The role of metabolic ecosystem in cancer progression - metabolic plasticity and mTOR hyperactivity in tumor tissues.

Despite advancements in cancer management, tumor relapse and metastasis are associated with poor outcomes in many cancers. Over the past decade, oncogene-driven carcinogenesis, dysregulated cellular signaling networks, dynamic changes in the tissue microenvironment, epithelial-mesenchymal transitions, protein expression within regulatory pathways, and their part in tumor progression are described in several studies. However, the complexity of metabolic enzyme expression is considerably under evaluated. Alterations in cellular metabolism determine the individual phenotype and behavior of cells, which is a well-recognized hallmark of cancer progression, especially in the adaptation mechanisms underlying therapy resistance. In metabolic symbiosis, cells compete, communicate, and even feed each other, supervised by tumor cells. Metabolic reprogramming forms a unique fingerprint for each tumor tissue, depending on the cellular content and genetic, epigenetic, and microenvironmental alterations of the developing cancer. Based on its sensing and effector functions, the mechanistic target of rapamycin (mTOR) kinase is considered the master regulator of metabolic adaptation. Moreover, mTOR kinase hyperactivity is associated with poor prognosis in various tumor types. In situ metabolic phenotyping in recent studies highlights the importance of metabolic plasticity, mTOR hyperactivity, and their role in tumor progression. In this review, we update recent developments in metabolic phenotyping of the cancer ecosystem, metabolic symbiosis, and plasticity which could provide new research directions in tumor biology. In addition, we suggest pathomorphological and analytical studies relating to metabolic alterations, mTOR activity, and their associations which are necessary to improve understanding of tumor heterogeneity and expand the therapeutic management of cancer.

In Situ Metabolic Characterisation of Breast Cancer and Its Potential Impact on Therapy.

In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases-accumulating genomic alterations and further heterogeneity-are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies.

Inhibition of Metabolic Shift can Decrease Therapy Resistance in Human High-Grade Glioma Cells.

The high-grade brain malignancy, glioblastoma multiforme (GBM), is one of the most aggressive tumours in central nervous system. The developing resistance against recent therapies and the recurrence rate of GBMs are extremely high. In spite several new ongoing trials, GBM therapies could not significantly increase the survival rate of the patients as significantly. The presence of inter- and intra-tumoral heterogeneity of GBMs arise the problem to find both the pre-existing potential resistant clones and the cellular processes which promote the adaptation mechanisms such as multidrug resistance, stem cell-ness or metabolic alterations, etc. In our work, the in situ metabolic heterogeneity of high-grade human glioblastoma cases were analysed by immunohistochemistry using tissue-microarray. The potential importance of the detected metabolic heterogeneity was tested in three glioma cell lines (grade III-IV) using protein expression analyses (Western blot and WES Simple) and therapeutic drug (temozolomide), metabolic inhibitor treatments (including glutaminase inhibitor) to compare the effects of rapamycin (RAPA) and glutaminase inhibitor combinations in vitro (Alamar Blue and SRB tests). The importance of individual differences and metabolic alterations were observed in mono-therapeutic failures, especially the enhanced Rictor expressions after different mono-treatments in correlation to lower sensitivity (temozolomide, doxycycline, etomoxir, BPTES). RAPA combinations with other metabolic inhibitors were the best strategies except for RAPA+glutaminase inhibitor. These observations underline the importance of multi-targeting metabolic pathways. Finally, our data suggest that the detected metabolic heterogeneity (the high mTORC2 complex activity, enhanced expression of Rictor, p-Akt, p-S6, CPT1A, and LDHA enzymes in glioma cases) and the microenvironmental or treatment induced metabolic shift can be potential targets in combination therapy. Therefore, it should be considered to map tissue heterogeneity and alterations with several cellular metabolism markers in biopsy materials after applying recently available or new treatments.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells.

BACKGROUND: Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. METHODS: In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. RESULTS: Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients' materials. CONCLUSIONS: Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.

GABA, glutamine, glutamate oxidation and succinic semialdehyde dehydrogenase expression in human gliomas.

BACKGROUND: Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions. METHODS: Metabolic characteristics of human glioma cell models - including mitochondrial function, glycolytic pathway and energy substrate oxidation - in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry. RESULTS: U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation). CONCLUSIONS: Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas.

In situ analysis of mTORC1/2 and cellular metabolism-related proteins in human Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease with features of a low-grade neoplasm. It is primarily caused by mutations in TSC1 or TSC2 genes. Sirolimus, an inhibitor of mTOR complex 1 (mTORC1), slows down disease progression in some, but not all patients. Hitherto, other potential therapeutic targets such as mTOR complex 2 (mTORC2) and various metabolic pathways have not been investigated in human LAM tissues. The aim of this study was to assess activities of mTORC1, mTORC2 and various metabolic pathways in human LAM tissues through analysis of protein expression. Immunohistochemical analysis of p-S6 (mTORC1 downstream protein), Rictor (mTORC2 scaffold protein) as well as GLUT1, GAPDH, ATPB, GLS, MCT1, ACSS2 and CPT1A (metabolic pathway markers) were performed on lung tissue from 11 patients with sporadic LAM. Immunoreactivity was assessed in LAM cells with bronchial smooth muscle cells as controls. Expression of p-S6, Rictor, GAPDH, GLS, MCT1, ACSS2 and CPT1A was significantly higher in LAM cells than in bronchial smooth muscle cells (P<.01). No significant differences were found between LAM cells and normal bronchial smooth muscle cells in GLUT1 and ATPB expression. The results are uniquely derived from human tissue and indicate that, in addition to mTORC1, mTORC2 may also play an important role in the pathobiology of LAM. Furthermore, glutaminolysis, acetate utilization and fatty acid ß-oxidation appear to be the preferred bioenergetic pathways in LAM cells. mTORC2 and these preferred bioenergetic pathways appear worthy of further study as they may represent possible therapeutic targets in the treatment of LAM.

[Anticancer drug research in Hungary, 1950-2000]. / Daganat-kemoterápiás kutatások Magyarországon az 1950−2000 közötti években.

The present review about the history of anticancer drug research in Hungary intends to call attention to the importance of studies on their mode of action. Several lines of evidence suggest that clinically usable oncopharmacological properties could be revealed by this way. Among the numerous compounds certain alkylating sugar alcohols and 2'-deoxyuridine derivatives were submitted to detailed investigations concerning their mode of action. Myelobromol with selective action on the myeloid elements of bone marrow has been justified for its application in chronic myeloid leukemia therapy and also in bone marrow ablation before transplantation. Mitolactol is able to cross bloodbrain barrier, consequently could control certain brain tumors. 5-etil-2'-deoxyuridine by reducing dihydropyrimidine dehydrogenase activity is able to increase 5-fluorouracil concentration in the blood, resulting in improved antitumor effect. In contrast, 5-hexil-2'-deoxyuridine, as an inhibitor of glycoconjugate pathway by reducing heparan sulfate production, has the ability to prevent metastasis. Noteworthy, the remarkable effects of vinca alkaloids, antiestrogens, and GNRH analogues were also presented in this review.

Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells.

BACKGROUND: Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming. METHODS: HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well. RESULTS: Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13C atoms from 13C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo. CONCLUSIONS: Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of tumours to increase the success of therapies.

[Measuring 14C-glucose and 14C-acetate oxidation in tumour cells and tumorous host organism]. / A bioenergetikai profil vizsgálata 14C-glükóz és 14C-acetát oxidációjának összehasonlításával tumorsejtekben és tumoros szervezetben.

Tumour cell metabolism can be influenced by alterations of the extracellular microenvironment and the tumour-promoting genetically changed mechanisms. There is increasing interest to introduce appropriate bioenergetic assays to describe the therapeutic effect and metabolic subtypes of tumours in clinical oncology. The analysis of 14C-glucose and 14C-acetate oxidation could be a suitable method to examine the metabolic/bioenergetic profiles of tumour cells and tumorous host organisms. The metabolic activity of tumour cells (in vitro cell lines, primary human lymphocytes and leukaemia cells) and the tumourous host organism were examined in vitro and in vivo by detecting the released CO2 levels derived from the radioactive carbon atom labelled energy substrates. We have found that the most cancer cells of solid tumours oxidised glucose more intensively than acetate. It was interesting that AML, CML and CLL cells isolated from blood preferred acetate as an energy substrate in vitro. Furthermore, based on our observations, tumours affected the glucose or acetate oxidation of the organism when applying bioenergetic substrates per os or iv. We provided the first data about the alterations in metabolic profiles of the tumour bearing organism in xenograft models. In summary, according to our results, comparison of the energy substrate oxidation can be an indicative method related to the metabolic profile analysis of tumour cells in vitro and tumorous host organism in vivo.

Extracellular matrix as target for antitumor therapy.

The aim of the present review is to survey the accumulated knowledge on the extracellular matrix (ECM) of tumors referring to its putative utility as therapeutic target. Following the traditional observation on the extensive morphological alteration in the tumor-affected tissue, the well-documented aberrant cellular regulation indicated that ECM components have an active role in tumor progression. However, due to the diverse functions and variable expression of proteoglycans, matrix proteins, and integrins, it is rather difficult to identify a comprehensive therapeutic target among ECM components. At present, the elevated level of heparanase and the prominent expression of αvß5 integrin are considered as promising therapeutic targets. The inhibition of glycosaminoglycan offers another promising approach in the treatment of those tumors which are stimulated by proteoglycans. It can be ascertained that a selective ECM inhibitor would be a great asset to control metastasis driven by ECM-mediated signaling.

Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism.

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10 min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1-100 µM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78 µM for the analytes concerned. To test the PGC-HPLC-MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-(13)C glucose or 1-(13)C acetate. Applying the MRM mode, the incorporation of (13)C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.

[History of the therapy of pernicious anemia]. / Az anaemia perniciosa terápiájának története.

Increased blood cell regeneration in exsanguinated experimental animals treated either with liver or with aqueous liver extracts was reported by Whipple and by Jeney and Jobling, respectively. These findings stimulated Minot and Murphy to provide evidence for the efficacy of liver against anaemia in clinical studies. After oral administration of liver (45-50 g per day) for 45 patients with anaemia perniciosa improvement of the hematological status was demonstrated. Consequently, for proving the therapeutic value of liver therapy Whipple, Minot and Murphy received Nobel price in 1934. The isolation of the antianemic factor from the liver has been succeeded in 1948 and designated as vitamin B12. At the same time Lucy Wills applied yeast for the treatment of pregnant women with anemia related to undernourishment. The conclusions of this study inspired the discovery of folate. The detailed investigation of the mode of action of vitamin B12 and folate enriched our knowledge in the area of pathophysiology and extended the clinical application of these two drugs.

Syndecan-1 enhances proliferation, migration and metastasis of HT-1080 cells in cooperation with syndecan-2.

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

Enhanced 5-fluorouracil cytotoxicity in high cyclooxygenase-2 expressing colorectal cancer cells and xenografts induced by non-steroidal anti-inflammatory drugs via downregulation of dihydropyrimidine dehydrogenase.

PURPOSE: To prove that 5-FU cytotoxicity could be increased by combination with low-dose non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin or NS-398) in high cyclooxygenase-2- (COX-2) expressing cells and xenografts through the modulation of dihydropyrimidine dehydrogenase (DPD) mRNA expression and/or enzyme activity. METHODS: HT-29 cells were grown on collagen IV coated plates (HT-29-C). The antiproliferative effect of 5-fluorouracil (5-FU) +/- NSAIDs was examined on non-COX-2 expressing HT-29 and COX-2-expressing HT-29-C cells by sulphorhodamine B assay. The COX-2 and DPD expressions were visualized by immunofluorescent staining, and prostaglandin E(2) levels were measured by ELISA kit. The HT-29 xenograft was established in SCID mice and treated with 5-FU +/- NSAIDs for 5 days. The tumor volume, enzyme activity, and DPD mRNA expression were investigated by caliper, radioenzymatic method, and real-time RT-PCR, respectively. The drug interaction was calculated for both combinations (5-FU + indomethacin and 5-FU + NS-398). RESULTS: Collagen IV up-regulated significantly the COX-2 and DPD mRNA, and protein expressions, and also their enzyme activities in HT-29 cells. NSAIDs enhanced in a synergistic manner the cytotoxic effect of 5-FU treatment both in vitro and in vivo. Downregulation of DPD was observed after 5-FU monotherapy, but the combined effect of NSAIDs and 5-FU on DPD mRNA expression, and enzyme activity was superior to the effect of 5-FU alone. CONCLUSIONS: Since 5-FU + NSAID treatment can alter the DPD enzyme activity resulting in an enhanced cytotoxic effect, further studies in clinical practice are warranted.

Differential inhibition of single and cluster type tumor cell migration.

For the control of tumor metastasis it is important to identify chemical compounds with antimigratory potency. Agents acting against single cell and cluster type migration are necessary for successful antimetastatic therapy. In the present study, the migration of HT-1080 fibrosarcoma cells and OSCORT osteosarcoma cells was compared in a Boyden chamber and in an extracellular matrix (ECM)-based three-dimensional cell culture (3-DCC) model system. The Boyden chamber offers a model of single tumor cell migration, whereas the 3-DCC model system demonstrates invasive growth in the form of a cluster. Since PD98059 (MEK inhibitor) exclusively reduced migration in the 3-DCC model, it may be plausible that the ERK/MAPK signaling pathway is essential for cluster type migration. Interestingly, single cell migration was stimulated upon blocking phosphatidylinositol 3-kinase (PI3K) and also p38-MAPK by treatment with LY294002 and SB203580 respectively. A remarkable reduction of single cell migration was observed following treatment with okadaic acid, a phosphatase 1 (PP1) and 2A (PP2A) inhibitor, which was rather intriguing. This study provided evidence that certain cytotoxic/cytostatic agents at appropriate concentrations were able to preferentially inhibit certain types of migration relative to cell proliferation. Single cell migration was selectively inhibited by taxol at very low subtoxic concentration, whereas 5-hexyl-2'-deoxyuridine (HUdR) exclusively inhibited the cluster type of migration. The borrelidin compound was able to inhibit both types of tumor cell migration, but single tumor cell migration was much less affected. It is interesting that migration was more reduced than proliferation by borrelidin, especially at the advanced growth stage. Taxol is recommended as an agent acting against single cell migration, as well as HUdR and borrelidin as leading compounds for developing antimetastatic drugs against cluster type migration.

Co-inhibition of cyclooxygenase-2 and dihydropyrimidine dehydrogenase by non-steroidal anti-inflammatory drugs in tumor cells and xenografts.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) may be able to enhance the antitumor effect of cancer drugs. Cyclooxygenase-2 (COX-2) is the best characterized target of NSAIDs. It was demonstrated that elevated dihydropyrimidine dehydrogenase (DPD) and COX-2 activities influence the response to 5-fluorouracil (5-FU). We previously showed that NSAIDs increased 5-FU sensitivity only in high COX-2-expressing cancer cells. MATERIALS AND METHODS: The effect of indomethacin and NS-398 on DPD activity and mRNA expression in a high COX-2-expressing (determined by Western blotting, immunoflourescence and immunohistochemistry) cell line (24-, 48-hour, 10-day treatment) and xenograft (3-week treatment) was investigated. RESULTS: The coexistence of high COX-2 and DPD activity or low activities of both enzymes were detected. After treatment with NSAIDs, a simultaneous and significant decrease of both activities was also demonstrated. CONCLUSION: NSAIDs could be promising modulators of fluorouracil-based chemotherapy, especially in high COX-2-expressing tumours.

Effect of somatostatin analogue octreotide in medulloblastoma in xenograft and cell culture study.

BACKGROUND: The effect and possible timing of nonradiolabeled somatostatin analogue octreotide are still not determined in the treatment of medulloblastoma, while the presence of somatostatin receptor type-2 (SSTR2) is proved in the majority of medulloblastoma by several authors. PROCEDURES: Daoy, SSTR2A positive medulloblastoma cell culture was tested with octreotide in monotherapy and combined with cisplatin, etoposide, and vincristine. Daoy medulloblastoma mice xenograft was treated with octreotide alone. RESULTS: In monolayer cell culture high-dose octreotide (44 microM) resulted in mitotic inhibition with parallel increment of apoptosis. Combination with cytostatic drugs did not result in additive or synergistic effect, but vincristine was partially antagonized. In medulloblastoma xenograft, octreotide monotherapy (100 microg/kg/day for 10 days) resulted in partial tumor growth inhibition. CONCLUSIONS: High concentration of nonradiolabeled octreotide may have role in the treatment of medulloblastoma by long-term administration. Concomitant administration of octreotide with widely used cytostatic drugs against medulloblastoma will not have beneficial impact.

Enhancement of 5-fluorouracil efficacy on high COX-2 expressing HCA-7 cells by low dose indomethacin and NS-398 but not on low COX-2 expressing HT-29 cells.

The antiproliferative effect of 5-fluorouracil (5-FU) in the presence of low dose non-steroidal anti-inflammatory drugs (NSAIDs) on high cyclooxygenase-2 (COX-2)-expressing HCA-7 and low COX-2-expressing HT-29 colon carcinoma cell lines was investigated. Pharmacogenetic parameters were studied to characterize the 5-FU sensitivity of the two cell lines. Thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms were determined by PCR analysis. Cell proliferation was measured by SRB assay, cell cycle distribution and apoptosis by FACS analysis. Cyclooxygenase expression was detected by Western blot and also by fluorescence microscopy. Prostaglandin E(2) (PGE(2)) levels were investigated with ELISA kit. The HT-29 cell line was found to be homozygous for TS 2R and 1494ins6 and T homozygous for MTHFR 677 polymorphisms predicting high 5-FU sensitivity (IC(50): 10 microM). TS 3R homozygosity, TS 1496del6 and MTHFR 677CT heterozygosity may explain the modest 5-FU sensitivity (IC(50): 1.1 mM) of the HCA-7 cell line. Indomethacin and NS-398 (10 microM and 1.77 microM, respectively) reduced the PGE(2) level in HCA-7 cells (>90%). Low concentrations of NSAIDs without antiproliferative potency increased the S-phase arrest and enhanced the cytotoxic action of 5-FU only in HCA-7 cells after 48-hours treatment. The presented data suggested that the enhancement of 5-FU cytotoxicity by indomethacin or NS-398 applied in low dose is related to the potency of NSAIDs to modulate the cell-cycle distribution and the apoptosis; however, it seems that this effect might be dependent on cell phenotype, namely on the COX-2 expression.

Extracellular matrix induces doxorubicin-resistance in human osteosarcoma cells by suppression of p53 function.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumor in childhood and adolescence. The several chemotherapy-resistant cases of osteosarcoma are at a higher risk of relapse and adverse outcome. OBJECTIVE: The aim of the current study was to determine the role of extracellular matrix in the resistance developed against chemotherapeutic treatments of human osteosarcoma cells. MATERIALS AND METHODS: A cell line, named OSCORT was established from the biopsy of a 17-year-old male patient with primary osteosarcoma. Cell proliferation, apoptosis and quantification of DNA damage after treatments with doxorubicin were investigated in classical and three-dimensional cell culture systems using an extracellular matrix gel. The experimental results were related to the clinical observations of the case. RESULTS: The cells cultured in extracellular matrix gel have shown resistance to doxorubicin similar to that seen in the clinical case, as demonstrated by their proliferation, apoptosis and doxorubicin-induced DNA damage characteristics. Among the extracellular matrix components, the heparan sulfate proteoglycan and-to a lesser extent-fibronectin were involved in the doxorubicin resistance. Laminin and nidogen did not decrease the cytoreductive effect of doxorubicin, while collagen IV even increased it. The extracellular matrix gel decreased the protein levels of p53 and abrogated its cell nuclear translocalization. The most frequent known mutations in the p53 gene were not found in OSCORT cells. CONCLUSION: The current study provides experimental evidence for an epigenetical, extracellular matrix-induced loss of p53 function, which lead to a potent chemotherapy resistance showing accordance with the clinical experience.

[Study of drugs against neoplastic metastasis]. / A tumorok áttétképzését korlátozó hatóanyagok kutatása.

Further progress in the therapy of malignant diseases is expected from the introduction of potent antimetastatic drugs. Surveying of the complex and multi-step behavior of the metastatic process, compounds showing inhibitory action against tumor cell migration may be ranked among the promising antimetastatic agents. Our present study indicate, however, that the antimigratory actions of certain antitumor drugs (doxorubicin, taxol), and inhibitors of signal transduction (PD-98059, LY-294002, SB-203580) are highly dependent on the assay applied (Boyden-chamber, 3D ECM cell culture). It has been proposed that agents interrupting cell-extracellular matrix contacts (hexyldeoxyuridine, borrelidin) and others interfering with the regulatory mechanism of gene translation (rapamycin, ribavirin) could be regarded as leading compounds in the antimetastatic drug development process. Nevertheless, for introducing diagnostically based targeted therapy the forthcoming tasks must include the further elucidation of the molecular mechanisms implicated in the amoeboid and cluster type of cell migration.