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Experimental Therapeutics, Preclinical Pharmacology |
Ribozyme Pharmaceuticals, Inc., Boulder, Colorado 80301 [P. A. Pa., K. S. Bo., A. M. G., A. A., K. S. Bl., S. L. G., K. L. J., L. E. A., F. E. W., M. A. R., T. J. P.], and Chiron Corp., Emeryville, California 94608 [P. A. Pi., R. J. T., C. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSKM12L4a Human Colorectal...RESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSKM12L4a Human Colorectal...RESULTSDISCUSSIONREFERENCES |
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Among the many known triggers of tumor angiogenesis, VEGF6 has emerged as a relatively specific effector (2 , 3) . In fact, VEGF expression has been observed in many human tumor types (4, 5, 6, 7, 8, 9, 10) , is up-regulated in response to hypoxia (11 , 12) , and has been specifically linked with tumor neovascularization (13, 14, 15) . Tumor cells engineered to express VEGF constitutively exhibit enhanced tumor growth and angiogenic phenotypes (16) . Conversely, treatments with anti-VEGF monoclonal antibodies have been shown to inhibit the growth of a variety of solid tumors in murine models (8 , 17 , 18) .
Inhibition of VEGF signaling at the receptor level has been proposed as a means of inhibiting VEGF-dependent tumor growth and metastasis (19, 20, 21) . The mitogenic selectivity of VEGF for endothelial cells is due to the relatively exclusive distribution of VEGF receptors in this cell type (2 , 22) . Two major human VEGF receptor subtypes have been identified and are known as Flt-1 (VEGFR-1) and KDR (VEGFR-2 or Flk-1 in the mouse) (23) . Although Flt-1 and KDR/Flk-1 receptors both appear to be involved in angiogenesis, their respective roles in this process are only partially elucidated.
Both Flt-1 and KDR/Flk-1 receptors may play important roles in VEGF-stimulated cell proliferation. For example, the expression of the murine Flk-1 receptor is associated with proliferating endothelial cells (24) , and human endothelial cells expressing the KDR receptor exhibit shape changes and enhanced DNA synthesis on stimulation with VEGF (25) . Human umbilical vein endothelial cells expressing Flt-1 receptor mutants are resistant to VEGF-stimulated proliferation (26) . In addition, Barleon et al. (21) demonstrated that purified VEGF and conditioned media from hypoxic tumor cell lines can up-regulate the expression of Flt-1 but not KDR/Flk-1 receptors in cultured endothelial cells.
Developmental models provide useful insight into the physiological roles of these receptors in vivo. Although mutations in either Flk-1 or Flt-1 are fatal to developing mouse embryos, their respective embryological abnormalities in vasculogenesis and angiogenesis differ (27, 28, 29) . Abnormal Flk-1 expression leads to defective endothelial cell development (28 , 29) , whereas mutant Flt-1 embryos show normally differentiated endothelial cells that form highly abnormal and disorganized vascular structures (27) . This body of evidence suggests that inhibition of VEGF receptor signaling could be an important target for therapeutic intervention in pathological angiogenesis.
Hammerhead ribozymes are trans-acting, RNA-based
enzymes that specifically bind and cleave target RNA. In recent years,
considerable effort has been directed toward the application of
ribozyme technologies to inhibit specific gene expression
(30)
. Stabilization chemistries have been developed to
render these molecules highly resistant to serum nucleases
(31)
and hence suitable for in vivo
applications. As a result, local delivery of chemically stabilized
ribozymes has been reported to inhibit specifically cytokine-stimulated
expression of stromelysin mRNA in a rabbit knee osteoarthritis model
(32)
and VEGF-stimulated angiogenesis in a rat corneal
pocket model (33)
. In addition, a stabilized ribozyme
targeting protein kinase C 
mRNA was shown to reduce sc implanted
glioma tumor growth in rats (34)
.
We reported previously on ribozymes that target specific sites in the mRNA of each of the two major VEGF receptor subtypes, Flt-1 and KDR (33) . Testing in cultured human microvascular endothelial cells was used as a way to choose the most effective anti-Flt-1 and KDR ribozymes to advance into animal studies. These cell culture studies resulted in the choice of one lead ribozyme site in each mRNA target. In particular, ribozymes targeting Flt-1 site 4229 or KDR site 726 were chosen from a panel of ribozymes targeting different sites spanning the length of the target mRNAs. These choices were based on the ability of the ribozyme to inhibit VEGF-stimulated cell proliferation and to decrease specifically their targeted mRNA in cell culture relative to attenuated controls. In subsequent in vivo studies, anti-Flt-1 and KDR ribozymes targeting these specific sites significantly decreased angiogenesis when tested in a corneal pocket model of VEGF-induced angiogenesis. In these two assays, controls that had the same binding arms as the ribozymes but reduced cleavage activity due to nucleotide changes in the catalytic core showed little, if any, effect. Here we report pharmacodynamic studies using systemic delivery of stabilized hammerhead ribozymes designed to inhibit the expression of the human VEGF receptors, Flt-1 or KDR. Each ribozyme is targeted to a conserved sequence in either Flt-1 or KDR mRNA that is also present in the mouse and rat, enabling rodent studies to be conducted with the same agent in these species. The Lewis lung carcinoma model was chosen for initial study because neovascularization in this tumor type is VEGF dependent (35 , 36) , and the highly metastatic line can be used to study the process of metastasis without resection of the primary tumor. In this model, both the anti-Flt-1 and the anti-KDR ribozyme inhibited primary tumor growth when administered iv by continuous infusion. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. In further testing of the anti-Flt-1 ribozyme in a model of human colorectal metastasis, the number of liver metastases was also significantly reduced.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSKM12L4a Human Colorectal...RESULTSDISCUSSIONREFERENCES |
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. The five remaining
unmodified ribonucleotides in the catalytic core are essential for
cleavage activity (31)
. The four phosphorothioate linkages
at the 5'-end and the 3'-3' inverted deoxyabasic residue at the 3'-end
provide additional resistance to exonucleases (31)
. The
attenuated ribozyme controls maintain the binding arm sequence of the
parent ribozyme and thus are still capable of binding the mRNA target.
However, they have two nucleotide changes in the core sequence (Table 1)
that substantially reduce their ability to carry out the cleavage
reaction (Fig. 1)
. Ribozymes were
designed to target Flt-1 or KDR/Flk-1
mRNA sites conserved in human, mouse, and rat.
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Animal Experimentation
All animal experimentation was performed in accordance with the
Guide for the Care and Use of Laboratory Animals (39)
and
the policies and procedures of the Ribozyme Pharmaceuticals, Inc. and
Chiron Corp. animal care and use programs.
LLC-HM
Tumor Inoculations.
Female 6–8-week-old C57BL/6 mice (Harlan Bioproducts, Indianapolis,
IN) were housed in groups of four and inoculated sc in the left flank
with 5 x 105 LLC-HM cells from brei
preparations of tumors exhibiting a highly metastatic phenotype in mice
(six passages; the original LLC-HM cell line was kindly provided by Dr.
Michael O’Reilly; Children’s Hospital and Harvard Medical School,
Boston, MA).
Ribozyme Administrations.
Three days after tumor inoculation, animals were anesthetized with 125
mg/kg ketamine and 8 mg/kg xylazine in sterile veterinary saline and
placed in a supine position on a 37°C warming pad. The left jugular
vein was exposed, and sc fat and fascia were cleared from the vessel.
After tying off the jugular vein distally, a small incision was made in
the jugular vein, and a catheter constructed from PE 50 polyethylene
medical tubing (prefilled with sterile saline) was introduced into the
vessel to approximately 0.5 cm. The vessel was secured to the catheter
proximal to the incision with three ligatures and anchored to the
surrounding muscle tissue using 4-0 silk. The catheter was fed through
a trochar implanted sc, passing from the neck wound dorsally toward the
nuchal region. When the catheter was in place and its volume had been
replaced with test solution, it was attached to an Alzet osmotic
minipump (flow rate, 12 µl/day). Pumps were filled with either
ribozyme, attenuated control, or saline solutions and implanted sc
through a dorsal incision to a final position caudal to the scapulae.
Animals were allowed to recover on 37°C warming pads.
Test substances were dissolved in saline at concentrations ranging from 1.7–167 mg/ml. Beginning 3 days after tumor inoculation, animals were dosed at 0, 1, 3, 10, 30, and 100 mg/kg/day for 14 days. These doses correspond to 0, 3, 9, 30, 90, and 300 mg/m2/day. Ten animals were used for each treatment group. Less than 10% of the 210 animals in the LLC-HM study were removed early, primarily because of catheter failures (i.e., detaching from the vessel or pump), or because the animal did not survive pump implantation. Thus, 7–10 animals remained in each group at the end of the study.
Measurement of Tumor Growth and Metastasis.
In pilot experiments, LLC-HM-injected animals began to become moribund
after about 24 days due to substantial lung tumor burden. To assess
both primary tumor growth and metastatic disease in the same animal,
the experiment was terminated on day 25, before control animals became
moribund. Primary tumor length and width were measured with
microcalipers every other day from postinoculum days 4–24. Tumor
volume was calculated using the following formula:
0.5(length)(width2). Twenty-five days after
inoculation, animals were killed, and tumors and lungs were removed.
The numbers of surface macrometastases on the lung were counted under a
dissecting microscope at a magnification of x25.
Data Analysis.
Group tumor volume means from each treatment group obtained from days
12–24 postinoculation were subjected separately to normality testing
and one-way ANOVA. Treatment group means for ribozymes and their paired
attenuated controls were compared using a Tukey-Kramer post
hoc test for significance ( = 0.05). The mean numbers of
pulmonary metastases from each treatment group (including the saline
control) were subjected to normality testing and one-way ANOVA,
followed by a Dunnett’s test for significance ( = 0.05) using
the saline group as the control.
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KM12L4a Human Colorectal Carcinoma/Liver Metastasis Model |
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The tumor cell inoculation was performed as follows:
(a) animals were anesthetized with 125 mg/kg ketamine and 8
mg/kg xylazine; (b) an abdominal incision of 
3 mm was
made to the left of the midline; and (c) the spleen was
gently exteriorized. The KM12L4a cell suspension was inoculated into
the spleen after slowly inserting a 28-gauge needle into the large pole
of the spleen until the bevel of the syringe became visible in the
central portion of the spleen just beneath the splenic capsule.
Ribozyme Administrations.
Three days after inoculation, animals were surgically implanted with sc
14-day Alzet osmotic minipumps (flow rate, 12 µl/day) filled with
either saline or anti-Flt-1 ribozyme solutions. Animals were
dosed at 0, 12, 36, and 100 mg/kg/day for 14 days. These administered
doses correspond to 0, 36, 108, and 300
mg/m2/day. On day 18, the initial pumps were
removed and replaced with fresh minipumps containing either saline or
the same doses of anti-Flt-1 ribozyme. Animals were observed
daily after each surgery, and healing of the surgical incision sites
was unaffected by any treatment as compared with the saline control
group. The study was ended on day 41.
Fifteen animals were included in each treatment group. If tumor cells successfully seed the liver, metastatic tumor formation in this xenograft model is usually rapid and aggressive; however, a poor tumor take is possible. Animals were randomly distributed before assignment to treatment groups to address the variable outgrowth properties of this model. Several animals were removed from the study as follows: (a) saline group, one animal was killed on day 33 because of the tumor burden; (b) 12 mg/kg/day group, one animal was killed early (day 17) because of continued loss of body weight, and one animal was found dead on day 35; (c) 100 mg/kg/day, one animal did not survive cell implantation, and one animal did not survive pump replacement. Thus, the final sample size for the saline-treated group and the 12, 36, and 100 mg/kg/day dose groups was 14, 13, 15, and 13 mice, respectively.
Measurement of Liver Weight and Liver Metastasis.
On day 41, all surviving animals were killed by
CO2 inhalation. Livers were removed, weighed, and
scored for liver metastases. In cases where the number of tumor foci
was too numerous to count (i.e., >100), a count of 101 was
applied.
Data Analysis.
All statistical testing was performed at the = 0.05 level. Because
the individual numbers of liver metastases in each treatment group were
not normally distributed, group means were compared using the
Kruskal-Wallis nonparametric rank test at 95% confidence limits.
Individual liver weight means were normally distributed. The liver
weight data were subjected to ANOVA followed by Dunnett’s test for
significance. Differences between treatment groups with respect to the
presence or absence of metastases were compared using the
2 test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSKM12L4a Human Colorectal...RESULTSDISCUSSIONREFERENCES |
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) that substantially reduced their ability to carry out the
cleavage reaction. These controls had negligible effect on the end
points measured previously in cell culture and only reduced
angiogenesis at the highest doses tested in the corneal model. Thus,
for direct comparison, similar controls were included in the initial
Lewis lung carcinoma studies reported here.
Although these initial in vivo data were established using
3-bp stem II ribozymes, the 4-bp stem II versions (Table 1)
were used
in the current studies. This decision was based, in part, on the higher
in vitro cleavage activity of the 4-bp stem II ribozymes
compared with that of the 3-bp stem II ribozymes (see below), although
their activities in cell culture (33)
and in the corneal
pocket model are comparable (data not shown).
Fig. 1
shows the results of in vitro catalytic cleavage
assays conducted for the 4-bp stem II anti-Flt-1 and
anti-KDR ribozymes, along with their paired attenuated
controls. The changes in the core resulted in attenuated analogues that
have in vitro cleavage rates that are at least 2 orders of
magnitude lower than those of the active ribozymes. For the
anti-Flt-1 ribozyme and its paired attenuated control, the
first-order rate constants (k1) were
0.464 ± 0.005 min-1 and 0.001 ±
4 x 10-5 min-1,
respectively. For the anti-KDR ribozyme and its paired
control, k1 values were 0.780 ±
0.071 min-1 and 0.0002 ± 2 x
10-4 min-1, respectively.
For comparison, k1 values for the 3-bp
stem II versions of these ribozymes reported previously in the corneal
model study (33)
were lower (0.081 ± 0.007 and
0.434 ± 0.024 min-1 for the
anti-Flt-1 and anti-KDR ribozyme, respectively).
These cleavage data, together with the original cell culture and
corneal model results (33)
, demonstrate that the effect
observed for active ribozymes occurs through a ribozyme-specific
(i.e., cleavage) mechanism.
Effects of Anti-Flt-1 or KDR
Ribozymes on LLC-HM Primary Tumor Growth.
Neovascularization of Lewis lung carcinoma tumors depends on VEGF
(35
, 36)
. A highly metastatic variant of Lewis lung
carcinoma (LLC-HM) was selected in the present study because it allows
for the assessment of primary tumor growth and metastasis without
resection of the primary tumor. Thus, both the antitumor and
antimetastatic effects of the anti-Flt-1 and
anti-KDR ribozymes (together with their respective
attenuated controls) were evaluated in the LLC-HM model after
continuous iv administration. Pilot studies conducted before the
current study clearly demonstrated that the anti-Flt-1 or
the anti-KDR ribozyme did not affect the healing of any
surgical wounds associated with jugular catheterization and/or
implantation of the Alzet minipump in mice (data not shown).
In general, both anti-Flt-1 and anti-KDR
ribozymes inhibited primary tumor growth within the 1–100 mg/kg/day iv
dose range. The effect on primary tumor growth observed immediately
after the dosing period (day 18) is given in Fig. 2
. The anti-Flt-1 ribozyme
(Fig. 2A)
reduced LLC-HM primary tumor growth in a
dose-dependent manner as compared with either saline or its
corresponding attenuated control. At the lowest dose (1 mg/kg/day),
both the anti-Flt-1 ribozyme and its attenuated control
reduced primary tumor growth similarly versus saline
controls (50%; P < 0.001 for both observations).
However, with increasing dose, active anti-Flt-1 ribozyme
reduced primary tumor growth to a greater extent than the attenuated
control (P < 0.001 for 10 and 30 mg/kg/day dose
groups). The attenuated control showed no further tumor growth
inhibition until the highest dose (100 mg/kg/day). The greatest
inhibition of tumor growth by the active anti-Flt-1 ribozyme
(92% as compared with saline; P = 0.0001) and the
largest significant difference between active Flt-1 ribozyme
and its attenuated control (89%; P = 0.0001) occurred
at a dose of 30 mg/kg/day. Mean tumor volumes for the active
anti-Flt-1 ribozyme and attenuated control groups at this
dose were 65.34 ± 37.18 and 585.45 ± 98.67
mm3, respectively, compared with 830.86 ±
82.53 mm3 for the saline control group.
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. At a dose of 1 mg/kg/day, the
anti-KDR ribozyme had no effect on primary tumor growth
versus the saline control. However, at all higher doses
(3–100 mg/kg/day), the anti-KDR ribozyme significantly
inhibited primary tumor growth as compared with the saline control by
about the same degree (68–75%; P < 0.0001 for all
observations). The antitumor dose relationship profile of the
attenuated control was more complicated. Doses of 1 and 3 mg/kg/day
showed significant tumor growth inhibition compared with saline
(54–57%; P = 0.0019 for both observations). In
contrast, higher doses of the attenuated control had no effect on tumor
growth. For the anti-KDR ribozyme, 10 mg/kg/day was the
lowest dose that resulted in significant inhibition of primary tumor
volume (68%; P = 0.0001) where the corresponding
attenuated control had no significant effect (versus
saline). A statistically significant difference between the effects of
the active anti-KDR ribozyme and its attenuated control was
detected at the 100 mg/kg/day dose (72%; P < 0.001).
A more complete view of the integrated difference between the active
ribozymes versus their attenuated controls is provided by
the primary tumor growth characteristics from the entire 24-day growth
period. Overall, the greatest difference between the ribozymes and
their controls, even after dosing was completed, was observed with a
dose of 30 mg/kg/day for the anti-Flt-1 ribozyme and a dose
of 10 mg/kg/day for the anti-KDR ribozyme. Primary tumor
growth curves for these two treatments are shown in Fig. 3
. For the anti-Flt-1 ribozyme
(Fig. 3A)
, the maximum tumor volume reduction compared with
the saline control was 92% immediately after the last day of dosing
(day 18) and 77% at day 24. Moreover, the maximum tumor volume
reduction for the anti-Flt-1 ribozyme compared with its
attenuated control was 89% on day 18 and 65% at day 24. It should be
noted that the reduction observed with the anti-Flt-1
ribozyme from day 18 through day 24 was significant compared with both
the saline and attenuated controls, even though ribozyme treatment was
discontinued on day 17 (P < 0.05).
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the maximal tumor volume reduction compared with saline was 68% on day
18 (P = 0.0001) and 60% on day 24 (P < 0.05). In fact, the reduction in tumor growth at this dose was
statistically significant when compared with saline from days 12–24
(P < 0.05 for all observations). In contrast,
LLC-HM tumor growth was not significantly reduced by treatment
with the anti-KDR attenuated control at this dose, except as
noted on day 22. Even so, the differences in tumor volume reduction
between the active anti-KDR ribozyme and its attenuated
control were statistically different only on a limited number of days,
e.g., day 20 where a 56% difference was noted
(P < 0.05).
Antimetastatic Activity of Anti-Flt-1 and
KDR Ribozymes in LLC-HM Tumor-bearing Mice.
The antimetastatic activities of the anti-Flt-1 and
anti-KDR ribozymes and their attenuated controls were also
determined (Fig. 4)
. For this assessment,
lung macrometastases were counted at the end of the study
(postinoculation day 25 and 8 days after the cessation of treatment).
As shown in Fig. 4A
, the anti-Flt-1 ribozyme reduced the
number of pulmonary metastases with respect to saline controls in a
dose-dependent manner. The greatest antimetastatic activity with
respect to saline controls (78% and 83% inhibition) was observed at
doses of 30 and 100 mg/kg/day anti-Flt-1 ribozyme,
respectively (P = 0.005 for both observations). In
contrast, no antimetastatic activity was observed with the
anti-Flt-1 attenuated control; the data were statistically
indistinguishable from saline controls at all doses and showed no
dose-dependent trend.
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, but not to the same
degree as the anti-Flt-1 ribozyme. For the dose range of
3–100 mg/kg/day, the anti-KDR ribozyme reduced the number
of metastases 50–60%, although this was not significantly
different compared with the saline control group. Interestingly, any
reduction in the number of pulmonary metastases present at the lowest
doses of the attenuated control was not observed at the higher doses.
No dose of the anti-KDR attenuated control resulted in a
statistical reduction of lung metastases (versus saline
control).
Inhibition of Liver Metastasis in a Model of Human Colorectal
Cancer.
Because the anti-Flt-1 ribozyme exhibited a slightly more
robust inhibition of primary tumor growth and also significantly
inhibited lung metastasis in the LLC-HM model, this ribozyme was tested
further in a metastatic model of human colorectal cancer. In this
model, human colorectal cancer cells (KM12L4a) were implanted into the
spleens of mice, and after 5 days, the spleens were removed. Continuous
sc administration of the anti-Flt-1 ribozyme was initiated 3
days after the splenic inoculation and continued for 28 days. At the
end of the study (day 41), livers were weighed, and spontaneous
metastases to the liver were counted. Although iv dosing was used in
the LLC-HM study reported above, subsequent pharmacokinetic studies
with the anti-Flt-1 ribozyme in mice also supported sc
dosing. The anti-Flt-1 ribozyme is 80% bioavailable
after sc administration, and its t1/2
in plasma after a single administration is similar to that seen with iv
dosing (40)
. Thus, a sc route of administration was used
in this xenograft model in an effort to avoid the surgical
complications associated with chronic vascular catheterization in
immunocompromised mice.
The effects on indices of liver metastasis of tumor-bearing animals
after treatment with 12, 36, or 100 mg/kg/day anti-Flt-1
ribozyme are listed in Table 2
and are
shown graphically in Fig. 5
. As seen in
Table 2
, there was no statistical difference in liver weight between
ribozyme-treated and control animals. However, there was a clear
decrease in metastatic incidence at 100 mg/kg/day (P =
0.03) as well as a dose-dependent reduction in the mean number of
spontaneous metastases to the liver (P = 0.03 for the
36 and 100 mg/kg/day doses). Moreover, treatment with the
anti-Flt-1 ribozyme at doses of 36 and 100 mg/kg/day reduced
the median number of liver metastases to 5 and 0, respectively
(P = 0.03 for both observations).
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. Hepatic metastases are visible as
darkened areas in photographs of livers from control animals
treated only with saline (Fig. 6B)
. In contrast, the gross
morphology of the livers treated with 100 mg/kg/day
anti-Flt-1 ribozyme is similar to that of normal livers
(Fig. 6A)
.
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DISCUSSION |
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In supporting cell culture studies (33) , a significant decrease in cell proliferation was demonstrated with the active anti-Flt-1 and anti-KDR ribozymes, but not with their corresponding attenuated controls. In addition, these controls behaved as expected in the corneal model of VEGF-induced angiogenesis in that their effect on decreasing angiogenesis was significant only at the higher doses, if at all. These results suggest a ribozyme-specific (cleavage) mechanism of action.
In the pharmacodynamic studies reported here, the active
anti-Flt-1 and KDR ribozymes exhibited antitumor
activity. Treatment with the anti-Flt-1 ribozyme reduced
LLC-HM primary tumor growth (Fig. 2A)
and the number of lung
metastases (Fig. 4A)
in a dose-dependent manner. Moreover,
the number of spontaneous liver metastases in the colorectal cancer
model was significantly reduced after treatment with the two highest
doses of the anti-Flt-1 ribozyme (Table 2
; Figs. 5
and 6
).
For the anti-KDR ribozyme, all doses except the lowest dose
significantly inhibited LLC-HM primary tumor growth (Fig. 2B)
. In stark contrast to the anti-Flt-1
ribozyme, there was no significant effect on lung metastasis at any
dose of the anti-KDR ribozyme (Fig. 4B)
.
Whereas the anti-Flt-1 attenuated control had minimal
effects on primary tumor growth and no effect on metastasis, some
unexpected effects of the anti-KDR attenuated controls were
noted on primary tumor growth, particularly at the lowest doses tested
(Figs. 2B and 4B)
. For the anti-KDR
attenuated control, the lower doses inhibited primary tumor growth, but
the higher doses showed no inhibition. It is important to note that the
anti-KDR control failed to inhibit cell proliferation in
cultured human endothelial cells (33)
and did not
statistically inhibit angiogenesis in the VEGF-induced rat corneal
model (data not shown). Additional studies would be necessary to
characterize any specific role of this modified oligonucleotide in the
complex processes involved in tumor growth.
The specific roles of each VEGF receptor in VEGF-induced endothelial cell proliferation and angiogenesis are poorly understood. It is clear that the KDR/Flk-1 receptor mediates VEGF-induced endothelial cell proliferation, shape, and motility changes (25 , 41) . KDR/Flk-1 is also involved in embryonic vasculogenesis and angiogenesis (24 , 28 , 29) . However, evidence is now emerging for the participation of the Flt-1 receptor in endothelial cell proliferation and angiogenesis. Barleon et al. (21) demonstrated that VEGF stimulation of Flt-1 receptors in cultured endothelial cells enhances DNA synthesis. In addition, Fong et al. (27) demonstrated that Flt-1 receptor mutants result in abnormalities characterized by normal endothelial cell differentiation but abnormal three-dimensional vessel structure in embryonic blood vessels. Previous results from our own work with anti-Flt-1 and anti-KDR ribozymes demonstrated that both Flt-1 and KDR/Flk-1 receptors participate in endothelial cell proliferation and angiogenesis (33) .
It is clear that VEGF is involved in tumor growth and metastasis. Studies have shown that enhanced VEGF expression in tumors leads to increased tumor growth (16) , whereas therapies that target VEGF itself reduce tumor growth and neovascularization as well as metastasis (8 , 17 , 18 , 42) . Thus, it is reasonable to assume that interfering with VEGF receptor function should also impact primary tumor growth and metastasis. Recently, the inhibition of VEGF receptor function has been demonstrated as an alternative means of cancer therapy. The expression of a tyrosine kinase-deficient Flk-1/KDR mutant resulted in reduced blastoma tumor growth and neovascularization in vivo (43) . Strawn et al. (20) have shown that small molecule Flk-1/KDR antagonists reduce angiogenesis in a chorioallantoic membrane model. Several of these tyrosine kinase inhibitors are currently in clinical trials for a variety of clinical indications. Additionally, Shibuya et al. (19 , 44) presented evidence that the Flt-1 receptor is involved in tumor angiogenesis. The results from the current study reinforce the hypothesis that targeting the VEGF/VEGF receptor system is a promising strategy for targeting solid tumor growth and metastasis by interfering with the process of neovascularization.
In addition to potential roles as therapeutics, ribozymes targeting VEGF receptor mRNA can also be used to determine the relative role(s) of each subtype in signal transduction and on downstream biological effects. The anti-Flt-1 and anti-KDR ribozymes provide unique tools for elucidating the functional roles of VEGF receptors in the angiogenesis, growth, and metastasis of VEGF-expressing tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place,
Boulder, CO 80301. Phone: (303) 449-6500; Fax: (303) 449-6995; E-mail: pavco{at}rpi.com 
2 Present address: Matrix Pharmaceuticals, 34700
Campus Drive, Fremont, CA 94555.
3 Present address: Elan Pharmaceuticals,
Avenue One Letchworth, Hertfordshire, United Kingdom SG62HU.
4 Present address: Incyte Microarrays, 6519
Dumberston Circle, Fremont, CA 94555.
5 Present address: Human Genome Sciences, Inc.,
9410 Key West Avenue, Rockville, MD 20850.
6 The abbreviations used are: VEGF, vascular
endothelial growth factor; LLC-HM, Lewis lung carcinoma-highly
metastatic variant.
Received 12/ 8/99; revised 2/23/00; accepted 2/23/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSKM12L4a Human Colorectal...RESULTSDISCUSSIONREFERENCES |
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ribozyme blocks glioma cell growth. Nat. Biotechnol., 16: 556-561, 1998.[CrossRef][Medline]
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) or the
anti-Flt-1 attenuated control (
) or
(B) the anti-KDR ribozyme (•) or the
anti-KDR attenuated control (
). The dose range is
1–100 mg/kg/day. For reference, the mean tumor volume ± SEM for
the saline-treated group (n = 10 animals) is
indicated by the gray bar in both panels. Animals
received continuous iv administration of ribozyme or attenuated control
for 14 days, beginning on day 3 after tumor inoculation. Thus, these
day 18 data represent tumor volumes as measured 1 day after the
cessation of treatment. Data for each group (n =
7–10 animals) are presented as mean tumor volume ± SEM. *,
P < 0.05 versus saline (Dunnett’s
test for significance); 
, P < 0.05 for active
ribozymes compared to their time- and dose-matched paired attenuated
controls (Tukey-Kramer post hoc test for significance).
Where statistically significant, the percentage of inhibition of tumor
growth versus saline is indicated on the graph. Direct
comparisons to the attenuated controls are as follows: for the
anti-Flt-1 ribozyme (A), doses of 10 and 30 mg/kg/day
resulted in a statistically significant 63% and 89% inhibition of
tumor growth relative to the attenuated control, respectively. For the
anti-KDR ribozyme (B), only the highest
dose (100 mg/kg/day) resulted in a statistically significant inhibition
of tumor growth relative to its paired attenuated control (72%
inhibition).
) is shown on both panels. Animals
received continuous iv administration for the 14-day treatment period
indicated in each panel, beginning on day 3 after tumor inoculation.
Data for each group (n = 9–10 animals) are
presented as mean tumor volume ± SEM. *, P < 0.05 versus saline (Dunnett’s test for
significance); 
