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Experimental Therapeutics |
Cancer Research Unit, University of Bradford, Bradford BD7 1DP, United Kingdom [R. M. P., P. M. L., C. M. J., D. J. S.], and Tumour Biology Center at the University of Freiburg, D-79106 Freiburg, Germany [A. M. B., H-H. F.]
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ABSTRACT |
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INTRODUCTION |
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MMC3 is a clinically active antineoplastic agent used to treat a variety of tumors and is regarded as the prototypical bioreductive drug (6 , 7) . Its mechanism of action is complex involving several reductase enzymes, some of which have yet to be identified (8, 9, 10, 11, 12, 13) . It is generally believed that the enzyme DTD [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2] is the major enzyme responsible for bioreductive activation of MMC under normal oxygenated conditions, whereas under hypoxic conditions, other enzymes (such as cytochrome P-450 reductase) assume a prominent role (14, 15, 16) . Attempts to elucidate the fine details of MMC activation in terms of identifying enzymes that determine cellular response have, however, generated conflicting and controversial results. This is particularly true in the case of DTD, where MMC has been shown to be a substrate for DTD at acidic pH, whereas under normal physiological pH conditions, MMC is not only a poor substrate but is also an inhibitor of enzyme activity (17 , 18) . Attempts to clarify the role of DTD in the activation of MMC using a variety of experimental models (e.g., transfected cells, MMC-resistant cell lines, and the use of dicumarol as an inhibitor of DTD) have not been successful in that conflicting reports for and against a major role for DTD in MMC activation have been published (19, 20, 21, 22, 23) . In terms of predicting responses to MMC in vitro based upon DTD activity, reports of good correlations (24) conflict with reports of poor correlations (25) . Only limited studies have been conducted in vivo, although a good correlation between DTD activity and antitumor activity has been reported in a panel of eight non-small cell lung cancer and small cell lung cancer xenografts (26) . There are, however, reports of poor correlations between DTD activity and MMC activity in vivo (27) ; therefore, the issue of whether tumor response to MMC in vivo can be predicted on the basis of DTD activity remains confused.
Recent studies by Cummings et al. (28 , 29) have suggested that the concept of enzyme-directed bioreductive drug development may need to be remodeled, on the basis that the mechanism of action of compounds such as MMC and the structurally related indoloquinone EO9 are too complex to allow for accurate predictions of response based upon the activity of a single enzyme. An alternative approach based upon the ability of homogenates of tumor tissue to metabolize bioreductive drugs has been proposed by Cummings et al. (28 , 29) , and good correlations between response and the rate of reduction of EO9 have been reported in a limited number of tumors (29) . To assess the relative merits of predicting tumor response on the basis of DTD levels or metabolism of MMC by tumor homogenates, this study has compared both end points in a large panel of human tumor xenografts. These xenografts have been established within the European Organization for Research and Treatment of Cancer as an in vivo-based screening facility to identify novel anticancer drugs and have been extensively characterized in terms of their response to several anticancer drugs including MMC (30) . In addition, in vitro chemosensitivity studies using the soft agar clonogenic assay have been conducted with the aim of assessing the relationship between DTD activity and chemosensitivity in vitro.
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MATERIALS AND METHODS |
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Tissue Collection.
Tissues of human tumor xenografts growing s.c. in thymus aplastic nude
mice (NMRI background) were collected from the Freiburg Tumor Bank.
This bank comprises over 350 human tumor xenograft models that were
established in serial passage in vivo, of which 60 are
intensively characterized with respect to morphology/histology,
chemosensitivity patterns (in vitro and in vivo),
and molecular targets (30)
. Approximately 500 mg of fresh
tissue of each tumor was subjected to the clonogenic assay for in
vitro chemosensitivity testing against MMC, and 1 g of tumor
was flash frozen in liquid nitrogen immediately after removal. Later,
tissues were stored at -80°C for determination of DTD enzyme
activity and MMC metabolism.
Clonogenic Assay/in Vitro MMC Sensitivity Testing.
Freshly removed xenograft tissues were minced and then incubated with
an enzyme cocktail (collagenase, 1.2 units/ml; DNase, 375 units/ml;
hyaluronidase, 29 units/ml) at 37°C for 30 min. Tumor homogenates
were washed twice with PBS, passed through sieves (200–50 µm), and
viable tumor cells were counted. The assay was performed according to a
modified two-layer soft agar assay in 24-well plates (31)
.
In brief, 4 x 104 to 8 x 104 cells were added to 0.2 ml Iscoves’
modified Dulbecco’s medium/20% FCS containing 0.4% agar and plated
on top of the base layer (0.75% agar containing Iscoves’ modified
Dulbecco’s medium plus 20% FCS). After 24 h, an additional 0.2
ml of medium (control) or medium containing MMC was added. Each plate
contained six untreated control wells, three vehicle controls, and six
different drug concentrations (0.1 ng-10 µg/ml) in triplicate.
5-Fluorouracil was used as positive control in a single concentration
(1000 µg/ml). Cultures were incubated at 37°C, 7%
CO2 for 5–15 days, and monitored closely for
colony growth. Vital colonies were stained with 50 µl/well
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1
mg/ml) 24 h prior to evaluation, and colonies >50 µm were
counted with an automated image analysis system (Omnicon FAS IV;
Biosys). Results were expressed as the concentration required to induce
70% growth inhibition/cell kill (IC70). Assays
were considered evaluable if control groups produced >20 colonies with
a diameter of >50 µm, and initial plate colony counts on days 0 or 2
were <20% of the final control group colony count. Plating
efficiencies were 
0.1% and are consistent with the low plating
efficiencies reported for primary human tumor cell cultures
(1)
.
Assessment of in Vivo Activity of MMC in Human
Tumor Xenograft Models.
The response of 43 human tumor xenografts to MMC in vivo has
been described previously in detail elsewhere (30)
and are
summarized below. Master stocks of all tumors in the Freiburg panel are
maintained in liquid nitrogen, and all chemotherapy studies are
conducted on tumors within 10 passages from recovery from master stocks
(32)
. Although the original chemotherapy studies were
conducted prior to 1992 (30)
, this policy should ensure
that the response and biochemical properties remain stable enough to
allow a valid retrospective study to be conducted. Nevertheless, to
ensure that chemosensitivity profiles to MMC were stable, chemotherapy
studies were repeated for 13 human tumor xenografts according to the
methodology described previously (30)
. Tumors were
implanted s.c. into both flanks of outbred athymic nude mice of NMRI
genetic background, and treatment started when the tumors reached a
median diameter of 6 mm. At this time (day 0), mice were randomly
assigned to either treatment or control groups with five to six mice
per group, and MMC was administered i.v. at the maximum tolerated dose
of 2 mg/kg on days 1 and 15. Antitumor effects were determined by
two-dimensional caliper measurements that were normalized relative to
tumor volume at day 0. Experiments were terminated when tumors reached
a size of 1.5 cm in diameter. Activity was expressed in terms of
percentage of optimal T/C (i.e., relative volume of treated
tumors divided by the relative volume of control tumors x 100 at the time of maximal drug effect) and classified as
complete regression (T/C <10%, +++), partial remission (T/C 11–25%,
++), minimal remission (T/C 25–50%, +), resistant (T/C >50%, -).
The response of these tumors was very similar (within 95% confidence
intervals) to tumor responses obtained in the original study, thereby
validating the experimental design of this study (data not shown). All
animal experiments were performed in accordance with German Animal
License Regulations (Tierschutzgesetz) identical to United Kingdom
Co-ordinating Committee on Cancer Research Guidelines for the Welfare
of Animals in Experimental Neoplasia (33)
.
Measurement of DTD Activity.
Tissues were homogenized (10% w/v homogenate) in sucrose (0.25
M) using an Ultra Turrax blender. Cytosolic fractions were
prepared by centrifugation of the homogenate at 18,000 x g for 4 min, followed by further centrifugation of
the supernatant at 110,000 x g for 1 h
at 4°C in a Beckman Optima TL ultracentrifuge. Activity of DTD in the
supernatant was determined spectrophotometrically (Beckman DU650
spectrophotometer) by measuring the dicumarol-sensitive reduction of
DCPIP at 600 nm (34)
. Each reaction contained NADH (200
µM), DCPIP (40 µM),
dicumarol (20 µM, when required), and a
cytosolic fraction of tissues (50 µl per assay) in a final volume of
1 ml of Tris-HCl buffer (50 mM, pH 7.4)
containing BSA (0.7 mg/ml). Rates of DCPIP reduction were calculated
from the initial linear part of the reaction curve (30 s), and results
were expressed in terms of nmol DCPIP reduced/min/mg protein using a
molar extinction coefficient of 21 mM/cm for
DCPIP. Protein concentration was determined using the Bradford assay
(35)
.
COMPARE Analysis.
We have developed a COMPARE algorithm based on the differential
activity of drugs against human tumors growing in soft agar in
vitro analogous to the National Cancer Institute-DTP COMPARE
computer program (36)
. This tool allows for comparison of
possible structural similarity and molecular targets (37)
.
Thus, DTD levels in human tumor xenografts were ranked and related to
their in vitro sensitivity against MMC using the Spearman
rank coefficient test (38)
.
Metabolism of MMC by Tumor Homogenates.
A limited panel of xenografts were selected for this aspect of the
study. The selection criteria were simply based upon extremes of tumor
response to MMC in vivo [i.e., sensitive (T/C
<10%) and resistant (T/C >50%)], and sufficient tumor specimens
were included in each group so that the full range of DTD activity was
represented (Table 3)
. Tumors were homogenized in ice-cold Tris-HCl (5
mM, pH 7.4) containing EDTA (0.5
mM) and sucrose (250 mM)
using an Ultra Turrax blender (Janke and Kunkel). Samples were
centrifuged at 1000 x g for 1 min to remove
tumor fragments. Each reaction consisted of tumor homogenate (10 mg/ml
protein), MMC (200 µM), NADH and NADPH (2
mM) in a final volume of 0.2 ml of homogenizing
buffer. Samples were incubated at 37°C, and at various time intervals
after the addition of MMC, 30 µl of reaction mix were removed and
added to 90 µl of acetonitrile containing the internal standard,
porfiromycin (50 µM), samples were mixed, the
solvent was evaporated in a Jouan evaporator, and the residue was
resuspended in 100 µl of mobile phase. Chromatographic separation of
MMC was achieved using a RP-18 end-capped LiChrospher column (5 µm,
250 x 4 mm; Phenomenex, Cheshire, United Kingdom). The
HPLC system consisted of a system Gold Beckman 126 Programmable Solvent
Module (Beckman Instruments UK Ltd., High Wycombe, United Kingdom), a
Beckman 507 Autosampler, which was cooled to 4°C by the Grant Cooling
Unit LTD6 (Grant Instruments, Cambridge, United Kingdom), and a Beckman
168 Photo Diode Array Detector. Dual wavelength detection used 365 nm
for MMC and 310 nm for the detection of metabolites with a flow rate of
1.2 ml/min, and data were processed using System Gold software
(Beckman). The mobile phase consisted of an 18 mM
phosphate buffer (pH 6.4):methanol mixture. The gradient program to
separate MMC from metabolites was 95% A up to 10 min and then 5% A by
30 min, where A was 95% buffer and B was 76% buffer. The half life of
MMC was determined from least squares log linear regression analysis
using the equation T1/2 = 0.693/Kd where
Kd is the decay rate constant (the
slope of the regression analysis x 2.303).
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RESULTS |
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. There was a broad spectrum of DTD activity both throughout the panel
of tumors and within each tumor type (with the exception of gastric
cancers where the range of DTD activity was 510.6–980.5 nmol/min/mg).
Responses to MMC have been determined for 43 human tumor xenografts,
and a broad spectrum of response exists with lung tumors (T/C <10% in
five of seven non-small cell lung cancer) being particularly sensitive
to MMC (Table 1)
. No correlation between DTD activity and the response
of xenografts to mitomycin C exists (Fig. 1)
.
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, a poor correlation exists (regression coefficient, 0.055). Similarly,
by using a COMPARE algorithm based on the rank of DTD levels and
IC70 of MMC, the poor relationship between DTD
activity and the response of xenografts to MMC was confirmed, which is
reflected in a Spearman rank coefficient of 0.24 (Table 2)
.
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. No metabolites of MMC were present immediately after the addition of
MMC (Fig. 3A)
. After 60 min incubation at
37°C, MMC was barely detectable, and several metabolites of MMC were
found. 2,7-DAM was identified (this coeluted with the products formed
as a result of the reaction of MMC with DTD and NADPH) and a very polar
metabolite (unknown identity) were the most prominent species detected
(Fig. 3B)
. Two other metabolites were tentatively identified
as 1,2-cis- and
1,2-trans-1-hydroxy-2,7-diaminomitosene, because these two
metabolites coeluted with the hydrolysis products formed after the
incubation of MMC with HCl (0.1 N) as reported by
Cummings et al. (39)
. No obvious correlation
between the presence of these metabolites and antitumor activity
(i.e., 2,7-DAM and the polar metabolite could be found in
poorly responding tumors that metabolized MMC) was observed (data not
shown). A broad spectrum of rates of MMC metabolism was observed in the
panel of tumors studied (Table 3)
, and examples of MMC metabolism by homogenates derived from resistant
(RXF 393) and sensitive tumors (LXFL 529) are presented in Fig. 4
. Half-lives of MMC in homogenates derived from RXF 393 and LXFL 529
tumors were 234 ± 52 and 34 ± 4 min,
respectively. The concentrations of MMC used in this study (200
µM) are above the plasma levels of MMC found
in vivo (20 µM). This
concentration was necessary to detect metabolites of MMC and in the
case of LXFL 529 tumors, no differences in terms of MMC half-lives were
observed when starting MMC concentrations of 200 or 20
µM were used (data not shown).
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and Fig. 5
. In both good (T/C <10%, score +++) and poor (T/C >50%, score -)
responders to MMC, a broad spectrum of DTD activity exists (Table 2)
.
Mean DTD activities for good and poor responders were 326.15 ± 340.21 and 355.4 ± 231.5 nmol/min/mg,
respectively. No significant differences exist between DTD activity and
response to MMC in this panel of tumors (Fig. 5A)
.
Similarly, no correlation existed between the rate of MMC metabolism by
tumor homogenates (expressed as T1/2
values) and DTD activity (Fig. 5B)
in the tumor xenografts
studied. MMC half-lives in tumor homogenates derived from sensitive and
poorly responsive xenografts were 75 ± 48.3 and
280 ± 129.6 min, respectively. A significant difference
[P < 0.005 (two tailed t test)]
existed between rates of MMC metabolism in poor and good responders to
MMC, although a subset of tumors exist that have the ability to
metabolize MMC yet do not respond well to MMC in vivo (Fig. 5C)
. Within this panel of tumors, in vitro
chemosensitivity data demonstrated that the majority of responsive
tumors had low IC70s with the exception of LXFA
289, which was relatively resistant to MMC in vitro (Fig. 5D
and Table 3
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The results clearly demonstrate that tumor responses
in vivo to MMC cannot be predicted on the basis of DTD
activity alone (Fig. 1)
. It should be noted that this conclusion is
based upon the assumption that tumor response to MMC should be
proportional to DTD activity. Recent studies using the BE human colon
carcinoma cell line transfected with NQO1 have demonstrated that both a
lower threshold of DTD activity is required to initiate toxicity to
streptonigrin and RH1 (>22 nmol/min/mg) and an upper threshold (>77
nmol/min/mg) beyond which no further increase in toxicity occurs
(40)
. The results presented in Fig. 1
clearly demonstrate
that the response of tumors to MMC in vivo is independent of
DTD activity and that thresholds of DTD activity cannot be applied in
this case with any degree of certainty. A comparison between in
vitro chemosensitivity and DTD activity also demonstrates that
responses to MMC at the cellular level cannot be predicted on the basis
of DTD levels (Fig. 2)
. These results support the findings of other
groups that the response of cells to MMC in vitro cannot be
forecast on the basis of DTD activity (25)
. Recent studies
using BE cells transfected with NQO1 have, however, suggested that the
influence of DTD activity on chemosensitivity in vitro may
be affected by the drug exposure conditions (i.e.,
acute or chronic) used in vitro (41)
. For
example, in the case of the aziridinyl benzoquinone compounds, MeDZQ
and RH1, a marked cytotoxic potentiation in DTD-rich BE cells occurred
when acute (24 h) drug exposures were used, whereas chronic exposures
(96 h) showed much less potentiation. In the case of MMC, however, the
duration of drug exposure is unlikely to influence the correlation
between MMC toxicity in vitro and DTD activity because
conflicting reports of good and poor correlations have been generated
using similar chronic drug exposure conditions (24
, 25)
.
In addition, the poor correlation between the rank order of responses
in vitro and DTD activity suggest that the use of short-term
drug exposures would not improve the predictive value of the assays.
The results of this study, in conjunction with the controversy
surrounding MMC activation by DTD, therefore support the view expressed
by Cummings et al. (29)
that the concept of
enzyme-directed bioreductive drug development needs to be remodeled in
the case of MMC. It is important to stress that this conclusion applies
only to MMC, and the concept may still be applicable for other
bioreductive drugs. MMC has a complex mechanism of action involving
several enzymes (29
, 42
, 43)
, but for compounds that have
a simpler mechanism of action where one enzyme predominates in the
activation process, this concept may still be valid.
The alternative approach as set out by Cummings et al.
(29)
is to determine the ability of tumor homogenates to
metabolize MMC on the basis that bioactivation of the drug is
determined by various enzymes present in the tumor. In a selected panel
of tumors that represent the extremes in terms of antitumor response to
MMC [i.e., responsive (T/C <10%), n = 7 and nonresponsive (T/C >50%), n = 11], the half-life of MMC in tumor homogenates was 75 ± 48.3 and 280 ± 129.6 min, respectively. The
difference in means between the two groups was statistically
significant (P < 0.005) and represented a
marked improvement over the correlation between DTD and antitumor
response in this panel of tumors (Fig. 5A)
. There was also a
poor relationship between DTD levels and MMC metabolism (Fig. 5B)
, which provides indirect evidence to suggest that other
enzymes are involved in MMC reduction. Cummings et al.
(29)
have proposed a model whereby several enzymes compete
for MMC on the basis of protein level as opposed to enzyme kinetics.
This is based upon the fact that Michaelis Menton affinity constants
for MMC are similar for various enzymes (8
, 10
, 29
, 44)
.
If this were the case, then DTD-rich tumors should metabolize MMC
rapidly, but this does not hold true for all DTD-rich tumors studied
(Table 3)
. In addition, it is of considerable interest to note that
some poorly responsive tumors (Table 3)
have the ability to metabolize
MMC (e.g., PRXF DU145, PAXF 736, and MEXF 535). There are
several possible explanations for this including the fact that within
these tumors, the disappearance of the parent compound is the result of
a detoxification pathway as opposed to bioreductive activation. In all
of these tumors, however, 2,7-DAM (which is a marker for bioactivation)
was detectable at levels comparable with tumors that were sensitive to
MMC (data not shown). Alternatively, other morphological features of
the tumor (i.e., poor blood supply resulting in poor drug
delivery) or cellular defense mechanisms (i.e., drug
resistance) may have a significant bearing on the outcome of
chemotherapy. With regard to possible drug resistance mechanisms, a
recent paper by Belcourt et al. (45)
have
demonstrated that the bacterial MCRA protein (which acts as a
hydroquinone oxidase, thereby oxidizing the reduced MMC back to the
parent compound) causes profound resistance to MMC under aerobic
conditions in Chinese hamster ovary cells transfected with the
mcrA gene. However, it is not clear whether
resistance to MMC could be caused by a MCRA-like mechanism in mammalian
cells. Intrinsic drug resistance may well be the case for PRXF DU145,
which has relatively high IC70s (24 ng/ml; Table 3
) but not for PAXF 736, which is quite sensitive to MMC in
vitro (IC70, 5 ng/ml). Further studies are
required to determine why these tumors are resistant to MMC, despite
their inherent ability to metabolize MMC.
The results of this study have demonstrated that antitumor responses to MMC cannot be forecast on the basis of either DTD activity or in vitro chemosensitivity, whereas a better correlation between MMC metabolism by tumor homogenates and tumor response in vivo exists. It is of interest to note that a discrepancy exists between the measures of chemosensitivity in vitro and metabolism by tumor homogenates in vitro in terms of predicting responses in vivo because conceptually, these end points should give similar results. There are, however, significant differences between the two methodologies that may explain these findings. The major difference relates to the fact that in vitro metabolism studies are short-term assays performed on tumor homogenates, whereas in vitro chemosensitivity assays described in this paper are relatively long term (conducted over 5–15 days). In the case of the later, it is conceivable that many biological and biochemical parameters may change during the culture period that may have either direct or indirect effects on the outcome of chemotherapy in vitro. For example, the selective growth of the anchorage-independent clonogenic cell population in soft agar effectively removes tumor cells from contact with normal stromal cells and the extracellular matrix. There is now a considerable body of evidence (46) pointing to the fact that many biological processes are influenced by the tumor microenvironment (i.e., the extracellular matrix and stromal components of tumors), and therefore, the results of the clonogenic assay in vitro may not reflect the response of tumors in vivo (as a result of altered cell kinetics or biochemical properties). In addition, the activity of several drug-metabolizing enzymes (e.g., members of the cytochrome P-450 family) is markedly decreased within a few hours of isolation from fresh tissue (47) . In vitro metabolism studies, on the other hand, are conducted within a time scale where changes in biochemical and biological parameters are unlikely to occur. The short-term nature of in vitro metabolism studies may therefore represent a more realistic "snapshot" of tumor biology/biochemistry compared with the chronic nature of the clonogenic assay described in this report.
In conclusion, this study has clearly demonstrated that antitumor responses to MMC cannot be predicted on the basis of DTD levels alone. In view of the size of the panel of tumors used, together with the fact that a broad spectrum of DTD levels exists in both responsive and nonresponsive tumors, it is clear that individualizing chemotherapy on the basis of DTD levels is not feasible with regard to MMC. Measurement of MMC metabolism by tumor homogenates, on the other hand, can distinguish between responsive and nonresponsive tumors in the majority of cases. No correlation was seen in terms of the major metabolite of MMC (2,7-DAM) and tumor response in this study, which would appear to conflict with the hypothesis put forward by Cummings et al. (29) . It is important to stress, however, that the generation of reactive metabolites would be technically challenging to measure accurately in view of the fact that these metabolites will bind covalently to cellular macromolecules. On the basis of this study, measurement of the disappearance of the prodrug maybe feasible in terms of predicting tumor response in vivo. If a sensitivity threshold for MMC metabolism (in terms of T1/2 values) of 200 min were imposed, the predictive value of the assay is: 7/7 true-positive predictions, 9/11 (81.8%) true-negative predictions, and 2/11 (18.8%) false-positive predictions. This represents a substantial improvement over an enzymological end point, but a subset of tumors exists that is capable of metabolizing MMC but does not respond to MMC in vivo. Tumor responses are determined by many factors (48) , and it is unlikely that any ex vivo assay will mimic all of these conditions. The key condition that has to be achieved by any predictive assay is that the incidence of false-negative predictions must be low. The results of this study suggest that by measuring the ability of a tumor homogenate to metabolize MMC, it could be possible to identify those tumors that have a good probability of responding. From a technical standpoint, the assay described in this report could be adapted to biopsy material because samples as small as 200 mg can be assayed reproducibly. Further studies are required using clinical material to assess whether this approach to predicting MMC activity has real clinical applications.
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FOOTNOTES |
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1 Supported by the Cancer Research Campaign and
the Association for International Cancer Research. 
2 To whom requests for reprints should be, at
Cancer Research Unit, University of Bradford, Bradford, BD7 1DP, United
Kingdom. Phone: 44-1274-233226; Fax: 44-1274-233234; E-mail: r.m.phillips{at}bradford.ac.uk
3 The abbreviations used are: MMC, mitomycin C;
DTD, DT-diaphorase; DCPIP, dichlorophenolindophenol; HPLC,
high-performance liquid chromatography; 2,7DAM,
2,7-diaminomitosene.
Received 1/13/00. Accepted 9/19/00.
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) and -resistant (RXF393, 
) human tumor
xenografts. Half-lives of MMC in LXFL 529 and RXF 393 tumor homogenates
were 34 and 234 min, respectively.