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Experimental Therapeutics |
Department of Chemistry, Institute for Lasers, Photonics, and Biophotonics, State University of New York, Buffalo, New York 14260 [L. J. K., X. W., H. E. P., E. J. B., P. N. P., C. L.]; Departments of Physiology and Biophysics [L. J. K., C. L.] and Oral Biology [L. J. K.], State University of New York, Buffalo, New York 14214; and Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70112 [A. V. S., A. N.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The targeting of cytotoxic agents linked to hydrophilic peptide hormone analogues could help overcome some of the shortcomings of chemotherapy. AN-152 was developed by coupling the frequently used antineoplastic agent, doxorubicin, to an analogue of the peptide hormone LH-RH,3 [D-Lys6 ]LH-RH (2) . This analogue was chosen to serve as a carrier peptide for targeting because many tumors express receptors for LH-RH (3, 4, 5) . In addition to gonadotrope cells in the anterior pituitary, the expression of LH-RH receptors has been demonstrated in various human cancers, including breast (6) , ovarian (7, 8, 9) , endometrial (3 , 8 , 9) , prostatic (4 , 10 , 11) , and pancreatic (12 , 13) . AN-152 is significantly less toxic and more potent against cancers with LH-RH receptors than doxorubicin (2 , 14) . The decreased toxicity is presumably caused by a slower entry into normal cells than doxorubicin, resulting in a different tissue distribution of the more hydrophilic peptide conjugate from that of the more lipophilic doxorubicin. The enhanced efficacy is likely attributable to the high-affinity binding of AN-152 to receptors for LH-RH (15) on these cancers. Thus, treatment of cancers expressing LH-RH receptors with AN-152 would be more selective and would exert a lower toxicity than conventional therapies.
It has been proposed that the expression of LH-RH receptors in cancers is mediated by the loss of balance in the tyrosine kinase growth control pathway seen in many of these cancers (16 , 17) . In the hamster cheek pouch carcinoma model of oral cancer, LH-RH receptors appear in a predictable manner during carcinogenesis (16) . This change takes place much more rapidly by enhancing selective tyrosine kinase activity through events mediated by EGF stimulation (16) . Previous studies using cell membrane preparations have shown that LH-RH receptors can be activated by phosphorylation and inactivated by dephosphorylation (17) . LH-RH receptor activation can be dramatically increased by EGF-stimulated tyrosine kinase activity (17) . The increase in the tyrosine kinase signal in cancer cells results in activation of latent receptors by phosphorylation and increases the number of functional receptors (17) . An analogue of somatostatin, RC-160, which activates tyrosine phosphatase activity, decreases the number of functional receptors through dephosphorylation (17) . The use of cell membrane extracts in these LH-RH binding experiments eliminated receptor synthesis or degradation as possible explanations for the modulation of receptor binding in response to EGF and RC-160 (17) . Lee et al. (18) reported that EGF specifically phosphorylates a tyrosine residue on a Mr 60,000 protein, which corresponds to the LH-RH receptor (19) , and that RC-160 dephosphorylates a tyrosine residue on that same protein (18) . These findings indicate that the modulation of LH-RH receptor binding in cell membrane preparations in response to EGF and RC-160 are attributable to tyrosine kinase and tyrosine phosphatase stimulation, respectively.
In this study, we show that acute treatment of cancer cells that express LH-RH receptors with EGF or RC-160 can sensitize or desensitize cells to AN-152 chemotherapy. To evaluate AN-152 action on cancer cells and modulation of its uptake by EGF and RC-160, the cytotoxic agent was conjugated to a two-photon fluorophore, C625, used to label AN-152 (AN-152:C625), [D-Lys6 ]LH-RH ([D-Lys6 ]LH-RH:C625), and doxorubicin (Dox:C625) (Ref. 20 ). Optical tracking of the AN-152:C625 or its components, doxorubicin and [D-Lys6 ]LH-RH:C625, provided valuable information about the mechanism of this new type of hormone-directed chemotherapy. This method of high-resolution optical tracking allowed us to determine the intracellular path of the drug and the effect of receptor regulation on drug entry into cells. We followed the entry through the membrane, into the cytoplasm and the nucleus, by TPLSM. Cytotoxicity assays were performed to examine whether variations in cell sensitivity to AN-152 parallel alterations produced by pretreatment with EGF or RC-160.
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MATERIALS AND METHODS |
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-amino group of D-lysine in
[D-Lys6
]LH-RH and to
label doxorubicin. All final products were purified by high-performance
liquid chromatography, and the conjugates were identified by
two-dimensional nuclear magnetic resonance and by fast atom bombardment
ionization mass spectrometry.
TPLSM.
The microscope used was a commercially available, MRC-1024 laser
scanning confocal microscope from Bio-Rad, modified for use as a TPLSM.
The light source at 800 nm for two-photon microscopy was a
mode-locked Ti:Sapphire laser pumped by an Argon ion laser, producing a
train of pulses of 80 fs duration at a frequency of 90 MHz. The average
power used was 15 mW. The images shown in this report were collected by
keeping the aperture fully open, making the system a TPLSM. This
enabled us to view biological processes without damaging the living
cells. The water immersion objective lens used for all biological
imaging was a Nikon Fluor-60X (NA = 1). A scan rate of
4000 µm2/s was used, which was found to be safe
for the living cells.
Doxorubicin shows some fluorescence with an excitation wavelength of 488 nm. Its entry into cells was monitored by one-photon confocal microscopy using the same equipment described above using an emission filter for 580 nm (band-pass filter, 580/32).
Cell Culture.
MCF-7 cells (American Type Culture Collection) were grown in improved
MEM supplemented with 10% fetal bovine serum. UCI-107 cells were
provided by Dr. P. Carpenter (University of California, Irvine Medical
Center, Irvine, CA) and were grown in improved MEM supplemented with
5% heat-inactivated fetal bovine serum. Both cultures were maintained
at 37°C in a humidified atmosphere with 5%
CO2.
Modulation of AN-152 Uptake by EGF and Somatostatin Analogue
RC-160.
Cells were pretreated (2 h) with 10 nM EGF (from Sigma) or
10 nM RC-160 or received no pretreatment, followed by
addition of 0.6 µM of one of the chromophore-labeled
compounds, AN-152:C625,
[D-Lys6
]LH-RH:C625, or Dox:C625, or
0.6 µM unlabeled doxorubicin. The competition assay was
performed by adding 1 or 0.1 µM
[D-Trp6
]LH-RH 10 min prior to
addition of AN-152:C625. For each experiment, cells were viewed through
the microscope at approximately x600. The field was viewed initially
as soon as the labeled drug was administered for 5 or 10 min, then
every 10 min for the first 90 min, and subsequently at 180 and 270 min,
and the images were saved. Cell viability was assessed throughout the
experiments by visual observation of cell morphology and maintenance of
attachment to the Petri dish.
Image Analysis.
Image analysis was performed on a PC computer using the public domain
Image/J program that was developed at the United States NIH and
available on the
Internet.4
The mean gray value (the sum of the gray values of all pixels in the
selection divided by the number of pixels in the selected area), a
measure of absorbance, was determined by selecting the area occupied by
the cell for analysis by the program. The mean gray value of the
background was determined and subtracted from that of the cells. The
mean gray value was determined for several cells and several background
positions in each experimental condition and averaged. The gray values
were divided by the number of minutes that the cells were exposed to
the drug to obtain a rate of increase in absorbance.
Regulation of AN-152 Efficacy by EGF and Somatostatin Analogue
RC-160.
Cells grown in multiwell plates were treated as follows: 1. control, no
treatment; 2. AN-152 (1 nM to 10 µM); 3. EGF
(10 nM); 4. pretreatment (2 h) with EGF (10
nM), followed by AN-152; 5. RC-160 (10 nM); 6.
pretreatment (2 h) with RC-160 (10 nM), followed by AN-152;
7. doxorubicin (1 nM to 10 µM); 8.
pretreatment (2 h) with EGF (10 nM), followed by
doxorubicin; and 9. pretreatment (2 h) with RC-160 (10 nM),
followed by doxorubicin. The cytotoxicity was assessed by a
colorimetric assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
tetrazolium salt (22)
, and automatic microplate scanning
spectrophotometry at 540 nm. Absorbance corresponds to cell survival,
with higher values indicating a greater number of viable cells
(23)
. Dose-response curves were constructed, and
ED50 values were extrapolated. Statistically
significant differences were determined using a two-tailed Student
t test for each array of data points for comparison to
doxorubicin treatment and to AN-152 treatment. Differences with
P 
0.01 or P 0.05 were reported as statistically significant differences.
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RESULTS |
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3 h with no visible
effect on cell viability. The drug was sequentially localized to the
cell membrane, cytoplasm, and nucleus by optical sectioning. From
computer analysis of the images collected, the average rate of
association of AN-152:C625 with the cells was calculated for cells that
received no pretreatment (control condition) or that had been
pretreated with EGF or RC-160 for 2 h (Fig. 1A
). Visualization of AN-152:C625 entry into each cell
compartment occurred markedly faster and with greater intensity in
MCF-7 cells that were pretreated for 2 h with 10
nM EGF than in cells with no hormone pretreatment
(Figs. 1A
and 2
). MCF-7 cells pretreated for 2 h with 10 nM
RC-160, followed by AN152:C625 treatment, showed a lower intensity of
labeling and a slower time course than EGF-pretreated cells or control
cells (Figs. 1A
and 2
), but the rate of uptake was not
statistically different from control cells. In all three trials, the
time course of drug entry was the same.
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and 3
). Pretreatment with EGF effectively up-regulated
[D-Lys6
]LH-RH:C625 entry,
and RC-160 down-regulated its entry. In this case, however, in all but
one of the trials,
[D-Lys6
]LH-RH:C625 was
not seen in the nucleus. Conjugation of the two-photon chromophore to
doxorubicin altered its lipophilic character (data not shown).
Therefore, unlabeled doxorubicin was visualized by one-photon confocal
laser scanning microscopy using 488 nm, but the localization
capabilities were limited. Doxorubicin entry into the cytoplasm and
nucleus could be visualized in MCF-7 cells after an average of 40 min,
but with no apparent accumulation on the cell membrane (image not
shown). Pretreatment of MCF-7 cells with EGF did not alter the entry of
doxorubicin (average of 41 min). Pretreatment with RC-160, however,
appeared to speed the time course of uptake of doxorubicin into cells,
with drug entry evident within an average of 17 min and slightly higher
intensity of labeling. However, when the rate of doxorubicin entry into
cells was compared for cells in each of the three conditions (control,
RC-160, and EGF) over 1 h, there was no difference (each condition
had a rate of increase in absorbance/min of 0.1 ± 0.03).
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). This was compared with cells under the same conditions in
the absence of
[D-Trp6
]LH-RH (Fig. 4B
).
[D-Trp6
]LH-RH effectively
competed for receptors for AN-152:C625, and the rate of drug
association with the cells was five times faster in the absence of
[D-Trp6
]LH-RH (Fig. 1A
). The intensity of labeling was significantly reduced by
competition with
[D-Trp6
]LH-RH, and with
higher [D-Trp6
]LH-RH
concentrations the entry of the drug decreased, as evidenced by a lower
intensity of fluorescence in the cells.
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). Hormone regulation of drug uptake, as
observed in MCF-7 cells, was not seen with the cells lacking LH-RH
receptors. Pretreatment of UCI-107 cells with EGF did not alter the
rate of AN-152:C625 entry, although RC-160-pretreated cells were more
intensely labeled, and the rate of drug entry was greater (Fig. 1A
).
Regulation of AN-152 Efficacy by EGF and Somatostatin Analogue
RC-160.
MCF-7 human breast cancer cells, which are reported to express LH-RH
receptors (2
, 15)
, were treated with EGF to up-regulate
the LH-RH receptors and somatostatin analogue, RC-160, to down-regulate
them (17)
. Although these peptide hormones are widely
known to have effects on the proliferation of cells, under our
experimental conditions, exposure of MCF-7 cells to this concentration
of EGF or RC-160 for 96 h had a relatively small effect on viable
cell number (Fig. 5
). When these cells are grown in media lacking fetal bovine serum, a
greater growth response to EGF and RC-160 has been
noted.5
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. The cells were more sensitive to AN-152 after exposure to EGF, as
reflected by a decrease in the ED50 from 2.4
µM for AN-152 to 0.4 µM for EGF exposure
prior to AN-152 treatment. The cells were somewhat less sensitive to
AN-152 after exposure to RC-160, although the difference was not
statistically significant. The ED50 for AN-152
was increased by pretreatment with RC-160 to 3.0 µM.
Exposure to the hormones prior to doxorubicin did not have the same
regulating effect on the efficacy of the drug. The
ED50 for doxorubicin was 9.7 µM,
and EGF pretreatment did not significantly alter the
ED50 (9.1 µM). RC-160 increased the
sensitivity to doxorubicin, as demonstrated by the lower
ED50, 5.7 µM. AN-152 was more
effective in killing MCF-7 cells than the conventional doxorubicin
(P = 0.024), and this could be still
significantly enhanced by EGF treatment prior to AN-152 exposure
(P = 0.004). AN-152 with RC-160 differed
significantly from the doxorubicin exposure (P = 0.016); doxorubicin with RC-160 also differed but not
significantly from exposure to doxorubicin alone
(P = 0.100).
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), which was reflected by an increased number of viable
cells treated with AN-152 or doxorubicin. Conversely, RC-160 slowed the
growth of the cells (Fig. 5
); therefore, upon exposure to AN-152 or
doxorubicin, the result was a lower cell number. The
ED50s for AN-152 or doxorubicin 96 h after
treatment were extrapolated from dose-response curves (Table 1)
.
Overall, the ED50s are lower in UCI-107 cells
than for MCF-7 cells. The ED50 for AN-152 was 0.4
µM and was increased by pretreatment with EGF to 2.9
µM. The ED50 for AN-152 was
decreased by pretreatment with RC-160 to 0.005 µM. The
ED50 for doxorubicin was 0.4 µM and
was increased by EGF pretreatment to 2.0 µM and decreased
by RC-160 to 0.001 µM. Killing of UCI-107 cells by AN-152
was not significantly different from the conventional doxorubicin.
Treatment with EGF prior to AN-152 exposure resulted in greater cell
survival and was significantly different from AN-152 alone
(P = 0.002) or doxorubicin alone
(P = 0.003). EGF treatment prior to
doxorubicin exposure also resulted in greater cell survival and was
significantly different from doxorubicin alone (P = 0.014) or AN-152 alone (P = 0.018).
AN-152 with RC-160 resulted in fewer surviving cells and differed
significantly from the exposure to doxorubicin alone
(P = 0.033) and to AN-152 alone
(P = 0.027). Similarly, doxorubicin with
RC-160 resulted in fewer surviving cells and differed significantly
from the exposure to doxorubicin alone (P = 0.026) and to AN-152 alone (P = 0.018). |
DISCUSSION |
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Up-regulation of LH-RH receptors by EGF, through tyrosine
phosphorylation of receptors, has been demonstrated previously
(17
, 18)
. In this study, we used EGF stimulated
up-regulation of LH-RH receptors to enhance the uptake and efficacy of
the targeted chemotherapeutic agent, AN-152, in responsive cancer
cells. Pretreatment with EGF resulted in more rapid cellular entry of
AN-152:C625 and more intense labeling in cancer cells that express
LH-RH receptors, such as the human MCF-7 breast cancer cells. Other
LH-RH receptor-positive cells under current investigation by our
laboratory, KB and HCPC (human and hamster oral carcinoma cells,
respectively), responded in a similar manner to hormone-induced
receptor
regulation.6
The enhanced uptake of AN-152 coincided with the increased binding
activity of LH-RH receptors (17)
. The increased entry of
AN-152:C625 after pretreatment with EGF, seen by TPLSM, mirrors the
heightened sensitivity of these cells. Our results showed that the rate
of association with the cells was 50% faster (Fig. 1A
), and
the ED50 decreased by 83% (Table 1)
. The rate of
AN-152:C625 association with the cells was 50% faster than the rate of
doxorubicin entry into cells, and the entry of doxorubicin was not
affected by EGF pretreatment. Accordingly, AN-152 was more effective
than doxorubicin in killing these cells (75% lower
ED50), and the efficacy of doxorubicin was not
significantly enhanced by EGF pretreatment, as the AN-152 was (Table 1)
. This indicates that simply altering the mitotic rate with EGF
cannot account for the increased efficacy seen in response to EGF
during AN-152 treatment. These results are consistent with the
regulation of activity of LH-RH receptors by EGF-mediated tyrosine
kinase phosphorylation (16, 17, 18
, 26)
to increase the
functional binding sites for AN-152.
Pretreatment of MCF-7 cells with RC-160 resulted in slower entry of
AN-152:C625 and reduced cytotoxicity of the AN-152. The rate that the
agent associated with the cells was 20% slower (Fig. 1A
),
and the cytotoxicity decreased by 20% but did not represent a
statistically significant decrease (Table 1)
. RC-160 has been shown to
down-regulate LH-RH receptors through stimulation of tyrosine
dephosphorylation (17
, 18)
. It follows that
pretreatment of cells with RC-160 decreases the functional binding
sites for AN-152. Conversely, RC-160 decreased the doxorubicin
ED50 by 41%. This corroborates the findings of
Lee et al. (27)
and Weckbecker et
al. (28)
that somatostatin analogues enhance the
entry and cytotoxicity of doxorubicin.
UCI-107 human ovarian cancer cells, which are devoid of LH-RH receptors
(14)
, were studied as a control cancer. These cells showed
little uptake of AN-152:C625, and pretreatment with EGF or somatostatin
did not appreciably alter the rate of uptake (Fig. 1A
). In
cytotoxicity studies, AN-152 and doxorubicin were almost equally
effective. Because of the lack of LH-RH receptors, internalization of
AN-152 does not occur, and in time doxorubicin is freed from the
peptide carrier through hydrolysis by serum carboxyl esterases
(EC.3.1.1.1) present in the medium. The short, 10-min half-life of
AN-152 in the serum of female nude mice supports this view that
explains a similar effect of AN-152 and doxorubicin in UCI-107 cells
(14)
. In the case of AN-152:C625, it is doxorubicin:C625,
which is insoluble in aqueous media, is cleaved by the enzymes, and
thus the entry of the labeled doxorubicin into cells does not occur
(Fig. 2G
). Our hormonal pretreatment results are also
consistent with an absence of LH-RH receptors. UCI-107 cells that had
been treated with EGF prior to AN-152 or doxorubicin displayed a
decrease in cytotoxicity (Table 1)
, likely accounted for by the
increase in the rate of growth in response to the EGF (Fig. 5
). RC-160
exposure prior to AN-152 or doxorubicin resulted in increased
cytotoxicity (Table 1)
. RC-160 exposure alone in this cell line
resulted in decreased cell survival; therefore, this result could be,
at least in part, attributable to the inherent antiproliferative
properties of RC-160 in this cell line (Fig. 5
). The lack of regulation
of drug uptake in this LH-RH receptor-negative cell line supports that
the action of AN-152 in LH-RH receptor-positive cells is mediated by
LH-RH receptor activity, as suggested previously in cytotoxicity assays
(2)
.
Optical tracking of AN-152, [D-Lys6 ]LH-RH, and doxorubicin, in combination with hormonal alteration of LH-RH receptors, provided important information about this new type of targeted chemotherapy. The localization capabilities afforded by this technique enabled us to determine that the labeled drug enters the cell and is later concentrated within the nucleus. Prior to this investigation, it was theorized that the agent associated with the membrane receptors and acted from outside of the cell (2) , possibly through the generation of cytotoxic free radicals. We now know that there is opportunity for nuclear interaction as well in LH-RH receptor-bearing cells. AN-152, like doxorubicin (29) , the cytotoxic base of AN-152, is likely to have several cytotoxic mechanisms of action. These could include membrane interactions, free radical production, topoisomerase II inhibition, apoptosis induction, and multiple interferences with DNA.
Our experiments tracking the entry of the drug and its components helped to clarify the cellular trafficking of AN-152 and the modulation of its effectiveness by hormonal pretreatment. The predictable pattern of uptake of [D-Lys6 ]LH-RH:C625 in response to EGF and RC-160 provides further evidence that action of AN-152 is indeed receptor mediated. Addition of [D-Trp6 ]LH-RH prior to AN-152:C625 administration demonstrated competition with AN-152:C625 for LH-RH binding sites. Labeling of cells with AN-152:C625 was significantly decreased by the presence of [D-Trp6 ]LH-RH, even after pretreatment with EGF. These results strongly support the hypothesis that LH-RH receptors mediate entry of AN-152.
Evaluation of doxorubicin uptake demonstrated that in contrast, the mechanism of doxorubicin action does not involve LH-RH receptors or binding to membrane receptors. The regulation of entry of AN-152 by hormonal modulation in MCF-7 cells, but not in UCI-107 cells, and the lack of regulation of doxorubicin uptake in either cell line lend additional support for the hypothesis that the sensitivity of MCF-7 cells to AN-152 is related to LH-RH receptor regulation.
Our results reinforce the view that the increase in the efficacy of a
targeted antineoplastic drug could help to circumvent the disadvantages
and limitations of conventional chemotherapy for some cancers. These
results also suggest that the elevated levels of EGF seen in certain
cancers may further facilitate a higher uptake of targeted cytotoxic
LH-RH analogues. Previous studies indicate that this occurs by
EGF-mediated tyrosine phosphorylation and activation of LH-RH
receptors. LH-RH receptors are present in 50% of breast cancers
(6)
, 80% of ovarian and endometrial cancers
(8)
, and 86% of prostate cancers (10)
,
making these cancers excellent candidates for this type of
chemotherapy.
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FOOTNOTES |
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1 This work was supported by the Margaret
Duffey-Cameron Troupe Foundation (Buffalo General Hospital), by the
Dentist Scientist Award Grant 5K16DE00158, and by the Directorate of
Chemistry and Life Sciences of the Air Force of Scientific Research
Contract F496209710454. 
2 To whom requests for reprints should be
addressed, at Institute for Lasers, Photonics, and Biophotonics,
Department of Chemistry, NSM Complex, Room 811, State University of New
York, Buffalo, New York 14260-3000. E-mail: liebow{at}acsu.buffalo.edu
3 The abbreviations used are: LH-RH, luteinizing
hormone-releasing hormone; AN-152, conjugate of
[D-Lys6]LH-RH and doxorubicin; EGF, epidermal
growth factor; TPLSM, two-photon laser scanning microscopy; C625,
4-(N,N-diphenylamino)-4'-(6-O-hemiglutarate)hexylsulfinyl
stilbene.
4 Internet address:
http://rsb.info.nih.gov/ij/.
Received 12/13/99. Accepted 5/24/00.
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