SEARCH

SEARCH BY CITATION

Keywords:

  • tryptophan hydroxylase;
  • 7-hydroxytryptophan;
  • carcinoid tumor;
  • small-cell lung carcinoma;
  • suicide prodrug

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

BACKGROUND

Carcinoids and small cell lung carcinomas stimulate their growth in an autocrine manner by releasing serotonin, an effect that is blocked by selective serotonergic receptor antagonists that, unfortunately, exert undesirable side effects on serotonergic central nervous function. Moreover, conventional chemotherapeutic agents, such as streptozocin, fluorouracil, cyclophosphamide, and doxorubicin, which target tumor cells directly, have produced disappointing results in the treatment of patients with these tumors in the advanced stage. Therefore, there is still a need for more specific and potent chemotherapeutic agents in the fight against serotonin-producing tumors.

METHODS

The authors synthesized 7-hydroxytryptophan to test its chemotherapeutic value in cell culture, using a system consisting of serotonin-producing and nonproducing cell lines.

RESULTS

The authors chose tryptophan hydroxylase, the rate-limiting enzyme of serotonin biosynthesis, which is expressed highly in small cell lung carcinomas and carcinoids, as a target for the induction of cellular suicide by chemotherapy. They found that this otherwise substrate specific enzyme was capable of metabolizing in situ a harmless tryptophan analogue, 7-hydroxytryptophan, to a potent toxin, 5,7-dihydroxytryptamine, a conversion blocked by the specific tryptophan hydroxylase inhibitor parachlorophenylalanine.

CONCLUSIONS

These data suggest that 7-hydroxytryptophan may be a highly specific chemotherapeutic compound against serotonin-producing tumors that also interferes with the autocrine capabilities of serotonin synthesis. Cancer 2002;94:3135–40. © 2002 American Cancer Society.

DOI 10.1002/cncr.10592

Carcinoid tumors are neuroendocrine tumors that are found predominantly in the gastrointestinal tract, where they arise from chromaffin cells of the crypts of Lieberkühn, but they also occur in the bronchiopulmonar system and in the pancreas.1 Conventional chemotherapeutic agents, such as streptozocin, fluorouracil, cyclophosphamide, and doxorubicin, which target tumor cells directly, have produced disappointing results in the treatment of patients with this disease in the advanced stage. Recently, hormonal therapy with somatostatin analogs and immunomodulation with interferon-α have yielded better results in decreasing tumor size.2, 3

However, between 3% and 18% of patients succumb to the severe hormonal syndromes associated with metastatic disease,1, 4 i.e., carcinoid syndrome, which is defined by a largely decreased availability of the rare essential amino acid tryptophan (Trp).2, 3 Trp is not only the precursor of nicotinamide and nicotinamide adenine dinucleotide+ but is also the substrate of tryptophan hydroxylase (TPH; EC 1.14.16.4), the rate-limiting enzyme in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine; 5-HT; 5), which explains the profound mood disorders found in patients with advanced carcinoid tumors. It is interesting to note that, in patients with carcinoid syndrome, it is believed that the high level of TPH expression in carcinoid tumors is responsible for the decreased Trp levels, because carcinoids synthesize exhaustive amounts of 5-HT,6 using up to 60% of the available substrate for central nervous system (CNS) and gastrointestinal 5-HT and nicotine amide synthesis, whereas normal serotonergic metabolism uses only about 1% of the dietary Trp.6 Therefore, both metabolic pathways are impaired profoundly in other tissues in patients with carcinoid syndrome, resulting in pellagra-like symptoms that can be reduced partially by Trp administration.6

Mitogenic effects of 5-HT have been described in patients small cell lung carcinoma (SCLC) cell lines7 as well as in a non-SCLC (carcinoid) human lung carcinoma cell line.8 Moreover, a similar autocrine role of 5-HT has been demonstrated in human pancreatic carcinoid tumors.9 Therefore, 5-HT should be added to the list of autocrine growth factors that are produced and released by these very aggressive tumors. Recently, Gilbert et al.10 presented a possible chemotherapeutic strategy based on the inhibition of aromatic amino acid decarboxylase (AAAD; EC 4.1.1.28) of carcinoid tumors with carbidopa. AAAD commits the second step in the biosynthesis of 5-HT. Therefore, their strategy was based solely on the interruption of the growth-stimulating autocrine loop effected by 5-HT on carcinoid tumors and other 5-HT-producing tumors, such as SCLC.

In vivo, AAAD inhibition by carbidopa, leading to an extensive decrease in peripheral 5-HT levels,11 may lead to several complications, especially concerning primary hemostasis and blood pressure. Therefore, we decided to elucidate the potential of a combined antitumor therapeutic strategy: reducing 5-HT biosynthesis in highly 5-HT-synthesizing, transformed cells by substrate competition for TPH and the simultaneous conversion of a harmless competitive inhibitor into a potent toxin using the catalytic activity of TPH.

For this purpose we tested 7-hydroxytryptophan (7-HTP) as a substance that may possess both mentioned properties, because it is a competitive inhibitor of TPH activity and is convertible into 5,7-dihydroxytryptamine (5,7-DHT), a very potent intracellular toxin,12 by TPH and AAAD in situ. It is noteworthy that 5,7-DHT, along with the analogous 5,6-DHT, does not have any extracellular toxicity but does possess high toxicity in the cytosol after it has been transported actively by the selective 5-HT reuptake transporter (5-HTT12), because redox-cycling of oxidation products of 5,7-DHT (and 5,6-DHT) produce highly cytotoxic hydroxyl radicals.12 Because 5-HTT is expressed in a wide range of cells, and not only in carcinoid tumors, direct chemotherapeutic application of DHTs is not admissible, because this would affect the viability of too many tissues, leading to expectably strong, undesirable side effects.

Herein, we report the establishment of a cell culture system based on TPH-expressing NG108-15 cells and non-TPH-expressing COS7 cells, which allowed the evaluation of TPH-activated suicide prodrugs. Furthermore, we scrutinized the potency of TPH-based chemotherapeutics detected by this system on 5-HT-synthesizing tumor cell lines. In this article, we report the specific inhibitory effect of 7-HTP on the proliferation of the 5-HT-producing cell lines NG108-15 (mouse neuroblastoma X rat glioma13), P815 (mouse mastocytoma14), and BON (human pancreatic carcinoid9).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

Synthesis of 7-HTP

A suspension of 500 mg of 7-benzyloxy-D,L-tryptophan (Sigma, Deisenhofen, Germany) and 250 mg of 5% palladium on charcoal (Fluka, Germany) in 10 mL of degassed reagent-grade water and 10 mL of absolute ethanol was hydrogenated at room temperature and atmospheric pressure for 3 hours. The catalyst was filtered off through a 0.25-μm membrane (Millipore, Germany), washed once with degassed reagent-grade water, and the filtrate was evaporated in vacuo, yielding 212 mg (60%) of pure 7-HTP. The product was identified first by intense violet coloration of a solution of 0.5% Fast Blue B salt (Sigma). Then, purity was assessed by high-pressure liquid chromatography on a C18 reverse-phase column (Vydac; type 218TP104) and fluorometric (Fig. 1A) and ultraviolet (UV)-visible light (VIS) detection, basically as reported previously.15 Briefly, the carrier buffer was 10 mM potassium phosphate buffer, pH 5.0, containing 5% methanol with a flow rate of 2 mL per minute. Continuous fluorescence measurement of the column eluate was performed at 345 nm with excitation at 295 nm, and UV absorption was measured at 295 nm.

thumbnail image

Figure 1. (A) A representative high-pressure liquid chromatography run of synthesized 7-hydroxytryptophan (7-HTP) with fluorescence detection. The reaction product elutes with a retention time of 3.2 minutes. No contaminants can be detected in the reaction product by using either fluorescence detection or ultraviolet-visible light detection (not shown). (B) The specific toxicity of 7-HTP on NG108-15 cells. The metabolism of 7-HTP to 5,7-dihydroxytryptamine (5,7-DHT) is committed in two steps by tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (AAAD) in vivo, in competition with tryptophan (Trp) for serotonin (5-hydroxytryptamine; 5-HT) biosynthesis. In contrast to 5,7-DHT, which is transported only by the 5-HT reuptake transporter (5-HTT), 7-HTP can be transported into virtually all cell types by the large neutral amino acids transporter (LNAA). Therefore, 7-HTP is toxic only in TPH-expressing cells. The major oxidative products of 5,7-DHT, 5-hydroxytryptamine-4,7-dione (5-HT-4,7-D) and 6,6′-bi(5-hydroxytryptamine-4,7-dione) (6,6 ′-bi(5-HT-4,7-D), which are formed only intracellularly, undergo redox cycling with concomitant superoxide radical anion, (O2) formation. Dismutation of O2 yields H2O2 which, as a result of transition metal ion-catalyzed Haber–Weiss chemistry, yields the cytotoxic hydroxyl radical, HO·.12

Download figure to PowerPoint

Cell Culture

COS7, P815, and NG108-15 cells were obtained from the American Type Culture Collection (Rockville, MD), and BON cells were kindly provided by Dr. Ahnert-Hilger (Charité, Berlin, Germany). BON and COS7 cells were grown in Dulbecco modified Eagle medium (DMEM) containing 1 mg/mL glucose, NG108-15 cells were grown in DMEM containing 4.5 mg/mL glucose and HAT medium supplement (Sigma, Deisenhofen, Germany), and P815 cells were grown in RPMI 1640 medium (all media were obtained from Life Technologies, Germany) in a humidified incubator and an atmosphere of 5% CO2 in air at 37 °C. All media were supplemented with 10% fetal calf serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin.

Cells were seeded in 24-well plates in 250 μL medium per well; and 7-HTP, 5,6-DHT, or 5,7-DHT solutions in phosphate-buffered saline (PBS; Life Technologies) or equal volumes of PBS as controls were added in the indicated final concentrations in triplicate. For continuous exposure, drugs were replaced daily. After the indicated times, aliquots of treated cells were taken directly from suspension (P815 cells) or after lifting (BON, NG105-15, and COS7 cells) with 0.05% trypsin and 0.53 mM ethylenediamine tetraacetic acid (Life Technologies) combined with media. Viable cells were then counted on a Neubauer hemocytometer after trypan blue staining.

Statistical Analyses

Experiments were performed independently in triplicate. After normalization to vehicle controls (100%), means and standard errors of the mean of all experiments were calculated, and statistical significance was calculated with type 3, two-sided Student t tests. A P value < 0.05 was considered significant.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

5,6-DHT and 5,7-DHT Are Potent Toxins for NG108-15 Cells but Not for COS7 Cells

To assess whether NG108-15 cells are suitable to test the toxicity of 7-HTP, we first examined the effect of the toxic metabolites 5,6-DHT and 5,7-DHT, on these cells. The toxicity of both compounds relies on their major oxidative products, which are formed intracellularly only and undergo redox cycling with the concomitant formation of the superoxide radical anion, O2·. Dismutation of O2· yields H2O2, which, as a result of transition metal ion-catalyzed Haber–Weiss chemistry, yields the cytotoxic hydroxyl radical, HO·.12 Although both compounds share a common mechanism of action, 5,7-DHT has only poor stability in cell culture media. Therefore, most experiments were conducted with 5,6-DHT. 5,6-DHT in μM concentrations (and 5,7-DHT in μM-to-mM concentrations; not shown) specifically kills NG108-15 cells in a dose dependent manner but has no adverse effect on the survival of COS7 cells, as expected (Fig. 2). The reason for this difference in susceptibility is the fact that NG108-15 cells, in contrast to COS7 cells, express 5-HTT13 (our unpublished results), underlining the above-mentioned need of active transport through 5-HTT and rapid intracellular accumulation of DHTs to exert their cytotoxic effect.

thumbnail image

Figure 2. Survival rates are shown as the percentage of vehicle controls of COS7 cells (A,C) and NG108-15 cells (B,D) after treatment with increasing doses of 5,6-dihydroxytryptamine (5,6-DHT) after 24 hours (A,B) and 96 hours (C,D). DHTs reduced the survival of NG108-15 cells in a dose dependent manner. Asterisks: P < 0.05 compared with untreated cells.

Download figure to PowerPoint

Our data are in agreement with the evidence that 5,6-DHT and 5,7-DHT are potent toxins inside the target cells but are inoffensive outside of the target cells (Fig. 1B12). Therefore, even the death and lysis of tumor cells killed by 7-HTP and the concomitant liberation of small amounts of formed 5,7-DHT should have only minor effects on other tissues in vivo. Furthermore, it has been shown that 5,7-DHT is not transported through the blood-brain barrier in adults16; thus, its synthesis and liberation in peripheral tissues can be expected to be of minor influence on CNS serotonergic function. It is interesting to note that 5-HT-producing SCLC cells take up exogeneous 5-HT by a zimelidine-sensitive transporter,7 which presumably is identical with the brain and platelet reuptake transporter,17 transporting DHTs into target cells. Therefore, it can be expected that 5,7-DHT liberated from 7-HTP-damaged tumor cells may act further locally on other tumor cells by 5-HTT dependent transport, potentiating the chemotherapeutic effect of 7-HTP.

7-HTP Is a Potent Toxin for NG108-15 Cells but Not for COS7 Cells

Because we assumed that TPH and AAAD metabolize 7-HTP to the toxin 5,7-DHT in competition with the conversion of Trp to 5-HT (Fig. 1B), we tested the effect of 7-HTP on NG108-15 and COS7 cells. Also, 7-HTP exerts a specific toxic effect and inhibits the proliferation and survival of the TPH-expressing NG108-15 cells, but it has no effect on the non-TPH-expressing COS7 cells (Fig. 3A,B).

thumbnail image

Figure 3. (A,B) Survival rates are shown as the percentage of vehicle controls after 24 hours at the indicated concentrations of 7-hydroxytryptophan (7-HTP), revealing that 7-HTP exerted a significant, specific toxic effect only on tryptophan hydroxylase (TPH)-expressing NG108-15 cells (B) but not on non-TPH-expressing COS7 cells (A). Asterisks: P < 0.05 compared with untreated cells. (C,D) The protection of serotonin (5-hydroxytryptamine; 5-HT)-producing cells against 7-HTP by TPH-inhibition with parachlorophenylalanine (PCPA). The viability of COS7 cells was not affected by 3 mM 7-HTP or by 2.5 μM PCPA after 18 hours (C). Although 3 mM 7-HTP alone reduced the survival rate of NG108-15 cells significantly, the presence of 2.5 μM PCPA resulted in rescue of the exposed cells (D). PCPA alone had neither a stimulatory influence nor an inhibitory influence on the proliferation of the cell lines NG108-15 and COS7. Asterisks: P < 0.05.

Download figure to PowerPoint

NG108-15 Cells Can Be Protected from the Toxicity of 7-HTP by Specific Inhibition of TPH

To ensure the TPH dependent toxification of 7-HTP, we tested to determine whether TPH inhibition is able to protect NG108-15 cells against the deleterious action of 7-HTP. Because parachlorophenylalanine (PCPA) is an established, highly specific, and irreversible inhibitor of TPH activity that is used frequently in TPH-related research,18 we assumed that this compound could protect TPH-expressing cells against 7-HTP.

In fact, the strong cytotoxic effect of 7-HTP on NG108-15 cells (Fig. 3B) is abolished by exposure of these cells in the presence of 2.5 μM PCPA (Fig. 3D), whereas COS7 cells are affected neither by 7-HTP nor by PCPA (Fig. 3A,C). Therefore, the TPH dependency of the mechanism of toxification of 7-HTP to 5,7-DHT is clearly visible from the protective effect of PCPA against the otherwise deleterious exposure of 5-HT-producing NG108-15 cells to 7-HTP, confirming the high specificity of our pharmacologic approach. These data establish NG108-15 cells as valuable tools for the investigation of TPH-based chemotherapeutic approaches.

7-HTP Also Exerts Specific Cytotoxicity on Other TPH-Expressing Cell Lines, such as Mouse Mastocytoma Cells (P815) and Human Pancreatic Carcinoid Cells (BON)

The wide spectrum of 7-HTP as a specific chemotherapeutic agent against 5-HT-producing tumors was established further by using completely different cell lines that had only TPH expression in common. It was found that all other investigated TPH-expressing cell lines, such as the mouse mastocytoma cell line P815 and the human carcinoid cell line BON, also are highly sensitive to the TPH dependent suicide toxin 7-HTP (Fig. 4A,B).

thumbnail image

Figure 4. The specific toxicity of 7-hydroxytryptophan (7-HTP) on tryptophan hydroxylase (TPH)-expressing tumor cell lines is demonstrated. (A) Mastocytoma P815 cells were sensitive to 7-HTP at high concentrations (up to 4 mM) and/or long exposure times (48 hours). (B) The human pancreatic carcinoid BON cells were sensitive similarly to both 3 mM 7-HTP and NG108-15 cells, resulting in < 5% survival after 24 hours (B). Asterisks: P < 0.05 compared with untreated cells.

Download figure to PowerPoint

This is the first specific cytotoxic approach based on TPH, the rate-limiting enzyme in the biosynthesis of 5-HT, for the selective ablation of 5-HT-producing tumor cells: nontoxic 7-HTP is metabolized to the potent toxin 5,7-DHT in situ, affecting the viability of 5-HT-producing tumor cells by its direct toxic effect and simultaneously interrupting the 5-HT dependent autocrine growth-stimulating loop by competitive inhibition of TPH for Trp conversion to 5-HT. Attempts to inhibit 5-HT-producing tumor cell proliferation by interfering with its autocrine loops have been performed both in experimental models7, 10, 19 and in patients;20 the data described above provide the possibility of a further approach to the control of the proliferation of 5-HT-producing tumors. The above data suggest that highly specialized tumors generally can be fought by using their distinct metabolic pathways as targets by delivery elsewhere of inoffensive substrates and forcing them to produce highly toxic derivatives by these pathways that cause the specific suicidal death of these cells only.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

The authors thank G. Böttger for her excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES