NOSH-aspirin is a potent inhibitor of colon cancer cell growth: Effects of positional isomerism

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Università degli studi di Pisa

Facoltà di Scienze Matematiche Fisiche e Naturali

Laurea magistrale in Biologia Applicata alla Biomedicina

TESI DI LAUREA

NOSH-aspirin is a potent inhibitor of colon cancer

cell growth: Effect of positional isomerism

A.A. 2013-2014

Relatori:

Prof. Aldo Paolicchi

Dr. Khosrow Kashfi

Candidata:

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1

1.0 Introduction

Colorectal cancer is a malignant tumor arising from the mucosa, the inner layer of the large intestine. (Fig.1)

Fig.1: Picture of colon (colorectal) cancer.

The most common colon cancer cell type is adenocarcinoma which accounts for 95% of cases, other, rarer types include lymphoma, carcinoid and sarcoma.

It is the second most common cause of cancer death in women, after breast cancer, and the third most common in men, after lung and stomach cancer. [1]

Globally incidences vary 10-fold with highest rates in the USA and Eastern Europe and lowest rates in developing countries (Africa and South-Central Asia).

Colon and rectum cancer represents 8.6% of all new cancer cases in the U.S. The American Cancer Society estimates that in 2013 about 142,820 people will be diagnosed with colorectal cancer and about 50,830 people will die of this disease. [2]

In Italy, it is estimated that there will be 27,000 new cases/year of colon and rectum cancer and an estimated 15,000 people will die of this tumor.

The median age at diagnosis is 69. Colorectal cancer rates are highest in people aged 75-84 years. There is no single cause of colon cancer. Nearly all colon cancers begin as noncancerous (benign) polyps, which slowly develop into cancer.

The most commonly mutated gene is the APC (Adenomatous Polyposis Coli) gene, which produces an increase signaling activity of the Wnt/β catenin pathway. Other mutations must occur for the cell to become cancerous. [3]

The p53 protein, produced by the TP53 tumor suppressor gene, normally monitors cell division and kills cells if they have Wnt pathway defects. Eventually, a cell line acquires a mutation in the TP53 gene and transforms the tissue from an adenoma into an invasive carcinoma.

Otherwise KRAS oncogene can acquire mutations, that result in over-activation of cell proliferation. [4]

Another apoptotic proteins commonly deactivated in colorectal cancers is DCC (Deleted in Colorectal Cancer), that has a role in migration of cancerous cells. [5] (Fig.2)

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3 Fig.2: Picture of colon cancer formation.

Risk factors for colorectal cancer include older age, presence of colon polyps, inflammatory bowel disease (Crohn's disease or ulcerative colitis) and a personal history of breast cancer. [6] Certain inherited diseases also increase the risk of developing colon cancer. Two of the most common are Familial adenomatous polyposis (FAP) and Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. The first familial syndrome is usually caused by APC mutation, the second one by mutation of DNA Mismatch Repair Genes.

Environmental factors that may promote carcinogenesis are a diet high in fat and low in fiber, consumption of beer and carcinogens, such as heterocyclic amines, products of bacterial metabolism. [7]

Symptoms of colorectal cancer typically include rectal bleeding and anemia.

Colon polyps and early cancer can have no symptoms. Therefore regular screening is important, so that precancerous polyps can be removed before they turn into cancer.

Screening tests can also find colorectal cancer early, when treatment works best.

In patients with advanced disease, the efficacy of currently available treatments is limited. Nonsteroidal anti-inflammatory drugs (NSAIDs) in general and aspirin (ASA) in particular are prototypical chemopreventive agents. [8] However, the use of NSAIDs is limited by their significant toxicity (gastrointestinal, renal, and cardiovascular). [9]

To overcome these limitations and to improve the safety profile of NSAIDs, numerous strategies have been employed. One such strategy has been the use of nitric oxide (NO), a gaseous mediators with physiological relevance, leading to generation of NO-releasing NSAIDs (NO-NSAIDs). The development of NO-NSAIDs was based on the observation that NO could enhance the local gastric mucosal defense mechanisms, thereby decreasing NSAID-induced gastric toxicity. [10;11]

Animal and human studies have shown that many NO-NSAIDs are indeed safer to the gastro-intestinal mucosa than the parent NSAID. [12–16]

Some NO-NSAIDs can form quinone methide intermediates [17–19], questioning the role of NO in their biological activity, others yet have very high IC50s for cell growth inhibition [20].

Hydrogen sulfide (H2S) is also produced by the gastric mucosa, and like NO, contributes to the

ability of this tissue to resist damage induced by luminal substances. [21]

This has led to a new class of NSAIDs possessing an H2S-releasing moiety (H2S-NSAIDs) that

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4 Given the evidence that both NO and H2S may have crucial roles in anti-inflammation, and the fact

that cancer may be viewed as an inflammatory disease [28], Dr. Kashfi of the Sophie Davis School of Biomedical Education - New York, postulated that a new hybrid that incorporated the active parts of each compound might be more potent than either one alone.

This hypothesis has proved to be correct leading to the synthesis and characterization of a new class of anti-inflammatory compounds termed NOSH-compounds (nitric oxide- and hydrogen sulfide-releasing compounds) that have IC50s for cell growth inhibition in the low nano-molar range. (Fig.3)

Fig.3: Representation of NOSH-ASA chemical structure.

The cell growth inhibitory properties of several NOSH-compounds were evaluated in eleven different human cancer cell lines of six different tissue origins (colon, breast, pancreas, lung, prostate and leukemia).The lead compound, NOSH–aspirin (NOSH–ASA, NBS-1120), although highly potent, showed very limited cyto-toxicity: lactate dehydrogenase (LDH) release at 4 times its IC50 for cell growth inhibition was less than 10% at 24h.

This compound also showed strong anti-inflammatory properties that were comparable to aspirin, as demonstrated by measuring the in vivo carrageenan-induced rat paw edema, and direct measurement of cyclooxygenase-dependent production of PGE2. [29]

In 2011, I tested the effect of NOSH-ASA in inhibiting colon cancer cell growth in vivo, working in the Dr. Kashfi’s lab as a volunteer.

The experiment was performed on established tumors in a xenograft model of human colon cancer cells. Male athymic SKID mice were implanted (with a subcutaneous injection) in the right flank with HT-29 (2 x 106) cells suspended in 50% Matrigel, after 10 days the animals were randomly divided into 4 groups (N = 7/group) and gavaged daily with NOSH-aspirin (25 or 50 or 100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 18 days of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies.

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5 The results showed that NOSH-aspirin had no effect on the weight of the mice even at the highest concentration used. In fact the average weight of the mice in each group was comparable at the beginning and end of the study, 21.8 ± 0.98 – 28.8 ± 1.9 g in the untreated mice and 21.7 ± 0.82 – 27.2 ± 1.1 g in the treated mice. There were no overt signs of toxicity.

NOSH-ASA dose-dependently reduced tumor mass by 50 ± 7%, 75 ± 5%, 90 ± 3 % at 25, 50, and 100 mg/kg, respectively. Tumor volume was also dose-dependently reduced as a function of time. (Fig. 4; 5; 6; 7) Some mice were also treated with aspirin (50 mg/kg) for comparsion reasons. At this dose, the salicilate content of aspirin was 1.3 -times that of NOSH-ASA at 100 mg/kg.

Fig.4 Fig.5

Fig.4: NOSH-ASA inhibits tumor xenograft growth. Athymic nude mice were injected

subcutaneously with HT-29 cells for the development of tumors. The reduction of tumor volume in Nude mice can be seen after treatment with different concentration of NOSH-ASA.

Fig.5: Graphic representation of the reduction of tumor volume in Nude mice as a function of time.

Fig.6 Fig.7

Fig.6: Representative tumor sizes for untreated and treated mice with different concentration of

NOSH-ASA, compared with aspirin.

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6 NOSH-aspirin inhibited growth of HT-29 colon cancer cells xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells). NF-kB, a transcription factor activated in control xenografts that can contribute to tumorigenesis, was significantly inhibited by NOSH-aspirin. [30] (Fig.8)

We are currently studying the molecular targets of these interesting compounds with respect to cell growth inhibition.

Some of the hypothetical targets which I investigated were FoxM1 and P53.

The Fox family of transcription factors is a large group of proteins that share a common DNA binding domain termed forkhead domain. The gene encoding human FoxM1 maps to chromosome 12p13.33. [31;32] The forkhead-family transcriptional activator is considered to be a master regulator of the cell cycle by controlling the expression of genes that are crucial for G1/S and G2/M

progression.

Cellular proliferation involves stimulation of the mitogen-activated protein kinase (MAPK) pathway, consisting of the Ras/Raf1/MEK/MAPK cascade, and activation of the phosphoinositol 3-kinase (PI3K) pathway, consisting of the PI3K/phosphoinositide-dependent 3-kinase 1 (PDK1)/Akt cascade. [33;34] However, cell division is tightly regulated at the G1/S (DNA replication) and G2/M

(mitosis) transitions of the cell cycle by temporal activation of multiple cyclin-dependent kinases (Cdks) complexed with their corresponding cyclin regulatory subunits. [35]

Cdk2-cyclin E or A and Cdk1-cyclin B kinase activity is essential for progression through the G1/S

and G2/M transitions of the cell cycle, respectively. Furthermore, Cdk activity is negatively

regulated by phosphorylation of Thr 14 and Tyr 15 by the dual specific Myt1 kinase (Wee1). [36] Cdk can be reactivated by a dual specificity phosphatase of the Cdc25 family (a and b). In addition, Cdk activity requires phosphorylation of Thr 160 by the Cdk-activating kinase (CAK) protein, which is also a direct target for MAPK phosphorylation. [37]

Fig.8: NOSH-ASA inhibits proliferation, induces apoptosis and decreases NF-kB

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7 Cdk2 activity in complex with either cyclin E or cyclin A cooperates with cyclin D-Cdk4 or Cdk6 to phosphorylate the retinoblastoma (RB) protein, which results in release of bound E2F transcription factor, allowing stimulation of genes required for S phase. [38] (Fig.9)

The CDK inhibitor proteins p21cip1 and p27kip1 are phosphorylated by the Cdk2-cyclin E complex and are then recognized by the specificity subunits ( Skp2 (S phase kinase-associated protein 2) and Cks1 (CDK subunit 1)) of the SCF (Skp1-Cullin1-F box) ubiquitin ligase complex, which targets them for ubiquitin-mediated proteasome degradation. [39;40]

The Cdk1-cyclin B complexes drive M-phase progression through phosphorylation of protein substrates that are essential for chromosome segregation, breakdown of the nuclear envelope, and cytokinesis.

FoxM1 stimulates trascription of multiple target genes critical for progression through the cell cycle including Cyclin B,Cdc25b phosphatase, Plk1 and Cenpf (centromere protein F: interactors of the kinetochore complex crucial for proper chromosome segregation). FoxM1 also reduces nuclear levels of the CDK inhibitor proteins p27 and p21 through the transcriptional activation of the specificity subunits Skp2 and Cks1 of the SCF ubiquitin ligase complex. [41] In addition FoxM1 is implicated in regulation of Survivin (apoptosis inhibitor) and Aurora B kinase (involved in the recognition of the correct chromosome attachment to spindle microtubules). [42]

The transcriptional activation function of FoxM1 depends upon phosphorylation of Thr 596 in the C-terminal activation domain by Cyclin/Cdks complex, which serves as priming phosphorylation for further phosphorylations by Polo-like kinase 1 (Plk1), which results in recruitment of the p300/CREB-binding protein (CBP) histone acetyltransferase proteins that act to increase the expression of their target genes. [43]

The transcriptionally active, phosphorylated FoxM1 accumulates as the cells progress through the cycle. At the end of M-phase, FoxM1 becomes dephosphorylated by the phosphatase PP2A, and in early G1-phase of the next cycle, it is polyubiquitinated by APC (anaphase-promoting complex)/E3

ubiquitin ligase for degradation by the proteasome. The degradation of FoxM1 in the early G1-phase

is important for regulated entry into S-phase. Thus, in proliferating cells, FoxM1 is synthesized and degraded in every cycle of cell division. Synthesis of FoxM1 in early G1-phase, or during a

transition from G0 to G1-phase, is stimulated by growth factors. (Fig.9)

Therefore, it is not surprising that FoxM1 expression is restricted to proliferating cells. Fig.9: Cdk/cyclin

complexes regulate Rb/E2F- and FoxM1-mediated transcription.

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8 In adult mammals, FoxM1 expression is detected mainly in the progenitor and regenerating tissues, and it is abundantly expressed in human carcinomas that originate from different tissues and drives tumor development by stimulating proliferation. FoxM1 expression is upregulated, and in many cases required, in several solid human cancers types including liver, breast, lung, prostate, cervix of uterus, colon, pancreas and brain. [44;45]

Overexpression of FoxM1 in various tumors indicates a strong dependence of the tumor cells on FoxM1, and that is explained partly by its role in cell proliferation. In addition FoxM1 has been implicated in several cancer-related process such as angiogenesis, tumor invasion and metastasis through induction of matrix metalloproteinase MMP as well as vascular endothelial growth factor VEGF. [46] These FoxM1 functions are partially inhibited by the tumor suppressor p19-arf, Cdk inhibitor belongs to the INK4 class. [47] (Fig.10)

FoxM1 expression is also induced by oncogenes. For example, activated RAS increases expression of FoxM1 by inducing the cellular levels of the reactive oxygen species. FoxM1 functions in a negative feedback loop to attenuate the levels of reactive oxygen species (ROS) by stimulating expression of the antioxidant genes Superoxide Dismutase (MnSOD), Catalase, and Peroxiredoxin 3 (PRDX3). [48] This ROS-regulatory function of FoxM1 protects proliferating normal or tumor cells from oxidative stress and promotes survival.

The functions of FoxM1 in expression of the cell division genes, and in attenuation of oxidative stress, are significant for cancer development and progression. Consistent with that, expression and the transcriptional activity of FoxM1 are regulated by the tumor suppressor genes. For example, expression of FoxM1 is negative regulated by p53. [49]

Fig.10: FoxM1 expression is stimulated by oncogenes (e.g.RAS) and growth factors and inhibited

by p53. Rb and p19Arf inhibit activity of FoxM1. FoxM1 stimulates expression of genes involved in cell division, attenuation of oxidative stress, tumorigenicity and various steps of tumor metastasis.

The TP53 gene, which resides on chromosome 17p13.1 and encodes the p53 protein, is a major tumor suppressor that plays an important role in response to damaged DNA, directing such processes as apoptosis, cell cycle arrest and senescence.

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9 The p53 protein functions as a trascription factor by binding specific DNA sequences and regulating trascription from promoters containing those sequences. [50]

In normal cells, DNA damage first induces an increase in p53 levels by inhibiting the normal rapid turnover of the protein, leading to a quick accumulation of p53 in stressed cells. Second, a conformational change forces p53 to be activated as a transcription regulator in these cells.

p53 is normally targeted for ubiquitin-dependent proteolysis by association with the Mdm2 protein, which transports p53 from the nucleus to the cytosol. [51] MAPK, ATM/ATR (ataxia telangiectasia mutant and Rad3-related protein kinase) and Chk1/Chk2 (checkpoint kinases) inhibit this association by phosphorylation of p53 on specific amino-terminal residues. Phosphorylation also facilitates acetylation of p53 carboxy-terminal residues by TIP60 (that belongs to the MYST histone acetyltransferases family).These modifications increase the affinity of p53 for its DNA binding site and hence increase its ability to activate trascription.

Activated p53 binds DNA and activates expression of several genes including p21, which binds to the CDK2 inhibiting the cell cycle progression into the S phase, acting as the “stop signal” for cell division and promoting the activation of DNA repair proteins. [52] The progression into the M phase also requires Cdc2, which can be inhibited by p21, GADD45 (Growth Arrest and DNA Damage) or 14-3-3 proteins. p53 regulates the expression of these inhibitory proteins to induce growth arrest. (Fig.11) Activation of the MAPK pathway via ERK can be induced by ROS production, leading to an increase of p21 protein and cell cycle arrest.

If DNA damage proves to be irreparable p53 can initiate apoptosis, the programmed cell death via the positive transcriptional regulation of its target Puma (Bcl2 binding component 3) belonging to the BH3-only subgroup of Bcl-2 family proteins. [53]

PUMA interacts with anti-apoptotic Bcl-2 family members such as Bcl-xL and Bcl-2 inhibiting their interaction with the pro-apoptotic molecules, Bax and Bak. When the inhibition of these is lifted, they result in the translocation of Bax and activation of mitochondrial dysfunction resulting in release of mitochondrial SMAC (small mitochondria-derived activator of caspases), cytochrome c and apoptosis-inducing factor (AIF).

SMAC binds to inhibitor of apoptosis proteins (IAPs) and deactivates them, preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed.

Once cytochrome C is released it binds with Apoptotic protease activating factor - 1 (Apaf-1) and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3 leading to cell death. [54] (Fig.12)

Fig.11: The role of

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P53 protein, in addition to its role as regulator of gene transcription, also has a pro-apoptotic effect on direct mitochondrion, participating in protein interactions with members of pro- and anti- apoptotic Bcl-2 family. p53 needs to be phosphorylated when it acts as a transcription factor while its post-translational modifications are not necessary when it promotes mitochondrial apoptosis through protein-protein interaction. [55]

Apoptosis needs to be highly regulated since defects in this process will lead to extended cell survival and may contribute to neoplastic cell expansion.

In fact TP53 is functionally inactivated in 50% of human cancers and altered by point mutations in other 50%.

The great majority of the missense mutations are clustered within the central most conserved region of p53 that spans the DNA-binding domain, and among these are a small number of ‘‘hot spot’’ residues that occur with unusually high frequency (i.e. 248, 273, 175, 245, 249 and 282). [56] Many of these stable mutant forms of p53 can exert a dominant negative effect on the remaining wild-type allele, serving to abrogate the ability of wild-type p53 to inhibit cellular transformation and to acquire functions that promote tumor growth.

Wild type p53 executes its tumor suppressor activity mainly via the negative transcriptional regulation of a number of transcription factors that control cell growth and survival, such as, c-Myc or FoxM1, or other genes such as Plk1. [57;58]

P53 is required for the downregulation of FoxM1. After DNA damage, p53 facilitates the repression of FoxM1 mRNA, which is accompanied by a decrease in FoxM1 protein levels.

Down regulation of FoxM1 inhibits cell proliferation and induces cell cycle arrest with reduced expression of cyclin B1, cyclin D1, Cdk2 and increased expression of the cell cycle inhibitors, p21 and p27.

Therefore inactivation of p53 in tumors could partially explain the phenomenon of FoxM1 overexpression in human cancers.

Modulation of FoxM1, as an oxidative stress associated gene, could enhance the efficacy of chemotherapeutic drugs which exert their cytotoxic effects by oxidative stress.

Since FoxM1 suppression is necessary for full p53-dependent responses to stress/treatments and appears to inhibit tumorigenesis, chemical compounds that target FoxM1 and P53 may act as anticancer drugs.

Fig.12: The role of

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2.0 Thesis Objective

My research was carried out in the Dr. Kashfi’s lab in the department of Physiology and Pharmacology of The Sophie Davis School of Biomedical Education at the City College of the City University of New York – U.S.

In the present study I evaluated the effect of 9 different NOSH-compounds on HT-29 and HCT-15 colon cancer cells growth, obtained with cell culture techniques.

Electron donating/withdrawing groups have been incorporated about the benzyl ring in order to evaluate the effect of substitution on stability and biological activity of these novel compounds. In particular, I compared the cell growth inhibitory properties of ortho-NOSH-aspirin (in which the two moieties that release NO and H2S are covalently linked at the 1, 2 positions of acetyl salicylic

acid) to that of the 1, 3 (meta-NOSH-aspirin) and the 1, 4 (para-NOSH-aspirin) positional isomers. Data was measured using a colorimetric MTT assay kit (Roche, Indianapolis, IN) and compared with biostatistical analysis.

I also analyzed the effect of the positional isomers of NOSH-aspirin on HT-29 colon cancer cell kinetics (proliferation, cell cycle transition and apoptosis) and ROS levels, in order to confirm the potential anti-growth activity of NOSH-ASA and define the lead compound.

Data was measured by ELISA immunoassay and flow cytometry using a FACS Calibur (BD Bioscience).

Finally, I identified some of the lead compound’s molecular targets (P53 and FoxM1), analyzing their expression in HT-29 colon cancer cells with the Western Blot technique.

These interesting data could be a platform to understand the molecular mechanisms of NOSH-aspirin action.

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3.0 Materials and Methods

3.1 Reagents

9 different NOSH-compounds are classified in numerical order on the basis of their chronological synthesis from the first one (ortho-NOSH-aspirin).

 NOSH-ASA (ORTHO-NOSH-aspirin) 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-1 (PARA-NOSH-aspirin) 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 4-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-2 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3-chloro-4-((4-(nitrooxy)butanoyl)oxy)benzoate

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13  NOSH-3 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 5-chloro-2-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-4 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-methoxy-5-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-5 (META-NOSH-aspirin) 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-6 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-chloro-3-((4-(nitrooxy)butanoyl)oxy)benzoate

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14  NOSH-7 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3-methoxy-4-((4-(nitrooxy)butanoyl)oxy)benzoate  NOSH-8 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 5-methoxy-2-((4-(nitrooxy)butanoyl)oxy)benzoate 3.2 Cell Culture

Most cell culture handling was carried out in Class II Biological safety cabinets in order to keep to a minimum the environmental contamination.

HT-29 (ATCC® HTB38™) and HCT-15 (ATCC® CCL-221™) human colon adenocarcinoma were obtained from American Type Tissue Collection (Manassas, VA).

The HT-29 cell line is positive for expression of both COX-1 (cyclo-oxygenase) and COX-2 enzymes, instead HCT-15 cells are COX null. [59]

In HT-29 line there is a G → A mutation in codon 273 of the p53 gene resulting in an Arg → His substitution. The p53 antigen produced by HCT-15 colorectal cell line has a C → T mutation resulting in Ser → Phe at position 241.

These “hot spot” missense mutations in TP53 gene are found in many malignancies with an higher frequency than expected by chance. This fact is often cited to support a gain of function hypothesis, which states that mutations in p53 confer novel activities to support oncogenic transformation. HT-29 and HCT-15 human colon cancer cells were grown as monolayer (adherent cultures) in McCoy 5A and RPMI-1640 media, respectively.

The culture medium is the most important component of the culture environment, because it

provides the necessary nutrients, growth factors, and hormones for cell growth, as well as regulating the pH and the osmotic pressure of the culture.

All media were supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin (50 U/ml) and streptomycin (50 µg/ml) (Invitrogen, Carlsbad, CA).

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15 Cells were seeded on culture dishes making a 1:5 dilution (1 unit volume of cells + 4 unit volume of medium)and incubated at 37°C in 5% CO2 and 90% relative humidity.

Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. In this case the medium needs to be removed directly by aspiration and changed.

Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density.Passaging (or splitting cells) involves transferring a small number of cells into new dishes. For adherent cultures, cells first need to be detached from the growth surface. This was obtained by trypsinization.

The medium was aspirated from the culture plate and a mixture of 0.05% trypsin / EDTA

(EthyleneDiamineTetraacetic Acid solution to remove all traces of serum which contains trypsin inhibitor) was added in it. Cells were incubated at 37°C for 3-5 min, monitoring periodically cell detachment under a microscope. The proteolysis reaction quickly was terminated by addition of complete medium containing serum. After spinning down cells in a culture tube with centrifuge (1200 rpm for 5 min), supernatant was removed and finally replaced with fresh medium.

Another cell harvesting method was obtained by scraping.

The old medium was aspirated from the colture plate and cells were washed with sterile 1X PBS (phosphate buffered saline) for two times. Cells in 1mL of PBS were harvested using a cell scraper and put into microcentrifuge tube to spin at 1200 rpm for 10 min at 4°C. The supernatant were discarded by decanting.

Scraping is often the recommended subculturing method for cell lines that have surface receptors or antigens that could be destroyed by using a digestive enzyme such as trypsin. It can be used when preparing cell lysates (liquid preparations of cell contents for analysis).

Cells were counted using a hemocytometer. 10µL of cells were placed on it and put under the microscope to observe the density and number of cells. The cells only in the outer four quadrants were counted. (Fig.13)

Cell density was measured as: total number of cells in 4 quadrants/ 4*104 and multiple by dilution factor.

For cryopreservation of the complete growth media, the final DMSO (dimethyl sulfoxide) concentration was adjusted to 1%.

Fig.13: Grid

system of hemocytometer.

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3.3 Determination of cell growth inhibition by MTT assay

Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population’s response to external factors.

Cell growth inhibitory effect of all NOSH-compounds was measured using a colorimetric MTT assay (Roche, Indianapolis, IN).

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity.(Fig.14) [60]

Since for most cell populations the total mitochondrial activity is related to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects of drugs on cell lines.

In particular, the yellow tetrazolium MTT is reduced by the action of NAD(P)H-dependent cellular oxidoreductase enzymes, largely in the cytosolic compartment of the cell, to generate its insoluble formazan, which has a purple color. The resulting intracellular formazan can be solubilized with a Detergent Reagent (usually a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) and quantified by spectrophotometric means. (Fig.15)

Fig.15: The chemical reaction produces a purple-colored Formazan within the cells.

Therefore, reduction of MTT dyes increases with cellular metabolic activity due to elevated NAD(P)H flux. In fact the MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The MTT Reagent yields low background absorbance values in the absence of cells. For each cell type the linear relationship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.

Fig.14: MTT reduction

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17 KIT components:

-MTT Reagent (25 mL)

-Detergent Reagent (2 x 125 mL)

Cancer cells were plated in 96-well plates at a density of 30000 cells/well. The cells were incubated for 24h with different concentrations of NOSH-compounds, following the protocol below.

1. After trypsinization resuspend the cells in fresh medium at 1x106 cells/mL.

2. Diluite drugs from the stock (100 mM) with DMSO (dimethyl sulfoxide) right before adding to

the cells. Make serial dilutions to obtain different concentration of the drugs: 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.1mM and 0.5 mM. (Fig.16)

3. Take 7 eppendorf (one for the control and the other six eppendorf for the different concentrations

of the drug) for each 9 NOSH-compounds.

4. Put 1 mL of cells+medium in each eppendorf 5. Add 1 µL of DMSO for the blanks

6. Add 10 µL of different concentration of drugs in the 6 eppendorf

7. Plate out, in triplicate, 100 µL of the dilutions into wells of a microtiter plate. Include three

control wells of medium-DMSO alone to provide the blanks for absorbance readings. (Fig.17)

8. Incubate the cells overnight

Fig.16: Serial

dilution of drug from the stock.

Fig.17: Example of

96 well-plate used for MTT assay.

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18 After the indicated time, 10 µL of MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, 5 mg/mL in phosphate buffered saline), was added to each well, and the plates were incubated for 2h at 37°C.

When the purple precipitate was clearly visible under the microscope, the media was aspirated, and 100 µL of the solubilization solution (Detergent Reagent: 10% SDS in 0.01 M HCl) was added to all wells to solubilize the formant crystals. The plate was covered in the dark for 1 hour.

The absorbance of the plates was measured on a spectrophotometric plate reader at a wavelenght of 570nm.

I expected that the absorbance values of the treated cells were lower than the control cells indicating a reduction in the rate of cell proliferation. To quantify the cell growth inhibitory effect of all NOSH-compounds I calculated the IC50s (the half maximal inhibitory concentration) of each NOSH-compounds and compared them to see if there was any difference between the compounds or different effects between HT-29 and HCT-15 cell lines.

IC50 represents the concentration of a drug that is required for 50% inhibition in vitro.

This quantitative measure indicates how much of each NOSH-compound is needed to inhibit the cell growth by half.

The IC50 of a drug can be determined by constructing a dose-response curve.

From triplicate readings of the absorbance, I determined the average and divided it by the average value for the blank, multiplied by 100. Then I arranged data with cellular metabolic activity on vertical axis and concentration of the drug on horizontal axis; the IC50 is the concentration at which the curve passes through the 50% inhibition level. (Fig.18)

Fig.18: Calculation of the IC50 for cell growth inhibition. For the example I used data obtained

from HCT-15 human adenocarcinoma cell line treated with different concentration of NOSH-ASA.

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19  Statistical analysis

In vitro data, that were obtained from all NOSH-compounds of the HT-29 and HCT-15 cell lines, are presented as mean ± SEM (standard error of the mean) for 3-5 different sets performed in triplicate.

Mean= (X1 + X2 + … + XN) / N

SEM= standard deviation (DS) / radq(n) DS= radq (variance)

VARIANCE= dev / degrees of freedom (n-1) Deviance= ∑(Xi – mean)^2

Comparison between groups (HT-29 and HCT-15 human adenocarcinoma cell lines) treated with positional isomers of NOSH-aspirin (ortho, para and meta NOSH-asa) was done using Student’s t tests; p-values <0.05 were considered significant.

In a statistical test, the p-value is the probability of getting the same value for a model built around two hypotheses, one is the "null" hypothesis (H0), the other is the hypothesis under testing (H1). If this p-value is less than or equal to the threshold value previously set (traditionally 5% or 1%), one rejects the null hypothesis and accepts the test hypothesis as valid. [61]

Such a result indicates that the observed result would be highly unlikely under the null hypothesis. P< 0.05 (or 0.01): strong presumption against null hypothesis

P> 0.05 (or 0.01): no presumption against the null hypothesis

The p-value does not in itself support reasoning about the probabilities of hypotheses, nor choosing between different hypotheses; it is simply a measure of how likely the data (or a more "extreme" version of it) were to have occurred by chance, assuming the null hypothesis is true.

Since the data are hypothesized to follow the normal distribution and the observations within each sample and groups were obtained indipendently, I can use unpaired t-test as one of the statistical hypothesis test.

The t test compares the size of the difference between means with the standard error (SE) of that difference:

t = difference between means / SE of difference

SE of difference= dev pooled * radq (1/n +1/n) VAR pooled= ∑deviance / ∑degrees of freedom

The degrees of freedom equal the number of pairs minus one: dF= (n-1) Deviance= ∑X^2

– TC TC (correction term)= (∑X)^2

/n

My H0 hypothesis is that there are no statistical differences beetween groups: ortho vs meta, ortho vs para, meta vs para.

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20 I can also compare the Student’s t tabulated value with the calculated one:

If |t|< |t(0.05, n-2)| I do not reject the H0 hypothesis.

3.4 Drug treatment

The HT-29 cells (1 x 106 cells/mL) were treated with the positional isomers of NOSH-ASA, according to their different IC50 for cell growth inhibition, for further analysis. (Fig.19)

For each compound (ortho, meta and para NOSH-aspirin) I took four falcon tubes and put in the new medium with different concentration of the drug corresponding to

- control (only DMSO) - ½ x IC50

- IC50 - 2 x IC50

The old medium was aspirated from colture dish and the mix (new medium+drug) was added in it; cells were incubated at 37°C in 5% CO2 and 90% relative humidity usually for 1-2 hours.

a b c Fig.19:Photos representing three phases of drug treatment: a- medium added b- drug added c- colture dishes that need to be treated with drug.

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21

3.5 Analysis of cell proliferation by PCNA ELISA immunoassay

The immunoassay techniques are used for detection or quantification of a substance based on an immunological reaction.

Proliferating cell nuclear antigen (PCNA) was determined using an ELISA (enzyme-linked immunosorbent assay) Kit (Calbiochem, La Jolla, CA), in accordance with the manufacturers protocol.

The protein encoded by PCNA gene is found in the nucleus and is a cofactor of DNA polymerase delta. In fact the homotrimeric PCNA and its adapter protein replication factor C (RFC) cooperate to form a moving clamp that is an attachment point for DNA polymerases δ, increasing the processivity of leading strand synthesis during DNA replication. [62]

The clamp loaders (RFC) function is to temporally open the sliding clamp to allow DNA strands to pass through. To transfer DNA into the center of the PCNA clamp, there is a common ATP dependent mechanism involved. If ATP hydrolysis occurs, a stable clamp on DNA is formed and interactions between RFC and PCNA become weak and are disrupted. If polymerase delta exists, its recruitment links with this step. Reverse reaction of this loading will unload PCNA from DNA. (Fig.20) Through these cycles, multiple PCNA molecules can be loaded at the 3' end of DNA by interaction with RFC.

When PCNA is loaded onto DNA, it is also capable of binding to other proteins involved in DNA replication, recombination and repair, including various DNA nucleases (such as Flap endonuclease 1 (FEN1) or the nucleotide excision repair protein XPG), DNA polymerase Epsilon, DNA ligase-I, the mismatch repair proteins MLH1 and MSH2 and the member of PRR (post replication repair) pathway RAD6. PCNA also has a function in post-replicative DNA processing such as methylation (interacting with DNA methyltransferase).

PCNA (most often under the negative control of P53) can commit cells to cell cycle arrest and repair of DNA damage, or, when repair is not possible, absence or low levels of functional PCNA may drive cells into apoptosis.

A role for PCNA in cell cycle control is recognized by its interaction with cell-cycle regulators that play multiple roles in cell-cycle progression and checkpoint control proteins such as cyclins (D and A), cyclin-dependent kinases (CDK2, CDK4 and CDK5) and the CDK-inhibitor p21 protein [63;64].

Fig.20: Loading and

unloading pathway of PCNA at the 3' end of DNA by interaction with RFC.

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22 In addition, the role of PCNA as a component of the cell cycle control apparatus is demonstrated by its association with the growth-arrest and DNA-damage inducible proteins gadd45 (p53-inducible) and MyD118. [65]

These varied interactions with DNA metabolic proteins imply that PCNA is a central factor for the coordination of DNA replication, DNA repair, epigenetic inheritance and cell-cycle control.

Since PCNA is present at very low levels in quiescent cells, it can be a significant marker for detecting the transition of a non-proliferating cell population to a proliferating cell population. [66] The PCNA ELISA is a “sandwich” enzyme immunoassay employing a primary rabbit polyclonal antibody and the secondary mouse monoclonal antibody clone PC10.

A polyclonal antibody, specific for the human PCNA protein (our analyte), has been immobilized onto the surface of microtiter wells provided in the kit.

Cells containg antigen in suspension buffer are pipetted into the wells followed by incubation with biotinylated detector monoclonal antibody for two hours, during which time any PCNA present binds to the capture and detecting antibodies. (Fig.21)

Unbound material is washed away and horseradish peroxidase-conjugated streptavidin is added, which binds to the detector antibody. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate TetraMethylBenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stop reagent), the intensity of which is proportional to the amount of PCNA protein in the sample.

The colored reaction product is quantified using a spectrophotometer. The absorbance of the solutions in the wells was measured at 450 nm.

Quantitation is achieved by the construction of a standard curve using known concentrations of PCNA (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of PCNA with that obtained from the standards, the concentration of PCNA in the sample can be determined.

Materials Provided:

• Coated Microtiter Plate: 96 wells coated with PCNA polyclonal antibody. • PCNA Standard: two vials containing lyophilized PCNA protein.

• Detector Antibody: biotinylated monoclonal anti-human PCNA antibody, clone PC10. • 400X Conjugate: Streptavidin-Peroxidase Conjugate; 400-fold concentrated solution.

Fig.21: Illustration

of the “sandwich” enzyme-linked immunosorbent assay (ELISA).

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23 • Conjugate Diluent: buffer for dilution of 400X Conjugate.

• Substrate: chromogenic substrate (TMB).

• Sample Diluent: buffer used to dilute standards and samples.

• 20X Plate Wash Concentrate: 20-fold concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.

• Antigen Extraction Agent (AEA): used for the extraction of PCNA from cell preparations. • Stop Solution: 2.5 N sulfuric acid.

• Plate Sealers: to cover plates during incubations.

Materials Required But Not Provided:

• Cell Resuspension Buffer: 50 mM Tris-HCl pH 8.0, containing 5 mM EDTA (Ethylenediaminetetraacetic acid), 0.2 mM PMSF (phenylmethylsulfonyl fluoride), 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin.

Preparation of the samples was obtained following the Cell Lysate Protocol:

1. Remove the medium from the plates with HT-29 cells (1 x 106 cells/mL) treated for 24 h with various concentrations of the positional isomers of NOSH-ASA. Wash cells twice with 1X PBS.

2. Add 200 µL of Antigen Extraction Agent (AEA provided) to 1 mL of Resuspension Buffer. 3. Aspirate PBS from the culture dishes and resuspend the cells in 400 µL of Resuspension Buffer +

AEA.

4. Harvest the cells by scraping and transfer extracts to microcentrifuge tubes. 5. Incubate 30 minutes on ice with occasional vortexing.

6. After this time, centrifuge for 5 minutes at 1500 rpm at 4°C.

7. Aliquot cleared lysate to clean microfuge tubes to avoid multiple freeze/thaws.

Samples must be frozen at –20°C for 18-24 hours prior to testing for PCNA levels in the assay. These samples are now ready for analysis.

PCNA ELISA protocol.

Each time an assay is performed, Lyophilized Standard needs to be reconstituite

1. PCNA standard must be diluted with 1 mL of sample diluent. Let the reconstituted standard sit

for 15 minutes at room temperature.

2. After reconstituting the PCNA Standard it should be diluted with Sample Diluent.

Obtain six tubes and label them 30, 15, 7.5, 3.75, 1.875 and 0 ng/mL.

Add 250 µL of Sample Diluent into each tube except the 30 ng/mL tube (first tube) which gets “undiluted” reconstituted standard.

Remove 500 µL from the original vial of lyophilized material and add it to the first tube.

Remove 250 µL from the first tube (30 ng/mL) and add it to the second tube (15 ng/mL) and mix gently. Repeat this procedure until you reach the fifth tube (1.875 ng/mL).

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24

3. All sample (cell lysate) must be diluited in 1:5 dilution with sample diluent.

(For 12 sample, add 250 µL of sample diluent to 50 µL of sample).

Samples found to contain greater than 30 units/ml PCNA (outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the PCNA concentration falls within the range spanned by the standard curve, and assayed again.

4. Take the 96-well plate and pipette 50 µL of the Detector Antibody into each well.

5. Add samples and each of the PCNA standards (in duplicate) by pipetting 50 µL into appropriate

wells using clean pipette tips for each sample.

6. Cover wells with a plate sealer and incubate at room temperature for 2 hours.

7. Prepare a working solution (1X) of Wash Buffer by adding 50 mL of the 20X concentrated

solution (provided), to 950 mL of deionized water or half dose of both reagents. Mix well.

8. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.

9. Dilute 400X by adding 30 µL into Conjugate Diluent solution provided (12 mL), mix gently. 10. Pipette 100 µL of the 1X Conjugate into each well, cover with a plate sealer and incubate at

room temperature for 30 minutes.

11. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely. 12. Flood entire plate with dH2O. Remove contents of wells by inverting over sink and tapping

on paper towels.

13. Add 100 µL of Substrate Solution to each well and incubate in the dark at room temperature

for 30 minutes.

14. Add 100 µL of Stop Solution to each well in the same order as the previously added

Substrate Solution.

15. Measure absorbance in each well using a spectrophotometric plate reader. A wavelength of 450

nm can be used. Wells must be read within 30 minutes of adding the Stop Solution.

Results were evaluated by constructing a standard curve.

I determined the average of the duplicate absorbance value for each standard, including zero, and all sample value and plotted the mean absorbance values for each of the standards on the Y axis,

versus the concentration of each standard (U/mL) on the X axis. (Fig.23)

The concentration of unknowns was determined by interpolation from the standard curve. For samples which have been diluted, the PCNA concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the PCNA value obtained from the standard curve must be multiplied by five).

Fig.22: Example

of the PCNA Standard dilution

with Sample Diluent.

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25

3.6 Flow cytometric analysis

Flow cytometry is a biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering. About my thesis work, flow cytometry has been used for apoptosis and cell cycle assays and for the determination of reactive oxygen species.

It is a rapid technique allowing simultaneous analysis of multiple cellular parameters, including DNA content. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits ﬂuorescence.

A ﬂow cytometer is made up of three main systems: ﬂuidics, optics, and electronics. (Fig.24)

The ﬂuidics system transports particles in a stream to the laser beam for sensing.

The optics system consists of lasers to illuminate the particles in the sample stream and optical ﬁlters to direct the resulting light signals to the appropriate detectors.

The electronics system converts the detected light signals into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deﬂect particles. [67]

Fig.24: Scattered and

emitted light signals are converted to electronic pulses that can

be processed by the computer.

Fig.23: Standard curve.

The mean signal of each standard run.

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26 The technique relies on a single-cell suspension being passed within a stream of sheath fluid through an optically focused excitation light source, either a laser or an arc lamp. Lasers are commonly used because they provide a single wavelength of coherent light while arc lamps produce a mixture of incoherent wavelengths that must be filtered.

The portion of the ﬂuid stream where particles are located is called the sample core. (Fig.25)

For optimal illumination, the stream transporting the particles should be positioned in the center of the laser beam. In addition, only one cell or particle should move through the laser beam at a given moment. To accomplish this, the sample is injected into a stream of sheath fluid within the flow chamber. The design of the flow chamber causes the sample core to be focused in the center of the sheath fluid where the laser beam will then interact with the particles. Based on principles relating to laminar flow, the sample core remains separate, but coaxial within the sheath fluid.

The ﬂow of sheath ﬂuid accelerates the particles and restricts them to the center of the sample core. This process is known as hydrodynamic focusing. [68]

The sample pressure is always higher than the sheath ﬂuid pressure, but sample flow rate can be regolated by changing the sample pressure relative to the sheath one.

A higher ﬂow rate is generally used for qualitative measurements such as immunophenotyping. The data are less resolved, since the cells are less in line in the wider core stream, but are acquired more quickly. A lower ﬂow rate decreases the width of the sample core and restricts the position of the cells to a smaller area. The majority of cells passes through the center of the laser beam; thus the light illuminating the cells and emitted from the cells is more uniform. A lower rate is generally used in applications where greater resolution is critical, such as DNA analysis.

When particles pass through the laser intercept, they scatter laser light depends on the physical properties of a particle, namely its size and internal complexity. Factors that affect light scattering are the cell's membrane, nucleus, and any granular material inside the cell.

The amount of light scattered in the forward direction is detected in the forward-scattered channel (FSC) and its intensity is roughly proportional to the size of the cells or particles. Light scattered perpendicular to the path of the laser is detected in the side scatter channel (SSC) and its intensity is more closely related to granularity. (Fig.26) [69]

Fig.25: The particles are randomly

distributed when sample solution is injected into a flow cytometer. The sample is ordered into a single particle stream then can be interrogated by the machine’s detection system.

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27 Correlated measurements of FSC and SSC can differentiate cell types in a heterogeneous cell population. For example major leucocyte subpopulations can be differentiated with this tecnique.

Fig.26: Light-scattering properties of a cell.

If the cells have been stained with fluorochromes they will also emit fluorescence intensities at levels that directly correspond to the number or density of fluorochrome molecules on or within the cell.

Fluorochromes are typically attached to a monoclonal antibody that recognises a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure.

A ﬂuorescent compound absorbs light energy over a range of wavelengths that is characteristic for that compound and emits wavelengths that will be longer than those absorbed.

The argon ion laser is commonly used in flow cytometry because the 488-nm light that it emits excites more than one fluorochrome. One of these fluorochromes is fluorescein isothiocyanate (FITC). More than one fluorochrome can be used simultaneously if each is excited at 488 nm and if the peak emission wavelengths are not extremely close to each other.

The fluorescence signal emitted by any specific fluorochrome is collected through separate channel detectors (photomultipliers) by means of a series of optical filters and mirrors that guide the beam of light. (Fig.24) The photodetectors convert the light signals to electronic signals (voltages).

The specificity of a detector for a particular fluorescent dye is optimized by placing a filter in front of the photomultipliers, which allows only a narrow range of wavelengths to reach the detector. [70] This spectral band of light is close to the emission peak of the fluorescent dye. Such filters are called bandpass (BP) filters.

For example, the ﬁlter used in front of the FITC detector is labeled 530/30. This number gives the characteristics of the spectral band transmitted: 530 ±15 nm, or wavelengths of light that are between 515 nm and 545 nm.

Other filters used in the flow cytometer are shortpass (SP) filters, which transmit wavelengths of light equal to or shorter than a specified wavelength, and longpass (LP) filters, which transmit wavelengths of light equal to or longer than a specified wavelength. (Fig.27)

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28 Fig.27: Light transmittance through longpass, shortpass, and bandpass filters.

Another type of photodetectors is the photodiode, which is less sensitive to light signals than the photomultipliers and thus is used to detect the stronger FSC signal.

Once light signals have been converted to electronic pulses by the ADC (Analog-to-Digital Converter), the data are collected and stored in the computer.

The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates". A gate can also be set on the FSC vs SSC plot to restrict the analysis to a speciﬁc population within the sample.

The samples were acquired automatically using the Loader with acquisition criteria of 10,000 events for each tube. Flow cytometry data can be analyzed by specific software, as FlowJo.

 Cell cycle analysis

Analysis of cellular DNA content by flow cytometry can reveal the distribution of cells in three major phases of the cycle (Fig.28):

-G0, resting, non dividing cells, or any cycling cells in G1

-S phase (when DNA is synthesized) -G2 or in the process of Mitosis (M).

Fig.28: Cell cycle

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29 At different stages of the cell cycle, cell nuclei contain different amounts of DNA.

For example, after receiving signals for proliferation, diploid cells exit the resting state Gap 0 (G0)

phase and enter the Gap 1 (G1) phase. At this stage, the diploid cells maintain their ploidy by

retaining two complete sets of chromosomes (2N). As the cells enter the synthesis (S) phase, DNA replication starts, and in this phase, cells contain varying amounts of DNA. The DNA replication continues until the DNA content reaches a tetraploid state (4N) with twice the DNA content of the diploid state. Tetraploid cells in the G2 phase start preparing for division and enter the mitosis (M)

phase when the cells divide into two identical diploid (2N) daughter cells. The daughter cells continue on to another division cycle or enter the resting stage (G0 phase). Based on DNA content

alone, the M phase is indistinguishable from the G2 phase, and G0 is indistinguishable from G1.

Therefore, when based on DNA content, cell cycle is commonly described by the G0/G1, S, and

G2/M phases. [71]

The first step in preparing cells for cell-cycle analysis is permeabilization of the cells’ plasma membranes. This is usually done by incubating them in a buffer solution containing a detergent such as NP-40 or by fixating them in ethanol.

Most fluorescent DNA dyes are not membrane permeable, that is, unable to pass through an intact cell membrane. Permeabilization is therefore crucial for the success of the next step, the staining of the cells. [72]

The procedure involves using the propidium iodide (PI) as the DNA fluorochrome, that can quantify how much DNA is present within the cells. In fact PI requires blue light as the excitation source and it is easly excited by 488 nm argon ion laser.

When the cells pass through the flow cytometer’s laser, a fluorescence pulse is generated that correlates with the amount of dye associated with the DNA and thus with the total amount of DNA in the cell.

Because PI also stains double-stranded RNA, which interferes with the DNA signal, all the RNA is removed by the addition of RNase A to the staining solution.

This is a rapid method to study the mechanism of the action of drugs by means of alterations in the cell cycle distribution.

Experimental procedure:

1. HT-29 cells (1 x 106 cells/mL) were treated with various concentrations of positional isomers of NOSH-ASA, corrisponding to the IC50 of each compound, and harvested by scraping. After centrifuging at 2000 rpm for 10 min, the pellet was saved and washed once with 1X PBS.

Cells were pelleted again by centrifugation, throwing the PBS out.

2. Cells were suspended in 0.5 mL of 1X PBS, and fixed in 70% ethanol (3 mL) dropwise while

vortexing. This suspension was transferred into centrifuge tubes at -20°C for at least 2 h (usually stored for 24h).

3. The ethanol-suspended cells were centrifuged at 2000 rpm for 10 min at 4°C. The ethanol was

fully decanted.

4. Cell pellet was washed in 1mL of PBS containing 1% FBS/0.5% NP-40 on ice for 10 min and

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30

5. After taking off the remaining PBS cells were suspended in 500 µL of PBS/1% FBS containing

40 µg/mL propidium iodide and 200 µg/mL RNase type IIA (PI/Rnase staining buffer, BD Pharmingen) and incubated at 37°C in the dark for 30 min.

6. Samples were transferred to the FACS tubes for flow cytometric analysis.

7. Cell cycle phase distributions of control and treated HT-29 cells were obtained using a Coulter

Profile XL equipped with a single argon ion laser. The percentage of cells in G0/G1, S and G2/M

phases was determined from DNA content histograms. [73]

 Assay for apoptosis

Apoptosis is a normal genetically programmed process that occurs during embryonic development, as well as in maintenance of tissue homeostasis, under pathological conditions, and in aging.

The process is characterized by specific morphologic features, including loss of plasma membrane asymmetry and attachment, plasma membrane blebbing, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA.

The increase in apoptotic cells is reflected by changes in the light scatter properties for the untreated and treated tubes (FSC-A vs SSC-A plots). (Fig.29) During apoptosis, cell shrinkage occurs, which is associated with a decrease in forward scatter. Further, the formation of apoptotic vesicles

in the cells during apoptosis leads to an increased side scatter profile. [74]

Fig.29: The image shows how the light scatter properties

change from viable cells to apoptotic cells.

An easy way to acquire and analyze data from apoptotic cells is provided by Annexin V FITC Apoptosis Detection Assay (BD BioSciences Pharmingen, San Diego, CA), usually used as screening for drug toxicity. The kit provides a set of reagents for the detection of apoptosis using flow cytometry.

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31 KIT components:

-Annexin V-FITC

-Propidium Iodide Staining Solution -Annexin V Binding Buffer

Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). [75]

In apoptotic cells, after loss of plasma membrane asymmetry, the phospholipid PS is translocated from the inner to the outer leaflet of the plasma membrane,marking cells as targets of phagocytosis. Therefore Annexin V can easily bind the PS exposed to the apoptotic cell surface.

For flow cytometric analysis, Annexin V conjugated to fluorochromes can be used as a sensitive probe for the detection of exposed PS in cells undergoing apoptosis.

PS translocation precedes the loss of membrane integrity, which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes.

Therefore, staining with Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI), that binds to DNA by intercalating between the bases. Since PI is membrane impermeant and generally excluded from viable cells, it is used for identification of early and late apoptotic cells. In fact viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. (Fig.30)

Fig.30: Phosphatidylserine, normally located on the cytoplasmic surface of the cell membrane,

becomes exposed to the extracellular environment. Annexin V (human vascular anticoagulant with high affinity for PS), allows the detection of PS.

Thus, cells that are considered viable are both Annexin V and PI negative, while cells that are in early apoptosis are Annexin V positive and PI negative, cells that are in late apoptosis are both Annexin V and PI positive and cells already dead are Annexin V negative and PI positive. (Fig.31)

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32 Experimental Procedure

1. HT-29 cells (1 x 106 cells/mL) were treated from 0 to 24 h with various concentrations of the positional isomers of NOSH-aspirin, that correspond to the IC50 of each compound.

The medium was aspirated from the colture plate and cells were washed with sterile 1X PBS (phosphate buffered saline) for two times. Cells were harvested by scraping, using a cell scraper. Cells in 1mL of PBS were put into microcentrifuge tube to spin at 1250 rpm for 10 min at 4°C. The supernatant was discarded by decanting. Pellet can be frozen at -20°C to be utilized the next day, adding 1mL of 1X PBS, centrifuging and removing the supernatant.

2. Cells were washed and resuspended in 500µL of 1X Binding Buffer (Annexin V binding buffer,

0.1 M HEPES/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2; BD BioSciences Pharmingen, San

Diego, CA).

3. 200 µL of cells with binding buffer were transferred to FACS tube. For the correct setting of the

flow cytometer I needed to two different FACS tube that corresponded to FITC and PI in addition to the the sample tube and the unstained one.

4. 10 µL of Annexin V-FITC was added to the FITC tube, 10 µL of Propidium Iodide Staining

Solution was added to the PI tube and both FITC (10 µL) + PI (10 µL) to the sample tube.

5. The cells were incubated at room temperature for 20 min in the dark.

6. 400 µL of 1X Binding Buffer were added to each tube and mixed with a vortex mixer.

7. Percentage of apoptotic cells were obtained using a Becton Dickinson LSR II equipped with a

single argon ion laser. Data were analyzed by Flow Jo software.

Fig.31: Annexin V FITC-A vs Propidium Iodide-A (PI-A) with gates for following populations:

Q4. Annexin V–/PI– Q3. Annexin V+/PI– Q2. Annexin V+/PI+ Q1. Annexin V–/PI+

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33  Determination of reactive oxygen species (ROS)

I examined whether positional isomers of NOSH-aspirin induced ROS levels. ROS are chemically reactive molecules containing oxygen.

Most reactive oxygen species are generated as by-products during mitochondrial electron transport. In addition ROS are formed as necessary intermediates of metal catalyzed oxidation reactions. Atomic oxygen has two unpaired electrons in separate orbits in its outer electron shell. This electron structure makes oxygen susceptible to radical formation. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including: superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion and nitric oxide. (Fig.32) [76]

Exogenous ROS can be produced from pollutants, tobacco, smoke, drugs, xenobiotics or radiation. ROS are produced intracellularly through multiple mechanisms and depending on the cell and tissue types, the major sources being the producers of ROS NADPH oxidase (NOX) complexes in cell membranes, mitochondria, peroxisomes and endoplasmic reticulum.

Under normal physiologic conditions, cells control reactive oxygen species levels by balancing the generation of ROS with their elimination by a scavenging system (set of antioxydant that may be both enzymatic and non enzymatic in nature, as superoxide dismutase, catalase, glutathione peroxidase or peroxiredoxin, glutaredoxin and vitamins C, E). However, under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids, and DNA, giving rise to fatal lesions in cells that, in turn, contribute to many human diseases, including cancer. [77]

ROS activate various transcription factors (e.g., nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), activator protein-1 (AP1), hypoxia-inducible factor-1α (HIF1)) resulting in the expression of proteins that control inflammation, cellular transformation, tumor cell survival, tumor cell proliferation and invasion, angiogenesis, and metastasis. Paradoxically, ROS also control the expression of various tumor suppressor genes (p53, Rb, and PTEN). [78]

These statements suggest both cancer-promoting and cancer-suppressing actions of the ROS.

On one hand, at low levels, ROS facilitates cancer cell survival since cell-cycle progression driven by growth factors and receptor tyrosine kinases (RTK) require ROS for activation and chronic inflammation, a major mediator of cancer, is regulated by ROS. On the other hand, a high level of ROS can suppress tumor growth through the sustained activation of cell-cycle inhibitor and induction of cell death as well as senescence by damaging macromolecules.

Fig.32: Electron structures of

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34 This paradox provides a great challenge for researchers whose aim is to exploit ROS stress for the development of cancer therapies. [79;80]

Many anticancer agents induce reactive oxygen species (ROS) as part of their mechanism of action. Cancer cells exhibit greater ROS stress than normal cells do, partly due to oncogenic stimulation, increased metabolic activity and mitochondrial malfunction. [81] Further increase of oxidative stress in cancer cells, having an higher basal level of ROS, induce cell death that would not occur in normal cells, protected by efficient antioxidant defense mechanisms. (Fig.33)

Therefore, it should be possible to preferentially accumulate ROS in cancer cells and kill them selectively.

Thus, establishing the precise role of ROS requires the ability to measure ROS accurately. Molecular probes can be used as indicators for reactive oxygen species in cells.

About my thesis work, the cell permeant reagent 2’,7’–dichlorofluorescein diacetate (DCFDA) was used as a probe for Hydrogen peroxide (H2O2) and other peroxides, while dihydroethdium (DHE) as

an intracellular probe for preferentially superoxide anion (O2-); (Molecular Probes, Life

Technologies, NY).

Chemically reduced and acetylated forms of 2’,7’–dichlorofluorescein (H2DCFDA) are

nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.

After diffusion in to the cell, H2DCFDA is deacetylated by cellular esterases to a non-fluorescent

compound (H2DCF), which is later oxidized by ROS into 2’, 7’–dichlorofluorescin (DCF). (Fig.34)

Oxidation of these probes can be detected by monitoring the increase in fluorescence with a flow cytometer using excitation sources and filters appropriate for fluorescein (FITC) since DCF has maximum excitation and emission spectra of 495 nm and 529 nm respectively.

a. b. c.

Fig.34: The molecular structures of (a) H2DCFDA, (b) deacetylated H2DCF and (c) the

deacetylated, oxidized product, DCF.

Fig.33: Model proposed to explain

the opposite effect on cell proliferation of the increased oxidative stress in normal cells and