. Pathway analyses and pharmacological examinations highlight the ER chaperone BIP as an upstream coordinator of this transcriptomic signature. Additional patient-based data support our overall conclusion that ER stress and UPR induction are shared, important factors in the response and adaptation of metastatic tumor cells to their micro-environment. Our studies pave the way for additional mechanistic investigations and offer new clues towards effective therapeutic interventions in metastatic disease.


Subject(s)
Adipocytes/metabolism , Prostatic Neoplasms/genetics , Animals , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Humans , Male , Mice , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA-Seq , Transcriptome , Unfolded Protein Response/genetics
7.
Adv Radiat Oncol ; 6(4): 100679, 2021.
Article in English | MEDLINE | ID: mdl-34286163

ABSTRACT

PURPOSE: This study aimed to evaluate a combination of radiation therapy (RT), androgen deprivation therapy (ADT), and pexidartinib (colony-stimulating factor 1 receptor [CSF1R]) inhibitor in men with intermediate- and high-risk prostate cancer. CSF1R signaling promotes tumor infiltration and survival of tumor-associated macrophages, which in turn promote progression and resistance. Counteracting protumorigenic actions of tumor-associated macrophages via CSF1R inhibition may enhance therapeutic efficacy of RT and ADT for prostate cancer. METHODS AND MATERIALS: In this phase 1 study, the treatment regimen consisted of pexidartinib (800 mg, administered as a split-dose twice daily) and ADT (both for a total of 6 months), and RT that was initiated at the start of month 3. RT volumes included the prostate and proximal seminal vesicles. The delivered dose was 7920 cGy (180 cGy per fraction) using intensity modulated RT with daily image guidance for prostate localization. The primary objective was to identify the maximum tolerated dose based on dose-limiting toxicities. RESULTS: All 4 enrolled patients who were eligible to receive RT had T1 stage prostate cancer, 2 were intermediate risk, and 2 were high risk. The median age was 62.5 years, and the prostate-specific antigen levels were in the range 6.4 to 10.7 ng/mL. The patients' individual Gleason scores were 3 + 3, 4 + 3, 4 + 4, and 4 + 5. All 4 patients reported ≥1 adverse events before RT. Grade 1 hypopigmentation was observed in 1 patient, and grade 3 pulmonary embolus in another. One patient experienced fatigue and joint pain, and another elevated amylase and pruritus (all grade 3 toxicities). Five of the 6 adverse events noted in 3 patients were all grade 3 toxicities attributable to pexidartinib, qualifying as dose-limiting toxicities and ultimately resulting in the study closure. CONCLUSIONS: The combination was not well tolerated and does not warrant further investigation in men with intermediate- and high-risk prostate cancer.

8.
Mol Cancer Res ; 17(12): 2508-2521, 2019 12.
Article in English | MEDLINE | ID: mdl-31562254

ABSTRACT

Adipocyte-tumor cell cross-talk is one of the critical mediators of tumor progression and an emerging facilitator of therapy evasion. Tumor cells that metastasize to adipocyte-rich bone marrow take advantage of the interplay between metabolic and inflammatory pathways to activate prosurvival mechanisms that allow them to thrive and escape therapy. Using in vitro and in vivo models of marrow adiposity, we demonstrate that metastatic prostate carcinoma cells engage bone marrow adipocytes in a functional cross-talk that promotes IL1ß expression in tumor cells. Tumor-supplied IL1ß contributes to adipocyte lipolysis and regulates a proinflammatory phenotype in adipocytes via upregulation of COX-2 and MCP-1. We further show that the enhanced activity of the IL1ß/COX-2/MCP-1 axis and a resulting increase in PGE2 production by adipocytes coincide with augmented hypoxia signaling and activation of prosurvival pathways in tumor cells, revealing a potential mechanism of chemoresistance. The major consequence of this interplay is the reduced response of prostate cancer cells to docetaxel, a phenomenon sensitive to the inhibition of lipolysis. IMPLICATIONS: Studies presented herein highlight adipocyte lipolysis as a tumor-regulated metabolic event that engages proinflammatory cross-talk in the microenvironment to promote prostate cancer progression in bone. Understanding the impact of bone marrow adipose tissue on tumor adaptation, survival, and chemotherapy response is fundamentally important, as current treatment options for metastatic prostate cancer are palliative.


Subject(s)
Chemokine CCL2/genetics , Cyclooxygenase 2/genetics , Interleukin-1beta/genetics , Prostatic Neoplasms/drug therapy , Adipocytes/drug effects , Animals , Biopsy , Bone Marrow Cells/drug effects , Dinoprostone/genetics , Docetaxel/pharmacology , Drug Resistance, Neoplasm/drug effects , Humans , Inflammation/drug therapy , Inflammation/genetics , Inflammation/pathology , Lipolysis/drug effects , Male , Mice , Prostate/drug effects , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Tumor Microenvironment/drug effects
9.
J Am Chem Soc ; 140(43): 14367-14380, 2018 10 31.
Article in English | MEDLINE | ID: mdl-30278123

ABSTRACT

Dual action agents containing a cysteine protease inhibitor and Ru-based photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Ru-based PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Ru-based complexes were synthesized with the formula [Ru(bpy)2(1)](O2CCF3)2 (3), where bpy = 2,2'-bipyridine and 1 = a bipyridine-based epoxysuccinyl inhibitor; [Ru(tpy)(NN)(2)](PF6)2, where tpy = terpiridine, 2 = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2'-bipyridine (4); 6,6'-dimethyl-2,2'-bipyridine (5); benzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (6); and 3,6-dimethylbenzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (7). Compound 3 contains a [Ru(bpy)3]2+ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds 4-7 were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes 5 and 7 undergo photoactivated ligand dissociation, whereas 6 and 7 generate singlet oxygen. Inhibitors 1-7 all potently and irreversibly inhibit CTSB. Compounds 4-7 were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that 4-7 potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound 7 causes efficient cell death under light conditions, consistent with its ability to release a Ru(II)-based photosensitizer and to also generate 1O2.


Subject(s)
Antineoplastic Agents/pharmacology , Coordination Complexes/pharmacology , Peptide Hydrolases/metabolism , Photosensitizing Agents/pharmacology , Protease Inhibitors/pharmacology , Ruthenium/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Drug Screening Assays, Antitumor , Humans , Kinetics , Photosensitizing Agents/chemical synthesis , Photosensitizing Agents/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Ruthenium/chemistry , Thermodynamics
10.
Inorg Chem ; 57(13): 7881-7891, 2018 Jul 02.
Article in English | MEDLINE | ID: mdl-29882662

ABSTRACT

Two new Re(I)- and Ru(II)-based inhibitors were synthesized with the formulas [Re(phen)(CO)3(1)](OTf) (7; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru(bpy)2(2)](Cl)2 (8; bpy = 2,2'-bipyridine), where 1 and 2 are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds 7 and 8 were characterized using various spectroscopic techniques and elemental analysis; 7 and 8 both show exceptionally long excited state lifetimes. Re(I)-based complex 7 inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor 1 with a second-order rate constant of 211000 ± 42000 M-1 s-1. Irreversible ligation of 7 to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-1-Re(CO)3(phen). Compound 7 was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with 7 followed by imaging via confocal microscopy showed substantial intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of 7 to label CTSL in living cells. Overall this study reveals that a Re(I) complex can be attached to an enzyme inhibitor and enhance potency and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due to long excited state lifetimes and non-native element composition.


Subject(s)
Cathepsin L/antagonists & inhibitors , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Rhenium/chemistry , Ruthenium/chemistry , Cathepsin L/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Humans , Luminescent Agents/chemistry , Luminescent Agents/pharmacology , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation
11.
Curr Osteoporos Rep ; 16(4): 443-457, 2018 08.
Article in English | MEDLINE | ID: mdl-29869753

ABSTRACT

PURPOSE OF REVIEW: Bone marrow adipocytes have emerged in recent years as key contributors to metastatic progression in bone. In this review, we focus specifically on their role as the suppliers of lipids and discuss pro-survival pathways that are closely linked to lipid metabolism, affected by the adipocyte-tumor cell interactions, and likely impacting the ability of the tumor cell to thrive in bone marrow space and evade therapy. RECENT FINDINGS: The combined in silico, pre-clinical, and clinical evidence shows that in adipocyte-rich tissues such as bone marrow, tumor cells rely on exogenous lipids for regulation of cellular energetics and adaptation to harsh metabolic conditions of the metastatic niche. Adipocyte-supplied lipids have a potential to alter the cell's metabolic decisions by regulating glycolysis and respiration, fatty acid oxidation, lipid desaturation, and PPAR signaling. The downstream effects of lipid signaling on mitochondrial homeostasis ultimately control life vs. death decisions, providing a mechanism for gaining survival advantage and reduced sensitivity to treatment. There is a need for future research directed towards identifying the key metabolic and signaling pathways that regulate tumor dependence on exogenous lipids and consequently drive the pro-survival behavior in the bone marrow niche.


Subject(s)
Adipocytes/metabolism , Bone Marrow Cells/metabolism , Bone Neoplasms/metabolism , Lipid Metabolism , Tumor Microenvironment , Adipocytes/cytology , Apoptosis , Bone Marrow Cells/cytology , Bone Neoplasms/secondary , Cell Respiration , Fatty Acids/metabolism , Glycerol/metabolism , Glycolysis , Humans , Mitochondria/metabolism , Oxidation-Reduction , Oxidative Phosphorylation , Oxidative Stress , Peroxisome Proliferator-Activated Receptors/metabolism , Signal Transduction , Tumor Hypoxia
12.
Sci Rep ; 8(1): 40, 2018 01 08.
Article in English | MEDLINE | ID: mdl-29311669

ABSTRACT

Metastatic tumor cells engage the local tumor microenvironment and activate specific pro-survival mechanisms to thrive and progress in the harsh bone marrow niche. Here we show that the major contributors to the survival of carcinoma cells that have colonized the bone marrow are the adipocyte-induced oxidative stress and ER stress pathways. We demonstrate that upon exposure to adipocyte-rich environments in vitro or in vivo, bone-trophic prostate and breast tumor cells upregulate the oxidative stress enzyme, HO-1. We also show that HO-1 levels are significantly increased in human metastatic prostate cancer tissues and that stable HO-1 overexpression in tumor cells promotes growth and invasiveness. Co-incident with the adipocyte-induced expression of HO-1, there is an upregulation of ER chaperone BIP and splicing of XBP1, indicating adipocyte-driven unfolded protein response, a process that we show to be sensitive to antioxidant treatment. Importantly, we also demonstrate that triggering of the oxidative stress and ER stress responses, or HO-1 induction by adipocyte exposure result in the activation of pro-survival pathways, involving survivin. Collectively, our findings reveal a new link between HO-1 and survivin expression in tumor cells, and provide a new insight into potentially targetable survival pathways in bone-metastatic disease.


Subject(s)
Adipocytes/metabolism , Bone and Bones/metabolism , Endoplasmic Reticulum Stress , Gene Expression Regulation , Heme Oxygenase-1/genetics , Inhibitor of Apoptosis Proteins/genetics , Oxidative Stress , Animals , Biopsy , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Survival/genetics , Heme Oxygenase-1/metabolism , Humans , Immunohistochemistry , Inhibitor of Apoptosis Proteins/metabolism , Male , Mice , Oligopeptides/metabolism , Survivin , X-Box Binding Protein 1/metabolism
13.
Chem Commun (Camb) ; 53(26): 3673-3676, 2017 Mar 28.
Article in English | MEDLINE | ID: mdl-28304025
14.
Oncotarget ; 7(40): 64854-64877, 2016 10 04.
Article in English | MEDLINE | ID: mdl-27588494

ABSTRACT

Metabolic adaptation is increasingly recognized as a key factor in tumor progression, yet its involvement in metastatic bone disease is not understood. Bone is as an adipocyte-rich organ, and a major site of metastasis from prostate cancer. Bone marrow adipocytes are metabolically active cells capable of shaping tumor metabolism via lipolysis and lipid transfer. In this study, using in vitro and in vivo models of marrow adiposity, we demonstrate that marrow fat cells promote Warburg phenotype in metastatic prostate cancer cells. We show increased expression of glycolytic enzymes, increased lactate production, and decreased mitochondrial oxidative phosphorylation in tumor cells exposed to adipocytes that require paracrine signaling between the two cell types. We also reveal that prostate cancer cells are capable of inducing adipocyte lipolysis as a postulated mechanism of sustenance. We provide evidence that adipocytes drive metabolic reprogramming of tumor cells via oxygen-independent mechanism of HIF-1α activation that can be reversed by HIF-1α downregulation. Importantly, we also demonstrate that the observed metabolic signature in tumor cells exposed to adipocytes mimics the expression patterns seen in patients with metastatic disease. Together, our data provide evidence for a functional relationship between marrow adipocytes and tumor cells in bone that has likely implications for tumor growth and survival within the metastatic niche.


Subject(s)
Adipocytes/pathology , Bone Marrow Cells/pathology , Bone Neoplasms/metabolism , Carcinogenesis/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Prostatic Neoplasms/metabolism , Walker-Warburg Syndrome/genetics , Adipocytes/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/secondary , Coculture Techniques , Glycolysis/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lipid Metabolism , Lipolysis/genetics , Male , Mice , Paracrine Communication , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Small Interfering/genetics , Transcriptome , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
15.
Article in English | MEDLINE | ID: mdl-27458427

ABSTRACT

Adipocytes are a major component of the bone marrow that can critically affect metastatic progression in bone. Understanding how the marrow fat cells influence growth, behavior, and survival of tumor cells requires utilization of in vitro cell systems that can closely mimic the physiological microenvironment. Herein, we present two new three-dimensional (3D) culture approaches to study adipocyte-tumor cell interactions in vitro. The first is a transwell-based system composed of the marrow-derived adipocytes in 3D collagen I gels and reconstituted basement membrane-overlayed prostate tumor cell spheroids. Tumor cells cultured under these 3D conditions are continuously exposed to adipocyte-derived factors, and their response can be evaluated by morphological and immunohistochemical analyses. We show via immunofluorescence analysis of metabolism-associated proteins that under 3D conditions tumor cells have significantly different metabolic response to adipocytes than tumor cells grown in 2D culture. We also demonstrate that this model allows for incorporation of other cell types, such as bone marrow macrophages, and utilization of dye-quenched collagen substrates for examination of proteolysis-driven responses to adipocyte- and macrophage-derived factors. Our second 3D culture system is designed to study tumor cell invasion toward the adipocytes and the consequent interaction between the two cell types. In this model, marrow adipocytes are separated from the fluorescently labeled tumor cells by a layer of collagen I. At designated time points, adipocytes are stained with BODIPY and confocal z-stacks are taken through the depth of the entire culture to determine the distance traveled between the two cell types over time. We demonstrate that this system can be utilized to study effects of candidate factors on tumor invasion toward the adipocytes. We also show that immunohistochemical analyses can be performed to evaluate the impact of direct interaction of prostate tumor cells with adipocytes. Our models underline the importance of using the appropriate culture conditions to mimic physiological interactions between marrow adipocytes and metastatic tumor cells. These systems have a potential to be utilized for analyses of various factors that may be regulated by the adipocytes in bone. Their application likely extends beyond metastatic prostate cancer to other tumors that colonize the bone marrow microenvironment.

16.
Biol Chem ; 397(6): 571-82, 2016 06 01.
Article in English | MEDLINE | ID: mdl-26901495

ABSTRACT

Collagenolytic activity of cathepsin K is important for many physiological and pathological processes including osteoclast-mediated bone degradation, macrophage function and fibroblast-mediated matrix remodeling. Here, we report application of a light-activated inhibitor for controlling activity of cathepsin K in a 3D functional imaging assay. Using prostate carcinoma cell line engineered to overexpress cathepsin K, we demonstrate the utility of the proteolytic assay in living tumor spheroids for the evaluation and quantification of the inhibitor effects on cathepsin K-mediated collagen I degradation. Importantly, we also show that utilizing the ruthenium-caged version of a potent nitrile cathepsin K inhibitor (4), cis-[Ru(bpy)2(4)2](BF4)2 (5), offers significant advantage in terms of effective concentration of the inhibitor and especially its light-activated control in the 3D assay. Our results suggest that light activation provides a suitable, attractive approach for spatial and temporal control of proteolytic activity, which remains a critical, unmet need in treatment of human diseases, especially cancer.


Subject(s)
Cathepsin K/antagonists & inhibitors , Imaging, Three-Dimensional , Light , Prostatic Neoplasms/pathology , Protease Inhibitors/pharmacology , Cathepsin K/genetics , Cell Line, Tumor , Collagen Type I/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Male , Prostatic Neoplasms/genetics , Proteolysis/drug effects , Proteolysis/radiation effects
17.
Inorg Chem ; 55(1): 10-2, 2016 Jan 04.
Article in English | MEDLINE | ID: mdl-26670781

ABSTRACT

Three complexes of the general formula [Ru(TPA)L2](PF6)2 [TPA = tris(2-pyridylmethyl)amine], where L = pyridine (1), nicotinamide (2), and imidazole (3), were prepared and characterized spectroscopically. X-ray crystallographic data were obtained for 1 and 3. Complexes 1-3 show strong absorption in the visible region and selective release of heterocycles upon irradiation with visible light. Time-dependent density functional theory calculations are consistent with the presence of singlet metal-to-ligand charge-transfer bands in the visible region in 1-3. Caged heterocycles 1-3 are highly stable in solution in the dark, including in cell growth media. Cell viability data show no signs of toxicity of 1-3 against PC-3 cells at concentrations up to 100 µM under light and dark conditions, consistent with Ru(TPA) acting as a nontoxic and effective photocaging group for aromatic heterocycles.


Subject(s)
Heterocyclic Compounds/chemistry , Light , Pyridines/chemistry , Ruthenium Compounds/chemistry , Crystallography, X-Ray , Models, Molecular
18.
Clin Exp Metastasis ; 32(4): 353-68, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25802102

ABSTRACT

Increased bone marrow adiposity is a common feature of advanced age, obesity and associated metabolic pathologies. Augmented numbers of marrow adipocytes positively correlate with dysregulated bone remodeling, also a well-established complication of metastatic disease. We have shown previously that marrow adiposity accelerates prostate tumor progression in the skeleton and promotes extensive destruction of the bone; however, the factors behind adipocyte-driven osteolysis in the skeletal tumor microenvironment are not currently known. In this study, utilizing in vivo diet-induced models of bone marrow adiposity, we reveal evidence for positive correlation between increased marrow fat content, bone degradation by ARCaP(M) and PC3 prostate tumors, and augmented levels of host-derived CXCL1 and CXCL2, ligands of CXCR2 receptor. We show by in vitro osteoclastogenesis assays that media conditioned by bone marrow adipocytes is a significant source of CXCL1 and CXCL2 proteins. We also demonstrate that both the adipocyte-conditioned media and the recombinant CXCL1 and CXCL2 ligands efficiently accelerate osteoclast maturation, a process that can be blocked by neutralizing antibodies to each of the chemokines. We further confirm the contribution of CXCR2 signaling axis to adiposity-driven osteoclastogenesis by blocking fat cell-induced osteoclast differentiation with CXCR2 antagonist or neutralizing antibodies. Together, our results link CXCL1 and CXCL2 chemokines with bone marrow adiposity and implicate CXCR2 signaling in promoting effects of marrow fat on progression of skeletal tumors in bone.


Subject(s)
Bone Neoplasms/secondary , Chemokine CXCL1/metabolism , Chemokine CXCL2/metabolism , Osteolysis/pathology , Prostatic Neoplasms/pathology , Adipocytes/metabolism , Adiposity , Animals , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Bone Remodeling/physiology , Bone and Bones/pathology , Cathepsin K/metabolism , Cell Differentiation , Cell Line, Tumor , Coculture Techniques , Humans , Male , Mice , Neoplasm Transplantation , Obesity , Osteoclasts/cytology , Osteogenesis/physiology , Transplantation, Heterologous , Tumor Microenvironment
19.
ChemMedChem ; 9(6): 1306-15, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24729544

ABSTRACT

Light-activated inhibition of cathepsin activity was demonstrated in a cell-based assay. Inhibitors of cathepsin K, Cbz-Leu-NHCH2 CN (2) and Cbz-Leu-Ser(OBn)-CN (3), were caged within the complexes cis-[Ru(bpy)2 (2)2 ]Cl2 (4) and cis-[Ru(bpy)2 (3)2 ](BF4 )2 (5) (bpy=2,2'-bipyridine) as 1:1 mixtures of Δ and Λ stereoisomers. Complexes 4 and 5 were characterized by (1) H NMR, IR, and UV/Vis spectroscopies and electrospray mass spectrometry. Photochemical experiments confirm that 4 releases two molecules of 2 upon exposure to visible light for 15 min, whereas release of 3 by 5 requires longer irradiation times. IC50 determinations against purified cathepsin K under light and dark conditions with 4 and 5 confirm that inhibition is enhanced from 35- to 88-fold, respectively, upon irradiation with visible light. No apparent toxicity was observed for 4 in the absence or presence of irradiation in bone marrow macrophage (BMM) or PC3 cells, as determined by MTT assays, at concentrations up to 10 µM. Compound 5 is well tolerated at lower concentrations (<1 µM), but does show growth-inhibitory effects at higher concentrations. Confocal microscopy experiments show that 4 decreases intracellular cathepsin activity in osteoclasts with light activation. These results support the further development of caged nitrile-based inhibitors as chemical tools for investigating spatial aspects of proteolysis within living systems.


Subject(s)
Cathepsin K/antagonists & inhibitors , Coordination Complexes/chemistry , Enzyme Inhibitors/chemistry , Light , Ruthenium/chemistry , 2,2'-Dipyridyl/chemistry , Animals , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Cathepsin K/metabolism , Cell Line , Cell Survival/drug effects , Coordination Complexes/chemical synthesis , Coordination Complexes/toxicity , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Mice , Microscopy, Confocal
20.
Cancer Metastasis Rev ; 33(2-3): 527-43, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24398857

ABSTRACT

Adipocytes are important but underappreciated components of bone marrow microenvironment, and their numbers greatly increase with age, obesity, and associated metabolic pathologies. Age and obesity are also significant risk factors for development of metastatic prostate cancer. Adipocytes are metabolically active cells that secrete adipokines, growth factors, and inflammatory mediators; influence behavior and function of neighboring cells; and have a potential to disturb local milleu and dysregulate normal bone homeostasis. Increased marrow adiposity has been linked to bone marrow inflammation and osteoporosis of the bone, but its effects on growth and progression of prostate tumors that have metastasized to the skeleton are currently not known. This review focuses on fat-bone relationship in a context of normal bone homeostasis and metastatic tumor growth in bone. We discuss effects of marrow fat cells on bone metabolism, hematopoiesis, and inflammation. Special attention is given to CCL2- and COX-2-driven pathways and their potential as therapeutic targets for bone metastatic disease.


Subject(s)
Adipocytes/metabolism , Bone Marrow/metabolism , Bone Marrow/pathology , Adiposity , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bone Marrow/drug effects , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Bone and Bones/metabolism , Bone and Bones/pathology , Homeostasis , Humans , Inflammation/metabolism , Male , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Tumor Microenvironment
SELECTION OF CITATIONS
SEARCH DETAIL
...