Fluoropyrimidines are among the most widely used antineoplastic
agents with activity against breast, gastrointestinal, and head and
neck malignancies. Since their synthesis by Heidelberger during
the 1950s, fluoropyrimidines have undergone extensive preclinical and
clinical evaluation. The antineoplastic activity of fluorouracil
(5-FU) is improved by biochemical modulation[2,3] and when
administered as a continuous infusion. Although the oral
absorption of 5-FU is erratic and results in unpredictable and
variable plasma levels in patients, 5-FU prodrugs are
pharmacologically more predictable. These compounds are being
evaluated clinically and will likely become important drugs in the
care of patients.
Fluorouracil and its analogs tegafur and capecitabine (Xeloda) are
shown in Figure 1. After
administration, 5-FU is rapidly catabolyzed by the rate-limiting
enzyme dihydropyrimidine dehydrogenase (DPD), which has its highest
level of activity in the liver. This accounts for the high clearance
of the drug, with only 5% to 10% entering anabolism into active
compounds.[6,7] In addition, approximately 2% to 4% of the population
is DPD-deficient due to genetic polymorphism in DPD activity.
The oral fluoropyrimidines may be divided into two categories: those
administered in combination with a DPD inhibitor and those
administered without. Inhibition of DPD activity may be competitive
with 5-FU, as in the case of uracil and 5-chloro-dihydropyridine;
5-ethynyluracil acts as a suicide inhibitor by inactivating DPD.
Capecitabine, a non-DPD 5-FU prodrug, becomes cytotoxic only after
conversion to 5-FU. Following oral administration, capecitabine is
metabolized in the liver by carboxyl esterase into
5-deoxy-5-fluorocytidine and by cytidine deaminase into 5´-deoxy-5-fluorouridine
(Figure 2). The last step of
conversion occurs with pyrimidine phosphorylase, which is shown to be
at a higher concentration in the tumor than in normal tissue, with
the potential advantage of greater tumor selectivity.
Tegafur is a prodrug that slowly metabolizes into 5-FU through the
activity of hepatic cytochrome P450 and systemic soluble
enzymes;[10,11] 5-FU is the end product of this reaction. In order to
increase the concentration of 5-FU, and prevent degradation of 5-FU
by DPD, uracil is added at a molar concentration of 1:4
(5-FU:uracil). In preclinical models, this molar combination has
been identified as the most efficient.
S1 is a combination of tegafur and two modulators that prevent
degradation. Tegafur, potassium oxonate, and 5-chloro-2,4-dihydropyridine
(CDHP), are combined in a molar ratio of 1:0.4:1.[13,14] CDHP is a
competitive, reversible DPD inhibitor that prolongs the half-life of
5-FU. Oxonic acid is a pyrimidine phosphoribosyltransferase inhibitor
that is added to prevent the phosphorylation of 5-FU in the digestive
tract with the aim of reducing 5-FUrelated gastrointestinal toxicity.
The intravenous (IV) formulation of 5-FU may be given as an oral drug
when it is combined with the oral DPD inhibitor ethynyluracil.
Pretreatment with ethynyluracil results in 100% oral bioavailability
and extends the half-life of 5-FU from less than 10 minutes to over
100 minutes. In these circumstances, 5-FU has linear
pharmacokinetics and clearance is primarily renal. Complete DPD
inhibition might last for several weeks after ethynyluracil is
discontinued,[17,18] thus preventing further conventional dosing of
5-FU for several weeks or even months.
All 5-FU prodrugs and 5-FU combined with DPD inhibitors exert their
antineoplastic activity in a similar manner. The biochemical
modulation of 5-FU is reviewed in Figure
3. 5-FU may act via thymidylate synthase (TS) inhibition through
its active anabolites such as fluorodeoxyuridine monophosphate
(FdUMP). This reaction is facilitated by the formation of a ternary
complex consisting of FdUMP, TS, and 5,10-methylene tetrahydrofolate
(5,10-MTHF). The intracellular concentration of 5,10-MTHF can be
increased through the addition of other reduced folates such as
leucovorin, thus promoting a greater stability of the ternary
complex. Thymidylate synthase catalyzes the conversion of dUMP to
TMP, which is a precursor of TTP, one of the four
deoxyribonucleotides required for DNA synthesis. Also, incorporation
of the anabolites of 5-FU into RNA and DNA contributes to the
antineoplastic activity of 5-FU.[19,20] In preclinical models,
interferon demonstrated the ability to enhance the cytotoxicity of
5-FU by several potential mechanisms, including affecting thymidine
kinase activity, depleting intracellular thymidine concentration,
and most likely, enhancing DNA damage.[22,23]
Pretreatment with methotrexate results in an increased intracellular
concentration of phosphoribosyl pyrophosphate (PRPP), the cofactor
for conversion of 5-FU into fluorouridine monophosphate (FUMP) and,
subsequently, into 5-fluorouridine 5´-tri-
phosphate (FUTP).[24,25] The administration of high-dose methotrexate
is usually accompanied by leucovorin rescue, which can lead to a dual
effect. Leucovorin will not only protect against methotrexate-induced
toxicity, but can also interfere with methotrexate uptake, thereby
abolishing its effect. In addition, administration of leucovorin will
result in direct modulation of 5-FU.[26,27] The net effect seems to
be a leucovorin-mediated modulation. Therefore, methotrexate was
substituted with trimetrexate, which, due to its lipophilicity, does
not require the reduced folate carrier. This combination was very
effective in vitro, and is currently being evaluated in the clinic.
Phosphonacetyl-L-aspartate (PALA) is an inhibitor of aspartate
transcarbamylase, an important enzyme in the de novo synthesis of
uridine and cytidine nucleotides. Pretreatment with PALA can yield a
higher incorporation of 5-FU nucleotides into cellular RNA, and a
depletion of dUMP, leading to enhanced inhibition of TS.
After transport of preformed extracellular thymidine into cells, it
is anabolyzed by thymidine kinase to thymidine triphosphate (dTTP)
and replete dTTP pools, thereby bypassing the 5-FUinduced
depletion of dTTP. Dipyridamole interferes with the cellular
uptake of nucleosides. In tumor cells, in which the inhibition of TS
is critical, dipyridamole results in depletion of dTTP pools by
blocking the facilitated transport of exogenous thymidine.
Unfortunately, in vivo application of dipyr-idamole is hampered by
its high protein binding, thus preventing a dipyridamole-induced
inhibition of nucleoside transport.
All of these biochemical strategies have been extensively studied in
clinical trials.[31,32] Methotrexate, PALA, alpha-interferon, DP have
all been investigated as biochemical modulators, but the biochemical
modulation by leucovorin appears to be most efficientresulting
in a doubling of the response rate.
The rationale for combining UFT (uracil and tegafur) plus oral
leucovorin (a combination being developed under the trade name Orzel)
derives from extensive evaluation of leucovorin modulation of 5-FU.
As outlined in Figure 4, UFT
plus leucovorin produces a double 5-FU modulation. A 1:4 molar
combination of the 5-FU prodrug tegafur with uracil as a DPD
inhibitor was developed two decades ago in Asia. Preclinical studies
demonstrated that tegafur plus uracil resulted in higher tumor/blood
ratios and greater tumor activity.[33,34]
In addition, tegafur plus uracil was associated with lower central
nervous system and gastrointestinal toxicity compared to tegafur
alone. As uracil is added to tegafur, the half-life of 5-FU is
prolonged due to the inhibited ability of DPD to degradate 5-FU into alpha-fluoro-beta-alanine.
Only about 20% of patients with advanced colorectal cancer respond to
the combination of 5-FU and leucovorin via bolus administration,
leaving approximately 80% who fail to respond. Additionally,
responses usually last for a short time, with most patients failing
to respond later due to possible secondary resistance. Resistance to
fluoropyrimidines is multifactorial and may be related to their
antipyrimidine or antifolate properties (Table
1). A general form of fluoropyrimidine resistance is the
induction of TS activity at the initiation of 5-FU treatment (Figure
5).[37-40] This phenomenon has been observed in a number of
model systems both in vivo and in vitro.
Using an in vivo murine colon tumor model, Van der Wilt and
coworkers[39,41] observed the development of resistance following
weekly bolus injections and after continuous administration of 5-FU
for 10 to 21 days in tumors that were initially sensitive to such
treatment. This resistance was associated with a rapid increase in TS
levels, while plasma 5-FU levels under these conditions were
comparable to the levels that had been achieved during the first days
of infusion. Interestingly, pretreatment with leucovorin depressed
this 5-FUinduced TS increase after the bolus injections.
Sobrero and coworkers observed that low-dose, continuous exposure of
5-FU almost immediately resulted in resistant clones of the HCT-8
colon tumor cell line, whereas short-term exposure to 5-FU required a
longer period to induce resistance. Cells resistant to
intermittent short exposure were sensitive to continuous exposure,
but cells resistant to continuous exposure were also resistant to
short-term exposure. The observation that the increase in TS was less
pronounced if cells were pretreated with leucovorin may serve as
an explanation for the higher response rate observed with 5-FU
therapy when modulated by leucovorin. Other investigators observed a
similar increase in TS levels after treatment with 5-FU in a tumor
cell model.[43,44] In this experiment, gamma-interferon was able to
significantly reduce TS induction.
Deregulation of Thymidylate Synthase Protein Synthesis
The increase in TS levels is most likely explained by a deregulation
of the normal TS protein synthesis. Under certain physiologic
conditions, TS protein synthesis is related to the cell cycle, with
high activity associated with the S-phase. The translation of TS
mRNA appears to be controlled by its end product, the TS protein, in
an autoregulatory manner. However, when TS is bound to a ternary
complex, the protein can no longer regulate its synthesis, leading to
the observed increase. Expression of TS appears to be managed by a
translational regulatory process during the cell cycle as well as in
response to cytotoxic agents.
The capacity of TS as an RNA-binding protein was demonstrated by Chu
and coworkers. Nine different cellular RNAs were shown to form a
ribonucleoprotein complex with TS in intact colon cancer H630 cells.
Several of these isolated RNA sequences display a high degree of
homology to those encoding the human p53 tumor suppressor
protein, the c-myc and l-myc family of transcription
factors, and the human zinc finger 8 transcription factor. p53 and
c-myc encode for nuclear phospho-proteins that play central
roles in the regulation of cell cycle progression, DNA synthesis, and apoptosis.[47,48]
Interesting insights into the more downstream events of 5-FU
antineoplastic activity have been reported by Houghton and
coworkers, who used a TS-negative (TS-) human GC3I TS-
cell clone that was deficient in TS-mRNA and TS protein and studied
the regulation of apoptosis following thymineless stress. Protection
from apoptosis induced by thymidine deprivation was achieved by the
monoclonal antibody NOK-1, which inhibits Fas and binds to the Fas
Fas is one of several cell surface death-receptors, a type I integral
membrane protein characterized by cysteine-rich residues. It
belongs to the tumor necrosis factor receptor superfamily with an
intracellular domain required for downstream signaling. FasL is a
type II transmembrane protein homologous to tumor necrosis factor and
related cytokines. Fas interacts with FADD, and the death
effector domain of FADD interacts with a similar domain of proteases
(FLICE, FLICE-2). These caspases then degrade other proteins within
the nucleus and activate endonucleases.
The work by Houghton[52,53] indicates that the Fas/FasL apoptotic
system may be involved in the cytotoxicity induced by 5-FU. In
further experiments, the functional significance of a transfected K-ras
oncogene in influencing apoptosis following thymidine
deprivation was examined in a TS-deficient cell line.
Interestingly, survival was conferred in TS- but K-ras+ clones.
No difference in Fas expression was detected among the cell
lines after thymidine deprivation, indicating that K-ras does
not interfere with Fas expression. Apoptosis in the presence
of wild-type Ras correlated with upregulated expression of Bak and
was not observed in TS- K-ras+ clones, whereas survival in these
clones correlated with upregulated expression of BCL-XL. Therefore,
induction of cell death after TS inhibition may be regulated in part
by Fas and FasL and other proteins involved in the apoptosis
Work by Pritchard and coworkers further highlights the role of p53
as a promoter of cell death after 5-FU application in p53 knock-out
mice, and that of BCL-2 as a death inhibitor delaying cell
death and inhibiting cell proliferation in murine intestinal
Recently, inhibition of angiogenesis by UFT and its metabolites has
been reported. A murine renal cell carcinoma (RENCA) cell line
was studied as an angiogenesis model using a dorsal air skin assay.
Blood vessels are formed by an angiogenic factor released from these
malignant tumor cells. UFT showed a strong angiogenesis inhibitory
effect whereas 5-FU and doxifluridine did not. The angiogenesis was
apparently mediated by the degradation products of tegafur, gamma-hydroxybutyric
acid, and gamma-butyrolactone. The inhibitory effect was amplified
even when these compounds were administered by continuous infusion.
While the clinical relevance of this observation remains to be
determined, it is important to note that, in addition to its
cytotoxic effects, UFT may have the potential to inhibit
angiogenesis. This might be of special interest in the adjuvant setting.
Oral fluoropyrimidines are interesting compounds in the treatment of
patients with solid tumors. As they are metabolized into 5-FU, their
mechanism of action is basically similar to that reported with IV
administration of 5-FU. Biochemical modulation also may apply to oral
fluoropyrimidines, such as the combination of UFT plus oral
leucovorin. Evidence is emerging that further downstream effects of
fluoro- pyrimidines are related to proteins involved in the
regulation of apoptosis. The effect of UFT on angiogenesis deserves
1. Heidelberger C, Chaudhuri NK, Danneberg P, et al: Fluorinated
pyrimidines, a new class of tumor-inhibitory compounds. Nature
2. Advanced Colorectal Cancer Meta-Analysis Project: Meta-analysis of
randomized trials testing the biochemical modulation of fluorouracil
by methotrexate in metastatic colorectal cancer. J Clin Oncol
3. Advanced Colorectal Cancer Meta-Analysis Project: Modulation of
fluorouracil by leucovorin in patients with advanced colorectal
cancer: Evidence in terms of response rate. J Clin Oncol 10:896-903, 1992.
4. Meta-analysis Group in Cancer: Efficacy of intravenous continuous
infusion of fluorouracil compared with bolus administration in
advanced colorectal cancer. J Clin Oncol 16:301-308, 1998.
5. Fraile RJ, Baker LH, Buroker TR, et al: Pharmacokinetics of
5-fluorouracil administered orally, by rapid intravenous and by slow
infusion. Cancer Res 40:2223-2228, 1980.
6. Grem JL, McAtee N, Murphy, RF, et al: A pilot study of interferon
alfa-2a in combination with fluorouracil plus high-dose leucovorin in
metastatic gastrointestinal carcinoma. J Clin Oncol 9: 1811-1820, 1991.
7. McDermott BJ, van den Berg HW, Murphy RF: Nonlinear
pharmacokinetics for the elimination of 5-fluorouracil after
intravenous administration in cancer patients. Cancer Chemother
Pharmacol 9:173-178, 1982.
8. Meinsma R, Fernandez-Salguero P, Van Kuilenburg AB, et al: Human
polymorphism in drug metabolism: Mutation in the dihydropyrimidine
dehydrogenase gene results in exon skipping and thymine uracilurea.
DNA Cell Biol 14:1-6, 1995.
9. Miwa M, Ura M, Nishida M, et al: Design of a novel oral
fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil
selectively in tumours by enzymes concentrated in human liver and
cancer tissue. Eur J Cancer 34:1274-1281, 1998.
10. Au JL, Sadee W: The pharmacology of ftorafur (R,
S-1-(tetrahydro-2-furanyl)-5- fluorouracil. Recent Results Cancer Res
11. Anttila MI, Sotaniemi EA, Kairaluoma MI, et al: Pharmacokinetics
of ftorafur after intravenous and oral administration. Cancer
Chemother Pharmacol 10:150-153, 1983.
12. Taguchi T: UFT: biochemical modulation for 5-fluorouracil (5-FU).
Chin Med J (Engl) 110:294-296, 1997.
13. Shirasaka T, Shimamato Y, Ohshimo H, et al: Development of a
novel form of an oral 5-fluorouracil derivative (S-1) directed to the
potentiation of the tumor selective cytotoxicity of 5-fluorouracil by
two biochemical modulators. Anticancer Drugs 7:548-557, 1996.
14. Shirasaka T, Nakano K, Takechi T, et al: Antitumor activity of 1
M tegafur-0.4 M 5-chloro-2,4-dihydroxypyridine-1 M potassium oxonate
(S-1) against human colon carcinoma orthotopically implanted into
nude rats. Cancer Res 56:2602-2606, 1996.
15. Takechi T, Nakano K, Uchida J, et al: Antitumor activity and low
intestinal toxicity of S-1, a new formulation of oral tegafur, in
experimental tumor models in rats. Cancer Chemother Pharmacol
16. Baccanari DP, Davis ST, Knick VC, et al: 5-Ethynyluracil
(776C85): A potent modulator of the pharmacokinetics and antitumor
efficacy of 5-fluorouracil. Proc Natl Acad Sci USA 90:11064-11068, 1993.
17. Adams ER, Leffert JJ, Craig DJ, et al: In vivo effect of
5-ethynyluracil on 5-fluorouracil metabolism determined by 19F
nuclear magnetic resonance spectroscopy. Cancer Res 59:122-127, 1999.
18. Spector T, Harrington JA, Porter DJ: 5-Ethynyluracil (776C85):
Inactivation of dihydropyrimidine dehydrogenase in vivo. Biochem
Pharmacol 46:2243-2248, 1993.
19. Rustum YM, Trave F, Zakrzewski SF, et al: Biochemical and
pharmacologic basis for potentiation of 5- fluorouracil action by
leucovorin. NCI Monogr 5:165-170, 1987.
20. Peters GJ, Hoekman K, van Groeningen CJ, et al: Potentiation of
5-fluorouracil induced inhibition of thymidylate synthase in human
colon tumors by leucovorin is dose dependent. Adv Exp Med Biol
21. Wadler S, Schwartz EL: Antineoplastic activity of the combination
of interferon and cytotoxic agents against experimental and human
malignancies: A review. Cancer Res 50:3473-3486, 1990.
22. Houghton JA, Morton CL, Adkins DA, et al: Locus of the
interaction among 5-fluorouracil, leucovorin, and interferon-alpha 2a
in colon carcinoma cells. Cancer Res 53:4243-4250, 1993.
23. van der Wilt CL, Smid K, Aherne GW, et al: Biochemical mechanisms
of interferon modulation of 5-fluorouracil activity in colon cancer
cells. Eur J Cancer 33:471-478, 1997.
24. Bertino JR, Mini E, Fernandes DJ: Sequential methotrexate and
5-fluorouracil: Mechanisms of synergy. Semin Oncol 10:2-5, 1983.
25. Cadman E, Heimer R, Davis L: Enhanced 5-fluorouracil nucleotide
formation after methotrexate administration: Explanation of drug
synergism. Science 205:1135-1137, 1979.
26. van der Wilt CL, Braakhuis BJ, Pinedo HM, et al: Addition of
leucovorin in modulation of 5-fluorouracil with methotrexate:
Potentiating or reversing effect? Int J Cancer 61:672-678, 1995.
27. Romanini A, Li WW, Colofiore JR, et al: Leucovorin enhances
cytotoxicity of trimetrexate/fluorouracil, but not
methotrexate/fluorouracil, in CCRF-CEM cells. J Natl Cancer Inst
28. Collins KD, Stark GR: Aspartate transcarbamylase. Interaction
with the transition state analogue N-(phosphonacetyl)-L-aspartate. J
Biol Chem 246:6599-6605, 1971.
29. Martin DS, Kemeny NE: Overview of N-(phosphonacetyl)-aspartate +
fluorouracil in clinical Trials. Semin Oncol 19(2 suppl 3):228-233, 1992.
30. Grem JL, Fischer PH: Augmentation of 5-fluorouracil cytotoxicity
in human colon cancer cells by dipyridamole. Cancer Res 45:2967-2972, 1985.
31. Köhne-Wömpner CH, Schmoll HJ, Harstrick A, et al:
Chemotherapeutic strategies in metastatic colorectal cancer: An
overview of current clinical trials. Semin Oncol 19:105-125, 1992.
32. Köhne CH, Kretzschmar A, Wils J: First-line chemotherapy for
colorectal carcinomawe are making progress. Onkologie
33. Fujii M, Murakami N, Matsuka Y, et al: Chemotherapy of
gastrointestinal cancer in elderly patientsevaluation of
combination therapy with mitomycin C and 5- fluorouracil [in
Japanese]. Gan No Rinsho 29:A-8, 129-32, 1983.
34. Fujii S, Kitano S, Ikenaka K, et al: Effect of coadministration
of uracil or cytosine on the anti-tumor activity of clinical doses of
1-(2-tetrahydrofuryl)-5-fluorouracil and level of 5-fluorouracil in
rodents. Gann 70:209-214, 1979.
35. Yamamoto J, Haruno A, Yoshimura Y, et al: Effect of
coadministration of uracil on the toxicity of tegafur. J Pharm Sci
36. Pinedo HM, Peters GJ: Fluorouracil: Biochemistry and
pharmacology. J Clin Oncol 6:1653-1664, 1988.
37. Peters GJ, van der Wilt CL, van Groeningen CJ: Predictive value
of thymidylate synthase and dihydropyrimidine dehydrogenase. Eur J
Cancer 30A:1408-1411, 1994.
38. Priest DG, Ledford BE, Doig MT: Increased thymidylate synthetase
in 5-fluorodeoxyuridine resistant cultured hepatoma cells. Biochem
Pharmacol 29:1549-1553, 1980.
39. Van der Wilt CL, Pinedo HM, Smid K, et al: Elevation of
thymidylate synthase following 5-fluorouracil treatment is prevented
by the addition of leucovorin in murine colon tumors. Cancer Res
40. Peters GJ, Köhne CH: Fluoropyrimidines as antifolate drugs,
in Jackman AL (ed): Antifolate Drugs in Cancer Therapy, pp 101-145.
Totowa, NJ, Humana Press Inc, 1999.
41. Codacci-Pisanelli G, van der Wilt CL, Pinedo HM, et al:
Antitumour activity, toxicity, and inhibition of thymidylate synthase
of prolonged administration of 5-fluorouracil in mice. Eur J Cancer
42. Sobrero AF, Aschele C, Guglielmi AP, et al: Synergism and lack of
cross-resistance between short-term and continuous exposure to
fluorouracil in human colon adenocarcinoma cells. J Natl Cancer Inst
43. Chu E, Zinn S, Boarman D, et al: Interaction of gamma interferon
and 5-fluorouracil in the H630 human colon carcinoma cell line.
Cancer Res 50:5834-5840, 1990.
44. Chu E, Koeller DM, Johnston PG, et al: Regulation of thymidylate
synthase in human colon cancer cells treated with 5-fluorouracil and
interferon-gamma. Mol Pharmacol 43:527-533, 1993.
45. Navalgund LG, Rossana C, Muench AJ, et al: Cell cycle regulation
of thymidylate synthetase gene expression in cultured mouse
fibroblasts. J Biol Chem 255:7386-7390, 1980.
46. Chu E, Cogliati T, Copur SM, et al: Identification of in vivo
target RNA sequences bound by thymidylate synthase. Nucleic Acids Res
47. Chu E, Takechi T, Jones KL, et al: Thymidylate synthase binds to
c-myc RNA in human colon cancer cells and in vitro. Mol Cell Biol
48. Chu E, Copur S, Jones KL, et al: Thymidylate synthase regulates
the translation of p53 mRNA (abstract). Proc Am Assoc Cancer Res
49. Houghton JA, Ebanks R, Harwood FG, et al: Inhibition of apoptosis
after thymineless stress is conferred by oncogenic K-Ras in colon
carcinoma cells. Clin Cancer Res 4:2841-2848, 1998.
50. Krammer PH: CD95(APO-1/Fas)-mediated apoptosis: Live and let die.
Adv Immunol 71:163-210, 1999.
51. Smith CA, Farrah T, Goodwin RG: The TNF receptor superfamily of
cellular and viral proteins: Activation, costimulation, and death.
Cell 76:959-962, 1994.
52. Houghton JA, Harwood FG, Tillman DM: Thymineless death in colon
carcinoma cells is mediated via fas signaling. Proc Natl Acad Sci U S
A 94:8144-8149, 1997.
53. Houghton JA, Harwood FG, Gibson AA, et al: The fas signaling
pathway is functional in colon carcinoma cells and induces apoptosis.
Clin Cancer Res 3:2205-2209, 1997.
54. Pritchard DM, Potten CS, Hickman JA: The relationships between
p53-dependent apoptosis, inhibition of proliferation, and
5-fluorouracil-induced histopathology in murine intestinal epithelia.
Cancer Res 58:5453-5465, 1998.
55. Yonekura K, Basaki Y, Chikahisa L, et al: UFT and its metabolites
inhibit the angiogenesis induced by murine renal cell carcinoma, as
determined by a dorsal air sac assay in mice. Clin Cancer Res