Hyperoxia decreases muscle glycogenolysis, lactate production, and lactate efflux during steady-state exercise

The aim of this study was to determine whether the decreased muscle and blood lactate during exercise with hyperoxia (60% inspired O2) vs. room air is due to decreased muscle glycogenolysis, leading to decreased pyruvate and lactate production and efflux. We measured pyruvate oxidation via PDH, muscle pyruvate and lactate accumulation, and lactate and pyruvate efflux to estimate total pyruvate and lactate production during exercise. We hypothesized that 60% O2 would decrease muscle glycogenolysis, resulting in decreased pyruvate and lactate contents, leading to decreased muscle pyruvate and lactate release with no change in PDH activity. Seven active male subjects cycled for 40 min at 70% VO2 peak on two occasions when breathing 21 or 60% O2. Arterial and femoral venous blood samples and blood flow measurements were obtained throughout exercise, and muscle biopsies were taken at rest and after 10, 20, and 40 min of exercise. Hyperoxia had no effect on leg O2 delivery, O2 uptake, or RQ during exercise. Muscle glycogenolysis was reduced by 16% with hyperoxia (267 +/- 19 vs. 317 +/- 21 mmol/kg dry wt), translating into a significant, 15% reduction in total pyruvate production over the 40-min exercise period. Decreased pyruvate production during hyperoxia had no effect on PDH activity (pyruvate oxidation) but significantly decreased lactate accumulation (60%: 22.6 +/- 6.4 vs. 21%: 31.3 +/- 8.7 mmol/kg dry wt), lactate efflux, and total lactate production over 40 min of cycling. Decreased glycogenolysis in hyperoxia was related to an approximately 44% lower epinephrine concentration and an attenuated accumulation of potent phosphorylase activators ADPf and AMPf during exercise. Greater phosphorylation potential during hyperoxia was related to a significantly diminished rate of PCr utilization. The tighter metabolic match between pyruvate production and oxidation resulted in a decrease in total lactate production and efflux over 40 min of exercise during hyperoxia.     

Targeting prostate cancer cells with PSMA inhibitor-guided gold nanoparticles

Prostate-specific membrane antigen (PSMA) is a notable biomarker for diagnostic and therapeutic applications in prostate cancer. Gold nanoparticles (AuNPs) provide an attractive nanomaterial platform for combining a variety of targeting, imaging, and cytotoxic agents into a unified device for biomedical research. In this study, we present the generation and evaluation of the first AuNP system functionalized with a small molecule phosphoramidate peptidomimetic inhibitor for the targeted delivery to PSMA-expressing prostate cancer cells. The general approach involved the conjugation of streptavidin-coated AuNPs with a biotin-linked PSMA inhibitor (CTT54) to generate PSMA-targeted AuNPs. In vitro evaluations of these targeted AuNPs were conducted to determine PSMA-mediated and time-dependent binding to PSMA-positive LNCaP cells. The PSMA-targeted AuNPs exhibited significantly higher and selective binding to LNCaP cells compared to control non-targeted AuNPs, thus demonstrating the feasibility of this approach.     

Targeting and cellular trafficking of magnetic nanoparticles for prostate cancer imaging

Antibody-conjugated iron oxide nanoparticles offer a specific and sensitive tool to enhance magnetic resonance (MR) images of both local and metastatic cancer. Prostate-specific membrane antigen (PSMA) is predominantly expressed on the neovasculature of solid tumors and on the surface of prostate cells, with enhanced expression following androgen deprivation therapy. Biotinylated anti-PSMA antibody was conjugated to streptavidin-labeled iron oxide nanoparticles and used in MR imaging and confocal laser scanning microscopic imaging studies using LNCaP prostate cancer cells. Labeled iron oxide nanoparticles are internalized by receptor-mediated endocytosis, which involves the formation of clathrin-coated vesicles. Endocytosed particles are not targeted to the Golgi apparatus for recycling but instead accumulate within lysosomes. In T(1)-weighted MR images, the signal enhancement owing to the magnetic particles was greater for cells with magnetic particles bound to the cell surface than for cells that internalized the particles. However, the location of the particles (surface vs internal) did not significantly alter their effect on T(2)-weighted images. Our findings indicate that targeting prostate cancer cells using PSMA offers a specific and sensitive technique for enhancing MR images.     

Targeting the DNA double strand break repair machinery in prostate cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.     

Targeting prostate cancer based on signal transduction and cell cycle pathways

Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness.     

Targeting prostate cancer cells with a multivalent PSMA inhibitor-guided streptavidin conjugate

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.     

Targeting NPRL2 to enhance the efficacy of Olaparib in castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) lacks effective treatment, and studies have shown that PARPi inhibitors, such as Olaparib, are somewhat effective; however, the efficacy of Olaparib in CRPC still needs to be further improved. Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and is closely related to the DNA repair pathway, which can affect the sensitivity of many chemotherapeutic drugs. However, there is no research on whether NPRL2 is associated with sensitivity to Olaparib. Hence, in the present study, we examined the NPRL2 expression levels in several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) by Western blot. In addition, we investigated the role of NPRL2 expression and silencing in cell proliferation and in the regulation of ataxia telangiectasia mutated (ATM), which can mediate DNA repair and sensitivity to Olaparib. Furthermore, we performed in vitro and in vivo experiments to determine the mechanism of action of NPRL2 in adjusting Olaparib sensitivity. Our findings demonstrated that the NPRL2 expression level was upregulated in PCa cells, especially CRPC cells. NPRL2 overexpression promoted growth and resistance to Olaparib, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to Olaparib, and increased CRRL2 expression in PCa cells. In addition, the Olaparib-induced growth delay in NPRL2-silenced PC3 tumors in mice correlated with ATM signaling downregulation, an apoptosis increase and migration/invasion suppression. Our results indicate that NPRL2 silencing enhances sensitivity to Olaparib treatment in CRPC and that NPRL2 may serve as a potential therapeutic target and predict resistance to Olaparib in CRPC.     

Human prostate stromal cells stimulate increased PSA production in DHEA-treated prostate cancer epithelial cells

Dehydroepiandrosterone (DHEA) is commonly used as a dietary supplement and may affect prostate pathophysiology when metabolized to androgens and/or estrogens. Human prostate LAPC-4 cancer cells with a wild type androgen receptor (AR) were treated with DHEA, androgens dihydrotestosterone (DHT), T, or R1881), and E(2) and assayed for prostate specific antigen (PSA) protein and gene expression. In LAPC-4 monocultures, DHEA and E(2) induced little or no increase in PSA protein or mRNA expression compared to androgen-treated cells. When prostate cancer-associated (6S) stromal cells were added in coculture, DHEA stimulated LAPC-4 cell PSA protein secretion to levels approaching induction by DHT. Also, DHEA induced 15-fold more PSA mRNA in LAPC-4 cocultures than in monocultures. LAPC-4 proliferation was increased 2-3-fold when cocultured with 6S stromal cells regardless of hormone treatment. DHEA-treated 6S stromal cells exhibited a dose- and time-dependent increase in T secretion, demonstrating stromal cell metabolism of DHEA to T. Coculture with non-cancerous stroma did not induce LAPC-4 PSA production, suggesting a differential modulation of DHEA effect in a cancer-associated prostate stromal environment. This coculture model provides a research approach to reveal detailed endocrine, intracrine, and paracrine signaling between stromal and epithelial cells that regulate tissue homeostasis within the prostate, and the role of the tumor microenvironment in cancer progression.     

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context of prostate cancer bone metastasis

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic because of bone remodeling leading to the formation of new bone. However, recent studies have shown increased osteolytic activity in the beginning stages of PCa bone metastases, suggesting that targeting both osteolytic and osteoblastic mediators would likely inhibit bone remodeling and PCa bone metastasis. In this study, we found that PCa cells could stimulate differentiation of osteoclasts and osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting bone remodeling. Interestingly, we found that formulated isoflavone and 3,3'-diindolylmethane (BR-DIM) were able to inhibit the differentiation of osteoclasts and osteoblasts through the inhibition of cell signal transduction in RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone and BR-DIM down-regulated the expression of miR-92a, which is known to be associated with RANKL signaling, EMT and cancer progression. By pathway and network analysis, we also observed the regulatory effects of isoflavone and BR-DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc. Therefore, isoflavone and BR-DIM with their multi-targeted effects could be useful for the prevention of PCa progression, especially by attenuating bone metastasis mechanisms.     

MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.     

Targeting acidity in cancer and diabetes

While cancer is commonly described as “a disease of the genes”, it is also a disease of metabolism. Indeed, carcinogenesis and malignancy are highly associated with metabolic re-programming, and there is clinical evidence that interrupting a cancer's metabolic program can improve patients' outcomes. Notably, many of the metabolic adaptations observed in cancer are similar to the same perturbations observed in diabetic patients. For example, metformin is commonly used to reduce hyperglycemia in diabetic patients, and has been demonstrated to reduce cancer incidence. Treatment with PI3K inhibitors can induce hyperinsulinemia, which can blunt therapeutic efficacy if unchecked. While commonalities between metabolism in cancer and diabetes have been extensively reviewed, here we examine a less explored and emergent convergence between diabetic and cancer metabolism: the generation of lactic acid and subsequent acidification of the surrounding microenvironment. Extracellular lactic acidosis is integral in disease manifestation and is a negative prognostic in both disease states. In tumors, this results in important sequela for cancer progression including increased invasion and metastasis, as well as inhibition of immune surveillance. In diabetes, acidosis impacts the ability of insulin to bind to its receptor, leading to peripheral resistance and an exacerbation of symptoms. Thus, acidosis may be a relevant therapeutic target, and we describe three approaches for targeting: buffers, nanomedicine, and proton pump inhibitors.     

A factor from pancreatic and colonic cancer cells stimulates glucose uptake and lactate production in myoblasts.

Patients with cancer cachexia exhibit increased glucose flux and lactate production in skeletal muscle. The aim of this study was to examine the direct effect of cancer cell-conditioned media on glucose metabolism in L6 myoblasts. Media from PANC-1 and Colo 320 cells caused a marked time-dependent and concentration-dependent increase of 2-deoxyglucose uptake in GLUT-4 transfected L6 myoblasts. This effect was greater than maximal acute stimulation by insulin and the effect of insulin was additive. Glucose utilization and lactate production increased in parallel to glucose uptake. The effect was inhibited by the protein synthesis inhibitor, cycloheximide and the glucose transport inhibitor, cytochalasin B. The bioactive factor had a molecular weight of approximately 5,000 and the biological activity was destroyed by proteinase K digestion. Radioimmunoassay and immunoneutralization studies indicated the major factor involved is not TNFalpha, IL-1beta, insulin, IGF-I or IGF-II. Further purification and characterization are needed to reveal the identity of this novel factor or factors which may have other metabolic effects that contribute to the cancer cachexia and insulin resistance.