Cancer The Leading Cause Of Death Worldwide Biology Essay

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Cancer is one of the leading causes of death worldwide. Despite extensive investment, investigation and research over decades, the currently available anti-cancer drugs lay behind expectations and therefore new, highly active and well-tolerated anti-cancer agents are strongly needed. Pyrazine-2-carboxylic acid N'- (7-fluoro-pyrrolo[1,2-α]quinoxalin-4-yl)-hydrazide-oxalic acid co-crystal, referred to as NVX-412, is a co-crystal of oxalic acid and NVX-144, its parental lead compound (Hebar et al., 2012). NVX-412 is a promising drug candidate for the treatment of a number of cancer types and my efforts undertaken to better understand the mechanisms of action underlying its remarkable anti-cancer activity are summarized in my thesis.

In developed countries cancer is the leading cause of death and in developing countries the second leading cause of death following heart diseases. Worldwide it is responsible for one in eight deaths (American Cancer Society, 2008). It is generally assumed that all cancers are a consequence of accumulation of genetic alterations that lead to uncontrolled, autonomous growth and proliferation of the affected cells. Malignant tumors are characterized by their ability to invade tissues and ultimately metastasize to distant organs. Cancer is not a single disease but encompasses more than 100 distinct diseases that have diverse histopathological characteristics, differ in their genetic aberrations, risk factors, and epidemiology and thus also in the clinical outcome (Stratton et al., 2009).

Cancer classifications

Tumors are classified into a great variety of histological subtypes by pathologists. Over the last decades a classification system based on the primary site (topography) and the histology (cellular morphology) of neoplasms has evolved. This forms the basis for the World Health Organization (WHO) classification of malignant tumors and the international terminology "International Statistical Classification of Diseases and Related Health Problems 10th Revision" (ICD-10) (WHO, 2010). The ICD-10 was specifically extended to the "International Classification of Diseases for Oncology" (ICD-O), which is currently in its 3rd revision (ICD-O-3) (WHO, 2000; Hiddemann and Bartram, 2010).

In addition to histological classifications, tumors are graded according to their extent of progression. The International Union Against Cancer (UICC) has developed the TNM Classification of Malignant Tumors (TNM), a cancer staging system (UICC, 2011).

However, these currently available classification systems do not sufficiently reflect how variable tumors are at the biological level; in fact it is assumed that each tumor is unique regarding its genetic and epigenetic alterations (Kamb et al., 2007).

Cancer pathogenesis

Causes and risk factors for cancer are diverse; it can be triggered and influenced by endogenous factors such as inherited mutations, hormone status, obesity and age but also by exogenous, environmental factors such as smoking, alcohol, radiation, ultraviolet (UV) light exposure, diet, infectious agents (viruses), and environmental toxins. Based on migration or identical twin studies it is now estimated that only a fraction of cancer cases, between 5-10%, are attributable to genetic defects. Environmental and lifestyle factors account for the remaining 90-95% of cancer cases (Anand et al., 2008). Tumorigenesis, the transformation of normal human cells into malignant cancer cells, is a complex multi-step process that can proceed for decades. It is believed to follow principles similar to Darwinian evolution; cells continuously acquire heritable genetic alterations that under the mechanisms of natural selection confer a growth advantage because of the resultant phenotype (Hanahan and Weinberg, 2000; Stratton et al., 2009). Over the last decades our knowledge on the evolution of cancer has enormously increased and it is now known that cancer cells progressively acquire mutations that via a number of intermediate steps of pre-malignant states develop into malignant and invasive cancer cells. Mutations that occur in proto-oncogenes will give rise to oncogenes, i.e. mutated forms of normal cellular genes that are capable of transforming a cell mostly by dominant gain of function. So called tumor suppressor genes that, when working properly would suppress tumor formation, are inactivated by mutations mostly by recessive loss of function. A number of such acquired mutations or capabilities are common and shared by most if not all cancers. An attempt to give the puzzling complexity of cancer signaling a structure resulted in the publication of the seminal review "The Hallmarks of Cancer" by Hanahan and Weinberg in 2000. They argue that there are common traits - hallmarks - of cancer that "enable tumor growth and metastatic dissemination" (Hanahan and Weinberg, 2000). This review and the presented concept is until now regarded as a substantial contribution to cancer research. In 2011 they expanded their concept by two new emerging hallmarks and two new enabling characteristics (Hanahan and Weinberg, 2011). The six core hallmarks of cancer are "self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis". Although there are more than 100 different types of cancer resulting in a literally infinite combination of mutations and aberrations it is generally believed that these six characteristics allow tumor cells to successfully overcome anti-cancer defense mechanisms of cells and tissues. This set was expanded by two "emerging hallmarks", i.e. "deregulating cellular energetics and the avoidance of immune destruction" and two "enabling characteristics", i.e. "genome instability and mutation and tumor-promoting inflammation". Furthermore, the concept of "tumor microenvironment" gets more and more important. Tumors are very heterogeneous and are a mixture of various distinct cell types including normal cells that are recruited by the tumor. It is thought that these stromal cells also influence the malignant cells by enhancing the acquisition of anti-cancer defense mechanisms (Hanahan and Weinberg, 2011). The interested reader is referred to the extensive reviews of Hanahan and Weinberg and also an excellent review of Michael Stratton on the cancer genome (Hanahan and Weinberg, 2000; Stratton et al., 2009; Hanahan and Weinberg, 2011) for detailed description of these concepts.

The hallmarks of cancer resulted from an attempt to classify the huge number of alterations that are acquired during tumorigenesis. There is no need to say that this concept does not cover every detail and is for sure not the only concept or way to describe the process of tumorigenesis. I chose this concept to explain cancer pathogenesis because it is widely accepted and quite extensive. However, I want to clarify that there also is criticism on the concept of the hallmarks of cancer. It was argued that only one hallmark, i.e. tissue invasion and metastasis, is characteristic for malignant tumors, while the other hallmarks are characteristics of benign tumors as well (Lazebnik, 2010).

Signaling pathways

In the following, molecular signaling pathways relevant for my PhD thesis are addressed in brief.

The cell cycle

The eukaryotic cell cycle is divided into four distinct phases, the G1, S, G2 and M phase in which cells perform all processes necessary for self-duplication. Initially, only the M phase and interphase were known; interphase was later identified to include the G1, S and G2 phases. In the S or synthesis phase cells replicate their DNA, in the M or mitosis phase the replicated chromosomes are segregated and cell division into two daughter cells occurs. In the two phases in between, the G or gap phases, cells prepare for entry in either S (G1) or M phase (G2) and important checkpoints control whether a cell can proceed further in the cell cycle. The M-phase is divided into prophase, prometaphase, metaphase, anaphase and telophase. There is also a G0 phase that cells can enter in G1; actually most non-proliferating cells of the human body are in this reversible quiescent state (Vermeulen et al., 2003; Campisi and d'Adda di Fagagna, 2007). Cellular senescence is a "state of permanent cell cycle arrest" upon specific stress (Campisi and d'Adda di Fagagna, 2007). Three major transitions need to be tightly controlled during the cell cycle - the G1/S cell cycle checkpoint at the onset of S phase, the G2/M DNA damage checkpoint to ensure entry into mitosis only after successful DNA replication and the mitotic spindle checkpoint at the exit of mitosis (Hochegger et al., 2008). Furthermore, there is evidence that DNA damage checkpoints also exist during S and M phase (Vermeulen et al., 2003; Bartek et al., 2004). Specific protein complexes comprised of cyclin-dependent kinases (CDKs) and cyclins mediate these complex transitions from one cell cycle phase to the next. Protein levels of CDKs remain constant throughout the cell cycle, whereas cyclin levels increase and decrease during the cell cycle hence cyclically activating the CDKs. Additionally, CDK activity can be regulated by phosphorylation and dephosphorylation events at specific amino acid sites and also by CDK inhibitors (CKI) (Besson et al., 2008). Activated CDKs phosphorylate distinct sets of substrates thereby promoting progression through the cell cycle.

Deregulations of these processes that are normally very tightly controlled result in unrestrained cell proliferation and can lead to development of cancer. Indeed in cancer, fundamental alterations in the regulation of the cell cycle are observed and therefore the cell cycle control proteins represent promising targets for anti-cancer therapy (Vermeulen et al., 2003).

DNA damage response and DNA repair

First experimental evidence for DNA repair was found in the 1930s, but the importance of DNA repair pathways in cancer development and as targets for anti-cancer therapy was not realized for years (Friedberg, 2008). Studies in xeroderma pigmentosum (XP) patients, which develop multiple skin cancers, have identified defects in DNA repair pathways. This finding attracted interest and paved the way for thorough research in this field (Kelley, 2012).

The DNA damage response (DDR) and DNA repair are closely linked to cell cycle regulation in so far that diverse DNA lesions can activate cell cycle checkpoints, inhibit DNA replication and promote cell cycle delay or arrest. These effects provide more time for repair of DNA damage. In some cases cells can repair small lesions rapidly and cell cycle arrest is not necessary (Lazzaro et al., 2009). After successful repair of DNA damage cells can progress in the cell cycle. In case the lesions are unrepairable, cells can either enter permanent cell cycle arrest or undergo apoptosis (Branzei and Foiani, 2008). DNA damage can have multiple causes. On the one hand exogenous sources such as chemical genotoxic agents (cancer therapy), smoking, UV light exposure or ionizing radiation (IR), and on the other hand endogenous processes such as base oxidation by reactive oxygen species (ROS), base mismatches due to replication defects or replication fork collapse. Every day about 104 spontaneous base losses and single strand breaks (SSB) damage the DNA in a cell (Kelley, 2012). The decision which of the different DNA repair pathways is activated depends on the type of DNA lesion and also on the cell cycle phase of the damaged cell. The following types of DNA repair mechanisms are known: mismatch repair (MMR) that replaces mispaired DNA bases with the correct bases, base excision repair (BER) that excises a single altered base, and nucleotide excision repair (NER) that removes oligonucleotides of about 30 bp containing the damaged base. SSBs are repaired by the single strand break repair (SSBR) pathways mainly via poly(ADP-ribose) polymerase (PARP) signaling. For repairing double strand breaks (DSB) there are different mechanisms, either non-homologous end joining (NHEJ) that relegates the DSBs in an error-prone manner or homologous recombination (HR) that makes use of the sister chromatids as a template for accurate, error-free DSB repair (Ciccia and Elledge, 2011). As already mentioned the mechanisms of sensing the DNA lesions, recruitment of DDR factors to the site of DNA damage and ultimately the type of DNA repair differ depending on the type of DNA lesion and cell cycle phase. However, the basic mechanisms are similar. Whenever DNA lesions are recognized by sensor proteins, these proteins recruit and activate further mediators and effectors by phosphorylation. This activates the checkpoint surveillance system to allow for repair.

Cell death

Cell death is a highly heterogeneous process; under normal physiological conditions there are a number of possibilities how cells can die. One of the best studied mechanism of cell death is apoptosis, a term coined by Kerr, Wyllie and Currie in 1972 describing death of cells with specific morphological features (Kerr et al., 1972). Apoptosis is a form of programmed cell death and its mechanisms follow tightly controlled cascade-like processes that result in minimal damage and disruption to neighboring cells and efficient recycling of cell debris by phagocytosis without triggering an immune response (Elmore, 2007; Taylor et al., 2008). It is accompanied by distinct morphological characteristics like rounding and shrinking of the cells, plasma membrane blebbing, formation of apoptotic bodies, chromatin condensation, nuclear and organelle fragmentation, and hydrolysis of DNA ("DNA laddering") (Taylor et al., 2008; Kroemer et al., 2009). Cysteine aspartic acid-specific proteases, also known as caspases, are key players in regulating the apoptotic process. Other very important proteins in regulating and mediating apoptosis are the members of the B-cell lymphoma-2 (BCL-2) family. It is the tightly regulated interplay between these proteins that triggers anti- or pro-apoptotic responses. Deregulated cell death is one of the hallmarks of cancer (Hanahan and Weinberg, 2000) and the ability to evade apoptosis is closely linked to tumorigenesis and also resistance to chemotherapy.

Among the non-apoptotic mechanisms are necrosis (recently also termed necroptosis), autophagy or mitotic catastrophe (Okada and Mak, 2004; Kroemer et al., 2009).

Unfolded protein response

Upon different types of stress (perturbations of cellular energy levels, changes in redox state or Ca2+ concentrations) unfolded or misfolded proteins accumulate and aggregate in the endoplasmic reticulum (ER) lumen because of a reduced ability of the ER to correctly fold proteins (Szegezdi et al., 2006; Ron and Walter, 2007). The unfolded protein response (UPR) is a protective strategy to restore normal ER function in these ER-stress conditions. The ER transmembrane receptor PKR-like endoplasmic reticulum kinase (PERK), also called eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), plays a central role in triggering and regulating the UPR (Szegezdi et al., 2006).

Drug discovery and anti-cancer therapeutics

According to a recent review approx. 40% of therapies tested in clinical trials in 2009 were related to cancer, indicating the importance of oncology research for pharmaceutical and biotechnology companies (Aggarwal, 2010). However, despite significant advances in our understanding of the biology and molecular mechanisms underlying cancer, paired with huge investment in oncology research, the success rate for oncology products is extremely low (approx. 10%). The reasons therefore are frequently debated. The intricate signaling events leading to cancer development and progression in combination with pitfalls of diverse drug development strategies are described as being the main contributors of high failure rates of anti-cancer therapies (Kamb et al., 2007; Hait, 2010).

The history of chemotherapeutic drugs dates back to ancient times; already back then first ideas of preventing and treating cancer were born. However, the mechanisms underlying tumorigenesis were not very well understood until the late twentieth century (Bode and Dong, 2009). In the 1940s nitrogen mustards and anti-folate drugs marked the beginning of the "modern era of chemotherapy", before that surgery was largely applied for treating cancer but obviously had its limitations. Radiotherapy emerged as valuable treatment strategy in the 1960s enabled by the invention of linear accelerators (Chabner and Roberts, 2005). In 1971 US president Nixon declared the "war on cancer" by signing the "National Cancer Act of 1971" that should provide better prerequisites for research in the US and should improve therapy (Kamb et al., 2007). Since then extensive investment, investigation and research in this field has taken place resulting in a great number of therapies routinely applied in clinics for the treatment of various cancers (Bode and Dong, 2009). About 200 drugs are currently approved for the treatment of cancer. These drugs were identified by different approaches and have a broad spectrum of mechanisms and targets, and are assigned to different classes of anti-cancer treatments such as radiomimetics, alkylating agents, anti-metabolites, anti-tumor antibiotics, topoisomerase inhibitors, replication inhibitors, mitotic inhibitors, targeted therapies or immunotherapies (Kamb et al., 2007; American Cancer Society, 2012)

However, 40+ years after the war on cancer was declared researchers still face huge hurdles in developing efficient cancer therapeutics and there is strong unmet medical need for new drugs, also with new mechanisms of action.

The investigational drug NVX-412

Preliminary data on the parental compound of NVX-412

NVX-144 (the non-oxalate form of NVX-412) is a small molecule compound that was discovered by Neamati and colleagues (Grande et al., 2007; Neamati, 2009). Originally, this nitrogen-containing heterocyclic compound was developed as a human immunodeficiency virus-1 (HIV-1) integrase inhibitor via rational drug design, but turned out to exhibit remarkable cytotoxicity (Grande et al., 2007; Oshima et al., 2009). For HIV-1 integrase no cellular homologue has been described (Pani and Marongiu, 2000), but Grande and colleagues suggested that HIV-1 inhibitors could target cellular enzymes with similar active site chemistry (Grande et al., 2007). These include for example topoisomerases I and II that show such a high similarity to the retroviral integrases, that they are routinely used to counter screen integrase inhibitors (Plasencia et al., 2005).

To better understand the mode of action of NVX-144, comparative gene expression profiling with six drugs with known mechanisms of action was performed (Error: Reference source not found) (Neamati, 2009).

A principal components analysis was performed with the gene expression data. Principal components analysis is a technique of multivariate statistics extracting the most important information from a large dataset resulting in the so-called principal components, which are new, orthogonal variables giving the dataset a new structure. Similarities of multiple observations can be displayed as dots within a co-ordinate system in a high-dimensional data space (Abdi and Williams, 2010). It was shown that NVX-144 (together with structurally related chemical compounds SC23 and SC24) forms a distinct cluster (Error: Reference source not foundA). A hierarchical cluster analysis supports the classification of the compounds into different groups, showing separate clustering of the NVX-144 compound and its related structures SC23 and SC24 as well (Error: Reference source not foundB) (Neamati, 2009).

A more detailed analysis of the gene expression profiles upon treatment with the drugs revealed significant overlap with etoposide, mitoxantrone and camptothecin (Neamati, 2009). Taken together, the results from gene expression profiling, three-principal components analysis and hierarchical cluster analysis revealed similarities to etoposide, mitoxantrone and camptothecin, topoisomerase II and I inhibitors, respectively; however, NVX-144 clusters separately therefore suggesting a different mode of action (Neamati, 2009).

NVX-412

NVX-144 was shown to exert strong cytotoxicity, as summarized above. However, efforts have been made to even improve the anti-cancer effects and also certain physico-chemical characteristics. NVX-412, a co-crystal of NVX-144 with oxalic acid, is the result of this improvement (Error: Reference source not found).

The fact that NVX-144 forms a co-crystal with oxalic acid is of special interest, since it has been shown that co-crystals of N-heterocyclic rings with carboxylic acids show advantages compared to the respective salts (Aakeroy et al., 2007). NVX-412 confirms this notion by showing increased cytotoxic activity compared to the parental compound NVX-144 in HT-29 and HCT116 colon carcinoma cell lines with an IC50 that is 3 to 4-fold lower (Grande et al., 2007; Hebar et al., 2012).

Importantly, NVX-412 fulfils the criteria of the Lipinski's rule of five, which gives a first hint on drug-like properties and on whether a putative drug candidate may be suitable as a medicinal product (Lipinski et al., 2001).

The criteria of the Lipinski's rule of five are:

≤ 5 donators of hydrogen bonds (e.g. OH- or NH-groups)

≤ 10 acceptors of hydrogen bonds (e.g. O- or N-atoms)

molecular mass of ≤ 500 g/mol

octanol-water partition coefficient of max. 5

With 4 donators and 10 acceptors of hydrogen bonds, a molecular mass of 412 g/mol and a predicted octanol-water partition coefficient (Molinspiration Cheminformatics, Nova ulica, SR) of 2.078 all the criteria are fulfilled. These data give a first hint that the investigational chemical compound is suitable for further development as a medicinal product concerning its bio-availability factors.

AIMs OF STUDY

As discussed in the introduction there is strong unmet need for new, highly active, well-tolerated and easy-to-use anti-cancer agents. Pyrazine-2-carboxylic acid N'-(7-fluoro-pyrrolo[1,2-α]quinoxalin-4-yl)-hydrazide oxalate (NVX-412) is a promising drug candidate for the treatment of a number of cancer types but the main target or mechanism of action of NVX-412 is not known to date. The aim of this thesis is to thoroughly investigate the underlying mechanisms of action of the cytotoxic activity of NVX-412.

Materials and Methods

Several of the methods have been described by Hebar et al. (Hebar et al., 2012).

Drugs

NVX-412 was obtained from Novelix Pharmaceuticals, Inc. (La Jolla, CA, USA). For in vitro studies, the compound was dissolved in DMSO (25 mM stock stored at -80°C) and diluted at the concentrations indicated. Tunicamycin (TUN) from Streptomyces sp., Nutlin-3, and (S)-(+)-Camptothecin (CPT) were purchased from Sigma-Aldrich (Vienna, AUT) and dissolved in DMSO (stocks: 1 mg/ml, 3.5 mM and 5 mg/ml, respectively). Chloroquine (CQ) was also purchased from Sigma-Aldrich and dissolved at a stock concentration of 10 mM in H2O. Doxorubicin (DOX) and 5-Fluorouracil (5-FU) were obtained from the in-house pharmacy department at stock concentrations of 2 mg/ml and 50 mg/ml, respectively. All solutions were freshly prepared before use.

NCI-60 developmental therapeutics program (DTP) human tumor cell line screen

NVX-412 was included in an anti-cancer activity screen by the National Cancer Institute (NCI) (Shoemaker, 2006). The compound was tested against 59 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney (for a complete list of cell lines please refer to Shoemaker et al. 2006 (Shoemaker, 2006)). The methodology of this in vitro cancer screen is described in detail on the NCI website (http://dtp.nci.nih.gov/branches/btb/ivclsp.html). Briefly, the human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% FCS and 2 mM L-glutamine in 96-well plates at densities ranging from 5,000 to 40,000 cells/well. After 24 hours the experimental drug was added at 5 concentrations plus control and cells were incubated for additional 48 hours. For determination of the growth-inhibitory effect of the compound a sulphorhodamine B assay was performed that employs a chemical fixation step at the end of the drug treatment and a subsequent staining for 10 minutes. After a washing step, absorbance was determined at 515nm. Three different dose response parameters are then calculated: growth inhibition of 50% (GI50), which defines the drug concentration leading to a 50% reduction in the net protein increase, the drug concentration resulting in total growth inhibition (TGI) and the 50% lethal concentration (LC50), indicating a net loss of cells following treatment (Shoemaker, 2006).

NCI COMPARE

With the bioinformatic algorithm of the COMPARE analysis it is possible to quantify interrelations of cellular responses with the large database of molecular target information that have been collected during NCI-60 DTP tumor cell line screens. The exact methodology and algorithm is presented on the NCI COMPARE website (http://dtp.nci.nih.gov/docs/compare/compare.html). Briefly, NVX-412 was compared to all compounds in the public database. The COMPARE algorithm searches the database for similarities in the response pattern of the 60 cell lines tested in the NCI screen (based on Pearson correlation coefficient) (Shoemaker, 2006). This type of analysis allows detection of compounds with a similar cytotoxicity profile in the NCI-60 screen and gives hints on possible mechanisms of action.

Cell lines

The following cell lines were used in this study (cell lines from NCI-60 DTP Human Tumor Cell Line Screen not included): SV40 Tag immortalized mouse embryonic fibroblasts (PERK+/+ or PERK-/-) were kindly provided by D. Ron (Cambridge University, UK) and were cultured in DMEM supplemented with 10% FCS, 1% Penicillin plus Streptomycin (Pen Strep), 1% Non-Essential Amino Acids and 0.1% β-ME (Harding et al., 2003). The isogenic human colorectal carcinoma cell lines HCT116 p53+/+ and p53-/- and RKO p53+/+ and p53-/- were purchased from Horizon Discovery Ltd. (Cambridge, UK) and were cultured in McCoy's 5A medium supplemented with 10% FCS, 1% Pen Strep and 2 mM L-Glutamine (whenever the p53 status of HCT116 cells is not specified p53+/+ cells were used). CLBL-1 (canine B-cell lymphoma) (Rütgen et al., 2010), OSW (Kisseberth et al., 2007) and CL-1 (canine T-cell lymphoma) (Momoi et al., 1997) and GL-1 (canine B-cell leukemia) (Nakaichi et al., 1996) cell lines were kindly provided by B. C. Rütgen (University of Veterinary Medicine Vienna, Vienna, AUT), and were cultured in RPMI 1640 supplemented with 10% FCS and 1% Pen Strep. The B-cell Non-Hodgkin lymphoma cell lines SU-DHL-6 and SU-DHL-8, purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, GER), were cultivated in RPMI 1640 supplemented with 10% FCS and 1% Pen Strep. The same culture conditions were used for all the other lymphoma and leukemia cell lines Raji (Dolstra et al., 1999; Liu et al., 2001), Ramos (Dolstra et al., 1999; Liu et al., 2001), KG-1 (Furley et al., 1986; Dolstra et al., 1999; Andersson et al., 2005; Rucker et al., 2006), KG-1a (Furley et al., 1986), HL-60 (Dolstra et al., 1999; Andersson et al., 2005; Rucker et al., 2006), BV173 (Dolstra et al., 1999; Liu et al., 2001), NALM-1 (Andersson et al., 2005; Rucker et al., 2006) and K562 (Dolstra et al., 1999; Andersson et al., 2005; Rucker et al., 2006), which were kindly provided by P. Valent (Medical University of Vienna, Vienna, AUT) and are all previously published cell lines available either from American Type Culture Collection (ATCC), Manassas, VA, USA) or DSMZ. Hs27, a normal fibroblast cell line, was provided by D. Barlow (Research Center for Molecular Medicine, Vienna, AUT) and was grown in DMEM with 10% FCS and 1% Pen Strep (Reyes-Reyes et al., 2010; Lamore and Wondrak, 2012). The Ewing's Sarcoma cell lines TC32 and TC71 were a kind gift from P. Sorensen (UBC, Vancouver, CAN) and were grown in RPMI 1640 with 10% FCS, 1% L-Glutamine (Werner et al., 2008). Hs578T were grown in Minimum Essential Medium-α (MEM) supplemented with 10% FCS, 1% Pen Strep and 1% L-Glutamine and were a kind gift from T. Grunt (Medical University of Vienna, Vienna, AUT) (Sheridan et al., 2006). Both cell lines, Hs27 and Hs578T, are available from ATCC. HUVEC normal human umbilical vein endothelial cells were purchased from Lonza (Walkersville, Maryland, USA) and were grown in Clonetics® EGM® BulletKit media, respectively. Human white preadipocytes (hWP) were purchased from PromoCell (Heidelberg, GER) and were cultured in preadipocyte growth medium provided by the company. The breast adenocarcinoma cell line MCF-7, the cervical carcinoma cell line HeLa, the hepatocellular carcinoma cell line HepG2 and the colorectal adenocarcinoma cell line HT-29 were obtained from ATCC and were cultured in DMEM supplemented with 10% FCS and 1% Pen Strep. If not indicated otherwise all cell culture reagents were purchased from Gibco (Grand Island, NY, USA).

Cytotoxicity assays

Cells were plated in 24-well plates. After cells were allowed to recover for 24 hours, NVX-412 was added in fresh growth medium. The maximal DMSO concentration reached in all experiments was below 0.01%. Respective control experiments at the highest DMSO concentration were performed in order to rule out DMSO-induced effects. After 72 hours of incubation the proportion of viable cells was determined by cell counting with a Z1 Coulter Particle Counter (Beckman Coulter, Vienna, AUT), a ViCell XR (Beckman Coulter, Vienna, AUT) or a CASY® Cell Counter (Schärfe, Reutlingen, Germany). Cytotoxicity was expressed as IC50 values that were derived from the corresponding dose-response curves.

Stability assay

For investigating the stability of NVX-412 one aliquot each of NVX-412 was stored at -80°C and at room temperature. After 1, 3 and 6 weeks the activity of the substances was tested in HT-29 cells. Therefore cells were plated (2x104 cells/well) in 24-well plates and treated with 300 nM NVX-412 (corresponding to the IC50) after a recovery period of 24 hours. After 72 hours of incubation the proportion of viable cells was determined by cell counting with a Z1 Coulter Particle Counter (Beckman Coulter, Vienna, AUT), and the activity of NVX-412 was calculated.

Determination of influence of cell density

Cells were plated at different densities in 24-well plates. Densities were chosen according to optimal proliferation conditions for the respective cell lines and according to the conditions of further experiments planned. HT-29 cells were plated at 5x103, 1x104 and 2x104cells/well, HL-60 and K562 cells at 1.25x104, 3.75x104 and 7.5x104 cells/ml and SU-DHL-6 and SU-DHL-8 cells at 2x105, 4x105 and 5x105 cells/ml. NVX-412 was added to the adherent HT-29 cells after a 24 hours recovery period and to all the suspension cells directly after seeding. Cells were treated with one concentration of NVX-412 (500 nM for HT-29, HL-60 and K562 cells, 400 nM for SU-DHL-6 and 100 nM for SU-DHL-8 cells). After 72 hours of incubation the proportion of viable cells was determined by cell counting with a Z1 Coulter Particle Counter (Beckman Coulter, Vienna, AUT).

Determination of influence of incubation time

HT-29 and MCF-7 cells were plated at a density of 2x104 and HepG2 cells at a density of 1.5x104 cells/well in 24-well plates, respectively, and 1 µM (HT-29 and HepG2) or 150 nM NVX-412 (MCF-7) was added after a recovery period of 24 hours. SU-DHL-6 and SU-DHL-8 cells were plated at a density of 8x105 or 5x105 cells/well in 6-well plates and NVX-412 was directly added to an end concentration of 400 or 100 nM, respectively. After the respective incubation times with NVX-412 (short time points: 5 and 30 minutes, 4 hours; long time points: 4, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours) adherent cells were washed three times with fresh medium to completely remove the drug and afterwards cells were cultured in normal growth medium. Suspension cells were centrifuged and washed to remove the drug and were cultured in normal growth medium in fresh plates. After a total of 72 hours of incubation (including the NVX-412 treatment period) the proportion of viable cells was determined by cell counting with a Z1 Coulter Particle Counter (Beckman Coulter, Vienna, AUT).

Clonogenic assays

For clonogenic assays, HT-29 and HepG2 cells were plated in 60 mm dishes at a density of 1000 cells per dish. After 24 hours NVX-412 was added at the indicated concentrations. After cultivation for 12-14 days (when assessable colonies were visible), colonies were fixed in 70% ethanol, stained with 0.5% Crystal Violet and counted manually.

Proliferation kinetics

HT-29 cells were plated (2x104 cells/well) in 24-well plates. After a recovery period of 24 hours, NVX-412 was added in fresh growth medium to final concentrations of 0, 300 and 1000 nM, respectively. After 24, 48 and 72 hours cell numbers were determined with a Z1 Coulter Particle Counter.

Morphology assays

To investigate potential morphological changes upon treatment with NVX-412, HCT116, HeLa and HT-29 cells were plated in 6-well plates and incubated for up to 72 hours with the indicated concentrations of NVX-412, DMSO as vehicle control and 1 µM camptothecin (CPT) as a positive control for apoptotic morphology. Control experiments were carried out to ensure that morphological changes are not due to differences in confluency. After 24, 48 and 72 hours pictures were taken with an Olympus IX71 inverted microscope and camera system (Olympus Color View III) at 20x magnification.

Quantification of cell sizes

Cell sizes were quantified using specialized imaging software (ImageJ 1.45d, US NIH, Bethesda, MD, USA). Average areas of all cells present within 5 different fields of view per sample were determined after 48 hours treatment with 0, 0.15 and 1 µM NVX-412. The non-parametric Mann-Whitney U test was used to assess the statistical significance of differences between the cell sizes, because the Kolmogorov-Smirnov test showed that the data was not normally distributed (GraphPad Prism 5.0, La Jolla, CA, USA).

Western Blot

Treated cells were directly lysed in NP40 cell lysis buffer (Invitrogen, Grand Island, NY, USA) containing protease and phosphatase inhibitors. Protein concentrations were measured colorimetrically (DC Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by SDS - polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (WhatmanTM, Vienna, AUT). Equal loading was checked by Ponceau S (SERVA, Heidelberg, GER) staining. Bound antigen was visualized with the enhanced chemiluminescence detection system (Roche Diagnostics, Indianapolis, IN, USA). These procedures were performed according to the manufacturer's protocols. Antibodies specific for the following proteins of interest were used: CDK4, CDK6, Cyclin D3, p15, p21, p27, Cyclin E2, Cyclin A, PERK, pPERK (Thr980), pChk1 (Ser296), p53, p-p53 (Ser15), PARP, LC3B, β-Tubulin and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA) at 1:1000 dilution. Secondary antibodies were peroxidase-tagged goat anti-rabbit or anti-mouse IgGs (Cell Signaling Technology) (1:2000).

Immunofluorescence staining

HCT116 and HeLa cells were seeded on glass cover slips in 6-well plates. 24 hours after plating, HCT116 cells were incubated with 0.15, 0.5 and 1 µM NVX-412 and 1 µM CPT for 24 hours for staining of p-p53 and pATM (same conditions also for HeLa cells). For light chain 3B (LC3B) staining HCT116 cells were incubated with 0.15 and 1 µM NVX-412 and 20 µM CQ. For investigating phosphorylated histone 2AX (γH2AX), HeLa cells were incubated with 0.21, 0.5 and 1 µM NVX-412 and 1 µM CPT for 3 or 24 hours. Additionally, cells were allowed to recover for 24 hours in normal growth medium after 24 hours of incubation with NVX-412 or CPT. After the respective treatments, cells were washed and fixed with 4% methanol-free formaldehyde (Polysciences Inc. Warrington, PA, USA) for 15 minutes at room temperature and then permeabilized with 0.2% Triton X-100/PBS for 2 minutes at room temperature. After blocking with goat serum (5%) in 1% BSA/0.3% Triton X-100/PBS, the respective primary antibody was added and cells were incubated at 4°C overnight. The following antibodies were used: mouse p-p53 (Ser15) (Cell Signaling Technology, Danvers, MA, USA) at 1:100 dilution, mouse p-H2AX (Ser139) (Millipore, Billerica, MA, USA) at 1:1000 dilution, mouse p-ATM (Ser1981) (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) at 1:400 dilution, and rabbit LC3B (Cell Signaling Technology) at 1:200 dilution in 1% BSA/PBS, respectively. Cells were then washed 3 times for 10 minutes with 0.25% BSA/0.1% Triton X-100/PBS and incubated with the Alexa Fluor 488 conjugated secondary anti-mouse antibody (1:1000 in 0.25% BSA/0.1% Triton X-100/PBS) for 1 hour at room temperature in the dark. Cells were washed 3 times for 10 minutes with 0.05% Tween-20/PBS containing Hoechst 33342 (Sigma Aldrich, Vienna, AUT) and mounted with mounting medium (Fluoprep, bioMérieux, Marcy l'Etoile, FRA). Fluorescence was immediately recorded on a Zeiss LSM700 laser scanning microscope.

Quantification of γH2AX

The common procedure for assessing γH2AX induction is to count γH2AX foci. Since with NVX-412 and the positive control CPT γH2AX activation was that strong, no distinguishable and countable foci were seen. Therefore, the average γH2AX fluorescence intensity per nucleus was determined. To this end, γH2AX intensities within a certain field were measured using Quantity One - 4.6.9 (Basic freeware version, Bio-Rad Laboratories, Hercules, CA, USA) and were divided by the number of nuclei in this field.

Methyl green competition assay

The methyl green assay was used to investigate intercalation of NVX-412 into DNA. Salmon sperm DNA was diluted in PBS to reach a concentration of 100 µg/ml. 15 µl methyl green solution were added per sample (final concentration 31.7 µM). PBS was added to add up to a total reaction volume of 1 ml (including the drug solution added later). After incubation for 1 hour in the dark at 37°C drug solutions were added. NVX-412 was used at concentrations of 0.1, 1 and 10 µM. Doxorubicin was used as a positive control for DNA intercalation and was added at a concentration of 20 and 50 µM. Furthermore, a blank without salmon sperm DNA and a negative control without drug were performed. After a further incubation for 2 hours in the dark at 37°C samples were transferred into 96-well plates in triplicates and absorption was measured at 642nm. Intercalating agents lead to a loss of blue color.

Comet assay

The alkaline comet assay was used according to the method previously described (Tice et al., 2000; Heffeter et al., 2009) to determine the induction of DNA strand breaks. Briefly, HCT116 cells were seeded at a density of 3x105 cells/well in 6-well plates. After a recovery period of 24 hours cells were treated with the drugs; the positive control H2O2 at 50 µM for 5 minutes and with 150, 500 and 1000 nM NVX-412 and 1000 nM CPT for 3 hours, respectively. Cell viability was determined by Trypan Blue staining and was >90%. Afterwards, 100 µl of 0.5% low melting point agarose were added to the cells at 37°C and mixed. This cell suspension was then dropped on microscope slides pre-coated with a layer of 1.5% normal melting point agarose. After 15 minutes, when the agarose had solidified, cells were lysed in ice-cold lysing solution (2.5 M NaCl; 100 mM Na2EDTA, 10 mM Tris-HCl, 1% Triton X-100; pH 10) at 4°C overnight. After lysis, the slides were rinsed with H2O and incubated for 20 minutes in the electrophoresis buffer (300 mM NaOH, 1mM Na2EDTA, pH 12.5) on ice, a step needed for DNA unwinding. Electrophoresis was performed in a cooled horizontal gel electrophoresis chamber for 20 minutes at 25 mV and 300 mA. After electrophoresis the slides were washed twice, each with neutralization buffer (0.4 M Trizma Base, pH 7.5) and water. In the end slides were allowed to dry overnight at room temperature. The next day ethidium bromide was used to stain the DNA and comet tails were visualized using a fluorescent microscope (Nikon). An automated image analysis system (Comet Assay IV, Perceptive Instruments Ltd. Haverhill, UK) was used for analysis. The parameter tail intensity, i.e. the fraction of total DNA in the tail, was chosen because of its linear relation to DNA break frequency (Collins et al., 2008). Per treatment group, two slides were prepared and 50 cells each were analyzed.

Determination of p53 status dependency

The isogenic colon carcinoma cells lines HCT116 p53+/+ or p53-/- and RKO p53+/+ or p53-/- were cultured in 12-well plates. After a recovery period of 24 hours, NVX-412 or Nutlin-3 was added in fresh growth medium at the indicated concentrations. Nutlin-3, a MDM2 antagonist and p53 pathway activator was used to prove the differential biological effects of the p53 status. After 24 or 72 hours cell numbers were determined with a Beckman Coulter ViCell XR.

Determination of caspase activity

Cells were grown in white 96-well plates at a density of 4x103 or 3 x103 cells/well for TC32 and TC71 or HCT116 p53+/+ and p53-/-, respectively. After cells were allowed to recover for 24 hours drugs were added in fresh growth medium. DMSO was added as solvent control for NVX-412 and Doxorubicin was used as a positive control for caspase activation. After 24 hours of treatment Caspase-Glo® assays (Promega, Madison, WI, USA) for caspases 3/7, 8 and 9 were performed according to the manufacturer's protocol. Briefly, the lyophilized Caspase-Glo substrate was dissolved in the respective buffer. In case of Caspase-Glo 8 and Caspase-Glo 9, the MG-132 inhibitor was added to a final concentration of 60 µM directly before use. Equal volumes of reagent were added to each well containing cells and medium. After 45 minutes incubation in the dark at 37°C chemiluminescence was measured using a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany). Caspase activities of the untreated control were arbitrarily set to 1 and fold changes of all other treatments were calculated.

Nuclear blebbing analysis

TC32 and TC71 cells were grown in white 384-well plates at a density of 1x103 cells/well. After cells were allowed to recover for 24 hours drugs were added in fresh growth medium. After 24, 48 and 72 hours of incubation cells were stained for 20 minutes at 37°C with Hoechst 33342 at a final concentration of 15 µM to identify viable cell nuclei and ethidium homodimer 1 (1 µM final) to identify dead cells. Images were taken with the automated image acquisition system IN Cell Analyzer 1000 (GE Healthcare Life Sciences, Waukesha, WI, USA) and were then analyzed with IN Cell Developer Toolbox (GE Healthcare Life Sciences). Nuclei showing morphological features typical for apoptosis such as nuclear blebbing or apoptotic bodies were counted. This experiment was performed during a research visit in the laboratory of Poul H. Sorensen at the University of British Columbia, British Columbia Cancer Research Center, Vancouver, Canada.

DNA replication rate

HeLa, HCT116 and HUVEC cells were plated in black 96-well plates. After cells were allowed to recover for 24 hours NVX-412 or CPT was added in fresh growth medium. After 24 or 48 hours of incubation a chemiluminescent 5-bromo-2'-deoxyuridine (BrdU) incorporation enzyme-linked immunosorbent assay (ELISA) (Roche Applied Science, Penzberg, Germany) was performed according to the manufacturer's protocol. Briefly, cells were labelled with BrdU (final concentration 10 µM BrdU) for approximately 2.5 hours. Afterwards cells were fixed for 1 hour and incubated with anti-BrdU-POD working solution for 1 hour. After 3 washing steps substrate was added to the samples and chemiluminescent measurements were performed using a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany). For investigating recovery of DNA synthesis HCT116 and HeLa cells were treated 24 hours with NVX-412 or CPT, respectively. Measurements were performed after 24 hours treatment; cells in the remaining plates were allowed to recover for 24 hours in normal growth medium before DNA replication was measured. To check whether a possible decline in DNA replication rate is not simply due to lower cell numbers because of cell death induction, the proportion of viable cells was determined in parallel by a MTT-based cytotoxicity assay according to the manufacturer's protocol (EZ4U, Biomedica, Vienna, Austria). Colorimetric measurements were performed after approximately 2.5 hours of incubation with the substrate solution at 492 and 620nm.

Flow cytometric cell cycle analyses

HT-29, HeLa, HCT116, MEF PERK+/+ and MEF PERK-/- cells were plated in 6-well plates. After cells were allowed to recover for 24 hours, NVX-412 was added in fresh growth medium at the respective IC50 concentration and 0.5 and 1 µM. CPT was used at 50 nM and 1 µM. To analyze the cell cycle distribution, cells were collected after 24 hours of incubation and washed with PBS. Cells were fixed in 70% ethanol for at least 2 hours. For analysis, cells were transferred into PBS, incubated with RNAse A (0.04 µg/ml final concentration) for 30 minutes at 37°C, treated with 40 µg/µl propidium iodide for 30 min at 4°C and then analyzed by flow cytometry using BD FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The resulting DNA histograms were quantified using ModFit LT (Verity Software House, Topsham, ME, USA).

Measurement of intracellular ROS levels

HCT116 and HeLa cells were seeded at a density of 8x105 or 6x105 cells/dish in 10 cm dishes, respectively. After cells were allowed to recover for 24 hours NVX-412 was added in fresh growth medium at a concentration of 1 µM and cells were incubated for 3, 24 or 48 hours with the drug. Staining with the probe 2',7'-dichlorodihydro-fluorescein diacetate (H2DCFDA, Invitrogen) was performed as described elsewhere (Sallmyr et al., 2008). Briefly, cells were trypsinized and centrifuged at 1450 rpm for 5 minutes at room temperature. The cell pellets were washed with PBS once and then resuspended in 1 ml PBS. Cells were incubated with 0.2 µM H2DCFDA (dissolved in 100% ethanol) for 20 minutes at 37°. Cells were centrifuged at 1450 rpm for 5 minutes and cell pellets were resuspended in 500 µl PBS. 10 µM H2O2 served as positive control for the induction of ROS and was added just before staining. Intracellular ROS levels were determined by flow cytometry using a FACSCalibur (Becton, Dickinson and Company) and histograms were analyzed using FlowJo (TreeStar Inc., Ashland, OR, USA).

Acridine orange staining

HCT116 cells were seeded at a density of 8x103 cells/well in 8-chamber slides (Lab-TekTM, Thermo Fisher Scientific Inc., Waltham, MA, USA). After cells were allowed to recover for 24 hours drugs were added in fresh growth medium. NVX-412 was used at a concentration of 1 µM, the positive control CQ at a concentration of 20 µM. After 16 hours of incubation cells were stained with acridine orange hemi (zinc chloride) salt (Sigma Aldrich). Briefly, cells were washed once with medium without serum. Afterwards cells were stained for 15-30' in the dark with freshly prepared acridine orange working solution (final concentration 1 µg/ml diluted in medium without serum). Fluorescence in FITC channel was immediately recorded on a Zeiss Axio Imager 2. Acridine orange is a green fluorescent dye and accumulates in acidic organelles in a pH-dependent manner leading to a color switch to orange or bright red.

Hoechst DNA staining

HCT116 p53+/+ and p53-/- cells were seeded at a density of 1x105 cells/well in 6-well plates. After cells were allowed to recover for 24 hours drugs were added in fresh growth medium. NVX-412 was used at the IC50 concentration of 150 nM and CPT at a concentration of 50 nM, respectively. After 24, 48 and 72 hours treatment staining with Hoechst 33342 was performed. Cells were washed once with PBS and fixed with methanol for 20 minutes. Afterwards, cells were incubated with Hoechst 33342 (final concentration: 10 µg/ml in PBS with 0.1% Triton X-100) for 15 to 20 minutes at 37°C in the dark. After washing cells twice with ddH2O they were prepared with mounting medium (Fluoprep, bioMérieux, Marcy l'Etoile, FRA) and images were taken with an Olympus IX71 inverted microscope and camera system (Olympus Color View III) at 20x magnification. Nuclear morphology with special regard to apoptotic bodies or nuclear blebbing was investigated in these samples.

Recovery assay

HCT116 and HeLa cells were seeded in 6-well plates at densities of 1x105 or 8x104 cells/well, respectively. After cells were allowed to recover for 24 hours NVX-412 was added in fresh growth medium at various concentrations. Error: Reference source not found depicts the concept and treatment scheme of this recovery assay. Briefly, after cells were incubated for either 48 hours (I, lower concentrations of NVX-412) or 6 hours (II, higher concentrations of NVX-412) with NVX-412, cell numbers and viability were determined with a ViCell XR (Beckman Coulter, Vienna, AUT). After washing to remove the drugs cells were re-seeded at specific densities (3.2x104 for HCT116 and 2x104 for HeLa) in 12-well plates. Viability at the time of re-seeding was >95%. After 72 hours of incubation cell numbers and viability were determined.

siRNA experiments

HT-29 cells (22x103 cells/cm2) were cultured in DMEM with 10% FCS and 1% Pen Strep. After 24 hours, sub-confluent cells were transfected with siRNA using siLentFectTm Lipid Reagent (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer´s protocol. I applied 0 or 20 nM PERK siRNA (Stealth Select RNAiTM siRNA #1299001, HSS114059, Invitrogen, Grand Island, NY, USA) and incubated the cells at 37°C for 48 hours. For control purpose, cells were kept in transfection reagent (1.6 µg/ml) without siRNA. The PERK siRNA-induced knock-down was confirmed by immunoblotting using the antibodies mentioned in the Western Blot analysis section. After incubation with siRNA, cells were treated with various concentrations of NVX-412 as indicated for 72 hours at 37°C and survival assays were performed as described above.

Determination of combination effects with radiation

For establishing dose-response curves HCT116 cells were seeded at a density of 7x104 cells/well in 6-well plates. After allowing the cells to recover for 24 hours NVX-412 was added in fresh growth medium or cells were irradiated using a Theratron 780 60Co radiotherapy unit (MDS Nordion, Ottawa, ON, CAN). After 72 hours of incubation viable cell numbers were determined with a ViCell XR (Beckman Coulter, Vienna, AUT). Based on the results of the dose-response curves, suitable doses of irradiation and NVX-412 concentrations were chosen for the combination assay. HCT116 cells were seeded at a density of 1x104 cells/well in 24-well plates, 24 hours later cells were irradiated with 3 Gy and directly after the irradiation NVX-412 was added at concentrations from 0 to 300 nM. After 72 hours of incubation viable cell numbers were determined with a ViCell XR (Beckman Coulter, Vienna, AUT).

Statistical analyses

If not indicated otherwise, two-way Analysis of Variance (ANOVA; GraphPad Prism 5.0) was used to assess the statistical significance of differences between the data.

Results

Preliminary data

Gene expression patterns induced by NVX-144 and topoisomerase inhibitors are similar

NVX-144, the parental compound of NVX-412, has been investigated by its inventors as outlined in Chapter 1.3.1. The results from gene expression profiling showed similarities of NVX-144 to topoisomerase I and II inhibitors; however, cluster analyses suggested a distinct mechanism of action. Although this information is not of primary importance it nevertheless contributed to my scientific approach for elucidating the mechanisms of action of NVX-412.

NVX-412 is stable at room temperature for at least 6 weeks

The stability of a chemical compound is an important factor in the pre-clinical development phase and needs to be determined under GLP conditions with specialized assays (EMA, 2003). I was interested to collect data on the stability of NVX-412 and therefore performed cytotoxicity assays with NVX-412 when stored for several weeks at room temperature compared to the recommended storage at -80°C (Error: Reference source not found). Notably, there are no differences between the activity of NVX-412 stored at room temperature and -80°C, suggesting NVX-412 is chemically stable at room temperature for at least 6 weeks and that its activity is not compromised.

NVX-412 exerts broad anti-cancer activity

NVX-412 was included in an anti-cancer activity screen by the NCI to obtain an overview of the range of its activity. The compound was tested against 59 different human tumor cell lines originating from diverse cancer types, representing leukemia, melanoma and cancers of the lungs, colon, brain, ovary, breast, prostate, and kidney. The NCI-60 DTP Human Tumor Cell Line Screen revealed very strong anti-cancer activity for NVX-412 in tumor cell lines of all cancer types with a mean 50% inhibitory concentration (IC50) of about 200 nM (Error: Reference source not found and Error: Reference source not found). Only two cell lines showed a reduced sensitivity towards NVX-412, the neuronal cancer cell line SNB-75 with an IC50 of 9900 nM and the breast cancer cell line Hs578T with 3100 nM. The leukemic cell lines were the most sensitive ones, with a mean IC50 of 62 nM. In addition to the NCI-60 DTP Human Tumor Cell Line Screen, further cell lines derived from various tumor entities and two different species including human and canine cells and also normal non-cancer cells were tested (Error: Reference source not found). In agreement with the data obtained in the NCI screen, these data showed anti-cancer activity in all cell lines tested across a variety of tumor types (Error: Reference source not found). Normal human endothelial cells (HUVEC) and human white preadipocytes (hWP) displayed a reduced sensitivity compared to cancer cell lines. For HUVECs the IC50 was determined to be 2.0 µM and for human white preadipocytes hWP 1.0 µM, respectively.

Generally, the IC50 values generated in the NCI screen and in our laboratory were similar, although for some cell lines a higher variation could be observed. These differences can be attributed at least to some extent to the different methods used for the determination of the IC50 values. However, the consistent and broad anti-cancer activity in cell lines of various histology could be reconfirmed (Hebar et al., 2012).

Cell density has no substantial influence on the activity of NVX-412

It is known that differences in cell density can affect the biological effect of certain chemotherapeutic agents, a phenomenon described as the inoculum effect (Ohnuma et al., 1986). I investigated the influence of cell density on the activity of NVX-412 in adherent cells (HT-29) and cells in suspension (HL-60, K562, SU-DHL-6, SU-DHL-8) with a fixed concentration of NVX-412. I investigated cell densities of 5x103 to 2x104 cells/well for HT-29 cells, 1.25x104 to 7.5x104 cells/ml for HL-60 and K562 cells, and 2x105 to 5x105 cells/ml for SU-DHL-6 and SU-DHL-8 cells. These cell densities were chosen based on the conditions planned for future assays. In adherent HT-29 cells there were no significant cell density-dependent differences in response to NVX-412 (Error: Reference source not foundA). HL-60 and K562 cells were significantly less responsive to NVX-412 treatment at the lowest tested cell density of 1.25x104 cells/ml compared to higher cell densities (Error: Reference source not foundB and C). Although not significant, the response of SU-DHL-6 cells was similar; cells were less responsive at the lowest cell density of 2x105 cells/ml (Error: Reference source not foundD). In SU-DHL-8 cells the response was different, these cells were significantly less responsive to NVX-412 treatment at the highest tested cell density of 5x105 cells/ml (Error: Reference source not foundE). It is concluded that the investigated range of cell densities does not have a substantial influence on response to NVX-412, based on the small differences in response to NVX-412 in the range of 5 to 11%.

The anti-proliferative effects of NVX-412 depend on incubation time

I was interested in the influence of the incubation time on the anti-proliferative activity of NVX-412. First I investigated cell survival after 5 minutes, 30 minutes and 4 hours of incubation in the presence of a high concentration (1 µM) of NVX-412 corresponding to the IC90 after 72 hours treatment in HT-29 and HepG2 cells (Error: Reference source not foundA and B). In these experiments cells were incubated for the respective time in the presence of NVX-412. Cells were then washed to completely remove NVX-412 and were incubated for 72 hours in normal growth medium. Surprisingly, in both cell lines, incubation times up to 4 hours with NVX-412 did not affect survival.

In another set of experiments I investigated longer incubation times of up to 24 hours (0, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours) with NVX-412 in cell lines MCF-7, SU-DHL-6 and SU-DHL-8 at the corresponding IC50 concentrations. In these experiments there was at least a tendency towards a decrease in viable cell numbers (manuscript in preparation).

NVX-412 exerts bi-modal activity

In order to differentiate between cytostatic and cytotoxic effects of NVX-412 (e.g. direct induction of cell death observable by a drop in viable cell numbers or cytostatic effects observable by limited proliferation), I performed a proliferation kinetics experiment. Therefore I determined viable cell numbers of HT-29 colon carcinoma cells over 3 days of treatment with 0, 300 and 1000 nM of NVX-412 (Error: Reference source not found). At the IC50 (300 nM), proliferation of cells was significantly decreased after 2 days compared to the untreated control cells. At concentrations above the IC50 (at 1 µM), cell numbers started to decline below the numbers of cells seeded at the beginning of the experiment, suggesting a direct induction of cell death rather than cell cycle arrest followed by delayed cell death. Hence, it is assumed that NVX-412 exerts its anti-neoplastic effects in a dose-dependent (bi-modal) manner (Hebar et al., 2012).

NVX-412 inhibits clonogenic survival

Colony formation assays (also termed clonogenic survival assays) determine the ability of cells to undergo "unlimited" division and hence investigate another endpoint in addition to the short-term cytotoxicity seen in proliferation assays (Franken et al., 2006). I performed clonogenic survival assays to test the clonogenic potential of HepG2 and HT-29 cells upon NVX-412 treatment (Error: Reference source not found). The cells were incubated with 0, 10, 30, 100, 300 and 1000 nM of NVX-412 for 12 or 14 days, respectively. NVX-412 reduced the ability of both HepG2 and HT-29 cells to form colonies in a dose-dependent manner. Of note, for both cell lines the concentration of NVX-412 needed to achieve half-maximal effects was comparable to the IC50 concentrations determined in the short-term (3 days) cytotoxicity assays (Hebar et al., 2012).

Cell sizes are increased upon NVX-412 treatment

I was interested whether NVX-412 would change the morphology of cells, since this could give hints on the cell fate. Based on the results from the proliferation kinetics showing a possible bi-modal activity of NVX-412 (see Chapter 4.6, Error: Reference source not found) I chose to investigate the effects at the IC50 in HCT116, HeLa and HT-29 cells that is assumed to act cytostatic, and at a higher concentration (1 µM), at which cytotoxic effects predominate (Panel A of Error: Reference source not found - Error: Reference source not found). Treatment with NVX-412 at the IC50 led to changes in the morphological appearance of all three investigated cell types within 24 hours; the cells appeared larger than untreated cells. DMSO alone as vehicle control did not change the morphological appearance (DMSO control not shown). To investigate this interesting phenomenon in more detail, cell sizes were quantified after 48 hours treatment with NVX-412. A highly significant increase in cell sizes was seen for all three tested cell lines (Panel B of Error: Reference source not found - Error: Reference source not found). Over the next 48 hours a decrease of cell densities could be observed for HCT116, HeLa and HT-29 cells at the IC50 concentrations, but hardly any detached or dead cells were observed. However, this picture changed dramatically when higher concentrations of NVX-412 were used. Again, cells displayed an altered morphological appearance and increased size compared to untreated cells (Error: Reference source not found). However, after 48 hours the number of detached and dead cells increased (data not shown), similar to the treatment with the cell death inducer camptothecin (CPT) (Hebar et al., 2012).

NCI COMPARE analysis suggests similarity to DNA replication-inhibiting and DNA damaging compounds

The NCI profile of NVX-412 was compared to that of standard agents and marketed drugs using the COMPARE analysis tool from NCI (Shoemaker, 2006). Only moderate correlations (Pearson's correlation coefficients 0.4 - 0.7) to other known chemotherapeutics were observed (Error: Reference source not found). However, most of the top-listed compounds exert their anti-neoplastic activity via inhibition of DNA synthesis or induction of DNA damage. Among these, many compounds also interfere with topoisomerase I and/or II, DNA polymerase or other DNA binding enzymes. These observations fit the initial assumption that the parental compound NVX-144 targets a DNA binding or processing enzyme (Plasencia et al., 2005). Furthermore, gene expression profiling of NVX-144-treated cells showed similarities to gene expression profiles after treatment with etoposide, mitoxantrone and camptothecin (Neamati, 2009), all of which are known topoisomerase I or II inhibitors. Taken together, the results from the COMPARE analysis are supported by these initial findings for the parental compound NVX-144. Concluding, the above-mentioned evidence that the anti-neoplastic activity of NVX-412 might be related to induction of DNA damage and/or inhibition of DNA replication prompted me to investigate these phenomena.

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