University of Texas Rio Grande Valley ScholarWorks @ UTRGV

Chemistry Faculty Publications and Presentations College of Sciences

2018

Key Enzymes in Cancer: Mechanism of Action and Inhibition With Anticancer Agents

Debasish Bandyopadhyay The University of Texas Rio Grande Valley, [email protected]

Gabriel Lopez The University of Texas Rio Grande Valley

Stephanie Cantu The University of Texas Rio Grande Valley

Samantha Balboa The University of Texas Rio Grande Valley

Annabel Garcia The University of Texas Rio Grande Valley

See next page for additional authors

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Part of the Chemistry Commons, and the Life Sciences Commons

Recommended Citation Debasish Bandyopadhyay, Gabriel Lopez, Stephanie Cantu, Samantha Balboa, Annabel Garcia, Christina Silva, Diandra Valdes. In Chemistry Research and Applications (Vol. 2): Organic and Medicinal Chemistry, Chapter 9; Key Enzymes in Cancer: Mechanism of Action and Inhibition with Anticancer Agents. 2018, Nova Science Publishers, Inc., Hauppauge, New York, USA (ISBN: 978-1-53614-855-8).

This Book is brought to you for free and open access by the College of Sciences at ScholarWorks @ UTRGV. It has been accepted for inclusion in Chemistry Faculty Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact [email protected], [email protected]. Authors Debasish Bandyopadhyay, Gabriel Lopez, Stephanie Cantu, Samantha Balboa, Annabel Garcia, Christina Silva, and Diandra Valdes

This book is available at ScholarWorks @ UTRGV: https://scholarworks.utrgv.edu/chem_fac/131 In: Organic and Medicinal Chemistry, Volume 2 ISBN: 978-1-53614-855-8 Editor: Bimal Krishna Banik © 2019 Nova Science Publishers, Inc.

Chapter!}

KEY ENZYMES IN CANCER: MECHANISM OF ACTION AND INHIBITION WITH ANTICANCER AGENTS

Debasish Bandyopadhyay ", Gabriel Lopezt, Stephanie Cantut, Samantha Balboat, Annabel Garciat, Christina Silvat and Diandra Valdest Department of Chemistry, The University of Texas Rio Grande Valley, Edinburg, Texas, US

A heartfelt tribute to the memory of Professor Asima Chatterjee, the legendary scientist-teacher-humanitarian, on the occasion of her birth centenary (1917-2017).

ABSTRACT

Several enzymes play significant role in different stages of cancer including proliferation, invasion, metastasis and angiogenesis. Mechanism of actions of a few key enzymes along with their inhibitors have been discussed with particular attentation to riboneucleotide reductase, thymidylate synthease, topoisomerase II, interleukins, cell survival proteins and aminopeptidase N.

Keywords: cancer, enzyme, riboneucleotide reductase, thymidylate synthease, topoisomerase II, cell survival proteins, aminopeptidase N, angiogenesis, apoptosis, metastasis

1. INTRODUCTION

According to the World Health Organization (WHO), cancer is the second leading cause of death worldwide, and was responsible for 8.8 million deaths only in 2015. Globally, nearly

• Co11"esponding Author Email: [email protected] or [email protected]. t TI1ese authors contributed equally. 358 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

1 in 6 deaths is due to cancer and is expected to increase approximately 70% in the next two decades [l]. It is well known that cancer is neither a single nor a modem disease. Based on tissue and/or cell type more than 400 form of cancers are possible. The description of cancer was found in many ancient literatures including Egyptian "Edwin Smith" and "George Ebers" papyri, written between the 3000 BC [2] to 1500 BC [3]. The "Edwin Smith" papyrus refers to 8 cases of ulcers (tumors) of the breast that were removed by cauterization with a tool called "the fire d1ill." It describes the disease as: "there is no treatment" [3]. A closer look into the fossils of over 700 dinosaurs confirmed the existence of bone cancer. Osteosarcoma (growths suggestive of the bone cancer) was also been noticed in the ancient human mummies in Egypt. The word oncos (in Greek, it means swelling) was introduced by a Greek physician Galen ( 130- 200 AD) to define tumors and accordingly the term oncology was developed. The ancient Greek physician Hippocrates (460-370 BC), also known as the "Father of Medicine," first differentiated the benign and malignant tumors by introducing the terms carcinos and carcinoma to describe non-ulcer forming (benign) and ulcer-forming (malignant) tumors. The word carcinoma in Greek stands for crab. The disease was symbolized with crab possibly because of its finger-like tendency to spread throughout the body. Celsus, an ancient Roman physician (28 BC- 50 AD) u·anslated the Greek word carcinoma into cancer (means crab in Latin). Since then, several hundred of years no notable progress on understanding and treatment of cancer was made until 1775 when Percivall Pott (a British physician) noticed that a number of young boys employed as chimney sweeps developed cancer of the scrotum in later life. He suggested the presence of something in the soot which caused cancer. Therefore, the concept of carcinogen (cancer causing agent) was developed [3- 6]. With the progress of research and consequently with better understanding the meaning of carcinoma has been modified and now­ a-days it denotes the cancer arising in epithelial cells that cover external and internal body smfaces; approximately 90% of all human cancers are of this type. Also many other commonly used terms have been developed e.g., sarcoma (cancer arising in mesenchyme-derived tissue (supporting tissue) which includes cancers of bone, cartilage, fat, connective tissues and muscle; lymphoma (cancer arising in the lymph nodes and tissues of the immune system, leukemia (cancer of the immature blood cells that grow in the bone marrow and tend to accumulate in large numbers in the bloodstream), adenoma (cancer of glandular epithelium, in Latin adeno means gland) etc. Cancer can spread in the body following two mechanisms: invasion and metastasis. According to National Cancer Institute, invasion denotes "the direct migration and penetration by cancer cells into neighb01ing tissues" whereas metastasis stands for "the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then invade nonnal tissues elsewhere in the body'' [7]. To have a clear understanding, these two mechanisms can be compared with the ways fire can spread in a locality. When fire spread from one house to its adjacent house the process can be compared with invasion where cancer tissue/cells invade from one tumor to its adjacent body organ and new tumor is formed in that organ. The spark of fire can also spread from one burning house to a remote house by air (carrier). This can be compared with the migration of cancer tissue/cells carrying out by blood or lymph to another part of the body (for example from lung to liver) and new tumor is formed in that organ. Invasion and metastasis are considered as one of the hallmarks in cancer biology. It has been found that several enzymes are primarily responsible for proliferation, invasion and metastasis of the cancer cells. In this chapter, the role of a few key enzymes in cancer has been discussed. Key En:ymes in Cancer 359

2. RIBONUCLEOTIDE REDUCTASE (RR)

The human body contains a specific level of Ribonucleotide reductase which is an enzyme that helps with the biosynthesis of deoxyribonucleoside triphosphates. This enzyme converts ribonucloside diphosphates to deoxyribonucleoside diphosphates for the synthesis of DNA [8]. Ribonucleotide reductase has three subunit proteins in which one is a large subunit and the others are small subunits that work differently in the body for which they have different effects on different cancers [7]. These subunits are known as RRMl , RRM2 and RRM2B and they contribute to cancer in different ways and have their own role in which some factors are still unclear. The three subunits affect organs such as: Lungs, colon, pancrease and the thyroid. There are also complications with the ribonucleotide reductase in phannaceutical levels and treatment because it can inhibit, block or having different affects toward the type of treatment being involve for the certain cancer types being involved. Like the protein subunit RRMl that interacts with chemotherapeutic agents like gemcitabine, and cisplatin in pancreatic cancer and in non-small cell lung cancer respectively. RRMl is known as the large subunit in which it said to behave during the cell transformation and tumor development as a suppressor and is still not clear in some cancers. The other two protein subunits are RRM2 and RRM2B that contribute to help in the repair and replication of DNA [8]. The small subunit RRM2 mechanism is not well-known in cancer but is known to have the role in cell proliferation and to repair DNA [9] . With the elevation or dysregulation of ribonucleotide reductase it creates a characteristic for many different types of cancer or can be associated with malignant transfonnation. Ribonucleotide reductase subunits play different roles in many organs like lungs, thyroid and colon as described as in the sequel. Ribonucleotide reductase actively participate in repairing DNA damage and varies in the cell cycle and the ribonucleotide reductase subunits help maintain the balance. For proper DNA repair and replication, to have stable dNTP pools otherwise it causes cell death and the subunits to help regulate by the RRMl and RRM2 in which the RRMl works as a regulator for S phase of the cell cycle transcription [8, 10].

2.1. Papillary Thyroid Cancer

Thyroid cancer is a type of cancer known to be curable in many occasions. Patients with this disease who are being treated about 10 to 30% relapse and resist to the radio iodine treatment and study shows that in depth mechanistic investigation is required to develop new treatments. In papillary thyroid carcinoma 95.2% of the cancer tissues expressed RRMl was higher compare to the normal tissues showing that RMM 1 co ffe lated with T-stage, lymph node metastasis, extra-thyroidal invasion, and TNM stage. The RRMl has been studied by using the high and low differentiated thyroid carcinoma cells lines called TPC-1 and SW579 respectively. It has been demonstrated that RRMl has promoted DNA synthesis and proliferation in both types of thyroid carcinoma cell lines and the high expression ofRRMl contributes to aggressive phenotype [8]. The expression of the RRM2 subunit in the thyroid carcinoma and the papillary thyroid carcinoma was high. Studies have suggested that RRM2 and RRM2B have no 360 Debasish Bandyopadhy ay, Gabriel Lope=, Stephanie Cantu et al.

correlation with the TNM stage, LNM stage, T stage and extra-thyroidal invasion stage. The RRM2 is overexpressed in other cancers also, such as gasu·ic, hepatocellular and colon cancer. RRMl might play role differently in thyroid cancer through ribonucleotide reductase pathway tlrnt still needs to be studied as well as the other protein subunits to develop effective treatment for thyroid carcinoma and papilla1y carcinoma.

2.2. Colorectal Cancer

In colorectal cancer the small subunit called RRM2 and the protein cA.t\lIP 1 (CREBl) are tl1e possible targets to find its treatment. The CREB 1 comes from a bZIP family which is known for the transcription factor that mediates the transduction between the upstream signal and downstream gene transcription. In colorectal cancer, the DNA repair and cell proliferation subunit called RRM2 becomes the promoter for CREB 1 and it works as a transcription factor for the subunit and enhances tumor aggressiveness. RRM2 is identified as a tumor promoter in many cancer cells and in colorectal cancer cells it behaves as a critical effector which makes CREB 1 to promote aggressiveness for the cells as well as it promotes RRM2 expression. A relationship between RRM2 and CRB 1 has been predicted and in reality, both of them promote proliferation, migration, and invasion of colorectal cancer cells [9]. Studies suggest that the RRMl subunit might have a potential for cancer treatment with the interaction of RRMl and the ubiquitous red ox protein called human thioredoxin 1. There been clinical studies for colorectal cancer in which human thioredoxinl and the RRl\11 subunit correlate to cancer malignancy stage and both produce a synergistic anti-cancer effect in the colorectal cancer cells. In the study of colorectal cancer, it has shown that the RRMl subunit and the human thioredoxinl have an interaction between each other that enhances DNA synthesis [11 ].

2.3. Lung Cancer

There have been different studies on treatment for lung cancer and one effective treatment could be done by gemcitabine (Figure 1). Gemcitabine, a deoxycytidine analog, is phosphorylated to 5' monophosphate by the kinase called deoxycytidine and a very effective therapeutic agent for not only non-small cell lung cancer (NSCLC) but also for the pancreatic cancer [12, 13]. It has been reported that the ribonucleotide reductase subunit! and the messenger RNA together could be a predictor marker for the treated patients with gemcitabine or cisplatin. Basically, the RRM 1 is involved witl1 the repair system and synthesis of deoxyribonucleic acid in which it participates in DNA strand of incision and damage recognition [12]. Studies have shown that the subunit RRMl may have effect towards the gemcitabine response because the subunit might be the intracellular target for this agent [13]. Two non- small cell cancer cell lines (H356-G200 and H460-G400) were selected for this study and the activity of the protein RRMl was analyzed even though with its high levels the ribonucleotide reductase did not increase in one of the cell lines (H356-G200) and on cloning the encoded RRM 1 from these cells did not show any mutation. It was seen that 48% of the Key En:ymes in Cancer 361

lung tumors being studied had a loss ofheterozygosity of the chromosomal region that had the ribonucleotide reductase subunit Ml in this case saying that it can act as a tumor suppressor [13]. f: HO I N~O

~OH F Gemcitabine Cisplatin

Figure 1. Strncture of ribonucleotide reductase inhibitors: Gemcitabine and Cis-platin.

2.4. Pancreatic Cancer

Pancreatic cancer is still known as one of the lethal types of solid tumor due to its high resistance toward chemotherapeutics. Pancreatic cancer is one of the leading causes of cancer­ related death for both men and women and is the only major cancer with a 5-year relative survival rate in the single digits, at just 7. 7% [ 15]. While overall can cer incidence and death rates are declining, the incidence and death rates for pancreatic cancer are increasing [ 16]. It is a matter of serious concern that pancreatic cancer is projected to move past colorectal cancer to become the 2nd leading cause of cancer-related death in the United States around 2020 [ 17]. The justifications behind this estimation are: (i) in comparison to other cancers, pancreatic cancer can progress very quickly from stage I (localized within the pancreas) to stage IV (metastatic disease) in an average of 1.3 years; (ii) cunently, there are no proven biomarkers, or clues detectable in the blood or other bodily fluids th at could indicate the presence of a pancreatic tumor; (iii) only fewer than 20% of pancreatic cancer cases are diagnosed th at meet the criteria for surgical intervention (often the Whipple procedure) although the disease recurs in approximately 80% of these surgical patients; (iv) for non-surgical patients (not suitable for surgery), chemotherapy (sometimes along with radiation) is typically offered but is not considered curative at all (v) around 95% of pancreatic tumors are driven by mutations in KRAS gene, which signifies a very aggressive and treatment-resistant tumor. Mutated KRAS has been dubbed "undmggable." Both in pancreatic and lung cancers, the subunit RRMl overexpression has poor survival than with low levels ofRRMl after the gemcitabine treatment which makes the level t.o be become an indicator for gem-resistance. There are various metabolic enzymes that supposed to play significant role in drng-resistance that include, but are not limited to, ribonucleotide reductase Ml subunit, deoxycyridine kinase, cytidine deaminase and the ribonucleotide reductase M2 subunit [14]. In the human pancreatic cancer cell lines the levels of RRMl are associated with the chemotherapeutic agent (gemcitabine) in five pancreatic cancer cells and the agent with the patients that had low RRMl was more 362 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

effective. This showed that patients with low expression ofRRMl can live longer than those having overexpression ofRRMl [14]. It has been suggested that ribonucleotide reductase can help in reducing the antitumor effect on gemcitabine since the ribonucleotide reductase increases the dNTP which is referred to deoxynucleoside triphosphates pool in the cells that produces a decrease of the deoxy ribonucleotide analogues like the triphosphorylated gemcitabine into DNA. The RMMl protein subunit of ribonucleotide reductase plays different roles in cancer but in pancreatic cancer it's a target for gemcitabine and the inhibition of the RRl\111 reduces sensitivity of gemcitabine [ 18].

3. THYMIDYLATE S YNTHASE (TS)

The enzyme Thymidylate Synthase (TS) produces an essential precursor used in DNA replication. By inhibiting the action of this enzyme, the production of deoxythymidine monophosphate ceases. Not only does this disrupt the production of this necessary precursor, but TS inhibition subsequently intenupts the DNA replication process altogether. This intenuption of DNA replication in rum leads to apoptosis or programmed cell death. It is a chain of events that makes Thymidylate Synthase an important point of study in anticancer research. By inhibiting DNA biosynthesis and causing cell death, the uncontrolled cellular division that is seen in many types of cancer can be slowed down. Thymidylate Synthase inhibitor drugs and prodrugs are commonly used for this purpose. Although this type of treatment is relatively effective against many types of cancer, including colorectal, gastric, pancreatic, and ovarian cancers, research is still being unde1taken to detennine whether it can be effectively used against other types of cancer. Thymidylate Synthase is a key enzyme in the synthesis of 2'-deoxythymidine-5'­ monophosphate, an essential precursor for DNA biosynthesis [19]. The progression of a cell through the cell cycle checkpoints ensures that the subsequent generation of cells have the components necessary to preserve homeostasis and developmental morphogenesis [20]. This is trne for both carcinogenic and non-carcinogenic cell lineages. With this being the case, the activity of an enzyme, in this instance TS, which produces an essential precursor for DNA biosynthesis has significant and direct implications on the completion of the S Phase in the cell cycle. For example, total inhibition of Thymidylate Synthase would interfere with the progression of tl1e cell cycle by disrupting the synthesis of 2' -deoxythymidine-5 ' - monophosphate (dTMP). It is well known that progression of the cell cycle and control of apoptosis (programmed cell death) are intricately linked processes [21, 22]. Research has confirmed that the inhibition of TS leads to the cessation of DNA replication and thymineless death of proliferating cells, which renders the enzyme as an attractive target for cancer chemotherapy [23]. However, in order to get a better understanding on why TS inhibition causes DNA replication to halt and the cell to die a thymineless death, it is necessary to understand the enzyme's strncture, tl1e mechanism through which the enzyme functions, as well as its place in the cell cycle. Thymidylate Synthase is an obligato1y homodimer which fonns a complex with dUMP (2'­ deoxyuridine-5 '-monophosphate) and a THF (tetrahydrofolate) cofactor [23]. TS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by transfer of a Key En:ymes in Cancer 363

methylene group from a cofactor to generate deoxythymidine-5'-monophosphate dTMP [24]. In this process, the enzyme TS takes dUMP, and uses 5,10-methylenetetrahydrofolate as a cofactor to donate a methyl group, forming dTMP. It should be noted that although dTMP is a molecule, found in the nucleotides which compose the strands of DNA, produced by TS is not immediately available for use in the process of DNA biosynthesis. The dTMP entity is further phosphorylated to the triphosphate state (d TTP), which is a direct precursor for DNA synthesis. By blocking the synthesis of2'-deoxythymidine-5'-monophosphate, there can be no formation of 2'-deoxythymidine-5'-triphosphate, the nucleotide which is used to incorporate a thymine nitrogenous base into a newly forming DNA molecule during S Phase. As it has been previously mentioned that the inhibition of TS leads to the cessation of DNA replication and thymineless death of proliferating cells, which renders the enzyme an attractive target for cancer chemotherapy [23]. Furthennore, TS is the rate-limiting enzyme in the de novo synthesis of deoxythymidine monophosphate from deoxyuridine monophosphate [25]. The interruption of DNA replication blocks progress through the cell cycle which is thought to be intimately linked to control of apoptosis [20]. Taken together, the inhibition of TS indirectly blocks the replication of DNA which in tum leads to apoptosis. Therefore, this enzyme is considered an attractive target for cancer chemotherapy. When viewed from a cettain perspective, inhibition of thymidylate synthase in cancer cells leads to the death of the cancer cells. These nitrogenous bases, also known as organic bases (Figure 2), bind in a specific and complementary way. One purine will bind to one pyrimidine. In both RNA and DNA, guanine (a purine) binds to cytosine (a pyrimidine) via three intermolecular hydrogen bonds. Similarly, adenine (a purine) will bind with either thymine in DNA (a pyrimidine) or uracil in RNA (a pyrimidine) through two intermolecular hydrogen bonds. Schematic depictions of the interactions between adenine hydrogen bonding with thymine, as well as adenine hydrogen bonding with uracil are depicted in Figure 3.

NH2 0 N~ N HN:XN';) lL /, ~ N N H2NA N H ~ Adenine Guanine Purines

Thymine Cytosine Pyrimidines

Figure 2. Purines and pyrimidines: the 'organic bases.' 364 Debasish Bandyopadhy ay, Gabriel Lope=, Stephanie Cantu et al.

Figure 3. Pictorial representation of H-bonding between Adenine-Thymine, Adenine-Uracil and Guanine-Cytosine.

Each of strand of DNA is a polymer composed of nucleotide monomers, which are joined together to form the chromosomes we see, noting that the longest chromosome is - 250 million nucleotides in length [26]. It should be noted that each of these nucleotides are composed of three primary structural elements:

1. 5-Carbon sugar molecule (ribose/deoxyribose for RNA/DNA, respectively) 2. Phosphate group bound to the 5' carbon of the sugar molecule 3. Nitrogenous base bound to the l ' carbon of the sugar molecule

Thymidine nucleotide is pictured in Figure 4. It should be noted that although dTMP is the final molecule incorporated into DNA, dTTP is its precursor molecule. The two extra phosphate groups on dTTP a.re hydrolyzed to provide the energy needed to incorporate dTMP into the newly forming strand of DNA.

OH H

Figure 4. Deoxythymidine-5'-phosphate ( dTMP, thymidylic acid). Key En:ymes in Cancer 365

5-Fulorouracil (5-FU)

Figure 5. Thymidylate Synthase inhibitor as anticancer drugs.

The 5' sugar and phosphate components are important in maintaining the strnctural integrity of DNA, and they compose its "backbone" [25] but it is the latter of these three structural components, the nitrogenous bases that is the most critical to the current discussion. There are two types of nitrogenous bases that constitute the nucleotide monomers found in DNA: The purines and the pyrimidines (Figure 2). For the purposes of this discussion, it is important to emphasize the structures of uracil and thymine, noting that they are identical with the exception of a methyl group at the 5 position. During the replication of DNA, the enzyme DNA polymerase takes advantage of the complementarity exhibited by the nucleotides which comprise the genetic code. By using one of the double helix's strands as a template, DNA polymerase enzymes add nucleotides in a complememary manner, binding guanine with cytosine, and adenine with thymine. This process is repeated um.ii two copies of the genome have been created from one initial copy. It should be noted that an event which damages the DNA during the course of genetic replication could result in DNA damage response and apoptosis [27]. Following the DNA biosynthesis in the S Phase, the cell enters a final growth phase to prepare for cellular division. Accordingly, the purpose of this discussion emphasizes on the S phase. The S Phase is where the action of TS becomes a factor in anticancer research. Although it does not directly participate in DNA biosynthesis, TS's activity has a direct impact on the cell's ability to replicate a copy of the genome, which in turn affects the fate of the cell. Several Thymidylate Synthase inhibitor anticancer drugs like 5-fluorouracil and raltitrexed as well as tl1e prodrng capecitabine have been approved by the FDA (Figure 5). The drug 5- fluorouracil (5-FU) is structurally similar to the pyrimidine base uracil. The similarity in composition allows 5-FU to act as a substrate in the same metabolic pathway as uracil, and after its conversion to 5-fluorodeoxyuridine monophosphate, 5-FU inhibits the enzyme thymidylate synthase, ultimately inhibiting DNA synthesis [28]. As previously discussed, this inhibition leads to apoptosis. It should be noted that the mechanism of 5-FU's cytotoxic activity, however, it appears insufficient to explain all of the pharmacological effectiveness of 5-FU and further investigations are required. The prodrug capecitabine follows a similar fluoropyrimidine based mechanism of inhibition [29]. In target cells it is converted to 5'-deoxy- 5-fluorouridine in a three-step sequential enzymatic pathway and eventually it is convened to 366 Debasish Bandyopadhy ay, Gabriel Lope=, Stephanie Cantu et al.

5-FU. Once capecitabine is transformed to 5-fluorouracil, the same mechanism of action detailed above was observed. Although, capecitabine is transformed into 5-FU, this pyrimidine analog method ofthymidylate synthase inhibition is not the only technique available to stop TS activity. The drng raltitrexed takes advantage of the fact that a folate cofactor is needed for TS to function [30]. Raltitrexed acts as an antifolate molecule which inhibits the activity of TS. This therapeutic effect is imparted by raltitrexed competitively binding TS in an irreversible manner. This disallows the enzyme to bind its necessary cofactor, and DNA biosynthesis is stopped. These drngs have proven to be effective in inhibiting TS activity, but it should be noted that one meta-analysis that investigated 2,442 (gastric cancer) patients in 25 studies observed that response rates for FUC (fluoropyrimidine based chemotherapy) were significantly lower in patients with high TS activity than in those with low expression [31]. This indicates that although the drngs can effectively inhibit TS activity, a higher level of TS expression may make it more difficult to combat cancer using this technique. Furthennore, the studies which composed the meta-analysis had va1ying results, so the mechanism between levels of thymidylate synthase expression vs. treatment with drngs that inhibit TS is still unclear. Despite being very effective in certain types of cancer, particularly colorectal and gastric, research is still being undertaken to determine the advantage of using thymidylate synthase inhibitors against many types of cancer known to the modem medical community.

4. TOPOISOMERASE II

DNA topoisomerase II is a validated target for chemotherapeutic agents because disruption of this enzyme damages the DNA. DNA topoisomerases are enzymes that regulate the winding of DNA during replication and transcription. When DNA is replicating or being transcripted, it wounds tightly ahead of the replication fork. Topoisomerases keep the double helix from becoming too tightly wound by binding to the double-stranded DNA and cutting the phosphate backbones of the strands. By cutting the backbones of the DNA strands, they can be untangled and prevented from winding too tightly. At the end of the process, the DNA strand is resealed. The name topoisomerase comes from the fact that as the enzyme works on the DNA, the untangled and tangled strands are isomers that only differ in their topology. Therefore, topoisomerases are isomerase enzymes that only change the topology of the DNA [32]. There are two types of DNA topoisomerases in mammals: topoisomerase I and topoisomerase II [33] in which topoisomerase II enzyme is used in regulating DNA super helicity. Unlike topoisomerase I, topoisomerase II requires the binding of ATP and hydrolysis in order to function correctly. Topoisomerase II functions by cutting and opening the duplex of one DNA strand, stringing a second duplex through the passage it created, and then repairing the damage [34]. DNA topoisomerase II has been using as a target for a variety of anticancer drugs such as anthracenediones, anthracyclines, amsacrines, ellipticines, and non-intercalating etoposides [35, 36]. The structure of four anthracyclines (doxorubicin, daunorubicin, idarubicin, and epirnbicin are shown in Figure 6. In order for these drugs to work effectively, they must be able to disrupt at least one step of the catalytic cycle of DNA topoisomerase II. Drugs that can stabilize the topoisomerase II complex are known as topoisomerase II poisons and other drugs Key En:ymes in Cancer 367 that disrupt any steps of the catalytic cycle are known as catalytic inhibitors. The most used "topoisomerase inhibitors" are actually topoisomerase II poisons, which are utilized for their antitumor activities [37]. Although they all share similar mechanisms as to how they disrupt the function of DNA topoisomerase II, they have different pharmacological and pharmacodynamics characteristics. There are also slight nuances as to the toxicological effects each of these drugs have, with the anthracyclines exhibit higher toxicity whereas elliticine and mitoxantrone showed reduced toxicity (Figure 7). Etoposide is a derivative of podophyllotoxin, a drug used for the treatment. of genital warts and molluscum contagiosum. Historically, podophyllotoxins were used to treat cancers of the skin in the 19th century [38]. After clinical studies, podophyllotoxins and some of its derivatives showed promising antineoplastic activity. Despite their promise as antineoplastic agents, podophyllotoxins and their derivatives showed toxicity toward normal cells/tissues. Researchers began synthesizing more podophyllotoxin derivatives in order to find one that did not have so much toxicity and came across 4 ' - demethylepipodophyllin benzylidene glucoside (DEPBG). Two more analogues of DEPBG that showed even more promising antineoplastic activity, etoposide and teniposide, were then developed [39]. Etoposide is used to treat various cancers as a topoisomerase II inhibitor. These cancers include leukemia, lung cancer, lymphoma, nemoblastoma, ovarian cancer, and testicular cancer. Often, it is administered in combination with other chemotherapeutic agents and even used as a conditioning agent to ready a patient for a bone man-ow transplant. Although there is no clear mechanism of action as to how etoposide damages DNA, it is hypothesized that the drng interferes with the cleaving and resealing of the topoisomerase II's function by forming a stable protein-ligand cleavable complex [40]. So far, caspases, protease enzymes that play critical roles in cell apoptosis, have been shown to be a link between etoposide's activity and the mitochondrial apoptosis pathway, particularly caspase 2 [41]. By activating caspase 2, caspase 8 is also activated, which damages the mitochondria and causes the successive downstream activation of caspase 9 and 3 [42]. Mutations in the caspases has shown to affect etoposide's ability to work. A lack of caspase 3 in cells causes it to be resistant to etoposide [I 7]. The TNF- related apoptosis-inducing ligand (TRAIL), a ligand that causes apoptosis, also changes how caspases are expressed when activated by etoposide. Other proteins, such as p53, c-Myc, and Baff, which control the cell-cycle have also been shown to be important in etoposide-activated apoptosis [42]. Multiple mechanisms exist that make tumors resistant to etoposides. These mechanisms include a mutation at ser-11 06 in topoisomerase II that annuls the enzyme's phospho1ylation and reduces the effectiveness of etoposide, rapid repair of DNA breaks, functioning FLT3, and possessing a multidrug resistance (MDR) phenotype [43-45]. To make etoposide work on MDR resistant tumors, it must be turned into a prodrug which can create analogs of etoposide. As stated before, despite its use as an anticancer drug, etoposide has some toxicity (side effects). Common side effects of administering etoposide include nausea, vomiting, bone man-ow suppression, and alopecia. When administered in high doses, etoposide can cause fever, chills, liver toxicity, anal cavity irritation, and Palmar-planter eruptions. The World Health Organization (WHO) has also classified etoposide as a carcinogenic agent because current clinical evidence suggests that administration of etoposide can eventually cause acute myelogenous leukemia [46]. Leukemia caused by etoposide administration occurs approximately 2 to 3 years after treatment. This can be caused by the translocation of the mixed­ lineage leukemia (MLL) gene which can result from the activation of the caspases. Toxicity was mostly observed in patients who kept the etoposide concentration above l.7 µM for over 368 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

8 hours, which suggests that drug plasma concentration must be kept at a sufficient level to produce therapeutic effects, but that extended high concentrations of plasma etoposide causes greater toxicity [49]. About one one-third of etoposide administered through I.V. is excreted through urine and less than 2% of intact drug is eliminated into bile [50]. Only 6-8% of etoposide does not bound to plasma proteins, making it highly bound when administered. Disorders that affect how much protein binding there is or decrease the amount of albumin in the bloodstream will cause an increase of the pharmacological effect of etoposide since free drug is still biologically active. This could lead to an increase in unwanted side effects. There are relatively few known drug interactions with etoposide. There are no substantial interactions with cisplatin and carboplatin (platinum agents) [ 51] and grapefruit juice, which can inhibit the cytochrome P450 metabolism, does not affect the kinetics of etoposide [52]. But, related use of prednisone can induce etoposide excretion which may be caused by the induction of P­ glycoprotein (PgP) [53]. A closely related analogue of etoposide in teniposide (Figure 7). It was approved for medical use 10 years after etoposide was approved. The drug has mostly been used to treat child leukemias and lymphomas and CNS malignancies. It is administered by I.V. and if it leaks under the skin, burns the patient. Like etoposide, it can be used in conjunction with other anticancer drugs. Its mechanism of action is similar to etoposide in that it damages the DNA by forming cleavable complexes with topoisomerase II. Its toxicity effects are also similar to etoposide by causing nausea, vomiting, bone ma1Tow suppression, and alopecia. However, hypersensitivity happens more often with infusions ofteniposide than etoposide. It is also more potent at killing malignant cells than etoposide, which may be caused by better cellular uptake. It has less water solubility, renal clearance, binds tighter to plasma proteins, has a longer half­ life, and a larger biliary clearance than that of etoposide [54] .

0 OH 0 0 OH 0 OH ·,0HCH3

:. H :. H OMeO OH 0 OMe 0 OH 0

Doxorubicin Fr! (Adriamycin) ~ Daunorubicin OH NH2 OH 2

0 OH 0 0 OH 0 OH ''OH ''OHCH3

: H .: H OMe 0 OH 0 0 OH Ho$id Epirubicin ldarubicin O,<:C' OH H2N NH2

Figure 6. FDA approved anthracyclines as topoisomerase II inhibitors.

Anthracyclines are a set ofdrugs that are used as chemotherapeutic agents. They are usually extracted from the bacteria Streptomyces and Streptomyces peucetius var. caesius. This class of drugs are effective against more types of cancer than any other anticancer agents and only Key En:ymes in Cancer 369 few cancers, such as colon cancer, are insensitive to these drugs, making them one of the most successful chemotherapeutic treatments ever done. Idarubicin, epirubicin, doxorubicin, and daunorubicin are anthracyclines currently approved for medicinal use as topoisomerase II inhibitors. Anthracyclines are used to treat an ass01irnent of neoplastic cancers such as lymphoma, breast cancer, Kaposi's sarcoma, Ewing's sarcoma, leukemia, and ovarian cancer. Of all the anthracyclines, doxorubicin is the most commonly used drug. There have been attempts to create an analog similar to doxorubicin, but they have failed to reproduce its potency against solid tumors. The epimer of doxorubicin, Epirubicin, has similar activity and is mainly used to treat breast cancer [55]. Daunorubicin, in contrast, has little activity against solid tumors, but it is an important medicine against acute leukemia [56]. The analogue of daunorubicin is idambicin and it is mainly used for acute myeloid leukemia (AML) treatment. There has been evidence that idarubicin has a survival advantage over daunorubicin, but the effects are minimal. However, it has significant bioavailability [ 57]. There are four mechanisms in which anthracyclines work. One is that they inhibit the synthesis of RNA and DNA by intercalating the base pairs of the DNA/RNA strand, which causes the prevention of cancer cells from rapidly replicating. The second mechanism is the generation of free oxygen radicals mediated by iron that damage components of the tumor such as its proteins, cell membrane, and DNA [58]. The third mechanism involves histone eviction from chromatin that disrupts the epigenome, transcriptome, and DNA damage response [59]. Finally, the mechanism most relevant for this chapter, anthracyclines can inhibit topoisomerase II in the same way that etoposide was previously described [60]. Myelosuppression is the dose-limiting toxicity of anthracyclines. Like etoposides, side effects include nausea, alopecia, vomiting, and mucositis. Administering anthracyclines can also cause severe tissue reactions if leakage occurs under the skin during intravenous infusion. The wounds can degrade further over a period of weeks, heal slowly, and may even require skin grafting. Cardiotoxicity is the most serious side effect that is associated with treatment with anthracyclines. Three types of cardiotoxicity have been observed based on how soon symptoms appear. Immediately after infusion, there is acute cardiotoxicity that can include arrhythmias and pericarditis. Months to years after treatment with anthracyclines, late-onset cardiomyopathy can appear. If children are treated with anthracyclines, subclinical cardiotoxicity can appear when the children become adolescents or adults [ 61]. This cardiotoxicity is thought to be a result of the production of free radicals that involve iron­ doxorubicin complexes that damage the membranes of cardiac cells [62]. In respective studies, the total dose and schedule of anthracycline that was administered with a bolus was found to cause a greater chance of heart failure than continuous infusion [63]. There have been several attempts to remedy anthracycline's propensity to cause cardiotoxicity. One strategy includes administering dexrazoxane, which can presumably prevents the iron-catalyzed free radicals from damaging cardiac cell membranes because it is an iron-chelating agent. Several studies have shown that administering dexrazoxane alongside anthracyclines can protect the heart without affection the anthracycline's antineoplastic abilities [64]. Another strategy included encapsulating the anthracycline in liposomes to prolong its half-life and try to make it accumulate in tumor tissue instead of cardiac tissue. Cardiotoxicity was found to be reduced from 21 to 6% in patients with metastatic breast cancer when liposomal doxorubicin was administered compared to straight doxorubicin [65]. It is imperative that the cardiotoxicity of anthracyclines be minimized because once developed, it is irreversible and may advance into congestive heart failure even after treatment [66]. 370 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

The anthracyclines are metabolized into 13-dihydro (alcohol) derivatives that are more toxic than the original compounds [67]. As mentioned before, encapsulating the drug with liposomes prolongs its half-life and can also change its pharmacodynamics characteristics. These deviations are based on the kinds of lipids used in the formulation of the liposome. There are two FDA-approved doxorubicin liposomal formulations. They are doxil and myocet. Doxil uses pegylated lipids in its formulation while Myocet uses phosphatidylcholine and cholesterol. Doxil is a slow acting drug, only releasing less than 10% of its doxorubicin within a day. This makes its half-life to be about 45-90 hours. It.s toxicity effects also change, causing palmar­ plantar e1ythrodysesthesia and mucositis. Myocet is a faster acting drug than Doxil. It releases half of the doxorubicin within an hour. Myocet's toxicity effects are myelosuppression and mucositis [68]. Mitoxantrone is the only representative of its class, anthacendiones, which is approved for medicinal use. (Figure 7). Anthacendiones are usally compounds that are used in the textile industry to create dyes, or in the pulp industry, or even as bird repellant. Unlike anthracyclines, it does not have the ability to fo1m the cardiotoxic free radicals [69]. Like most topoisomerase II poisons, mitoxantrone binds to the enzyme and forms a stable, cleavable complex that causes the DNA strands to break. This causes apoptosis, killing the cell [70]. There are several ways that tumors can become resistant to mitoxantrone. Several mechanisms include a change in topoisomerase II activity, less intracellular drug accumulation, an increase in glucuronidation, and a change in nuclear/cytoplasmic distribution of the drug [71]. Mitoxanrrone has a large volume of distribution, 1000-4000 1/.1112, and binds to protein tightly, 78% [72]. Its main mechanism for clearance is through the liver with 6-11 % of the drug cleared by the kidneys [73].

<;:H3 0/'-..0 2s HO H ·,,H OHO HO HO'.. O ·,,H 0 HO'.. O 0 , '.::::: OCH3 H I ·,,,NOCH3 ~ OH

OCH3 ~ OH Etoposide OCH3 H Teniposide ~,,--___,,N~OH

~ OH O HN~ N~OH H3C M1 toxantrone H Ellipticine

Figure 7. Topoisomerase II inhibitors as anticancer drngs. Key En:ymes in Cancer 371

The usual cancers treated with mitoxantrone are prostate cancer, leukemia, lymphoma, and breast cancer. Since there is overall less toxicity with mitoxantrone compared to doxorubicin, it is used for patients who may be sensitive to the toxic effects of doxorubicin. Mitoxantrone has shown no advantage over any of the anthracyclines. Mitoxantrone can also be administered in conjunction with prednisone to delay disease and improve the patient's quality oflife without affecting survival length. Ellipticine is a natural, simple alkaloid that is a known DNA topoisomerase II inhibitor. It has shown to have therapeutic effects against a variety of cancers. There is a strong interest in ellipticene and its derivatives because they have a high efficiency against a variety of cancers, with less effects, and a lack of hematologic and hepatic toxicity [74]. One negative aspect about ellipticine is its propensity to mutate. Ellipticine is also mutagenic to Salmonella typhimurim, Neurospora crassa, bacteriophage T4, and mammalian cells. The exact mechanism in which ellipticine causes apoptosis is unknown. However, it is theorized that DNA damage can be caused by either the interaction with DNA topoisomerase II or the intercalation into DNA [75]. A further study has shown that ellipticine can stimulate a topoisomerase II-mediated DNA breakage. The study consisted of using a yeast genetic system to determine that the target for ellipticine was indeed topoisomerase II. The study provided a model that seemed to indicate that a topoisomerase II-ellipticine-DNA complex is formed when the drug initially binds to the enzyme or the DNA itself [76].

5. INTERLEUKINS

Interleukins a.re low molecular weight cytokines that a.re secreted by white blood cells (leukocytes) and a.re one of the most essential to the immune system response by eliminating wounds, such as: infections, foreign bodies and other substances in order to begin the healing process. Some majorly important interleukins would be IL-1 , 2, 3, and 6, which signal the attraction of phagocytes and other correcting cells to the sight of the wound or infection. The major importance of interleukins is the ability to develop new blood vessels, which is a process called angiogenesis. Interleukins have the ability to inhibit or promote the ability to create new blood vessels. Interleukin-I (IL-1) contains pro inflammatory cytokines, which function to protect inflammatory responses. IL-6 has been known to induce vascular endothelial growth factor (VEGF) expression, which then enables the promotion of the creation of new vessels whereas IL-3 supports the growth and differentiation of bone marrow stem cell. Interleukin-2 is produced exclusively by T-lymphocytes by the activation of antigen and mitogens. Human. To induce biological activities, interleukin-2 binds to a fixed receptor on the surface of extremely delicate cells. The fixed receptor complex consists of three membrane-spanning polypeptide chains (alpha, beta, gamma chains). The alpha chain binds interleukin-2 with low affinity, while gamma subunit does not interact directly. Alpha and beta chains play a role in intracellular signaling and signal transduction, respectively. However, the role of gamma chain is still unclear but critical. IL-2 enhances antibody production and secretion and it has the ability to stimulate the growth and differentiation of natural killer cells which then form lymphokine­ activated killer cells, leading to its ability to eliminate tumor cells or virally infected cells directly. Treatments to cancer immunotherapy have therefore been formulated to increase the survival rate of patients, many involving the utilization ofinterleukin-2 as the primary immune- 372 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al. stimulant, including stimulated natural killer cells, LAK (lymphokine-activated killer) cells activated in vitro, and stimulation of cytotoxic T-cells, which promote tumor regression. In the T cells stimulation, interleukin-2 increases the number ofT-cells, then activates and boosts cell production. The specific T-cells would therefore inhibit cancers. The administrations of a significantly high doses of interleukin-2 by itself could initiate a similar response, but will sustain side effects. The ability of interleukin-2 to trigger the stimulation of T-cells is essential in the treatment of infectious diseases. Administrations of interleukin-2 can induce side effects, such as cardiovascular, hepatic or pulmonary complications. The side effects may be produced not only directly by interleukin-2, but also by plenty of other additional cytokines whose synthesis is amplified by IL-2 administration. The additional cytokines are interleukin-3, 4, 5, 6, tumor necrosis factor, and interferon-y. Many medical conditions are caused by the immune system itself. A treatment strategy to fight cancer might include giving high dose interleukin 2 or IL-2 through and intravenous IV infusion. IL-2 is a substance that tells the immune system to fight against ailments. T-cells clutch on to a few cancer-causing proteins and become activated and cause the cancer cells to break apart. Receiving IL-2 through an IV allows it to move throughout the body and act on cancer cells that may have spread. High dose ofIL-2 is a standard tTeatment that was approved to treat metastatic melanoma and metastatic kidney cancer. High dose IL-2 is given to patients with advanced melanoma or those with metastatic kidney cancer who cannot be treated with surge1y. The role of interleukin 2 therapy made a big splash when it was introduced for kidney cancer was approved by the FDA for this indication back in 1992. Since then there's been a lot of debate whether or not IL-2 or interleukin 2 represent the most ideal therapy for patients with metastatic or distantly spread kidney cancer although this therapy may cause severe cardiac and lung toxicities [77].

6. CELL SERVIVAL PROTEINS

Cells live in competition with each other; healthy cells embrace tissue while competing with those cells that m·e older/weaker. Once the "healthy" cells seize they would get rid of the older cells by a process called apoprosis. Apoptosis is good in the sense that it gets rid of unwanted cells but has also been linked in developing certain cancers. The body is built like a large machine when it feels that it has things that are old or that it does not need it will "upgrade" to get rid of the old things to bring new things in such as regulating stem cells, and tissue re­ population. The cell survival proteins in the human body play import.ant role in cell survival/death programs, proliferation and the elimination of damaged cells. When cells feel, they are being "invaded" they tly and figure out the cause of it, when they do not seem to find the root of the problem they activate different mechanisms to get rid of whatever is out of the nonn. When they still feel threatened, they go to activate the cell death programs or cell death signaling pathways to remove the damaged cells. There are two types of cell injury, that is reversible cell injury which is harm done to the cell that can be reversed once the stress has been relieved. Irreversible cell injury, which causes the cell to die or go through cell suicide. Certain things that affect the cell positively or negatively can be stress induced leading the cell suicide. It can be good in the sense that it is getting rid of the infected cells, cancer cells, and Key En:ymes in Cancer 373

damaged cells. Cell death programs must be kept in check since unregulated cell death is linked with the production of different diseases. There are various ways of getting rid of damaged cells, the cell death program is broken up into various components such as apoptosis (type 1), which causes shrinkage. In the human body, there are about 50-70 billion cells th at die each day. It is a normal process during embryological development and a normal process during the development of our immune system. As it has been stated before, cancer is caused by the uncontrolled proliferation of cells, cancer cells are hungry in order to meet their demand for the nutrients and cellular building blocks that enable them to grow and divide their metabolism rapidly. New insights into these processes have sparked interest in developing drugs that target the unique metabolic characteristics of cancer cells. Such drugs have the potential to treat cancers arising from many different tissues, while leaving normal cells mostly unaffected. The altered metabolism of cancer cells includes an increased uptake of nutrients such as glucose and glutamine. They also start to metabolize glucose in a slightly different way, a phenomenon called the Warburg effect. In all cells, glucose is broken down in several steps to pyruvate. In nonnal cells most of the pyruvate enters the citric acid cycle in the mitochondria, which effectively acts as the energy factories of the cell. In this way, most of the glucose that is taken up is completely oxidized to carbon dioxide. In the 1920's Otto Warburg discovered that cancer cells unlike healthy cells metabolize most of their glucose using biochemical pathways that do not require oxygen regardless of whether oxygen is available. Excess glucose is taken up and like in nonnal cells broken down to pyruvate, however instead of entering the citric acid cycle most of the pyruvate is converted to lactate, which is secreted from the cell. It is still controversial why tumor cells employ this relatively ineffective way of extracting energy from glucose. One theo1y is that the process reflects a metabolic program that cells use to conve1i nutrients into the building blocks of macromolecules. Cells can be viewed as molecular factories; glycolysis is not the only aspect of the cells metabolism that is altered [78]. Cancer cells undergo a general shift from catabolic to anabolic metabolism in order to produce the greatly increased amounts of proteins, nucleic acids and liquids they need to supp01i their high rates of proliferation. Cancer cells also take up amino acids, amongst these, glutamine is of particular importance, it acts as a key source of nitrogen which is required for nucleotide and new amino acid synthesis. In some cancer cells glutamine also acts as a critical source of carbon, which is required for replenishing the components of the citric acid cycle. Many anti-cancer drugs on the market have metabolic targets in the cells although for a large proportion of them the metabolic target was only recognized long after the drug was developed. For example, the widely used chemotherapy drugs 5-flurouracil and gemcitabine interfere with DNA synthesis. Today there are many approaches in development that aim to target different aspects of cancer cell metabolism. The possibility of exploring of blocking glutamine to inte1rupt the supply of carbon and nitrogen. They are also considering other enzyme targets that might limit the use of nutrients to make key building blocks. Some strategies are aimed at interfering with the high rates of glucose metabolism and lactate secretion as described by Otto Warburg. Anticancer approaches targeting tumor metabolism faced many challenges a major one is finding the therapeutic window to prevent toxicity to no1111al tissues. However, it has become clear that the genetic changes associated with cancer can create addictions to specific metabolic pathways, which can 374 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

be therapeutically exploited. Moreover, cancer cells can be more sensitive to metabolic alarms than nonnal cells due to the loss of cell cycle checkpoints. A better understanding of how metabolism is altered in specific genetic context that lead to cancer will help identify enzymes or combination of enzymes to target for intervention and hopefully allow the introduction of more effective anti-cancer drugs. Cancer cells form blood vessels around them to receive oxygen and nutrients. However, cancerous blood vessels are structurally and functionally abnormal, so blood or oxygen can flow poorly compared to normal blood vessels, which hampers the effects of anticancer drugs. It has also been rep01ied that that normalizing the blood vessels increased the oxygen supply in cancerous tissues and slowed the growth of cancer. The drug efficacy is enhanced by the delive1y and effectiveness of existing drugs, giving a lower does is sufficient for treatment. For example, cis-platin is an important anti-cancer drng which is an in credibly useful complex, it is used to treat various cancers. In particular, it is very useful that it has given a_greater than 90% survival rate in testicular cancer. The functions of cis-platin bonds to DNA and it bonds to the nitrogen on guanine one of the nucleotides in DNA. If the lone pairs of electrons on the nitrogen that allows nitrogen to fonn date of covalent or coordinate bonds with the cis-platin complex that fo1ms bonds with the DNA nucleotides and it is actually a ligand substitution reaction, the guanine is substituted for the chloride ligands, since guanine is a better ligand to serve this purpose. Cis-platin is bound to our DNA and it distorts the DNA and prevents the effective replication cis-platin prevents the cells from multiplying and this DNA from copying and that's what allows it to act as an effective anti­ cancer drug, and cancer cells multiply ve1y fast which is what allows them to be super dangerous on the tumors to grow and if the drug can prevent the application it can be useful in the treatment of different cancers [78, 79].

7. AMINOPEPTIDASE N

Aminopeptidase N (APN, also known as CD-13) is a widely expressed type II trnnsmembrane protease that is heavily correlated with various solid and hematological malignancies in humans. APN degrades preferentially proteins and peptides with a N-tenninal neutral amino acid. It is a tumor marker that is overly expressed on the cell surface of almost all major tumor fonns including skin, ovary, lung, stomach, colon, kidney, bone, prostate, renal, pancreatic, thyroid, and breast cancers. APN is involved in angiogenesis; the inhibition of new blood vessel formation which is an effective means of limiting both the size and metastasis of solid tumors. The expression of APN is higher in a variety of metastatic cancers than in normal tissues. The enzyme Aminopeptidase N (APN) is located on the gene ANPEP with its cytogenetic location of 15q26.1. Aminopeptidase N is a type II metalloprotease that belongs to the M 1 family of the Ma clan. Ma clan consists of 967 amino acids with a short N-temlinal cytoplasmic domain, a single transmembrane palt, and a large cellular ectodomain containing the active site. APN has a molecular weight of 110,000 kDa. APN exists as membrane aminopeptidase N and soluble aminopeptidase N. It also can act as a receptor for human coronavirus [80]. APN is involved in several key functions such as the final digestion of peptides generated from Key En:ymes in Cancer 375

hydrolysis of proteins by gastric and pancreatic proteases which help to digest different kinds of proteins. APN plays a role in the processing of a variety of peptides. APN is involved in the cleavage of peptides bound to a major histocompatibility complex, angiogenesis, and promotes cholesterol crystallization. APN has catalytic activity on N-terminal amino acid, amide, or aiyl amide. It is seen in the epithelial cells of the kidney, small intestine and respirato1y tract as well as other plasma membranes. APN is found in the vasculature of tissues that undergo angiogenesis, in malignant gliomas, and lymph node metastases from multiple tumor types but not in blood vessels of nonnal tissues. A soluble fonn has been found in plasma and has been found to be elevated in plasma and effusions of cancer patients [8 1]. APN is a potent angiogenic regulator. It regulates angiogenesis. Accordingly, it is a potential target in cancer chemotherapy. Angiogenesis is the blood vessel formation from pre-existing vessels. Tumor angiogenesis described as the growth of new blood vessels that tumors need to grow. The ultimate purpose of angiogenesis is to supply nutrients and oxygen to the cells to proliferate uncontrollably. It is also imponant for the removal of waste products. Cancer cells can also spread to other pan s of the body through the blood and lymph systems. Tumor growth and metastasis are triggered by chemical signals from tumor cells in a phase of rapid growth and depend on angiogenesis and lymphangiogenesis, which means many tumor types first metastasize via lymphatic vessels to their regional lymph nodes [82, 83]. Cancer metastasis is an important batTier to cancer treatment and the cause of patient death. It is considered as a major cause of operation failure, postoperative relapse, and ultimately death. As stated earlier, metastasis is a complex, multistep process, where cancer cells spread from primary sites to new places. APN is being pursued by many as an important target against cancer metastasis and angiogenesis which suggests that synthetic APN inhibitors have the potential to overcome cancer metastasis and angiogenesis [84].

7.1. APN Expression in Normal Tissues and Malignant Neoplasms

Arninopeptidase N can he expressed in hoth cancer cells and nonmalignant cells but it is still unclear where and how APN acts to regulate tumor angiogenesis. Reduction in angiogenesis more than likely accounts for the tumor growth defect that results from enzyme inactivation. APN is expressed not only by endothelial cells, but also by pericytes, myeloid, and mesenchymal stromal cells. These are central components of the environment that is needed for tumor to grow. Vascular endothelial growth factor (VEGF), an angiogenesis regulator, reduces the expression of APN at an early stage of tumor growth [85]. APN is also expressed in many tissues but especially on the membrane of the small intestine where it has an important role in peptide digestion. It can also be found in proximal tubules, on synaptic membranes of the central nervous system, on the surface of granulocytes and microphages. Positive APN expression has been found in non-haematopoietic cells such as fibroblasts, osteoclasts, and the epithelium that line the renal proximal tubules, intestine and in the bile duct canaliculi. In addition, other positive APN expression has been detected in hepatocellular, gall bladder, and renal carcinoma cell line. This wide distribution of APN proposes that it might be site dependent [86]. Aminopeptidase N has been linked specifically to cancer, because it has been identified as a cell-surface marker for malignant myeloid cells. 376 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

It has reached high levels of expression in association with the progression of tumors such as, breast, ovarian, and prostate cancer. APN expression was also detected in the n01mal colonic epithelium and in 30% of colonic carcinomas, acute myeloblastic leukaemia (AML) and some cases of acute lymphoblastic leukaemia (ALL) as well. High levels of APN expression in these two types of neoplasm has been related to the reduced success rate in reducing remission and shortened survival time especially in patients with ALL. After all the collaborative work conducted, APN is now considered as a potential marker for drug resistance [86]. It has been shown (in vivo) that inhibition of APN expression reduces prima1y tumor growth. They had tumor growth in WT-mice (positive APN) and APN-null mice. Control­ shRNA and APN-shRNA were delivered into the mice through short hairpin RNA (shRNA) which is primarily utilized during transient in vitro studies. Then B 16F 10 cells expressing control-shRNA and APN-shRNA were introduced subcutaneously into APN-mice and APN-null mice. After 11 days, the control-shRNA tumors showed a marked growth in WT mice. As supposed, these tumors showed reduced tumor volume and weight in the APN-null mice. To evaluate whether decreased vascularization resulting from APN deficiency accounted for the observed inhibition of tumor growth the tumor vasculature was subsequently analyzed by using antibodies against the endothelial marker, "CD31." This would provide insight if inhibition of tumor angiogenesis was associated with the loss of APN enzymatic activity. The results showed that large blood vessels were at first numerous when both malignant and host cells expressed APN in the LLC model. The results showed that APN clearly reduced blood vessel size and blood vessel density. Anti-APN inununofluorescence was also reduced tumors when the APN gene was deleted or knocked down in malignant cells. Finally, APN detection was located in tumor cells and was associated almost only with CD3 l-positive endotl1elial cells in control-shRNA tumors [85].

7.2. Targeted Drug Therapy

Targeted cancer therapies are sometimes called "molecularly targeted dmgs/precision medicines" according to ilie National Cancer Institute (NCI). Targeted cancer tl1erapies use prodrugs that are widely used in the targeted delivery of cytotoxic compounds to cancer cells that block the growth and spread of cancer by interfering with specific molecular targets that are involved in the growth and spread cancer. The development of targeted therapies requires the identification of good targets that play a key role in cancer cell growth and survival such as APN. Metalloproteases such as APN, that are present in cancer cells but not nonnal cells that are more abundant in cancer cells would be potential targets. This is especially impo1tant if they are known to be involved in cell growth or survival. Targeted therapies do have some limitations. One is that cancer cells can become resistant to them over time by developing resistance. Resistance can occur in two ways: the target itself changes through mutations that are adapted since they increases the survival of the cancer and so the targeted therapy no longer interacts well with it or the tumor finds a new pathway to achieve tumor growth that does not depend on the target being treated [87]. Key En:ymes in Cancer 377

7.3. The Use of APN as a Target for Cancer Therapy

APN is a ubiquitous enzyme that proliferates in a variety of tissues, muscles, and organs. It is described but not limited to as a multifunctional protein with enzymatic functions. Its other functions include operating as an antigen presentation and a receptor for some human viruses. Aminopeptidase N (APN) has two functions unrelated to its enzymatic activity. These include mediating tumor cell motility and serving as a receptor for tumor horning peptides (peptides that bring anti-cancer drugs to tumor cells). APN has recently emerged as one of the most promising approaches for cancer treatment. By being discovered as a receptor for a tumor­ horning peptide motif, NGR (Asp-Gly-Arg), which is capable of horning selectively to the tumor vasculature [88]. The tumor homing therapy (also known as targeted drug therapy) is the concept which is linked to anti-tumor drugs to a tumor-homing peptide. It specifically recognizes a receptor that is uniquely or overly expressed on tumor cell surfaces and actively guides the drugs to the tumor cell surfaces. APN and integrins are two of the most promising tumor-homing receptors. APN and integrin based tumor horning therapies are the only ones that are currently in clinical trials [89]. Curcumin, a gold-colored spice commonly used in the Indian subcontinent for its variety of uses such health care, belongs to the class "curcuminoids" (Figure 8), has been shown to exhibit antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activities. Therefore, it has the potential to fight against various malignant diseases, diabetes, allergies, arthritis, Alzheimer's disease, and other chronic illnesses. Curcumin exhibits activities like recently discovered tumor necrosis factor blockers [90]. Once curcumin is paired with APN, it binds to APN and irreversibly inhibits its activity. The direct interaction between curcumin with APN was confirmed both in vitro and in vivo studies. The screening of APN inhibitors was carried out using 3000 compounds from natural and chemical products. During the screening, curcurnin was quickly identified as a potent inhibitor of APN. Curcumin and its derivatives like demethoxycurcumin, bis-demethoxycurcumin, hydrazinocurcumin, and hestatin were testeci for their ability to block APN in vivo stuciies [88) In vivo enzyme assays that were carried out by monitoring the enzymatic degradation of a fluorescent substrate using cultured cells as a source of enzyme showed curcumin inhibited the enzymatic activity of APN. The APN activity of drug-untreated control was then normalized to 100% of the enzyme activity. The data suggest that the inhibition of APN by curcumin is irreversible and that the mode of inhibition of APN by curcumin is different from that of bestatin, which is a reversible and competitive inhibitor. Curcumin and other known APN inhibitors strongly inhibit APN-positive tumor cell invasion and basic fibroblast growth factor­ induced angiogenesis and when taken together results in the novel feature of exhibiting its irreversible inhibitor propenies that can be beneficial in the goal of inhibiting angiogenesis [88]. Bestatin (also known as Ubenimex), derived from Streptomyces olivoreticuli (Figure 9) inhibits a known 12 kinds of APN's. The fi.mction ofbestatin is to inhibit tumor cell invasion, and aminopeptidase activities of human metastatic tumor cells. This is an effect attributed to the inhibition of APN. A proximate study revealed that APN is the principal receptor for the NGR peptide motif and it was illustrated that this receptor is expressed only on the endothelial cells of angiogenic vasculature but not normal vasculature. Further studies have established that APN is an impottant regulator of endothelial morphogenesis during angiogenesis. 378 Debasish Bandyopadhyay, Gabriel Lope=, Stephanie Cantu et al.

yH3 0 0 yH3 ,o o, ~ ~ H H ~ H H # Predominates Predominates ,9 / 'o o/ 'o 0 in solid phase in solu1ion phase and solu1ion (pH ,;; 7) Curcumin and solution (pH :1: 8)

/H,, 0 0 o ·o ::::,.._I ~ ~

# HO HO Demctboxycurcumin l -._ 0 0 0 0 ::::,.._ I

.,,,:; OH - HO -- HO OH Bis-demelhoxycurcumin

Figure 8. Curcuminoids as APN inhibitors.

I ~ N- NH Bestatin (Ubenimex) Hydrazinocurcumin HO,N¼~6-, 0 OD H OH 0 :r I Amastatin ::::,... Tosedostat

Figure 9. Some significant inhibitors of APN.

Aminopeptidase N is dysregulated in human malignancy which contributes to the neoplastic properties which interferes APN expression. Also, including the function or signaling that may lead to the development of novel anticancer drugs. This approach has been evaluated for some time and several investigational new drugs are cuITently under clinical investigation. The target may be used in several ways to employ anti-tumoral effects even if the targets are principally different. APN inhibition is being explored as a target for cancer therapy by exploring APN inhibitors that stop proliferative effects. They generally have broad and overlapping substrate specificity and inhibitors that are not expected to be specific. Some of these APN inhibitors with APN-inhibiting properties have entered early clinical trials. Such as, CHR-2797 (Tosedostat, Figure 9) which is a novel metalloenzyme inhibitor that is Key En:ymes in Cancer 379 converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of several intracellular aminopeptidases which exerts anti-proliferative effects against a variety of tumor cell lines in studies. APN is among other several compounds that are under preclinical development. These include novel synthetic compounds or structure analogs such as bestatin, dimethylaminoethyl ester (LYP). There are other more surprising compounds such as curcumin, a phenolic natural product, it has been noted an ineversible inhibitor of APN among its other many functions. Silencing APN showed evidence that it reduces the potency to metastasize. This new idea created of the use of differential enzyme expression as a basis for cancer therapy shows promise. However, the prodrng concept has also been thoroughly investigated but so far the results are mainly restricted to preclinical studies. Only with time will this area be developed further [91]. The battle to find better treatments to end the high failure rates in patients with advanced metastatic cancers continues. Targeting matrix metalloproteinases (MMPs) when treating cancer metastasis has large unsatisfactory results which leads to APN resulting as one of the most studied cancer therapeutic targets against cancer metastasis and angiogenesis. The extracellular mau·ix (ECM) is the principal banier of malignant cell dissemination. The ECM is a substrate of APN and matrix metalloproteinases MMPs. The way this multistep process occurs as APN degrades ECM to promote malignant cell invasion into the bloodstream. In the angiogenesis process, neo-endothelial cells invading through the ECM also need APN. Therefore, APN would be a key target to inhibit cancer metastasis and angiogenesis. Dysregulated expression and high-level exopeptidase activity of APN are found in various mammalian cancer cell lines in addition to neo-endothelial cells. This cancer cell lines include melanoma, prostate, ovarian, colon, renal and pancreas carcinomas. These could potentially benefit from treatment if breakthrough in evaluating APN and cell proliferation inhibition could be determined and carried through more in populations [84].

7.4. Expression of APN in Human Pancreatic Carcinoma

The significance of Aminopeptidase N is expansive as it is involved in a variety of cancers and it is not limited to only one kind. This can be useful as new techniques and treatments become available they can be applied and administered on a broad variety ofcancers. Pancreatic carcinoma is one ofthe dreadful cancers where APN plays a significant role in. Through studies using monoclonal antibody (MH8-11) that recognized APN inhibited human umbilical vein endothelial cells migration and capillary like fo1mation in the umbilical vein endothelial cells. It was investigated the expression of APN and the intratumor microvessel density in patients with pancreatic carcinoma. APN gene expression was detected by reverse transcriptase-PCR that was positive in 50% of the tumors and the APN protein was positive and detected by immunohistochemistry in 48% of tumors investigated in the 50 patients with pancreatic carcinoma. The median survival for patients with APN expression was significantly shorter than those without this expression. This concludes that APN can be used a new possible marker for these types of patients with pancreatic carcinoma. APN is positively correlated with angiogenesis for this cancer [92]. 380 Debasish Bandyopadhy ay, Gabriel Lope=, Stephanie Cantu et al.

7.5. Expression of APN in Human Colorectal Cancer

In recent studies in colorectal cancer the significance of APN expression is being discovered. Study showed the activity of APN in tissue and plasma of colorectal cancer patients was higher it decreased overall survival of the patient. It was reported that mRNA levels of APN were lower in colorectal adenomas and adenocarcinomas than in the surrounding mucosa that was not involved. Higher plasmatic AON activity was therefore negatively correlated as previously mentioned. The detenninations of APN activity levels can be used as a possible marker that is inexpensive, minimally invasive way ro define the aggressiveness of colorectal cancer. This makes APN a good candidate to be a target molecule. Hopefully, this will initiate more research into better ways handle the diagnosing and treatment of colorectal cancer [93].

7.6. Aminopeptidase Nin Human Cervical Cancer

Cervical cancer sta1ts in the cells lining the cervix. It is one of the most aggressive types of cancers. Cell surface APN/CD 13 activity in HeLa cells was used in the experiment and was discovered that the suppression of APN/CD 13 using bes ta tin, an inhibitor of APN/CD 13 activity, could affect tumor radio-sensitivity in cervical cancer cells both in vitro and in vivo studies. Bestatin enhanced the effectiveness of radiotherapy and apoptosis by acting as a radiosensitizer both in vitro and in vivo studies. In colony fonnation assays, a significant decline in clonogenic survival was observed in bestatin treated cells. A combination of radiation and bestatin showed a significant prolongation of the tumor-doubling time compared with the control or radiation-alone groups. As radiation increased, APN/CD 13 activity the potential of bestatin to act as a radiosensitizer increased. The survival curves obtained for radiation alone and bestatin treated cells in combination with irradiation. A significant decline in clonogenic survival was observed in bestatin treated cells in combination with radiation. Funher studies are needed but there is a positive outlook in the possible use of bes tatin to act as a radiosensitizer in ce1v ical cancer treatment. The inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficiency of radiotherapy for uterine cervical cancer [94]. The expansive role of cancer research is breaching new frontiers to a problem in stagnancy. Finding new and innovative ways to approach such a malicious disease such as cancer is crucial for the overall well-being of the populous. With its unique propenies of being a zinc-dependent metalloproteinase readily involved with angiogenesis the life-line for tumor generation. New approaches exploring the role of APN such as being used as a cancer stem cell markers and becoming a leader in the development of anti-cancer and anti-inflammatory drugs and can serve as a target for delivering drugs into tumors and inhibiting angiogenesis. APN can even be involved in unonhodox approaches such as using pairing it curcumin to achieve irreversible effects on angiogenesis. Natural and synthetic inhibitors of APN activity have been characterized to inhibit angiogenesis. These inhibitors have revealed that APN can modulate bioactive peptide responses (pain management, vasopressin release) and to influence in1I11une functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). Thus, APN inhibition may lead to the development of anti-cancer drugs primarily inhibiting . . ang1ogenes1s. Key En:ymes in Cancer 381

CONCLUSION

Till date more than 73 proteins have been identified that play various roles in human cancers and the research is still under progress. Only a few key proteins have been discussed in this chapter along with their notable inhibitors. More research is necessary to identify different mechanism of actions of cancers as well as to develop more potent drugs with better potency (toxicity) towards cancer cells and less toxicity (side effects) towards normal cells. Research programs should be cooperated with awareness programs to fight against cancer worldwide in a meaningful manner.

ACKNOWLEDGMENTS

DB is thankful to the Kleberg Foundation of Texas and the University of Texas Rio Grande Valley.

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