Apalutamide radio-sensitisation of prostate cancer.

INTRODUCTION: The combination of radiotherapy with bicalutamide is the standard treatment of prostate cancer patients with high-risk or locally advanced disease. Whether new-generation anti-androgens, like apalutamide, can improve the radio-curability of these patients is an emerging challenge. MATERIALS AND METHODS: We comparatively examined the radio-sensitising activity of apalutamide and bicalutamide in hormone-sensitive (22Rv1) and hormone-resistant (PC3, DU145) prostate cancer cell lines. Experiments with xenografts were performed for the 22Rv1 cell line. RESULTS: Radiation dose-response viability and clonogenic assays showed that apalutamide had a stronger radio-sensitising activity for all three cell lines. Confocal imaging for γΗ2Αx showed similar DNA double-strand break repair kinetics for apalutamide and bicalutamide. No difference was noted in the apoptotic pathway. A striking cell death pattern involving nuclear karyorrhexis and cell pyknosis in the G1/S phase was exclusively noted when radiation was combined with apalutamide. In vivo experiments in SCID and R2G2 mice showed significantly higher efficacy of radiotherapy (2 and 4 Gy) when combined with apalutamide, resulting in extensive xenograft necrosis. CONCLUSIONS: In vitro and in vivo experiments support the superiority of apalutamide over bicalutamide in combination with radiotherapy in prostate cancer. Clinical studies are encouraged to show whether replacement of bicalutamide with apalutamide may improve the curability rates.

SMER28 is a mTOR-independent small molecule enhancer of autophagy that protects mouse bone marrow and liver against radiotherapy.

Effective cytoprotectors that are selective for normal tissues could decrease radiotherapy and chemotherapy sequelae and facilitate the safe administration of higher radiation doses. This could improve the cure rates of radiotherapy for cancer patients. Autophagy is a cytoplasmic cellular process that is necessary for the clearance of damaged or aged proteins and organelles. It is a strong determinant of post-irradiation cell fate. In this study, we investigated the effect of the mTOR-independent small molecule enhancer of autophagy (SMER28) on mouse liver autophagy and post-irradiation recovery of mouse bone marrow and liver. SMER28 enhanced the autophagy flux and improved the survival of normal hepatocytes. This effect was specific for normal cells because SMER28 had no protective effect on hepatoma or other cancer cell line survival in vitro. In vivo subcutaneous administration of SMER28 protected mouse liver and bone marrow against radiation damage and facilitated survival of mice after lethal whole body or abdominal irradiation. These findings open a new field of research on autophagy-targeting radioprotectors with clinical applications in oncology, occupational, and space medicine.

Comparison of the effect of the antiandrogen apalutamide (ARN-509) versus bicalutamide on the androgen receptor pathway in prostate cancer cell lines.

Apalutamide (ARN-509) is an antiandrogen that binds selectively to androgen receptors (AR) and does not show antagonist-to-agonist switch like bicalutamide. We compared the activity of ARN versus bicalutamide on prostate cancer cell lines. The 22Rv1, PC3, and DU145 cell lines were used to study the effect of ARN and bicalutamide on the expression cytoplasmic/nuclear kinetics of AR, AR-V7 variant, phosphorylated AR, as well as the levels of the AR downstream proteins prostate-specific antigen and TMPRSS2, under exposure to testosterone and/or hypoxia. The effects on autophagic flux (LC3A, p62, TFEB, LAMP2a, cathepsin D) and cell metabolism-related enzymes (hypoxia-inducible factor 1α/2α, BNIP3, carbonic anhydrase 9, LDHA, PDH, PDH-kinase) were also studied. The 22Rv1 cell line responded to testosterone by increasing the nuclear entry of AR, AR-V7, and phosphorylated AR and by increasing the levels of prostate-specific antigen and TMPRSS2. This effect was strongly abrogated by ARN and to a clearly lower extent by bicalutamide at 10 µmol/l, both in normoxia and in hypoxia. ARN had a stronger antiproliferative effect than bicalutamide, which was prominent in the 22Rv1 hormone-responsive cell line, and completely repressed cell proliferation at a concentration of 100 µmol/l. No effect of testosterone or of antiandrogens on autophagy flux, hypoxia-related proteins, or metabolism enzyme levels was noted. The PC3 and DU145 cell lines showed poor expression of the proteins and were not responsive to testosterone. On the basis of in-vitro studies, evidence has been reported that ARN is more potent than bicalutamide in blocking the AR pathway in normoxia and in hypoxia. This reflects a more robust, dose-dependent, repressive effect on cell proliferation.

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism.

This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy.

BACKGROUND: The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation. METHODS: A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1(nu) was evaluated. RESULTS: Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours. CONCLUSIONS: The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.

Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer.

INTRODUCTION: Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. MATERIALS AND METHODS: In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. RESULTS: Using confocal microscopy on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a 'highLC3A/lowLAMP2a' phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. CONCLUSIONS: High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy.

Effect of mitochondrial metabolism-interfering agents on cancer cell mitochondrial function and radio/chemosensitivity.

Abnormal mitochondrial function is common in cancer cells and activates metabolic pathways suppressed in normal tissues. Experimental and clinical studies suggest that mitochondria might serve as targets for novel anticancer therapies. We investigated whether mitochondrial metabolism-interfering agents (MMIAs) available currently in clinical practice affect cancer cell mitochondrial metabolism and synergize with chemotherapy and radiotherapy. Two cancer cell lines A549 (lung cancer) and DU145 (prostate cancer) were treated with a variety of MMIAs (metformin, nimodipine, memantine, oxytetracycline, amiodarone, and sodium azide) and their response was assessed using a resazurin reduction method and confocal microscopy. Focusing on amiodarone and metformin, we investigated their potential sensitizing effect on cancer cells when treated with ionizing radiation, cisplatin, and docetaxel. Resazurin reduction was increased by metformin and decreased by amiodarone at nontoxic concentrations. Amiodarone induced mitochondrial swelling, whereas metformin exerted no apparent effect on their morphology. Amiodarone and metformin exerted a weak radiosensitization effect on A549, whereas a synergetic activity with cisplatin and docetaxel was evident in both cell lines. It can be concluded that amiodarone and metformin, being well-established drugs in clinical practice, constitute two potential drugs for further experimental and clinical evaluation as cancer cell sensitizers to chemotherapy and radiotherapy.

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

CYP17A1 and Androgen-Receptor Expression in Prostate Carcinoma Tissues and Cancer Cell Lines.

BACKGROUND: CYP17A1 is involved in the steroidogenesis of dehydroepiandrosterone and androstenedione. CYP17A is a target for the hormonal treatment of prostate cancer (PCa). OBJECTIVES: To investigate the role of CYP17A1 as a driver of PCa growth. MATERIALS AND METHODS: We examined the expression of CYP17A1 and of androgen receptors (AR) in PCa specimens and in PCa cell lines. RESULTS: CYP17A1 was strongly expressed in the cytoplasm of PCa cells (median 50% of cancer cells, range 0-100%). The nuclear AR expression in cancer cells was directly related with CYP17A1 (p < 0.0001, r = 0.51). The hormone dependent 22Rv1 cell line expressed the CYP17A1 and AR protein and mRNA, in contrast to the PC3 and DU145 cell lines (p < 0.0001). Testosterone and dexamethasone induced nuclear expression of AR and this effect was abolished by abiraterone. CYP17A1 levels were not affected by the incubation with testosterone, while abiraterone significantly reduced its expression. Abiraterone reduced the growth rate and migration of testosterone stimulated 22Rv1 cells. CONCLUSIONS: CYP17A1 is strongly expressed in half about of human prostate carcinomas, implying an intracellular androgen synthesis by cancer cells. Abiraterone effectively blocked nuclear accumulation of AR and suppressed CYP17A1 expression. CYP17A1 may function as a biomarker to select the best hormonal anticancer therapy.

Autophagic flux response and glioblastoma sensitivity to radiation.

OBJECTIVE: Glioblastoma is the most common primary brain tumor in adults and one of the most lethal human tumors. It constitutes a unique non-metastasizing human tumor model with high resistance to radiotherapy and chemotherapy. The current study investigates the association between autophagic flux and glioblastoma cell resistance. METHODS: The expression kinetics of autophagy- and lysosome-related proteins following exposure of two glioblastoma cell lines (T98 and U87) to clinically relevant radiation doses was examined. Then, the response of cells resistant to radiotherapy and chemotherapy was investigated after silencing of LC3A, LC3B, and TFEB genes in vitro and in vivo. RESULTS: Following irradiation with 4 Gy, the relatively radioresistant T98 cells exhibited enhanced autophagic flux. The more radiosensitive U87 cell line suffered a blockage of autophagic flux. Silencing of LC3A, LC3B, and TFEB genes in vitro, significantly sensitized cells to radiotherapy and temozolomide (U87: P < 0.01 and < 0.05, respectively; T98: P < 0.01 and < 0.01, respectively). Silencing of the LC3A gene sensitized mouse xenografts to radiation. CONCLUSIONS: Autophagy in cancer cells may be a key factor of radio-resistance and chemo-resistance in glioblastoma cells. Blocking autophagy may improve the efficacy of radiochemotherapy for glioblastoma patients.

Amifostine Protects Mouse Liver Against Radiation-induced Autophagy Blockage.

BACKGROUND/AIM: Amifostine is the only selective normal tissue cytoprotector, approved for the protection against platinum toxicities and radiotherapy-induced xerostomia. Free radical scavenger and DNA repair activities have been attributed to the drug. MATERIALS AND METHODS: We investigated the effect of amifostine on autophagy, lysosomal biogenesis and lipophagy of normal mouse liver exposed to clinically relevant doses of radiation. RESULTS: The study provides evidence that ionizing radiation blocks autophagy activity and lysosomal biogenesis in normal mouse liver. Amifostine, protects the liver autophagic machinery and induces lysosomal biogenesis. By suppressing autophagy, ionizing radiation induces lipid droplet accumulation, while pre-treatment with amifostine protects lipophagy and up-regulates the TIP47 protein and mRNA levels, showing a maintenance of lipid metabolism in the liver cells. CONCLUSION: It is concluded that amifostine, aside to DNA protection activity, exerts its cytoprotective function by preventing radiation-induced blockage of autophagy, lysosomal biogenesis and lipophagy.

Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity.

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.

Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis.

BACKGROUND: Disrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. METHODS: The expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. RESULTS: Overexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P < .0001, r = 0.53), cathepsin D (P = .0002, r = 0.36), light chain 3A (LC3A) (P = .02, r = 0.22), and hypoxia-inducible factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. CONCLUSION: Intense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies.

Inhibition of IKK-NFκB pathway sensitizes lung cancer cell lines to radiation.

OBJECTIVE: : Cancer cell radioresistance is a stumbling block in radiation therapy. The activity in the nuclear factor kappa B (NFκB) pathway correlates with anti-apoptotic mechanisms and increased radioresistance. The IKK complex plays a major role in NFκB activation upon numerous signals. In this study, we examined the interaction between ionizing radiation (IR) and different members of the IKK-NFκB pathway, as well as upstream activators, RAF1, ERK, and AKT1. METHODS: : The effect of 4 Gy of IR on the expression of the RAF1-ERK-IKK-NFκB pathway was examined in A549 and H1299 lung cancer cell lines using Western blot analysis and confocal microscopy. We examined changes in radiation sensitivity using gene silencing or pharmacological inhibitors of ERK and IKKß. RESULTS: : IKKα, IKKγ, and IκBα increased upon exposure to IR, thereby affecting nuclear levels of NFκB (phospho-p65). ERK inhibition or siRNA-mediated down-regulation of RAF1 suppressed the post-irradiation survival of the examined lung cancer cell lines. A similar effect was detected on survival upon silencing IKKα/IKKγ or inhibiting IKKß. CONCLUSIONS: : Exposure of lung cancer cells to IR results in NFκB activation via IKK. The genetic or pharmacological blockage of the RAF1-ERK-IKK-NFκB pathway sensitizes cells to therapeutic doses of radiation. Therefore, the IKK pathway is a promising target for therapeutic intervention in combination with radiotherapy.

Thermogenic protein UCP1 and UCP3 expression in non-small cell lung cancer: relation with glycolysis and anaerobic metabolism.

Uncoupling protein 1 (UCP1) is a proton transporter/channel residing on the inner mitochondrial membrane and is involved in cellular heat production. Using immunohistochemistry, we investigated the expression of UCP1 and UCP3 in a series of 98 patients with non-small cell lung cancer (NSCLC) treated with surgery. Expression patterns were correlated with histopathological variables, prognosis, and the expression of enzymes/proteins related to cell metabolism. Bronchial epithelium did not express UCP1 or UCP3, while alveolar cells strongly expressed UCP1. In tumors, strong expression of UCP1 and UCP3 was recorded in 43/98 (43.8%) and 27/98 (27.6%) cases, respectively. UCP1 was significantly associated with squamous cell histology (P = 0.05), whilst UCP3 was more frequently overexpressed in large cell carcinomas (P = 0.08), and was inversely related to necrosis (P = 0.009). In linear regression analysis, UCP1 was directly related to markers of glycolysis [hexokinase (HXKII) and phosphofructokinase (PFK1)] and anaerobic glucose metabolism [pyruvate dehydrogenase kinase (PDK1) and lactate dehydrogenase (LDH5)]. UCP3 was directly linked with a glucose transporter (GLUT2), monocarboxylate transporter (MCT2), glycolysis markers (PFK1 and aldolase), and with the phosphorylation of pyruvate dehydrogenase (pPDH). Kaplan-Meier survival analysis showed that UCP3 was significantly related to poor prognosis in squamous cell carcinomas (P = 0.04). UCP1 and UCP3 are overexpressed in a large subgroup of non-small cell lung tumors and their expression coincides with increased glucose absorption, intensified glycolysis, and anaerobic glucose usage. Whether UCPs are targets for therapeutic interventions in lung cancer is a hypothesis that demands further investigation.

Trachycladines and Analogues: Synthesis and Evaluation of Anticancer Activity.

The synthesis of four new analogues of marine nucleoside trachycladine A was accomplished by direct regio- and stereoselective Vorbrüggen glycosylations of 2,6-dichloropurine and 2-chloropurine with a d-ribose-derived chiron. Naturally occurring trachycladines A and B and a series of analogues were examined for their cytotoxic activity against a number of cancer cell lines (glioblastoma, lung, and cervical cancer). Parent trachycladine A and two analogues (the diacetate of the 2,6-dichloropurine derivative and N-cyclopropyl trachycladine A) resulted in a significant decrease in cell viability, with the latter exhibiting a stronger effect. The same compounds enhanced the cytotoxic effect of docetaxel in lung cancer cell lines, whereas additional experiments revealed that their mode of action relies on mitotic catastrophe rather than DNA damage. Moreover, their activity as autophagic flux blockers was postulated.

Hypoxia-inducible proteins HIF1α and lactate dehydrogenase LDH5, key markers of anaerobic metabolism, relate with stem cell markers and poor post-radiotherapy outcome in bladder cancer.

PURPOSE: To assess whether anaerobic metabolism, proliferation activity and stem cell content are linked with radioresistance in bladder cancer. MATERIALS AND METHODS: Tissue sections from 66 patients with invasive transitional cell bladder cancer treated with hypofractionated accelerated radiotherapy, was immunohistochemically analyzed for the Hypoxia-Inducible Factor 1α (HIF1α) and the anaerobic glycolysis enzyme lactate dehydrogenase 5 (LDH5). Proliferation index (Ki-67) and stem-cell marker (cluster of differentiation CD44, aldehyde dehydrogenase ALDH1) expression was also examined. RESULTS: Both HIF1α and LDH5 expression were linked with high CD44 stem cell population (p = 0.001 and 0.05, respectively), while high Ki-67 proliferation index was linked with nuclear LDH5 expression (p = 0.03) and high histological grade (p = 0.02). A strong significant association of HIF1α (p = 0.0009) and of LDH5 (p < 0.0001) with poor local relapse free survival (LRFS) was noted, which was also confirmed in multivariate analysis. A significant association with overall survival was also noted. Silencing of lactate dehydrogenase LDHA gene in the human RT112 bladder cancer cell line, or exposure to oxamate (LDH activity inhibitor), resulted in strong radio-sensitization. CONCLUSIONS: HIF1α and LDH5 are markers of poor outcome in patients with bladder cancer treated with radiotherapy. Blockage of anaerobic metabolism may prove of importance in clinical radiotherapy.

Normal tissue radioprotection by amifostine via Warburg-type effects.

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.

Fever-range hyperthermia vs. hypothermia effect on cancer cell viability, proliferation and HSP90 expression.

PURPOSE: The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression. MATERIALS AND METHODS: A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy. RESULTS: Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines. CONCLUSIONS: The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.