Modulation of Carbonic Anhydrase 9 (CA9) in Human ...

Current Pharmaceutical Design, 2010, 16, 000-000

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Modulation of Carbonic Anhydrase 9 (CA9) in Human Brain Cancer Harun M. Said1, Claudiu T. Supuran2, Carsten Hageman3, Adrian Staab1,4, Buelent Polat1, Astrid Katzer1, Andrea Scozzafava2, Jelena Anacker5, Michael Flentje1 and Dirk Vordermark1,6 1

Dept of Radiation Oncology, Medical Faculty, University of Wuerzburg, Germany, 2Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Università di Firenze188,Via Lastruccia 3, I-50019, Firenze, Italy, 3Dept of Neurosurgery, Medical Faculty, University of Wuerzburg, 4Paul Scherer Institute, Villingen, Switzerland, 5Gynaecology and Obstetrics, University of Wurzburg, 6 Dept of Radiation Oncology, Medical Faculty, University of Halle Wittenberg, Germany Abstract: Hypoxia is a crucial factor in tumour aggressiveness and its treatment resistance, particularly in human brain cancer. Tumour resistance against radiation- and chemotherapy is facilitated by oxygenation reduction at tumour areas. HIF-1 regulated genes are mostly responsible for this type of resistance. Among these genes, carbonic anhydrase isoform 9 (CA9) is highly overexpressed in many types of cancer especially in high grade brain cancer like GBM. CA IX contributes to tumour environment acidification by catalyzing the carbon dioxide hydration to bicarbonate and protons, leading to the acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs and therefore to inadequate application of radio-therapeutic or chemotherapeutic anti-cancer treatment strategies. Inhibition of this enzymatic activity by application of specific chemical CA9 inhibitors (sulphonamide derivative compounds) or indirect inhibitors like HIF-1 inhibitors (chetomin) or molecular inhibitors like CA9-siRNA leads to reversion of these processes, leading to the CA9 functional role inhibition during tumourigenesis. Hypoxia significantly influences the tumour microenvironment behaviour via activation of genes involved in the adaptation to the hypoxic stress. It also represents an important cancer prognosis indicator and is associated with aggressive growth, malignant progression, metastasis and poor treatment response. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Sulfonamide derivative compounds with CA9 inhibitory characteristics represent one of the optimal treatment options beside other CA9 inhibitory agents or chemical inhibitory compounds against its main regulating transcription factor which is the hypoxia induced HIF-1 when applied against human cancers with hypoxic regions like GBM, bearing potential for an effective role in human brain tumour therapeutic strategies. Glycolytic inhibitors, when added in controlled doses under hypoxia, lead to a reduced accumulation of HIF-1 and can function as indirect hypoxia regulated genes inhibitors like CA9. These may be used as alternative or in conjunction with other direct inhibitors like the sulphonamide derivate compounds, chetomin or specific siRNAs, or other different chemical compounds possessing similar functionality making them as optimal tools for optimized therapy development in cancer treatment, especially against human brain cancer. Further experimental analysis towards the tumour stage specific inhibitory CA9 characteristics determination are necessary to find the optimal therapeutic solutions among the different available modalities; whether they are direct or indirect chemical, molecular or natural inhibitors to be able to set up successful treatment approaches against the different human tumour diseases.

Keywords: Brain Tumour, GBM, LGA, HIF-1, CA9, Tumour Therapy, Glycolysis, Sulfonamide derivatives, CA inhibitors (CAI). INTRODUCTION Hypoxia plays an important role within the solid tumour microenvironment and significantly influences the tumour cells behaviour via activation of genes encoding proteins involved in adaptation to hypoxic stress. Also, it plays an important role in tumour progression and it is selective for cells with enhanced glycolytic activity, causing production of large amounts of lactic acid, one of the most common features of tumour cells (Warburg effect) which has attracted much attention owing to its significant correlation with tumour progression, treatment results and the overall disease prognosis [1, 2]. Tumour tissue growth requires a sufficient O2 and nutrients supply. However, proliferating tumour cells quickly grow beyond the O2 diffusion distance from the nearest blood vessel (100150 μm), leading to a highly irregular and tortuous tumour vasculature containing arteriovenous shunts, blind ends and incomplete endothelial linings. This has an effect on blood flow, which is less efficient than in normal tissues [3, 4]. Tumour expansion is characterized by rapid growth of cancer cells when tumours establish themselves in organs host tissues. Rapid tumours growth is accompanied by cancer cell microenvironment alterations caused by the inability of local vasculature to supply enough O2 and nutrients to the rapidly dividing tumour cells [5], making hypoxia one common feature of solid tumours [6]. *Address correspondence to these authors at the Department of Radiation, Oncology, University of Wurzburg, Josef-Schneider Str. 11, D-97080 Wurzburg, Germany; Tel: ?????????; Fax: ?????????; E-mail: [email protected]

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GBM are highly invasive brain tumours [7]. Transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of tumour cell adaptation and survival under hypoxic oxygenation conditions [8]. It regulates the expression of several genes related to O2 homeostasis in response to hypoxic stress [9]. Human GBM cells vary in their ability to survive under hypoxic conditions. Tumour hypoxia is a complex entity which is a function of O2 supply and demand. A shift towards anaerobic metabolism would decrease O 2 consumption rates leading to the improvement in tumour oxygenation [10, 11]. Under O2-limiting conditions, hypoxia-tolerant cells decrease their O2 consumption rate, whereas, hypoxia-sensitive cells continue to consume O2 at a relatively steady rate until the O 2 supply becomes exhausted [12, 13]. Tumour hypoxia is associated with adverse outcome in many malignancies. Tumour oxygenation is directly measured using microelectrodes or RuO2 microprobes, however, these techniques possess an invasive nature, restricting their use to accessible tumours [14]. Additional diagnostic tumour markers whose expression is induced by the tumour hypoxic microenvironment are necessary for definite advanced tumour characterisation according to its oxygenation status. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that consists of two subunits, HIF-1 and HIF-1 that are both bHLH proteins containing a PAS domain [15]. In normoxia, the von Hippel- Lindau tumour suppressor, which is the E3 ubiquitin ligase complex recognition component, targets HIF-1 [16, 17] leading to its ubiquitylation and consequent proteasomal degradation [16-21]. As a multi-subunit protein it regulates transcription at hypoxia response elements (HREs) containing a hypoxia binding sequence © 2010 Bentham Science Publishers Ltd.

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(HBS) and is composed of two basic helix-loop-helix proteins: a subunit, HIF-1, and the constitutively expressed HIF-1 subunits that is a non- O2 responsive nuclear protein playing various roles in transcription. Due to HRE positioning in the distal promoter region, HIF-1 also acts as a transcriptional enhancer. During hypoxia, the HIF-1/ heterodimer binds to the core pentanucleotide HBS (RCGTG) in the HREs of the target genes. [15, 22-26]. Among these genes, CA9 is one of the most strongly hypoxia-inducible [27]. CA IX protein is a transmembrane N-glycosylated isoenzyme localized at the cell surface in the form of dimers composed of monomeric subunits of 58/54 kDa [26, 28, and 29]. The -carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes in higher vertebrates including humans [29]. Hypoxia, via the HIF-1 cascade, leads to strong overexpression of CA IX in many tumours with the overall consequence that the imbalance in pH in the tissue is increased. Indeed, most hypoxic tumours are acidic (pH6), in contrast to normal tissues (pH7.4). The role of CA IX in the acidification processes of hypoxic tumours has been demonstrated by the research work of the Pastorekova Group [30, 31]. Numerous potent (and sometimes selective) CA IX inhibitors have been developed in the past few years on the basis of the idea that tumour acidification processes inhibition and re-establishment of a more normal pH might lead to regression of the tumour, especially when used in combination with classical anticancer drugs. Results confirmed this hypothesis establishing CA IX as a novel drug target for the development of both diagnostic tools and therapeutic agents [32-34]. CA IX is frequently expressed in different tumour types and absent from their normal counterparts including bladder, kidney, breast, lung, head and neck, and cervix uteri tumours [29, 31] and slightly expressed in human brain [35-37]. Since HIF-1 subunits are highly inducible by different oxygenation conditions in human GBM cells, HIF-1 acts as a master regulator of numerous hypoxia inducible genes related to angiogenesis, cell proliferation/ survival, and glucose/iron metabolism including CA9. Hypoxia induced CA IX expression is usually concentrated in the perinecrotic tumour regions in GBM [38] and is associated with bad anti cancer therapeutic applications outcome especially chemotherapeutic and radiotherapeutic treatment modalities. HUMAN BRAIN TUMOUR CELLS METABOLISM Tumour cell proliferation requires rapid macromolecules synthesis including lipids, proteins, and nucleotides. Many tumour cells exhibit rapid glucose consumption, with most of the glucosederived carbon being secreted as lactate despite abundant O2 availability (the Warburg effect) [39]. Ordered pattern of metabolic and morphologic changes occurs during neoplastic cell transformation. Apparently, an increase in the flux of certain metabolic pathways such as the hexose-monophosphate shunt and glycolysis develops during transformation of many cell types. This metabolic aberration is conventionally explained as a consequence of a higher metabolic requirement [40]. Gliomas represent 50% of primary brain tumours, and their prognosis remains poor despite the advances in diagnosis and therapeutic strategies [41]. Positron emission tomography is used to study abnormalities in the glucose oxidative metabolism in human cerebral gliomas. Tumour regional cerebral glucose consumption was not depressed and regional glucose extraction ratios were similar for tumour and brain tissue, [42, and 43]. In some other experimental analysis, it was observed in human xenografted gliomas that Lactate / pyruvate ratios increased 3-4 fold and HK activity was of 2-4 fold lower than that of normal rat brain tissue, used as the control. The mitochondria-bound HK (mHK) fraction varied considerably and represented 9 to 69% of the total HK of that normal rat brain [44]. In another experimental series Lactate to pyruvate ratio was >1, suggesting that energy metabolism in LGG is glycolytic in nature, particularly in the tumours centre. Peripheral tumours samples showed increased glucose consumption and cytochrome c-oxidase activity [41]. HK binds to a mitochondrial porin involving peripheral benzodiazepine receptors. HK and peripheral

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benzodiazepine receptors inhibition by lonidamine and diazepam led to synergistic anti tumoural activity in xenografted gliomas. These two receptors co-inhibition will lead to a decrease in glycolysis, often elevated in these tumours, without modifying energetic normal cells metabolism [45]. POTENTIAL ROLE OF GLYCOLYTIC INHIBITORS IN HUMAN BRAIN CANCER TREATMENT Glycolysis is the transformation of glucose to pyruvate which is a key step of ATP acquisition in all mammalian cells, including cancer tissues. Glucose transporters are commonly overexpressed in human malignancies enhancing glucose influx in the proliferating cancer cells [46]. Glycolytic inhibitors are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with cellular resistance to conventional anticancer drugs and radiation therapy [47]. The energy metabolism process is considered for brain cancer treatment through metabolic targeting in the normal orthotopic tissue. Glucose transporter, GLUT-1, is enriched in the brain capillary endothelial cells and mediates facilitated glucose diffusion through the blood brain barrier. Glucose is metabolized mostly, to pyruvate, which enters neurons and glia mitochondria and is converted to acetyl-CoA before entering the TCA cycle. In well-oxygenated normal cells, pyruvate enters the mitochondria and by the enzymic activity of pyruvate dehydrogenase, it is transformed to acetyl-Co A, the ATP production substrate through the Krebs cycle [48]. Under normal conditions, 13% of glycolytic pyruvate is converted to lactate [49]. Because intracellular acidosis triggers apoptosis blocking, increased glycolytic activity by down regulating HIF-1 may reduce apoptosis of the hypoxic cells [50]. Within this context, experimental results showed that iodide acetate minimized or inhibited both HIF-1 and CA IX protein and mRNA expression in GBM cells under long term in vitro hypoxia experimental cell oxygenation conditions [35-37] as well as in other tumour cells of different origin [51]. It has previously shown that CA IX may be a therapeutic target for cancer, since, inhibition of carbonic anhydrase isoenzymes with bacteriostatic or non-bacteriostatic sulfonamides e.g. acetazolamide results in either reduced tumour invasiveness or blocked tumour growth [34, 52]. Furthermore, CA isoenzyme antagonism has been observed to augment the cytotoxic effects of various chemotherapeutic agents, including platinum- based drugs. There is a clear glucose concentration involvement in the HIF1 and CA IX expression regulation, since glycolsis inhibitor iodide acetate application result in a minimized expression of both of them in different GBM cell lines. Accumulation of HIF-1 depends on Glucose concentration, therefore, glycolysis inhibitors, when added under hypoxia, lead to a reduced HIF-1 accumulation. It has also been shown that inhibition of mitochondrial respiration leads to the HIF-1 stabilization inhibition at low O2 concentrations [53]. Interference in the glycolysis path of GBM by IAA can represent a therapeutic alternative in CA IX involved therapeutic approaches and potentially other HIF-1 regulated hypoxia induced gene. On the other hand we can use CA IX status for diagnostic purposes to potentially aid in the selection of patients who might benefit from CAIX-targeted therapies [54]. CA9 can represent an optimal target for therapeutic applications in hypoxia related GBM tumours. In GBM inhibition of HIF-1 regulated hypoxia induced genes like CA9 is accomplished via the functional interference into the tumour cell glycolysis pathway via IAA and chetomin. Cancer cell energy metabolism deviates from that of normal tissues by maintaining high glycolytic rates which has also been associated with disease progression in several tumour entities [55-57]. We and others have shown that the hypoxic accumulation of HIF-1 occurs in a celltype-specific manner [35, 51] and is strongly dependent on glucose availability [58- 60]. Glucose metabolism modulation investigation using the glycolysis inhibitors iodoacetate (IAA) or 2-deoxyglucose (2-DG), affects the hypoxia influenced HIF-1 accumulation. This hypoxic HIF-1 accumulation depends strongly on the glucose

Modulation of Carbonic Anhydrase 9 (CA9) in Human Brain Cancer

availability [58, 60] beside the strong evidence shown that HIF-1 regulates glucose metabolism and maintenance of tumour growth [61, 62]. One explanation of this effect is the increased O2 availability for prolyl hydroxylation of HIF-1 when mitochondrial O2 consumption is reduced such that hypoxia is not recognized by prolyl hydroxylases [63]. Glycolysis inhibitors, when added under hypoxia, lead to a reduced accumulation of HIF-1. The regulation of HIF-1 by inhibition glycolysis is independent of the activation by prolyl hydroxylases in HT1080 cells. Furthermore, pyruvate was not able to increase hypoxic HIF-1 levels when glycolytic inhibitors were added to HT1080 cells, suggesting that the lack of pyruvate is not the reason for the reduced accumulation of HIF-1 under hypoxic conditions when glycolysis was inhibited. In contrast to previous reports, no evidence was found for a key role of pyruvate as a glycolytic metabolite promoting HIF-1 accumulation [64]. To further investigate the mechanism by which the glucose availability or glucose metabolites interact with HIF-1, we performed realtime RT-PCR to quantify the mRNA expression of the HIF-1 gene in cells under normoxia or hypoxia treated with IAA or 2-DG. We did not observe any significant changes in HIF-1 gene expression profiles after incubation of HT1080 cells with IAA or 2-DG. These findings corresponds to previous reports showing that hypoxia inhibits mRNA translation by suppressing multiple key regulators [65, 66] and limited nutrient availability can lead to drastically reduced protein synthesis [67]. We therefore presume that glucose levels affect the expression of HIF-1 on a translational level or by phosphorylation instead of transcriptional regulation. A likely explanation for the reduced hypoxic HIF-1 accumulation after glycolysis inhibition, as compared to full glucose availability, is the glucose dependence of mRNA translation. Interaction of glycolysis and the HIF-1 pathway may explain in part the effects of glycolysis inhibitors shown in preclinical and clinical studies where such agents have increased the efficacy in chemotherapy protocols and after radiation treatment [68, 69]. One can speculate that this effect depends on a reduced intra-tumoural accumulation of HIF-1 and thereby reduced expression of HIF-1- regulated genes. Previously, it was shown that glucose deprivation leads to an activation of multiple signal transduction pathways, changes in gene expression and induction of oxidative stress which mediate glucose-deprivationinduced cytotoxicity and metabolic oxidative stress in human cancer cells [70, 71]. Glycolysis modulation reduces the hypoxic accumulation of HIF-1 protein in human tumour cells by a translational or post-translational regulatory process. Therefore, tumour glucose levels manipulation represents a potential approach to therapeutically target HIF-1. A clear involvement of glucose availability in the hypoxic HIF-1a and CA IX expression in malignant glioma cells exist since application of the glycolysis inhibitor iodoacetate led to a sharply reduced expression of both proteins and CA9 mRNA in all cell lines tested. Most previous reports have focused on the effects of HIF-1 on glucose metabolism, rather than vice versa: Glycolytic enzymes are induced by hypoxia and lactate production by glycolysis is a major cause of the acidic extracellular pH of tumours [72]. Suboptimal O2 availability switches on cellular metabolism to anaerobic pathways for ATP production, which occurs through pyruvate transformation to lactic acid via the catalytic activity of lactate dehydrogenase 5 [73, 74]. Because intracellular acidosis triggers apoptosis blocking, increased glycolytic activity by down regulating HIF-1 may reduce apoptosis of hypoxic tumour cells. It was previously shown in non-brain tumour cell lines that the hypoxic accumulation of HIF-1 and expression of CA IX in vitro depend on the glucose concentration in the medium [60, 75]. As a consequence glycolysis inhibitors, when added under hypoxia, led to a reduced accumulation of HIF-1 via a translational or post-translational effect [51]. Interference with the glycolysis of malignant gliomas by IAA may therefore represent a therapeutic approach in targeting HIF-1 or CA IX. HIF-1 inhibition has been shown to slow tumour growth in vitro and in vivo tumour

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models [76, 77] and to act synergistically with other treatment modalities such as radiotherapy [78]. POTENTIAL ROLE OF CHEMICAL INHIBITORS IN HUMAN BRAIN CANCER TREATMENT Most anticancer drugs are transported by either active transport or passive diffusion into cells, where they frequently undergo further metabolism (79). It is well known that CA IX is highly over expressed in many types of cancer. Its expression, which is regulated by the HIF-1 [35, 80] transcription factor, is induced by hypoxia and correlates with a poor response to classical anti cancer therapeutic approaches like chemo- and radiotherapies (37). This chemo- and radio resistance occur due to the CA IX contribution to the tumour environment acidification by efficiently catalyzing the carbon dioxide hydration to bicarbonate and protons leading to metastatic phenotypes acquisition and chemoresistance to weakly basic anticancer drugs [81]. Among them are CA IX selective inhibitors, which can be inhibited via potent inhibitors derived from acetazolamide, benzenesulfonamides and ethoxzolamide which have been shown to inhibit the growth of several tumour cells invitro and in vivo [82, 83]. These drugs are pH sensitive, therefore, it is suggested that their cytotoxic activity depend on both intracellular pH (pHi) and pHe [84]. Targeting CAIX with such specific CA IX inhibitors [31], or also antibodies [85] should contribute, on one hand to the enhancing action of weakly basic drugs and on the other hand, to reduce the acquisition of metastatic phenotypes by pH imbalance controlling in the tumour cells. [86]. Selective CA IX inhibitors could prove useful for elucidating the CA IX role in hypoxic cancers, for pH imbalance in tumour cells controlling and for developing diagnostic or human tumour disease management therapeutic applications. CA9 specific enzymatic inhibition activity by specific inhibitors belonging to the group of sulphonamides like indisulam, reverts these processes, establishing a clear-cut role for CA IX in tumourigenesis. [86]. Practically, CA inhibitors have been previously shown to elicit synergistic effects when used in combination with other chemotherapeutics agents in animal models [87]. The CA inhibitors antiproliferative effect might also be due to their effect on other CA isoforms such as CA II or CA V, which provide the bicarbonate substrate for cell growth in carboxylation reactions involved in lipogenesis, nucleotide biosynthesis and gluconeogenesis, among others, thereby limiting the unrestrained tumour cells proliferation [33, 88, and 89]. Inhibition of this enzymatic activity by specific inhibitors, such as the sulfonamide indisulam reverses these processes, establishing a clear-cut role for CA IX in tumourigenesis. Thus, selective CA IX inhibitors could prove useful for elucidating the CA IX role in hypoxic cancers, for controlling the pH imbalance in tumour cells and for developing diagnostic or therapeutic applications for tumour management. CA IX inhibitors are undergoing continuous development in order to reach a high selectivity of these drugs and to avoid any side effects by other CA isozymes inhibition and avoiding them to play their physiological roles [32]. GLUCOSE METABOLISM IN HUMAN CANCER CELLS Glucose metabolism process may occur in nature either aerobically or anaerobically. In aerobic metabolism, glucose is converted to CO2 and H2O via the TCA cycle with the generation of about 36 moles of ATP per mole of glucose consumed. In anaerobic glycolysis, glucose is metabolized to lactic acid, producing 2 moles each of ATP and H+ ions per mole of glucose [90]. For fundamental thermodynamic reasons [91], the efficiency of aerobic metabolism is achieved at the cost of decreased maximum rate, and ATP production by the respiratory pathway rapidly saturates at high levels of glucose or limited O2 supply. In the lower-yield anaerobic pathway, more of the energy from glucose degradation is used to drive the reaction, allowing a greater maximum rate of metabolism. The net ATP production rate during the anaerobic pathway, in the presence of adequate glucose, can be similar to that of the aerobic route de-


spite the relative inefficiency. Malignant brain tumours from either humans or animal models lack metabolic flexibility, in contrast to normal brain that oxidizes glucose as well as ketone bodies for energy. They are largely dependent on glucose for energy [45, 9297]. Enhanced glycolysis produces excess lactic acid that can return to the tumour as glucose through the Cori cycle [98]. Normal mammalian cells under physiological conditions utilize high-yield aerobic glucose metabolism, but can adapt to periods of hypoxia by elevating the anaerobic pathway, provided the transition to hypoxia is gradual and allows for induction of response mechanisms such as HIF. The energy cost of this transition is substantial, as the output of ATP per mole of glucose is reduced by over 90%. To compensate this decreased efficiency, glycolytic flux must increase severalfold. Warburg first demonstrated tumour glucose metabolism alteration [99]. Transformed cells in vivo and in vitro typically rely on anaerobic pathways to generate ATP from glucose even in the presence of abundant O2. A rough correlation between malignancy degree and glycolytic rate has long been noted [100]. The decreased anaerobic metabolism efficiency is compensated by increased glucose, flux, maintaining energy production sufficiently in excess of basal metabolic demands to allow for cellular proliferation. METABOLIC CONTROL ANALYSIS IN HUMAN BRAIN TUMOURS Metabolic control analysis evaluates the degree of flux in metabolic pathways and can be used to analyze and treat complex diseases [101, 102]. This approach is based on findings that compensatory genetic and biochemical pathways regulate the tumour cells phenotype and bioenergetic potential [101- 103]. As rate-controlling enzymatic steps in biochemical pathways are dependent on the physiological system metabolic environment, the management of disease phenotype depends more on the flux of the entire system than on the expression of any specific gene or enzyme alone [103105]. Complex disease phenotypes can be managed through selforganizing networks that display system wide dynamics involving glycolysis and respiration. Global manipulations of these metabolic networks can restore orderly adaptive behaviour to widely disordered states involving complex gene-environmental interactions [103-106]. HYPOXIA INDUCED GENE EXPRESSION DEPENDENCY ON GLUCOSE AVAILABILITY Glycolytic enzymes are induced by hypoxia and lactate production by glycolysis is a major cause of the acidic extracellular pH of tumours [72]. Suboptimal O2 availability switches on cellular metabolism to anaerobic pathways for ATP production which occurs through pyruvate transformation to lactic acid via the catalytic activity of lactate dehydrogenase 5 [73, 74]. Because intracellular acidosis triggers apoptosis blocking, increased glycolytic activity by down regulating HIF-1 may reduce apoptosis hypoxic tumour cells [50]. We could previously show in non-brain tumour cell lines that hypoxic HIF-1 accumulation and CA IX expression in-vitro depend on the glucose concentration in the medium [60, 75]. Glycolysis inhibitors, when added under hypoxia, led to a reduced HIF1 accumulation via a translational or post-translational effect [51]. Interference with the glycolysis of malignant GBM by IAA may therefore represent a therapeutic approach alternative. In GBM, there is a clear involvement of glucose availability in hypoxic HIF1 and CA IX expression in malignant GBM cells since application of the glycolysis inhibitor IAA led to a sharply reduced expression of both proteins and CA9 mRNA in all cell lines tested. Most previous reports have focused on the effects of HIF-1 on glucose metabolism, rather than vice versa. REGULATION VIA OTHER HYPOXIA GENE REGULATORS Early growth response factor 1 (Egr-1) is a transcription factor that triggers transcription of downstream genes within 15-30 min of

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various stimulations [107]. In several publications [108, 109], it has been shown that the transcription factor Egr-1 is regulated via hypoxia, and hypothesized to be responsible for the hypoxia induced regulation of the NDRG1 gene in human tumours. The von HippelLindau tumour suppressor (pVHL), which is the recognition component of an E3 ubiquitin ligase complex, targets HIF-1 in normoxia [15, 16, and 17], leading to its ubiquitylation and consequent proteasomal degradation [17-19, 21]. Within this context, HIF-1 is responsible of several hypoxia induced genes regulation. These genes are expressed rapidly through specific promoter activation as addressed with its acting elements and regulative factors here previously. HIF-1 subunits are highly inducible and hypoxia responsive element (HRE) are bound by nuclear HIF-1 in human GBM cells in vitro under different oxygenation conditions, Also, the clear enhanced binding of nuclear extracts from GBM cell samples exposed to extreme hypoxic conditions confirms the HIF-1 western results [35-37, 110] and the NDRG1 regulation by HIF-1. Our findings [110] demonstrated that Egr-1 was not up-regulated in response to the extreme hypoxic (0.1 %O2) or even reoxygenative conditions after hypoxia in human GBM. As a consequence, HIF1 can still be considered as one of the promising targets for new therapeutic strategies in cancer research, especially therapeutic modulation of the adaptive hypoxic response. At the same time, we believe, based on the data that resulted from the research work of the different research groups worldwide, that Egr-1 could not be a new target for therapeutic modulation of the adaptive hypoxic response, at least in human GBM. SULPHONAMIDE DERIVATIVES AND THEIR ACTIVE AND POTENTIAL Role in the Treatment of Different Diseases Today, different chemical compounds exist either in nature or are synthetically developed possessing the functional ability to act as medical treatment agents that are of various natural appearance and chemical activity. Sulphonamide derivatives are among such compounds that play such an important role since more than half a century [88, 89]. The spectrum of diseases of different pathological origin or disease pathology that can be treated by them or can be the potentially treated by them is large. A group of such compounds function as anti-epileptic agents [111], anti-glaucoma agents [112], anti-Alzheimer’s disease agents [113], Diuretics [114], inhibition of the malaria parasite pathogenicity [115], migraine therapy [116], chronic parodontitis treatment [117], diabetic retinopathy prevention [118], and neuropathic pain inhibition [119] beside the treatment or potential treatment of many others diseases different other that need to be determined. Sulphonamide compounds beside other chemical compounds can function as diagnostic or medical treatment agents against cancer diseases in human especially as anti CA agents [120]. POTENTIAL ROLE OF CA9 INHIBITION BY CAI IN TUMOUR THERAPY CA9 inhibition is realized at various regulatory levels; while the indirect inhibitory occurs by the inhibition of the hypoxia induced regulatory transcription factor HIF-1, both on the protein and mRNA level [35-37, 51], the direct inhibition occurs after CA9 expression at CA IX protein or CA9-mRNA and level with the consequence that inhibitory power activity is in general different depending on the mode of inhibition, the level of inhibition-entry and the cell type where the functional inhibition take place. In general are two different alternative regulatory pathways how hypoxia induced CA9 regulative activity can be inhibited, direct or indirect manner. As shown in (Fig. 2). Indirect CA9 inhibition is mostly achieved via the hypoxia induced HIF-1 activity inhibition. This inhibition can be achieved either via the application of chemical substances like; CA inhibition via IAA, inhibition via Isoflavons [121], inhibition via Isothyocyanate [122], inhibition via Melotonin



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Fig. (1). HIF-1 induced regulation of hypoxia induced CA9 expression in human tumour cells A) Under normoxic oxygenation conditions in the tumor cell microenvironment, HIF-1 is rapidly degraded via the von Hippel-Lindau tumour suppressor gene product (pVHL)-mediated ubiquitin proteasome pathway. B) When the tumor environment aeration conditions shifts from normoxic to hypoxic aeration conditions, HIF-1 subunit becomes stable, translocates into the cellular nucleus and interacts with co-activators of which its transcription machinery is consisted such as p300 / CBP to modulate the transcriptional activity of numerous hypoxia inducible genes, like CA9 in our case and about 61 other hypoxia induced genes [136, 137].


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Fig. (2). Overview of the potential CA9 inhibition alternatives in human tumour cells In general, there are two different possible alternatives in which hypoxia CA9 regulative activity can be inhibited in a direct or indirect manner. The indirect CA9 is mostly achieved via inhibition of the hypoxia induced HIF-1 activity. This inhibition can be achieved either via the application of chemical substances like; CA9-inhibition via IAA, inhibition via Isoflavons, Inhibition via Isothyocyanate, inhibition via Melotonin, inhibition via Epithidiketopiperazines, CA9inhibition via chetomin or direct functional molecular Inhibition via HIF-1-siRNA. Direct hypoxia induced CA9-inhibition can be achieved via the direct inhibitory activity of mainly chemical derivatives of sulphonamide compunds or via direct inhibition by the inhibitory activity of CA9-siRNA molecules.

[123], inhibition via Epithidiketopiperazines [124]. CA9 expression inhibition via chetomin is another inhibitory option, were HIF-1 targeting human fibrosarcoma cell line by chetomin (150 nM) leads to transcriptional response to hypoxia suppression and reduces hypoxic radioresistance in these tumours, in vitro, showing increased radiosensitivity of hypoxic cells in vitro in response to chetomin as a premier observation [125]. Experimental conditions chosen were based on previous observation that a near-maximal HIF-1 expression occurs after 12h of hypoxia at an O2 concentration of 0.1% O2, which represents a level of hypoxia that is frequently observed in solid tumours and radiobiologically relevant [60, 75]. At the dose level of 150nM, chetomin exhibited a maximum specific effect on HRE-regulated activity inhibition. Other available options are the direct functional molecular inhibition via HIF-1-siRNA [126-131]. Confirmed direct hypoxia induced CA9 inhibition can be achieved either via the direct inhibitory activity of mainly chemical derivatives of sulphonamide compounds [120, 132-134] or via direct inhibition by the inhibitory activity of CA9-siRNA molecules [121]. COMPERATIVE EXPERIMENTAL ANALYSIS OF CA9 EXPRESSION AND ITS Expression Inhibition in Human Glioblastoma Cells In different in vitro CA9 expression analysis series, [35-37], we could observe that different molecular mRNA analysis methods resulted in experimental results with similar CA9 expression tendency and repetitive regulative CA9 expression values. Whether

northern blotting or RT- PCR was used to examine the differences on hypoxia dependent CA9 mRNA expression levels between different GBM cells under different standardized aeration and time conditions (normoxic or hypoxic aeration conditions). Interesting differences on CA9 mRNA expression levels between these GBM cells depending on the tumour micromilieu aeration conditions existed and where displayed in a form of a clear O2 concentration dependent CA9-mRNA expression, while HIF-1 mRNA showed no expression regulation due to hypoxic development in cell lines examined. On the protein level [35] a weak CA IX protein expression pattern was shown under normoxic oxygenation conditions, in U251, U373 and GaMG, while, in U87-MG it reached the positive control expression level. Under different hypoxia conditions examined, an increase in CA IX till 24h and a relative stability over 48h after reoxygenation without differences in the O2 concentration occurred, with respect to the difference in the CA IX expression levels between the different GBM cell lines. Incubation under 24h of hypoxia, with 50M IAA (0.1% O2) minimized the CA IX expression 90 % when compared to the basic normoxic expression level. Expression of CA IX and HIF-1 were stable and comparable to the expression pattern of hypoxic and reoxygenation conditions after hypoxia. In a comparative in vivo CA9 expression analysis series from two groups of tumour specimens collected from brain tumour patients suffering from either LGA or GBM [37], this, semi quantitative RT-PCR results showed a tumour grade associated CA9 expression pattern. CA9-mRNA was found uniformly upregulated in GBM, compared to normal brain and to LGA. An increase


of at least 2 fold in CA9 expression was shown in 3/15 patients (LGA) and 12/15 patients in (GBM). HIF-1 mRNA showed no regulation in vivo and in vitro due to hypoxic development in all cell lines examined. In parallel, expression analysis experiments on protein level in this two human patient groups analyzed showed that CA9 and HIF1 were expressed in GBM at a higher rate than in LGA. On the other hand, in another experiments series published here for the first time, we were able to determine the physiological behaviour of hypoxia induced CA9 in response to the regulative effect of sulphonamide derivative CAI in GBM (135). CA IX protein level expression under normoxic conditions was detectable in a cell-type specific manner. U87- MG exhibited a strong aerobic expression, U251 and U373 had moderate and GaMG very weak normoxic CA IX protein expression pattern as shown in Fig. 3. We could demonstrate that sulphonamide derivative CAI with a molecular weight of 157.20 g/mol [135] at functional concentrations between (3500 nM8000 nM) displayed inhibitory characteristics against hypoxia in-


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duced CA9 expression in the four GBM cell lines examined under 24h of hypoxia (0.1 % O2). Parallel experiments with the same tumour cell lines where CA9-siRNA under similar conditions applied confirmed these results. Also, we could demonstrate the ability of HIF-1 inhibitor chetomin in the hypoxia induced HIF-1 regulated CA9 expression in GBM (Fig. 4A, Fig. 4B), under similar oxygenation conditions at concentrations of chetomin between (150nM-500nM). These CA9 inhibition results especially via a functional CAI sulphonamide derivatives inhibition confirms the functional capability such compounds to regulate the expression of these important molecules towards the complete inhibition of these molecules, due to the cell proliferative inhibitory characteristics of these compound [120]. This parallel inhibition via other compounds like chetomin or alternative CA9-siRNA in this approach or the inhibition via IAA [35] under similar comparable conditions show that is possible to obtain such an hypoxia induced CA9 inhibitory function paired with a different level of inhibition with all the accompanied consequences. These sufonamide derivatives CAI repre-

Fig. (3). Comparative analysis of CA IX protein expression under in vitro hypoxia including hypoxia induced CA9 inhibition via chemical or non chemical alternatives-in vitro Early-passage U373, U251 and U87-MG human malignant GBM from the American Type Culture Collection (ATCC, Rockville, MD, USA) and GaMG, a cell line established from a patient with GBM (Gade Institut of the University Bergen, Norway) [138], were grown on glass Petri dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids, penicillin (100 IU/ml)/streptomycin (100 μg/ml) and 2 mM L-glutamine. Examined cells were treated with in vitro hypoxia for 1, 6, or 24 h at 5, 1 or 0.1% O2 as indicated in a Ruskinn Invivo2 hypoxic workstation (Cincinnatti, OH, USA), western blot analysis was performed as previously described [35] and detection was withn the M75 mouse monoclonal antibody against CA IX (Bayer Healthcare Co., diluted 1: 7200). The CA9-siRNA construct (Santa Cruz Biotechnology) were transfected into U373, U251, U87MG and GaMG GBM cells lines, these transient transfections were performed using Fugene6 transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Post-transfection cells were incubated for 8h under standard normoxic conditions (21% O2, 5% CO2) post transfection with further incubation under hypoxic conditions (0.1%) for 24 h and was able to completely inhibit the extreme hypoxia induced CA9. Densitometric evaluation of signal strengths in Western blotting or in semi-quantitative RT-PCR was performed with 1D Kodak Image Analysis Software. The proteins amount gave signals that were measured in Kodak light units (KLU) and divided by the loading control ß-actin corresponding signals. Also in parallel these cells were incubated under hypoxia with addition of (250nM-8000nM) of the CAI sulphonamide derivative with a molecular weight of 157.20 g/mol (135), or with chetomin (150-500nM). While in GaMG cells the CA9 inhibition was at comparatively low concentration of the CAI sulphonamide derivative compounds (4000nM) followed by U373 and U251, a complete inhibition was not possible in the PTEN mutated U87-MG. On the other hand the HIF-1 inhibitor chetomin was only able to down-regulate CA IX protein expression in GaMG to a basic expression level and did not change the expression substantially in the other three tumor cells analysed. Experiments were repeated for three times and the figure represents one representative experiment out of three. Treatment with 100 M DFO under aerobic conditions served as a positive control while cells incubated under aerobic conditions as negative control and -actin as a loading control.


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Fig. (4). Hypoxia induced CA9 gene mRNA expression analysis under in vitro hypoxia including the hypoxia induced CA9 inhibition via chemical or non chemical alternatives-in vitro Cell Incubation, oxygenation, treatment and transfection methods were the same as for the protein analysis and in a parallel approach. To compare the expression of the individual genes examined, , RNA was iolated, quantified and its quality was examined, RT-PCR was performed using primers designed as previously described [35, 37, 137], published information on -actin and CA9 mRNA sequences were obtained from GenBank. PCR was performed, and the PCR products were separated on agarose gels as previously described [37]. Densometric evaluation was similar as for the protein but off coarse the DNA signals were measured here and -actin served as house keeping gene. Also in parallel, these cells were incubated under hypoxia with addition of (250nM-5000nM) of the CAI sulphonamide derivative or with chetomin (150-500nM). While in GaMG cells the CA9 inhibition was at comparatively low concentrations of the sulphonamide derivative CAI (4000nM) followed by the other U373, U251 and U87-MG at 4500nM, on the other hand the HIF-1 inhibitor chetomin downregulated CAIX protein expression in GaMG at 400nM while in the other three tumor cells analysed it was possible at 300nM. Experiments were repeated for three times and the figure represents one representative experiment out of three. Treatment with 100 M DFO under aerobic conditions served as a positive control while cells incubated for 24 h under aerobic conditions as negative control and -actin as a loading control.

sent interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumours. We have to mention here that brain cell GBM is characterized by both, a very high CA IX expression and radiotherapy as well as chemotherapy unresponsiveness, rendering this CAI sulphonamide derivative be the leading candidate for such novel anti cancer therapeutic approaches. The CA9 inhibitory capacity of the sulphonamide derivative CAI, despite previous discussion about CA9 inhibition specificity achieved by them [120] showed from their inhibition results when they were compared to the parallel CA9 inhibition results of other modalities examined until now a similar inhibition tendency but with a different expression level inhibition and in a

tumour cell type specific manner as it is the case for the hypoxia induced expression of different cells [35-37, 51, 125], that we can say at least in GBM they are coming closer to the CA9 inhibition specificity point. Further experimental series are necessary to determine the CA9 inhibition specificity of each different CAI sufonamide derivative with taking in account the difference in both their functional activity and its specificity level depending on the tumour cell type examined. CONCLUSIONS Despite the ongoing research by the various research groups around the world, there is a further necessity towards identification


and characterisation of substances that posses at least similar or as an optimal goal to be achieved better functional characteristics and that can function against a typical disease pattern of this nature where the patient treatment can not be positively affected by a such medical drugs. Interference in the GBM glycolysis pathway in addition to the other alternative approaches can represent a therapeutic alternative in CA9 involved therapeutic solutions, since CA9 represent a stable marker gene as well as one of the optimal target for therapeutic applications of human brain cancer, therefore, the expression modulation of CA9 and potentially other HIF-1 regulated genes beside CA9 cab be achieved by alternative therapeutic approaches of this nature. We were able here to provide the elementary information necessary for optimizing the human cancer treatment outcome and life quality of human brain cancer patients. Alternative optimizer of human brain cancer treatment modalities, especially further drug administration with further phase I, II and III series of clinical studies are necessary for determination of the minimal effective inhibitory concentration in brain and in other human organ tissues suffering from cancer diseases with no or non harmful minimal side effects on the treated human patient are required. Controlled CA9 expression with down-regulation to expression levels that do not affect the basic and essential cellular functions and that are able to neutralize the non beneficiary effect of the local CA9 expression in human brain tumours environment is necessary in order to improve human radio-therapeutic and / or chemotherapeutic treatment modalities to be applied in the human brain cancer patient treatment. In GBM therapy, the main aim, as with all cancer diseases is to either eradicate the tumour or convert it into a controlled, quiescent chronic disease. Angiogenesis and hypoxia induced, HIF-1 regulated genes inhibition remains the main parts of therapeutic approaches in human oncology. It is well known that cancer cell metabolism can be perturbed specifically at the level of glycolysis leading to interesting therapeutic activities in cancer that can be displayed. CA IX and also its main regulator HIF-1 (what is not the case for Egr-1, at least in GBM) represent interesting targets for anticancer drug development with considering the fact that the CAI design and effective CA9 targeting for anti-cancer purposes requires further knowledge about their molecular characteristics, functional regulation mechanisms and their distribution beside the different factors that might be unknown until now that are favouring this process.


NDRG1 siRNA PAS

= = =

pVHL RuO2 TCA

= = =

N-myc Down-regulated Gene 1 Signal Inhibitory RNA Protein domain contained in many signalling proteins Von Hippel-Lindau tumour suppressor Ruthenium Oxide Microprobes Tricarboxylic acid

CONFLICTS OF INTERESTS STATEMENT The authors declare that they have no conflicts of interests related to the contents of this manuscript AKNOWLEDGEMENTS The authors would like to thank the University of Würzburg, Medical Faculty, Department of Radiation Oncology and the IZKF Würzburg (B25) to CH, GHV for financing this research and Bayer Healthcare Co. for provision of the M75 monoclonal antibody. Also, the authors would like to acknowledge the efforts & contributions made by the different research groups in the different aspects of the Carbonic Anhydrases research and tumour hypoxia signalling and regulation with their contributions, globally. REFERENCES [1] [2]

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ABREVATIONS [2- D- G] = 2-Deoxy-D-glucose bHLH = Basic Helix-Loop-Helix Protein Transcription Factors CAI = Carbonic Anhydrase Inhibitors CA9 = Carbonic Anhydrase 9 DFO = Desferrioxamine Egr-1 = Early Growth Response gene 1 GBM = Gliobastoma Multiforme h = Incubation or exposition period in hours HBS = Hypoxia Binding Sequence HIF-1 = Hypoxia Induced Factor -1 (Hypoxia activated subunit) HIF-1 = Hypoxia Induced Factor -1 (constitutional subunit) HRE = Hypoxia Responsive Element of the gene promoter region HK = Hexokinases Hypoxia = O2 Concentration (0.1-5%) IAA = Iodide Acetate Normoxia = O2 Concentration (21%)

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Received: July 5, 2010

Accepted: July 29, 2010

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