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Department of Oncology, Division of Developmental Oncology Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905
ABSTRACT
The
carcinoid tumor is an uncommon neuroendocrine neoplasm the hallmark of
which is excessive serotonin production. In studying kinetics of
tryptophan hydroxylase and aromatic-L-amino acid
decarboxylase (AAAD) in human carcinoid hepatic metastases and adjacent
normal liver (J. A. Gilbert et al., Biochem.
Pharmacol., 50: 845–850, 1995), we identified one
significant difference: the Vmax of
carcinoid AAAD was 50-fold higher than that in normal liver. Here, we
report Western and Northern analyses detecting large quantities of AAAD
polypeptide and mRNA in human carcinoid primary as well as metastatic
tumors compared with normal surrounding tissues. To assess the
feasibility of targeting these high AAAD levels for chemotherapy, AAAD
inhibitors carbidopa (
-methyl-dopahydrazine),
-monofluoromethyldopa (MFMD), and 3-hydroxybenzylhydrazine
(NSD-1015) were incubated (72 h) with NCI-H727 human lung carcinoid
cells. Carbidopa and MFMD were lethal (IC50 = 29 ± 2 µM and 56 ± 6 µM, respectively);
NSD-1015 had no effect on proliferation. On exposure to other human
tumor lines, carbidopa was lethal only to NCI-H146 and NCI-H209 small
cell lung carcinoma (SCLC) lines (IC50 = 12 ± 1
µM and 22 ± 5 µM, respectively).
Carbidopa (100 µM) decreased growth of (but did not kill)
SK-N-SH neuroblastoma and A204 rhabdomyosarcoma cells and did not
affect proliferation of DU 145 prostate, MCF7 breast, or NCI-H460 large
cell lung carcinoma lines. The rank order of lines by AAAD activity was
NCI-H146 > NCI-H209 > SK-N-SH > NCI-H727, whereas
A204, DU 145, MCF7, and NCI-H460 had no measurable activity. For lung
tumor lines (carcinoid, two SCLC, and one large cell lung carcinoma),
AAAD activity was correlated with the potency of carbidopa-induced
cytotoxicity. However, carcinoid cell death was not solely attributable
to complete inhibition of either AAAD activity or the serotonin
synthetic pathway. In further evaluating potential applications of
these findings with carbidopa, we determined that sublethal doses of
carbidopa produced additive cytotoxic effects in carcinoid cells in
combination with etoposide and cytotoxic synergy in SCLC cells when
coincubated with topotecan.
INTRODUCTION
The carcinoid tumor is the most frequently occurring neoplasm in the small bowel (1) . Surgical resection is usually curative for primary small bowel lesions <2 cm, whereas larger primaries generally metastasize to the liver and are usually lethal. Furthermore, metastatic lesions in the liver are often associated with symptoms of the malignant carcinoid syndrome, including flushing, diarrhea, and eventually carcinoid heart disease. Among the molecules associated with these symptoms is 5-HT,3 synthesized in the tumor from trp via the sequential action of TPH (EC 1.14.16.4) and AAAD (EC 4.1.1.28). The second most common site for carcinoid primaries is the lung (2) , where large primary tumors can produce the carcinoid syndrome in the absence of metastases (3) . In addition to the carcinoid neoplasm, other tumors are known to produce 5-HT and have high levels of AAAD activity, e.g., SCLC (4 , 5) . There is currently no effective treatment for either carcinoid disease or SCLC at advanced stages.
In a previous study characterizing the enzyme kinetics of TPH and AAAD in human carcinoid hepatic metastases and adjacent normal liver, we determined that the Km and Vmax of TPH and the Km of AAAD were similar in carcinoid and normal tissue (6) . In contrast, the Vmax for carcinoid AAAD was 50-fold higher than that of normal liver AAAD. In the present study, we investigated whether this difference was regulated at the transcriptional/translational level and whether it was characteristic of primary (ileal) carcinoid neoplasms in comparison with normal tissue as well as hepatic carcinoid metastases.
5-HT induces proliferation of a variety of cultured cells, including
aortic endothelial and smooth muscle, pulmonary artery and cerebral
vessel smooth muscle, and renal mesangial, lung fibroblast, pancreatic
carcinoid, and small cell lung carcinoma cells (see reviews, Refs.
7
and 8
). We examined AAAD inhibition as a
possible antitumor therapeutic strategy and here report the effects of
the AAAD inhibitors carbidopa (-methyl-dopahydrazine), MFMD, and
NSD-1015 on the proliferation of NCI-H727 pulmonary carcinoid cells.
Carbidopa is typically coadministered with L-Dopa
for the treatment of Parkinson’s disease to inhibit peripheral AAAD.
MFMD is a selective, enzyme-activated, irreversible inhibitor of AAAD
(9)
; NSD-1015 is a commonly used AAAD inhibitor in
vitro. Extending the above approach, we evaluated carbidopa
exposure in a series of human tumor cell lines with a wide range of
AAAD levels to determine the relationship between tumor cell AAAD
activity and the effect of carbidopa on cell proliferation. Included in
this series were SCLC cell lines (NCI-H146 and NCI-H209) with very high
levels of AAAD activity (10
, 11)
and mRNA (11
, 12) . Preliminary accounts of this work have been presented in
abstract form (13
, 14)
.
MATERIALS AND METHODS
Materials.
Molecular biology grade reagents were purchased from Sigma (St. Louis,
MO) except for: 50x Denhardt’s solution (United States
Biochemical, Cleveland, OH); agarose (FMC Bioproducts, Rockland,
ME); 3 M sodium acetate (pH 5.2) and 10x MOPS
(Amresco, Solon, OH); and AG 501-X8(D) (Bio-Rad, Hercules, CA). Poly(A)
was purchased from Boehringer Mannheim (Indianapolis, IN);
high-performance liquid chromatography grade methanol was from
EM (Gibbstown, NJ) and n-propyl alcohol from Burdick
& Jackson (Muskegon, MI). DTT, 5-HT, 5-OH-trp, carbidopa,
NSD-1015, and VP-16 were purchased from Sigma Chemical Co. (St. Louis,
MO). N-Methyl-N-propargylbenzylamine
hydrochloride and triethylamine were obtained from Aldrich
Chemical Co. (Milwaukee, WI), and pyridoxal-5-phosphate was from the
Calbiochem Corp (San Diego, CA). Topotecan was provided by the National
Cancer Institute; MFMD was a kind gift from Merrell Dow Research
Institute (Cincinnati, OH). All of the other chemicals used were
reagent grade.
Human Tissues.
Specimens were flash-frozen after excision and stored at -70°C.
Western Analysis.
Preparation of tissue homogenates (100,000 x g
supernatants), protein assays, and Western analyses were performed as
described previously (6)
. Rabbit anti-AAAD antibody was
supplied by Eugene Tech International, Inc. (Ridgefield Park, NJ).
Northern Analysis.
A 40mer probe (5'-TTT GCC ATC TGT TCT CGC ACG GTG GAA TCT GCC CAT GTG
C-3') complementary to coding sequence of human AAAD cDNA
(Ref.15
; GenBank accession no. M88700) was synthesized by
Mayo Molecular Biology Core Facility.
Total RNA was isolated from frozen tissue with RNA Stat-60 reagent
(Tel-Test "B"; Friendswood, TX).
Poly(A)+ RNA was then isolated with the Fast
Track kit (Invitrogen, San Diego, CA) and quantitated by measuring
A260. RNA was electrophoretically
fractionated in 1.2% agarose formaldehyde gels followed by downward
capillary transfer (Turboblotter; Schleicher & Schuell, Keene, NH) in
20x SSC to Nytran nylon membranes (Schleicher & Schuell). After UV
cross-linking, RNA was stained with methylene blue (16)
.
The blots were prehybridized (24 h at 42°C) in: 5x SSC, 10x
Denhardt’s solution, 250 µg/ml denatured and sheared herring sperm
DNA, 10 µg/ml poly(A), 0.1% SDS, and 50% deionized formamide. The
blots were hybridized with 25 x 106 cpm/ml
of 40mer radiolabeled with [-32P]dATP
(DuPont NEN, Boston, MA) with the DNA Tailing Kit (Boehringer Mannheim)
and purified on a NENSORB 20 cartridge (DuPont NEN). Posthybridization
washes were: 5x SSC (twice for 2 min at 24°C); 2x SSC, 0.1%
SDS (four times for 5 min at 24°C); and 1.0x SSC, 0.1% SDS (three
times for 15 min at 45°C). Autoradiography was for 4–10 days
at -70°C.
After removal of the probe with 2x saline-sodium phosphate-EDTA
buffer, 55% formamide, 1% SDS (1 h at 65°C), the blot was
prehydridized and reprobed as above with 5 x
106 cpm/ml of a 1.0-kb human GAPDH cDNA
fragment (Clontech, Palo Alto, CA) radiolabeled with
[-32P]dCTP (Amersham, Arlington Heights, IL)
with the Rediprime DNA Labeling System (Amersham) and purified on a
NENSORB 20 cartridge. Posthybridization washes were: 5x SSC (twice for
2 min at 24°C); 2x SSC, 0.1% SDS (four times for 5 min at 24°C);
and 0.1x SSC, 0.1% SDS (three times for 15 min at 50°C).
Autoradiography was for 1–6 days at -70°C.
Cell Culture.
All of the cell lines were purchased from American Type Culture
Collection (Manasses, VA), and cultured at 37°C in a humidified
environment of 95%:5% air:CO2 in the following
media (Life Technologies, Inc., Grand Island, NY) supplemented with
10% qualified fetal bovine serum (Life Technologies, Inc.): NCI-H146,
-H209, -H727, and -H460 lines in RPMI 1640; A204 and MCF7 in DMEM; and
Du 145 and SK-N-SH in MEM with Earle salts, 1 mM sodium
pyruvate, and 2 mM L-glutamine.
Trypan Blue Exclusion Assays.
Cells (50,000) were seeded in 60-mm plates (Falcon, Lincoln Park, NJ)
in 3 ml of growth medium supplemented with 50 units/ml of penicillin
and streptomycin (Life Technologies, Inc.). After incubation overnight,
the appropriate AAAD inhibitor concentration or diluent was added to
the plate of a suspension cell line or within 3 ml of fresh media to a
monolayer cell line (20 mM stock inhibitor solutions were
prepared in DMSO and stored at 4°C before use). For continuous
exposure, drug was replaced daily. Treated cells were collected by
centrifugation for 5 min at 4°C and 200 x g:
suspension cell lines, directly; and attached cell lines, on
combination of media with monolayer cells lifted with 0.05% trypsin,
0.53 mM EDTA (Life Technologies, Inc.). After
resuspension in culture medium, viable cells were counted on a
hemocytometer after a 5-min incubation in 0.2% trypan blue (Life
Technologies, Inc.). IC50 values were calculated
from dose-response plots by the method of nonlinear least-squares
regression to a logistic function. These values represented the
concentration of inhibitor required to bring the number of viable,
treated cells to 50% of the control cell number upon 72 h of
incubation. Data from experiments assessing combined drug effects were
analyzed by the median effect method of Chou and Talalay
(17)
, both as though drug actions were mutually
nonexclusive (i.e., the mechanisms were independent of one
another) as well as though they were mutually exclusive.
Cell Homogenates.
Homogenates were prepared at 4°C. Suspension cells were washed twice
with PBS (Life Technologies, Inc.) prior to collection by
centrifugation; monolayer cells were detached from flasks after washing
by scraping (Nunc 179707; Rochester, NY) into PBS prior to collection.
A volume of 0.3 M Tris-acetate, 5 mM DTT (pH
7.6) equal to the weight of the cellular pellet was added prior to
probe-sonication (Braun-sonic 1510; B. Braun, Melsungen,
Germany) at 100 W for two 15-s bursts on ice. After
ultracentrifugation at 100,000 x g for 1 h, the
supernatant was divided into aliquots and stored at -70°C until use.
AAAD Assay.
The assay conditions described previously (6)
were used
for homogenates.
To measure AAAD activity in intact cells after exposure to an AAAD inhibitor, cells (50,000) were seeded in 60-mm plates in 3 ml of growth medium, treated with the appropriate AAAD inhibitor concentration as described above, and incubated at 37°C for the appropriate length of time. Cells from six replicate plates were collected by centrifugation, monolayer cells first being lifted with 0.05% trypsin, 0.53 mM EDTA. One hundred fifty µl of 0.3 M Tris-acetate (pH 7.6) was added prior to probe-sonication; the broken cell preparation was then assayed for activity as described above. IC50 values were calculated from dose-response plots as the concentration of inhibitor required to bring the activity in homogenate from treated cells to 50% of that in control cell homogenate upon 10 min of incubation prior to cell harvesting.
Electron Microscopy.
NCI-H727 carcinoid cells were plated as for trypan blue assays and
incubated in experimental medium, alone or with 25 µM
carbidopa for 24 h. Adherent and nonadherent cells were then
collected separately, washed in PBS, and resuspended in Trumps fixative
[1% glutaraldehyde and 4% formaldehyde in 0.1 M
phosphate buffer (pH 7.2)]. Samples were postfixed in
phosphate-buffered 1% OsO4 and stained with 2%
uranyl acetate. Samples were dehydrated and embedded in Spurr’s resin.
Sections (90 nm) were cut on a Reichert Ultracut E ultramicrotome,
placed on 200 mesh copper grids, and stained with lead citrate.
Micrographs were taken on a JEOL 1200 EXII electron microscope.
RESULTS
Analysis of AAAD in Hepatic Carcinoid Metastases and Adjacent
Normal Liver Samples.
Our previous studies (6)
indicated, somewhat surprisingly,
that AAAD activity was significantly increased in carcinoid tumor
compared with surrounding normal tissue. To evaluate the basis for this
increase, we examined levels of AAAD polypeptide by immunoblotting and
AAAD message by Northern analysis in a series of matched tissue sets.
Western analysis with anti-AAAD antibody revealed a single band with a
mobility pattern consistent with the reported
Mr 50,000 of human
pheochromocytoma AAAD on SDS-PAGE (18)
. Fig. 1
A illustrates representative
data from one of three matched sets of hepatic carcinoid metastases and
surrounding normal liver tissues. AAAD bands on Western blots were
markedly enhanced in the tumors compared with the adjacent normal liver
samples.
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2.1 kb, consistent with the reported size of AAAD message in
human pheochromocytoma (19)
, was easily detected in mRNA
isolated from the three respective hepatic carcinoid metastases.
Representative data from one matched set of tissues, the same pair
examined by immunoblotting in Fig. 1
A, are illustrated in
Fig. 2
A. Some nonspecific
binding of the 40mer probe to residual 18S and 28S rRNA in the mRNA
preparations was also seen. Reprobing of the blot with a
[32P]-labeled GAPDH cDNA fragment confirmed the
absence of mRNA degradation and was used for normalizing (via
AAAD:GAPDH densitometric ratios) the mRNA loading in each lane.
|
B). The AAAD polypeptide level in the tumors was
markedly higher than in their adjacent normal tissues
(n = 3). Because the tissue quantity obtained in pairs
of primary ileal carcinoid tumors and surrounding normal ileum was
limited, Northern analysis was performed in a different triplicate of
matched tissue sets. No AAAD mRNA was detectable in two normal ileum
samples, and a very weak 2.1-kb band was detected in the third. A
major band consistent with human AAAD mRNA was readily detected in all
of the three respective primary ileal carcinoid tumors (Fig. 2
B).
Effect of Exposure to AAAD Inhibitors on Proliferation and
Intracellular AAAD Activity of Lung Carcinoid Cells.
The NCI-H727 human lung carcinoid line has abundant AAAD activity
(8)
and polypeptide content (Fig. 3
A). To assess the effect of AAAD
inhibition on carcinoid tumor growth, three AAAD inhibitors, carbidopa,
MFMD, and NSD-1015, were incubated with NCI-H727 cells for 72 h,
and the loss of viability was determined by trypan blue uptake assays.
Carbidopa and MFMD were cytotoxic to the carcinoid cells, whereas
NSD-1015 had no effect on growth. A time course of cytotoxicity induced
by 100 µM carbidopa (Fig. 3
B) indicated that
carcinoid cell death was detectable at 24 h, whereas total cell
kill occurred at 48–72 h. Dose-response curves of MFMD- and
carbidopa-induced cell death in the carcinoid cell line are shown in
Fig. 3
, C and D. Table 1
summarizes cytotoxicity data at 72 h on carcinoid cells exposed to the three AAAD inhibitors. For
comparison of this effect with that of a known chemotherapeutic agent,
NCI-H727 carcinoid cells were exposed to doxorubicin, lethal to
sensitive human tumor cell lines at nM
IC50 values (20)
. In contrast,
doxorubicin (
100 µM; n = 2)
was not lethal to NCI-H727 cells.
|
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and carbidopa-induced
AAAD inhibition in Fig. 3
E). At the identical concentrations
and exposure times, NSD-1015 did not completely inhibit intracellular
carcinoid AAAD activity, nor was it lethal to NCI-H727 cells. However,
when solubilized directly in media, NSD-1015 could be dissolved at 1000
µM. At this concentration, NSD-1015 totally
inhibited AAAD activity in carcinoid cells (n = 2) but
still had no growth inhibitory effect (n = 2). Because
AAAD inhibition would block the biosynthesis of 5-HT, we further
examined a possible role for 5-HT and the precursor 5-OH-trp in the
cytotoxicity of AAAD inhibitors. No effect on carcinoid cell
proliferation was produced by the addition of the AAAD substrate
5-OH-trp, which presumably would accumulate in cells as a consequence
of AAAD inhibition (100 µM; n = 2). Furthermore, the specific TPH inhibitor
p-chlorophenylalanine did not alter NCI-H727 cell
growth (1000 µM; n = 2).
Effect of Carbidopa on Proliferation of Human Tumor Cell Lines
Compared with the Maximal AAAD Activity of the Cell Lines.
The most potent cytotoxic AAAD inhibitor, carbidopa, was incubated with
seven additional human tumor cell lines exhibiting a wide range of AAAD
activity levels. Micromolar concentrations of carbidopa were lethal to
three of eight lines, all of lung origin: SCLC NCI-H146, SCLC NCI-H209,
and carcinoid NCI-H727 cells (Table 2)
. A
moderate decrease in cellular growth (but not complete cell death) was
observed in the SK-N-SH neuroblastoma and A204 rhabdomyosarcoma tumor
cell lines, whereas no effect was observed in the remaining three cell
lines. Table 2
also summarizes the maximal AAAD activity determined in
homogenates from all of the eight human tumor cell lines. The
relationship between the potency of carbidopa-induced cytotoxicity and
the maximal AAAD activity for the four lung tumor lines is illustrated
in Fig. 4
.
|
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A), NCI-H727 cells treated
with 25 µM carbidopa for 24 h consisted of
normal-looking cells as well as cells having a "foamy" appearance
resulting from the presence of rounded vacuoles and/or loss of
cytoplasmic integrity (Fig. 5
B). These carbidopa-treated
cells maintained relatively normal nuclear morphology and intact plasma
membranes.
|
A). However, a 1:30
combination of VP-16-with-carbidopa induced carcinoid cell death.
Analysis of these combination experiments with the median effect method
of Chou and Talalay (17)
indicated that the cytotoxic
effects on carcinoid cells of VP-16 and carbidopa coincubation were
additive (data not shown). Topotecan alone decreased the growth of SCLC
cells (Fig. 6
B). When combined 1:3000 with carbidopa, the
cytotoxic effect was greater than that produced by either drug alone.
Analysis of these results with the median effect method (Fig. 6
C) indicated that topotecan and carbidopa acted
synergistically (CI<1) in SCLC cells when the fraction of cell kill
was >65%.
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This study focuses on AAAD, the second enzyme of the 5-HT biosynthetic pathway, which, in carcinoid hepatic metastases, displays high levels of activity compared with that in surrounding normal tissue (6) . Here we describe the large quantities of AAAD polypeptide found in both hepatic carcinoid metastases and primary ileal lesions but not in surrounding normal tissues. Furthermore, high levels of AAAD mRNA were detected in metastatic and primary tumors, whereas low-to-undetectable levels were found in respective normal tissues. The inability to measure AAAD mRNA in the majority of normal tissue samples examined was not unexpected, given the very low abundance of AAAD mRNA (from 0.0005% of total RNA in brain to 0.0035% of total RNA in kidney) found in normal rat tissues (21) . Collectively, these results indicate that the increased AAAD activity observed in carcinoid tumors reflected increased steady-state mRNA levels.
Historically, AAAD was believed to be an unregulated enzyme present in excess of TPH in serotonergic tissues and in substantial quantities in nonserotonergic neurons and in peripheral organs, in which the function of the enzyme is not fully understood. Our studies, demonstrating high levels of AAAD protein and mRNA in human carcinoid primary and metastatic tumors relative to surrounding normal tissue, are consistent with recent evidence suggesting that AAAD may undergo transcriptional, translational, and posttranslational regulation (see reviews, Refs. 22 and 23 ).
In assessing tumor AAAD as a selective target for chemotherapeutic agents, we found that the AAAD inhibitors carbidopa and MFMD were lethal to NCI-H727 human lung carcinoid cells. This strategy was extended to a number of human tumor cell lines with varying amounts of cellular AAAD. The comparison of the IC50 value for carbidopa cytotoxicity with AAAD activity in each cell line indicated that, in general, cell lines with higher AAAD levels were more susceptible to carbidopa-induced cytotoxicity. The two exceptions were the neuroblastoma SK-N-SH line, which did not exhibit complete carbidopa-induced cell kill although having relatively high levels of AAAD activity, and the rhabdomyosarcoma A204 line, which demonstrated a moderate response to carbidopa although having no detectable AAAD. Carbidopa was lethal to only three lung lines (the NCI-H727 carcinoid line and the NCI-H146 and NCI-H209 SCLC lines), all three with abundant AAAD activity. NCI-H146 and NCI-H209 SCLC cells, in fact, have among the highest AAAD mRNA levels of the 38 (12) and 27 (10) neuroendocrine lines compared in the literature. We found a direct correlation between the potency of carbidopa-induced cytotoxicity and level of AAAD activity in the lung tumor lines, including NCI-H460 large cell lung carcinoma, which lacked AAAD activity and was unresponsive to carbidopa. Carbidopa was not lethal to the non-lung tumor lines tested.
There are several potential mechanisms for carbidopa- (and MFMD-) induced cytotoxicity to carcinoid and SCLC cells. Through AAAD inhibition, carbidopa might inhibit formation of a factor required for the growth/survival of these cells, e.g., 5-HT. However, no cytotoxicity to carcinoid cells was observed after complete inhibition of AAAD by NSD-1015, and no effect on growth was observed on incubation of these cells with TPH inhibitor, p-chlorophenylalanine. Furthermore, apoptotic cell death is often correlated with an interruption of cell-signaling pathways critical to cell survival. Investigation of carcinoid cells exposed to carbidopa (25 µM for 24 h) did not reveal, by Hoechst staining or transmission electron microscopy, the morphological changes typically associated with either apoptosis or necrosis. Thus, the 5-HT biosynthetic pathway appears not to be the mechanism of carbidopa-induced cell death.
Another possible mechanism might involve AAAD inhibition directly without relation to any associated effect on 5-HT synthesis. However, some of our results suggest that the relationship between AAAD expression and inhibitor cytotoxicity is not straightforward. Two of the eight cell lines did not exhibit a direct relationship between AAAD content and susceptibility to carbidopa-induced cell death (neuroblastoma SK-N-SH and rhabdomyosarcoma A204). In addition, of the two toxic AAAD inhibitors, carbidopa was more potent in inducing carcinoid cell death, whereas MFMD was the better AAAD inhibitor. Finally, carbidopa did not completely inhibit AAAD activity in intact NCI-H146 SCLC cells at concentrations that were lethal to the cells (data not shown), whereas concentrations of NSD-1015 high enough to shut down AAAD activity in intact NCI-H727 cells did not cause death in these cells, which indicated that complete inhibition of AAAD was not required for carbidopa-induced cell death in the carcinoid and at least one SCLC line.
Mechanisms other than interruption of 5-HT synthesis or direct inhibition of AAAD may well be responsible for the selective cytotoxicity of carbidopa. Carbidopa may be metabolized to a toxic product by certain cell lines with high levels of AAAD. Interestingly, a previous report indicated that Chinese hamster ovary fibroblasts transfected with bovine AAAD cDNA had increased intracellular catechol levels and increased sensitivity to the lethal combination of L-Dopa and manganese relative to wild type fibroblasts. This was attributed to AAAD decarboxylation of intracellular L-Dopa accumulated from growth medium to form dopamine, the proposed protoxin for manganese-induced intracellular oxidation (24) . Our data suggest that high levels of AAAD might be a necessary although not sufficient requirement for lung tumor lines to be susceptible to carbidopa-induced cytotoxicity, with abundant AAAD perhaps serving only as a marker for cells sensitive to this effect.
The selective carbidopa cytotoxicity toward human pulmonary carcinoid and SCLC cell lines suggests a possible new therapeutic approach for these two neoplasms, both presently untreatable in advanced stages. To further capitalize on these findings, we conducted studies of carbidopa in combination with several classes of antitumor agents. Preliminary data were most favorable for topoisomerase inhibitors (data not shown). The combination of VP-16 and carbidopa was cytotoxic in an additive fashion to carcinoid cells, a tumor generally unresponsive to traditional chemotherapeutic drugs (1 , 3) . In addition, sublethal concentrations of carbidopa (closer to those typically coadministered in Parkinson’s disease), displayed cytotoxic synergy in combination with topotecan in SCLC cells. The mechanism of carbidopa-induced cell killing, alone and in combination with known chemotherapeutic drugs, and the role played by AAAD expression and carbidopa metabolism in this cytotoxicity are currently under further investigation.
ACKNOWLEDGMENTS
We thank Drs. Scott H. Kaufmann and Richard Weinshilboum for review of the manuscript and Wanda Rhodes for its preparation.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by NIH Grant CA 58450,
National Cancer Institute, Department of Health and Human
Services. 
2 To whom requests for reprints should be
addressed, at Department of Oncology, Division of Developmental
Oncology Research, Guggenheim 13, Mayo Clinic and Foundation, 200 First
Street S.W., Rochester, MN 55905. Phone: (507) 284-2424; Fax: (507)
284-3906.
3 The abbreviations used are: 5-HT,
5-hydroxytryptamine or serotonin; trp, tryptophan; 5-OH-Trp,
5-hydroxytryptophan; TPH, trp hydroxylase; AAAD,
aromatic-L-amino acid decarboxylase; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; MFMD,
-monofluoromethyldopa; NSD-1015, 3-hydroxybenzylhydrazine; SCLC,
small cell lung carcinoma; VP-16, etoposide; CI, combination index.
Received 11/22/99; revised 9/ 1/00; accepted 9/ 5/00.
REFERENCES
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