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Experimental Therapeutics |
Section of General Internal Medicine [S-C. J. Y.] and Section of Endocrine Neoplasia and Hormonal Disorders, Department of Medical Specialties [S-C. J. Y., G. X., J. P., M. C., A. B.], M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One possibility is the use of a group of compounds with potential cancer therapeutic effect called FPT inhibitors (7 , 8) . These compounds were originally developed with the intention of blocking ras oncogene function. Ras, the protein product of the ras proto-oncogenes, is synthesized as a cytosolic precursor, and it requires posttranslational modification by conjugation of a farnesyl (15-carbon isoprenyl group) moiety to the COOH-terminal region. After farnesylation, Ras is localized to the inner surface of the cell membrane, in which it becomes functional in transducing the mitogenic signals of tyrosine kinase receptors.
There are many isoprenylated proteins other than Ras. Each isoprenylated protein has the characteristic COOH-terminal sequence of CAAX, XXCC, or XCXC, in which C is cysteine, A is any aliphatic amino acid, and X is any other amino acid (9, 10, 11) . Four posttranslational processing steps have been described for the CAAX sequence: (a) addition of either a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group to the cysteine residue; (b) proteolytic cleavage of the AAX peptide; (c) carboxymethylation of the farnesylcysteine; and (d) except for K-Ras, palmitoylation of cysteine residues located upstream of the CAAX motif. Proteins with the XXCC or XCXC motif are modified by geranylgeranylation and do not require an endopeptidase step. K-Ras and N-Ras can be alternatively geranylgeranylated when farnesylation is inhibited (12 , 13) , but nonfarnesylated oncogenic H-Ras can exert a dominant negative effect and, therefore, inhibit the function of membrane-bound Ras in some circumstances. Thus, although inhibition of FPT would be expected to affect only H-Ras, the dominant negative effect would inhibit the Ras-transforming pathway. Because wild-type Ras does not display the dominant negative phenotype, the observed inhibition would be selective for tumor cells. Interestingly, soluble complexes of nonfarnesylated oncogenic H-Ras and Raf can be isolated from cells, thus supporting the concept that a dominant negative Ras may sequester Ras effector proteins and prevent them from interacting with membrane-associated Ras.
FPT inhibitors abolish the function of Ras and block the mitogenic action of ras oncogenes (9, 10, 11) . One example is manumycin A, a natural product of Streptomyces, identified by random screening. This inhibitor has competition with farnesyl PPi (FPP) groups as its mechanism of inhibition and has shown antitumor activity in cell culture (14, 15, 16) and in nude mouse xenograft models (17) .
However, FPT inhibitors may also exert anticancer activity via mechanisms other than blocking the function of ras. For example, in a cell culture study, 31 of 42 cancer cell lines derived from various tumor types and of various oncogenic make-ups (including wild-type ras) were sensitive to a peptidomimetic FPT inhibitor (L-744832; 18 ). In a study using NIH3T3 cells transfected with various oncogenes, manumycin A exhibited action against cells transformed by non-ras oncogenes (19) . The authors of that study also concluded that the antiproliferative effect of manumycin was not necessarily directly related to interference with Ras processing. In addition, more than 10 unidentified isoprenylated proteins are affected by FPT inhibitors (19) . Among the known farnesylated CAAX proteins other than Ras, the G-proteins (for example, rap-1, rab, and rho), lamin A and B, and inositol triphosphate 5-phosphatase type I may have relevance to intracellular signaling and apoptosis. At present, however, the roles of these proteins in the antineoplastic activity of FPT inhibitors are not clear.
To explore the potential application of manumycin A in the therapy of ATC, we investigated the drug’s antiproliferative effect on six human ATC cell lines when used alone and in combination with paclitaxel, cisplatin, and doxorubicin, which are chemotherapeutic drugs frequently used for ATC. The hypothesis that these drug treatments cause apoptosis in ATC cells was tested by assessing the cells for increase in caspase-3 activity, internucleosomal DNA fragmentation, and specific cleavage of PARP in drug-treated cells. The potential for therapeutic application was also assessed using a nude mouse xenograft model with particular attention to the possibility of increased toxicity when manumycin and paclitaxel were combined.
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MATERIALS AND METHODS |
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Cell Culture.
Six human ATC cell lines were used: ARO, DRO, KAT-4, KAT-18, C643, and
Hth-74. All of the cells were cultured in RPMI 1640 with heat-treated
bovine serum (10%), penicillin (50 units/ml), streptomycin (50
µg/ml), MEM nonessential amino acids (1X), pyruvate (1
mM), glutamine (2 mM), and amphotericin
(2.5 µg/ml) at 37°C in a water-saturated atmosphere with 5%
CO2.
Late log-phase cultures were trypsinized, and 750-1500 cells were plated in each well of a 96-well tissue culture plate. The next morning, medium in each well was replaced with fresh medium or medium containing various concentrations of drugs, and the cells were then incubated for specified periods of time. In experiments that involved protein or DNA isolation, cells were cultured in six-well plates, and experimental treatments were applied when the cells were about 70–80% confluent.
Colorimetric Measurement of Viable Cells.
The number of viable cells was measured by a colorimetric technique
based on the cleavage of tetrazolium salts added to the culture medium
[Cell Proliferation Kit II (WST-1), Boehringer-Mannheim]. In this
technique, the tetrazolium salt WST-1 is broken down to the colored
product, formazan, by the "succinate-tetrazolium reductase" system
of the mitochondrial respiratory chain in viable cells. Thus, the
amount of formazan dye, measured colorimetrically as absorbance at
wavelength 450 nm (A450 nm)
with reference at wavelength 690 nm
(A690 nm), directly correlates with
the number of metabolically active cells.
During the last h of experimental treatment, 10 µl of WST-1 dye was
added to 100 µl of culture medium in each well. After 1 h of
incubation at 37°C,
A450 nm–A690 nm
was measured in each well using a microtiter plate spectrophotometer
(Dynex Tech.). All of the experiments were performed with the
absorbance values within the linear range of this colorimetric assay.
Viability was defined as follows:
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Colorimetric Assay of Caspase-3 Activity.
The CPP32/caspase-3 colorimetric protease assay kit from Chemicon
International, Inc. (Temecula, CA) was used. Two x 106 cells were plated in each vessel. After attachment to
the vessels overnight, the experimental groups were treated with
manumycin, paclitaxel, or manumycin plus paclitaxel, and the control
group was treated with DMSO 0.1% in culture medium. After cell lysis,
caspase-3 activity was measured in each cytosolic extract according to
the manufacturer’s protocol using DEVD-pNA as substrate. Absorbance
was measured at 405 nm using a microplate spectrophotometer (Dynex
Tech.).
SDS-PAGE and Immunoblotting.
After experimental treatments, cells floating in the culture medium
were pelleted by centrifugation. Cells that had become attached to the
well were rinsed with PBS. Both the cell pellet and the cells attached
to the well were lysed in a total of 150 µl of sample buffer [25
mM Tris (pH 6.8), 6 M urea, 2% 2-mercaptoethanol, 1% SDS,
0.002% bromphenol blue, 10 mM EDTA, 1 mM
phenylmethylsulfonylfluoride, and Complete Protease Inhibitor Mix
(Boehringer-Mannheim, 1 tablet per 50 ml)]. The DNA in the lysate was
sheared by rapidly passing the lysate five times through a 23-gauge
needle. SDS-PAGE was performed with standard methods. Kaleidoscope
prestained standards (Bio-Rad) were used for molecular weight
calibration. Immunoblotting (western blotting) was performed using
supported nitrocellulose membranes. Blocking was performed in 0.3%
Tween 20 (V/V) and 5% nonfat dry milk in PBS. The primary antibody was
rabbit polyclonal anti-PARP serum (Boehringer-Mannheim), and the
secondary antibody was antirabbit IgG-peroxidase conjugate
(Boehringer-Mannheim). Washing was performed with 0.3% Tween 20 (V/V)
in PBS. Kodak X-AR film was used to record the image generated by
enhanced chemiluminescence using the ECL kit (Amersham).
DNA Isolation and Electrophoresis.
The cell samples were processed for observation of DNA fragmentation
according to standard methods. Briefly, the cells were trypsinized and
detached from the culture well. Both detached cells and cells floating
in the medium were pelleted by centrifugation, and the cell pellet was
washed with PBS. Then the cell pellet was lysed in 40 µl of buffer
containing 0.25% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1
mM EDTA, and 2 mg/ml RNase A. After gentle mixing and
incubation at 37°C for 20 min, 5 µl of proteinase K (20 mg/ml) was
added to the sample and further incubated at 37°C until the solution
cleared. Then loading buffer was added and the DNA was electrophoresed
in a 2% agarose gel at 36 V in a buffer containing 40 mM
Tris acetate and 2 mM EDTA. Ethidium bromide-stained DNA
was visualized by transillumination with UV light, and the image was
recorded by a digital video gel documentation system (Fotodyne
Foto/Analyst Visionary gel documentation system).
Nude Mouse Xenograft Model.
One x 106 human ATC cells
suspended in RPMI 1640 were injected s.c. on a flank of each 7-week-old
nude mouse (nu/nu BALB-c mice bred at the animal facility of
University of Texas M. D. Anderson Cancer Center). The mice were
housed in barrier facilities on a 12-h light dark cycle with food and
water available ad libitum. s.c. tumors were measured every
2–3 days with calipers. Tumor volumes were calculated by the formula:
a2 x b x 0.4, where a is the smallest diameter and b is the diameter
perpendicular to a. After the tumors reached at least 10
mm3, the mice were randomly assigned into
experimental or control groups.
Drug solutions were injected i.p. on days 1 and 3 of a 7-day cycle for three cycles. The drugs were dissolved in tissue culture grade DMSO before dilution in tissue culture medium. The final concentration of DMSO was 0.1%. Mice in the control group received 2 injections with DMSO 0.1% in tissue culture medium. Mice in the manumycin group received 1 injection with DMSO 0.1% in tissue culture medium and 1 injection with manumycin. Mice in the paclitaxel group received 1 injection with DMSO 0.1% in tissue culture medium and 1 injection with paclitaxel. Mice in the manumycin plus paclitaxel group received 1 injection with manumycin and 1 injection with paclitaxel.
The tumor volume in each animal was calculated every 2 to 3 days. The logarithm of the tumor volume divided by the original tumor volume was calculated and averaged in each group and plotted against time since treatment began. The body weight, feeding behavior and motor activity of each animal were monitored as indicators of general health. At the end of the experiments when the animals were killed, blood samples were collected by intracardiac puncture, and lactate dehydrogenase, aspartate and alanine aminotransferases, alkaline phosphatase, and complete blood count with differential were measured in the veterinary clinical laboratory at University of Texas M. D. Anderson Cancer Center.
Statistical Analysis.
The statistical significance of differences between two groups was
assessed using Student’s t test with P < 0.05. The statistical significance of interaction between two
treatments (i.e., nonadditivity) was assessed using two-way
ANOVA. The computer software, SigmaStat for Windows 95 (Version 2.0,
Jandel Scientific), was used to facilitate calculations. Results were
reported at the 95% confidence level.
The cytotoxic interaction between manumycin and paclitaxel was also assessed using the median-effect method of Chou and Talalay (20) . The method has been described in detail elsewhere (21) . Briefly, dose-response curves were obtained for manumycin and paclitaxel, and for multiple dilutions of a fixed-ratio combination of the two drugs. The CI was the ratio of the combination dose to the sum of the single-agent doses at an isoeffective level. Therefore, CI <1, synergy; CI >1, antagonism; and CI = 1, additivity.
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RESULTS |
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Correlation of Viable Cell Number with Absorbance.
In experiments done to correlate cell numbers with absorbance obtained
by spectrophotometric assay of viable cells and to define the linear
range of the assay, the number of viable cells and
A450 nm–A690 nm
formed a tight correlation up to about 50,000 cells per well (data not
shown). The number of heat-killed cells per well (killed by incubating
at 70°C for 15 min) caused no significant change in the absorbance.
Therefore, the spectrophotometric method using WST-1 dye conversion by
mitochondrial enzymes was a valid technique for measuring the number of
viable cells. All of the experiments performed were within the linear
range of the assay.
Effect of Manumycin on Cell Number and Shape.
The effect of manumycin on ATC cells was studied in cell culture, with
the number of viable cells being measured spectrophotometrically as
described above. At the end of the treatments, the cells were also
observed under a microscope and photographed. In brief, manumycin
inhibited the growth and decreased the number of viable cells in all
six of the ATC cell lines, as confirmed by both spectrophotometry and
microscopy. Increasing concentrations of manumycin induced
morphological changes in the ATC cells (data not shown). Most control
cells, which had not been exposed to manumycin, grew attached to the
well, but a significant portion (i.e., rounded cells) grew
on top of the other cells without directly attaching to the well. As
the concentration of manumycin increased, the number of cells seen per
high-power field decreased. At concentrations that affected the
viability of the cells (>5 µM), the cells
became rounded. Additional increases in the concentration of manumycin
led to a condensed appearance of the cells.
Effects of Antineoplastic Drugs on Viability of ATC Cells.
Manumycin affected the viability of ATC cell lines, as shown by the
dose-response curves in Fig. 1A
. The viability of each cell line (as defined in
"Materials and Methods") was plotted against the concentration of
manumycin used in a 48-h treatment, with each data point representing
the geometric mean of at least three independent experiments. In brief,
manumycin decreased viability in all six of the ATC cell lines tested.
However, at the concentration of 54 µM, DRO and
C643 cells were the least sensitive of all of the cell lines. All of
the curves began to diverge from baseline at about 5
µM, a finding that agrees with the
concentration (IC50) of manumycin to inhibit 50%
activity of farnesyl:protein transferase as reported in the literature
(22)
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, paclitaxel
decreased viability in all six of the ATC cell lines. Paclitaxel
achieved >50% cell kill at 27 µM in all of
the cell lines except KAT-4. As shown in Fig. 1C
, cisplatin
had little effect on KAT-4 and KAT-18 cells (only about 10% of
cells were killed). The dose-response curves for ARO, Hth-74, and C643
cells were almost identical to one another. DRO cells, however, were
particularly sensitive to cisplatin. As shown in Fig. 1D
,
doxorubicin decreased viability in all six of the ATC cell lines. DRO
and KAT-18 cells were the most susceptible (about 70% of cells were
killed at a concentration of 10 µM). In the
other four cell lines, doxorubicin killed about 30% of cells at
the same concentration.
Effects of Manumycin in Combination with Paclitaxel, Cisplatin, or
Doxorubicin.
To explore whether manumycin could enhance the effect of the
chemotherapeutic agents currently used to treat ATC, the effects of
48-h treatments with manumycin plus paclitaxel, manumycin plus
cisplatin, and manumycin plus doxorubicin were examined. For each drug
combination, the effect was compared with the effects on ATC cells
treated with either drug alone and on control cells incubated without
the drugs. Each drug in a pair was used at a less than maximal
concentration so that its effect would not mask enhancement. In brief,
manumycin enhanced the effect of paclitaxel (Fig. 2A)
. The interaction (nonadditivity) of the two drugs was
significant (P < 0.05) in all six of the
cell lines (Fig. 2)
.4
In contrast, manumycin enhanced the effect of cisplatin (Fig. 2B)
only in DRO and KAT-18 cells and the effect of
doxorubicin (Fig. 2C)
only in C643, Hth-74, and KAT-4 cells.
The enhanced cytotoxic effect of manumycin plus paclitaxel was also
observed in PANC-1 and SK-Br3, a pancreatic carcinoma cell line and a
breast carcinoma cell line, respectively (data not shown).
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.) We conclude, therefore, that paclitaxel and manumycin enhanced the
cytotoxic effect of each other.
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, which indicated synergism in the combination of manumycin and
paclitaxel.
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Manumycin plus paclitaxel caused enhanced activation of caspase-3 (Fig. 5)
. Manumycin plus paclitaxel activated caspase-3 to a higher degree than
manumycin by itself, whereas paclitaxel did not significantly increase
caspase-3 activity compared with the control. Two-way ANOVA showed that
the interaction between manumycin and paclitaxel was significant
(F test, P < 0.01).
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, upper panel) and a Mr
24,000 fragment (data not shown) as detected by immunoblotting with
anti-PARP antiserum. In contrast, neither paclitaxel alone nor
doxorubicin alone had any significant effect on PARP (i.e.,
no change from the "no-drugs" control). Manumycin alone and
manumycin plus doxorubicin produced a pattern of PARP cleavage that was
not characteristic of apoptosis. Internucleosomal cleavage of DNA to
form a DNA ladder was observed in the cells treated with manumycin plus
paclitaxel (Fig. 6
, lower panel). The control and other
treatments (manumycin, doxorubicin, manumycin plus doxorubicin, and
paclitaxel) did not cause significant internucleosomal cleavage of DNA
after an 18-h incubation, as detected by ethidium bromide staining.
However, manumycin or paclitaxel alone did cause DNA laddering
detectable by ethidium bromide staining after incubating for 48 h
(data not shown). This specific cleavage of PARP and internucleosomal
cleavage of DNA supported the hypothesis that manumycin plus paclitaxel
increased cell death by enhancing apoptosis.
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, left panel) and KAT-4 cells (Fig. 7
, right
panel). The combination of manumycin plus paclitaxel seemed to
have an effect on tumor inhibition similar to that of manumycin against
ARO cells but showed an additional inhibitory effect against KAT-4
cells compared with either drug alone.
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DISCUSSION |
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Nevertheless, several observations argue against a nonspecific toxic
effect on living cells in general: (a) different degrees of
sensitivity to manumycin were manifested by different ATC cell lines
(Fig. 1A)
; (b) the manumycin concentration range
that inhibits the viability of ATC cells correlates with the
concentration range reported in the literature for inhibition of FPT.
The IC50 of manumycin against FPT is about 5
µM (22)
, and the inhibitory
effects of manumycin on ATC cells in the present study began to emerge
at about 5 µM, as evident on the dose-response
curves (Fig. 1A)
; and (c) manumycin induced
minimal toxicity in nude mice in an in vivo study using
xenograft models (17)
and in this report. Therefore, the
effect of manumycin on the ATC cells is likely to be, at least in part,
mediated by its blocking of Ras function and, thus, the proliferation
signal through the tyrosine kinase receptor pathway.
Manumycin also consistently enhanced the effect of paclitaxel in vitro in all six of the human ATC cell lines tested. In contrast, combinations of manumycin with doxorubicin and cisplatin resulted in synergism in some but not all of the ATC cell lines. Enhancement of the cytotoxic effect of paclitaxel by a different peptidomimetic farnesylation inhibitor (called L-744832) has been reported in other cell lines (23) , and our results corroborate and extend those findings. However, manumycin inhibits FPT by competition against farnesylpyrophosphate, whereas L-744832 inhibits FPT by mimicking the CAAX amino acid sequence. Nevertheless, their distinctly different chemical structures imply that potentiation of paclitaxel is caused by farnesylation inhibition. This potentiation has been observed previously in four breast cancer cell lines and one prostate cancer cell line, and now in six ATC cell lines, another breast cancer cell line, and one pancreatic cancer cell line. This phenomenon of enhancement between FPT inhibitors and paclitaxel may be general and, therefore, important because this synergistic combination of drugs may have potential application in a wide variety of tumors.
Our study also showed that manumycin plus paclitaxel induced apoptosis in ATC cells after an 18-h incubation, whereas neither drug alone induced a detectable degree of apoptosis as assessed by observing internucleosomal DNA fragmentation and specific cleavage of PARP in drug-treated cells. Synergistic activation of caspase-3 activity by manumycin plus paclitaxel was also observed. These observations suggest that the synergistic decrease in cell viability seen when manumycin and paclitaxel are combined may be accounted for, at least in part, by the synergistic induction of apoptosis after treatment with this drug combination. Paclitaxel induces apoptosis in many types of tumors, generally after 24–36 h (24 , 25) , and FPT inhibitors also induce apoptosis in cancer cells (26 , 27) . However, whereas the pathway by which paclitaxel induces apoptosis has been a focus of apoptosis research (25 , 28, 29, 30, 31, 32, 33) , little is known about the pathway by which FPT inhibitors induce apoptosis. It is open to speculation as to whether manumycin and paclitaxel perturb different regulators of apoptosis and lead to synergistic induction of apoptosis.
Whether manumycin plus paclitaxel would cause severe toxicity to normal cells is a critical question that needed to be addressed. We addressed this question by the in vivo studies using the nude mouse xenograft model. Toxicity to normal cells and the animal as a whole was carefully monitored by the observation of motor and feeding behavior, measurement of body weight, complete blood count with differential, and liver enzyme panel. The combination of manumycin and paclitaxel did not result in increased toxicity to normal cells or the host animal as a whole.
At least in the KAT-4 cell line, the combination of manumycin and paclitaxel produced more antitumor effect in vivo than either drug alone. With the particular dose regimen and administration schedule, synergism was not observed in vivo. One possible explanation for the lack of synergism in vivo may be the ceiling effect. Additional in vivo experimentation is justified to define the optimal way to combine manumycin and paclitaxel to maximize the therapeutic efficacy. In the search for more efficacious therapies for ATC, the discovery of synergism between manumycin and paclitaxel is one small step forward, and efficacy in vivo may justify future clinical trials.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a grant from the Physician
Referral Service of University of Texas M. D. Anderson Cancer Center
and by American Cancer Society Grant RPG-99-154-01-CDD (to
S-C. J. Y.). 
2 To whom requests for reprints should be
addressed, at Department of Medical Specialties, University of Texas
M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 40,
Houston, TX 77030. Phone: (713) 792-2840; Fax: (713) 794-4065; E-mail: syeung{at}notes.mdacc.tmc.edu
3 The abbreviations used are: ATC, anaplastic
thyroid cancer; FPT, farnesyl:protein transferase; PARP,
poly-(ADP-ribose)-polymerase; CI, combination index.
4 P < 0.05 by
F test for interaction between two factors in two-way
ANOVA.
Received 12/ 1/98. Accepted 12/ 1/99.
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) is shifted to the left of the control dose-response
curves in the absence of paclitaxel (•). In the lower 2
graphs, the dose-response curves of paclitaxel in the presence
of manumycin (
