BIOCHEMISTRY: A TEXTBOOK FOR HIGHER EDUCATION - E. S. Severin - 2004

CHAPTER 16. ONCOGENESIS

VII. Basic Principles of Tumor Diagnostics and Cancer Treatment

Despite extensive and multifaceted research in recent years, oncology still faces numerous challenges primarily related to the early Diagnosis and Treatment of specific nosological forms of the disease. One of the key areas in tumor Diagnostics is the search for and development of Methods to detect indicators of the neoplastic process, known as tumor markers.

A. Tumor Markers

Tumor markers (TMs) are compounds (Proteins, biologically active Peptides, Hormones, Enzymes, and metabolites) synthesized either by Cancer Cells or by normal tissue cells in response to tumor development. Ideally, they should be produced exclusively within the body of a tumor-bearing Organism and remain absent in normal cells, as they are products of the aberrant expression of the cancer Cell genome. TMs are typically detected in Blood or other biological Body Fluids and are used for population screening for tumor carriage, as prognostic factors, for assessing patient status during clinical staging, for treatment monitoring, and for detecting disease recurrence.

According to the modern Classification, TMs are divided into three main groups:

✵ primary tumor-associated markers;

✵ secondary tumor-produced markers (specific and nonspecific);

✵ secondary markers induced by the tumor process.

This classification is not without flaws, as the exact same compound can be synthesized by tumor cells and produced by normal organ cells in response to tumor invasion.

Most currently known TMs have certain limitations. In almost all cases, under various pathological conditions such as Inflammatory Diseases of the Liver, Pancreas, and Lungs, a nonspecific and often minor elevation in marker levels is observed; additionally, TMs are sometimes undetectable in the Cytology/cytology/16.html">Early stages of the disease.

Oncofetal Proteins

In clinical practice, the most frequently used assays involve proteins found in human embryonic Tissues and blood during prenatal development. These proteins either disappear completely or persist only in trace amounts after birth. During tumor progression, their synthesis resumes, and they are secreted into the bloodstream.

Carcinoembryonic antigen (CEA) — a single-chain glycoprotein with a molecular weight ranging from 150 to 300 kD, with the carbohydrate component accounting for 45% to 57% of its molecular weight. The carbohydrate moiety contains significant amounts of fructose, mannose, galactose, and N-acetylglucosamine. Detection of this TM is most commonly performed for the diagnosis of Colorectal Cancer and for monitoring patient status in the postoperative period. Following the complete and successful surgical removal of the tumor, CEA concentrations decrease. A subsequent rise in this parameter in operated patients indicates disease recurrence and a high probability of metastasis.

α-Fetoprotein (α-FP) — a glycoprotein with a Molecular Weight of 61–70 kD, structurally similar to albumin. The carbohydrate fraction accounts for ~5% of the total protein mass. It is a normal fetal serum protein synthesized in the liver, yolk sac, and gastrointestinal tract, and secreted into the bloodstream. The highest concentration of this protein is observed during Embryogenesis and fetal development. After birth and During the first year of a child's life, the Synthesis and Secretion of α-FP drop sharply, reaching an adult concentration of only 20 ng/ml. Blood concentrations of α-FP rise during The Development of liver cancer, making its measurement valuable for diagnosis and subsequent evaluation of treatment efficacy.

Human chorionic gonadotropin, placental alkaline phosphatase, and certain other placental proteins are also frequently utilized as tumor markers. β-Human chorionic gonadotropin (β-hCG) is a glycoprotein placental hormone with a molecular weight of 45 kD, composed of α and β subunits. Under normal conditions, it is either undetectable or present in negligible concentrations. During Pregnancy, the hormone begins to be synthesized and secreted into the blood, peaking around the 12th week of gestation (The basis of the pregnancy test). Its levels then slowly decline and remain very low both before and after childbirth.

In ovarian and testicular tumors, the concentration of the hormone—and in some cases, solely its β subunit—increases. Because the C-terminal region of the hCG β-subunit is immunoreactive, immunohistochemical detection of the hormone serves as an effective tumor marker in the diagnosis and treatment monitoring of hereditary and sporadic tumors. Measuring β-hCG levels in CEREBROSPINAL FLUID AIDS in diagnosing Brain and Central Nervous system metastases.

Differentiation Antigens are also employed as TMs; these represent organ- or tumor-specific lymphocyte Glycoproteins (such as tissue polypeptide antigen, tissue polypeptide specific antigen, and others) that are quantified in the blood using Monoclonal Antibodies.

For prostate cancer, prostate-specific antigen (PSA) is the most sensitive TM. It is practically undetectable in women, normally remains below 2 ng/ml in men, but increases substantially in both malignant and benign prostate tumors.

Hormones and their receptors (estrogens and androgens, parathyroid hormone, Calcitonin, Growth Hormone, Insulin, Glucagon, ACTH, catecholamines, serotonin) serve as TMs for hormone-producing Organs. Their quantification is widely applied in clinical practice (Table 16-2).

Class="center">Table 16-2. Hormones as tumor markers for specific types of tumors

The detection of Hormone Receptors as tumor markers has proven to be a crucial test for identifying patients who face a high risk of disease recurrence following surgery and who therefore require Chemotherapy.

For instance, in breast cancer patients, determining estrogen and progesterone receptor status is a paramount prognostic factor for the subsequent course of the disease. The presence of these receptors yields positive outcomes in a high percentage of cases (50–75%) when treated with the antiestrogen tamoxifen and significantly enhances patient survival.

Some enzymes and proteins are used for diagnostic purposes and to monitor therapeutic efficacy. For instance, in various morphological types of lung cancer, the most promising markers are neuron-specific enolase and the soluble cytokeratin fragment, a structural component of the bronchial epithelial Cytoskeleton.

High cathepsin D activity in biopsy material indicates a high metastatic potential of the tumor and correlates with low survival rates in cancer patients. Another tumor marker indicating an unfavorable disease course is high activity of Serine protease, specifically the urokinase-type plasminogen activator. This enzyme catalyzes The formation of plasmin, which participates in the activation of metalloproteinases and promotes invasive processes and metastasis.

Tumor markers that appear in the patient's body in response to tumor progression include acute-phase inflammatory proteins: ferritin, ceruloplasmin, haptoglobin, C-reactive protein, as well as LDH and creatine kinase isoforms.

B. Treatment of Cancer Patients

Therapeutic interventions are employed upon tumor detection or at later stages, utilizing chemotherapy, radiotherapy, and symptomatic treatment. A vital condition for successful treatment is the radical surgical removal of the tumor. Treatment strategy must satisfy two main requirements: to exert cytostatic (preventing proliferation) and cytotoxic (destroying tumor cells) effects. However, chemotherapy halts DNA Synthesis AND Cell Division via mechanisms common to all cells, hence its toxicity and numerous side effects on healthy, rapidly proliferating cells such as Hair follicles, hematopoietic cells, and the intestinal epithelium. Treatment success relies on the greater drug sensitivity of neoplastic cells compared to normal, unaltered cells, reflecting a compromise between antitumor efficacy and toxicity to healthy tissues. Pharmacological agents used in chemotherapy include DNA-damaging alkylating agents, antimetabolites that inhibit nucleic acid synthesis, Antibiotics, hormones, and natural compounds with diverse effects.

Alkylating Agents

Alkylating agents form bonds with bases in the DNA molecule and disrupt Replication. Most alkylating agents (cyclophosphamide, cisplatin, carboplatin, etc.) possess two functional groups, each capable of interacting with DNA bases to form intra- and interstrand cross-links within the DNA double helix. These bonds can form at any stage of the Cell Cycle, making the action of alkylating agents cell-cycle phase-nonspecific (Fig. 16-17).

Fig. 16-17. Formation of cross-links between alkylating agents and guanine residues in the DNA molecule.

Antimetabolites

Among antimetabolites, methotrexate, 5-fluorouracil, and cytosine arabinoside are most frequently used in clinical practice (see Section 10).

Methotrexate is a Folic acid derivative that competitively inhibits Dihydrofolate Reductase, an enzyme whose activity maintains the required rate of purine and pyrimidine nucleotide synthesis in cells, and ultimately of RNA and DNA.

5-Fluorouracil (5-FU) can be converted into a nucleoside triphosphate and incorporated into RNA. RNA modified in this way becomes functionally inactive. Additionally, 5-FU can be synthesized into 5-FdUMP, which forms an inactive ternary complex with N⁵, N¹⁰-methylene-H₄-folate and thymidylate synthase, thereby disrupting the supply of thymidylate NUCLEOTIDES to The Cell and inhibiting DNA synthesis.

Cytosine arabinoside (Ara-C) can be phosphorylated to Ara-CTP, which, on the one hand, acts as an inhibitor of DNA polymerase alpha, and on the other hand, is partially incorporated into DNA. Both effects of Ara-C block DNA synthesis during the S-phase of the cell cycle.

Anthracycline antibiotics: doxorubicin, carminomycin, and rubomycin (see Section 4) are widely used in the treatment of leukemias and solid tumors such as breast, lung, and Ovarian cancer. These polycyclic compounds exert multifaceted effects on Introduction/20.html">DNA Structure AND synthesis: they initiate and cause partial unwinding of The Double Helix; promote the formation of single- and double-strand breaks; and bind to topoisomerase II, which is involved in advancing the Replication fork along the DNA template. Furthermore, they generate free radicals that increase the number of breaks in the DNA molecule.

Vinca Alkaloids — vincristine and vinblastine. Plant alkaloids that bind to the microtubule protein tubulin and prevent its polymerization. They are regarded as mitotic poisons that arrest cell cycle progression at metaphase. Vinca alkaloids disrupt all types of cellular and organelle motility associated with the contraction or relaxation of microtubules. Vinblastine and vincristine are used clinically in the treatment of acute lymphoblastic leukemia, breast cancer, neuroblastoma, soft tissue sarcomas, melanoma, and ovarian tumors.

Horomonal Therapy

Although malignant transformation disrupts several mechanisms controlling tissue growth and differentiation, A number of tumors do not completely escape the body's regulatory influence, retaining hormone and neurotransmitter receptors on their cell surface or intracellularly. These primarily include tumors originating from hormone-dependent tissues: the breast, Uterus, Ovaries, Pituitary Gland, Thyroid Gland, Adrenal Glands, Prostate Gland, and certain others.

Since the synthesis of most Steroid Hormones is regulated by the Hypothalamus-pituitary system, blocking the mitogenic effect of sex and corticosteroid hormones is possible at several stages (Fig. 16-18), for example: at the stage of gonadotropin-releasing hormone action on the anterior pituitary gland; by inhibiting steroid hormone synthesis at the stage of testosterone conversion to estradiol via the enzyme aromatase; or through the binding of antagonists to steroid hormone receptors.

Fig. 16-18. Therapy for hormone-dependent cancer.

Tamoxifen is the most widely used agent (Fig. 16-19). Along with other classes of steroid hormones (progestogens, glucocorticoids, androgens), it is highly effective in treating breast cancer.

Fig. 16-19. Structures of estradiol and tamoxifen.

More than half of breast, ovarian, and endometrial tumors contain estrogen and progesterone receptors and respond to hormone therapy with estrogens, antiestrogens, and progestins, as well as combinations of hormones with cytostatic agents. Receptor-negative tumors are poorly responsive to hormone therapy; therefore, determining estrogen and progestin receptor levels is widely used to predict the efficacy of hormone therapy in breast, ovarian, and uterine tumors.

B. Drug resistance of tumor cells

Despite significant advances in the DEVELOPMENT OF NEW drugs and treatment approaches, chemotherapy often remains ineffective. The primary factor limiting the success of antineoplastic agents is drug resistance, which can be:

✵ primary, meaning it is inherent to malignant cells before treatment with specific agents begins;

✵ secondary, which develops in response to drug administration.

In the latter case, a positive response is observed initially, but it wears off after some time. Due to the high mutational plasticity of tumor cells and continuous Selection pressure favoring greater malignancy, initial drug therapy successfully reduces tumor size. Later, the remaining cell subpopulation becomes resistant to an entire group of drugs simultaneously, leading to so-called multidrug resistance (MDR), which is accompanied by disease relapse and metastasis. For example, treatment with vincristine can induce tumor cell resistance not only to vincristine itself, but also to structurally and functionally unrelated agents such as doxorubicin and etoposide. There are various mechanisms underlying MDR (Table 16-3).

Table 16-3. Mechanisms of multidrug resistance

Some of the Biochemical Mechanisms of drug resistance identified in tumor cells are caused by:

✵ decreased intracellular drug accumulation;

✵ emergence of alternative drug metabolic pathways;

✵ structural alterations in target cells for a given drug;

✵ attenuation of apoptosis—the programmed cell death pathway utilized by the body to eliminate altered cells.

Membrane transport and P-glycoproteins

In many cases, MDR is driven by an almost 1000-fold upregulation of P-glycoprotein, a membrane transport protein, in tumor cells (see Section 12). This protein acts as an energy-dependent pump that actively extrudes drugs from cells in an ATP-dependent manner, preventing them from reaching intracellular cytotoxic concentrations. The onset of MDR is triggered by amplification and/or point Mutations in mdr genes. The Emergence of MDR strongly correlates with a poor prognosis.

Investigations into P-glycoprotein inhibitors have demonstrated that the Ca²⁺ channel blockers verapamil and cyclosporine can compete with vincristine for the active sites of this protein, thereby reducing cellular drug resistance.

Consequently, clinical practice has shown that single-drug therapy is rarely curative. As a rule, chemotherapy is combined with radiotherapy, which induces free radical-mediated DNA strand breaks and apoptosis in irradiated tissues.

G. New directions in tumor therapy

Photodynamic therapy has emerged as a promising approach in cancer treatment. The principle relies on destroying the tumor using administered agents that become activated upon laser irradiation. Exposure to light in the presence of molecular oxygen generates free radicals. The cytotoxic effect is mediated primarily through the destruction of cell membranes rather than DNA damage, as photodynamic therapy is non-mutagenic.

Targeted drug delivery to target cells is based on the differential expression of membrane antigens (growth factor receptors, carcinoembryonic antigen, and antibodies) On the surface of tumor versus normal cells. The density of such proteins on The Plasma Membrane of cancer cells is dramatically elevated compared to normal cells.

Ligands directed against these proteins are used to deliver either an enzyme that catalyzes The conversion of a prodrug into its active form directly on the tumor cell surface, or a cytotoxic agent that enters the target cell via receptor-mediated endocytosis, thus bypassing MDR mechanisms (Fig. 16-20).

Fig. 16-20. Use of monoclonal antibodies for targeted delivery of drugs or toxins to tumor cells.

Inhibition of angiogenesis, or the formation of new Blood Vessels, halts tumor growth. Capillary endothelial cell proliferation can be suppressed using recently discovered angiogenesis inhibitors, such as angiostatin or thrombospondin.

Synthetic peptides acting as matrix metalloproteinase inhibitors hold significant therapeutic promise. In animal studies, such compounds have successfully inhibited the growth and metastasis of colorectal cancer.

Gene Therapy represents a cutting-edge field utilizing genes to treat hereditary and neoplastic disorders (see Section 4). This therapeutic modality is becoming increasingly feasible thanks to advances in Genetic Engineering that enable The production of recombinant DNA containing the therapeutic gene. The primary challenge remains the development of effective delivery systems to transport the therapeutic genes into the patient's body.

Clinical trials are underway for the treatment of patients with melanoma and colorectal cancer. In this Procedure, a patient's tumor or Bone Marrow cells are extracted and irradiated. A "therapeutic" gene (such as the interleukin-2 or tumor necrosis factor gene) is introduced into the irradiated cells, which are then re-implanted into the patient. The products of these "therapeutic" genes enhance tumor immunogenicity and facilitate the destruction of both the primary tumor and its metastases.

Viruses are also administered as vectors for "therapeutic" genes. Most commonly, adenoviral and retroviral constructs are used, as they readily integrate into the DNA of transformed cells. The viruses are stripped of the genetic material essential for viral replication and the inactivation of p53, and the "therapeutic" gene is inserted in its place.

All of the above gives hope that in the coming years, humanity will be able to effectively combat cancer and secure the lives and health of patients.