Basic Approaches

The basic mechanisms by which antineoplastic drugs kill tumor cells are summarized in Figure 54-3. Only compounds that show some selectivity for neoplastic cells are used clinically. In general, antimetabolites inhibit DNA synthesis, whereas alkylating agents, intercalators, and antibiotics damage or disrupt DNA, interfere with topoisomerase activity, or alter ribonucleic acid (RNA) structure. Steroids interfere with transcription, several plant alkaloids disrupt mitosis, agents such as asparaginase destroy essential amino acids needed for translation, and other drugs act through important growth factor signal transduction pathways. Many clinically used antineoplastic drugs must undergo either chemical or enzymatic modification to become actively cytotoxic.

FIGURE 54–3 Basic mechanisms by which antineoplastic drugs selectively kill tumor cells. E stands for enzymes, some of which are inhibited by these drugs. Inhibition of DNA or RNA synthesis or replication, production of miscoded nucleic acids, and formation of modified proteins are key mechanisms of action for many of these drugs.

Alkylating Agents

Alkylation refers to the covalent attachment of alkyl groups to other molecules. Alkylating agents came to be used for cancer therapy as a result of observations of the effects of the mustard gases on cell growth. Although these compounds are too toxic for clinical use in cancer, the first effective antineoplastic agents, including mechlorethamine, were developed from related nitrogen mustard alkylating agents and are still used today.

Alkylation takes place through chemical formation of a positively charged carbonium ion that reacts with an electron-rich site, particularly on DNA or RNA, to form modified nucleic acids. Most clinically used alkylating drugs have two active groups, which enable them to form covalent links between adjacent nucleic acid strands that are more difficult to repair than monofunctional adducts. These cross-links also prevent separation of the dual strands of DNA during cell cycling. For maximal kill, it is important to administer the maximally tolerated dose. The alkylation sequence for mechlorethamine reacting with the N-7 position of deoxyguanylate is shown in Figure 54-4. Although many other nucleophilic constituents, including RNA, proteins, and membrane components, become alkylated within cells, it is generally believed that the primary cytotoxic events occur through alkylation of DNA, especially by coupling to the N-7 position of the deoxyguanylates of either single- or double-stranded DNA.

FIGURE 54–4 Alkylation by mechlorethamine, showing positively charged intermediate ion and its covalent attachment to the N-7 position of two deoxyguanylate nucleotides of DNA.

Structures of several clinically used alkylating agents are shown in Figure 54-5. Cyclophosphamide undergoes a combination of enzymatic and chemical activation to form the active phosphoramide mustard alkylating agent (Fig. 54-6). Exposure of cells to cyclophosphamide and other alkylating agents can also lead to carcinogenesis. For example, leukemia is a well-known long-term complication in patients with Hodgkin’s disease treated with a regimen including mechlorethamine.

FIGURE 54–5 Structures of nitrogen mustards and busulfan.

FIGURE 54–6 Mechanism of enzymatic and chemical activation of cyclophosphamide to form active phosphoramide mustard. Acrolein has some antitumor activity but much less than that of the phosphoramide mustard.

Another group of antineoplastic alkylating agents in clinical use is the nitrosoureas. The structures and primary mechanisms of activation for these compounds are shown in Figure 54-7. In addition to alkylation of DNA, the nitrosoureas also cause carbamoylation of proteins, which may play a role in cytotoxicity. The alkylation route, however, is the major source of cytotoxicity. Nitrosoureas are lipophilic and can cross the blood-brain barrier, so they are often used to treat brain tumors.

FIGURE 54-7 Structures and activation pathways for nitrosourea alkylating agents. Carbamoylation of proteins also occurs but is believed to be a lesser cause of cell cytotoxicity than is alkylation of DNA.

Temozolomide is the first new alkylating agent approved for treatment of malignant gliomas in decades. It has a structure similar to dacarbazine and is rapidly absorbed after oral administration. Unlike many other alkylating agents, temozolomide crosses the blood-brain barrier. It methylates guanine and adenine, resulting in misincorporation of thymidine (across from the methylated guanidine), which cannot be easily corrected by the mismatch repair system. Resistance occurs by up regulation of components of the DNA mismatch repair system. Other compounds that form covalent bonds with DNA are cisplatin and its analog carboplatin, whose structures are shown in Figure 54-8. Cisplatin is a square planar complex of platinum with two ammonia molecules and two chloride ions at the corners of the plane. The reaction sequence of the active species is complex and not completely understood. Replacement of a chloride with a hydroxyl must occur before the platinum-nitrogen bond can interact with DNA. Subsequently, the second chloride is aquated and reacts with DNA. The stereochemistry of the complex enables the cis, but not the trans isomer, to form two covalent platinum-nitrogen bonds, primarily at two adjacent deoxyguanylates of DNA. This intrastrand cross-link prevents DNA replication and is cytotoxic. Cisplatin also forms interstrand cross-links and protein-DNA cross-links. Similar DNA cross-links are formed with carboplatin. Oxaliplatin is an organoplatinum compound constructed to overcome resistance to cisplatin by binding the platinum atom to 1,2 diaminocyclohexane. Oxaliplatin undergoes conversion to reactive metabolites that covalently bind to either adjacent guanines, adjacent adenine-guanines, or guanines separated by an intervening nucleotide. This creates interstrand and intrastrand cross-links and inhibits DNA replication and transcription. Oxaliplatin is approved for treatment of metastatic colorectal carcinoma, has activity in lung cancer, and may have activity in breast and esophageal cancers and lymphoma.

FIGURE 54-8 Square planar complex of cis-diamminedichloroplatinum (II), cisplatin, and the platinum derivative, carboplatin.

Several other clinically useful alkylating agents include busulfan, dacarbazine, procarbazine, ifosfamide, and melphalan. The busulfan type of compounds alkylate nucleic acid bases, primarily at the N-7 of guanine, and also alkylate-SH groups of glutathione and protein thiols.

The specific reaction sequences by which dacarbazine or procarbazine alkylate DNA are not well understood. Melphalan is a phenylalanine derivative and is actively transported into the cell by the carriers that transport leucine and glutamine. Melphalan is associated with induction of secondary leukemias. Chlorambucil is structurally similar to melphalan and is used primarily in treatment of chronic lymphocytic leukemia. Ifosfamide, like its analog cyclophosphamide, is activated by hepatic microsomes. Early studies with ifosfamide showed its use was associated with a significant incidence of hemorrhagic cystitis. Ifosfamide is now administered with a systemic thiol, sodium-2-mercaptoethane sulfonate (MESNA). MESNA becomes a free thiol after glomerular filtration and combines with the products responsible for causing the cystitis. Ifosfamide is active against several cancers, including small-cell lung cancer, sarcomas, lymphomas, testicular carcinoma, and gynecological cancers.

Antimetabolites

The antimetabolites are compounds that mimic the structures of normal metabolic constituents including folic acid, pyrimidines, or purines. The antimetabolites inhibit the enzymes necessary for folic acid regeneration or the pyrimidine or purine activation of DNA or RNA synthesis in neoplastic cells. Antimetabolites frequently kill cells in the S phase (see Fig. 54-1). Methotrexate (MTX), 5-fluorouracil (5-FU), cytarabine (Ara-C), 6-mercaptopurine (6-MP), gemcitabine, and 6-thioguanine (6-TG) are the primary antimetabolites used clinically.

Folic acid is essential for enzymatic reactions that transfer methyl and related groups during purine and pyrimidine synthesis. The antimetabolite MTX competitively inhibits the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate (FH₄; Fig. 54-9). As a consequence, FH₄ regeneration is blocked, and the synthesis of purines and pyrimidines is prevented. MTX is specific for cells in the S phase of the cell cycle. Intracellular addition of several glutamates to MTX greatly enhances its inhibitory activity and also prevents cellular efflux. MTX is more toxic to tumor cells than normal cells, in part because of the greater polyglutamating enzyme activity in tumor cells. Thus a higher concentration of the more active MTX covalently linked to multiple glutamates is trapped within tumor cells, where it acts as an antimetabolite.

FIGURE 54–9 Structures of dihydrofolic acid (FH₂), tetrahydrofolic acid (FH₄), and methotrexate. The reaction shown is a pyrimidine synthesis (thymidine monophosphate from deoxyuridylic acid) catalyzed by thymidylate synthase and requiring FH₄ as cofactor. E is dihydrofolate reductase, which is reversibly inhibited by methotrexate, thus preventing regeneration of FH₄ from dihydrofolate (FH₂). The rescue path is discussed in the text. The pyrimidines are needed for DNA formation.

Transport of MTX into cells is carrier mediated, and reduced MTX uptake is a prominent mechanism of tumor cell resistance. To overcome limitations of carrier uptake and enhance drug entry by passive diffusion, some investigators have infused high-dose MTX intravenously (IV) over several hours. When high-dose MTX is administered, it is mandatory that it be followed by a “rescue process” of leucovorin (citrovorum factor of N6-formyl-FH₄). This is a substitute for FH₄, which is believed to enter nonmalignant cells by a carrier-mediated process, enabling purine and pyrimidine synthesis to proceed. The clinical efficacy of this high-dose MTX-leucovorin rescue approach, however, is still under debate.

Pemetrexed is an MTX analog that inhibits several folate-dependent enzymes involved in synthesis of thymidine and purine nucleotides. Pemetrexed is preferentially converted to polyglutamate forms in malignant, as compared with normal cells. Because polyglutamated metabolites have an increased half-life, pemetrexed is most active in malignant cells. Pemetrexed is approved for the treatment of malignant mesothelioma in combination with cisplatin. It also appears, like MTX, to have activity in cervical and breast cancer.

Another antimetabolite, 5-FU, acts primarily by inhibiting pyrimidine synthesis and thus DNA formation. Its structure is shown in Figure 54-10. 5-FU is metabolized to the 5-fluoro analog of deoxyuridylic acid, which inhibits thymidylate synthase by covalent coupling. Capecitabine, an oral fluoropyrimidine, is an inactive precursor of 5-FU. It is converted to 5-FU selectively in the liver and tumor tissues. Evidence suggests that thymidine phosphorylase, the enzyme responsible for the final step in conversion to active 5-FU, is overexpressed in neoplastic tissues.

FIGURE 54–10 Structures of prototype purine and pyrimidine antimetabolites.

Ara-C also acts by inhibiting pyrimidine synthesis but through a more complex pathway (Fig. 54-11). The drug must undergo enzymatic conversion to the active cytosine triphosphate derivative, which is incorporated into DNA. At high doses, Ara-C also binds to and inhibits DNA polymerase competitively. Cytidine deaminase activity is high and deoxycytidylate kinase activity is low in some patients, resulting in considerable inactivation of the drug before conversion to its active form.

FIGURE 54–11 Competing activation and deactivation pathways for conversion of cytosine arabinoside to the active form that inhibits DNA polymerase.

Gemcitabine is another pyrimidine antimetabolite (see Fig. 54-10). It is a prodrug that, once transported into cells, must be phosphorylated by deoxycytidine kinase to an active form that inhibits DNA synthesis. Cell death most likely occurs as a result of blockade of DNA strand elongation. Gemcitabine appears to have activity against adenocarcinoma of the pancreas.

The purine analogs 6-MP and 6-TG (see Fig. 54-10) also must undergo activation to form nucleotides, which then act as competitive inhibitors of several enzymes in purine synthesis pathways. The adenosine deaminase inhibitor pentostatin (2-deoxycoformycin) is highly active against hairy cell leukemia.

Antibiotics

Several antibiotics of microbial origin are very effective in treatment of certain tumors. These antibiotics include doxorubicin, daunorubicin, bleomycin, actinomycin D, and mitomycin. The anthracycline structures of daunorubicin and doxorubicin are shown in Figure 54-12.

FIGURE 54–12 Structures of daunorubicin and doxorubicin.

Bleomycin is a mixture of several basic glycopeptides, with one called A2 predominating. Among the common anticancer drugs, it has a unique mechanism of action in that it forms a tertiary complex with O₂ and Fe⁺⁺ to cause sequence-specific single- and double-stranded DNA cleavage. The double-stranded DNA cleavage that results is thought to be lethal. Doxorubicin and daunorubicin intercalate between the bases in double-stranded DNA, poison topoisomerase II, generate free radicals, and possibly disrupt the functioning of the cell membrane. It is generally believed that their poisoning of DNA topoisomerase II constitutes their major antitumor action (Fig. 54-13). DNA topoisomerase II is essential for DNA replication and catalyzes the uncoiling and breakage of both strands of double-stranded DNA to modify the number and the types of linkage twists. Doxorubicin and daunorubicin inhibit the enzyme by stabilizing a covalent complex of an enzyme-DNA intermediate, preventing the DNA breaks from rejoining and leading to cell death. Doxorubicin is the single most active agent against breast cancer; daunorubicin and idarubicin are frequently used to treat leukemias.

FIGURE 54–13 Mammalian DNA topoisomerase II mechanism and anticancer drug action. Mammalian DNA topoisomerase II binds to DNA (A) forms two different types of protein-DNA complexes that are in rapid equilibrium: the noncleavable complex (B) and the cleavable complex (C). These complexes can be identified in vitro by the ability of detergent or alkali to separate DNA strands. The cleavable complex is transient but is stabilized by doxorubicin, daunorubicin, etoposide, and actinomycin (D). In the absence of drug, DNA strand passage occurs, whereas drugs block DNA strand passage and DNA replication.

Actinomycin D also intercalates into DNA, thus blocking transcription, which is a major source of its antitumor activity. Actinomycin D also causes single-stranded DNA breaks, possibly through production of free radicals, and it prevents synthesis of RNA. Mitomycin undergoes chemical activation in cells, resulting in formation of a derivative that cross-links DNA by alkylation.

Plant Alkaloids

The primary plant alkaloids, vincristine and vinblastine, bind avidly to tubulin, block microtubule polymerization, and disrupt mitotic spindle formation during mitosis at the M phase of the cell cycle (see Figs. 54-1 and 54-14). Cell death results from an inability to segregate chromosomes properly. Paclitaxel, which acts as a mitotic inhibitor, binds specifically and reversibly to tubulin, but unlike other antitubule drugs, it stabilizes microtubules in the polymerized form. Paclitaxel is active against solid tumors, including ovarian carcinomas. Etoposide is a semisynthetic derivative of podophyllotoxin that is prepared from the mandrake plant (mayapple). Etoposide and teniposide, a close analog, also inhibit topoisomerase II. Etoposide has significant activity against small-cell cancer of the lungs and testicular carcinoma and is used in most first-line regimens for these diseases. Teniposide is active against acute leukemias in children.

FIGURE 54–14 Microtubule dynamics in the presence of vincristine, vinblastine, or paclitaxel. A dynamic steady-state exists, with microtubule assembly occurring at one end and disassembly at the other (A). Vincristine and vinblastine (B) bind to tubulin dimers and block polymerization, allowing disassembly to predominate. In contrast, paclitaxel (C) blocks disassembly, causing stable microtubules to form even in the absence of normally essential cofactors. Cells treated with any of these agents are blocked in mitosis.

Topotecan is a semisynthetic plant alkaloid that inhibits topoisomerase I, thereby leading to single-stranded DNA breaks. Topotecan is used for refractory ovarian cancer and may have activity against small-cell lung cancer.

Irinotecan, a camptothecin derivative, is a prodrug requiring hydrolysis to form an active metabolite that binds to the topoisomerase I-DNA complex. Topoisomerase I relieves strain in DNA by reversibly breaking single strands of the double-stranded DNA helix. Camptothecins are cytotoxic because they combine with the topoisomerase I-DNA complex, stabilizing the structurally protective single-strand breaks induced by topo I and preventing their reconnection. This defect cannot be repaired by replication enzymes, and DNA synthesis is therefore prevented. Irinotecan is approved for treatment of metastatic colorectal carcinoma and may have activity in cervical, non-small-cell lung and gastric cancer.

Others

L-Asparaginase is administered to hydrolyze asparagine, required for growth in higher amounts by tumor cells than by normal cells. Depletion of asparagine shuts off protein and eventually nucleic acid synthesis. This approach is selective for neoplastic cells devoid of asparagine synthetase that are unable to synthesize the essential asparagine.

Hydroxyurea inhibits ribonucleotide reductase, which reduces ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. It presumably complexes with the non-heme Fe⁺⁺ required by the enzyme for activity and is an S phase-specific agent.

The mechanism of action of arsenic trioxide remains unclear, although the cellular changes it induces suggest that it causes apoptosis. Arsenic trioxide is metabolized by arsenate reductase to trivalent arsenic, which undergoes methylation predominantly within the liver. Trivalent arsenic is excreted in the urine. Arsenic accumulates mainly in liver, kidney, heart, lung, hair, and nails and is approved for therapy of anthracycline-resistant acute promyelocytic leukemia characterized by the presence of specific markers. Arsenic trioxide also appears to have activity in multiple myeloma.

Mechanisms of Resistance

Unfortunately, some patients initially respond favorably to antitumor drugs, but later the tumor may return and the same drugs may be ineffective. In other patients a drug protocol may show few positive results, even though the same protocol has proved beneficial in others. These situations are typical of resistance to antitumor drugs.

In resistant subjects the reduced effectiveness can often be attributed to a decreased intracellular concentration of drug, repair of drug-induced damage, or a modification of drug targets. Increased expression of proteins that block the energy-dependent process of apoptosis, including oncogenes such as BCL2, can also cause resistance to many agents. Several mechanisms account for these differences, as indicated in Box 54-1.

One mechanism of resistance is decreased drug uptake by cells, especially to drugs such as MTX, which requires carrier proteins for transmembrane transport. Actinomycin D resistance also results from decreased uptake. A second mechanism is lack of drug activation. Cyclophosphamide requires metabolic activation, and in the absence of this pathway, tumor cells can be resistant. A third mechanism is the enhanced conversion of the active agent to an inactive metabolite. For example, increased activity of aldehyde dehydrogenase leads to enhanced metabolism of cyclophosphamide and drug resistance.

Enhanced cellular efflux of drug is a fourth mechanism. Mammalian cells possess a large phosphoglycoprotein called P-glycoprotein that acts as an adenosine triphosphate-driven transmembrane transport protein. This P-glycoprotein functions to transport hydrophobic compounds with aromatic and basic properties out of the cell. Doxorubicin, daunorubicin, actinomycin D, etoposide, teniposide, vincristine, and vinblastine are all antitumor drugs to which resistance is manifest by cells possessing elevated concentrations of the multidrug-resistant P-glycoprotein. Intracellular drug concentrations are decreased because of energy-dependent removal by P-glycoprotein. Efforts are underway to develop compounds that can block the action of the P-glycoprotein pumps and circumvent multidrug resistance. Verapamil and other Ca⁺⁺-channel blockers block the P-glycoprotein pump, but only at unacceptably high doses. Analogs of cyclosporine that lack immunosuppressive properties may be more promising.

A fifth mechanism of resistance is exemplified by bleomycin resistance, in which cells rapidly repair the DNA breaks caused by this drug. DNA repair mechanisms may also be a source of resistance to other DNA-directed antitumor drugs. For example, cells with increased intracellular concentrations of dihydrofolate reductase as a result of gene amplification are resistant to MTX. MTX resistance may also be due to the presence of an altered enzyme that is still enzymatically active but has a lower binding affinity for the drug. For example, MTX is not conjugated with polyglutamates and is therefore not retained within the tumor cell. A higher unconjugated MTX concentration is required to inhibit dihydrofolate reductase. Thus the enzyme is no longer inhibited to the same degree by the usual concentration of MTX.

Sulfhydryl compounds, including glutathione and metallothioneins, act as cellular protective groups scavenging highly reactive compounds. Increased concentrations of these endogenous scavengers may render resistance particularly against alkylating agents. Last, resistance may be attributed to decreased available targets by tumor cells. For example, a decrease in topoisomerase II activity leads to resistance to etoposide and teniposide. Unfortunately, cancer cells often possess multiple pathways for drug resistance that may make therapeutic efforts to block one or two pathways ineffective clinically in reversing resistance.