Use of Natural Compounds to Increase Drug Accumulation or Reduce Drug Resistance

Cancer cells are better able to adapt to stress than normal cells. In cancer treatment, this adaptation results in tumor cells that develop resistance to chemotherapy drugs, which is a primary obstacle to treatment success. Cancer cells often develop resistance not only to the drug to which they have been exposed but also to other drugs and noxious agents they have not encountered. Development of this nonspecific or multidrug resistance might be likened to a battleship crew placed on "battle stations" after a surprise attack; in this state of alertness, they are prepared for any new onslaught, whatever its form. Once a cancer cell becomes stressed and develops multidrug resistance, it is not easily harmed.

To devise strategies for reversing multidrug resistance with natural compounds, we must first understand how resistance develops. At least four mechanisms have been described that mediate multidrug resistance:33

1. Decreased drug sensitivity. A cancer cell's sensitivity to a drug can be reduced if the cell produces mutated targets of the drug, such as a mutated enzyme, that are no longer sensitive to the drug's actions. Sensitivity can also be reduced if the cell overproduces the intended target (again, such as an enzyme). Lastly, it can be lowered if the cancer cell overproduces certain proteins that protect the cell from damage. Examples include altered production/activity of the antiapoptotic protein Bcl-2 (see Table 2.1), topoi-somerase II (see Chapter 2), and heat-shock proteins. The latter are produced in response to stress and prepare the cell for additional stress.

2. Increased repair of intended drug targets. Drug resistance can develop if the cancer cell increases the repair of drug targets. For example, many drugs target DNA, and some cancer cells can increase DNA repair. In some cases, this may take the form of greater p53 expression, since p53 facilitates repair of damaged genes.

3. Increased drug expulsion. Drug resistance can develop if export of the drug from the cell is increased. The required pumping action is commonly mediated through two proteins called P-glycoprotein and the multidrug resistance protein (MRP).a When one or both these proteins are overactive, drug concentra a A series of MRP proteins exist named MRP1, MRP2, and so on.

tions within the cell are minimized. Note that the functions of these proteins are still not fully understood, and reports on their activities vary.34

4. Altered metabolism and drug detoxification. Cancer cells can gain resistance by increasing their ability to detoxify drugs. A common example is increased resistance due to enhanced activity of the glutathione S-transferase drug detoxification system; recall that this enzyme catalyzes the formation of drug-glutathione conjugates (see Figure 18.1). Once formed, these water-soluble conjugates can be expelled from the cell.a Increased glutathione concentrations and/or glutathione S-transferase activity have been associated with resistance to many drugs. Multiple means are available by which the conjugates can be expelled from the cell; the primary mechanism in most appears to be via the glutathione-xenobiotic (GSH-X) pump. Increasing evidence indicates this pump is closely related to, if not identical with, the multidrug resistance protein (MRP) mentioned above. If they are indeed identical, this implies that MRP is able to expel both conjugated and unconjugated compounds.

Inhibition of drug resistance has a strong parallel with inhibition of cancer in general. Multiple mechanisms are at work both in cancer cell survival and multidrug resistance, and neither has yet been adequately inhibited by targeting a single mechanism. For example, two drugs, verapamil and cyclosporin, have received the most study as modifiers of multidrug resistance. Both inhibit drug resistance primarily through a single mechanism, inhibition of P-glycoprotein.35'36 The results of clinical studies using these drugs in patients with solid tumors have been disappointing, although some promising results were seen with hematological cancers.37 A recent review postulates that the poor results were likely due to the multitude of mechanisms occurring in multidrug resistance and the fact that it is affected by other factors such as cell proliferation, angiogenesis, and apoptosis, as well.37

The complexity of multiple mechanisms in drug resistance has been investigated by other authors. In a study on leukemia patients, no relationship was found between the resistance to chemotherapy and the expression of any single protein involved in drug resistance (MRP, p53, heat-shock protein, P-glycoprotein, and so on). A correlation to resistance was found, however, when groups of two or more of these proteins were analyzed together, indicating that a number of events occur simul-

a In some cases, glutathione conjugates can form spontaneously, without the activity of glutathione-S-transferase, but the reactions occur more readily when it is present.

taneously to confer multidrug resistance.38 Consequently, combinations of compounds that can inhibit drug resistance through multiple pathways may provide the greatest effect.

Although each of the four mechanisms listed could conceivably be affected by natural compounds, most research has focused on three areas: inhibition of P-glycoprotein, inhibition of the glutathione S-transferase drug detoxification system, and inhibition of heat-shock proteins. Each of these is discussed separately below.

Inhibition of P-glycoprotein

As mentioned, P-glycoprotein acts as a pump to export drugs and other noxious compounds from a cell. It has at least three characteristics that make it susceptible to inhibition by natural compounds. First, its actions are regulated largely through PKC activity, and as we know, many natural compounds inhibit PKC. Tumor cell variants that overexpress mdr (the gene that encodes for P-glycoprotein) tend to have several-fold higher levels of PKC activity compared to their drug-responsive counterparts. Consequently, PKC inhibitors reduce multidrug resistance in a variety of cell lines.39 40 For example, one study reported that PKC inhibitors reduced resistance of brain cancer cells to vincristine in vitro.41

A similar PKC-dependent effect is seen in cells surviving radiation exposure. Like chemotherapy, radiation is a noxious agent that stimulates multidrug resistance. Ionizing radiation rapidly and transiently activates PKC in a dose-dependent fashion.40 Since PKC activity is important for cell survival following radiation, PKC inhibitors can sensitize cells to radiation-induced kill-ing.42 For example, hypericin, which decreases PKC activity, increased the sensitivity of brain cancer cells to radiation in vitro.43

The second means by which natural compounds can inhibit P-glycoprotein is by blocking ATP binding. ATP is the energy source within cells. As discussed in Chapter 4, the pluripotent activity of several natural compounds against various ATP-dependent enzymes such as PTK and PKC may be due to their ability to decrease the binding of ATP, thereby reducing the enzyme's energy source. Other ATP-dependent enzymes like MRP/GSH-X might also be inhibited with these natural compounds.

The third means of inhibiting P-glycoprotein is through inhibition of NF-kB. In some cancer cell lines, the expression of the mdr gene is associated with prior increases in NF-kB activity and can be diminished by NF-kB antagonists.44,45 NF-kB activity can be reduced by a number of natural compounds, including antioxi-dants (see Chapter 5).

Inhibition of the Glutathione S-Transferase Drug Detoxification System

As already stated, the glutathione S-transferase drug detoxification system involves the formation of drug-glutathione conjugates and expulsion of these conjugates from the cell. Adequate glutathione must therefore be present for this system to function, and it is possible to reduce this form of drug resistance by lowering intracel-lular concentrations of glutathione. Compounds such as glutamine and whey may be well suited for this task (see Chapter 18). It is true that by lowering glutathione concentrations, cancer cells might come under considerable oxidative stress, and for several reasons we do not advocate the production of oxidative stress as a treatment strategy. If chemotherapy is being used, however, a commitment has likely already been made for a prooxi-dant therapy.

Drug resistance may also be lowered by reducing the activity of the MRP/GSH-X pump. Since this protein is dependent on ATP, a number of natural compounds may be able to affect its activity, as discussed previously.

Inhibition of Heat-shock Proteins

Heat-shock proteins are those that protect the cell from death due to adverse conditions. Heat-shock proteins are induced by stress, including that caused by chemotherapy drugs. Once produced, these proteins assist the cell to withstand future insults. The production of heat-shock proteins is stimulated by the binding of a transcription factor (called heat-shock factor) to DNA.

Natural compounds can inhibit heat-shock proteins through at least two mechanisms. First, some natural compounds, including quercetin, prevent heat-shock factor from activating gene transcription, thereby averting the production of heat-shock proteins.46,47 Second, increased heat-shock factor activity and gene expression are positively associated with increased NF-kB activity.48, 49,50 It follows then that inhibitors of NF-kB activity could inhibit production of heat-shock proteins.

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