CAMPUS DE LUGO

V

E

Vitamin E resides in the lipid domain of biological membranes and plasma lipoprotein, where it prevents lipid peroxidation of PUFA. Because vitamin E is the most important antioxidant for

preventing lipid peroxidation, vitamin E adequacy may significantly impact the rate of cancer cell proliferation and the response to cancer chemotherapy. Vitamin E has been shown to effectively reverse the inhibitory effect of lipid peroxidation on cell proliferation.

Vitamin E, in vitro, has been shown to enhance the cytotoxic effect of several anticancer drugs, including 5-fluorouracil (5-FU), doxorubicin, vincristine, dacarbazine, cisplatin, and tamoxifen.

V

C

Vitamin C is a versatile water-soluble antioxidant. It protects against lipid peroxidation by scavenging ROS in the aqueous phase before they can initiate lipid peroxidation.

In vitro studies with several tumor cell lines have shown vitamin C to enhance the cytotoxic activity of doxorubicin, cisplatin, paclitaxel, dacarbazine, 5-FU, and bleomycin. Vitamin C has also been shown to increase drug accumulation and to partially reverse vincristine resistance of human nonsmall-cell lung cancer cells.

C

Q

(U

)

Coenzyme Q10 (CoQ100) is an indispensable cofactor in the electron transport chain of mitochondria, functioning as an electron carrier between the enzyme complexes of the respiratory chain. CoQ10 is also a lipid-soluble antioxidant that scavenges lipid radicals within biological membranes. CoQ10 within mitochondria may play an important role in preventing lipid peroxidative damage of mitochondrial membranes.

A potentially important application of CoQ10 supplementation during chemotherapy is for the prevention of doxorubicin-induced cardiotoxicity, most importantly the chronic form, which is not prevented by other dietary antioxidants. The postulated mechanism of dose-limiting chronic cardiotoxicity, a complication that occurs to a greater or lesser degree with all anthracyclines, involves the production of oxidizing agents through an iron-dependent process. Consistent with this hypothesis is the generation of free radicals by doxorubicin, resulting in mitochondrial lipid perox- idation within myocardial cells. However, other effects of doxorubicin on the mitochondria of cardiac myocytes may be equally or more important. These effects of doxorubicin include (1) reduction of the CoQ 10 content of

Dietary Antioxidan ts 117

mitochondrial membranes, (2) inhibition of mitochondrial biosynthesis of CoQ 10-dependent enzymes, which interferes with the aerobic generation of ATP, and (3) inhibition of mitochondrial biosynthesis of CoQ10. These effects of doxorubicin on CoQ10 biosynthesis and function may explain the acute and chronic forms of doxorubicin-induced cardiotoxicity.

Acute doxorubicin-induced cardiotoxicity, a reversible side effect, is observed soon after a single dose of doxorubicin. It can be attributed to inhibition of respiratory enzymes by doxorubicin, which may result from competition between CoQ10 and doxorubicin for the enzymatic sites of the coenzyme, because both compounds contain a quinone group. Enzyme inhibition may also result from oxidation of CoQ10 by doxorubicin or doxorubicin-induced ROS, thus preventing CoQ10 from functioning as a cofactor for electron transport. Because the respiratory generation of ATP is essential to myocardial function, inhibition of this function by doxorubicin would interfere with the electrophysiological activity of the heart, resulting in electrocardiographic changes and reduction of ejection fraction, which are characteristic of acute doxorubicin-induced cardiotoxicity.

Chronic doxorubicin-induced cardiotoxicity, which results from repeated doses of doxorubicin, may be attributable to depletion of mitochondrial CoQ10, which disrupts electron transport and mitochondrial respiratory bioenergetics, and may ultimately lead to loss of mitochondrial integrity and necrosis of cardiac myocytes. Studies in rabbits that demonstrate that mitochondrial degeneration is the earliest and most prominent ultrastructural change associated with the chronic cardi- omyopathy induced by doxorubicin support this mechanism as the primary etiology. This mechanism is also consistent with the following clinical observations: chronic doxorubicin-induced cardiotoxicity is not reversible, the associated congestive heart failure is not responsive to digitalis, and antioxidants such as vitamin E and vitamin C do not prevent it.

P-C

P-Carotene is one of the more than 600 carotenoids that are produced by microorganisms and plants.

P-Carotene, a lipid-soluble antioxidant, occurs naturally as a mixture of cis and trans isomers. Cis P-Carotene is a more effective antioxidant than trans P-carotene.

P-Carotene has been shown to enhance the cytotoxicity of melphalan and BCNU on human squamous carcinoma cells and of cisplatin and dacarbazine on melanoma cells. In mice with transplanted mammary carcinoma, P-carotene enhanced the antitumor effect of cyclophosphamide, and in mice transplanted with FsaII fibrosarcoma or SCC VII carcinoma, P-carotene enhanced the antitumor effect of melphalan, BCNU, doxorubicin, and etoposide. P-Carotene (5 to 50 mg/kg) has been shown to reduce the genotoxicity of cyclophosphamide in mice and of mitomycin C, methyl methanesulfonate, and bleomycin in cultured cells. P-Carotene also reduced the rate of tumor induction in animals receiving chronic low doses of cyclophosphamide.

G

(GSH)

G

E

GSH, a tripeptide of glutamic acid, cysteine, and glycine (GluCysGly), is the major water- soluble antioxidant in the cytoplasm, nuclei, and mitochondria of cells. Many of the critical antioxidant functions of GSH require GSH peroxidase, which exists in several forms. Reduction of oxidized GSH (GSH disulfide), which is produced by reactions involving GSH peroxidase, requires GSH reductase.

GSH is not transported into cells. For circulating GSH to increase intracellular GSH concentrations, it must fi rst be hydrolyzed to Glu and CysGly, which are subsequently transported into the cell and serve as substrates for GSH synthesis. Thus, GSH administered orally or parenterally, and that produced by the liver and released into the circulation enhance tissue levels of GSH by providing a source of its constituent amino acids. In contrast, GSH monoesters, which are well absorbed after oral administration, as is GSH, are readily transported to cells and then hydrolyzed to GSH and the corresponding alcohol.

Thus, higher cellular levels of GSH result from oral administration of GSH monoesters than from oral administration of comparable doses of GSH.

118 Pharmacodynamic Basis of Herbal M edic ine

GSH has been investigated for its protective effect against cisplatin-induced nephrotoxicity and peripheral neuropathy. Although oxidative damage most likely contributes to these toxicities, the protective effect of GSH can be accounted for by a chemical interaction between GSH and cisplatin instead of the antioxidant properties of GSH. GSH contains a thiol (sulfhydryl, an -SH moiety) group.

Thiols are strongly nucleophilic and form stable covalent compounds with electrophilic compounds, such as the platinum coordination complexes cisplatin and carboplatin. Formation of the thiol-platinum complex inactivates the antineoplastic agent, which blocks not only its ROS- generating activity but also its cytotoxic effects. If inactivation occurs within the circulation before uptake of the drug by tumor cells, interference with the antineoplastic activity of cisplatin or carboplatin may occur. A similar concern exists if GSH is administered with alkylating agents, which are also strong electrophiles, because thiols can compete with DNA for alkylation, resulting in inhibition of antineoplastic activity. Thus, caution should be employed when GSH or any thiol compound is administered with platinum coordination complexes or alkylating agents.

Studies in laboratory animals have shown that intravenous administration of a high dose of GSH (up to 500 mg/kg) within 30 min of cisplatin injection protects against cisplatin-induced neurotoxicity and nephrotoxicity. Subcutaneous injection of GSH or GSH monoisopropyl ester 2.5 h before injection of cisplatin also protected mice against nephrotoxicity and the acute lethal toxicity of cisplatin, although the GSH ester was far more effective than GSH itself. In these studies, treatment with GSH or GSH ester did not interfere with the antitumor effectiveness of cisplatin, which can be explained by the characteristics of uptake of GSH and cisplatin. GSH and cisplatin are cleared rapidly from the circulation.

N-A

^-Acetylcysteine (NAC) is well absorbed after oral administration and readily transported into cells, where it is deacetylated. Although NAC, a thiol compound, is a free-radical scavenger, its more important antioxidant role is providing an intracellular source of cysteine, a substrate for GSH synthesis.

However, because of its nucleophilic thiol group, NAC (similar to GSH) can form stable covalent compounds with electrophilic alkylating agents and platinum coordination complexes, which inactivate the anticancer drugs. In this regard, NAC has been shown to block the cytotoxicity and ROS-generating capability of cisplatin in vitro. Thus, as with GSH, one should exercise caution when administering NAC during chemotherapy with any electrophilic antineoplastic agent.

In animal studies, NAC has been shown to prevent hemorrhagic cystitis that results from administration of cyclophosphamide or its position isomer ifosfamide. Hemorrhagic cystitis results from the toxic effect of acrolein, a metabolic product of cyclophosphamide or its position isomer ifosfamide. The mechanism whereby NAC prevents this toxicity may be prevention of the intra- cellular depletion of antioxidants, such as GSH, by acrolein. Concomitant administration of NAC with cyclophosphamide or ifosfamide does not impair antineoplastic activity, because both anti- cancer drugs are inactive until they are metabolized by the liver to their phosphoramide mustard metabolites.

NAC (50 to 2000 mg/kg), by means of free-radical scavenging or by enhancing intracellular levels of GSH, protects mice against acute doxorubicin-induced cardiotoxicity without interfering with the antitumor activity of the drug.

G

Glutamine is a conditionally essential amino acid. Although it does not possess antioxidant activity, it serves as a source of glutamate for GSH synthesis, thus supporting cellular antioxidant systems. Because glutamine is a primary fuel source for the rapidly proliferating enterocytes of the gastrointestinal tract, an important role of glutamine supplementation during chemotherapy is to reduce gastrointestinal injury that results from administration of antineoplastic drugs, especially the severe mucositis that results from treatment with antimetabolites such as 5-FU and methotrexate.

Glutamine has been shown to enhance the antitumor effectiveness of methotrexate in laboratory animals, an effect that may be attributed to the increase of the intracellular tumor concentration of methotrexate by glutamine. Oral glutamine, but not intravenous glutamine, has also been shown to

Dietary Antioxidan ts 119

reduce the bacteremia and mucosal injury associated with methotrexate-induced enterocolitis of rats.

Glutamine, administered by intragastric infusion, accelerates healing of the gut mucosa in rats receiving 5-FU.

S

Although inorganic selenium does not have antioxidant properties, selenium has an important role in cellular antioxidant defenses as a necessary component of selenoproteins. Selenium is incorporated into selenoproteins as selenocysteine. The glutathione (GSH) peroxidases are the best-characterized selenoproteins, although other circulating selenoproteins also have antioxidant functions.

In laboratory animals, parenteral administration of organic and inorganic selenium (210 to 12,000

|lg/kg) has been shown to protect against cisplatin-induced nephrotoxicity. Protection occurs without apparent inhibition of the antineoplastic activity of cisplatin, although this may be attributed to the fact that selenium administration allows for higher doses of cisplatin to be used. Additionally, selenium administration reduces cisplatin-induced myelosuppression. This raises a concern similar to that with administering cisplatin with thiol compounds, i.e., that the reduction of myelosuppression may indicate that selenium can also interfere with the antitumor activity of cisplatin. Selenium, with chemical properties similar to those of sulfur, can bind with platinum and inactivate the antineoplastic platinum coordination complexes. Thus, caution should be used when administering selenium during chemotherapy with cisplatin and carboplatin.

Parenteral administration of 27 to 60 |lg/kg of selenium to laboratory animals has been shown to inhibit doxorubicin-induced decreases in myocardial vitamin E and GSH peroxidase levels and to reduce changes in myocardial function that are consistent with acute doxorubicin-induced cardiotoxicity. Oral supplementation of sodium selenite also protects against acute doxorubicin- induced cardiotoxicity in rabbits.

G

D

Soybeans contain a number of isoflavones, including genistein and daidzein, which are the most extensively studied. Soybean isoflavones enhance cellular antioxidant status by scavenging ROS and by increasing the activity of antioxidant enzymes including GSH peroxidase, GSH reductase, and superoxide dismutase. Genistein has also been shown to abrogate chemical- and ligand- induced generation of ROS in vivo and in vitro, although these effects result, in part, from activities of genistein other than its antioxidant activities. In addition to its antioxidant properties, genistein has other activities that may enhance the antineoplastic effects of cancer chemotherapy. Although clinical studies are yet to be done to assess the impact of soy isoflavones on cancer chemotherapy, a substantial number of preclinical studies have been performed.

Genistein is an inhibitor of topoisomerase I and II. Inhibition of topoisomerase II by genistein has effects similar to those of doxorubicin and etoposide, i.e., enhanced topoisomerase- Il-mediated DNA cleavage, the formation of stable covalent protein-DNA cleavage complexes, and cell-cycle arrest at the G2-M junction. Genistein also inhibits the binding of ATP to its binding site on the enzyme, an activity not possessed by doxorubicin or etoposide. Thus, administration of genistein during chemotherapy with either of these drugs may enhance antineoplastic activity. Enhanced antineoplastic activity may also result from inhibition of topoisomerase I by genistein, although this effect, which results in arrest of the cell cycle in the S phase, requires a higher concentration of genistein than is necessary to inhibit topoisomerase II. Genistein has been shown to induce apoptosis in a number of tumor cell lines.

Although apoptosis can be explained by inhibition of topoisomerase II activity, inhibition by genistein of protein tyrosine kinases (PTKs) may also contribute to the process.

Q

Quercetin, a ubiquitous polyphenolic flavonoid, is one of the most potent natural antioxidants. In addition to its antioxidant properties, quercetin exhibits other activities that may enhance antineo- plastic activity.

120 Pharmacodynamic Basis of Herbal M edic ine

As with genistein, clinical studies are yet to be performed to determine the impact of quercetin on cancer chemotherapy.

Quercetin inhibits topoisomerase II activity. Similar to genistein, it enhances topoisomerase- II-dependent DNA cleavage complexes. It also inhibits topoisomerase-II-catalyzed ATP hydrolysis.

Because quercetin is an intercalative compound, it may enhance DNA cleavage by a mechanism similar to that of doxorubicin. Quercetin also inhibits several PTKs, with IC50 values that are comparable to or lower than those of genistein.

Quercetin has been shown to inhibit the growth of several human and animal cancer cell lines in vitro.

However, the sensitivity of different cell lines varies considerably. In human breast cancer cells the IC50

of quercetin for inhibition of growth was 23 ||M (7 |g/ml), whereas in human gastric cancer cells the IC50

was 32 to 55 ||M A head and neck squamous cell carcinoma line is unaffected by quercetin at <110 ||M Because growth inhibition of sensitive cells results from arrest of cell- cycle progression at the GrS boundary, this effect of quercetin is likely to be independent of its inhibition of topoisomerase II.

Quercetin has been shown to enhance the cytotoxicity of several antineoplastic agents. In multidrug-resistant cancer cells, quercetin markedly enhanced the growth-inhibitory effects of doxorubicin, although it did not affect the cytotoxicity of doxorubicin in drug-sensitive cells. In drug-sensitive cancer cells, quercetin has been shown to enhance the antiproliferative activity of cisplatin, nitrogen mustard, busulfan, and cytosine arabinoside. Quercetin also enhances the antitumor activity of cisplatin in athymic nude mice implanted with a human large-cell lung cancer.

CONCLUSIONS

Dietary supplementation with antioxidants may provide a safe and effective means of enhancing the response to cancer chemotherapy. Vitamin E may prove to be an important nutrient for enhancing antineoplastic activity because of its role in preventing lipid peroxidation, thus maintaining the rapid rate of proliferation of cancer cells. Other antioxidants may be important because of their antioxidant properties, as well as for activities such as inhibition of topoisomerase II and PTKs.

The quality of life of patients after chemotherapy may be improved by dietary supplementation with antioxidants that reduce or prevent chemotherapy-induced side effects. Although approved cytoprotectants are available, including dexrazoxane for doxorubicin-induced cardiotoxicity, amifostine for cisplatin-induced nephrotoxicity, and mesna for ifosfamide-induced hemor- rhagic cystitis, these agents are not without adverse effects. For example, dexrazoxane can reduce the antineoplastic activity of doxorubicin. This most likely results from complex formation and inactivation of doxorubicin, which may also prevent doxorubicin from interfering with coenzyme Q10 (CoP10) biosynthesis and function, thus explaining the cardioprotective effect of dexrazoxane. Dexrazoxane is also myelosuppressive and may increase the risk of secondary malignancies. Amifostine can induce hypotension, hypocalcemia, and nausea, and administration of mesna is associated with nausea, vomiting, and diarrhea. In contrast, certain dietary antioxidants, in doses that are without adverse effects, can ameliorate some side effects of cancer chemotherapy. In this regard, CoQ10 may prove to be an effective means of preventing cardiotoxicity without compromising antineoplastic activity when chemotherapy employs the versatile and highly effective drug doxorubicin. However, much more work is needed to establish a clear role for the use of dietary supplements as an adjunct to cancer chemotherapy (Conklin, 2000; and General References).

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Herbal Drugs and Their High