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Advances in Brief |
Calydon, Incorporated, Sunnyvale, California 94089
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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We previously developed an adenovirus variant, CV787, that replicates preferentially in prostate cells that produce PSA.2 This was accomplished by engineering two prostate-specific transcription response elements into the adenovirus genome to control the expression of essential viral genes (7 , 8) . Animal studies have demonstrated that a single i.v. administration of CV787 at a dose of 1 x 1011 particles/animal can eliminate distant prostate tumor xenografts in Balb/C nu/nu mice (7) . This virus has entered Phase I/II clinical trials for the treatment of locally recurrent prostate cancer by intraprostatic injection and of metastatic disease by i.v. administration.
Our animal studies have also indicated that the lethal dose of CV787 (LD100) for Balb/C nu/nu mice is 3 x 1011 particles. Because the curative dose of CV787 administered i.v. as a single bolus is 1 x 1011 particles, the curative therapeutic window is narrow. Although conventional cancer therapies (surgery, chemotherapy, and radiation) are often effective at curing early-stage disease, few human metastatic cancers are curable with a single modality. In an attempt to widen the single-dose curative therapeutic window of CV787, we explored a neoadjuvant therapy that consisted of combining CV787 with chemotherapeutic agents.
In the present study, we examined the efficacy of CV787 in combination with taxane (paclitaxel or docetaxel) against human prostate cancer cells using single doses. Synergistic efficacy was observed both in vitro and in vivo. Our in vivo studies show that the combination of CV787 with docetaxel at human therapeutic doses can eliminate LNCaP xenografts within 3–4 weeks using 1 x 108 particles of CV787. This result opens a single-dose curative therapeutic window for CV787 of over 1000:1 in this prostate cancer model.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Chemotherapeutic Agents and Virus.
Paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) and docetaxel
(Taxotere; Rhone-Poulenc Rorer Pharmaceuticals, Inc., Collegeville, PA)
were purchased from the Stanford Pharmacy (Stanford Medical Center,
Palo Alto, CA). These agents were diluted with medium without fetal
bovine serum just before use for in vitro studies and with
0.9% NaCl for in vivo studies.
CV787 is a prostate-specific replication competent adenovirus variant with the rat probasin promoter driving the Ad5 E1A gene and with the human prostate-specific antigen promoter and enhancer driving the Ad5 E1B gene (7) .
Cell Viability.
MTT assays were performed by seeding LNCaP, HBL-100, OVCAR-3, HepG2,
and 293 cells at 5000 cells/well in a 96-well plate (Falcon) 48 h
before infection as described previously (9)
, with
modifications. Cells were either infected with CV787 at an MOI of 2
PFU/cell or treated with the indicated chemotherapeutic agents
(paclitaxel at 6.25 nM and docetaxel at 3.12
nM). Cell viability was measured at the times indicated by
removing the media and replacing it with 50 µl of a 1 mg/ml solution
of MTT (Sigma Chemical Co., St. Louis, MO) and incubating for 3 h
at 37°C. After removing the MTT solution, the crystals that remained
in the wells were solubilized by the addition of 50 µl of isopropanol
followed by vigorous shaking. The absorbency was determined using a
microplate reader (Molecular Dynamics) at 560 nm (test wavelength) and
690 nm (reference wavelength). The percentage of surviving cells was
estimated by dividing the
A550 nm - A650 nm of virus-infected cells by
the A550 nm - A650 nm of mock infected
cells. Twelve replica samples were taken for each time point, and each
experiment was repeated at least three times.
Statistical Analysis.
The dose-response interactions between taxane and CV787 at the point of
IC50 were evaluated by the isobologram method of
Steel and Peckham (10)
as modified by Aoe et
al. (11)
. The IC50 was defined
as the concentration of drug that produced 50% cell growth inhibition,
i.e., 50% reduction in absorbance. Cells were exposed to
drugs sequentially for 24 h, and cell viability was determined by
the MTT assay after 6 days. The dose-response curves were plotted with
CurveExpert (Version 1.34) on a semilog scale as a percentage of the
control, the absorbance of which was obtained from the samples not
exposed to the drugs. IC50 value of CV787 and
taxane in LNCaP was then determined. On the basis of the dose-response
curves of CV787 alone and taxane alone, isobolograms (three isoeffect
curves, mode 1 and mode 2 lines) were computed. The envelope of
additivity, surrounded by mode 1 and mode 2 isobologram lines, was
constructed from the dose-response curves of CV787 alone and taxane
alone. The observed data were compared with the predicted maximum and
minimum data for the presence of synergism, additivity, or antagonism
by a statistical analysis using the Stat View 4.01 software program
(Abacus Concepts, Berkeley, California). When the data points of the
drug combination fall within the area surrounded by mode 1 and/or mode
2 lines (i.e., within the envelope of additivity), the
combination is described as additive. A combination that gives data
points to the left of the envelope of additivity can be described as
supraadditive (synergism), and a combination that gives data points to
the right of the envelope of additivity can be described as subadditive
(antagonistic; Ref. 12
). Fractional tumor volume relative
to untreated controls was determined as described previously
(13)
.
One-step Growth Curve and Virus Yield.
One-step growth curves of CV787 in the presence or absence of docetaxel
were performed in LNCaP and 293 cells as described (8)
.
Immunoblots.
LNCaP cells treated with CV787, taxane, or both CV787 and taxane were
incubated for the indicated times. Cells were washed with cold PBS and
lysed for 30 min on ice in 50 mM Tris (pH8.0), 150
mM NaCl, 1% IGEPAL CA360
[(octyphenoxy)polyethoxyethanol] (NP40 equivalent from Sigma), 0.5%
sodium deoxycholate, and protease inhibitor cocktail (Roche, Palo Alto,
California). After 30-min centrifugation at 4°C, the supernatant was
removed and protein concentration was determined by the ESL protein
assay kit (Roche). Fifty µg of protein/lane were separated on 8–16%
SDS-PAGE and electroblotted onto Hybond enhanced
chemiluminescence membranes (Amersham Pharmacia,
Buckinghamshire, England). The membranes were blocked overnight in PBS
with 0.1% Tween 20 supplemented with 5% nonfat dry milk. Primary
antibody incubation was done at room temperature for 2–3 h with PBS
with 0.1% Tween 20/1% nonfat dry milk-diluted antibody, followed by
wash and 1-h incubation with diluted horseradish peroxidase-conjugated
secondary antibody. Enhanced chemiluminescence (Amersham Pharmacia) was
used for detection. Antibodies for p53 and poly(ADP-ribose) polymerase
were from Roche. Antibodies against Fas/Fas-L, caspase 7, Bcl-2,
Bcl-XL, Bax, and secondary antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, California). All of the antibodies were
used according to manufacturer’s instructions. For quantifying the
bands, the gels were scanned and bands were analyzed by Multi-Analyst
software (Bio-Rad).
In Vivo Antitumor Efficacy.
LNCaP xenografts were established as described (7
, 14)
.
Mice that bore tumors were randomized into groups of four. The first
group received 1 x 1010 particles
of CV787 at day 1 via the tail vein i.v. CV787 was diluted in 0.1 ml
lyophilized buffer (5% sucrose, 1% glycine, 1 mM
MgCl2, 0.05% Tween 80 in 10 mM Tris
buffer) and injected into the tail vein using a 28-gauge needle. The
second group was given taxane only. Paclitaxel was i.p. administered at
a dose of 20 mg/kg, daily for 4 days starting at day 2. Docetaxel was
i.v. administered at a dose of 5 or 12.5 mg/kg at day 2, 5, and 8. The
third group was given CV787 (i.v.) at day 1 and taxane at the same
doses and schedule as the second group. As a control, a fourth group
was treated with 0.1 ml of normal saline (i.e., control)
i.v. at day 1 and then i.p. or i.v. for 4 days. The dose and route of
administration of paclitaxel were selected according to studies in nude
mice (15
, 16)
. For docetaxel, the dose was selected based
on the human clinical dose (Rhone-Poulenc Rorer Pharmaceuticals, Inc.)
and determined by a dose-range finding study in nude mice (see
"Results"). Tumor volume and PSA levels were measured as described
previously (7
, 14)
. Federal and institutional guidelines
for animal care were followed.
Immunohistochemistry.
Four groups of mice (n = 6) were treated with
vehicle, CV787 (1 x 1010
particles/animal), paclitaxel (15 mg/kg), or a combination of CV787 and
paclitaxel. Half of the animals were sacrificed on day 9 and the other
half on day 16. Histology methods for adenovirus were as described
(7)
.
Apoptotic cells were detected using M30 monoclonal antibody with reagents from the M30 CytoDEATH kit (Roche Molecular Biochemicals, Indianapolis, IN) as suggested by the manufacturer. Paraffin-embedded tumor sections were heated in citric acid buffer for 15 min to retrieve antigen, hybridized with M30 antibody, then counterstained with Harries hematoxylin (Roche Molecular Biochemicals). The stained sections were analyzed under a light microscope, and pictures of representative sections were taken.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Combination of CV787 with Paclitaxel or Docetaxel Is Synergistic in
LNCaP Cells in Vitro.
To study the potential interaction between CV787 and chemotherapy
in vitro, the effectiveness of the combined treatment of
several concentrations of paclitaxel or docetaxel with CV787 at various
MOI was evaluated in the PSA-producing prostate carcinoma LNCaP cell
line. Cells were treated with CV787 and paclitaxel or docetaxel, and
the cell viability was determined at various time points by the MTT
assay. Several concentrations of docetaxel and paclitaxel were tested
before the optimal concentrations were determined for further study,
6.25 nM for paclitaxel and 3.12
nM for docetaxel. Taxane concentrations were
chosen based on the dose-response curves for each cell line, such that
the selected drug concentrations show greater combined efficacy with
CV787 but minimal cell killing when treated with the same dose of
taxane alone. For example, infecting LNCaP cells with CV787 at an MOI
of 0.01 resulted in 80% cell survival 8 days after infection, whereas
paclitaxel at a dosage of 6.25 nM resulted in
80% survival 8 days after treatment. However, when CV787 and
paclitaxel were combined at these concentrations, cell survival dropped
to 12% 8 days after treatment (Fig. 1A)
.
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shows isobologram
representation of the statistical modeling used to analyze the drug
interactions between CV787 and paclitaxel. The combined data points
fell within the envelope of additivity or were smaller than that of the
predicted minimum data, which indicates that sequential exposure to
CV787 followed by paclitaxel produced synergistic effects.
Enhanced cytotoxicity was also observed in the combination treatment of
CV787 and docetaxel. LNCaP cells were first incubated with CV787 at an
MOI of 0.01 and 24 h later were treated with docetaxel at 3.12
nM. Cell viability was determined by MTT as shown in Fig. 1C
. Combination treatment showed 10% cell survival at day 8
after treatment, whereas CV787 alone produced 80% cell survival and
docetaxel alone showed 86% cell survival in prostate carcinoma LNCaP
cells.
Isobolograms were also generated to show the synergy between CV787 and
docetaxel. Dose-response curve analysis indicated that the
IC50 at day 5 in LNCaP cells for CV787 and
docetaxel was 0.368 MOI and 8.14 nM, respectively. Fig. 1D
shows isobologram representation of the statistical
modeling used to analyze the interaction between CV787 and docetaxel.
The combined data points fell to the left of the envelope of
additivity, or, restated, the IC50 in LNCaP cells
of CV787 in combination with docetaxel occurred at smaller doses than
that predicted from the use of CV787 or docetaxel alone. Thus,
sequential exposure to CV787 followed by docetaxel produced synergistic
effects.
Taxane Increases CV787 Burst Size in LNCaP Cells.
Paclitaxel and docetaxel are antineoplastic agents belonging to the
taxane family. They are novel antimicrotubule agents that promote the
assembly of microtubules from tubulin dimers and stabilize microtubules
by preventing depolymerization. This stability results in the
inhibition of the normal dynamic reorganization of the microtubule
network that is essential for vital interphase and mitotic cellular
functions (17)
. In addition, the taxanes induce abnormal
arrays or "bundles" of microtubules throughout the cell cycle and
multiple asters of microtubules during mitosis (18
, 19)
.
One possible explanation for the synergy seen with taxane and CV787 is
that taxane may augment the ability of CV787 to replicate in LNCaP
cells.
To examine the effect of paclitaxel and docetaxel on virus replication,
we performed the one-step growth curve. LNCaP cells were infected with
CV787 at an MOI of 1 for 4 h, followed by incubation in RPMI 1640
that contained docetaxel at a final concentration of 3.12
nM. Cells were harvested at various times after infection,
and the number of infectious virus particles was determined on 293
cells by standard plaque assay (7)
. As shown in Fig. 2
, although the initial rate of increase of CV787 in cells treated with
CV787 and docetaxel was similar to that of cells treated with CV787
alone, a plateau was reached for CV787 at approximately 72 h after
infection and at approximately 96 h after infection for CV787 and
docetaxel. Cells treated with CV787 and docetaxel produced 30,000
PFU/cell, whereas the cells infected with CV787 alone generated about
15,000 PFU/cell. Thus, docetaxel does not inhibit CV787 replication but
actually increases virus replication efficiency. A similar result was
obtained in a parallel study with paclitaxel (data not shown).
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. The combination of CV787 and paclitaxel was toxic to 293 and LNCaP
cells but not to HBL-100 and OVCAR-3 cells. There were few surviving
LNCaP cells 9 days after infection. In contrast, the viability of
HBL-100 and OVCAR-3 cells treated with CV787 and paclitaxel was nearly
90% throughout the course of the experiment, similar to that of cells
treated with paclitaxel alone.
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show that prostate cancer cells (LNCaP) treated with CV787 and
paclitaxel or docetaxel produced a similar burst size to the cells
infected with CV787 alone, which produced about 1000 PFU/cell, again
suggesting that taxane does not inhibit CV787 replication. As expected,
CV787 replicated poorly in the nonprostate cancer cells (HepG2,
HBL-100, and OVCAR-3), producing 5,000–10,000-fold lower virus yield
compared with the burst size in LNCaP cells. The same is true in the
combination treatment group. Nonprostate cancer cells treated with
CV787 and taxane also produced 5,000–10,000-fold lower virus, similar
to the nonpermissive cells infected with CV787 alone. These experiments
indicate that CV787 in the presence of taxane replicates efficiently in
prostate cancer cells but retains its attenuated phenotype in
nonprostate cancer cells.
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Synergistic Efficacy of Taxane with CV787 in Vivo.
The in vivo antitumor efficacy of CV787 in combination with
taxane was assessed in the LNCaP mouse xenograft model. We have shown
previously that a single i.v. administration of CV787 at 1 x 1011 particles/animal can eliminate s.c.
xenograft tumors in 6 weeks (7)
. These data were extended
using studies up through 10 weeks. Established human prostate tumors
(LNCaP cells) were treated with vehicles, CV787 (1 x 1010 particles/animal), paclitaxel (20
mg/kg), or both CV787 and paclitaxel. For the combination treatment,
animals received i.v. injections with either CV787 or vehicle, and
24 h later, paclitaxel was administered i.p. daily for 4 days. The
tumor volume data presented in Fig. 6A
show that there was a significant decrease in tumor volume
between control and all of the treatment groups. In this study, single
doses of CV787 or four doses of paclitaxel over 4 days were effective
in producing partial tumor regression 7 weeks or 2 weeks after
treatment, whereas the combination produced a near complete regression
within 2 weeks (Fig. 6A)
. Four weeks after treatment,
relative tumor volume decreased to 3% of baseline (from 418
mm3
to 14 mm3
) for the
combination treatment group and 31% of baseline for the paclitaxel
group, but it increased to 216% of baseline for the vehicle-treated
group and 162% of baseline for the CV787 group. These changes were
statistically significant by Student’s t test
(P < 0.05) for the comparison of the
combination treatment of CV787 with paclitaxel to any of the vehicles,
CV787 or paclitaxel alone. Additionally, serum PSA levels in mice that
received injections with vehicle increased, whereas the levels in mice
that received injections with CV787 and paclitaxel decreased to 
2%
of their starting values within 4 weeks (data not shown).
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summarizes relative tumor volume of control and treated groups on four
different time points. Combination therapy showed more than additive
effect (e.g., synergy) on tumor growth inhibition. On day
21, there was 4.4-fold improvement in antitumor activity in the
combination group when compared with the expected additive effect. At
this time point, CV787 alone or paclitaxel alone inhibited tumor growth
by 20% or 70%, respectively (fractional tumor volume, 0.815
mm3
and 0.287 mm3
,
respectively) when compared with the control group (Table 1)
. With
time, there was a progressive improvement in antitumor activity. On day
42, the CV787 and paclitaxel combination group showed a 9.2-fold higher
inhibition of tumor growth over additive effect (expected fractional
tumor volume). These data demonstrated a synergistic efficacy between
CV787 and paclitaxel in LNCaP xenografts.
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shows
results from LNCaP prostate tumor xenografts treated with CV787 and
docetaxel, both administered i.v. In the combination treatment group,
animals received i.v. injections with docetaxel (5.0 mg/kg) on day 2,
5, and 8, after a single i.v. injection of CV787 (1 x 1010 particles/animal) on day 1. Both CV787 and
docetaxel appear to be effective in producing stabilization of tumor
growth in the LNCaP mouse model, whereas a combination of the two
produces a complete regression within 5 weeks (Fig. 6B)
.
Analysis on fractional tumor volume, presented in Table 1
, indicated a
synergistic effect between CV787 and docetaxel in LNCaP xenografts. For
example, on day 42, the CV787 and docetaxel combination group showed a
6.4-fold higher inhibition of tumor growth over an additive effect
(Table 1)
.
To further investigate the dose range for CV787 treatment in
combination with docetaxel, we fixed the dose of docetaxel at 12.5
mg/kg and varied the dose of CV787 from 1 x 108 and 1 x 109 to 1 x 1010 particles/animal. Fig. 6C
shows
that treatment with CV787 alone or docetaxel alone resulted in tumor
growth inhibition. However, the combination of CV787 and docetaxel had
the greatest effect of the treatments tested. Complete regression was
achieved in the animals treated with docetaxel and CV787 at a dose of
either 1 x 1010, 1 x 109, or 1 x 108 particles. Synergy of antitumor activity was
also evident using 1 x 107
particles/animal, but complete regression was not observed (data not
shown). These changes were statistically significant by Student’s
t test for the comparison of combination treatment of CV787
and docetaxel with any of the vehicle, CV787 alone or docetaxel alone
treatments, with no statistical difference between the three
combination treatment groups. The complete response dose of CV787 alone
is 1 x 1011 particles/animal
(7)
. Thus, the combination of CV787 and docetaxel produces
a complete response with 1000-fold less virus, compared with the use of
CV787 alone.
Virus replication within LNCaP tumors was documented by
immunohistochemical staining of tumor sections using polyclonal
antibodies to Ad5 (14)
. Fig. 7
contains representative tumor sections from four animals 9 days after
treatment with either paclitaxel, CV787, or CV787 and paclitaxel. No
evidence of virus replication was found in the tumors treated with
either vehicle or paclitaxel, whereas evidence of necrosis and
multifocal inflammation was observed in a small portion of tumors
treated with paclitaxel (Fig. 7A, L)
. In the
CV787-treated tumors, although positively stained cells were visible
throughout the tumors, infected cells were predominantly located near
the tumor vasculature (Fig. 7A, M)
. The most
intriguing phenomena were in the samples treated with both the virus
and paclitaxel. Although few virus-infected cells were detected, most
of the cells in the sections were empty and virtually devoid of
cellular content. The remaining cells were much smaller and appeared to
have undergone a morphological change (Fig. 7A, R)
.
|
. Few
apoptotic cells were detected in the paclitaxel-treated tumor (Fig. 7B, L)
, although a significant number of
apoptotic cells along the blood vessel were present in the
CV787-infected tumors (Fig. 7B, M)
. However,
combination treatment produced more apoptotic cells than in any of the
other samples (Fig. 7B, R)
. In conclusion, the
immunohistochemical analysis of CV787-treated tumors suggests that both
virus replication-dependent cytolysis and apoptosis contribute to the
antitumor effect of CV787 and taxane.
Finally, there are two other results of clinical significance.
First, healthier animals, characterized by body weight, were observed
in the combination treatment group as compared with groups treated with
either agent alone (Table 2)
. Of particular interest is the transient weight loss caused by
using docetaxel alone, from which animals are protected by the use of
CV787 in combination with docetaxel. Indeed, animals treated with both
CV787 and Taxotere gain 24% more weight than untreated control animals
do (Table 2)
. Second, formal toxicology studies in BALB/c mice failed
to show synergistic toxicity from the combined use of docetaxel and
CV787 (data not shown).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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A synergistic decrease of cell viability of LNCaP prostate cancer cells was observed when CV787 was combined with taxane. LNCaP cells cultured with paclitaxel or docetaxel for 24 h before or after infection with CV787 had significantly decreased viability than cells treated with either agent alone. LNCaP cells treated with taxanes exhibited a greater burst size of CV787, whereas no significant effect on viral growth kinetics was seen. No significant difference in the effectiveness of the combined therapy of taxane and CV787 infection was observed by varying the time of taxane administration, whereas varying the administration schedule of paclitaxel with Ad-p53 gene therapy can modulate the synergistic activity between these two agents in ovarian cancer (26) .
In addition, combination treatment of CV787 with taxane did not alter the specificity of replication-mediated cytotoxicity. Cell viability assays indicated that CV787 in combination with taxane remains fully selective. This result was confirmed using the one-step growth curve. CV787 has been shown previously to replicate preferentially in PSA-producing human prostate cancer cells 10,000 times more efficiently than in non-PSA-producing cells (7) . In the presence of paclitaxel or docetaxel in the culture medium, CV787 replicated to the same degree of specificity in the non-PSA-producing human cell lines versus PSA+ cells.
A synergistic antitumor efficacy of combination therapy with CV787 and taxane was also observed in prostate LNCaP xenografts. Previous studies have demonstrated that tumors were eliminated within 6 weeks by a single i.v. administration of CV787 at a dose of 1 x 1011 particles (7) . Combination treatment of CV787 and taxane eliminated tumors within 4 weeks, whereas CV787 alone at the same dose of 1 x 1010 particles/animal could only slow down tumor growth. Statistical analysis of the in vivo studies indicated that the CV787 and taxane combination group showed a significant synergy with a 6.4–9.2-fold higher inhibition of tumor growth over additive effect.
In combination with a 12.5 mg/kg dose of docetaxel, 1 x 108 particles of CV787 led to a complete elimination of tumors within 4 weeks. Thus, the dose of CV787 required for complete remission has been reduced by 1000 times from 1 x 1011 to 1 x 108 particles. Thus, the combination of docetaxel with CV787 has increased the potential therapeutic window from 1 to 1000.
Investigations into the efficacy of virus-mediated gene therapy in combination with anticancer drugs have been published recently by other groups and demonstrated that paclitaxel had a synergistic or an additive effect when combined with Ad-mediated p53 gene therapy in several cancer models (3 , 26) . Cheon et al. (27) combined an Ad vector, which contained the HSV thymidine kinase (HSV-TK) gene driven by the osteocalcin promoter, with a low dose of methotrexate and demonstrated a significant increase in efficacy and prolonged survival in a murine osteosarcoma model (27) . A replication-selective adenovirus, ONYX-015, in combination with 5-fluorouracil or cis-DDP was shown to have a greater effect than either individual modality and prolonged survival (28 , 29) . Furthermore, a synergistic efficacy was also observed in the combination of a tumor-specific HSV mutant (HSV-1716) with chemotherapeutic agents in human non-small cell lung cancer (30) . Thus, the combination of virus-based therapy with chemotherapy for the cancer treatment appears to be promising.
From a clinical point of view, two additional observations are significant. First, healthier animals, characterized by body weight, were observed in the combination treatment group as compared with groups treated with either agent alone. Second, formal toxicology studies in Balb/C mice failed to show synergistic or additive toxicity from the combined use of docetaxel and CV787.
The mechanism(s) of synergistic activity in the combination of taxane
with CV787 is unknown at this time; however, our experiments suggest a
few hypotheses. First, taxane at the synergistic dose may be augmenting
viral replication. It has been previously shown that a low
concentration of paclitaxel (1–14 nM) increased the number
of cells transduced by recombinant adenovirus 3–35% in a
dose-dependent manner (26)
. Indeed, our data show that
although the synergistic dose of paclitaxel or docetaxel did not alter
virus replication kinetics (Fig. 2)
, the chemotherapy drugs slightly
increased the burst size of CV787 in LNCaP cells. Secondly, CV787 may
be augmenting the antitumor activity of taxane. E1A gene
expression has been shown to increase cellular sensitivity to
chemotherapeutic agents (31)
, and this enhanced
sensitivity is partially caused by the induction of p53-dependent
apoptosis by the E1A-induced sensitization of the cells
(32)
. Recently, Ueno et al. (33)
found that human ovarian cancer cells that were originally resistant to
paclitaxel became paclitaxel-sensitive in E1A down-regulated HER-2/neu
cells (33)
. In CV787, the E1A gene is intact
and may be overexpressed in PSA-producing LNCaP cells. E1A is a potent
inducer of p53 protein expression in infected cells (34)
.
p53 levels may increase after infection, thereby increasing cell
sensitivity to chemotherapy-induced apoptosis. Indeed, 2–8-fold more
p53 protein was detected in the CV787 and docetaxel-treated LNCaP cells
than that in the cells treated either with docetaxel alone or CV787
alone. This is consistent with the observation that more apoptotic
cells were seen in the LNCaP tumors that received combination treatment
than in tumors that received either agent alone (Fig. 7B)
.
The actions of the two agents may be occurring at two distinct points
in the same pathway. The activities of taxane and CV787 may be
affecting distinct points in the same or different cell death pathways
used in LNCaP cells. Further investigation of the possible mechanism(s)
of synergistic activity in the combination of taxane with CV787 is
under way.
Certainly, LNCaP mouse xenografts are likely to be more susceptible to docetaxel and CV787 chemotherapy than prostate cancer patients. For example, repeated injection of docetaxel can cure mice of LNCaP tumors3 (15) but cannot cure men of prostate cancer, and preexistent antibody to adenovirus can abrogate adenovirus-mediated antitumor activity (14) .
In summary, we have developed a novel therapeutic strategy for the treatment of prostate cancer with a prostate cancer-specific adenovirus mutant, CV787, in combination with conventional chemotherapeutic agents. This combination therapy produced an additional therapeutic benefit over either individual modality. Low-dose taxane produced a synergistic effect with CV787 both in vitro and in vivo, which suggests an important possible neoadjuvant therapy for the treatment of prostate cancer. The combination of taxane and CV787 created a single dose curative in vivo therapeutic window in the LNCaP Balb/C nu/nu xenograft model of over 1000:1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Calydon Incorporated, 1324 Chesapeake Terrace, Sunnyvale,
CA 94089. Phone: (408) 734-0733; Fax: (408) 734-2808; E-mail: dhenderson{at}calydon.com 
2 The abbreviations used are: PSA,
prostate-specific antigen; Ad, adenovirus; MOI, multiplicity of
infection(s); PFU, plaque-forming units; MTT,
3-[4,5-dimethylthiazole-2–4]-2,5-diphenyl-2H-tetrazolium bromide;
HSV, herpes simplex virus.
Received 8/ 8/00. Accepted 11/29/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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-induced apoptosis in the human prostatic carcinoma cell line LNCaP. Oncogene, 19: 1959-1968, 2000.[Medline]
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 K. Mantwill, N. Kohler-Vargas, A. Bernshausen, A. Bieler, H. Lage, A. Kaszubiak, P. Surowiak, T. Dravits, U. Treiber, R. Hartung, et al. Inhibition of the Multidrug-Resistant Phenotype by Targeting YB-1 with a Conditionally Oncolytic Adenovirus: Implications for Combinatorial Treatment Regimen with Chemotherapeutic Agents. Cancer Res., July 15, 2006; 66(14): 7195 - 7202. [Abstract] [Full Text] [PDF]
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N. Ramesh, Y. Ge, D. L. Ennist, M. Zhu, M. Mina, S. Ganesh, P. S. Reddy, and D.-C. Yu CG0070, a Conditionally Replicating Granulocyte Macrophage Colony-Stimulating Factor-Armed Oncolytic Adenovirus for the Treatment of Bladder Cancer Clin. Cancer Res., January 1, 2006; 12(1): 305 - 313. [Abstract] [Full Text] [PDF]
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R. L. Chu, D. E. Post, F. R. Khuri, and E. G. Van Meir Use of Replicating Oncolytic Adenoviruses in Combination Therapy for Cancer Clin. Cancer Res., August 15, 2004; 10(16): 5299 - 5312. [Abstract] [Full Text] [PDF]
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Q. Huang, X. Zhang, H. Wang, B. Yan, J. Kirkpatrick, M. W. Dewhrist, and C.-Y. Li A Novel Conditionally Replicative Adenovirus Vector Targeting Telomerase-Positive Tumor Cells Clin. Cancer Res., February 15, 2004; 10(4): 1439 - 1445. [Abstract] [Full Text] [PDF]
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 L. Barzon, M. Boscaro, and G. Palu Endocrine Aspects of Cancer Gene Therapy Endocr. Rev., February 1, 2004; 25(1): 1 - 44. [Abstract] [Full Text] [PDF]
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P. S. Holm, H. Lage, S. Bergmann, K. Jurchott, G. Glockzin, A. Bernshausen, K. Mantwill, A. Ladhoff, A. Wichert, J. S. Mymryk, et al. Multidrug-resistant Cancer Cells Facilitate E1-independent Adenoviral Replication: Impact for Cancer Gene Therapy Cancer Res., January 1, 2004; 64(1): 322 - 328. [Abstract] [Full Text] [PDF]
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N. A. Lanson Jr., P. L. Friedlander, P. Schwarzenberger, J. K. Kolls, and G. Wang Replication of an Adenoviral Vector Controlled by the Human Telomerase Reverse Transcriptase Promoter Causes Tumor-Selective Tumor Lysis Cancer Res., November 15, 2003; 63(22): 7936 - 7941. [Abstract] [Full Text] [PDF]
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K. Hamada, S. Kohno, M. Iwamoto, H. Yokota, M. Okada, M. Tagawa, S. Hirose, K. Yamasaki, Y. Shirakata, K. Hashimoto, et al. Identification of the Human IAI.3B Promoter Element and Its Use in the Construction of a Replication-selective Adenovirus for Ovarian Cancer Therapy Cancer Res., May 15, 2003; 63(10): 2506 - 2512. [Abstract] [Full Text] [PDF]
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L. M. Wein, J. T. Wu, and D. H. Kirn Validation and Analysis of a Mathematical Model of a Replication-competent Oncolytic Virus for Cancer Treatment: Implications for Virus Design and Delivery Cancer Res., March 15, 2003; 63(6): 1317 - 1324. [Abstract] [Full Text] [PDF]
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 W. Yan, G. Kitzes, F. Dormishian, L. Hawkins, A. Sampson-Johannes, J. Watanabe, J. Holt, V. Lee, T. Dubensky, A. Fattaey, et al. Developing Novel Oncolytic Adenoviruses through Bioselection J. Virol., February 15, 2003; 77(4): 2640 - 2650. [Abstract] [Full Text] [PDF]
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K. Bernt, M. Liang, X. Ye, S. Ni, Z.-Y. Li, S. L. Ye, F. Hu, and A. Lieber A New Type of Adenovirus Vector That Utilizes Homologous Recombination To Achieve Tumor-Specific Replication J. Virol., November 1, 2002; 76(21): 10994 - 11002. [Abstract] [Full Text] [PDF]
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T. Reid, E. Galanis, J. Abbruzzese, D. Sze, L. M. Wein, J. Andrews, B. Randlev, C. Heise, M. Uprichard, M. Hatfield, et al. Hepatic Arterial Infusion of a Replication-selective Oncolytic Adenovirus (dl1520): Phase II Viral, Immunologic, and Clinical Endpoints Cancer Res., November 1, 2002; 62(21): 6070 - 6079. [Abstract] [Full Text] [PDF]
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K. M. Bernt, D. S. Steinwaerder, S. Ni, Z.-Y. Li, S. R. Roffler, and A. Lieber Enzyme-activated Prodrug Therapy Enhances Tumor-specific Replication of Adenovirus Vectors Cancer Res., November 1, 2002; 62(21): 6089 - 6098. [Abstract] [Full Text] [PDF]
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K. Suzuki, R. Alemany, M. Yamamoto, and D. T. Curiel The Presence of the Adenovirus E3 Region Improves the Oncolytic Potency of Conditionally Replicative Adenoviruses Clin. Cancer Res., November 1, 2002; 8(11): 3348 - 3359. [Abstract] [Full Text] [PDF]
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J. Zhang, N. Ramesh, Y. Chen, Y. Li, J. Dilley, P. Working, and D.-C. Yu Identification of Human Uroplakin II Promoter and Its Use in the Construction of CG8840, a Urothelium-specific Adenovirus Variant That Eliminates Established Bladder Tumors in Combination with Docetaxel Cancer Res., July 1, 2002; 62(13): 3743 - 3750. [Abstract] [Full Text] [PDF]
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T. L. DeWeese, H. van der Poel, S. Li, B. Mikhak, R. Drew, M. Goemann, U. Hamper, R. DeJong, N. Detorie, R. Rodriguez, et al. A Phase I Trial of CV706, a Replication-competent, PSA Selective Oncolytic Adenovirus, for the Treatment of Locally Recurrent Prostate Cancer following Radiation Therapy Cancer Res., October 1, 2001; 61(20): 7464 - 7472. [Abstract] [Full Text] [PDF]
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Y. Li, D.-C. Yu, Y. Chen, P. Amin, H. Zhang, N. Nguyen, and D. R. Henderson A Hepatocellular Carcinoma-specific Adenovirus Variant, CV890, Eliminates Distant Human Liver Tumors in Combination with Doxorubicin Cancer Res., September 1, 2001; 61(17): 6428 - 6436. [Abstract] [Full Text] [PDF]
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Y. Chen, T. DeWeese, J. Dilley, Y. Zhang, Y. Li, N. Ramesh, J. Lee, R. Pennathur-Das, J. Radzyminski, J. Wypych, et al. CV706, a Prostate Cancer-specific Adenovirus Variant, in Combination with Radiotherapy Produces Synergistic Antitumor Efficacy without Increasing Toxicity Cancer Res., July 1, 2001; 61(14): 5453 - 5460. [Abstract] [Full Text] [PDF]
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