β-carboline alkaloid-enriched extract from the amazonian rain forest tree pao pereira suppresses prostate cancer cells

Abstract

Bark extracts from the Amazonian rain forest tree Geissospermum vellosii (pao pereira), enriched in alpha-carboline alkaloids have significant anticancer activities in certain preclinical models. Because of the predominance of prostate cancer as a cause of cancer-related morbidity and mortality for men of Western countries, we preclinically tested the in vitro and in vivo effects of a pao pereira extract against a prototypical human prostate cancer cell line, LNCaP. When added to cultured LNCaP cells, pao pereira extract significantly suppressed cell growth in a dose-dependent fashion and induced apoptosis. Immunodeficient mice heterotopically xenografted with LNCaP cells were gavaged daily with pao pereira extract or vehicle control over 6 weeks. Tumor growth was suppressed by up to 80% in some groups compared with tumors in vehicle-treated mice. However, we observed a striking U-shaped dose-response curve in which the highest dose tested (50 mg/kg/d) was much less effective in inducing tumor cell apoptosis and in reducing tumor cell proliferation and xenograft growth compared with lower doses (10 or 20 mg/kg/d). Although this study supports the idea that a pao pereira bark extract has activity against human prostate cancer, our in vivo results suggest that its potential effectiveness in prostate cancer treatment may be limited to a narrow dose range.

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ARTICLE
Journal of the Society for Integrative Oncology, Vol 7, No 2 (Spring), 2009: pp 59–65 59
© 2009 BC Decker Inc
ARTICLE
bark extract is enriched for alkaloids of the B-carboline fam-
ily. These types of alkaloids have been shown to be cytotoxic
for cancer cells, and their mechanism of action may involve
the targeting of cyclin-dependent kinases (CDKs).3–5 None
of the previous studies on pao pereira bark extracts involved
human prostate cancer cell lines, so here we report our pre-
clinical studies to test whether a standardized extract of pao
pereira bark might affect the in vitro or in vivo growth of a
prototypic human prostate cancer cell line, LNCaP.
Our focus on prostate cancer derives from the predomi-
nance of this cancer as a health concern for men in Western
countries. Prostate cancer is the most frequently diagnosed
malignancy in males and a leading cause of cancer deaths in
men.6 Given the relatively high frequency with which pros-
tate cancer occurs, prevention offers the most likely means
to reduce the health risk to men posed by the disease. If pao
pereira bark extract has tumor-suppressing activity for pros-
tate cancer without overt toxicity, one can consider the pos-
sibility that it might be used as a preventive agent as a dietary
supplement. Moreover, there is a great need for better thera-
peutic agents to treat advanced (metastatic) prostate cancer.
Although hormone therapy is the standard for men with this
stage of disease, it is mainly a palliative treatment that loses
effectiveness over time. Once prostate cancer progresses to
Numerous chemotherapeutic agents used in the
treatment of cancer were originally derived from
plants. Such agents include the vinka alkaloids, extracted
from the Madagascar periwinkle; taxanes, extracted from
Pacific yew tree bark; etoposide, extracted from the may-
apple plant; and irinotecan and topotecan, extracted from
Camptotheca acuminata. In a similar manner, Beljanski and
Crochet proposed that extracts of the bark of an Amazonian
rain forest tree, Geissospermum vellosii Allemão (familiarly
known as pao pereira), used medicinally by South American
Indian tribes, might have activity against human tumors.1–3
In preliminary investigations, Beljanski and Crochet demon-
strated that pao pereira bark extract suppressed the in vitro
growth of several human cancer cell lines, including ones
derived from melanoma and glioblastoma.1–3 Pao pereira
B-Carboline Alkaloid–Enriched Extract from the
Amazonian Rain Forest Tree Pao Pereira Suppresses
Prostate Cancer Cells
Debra L. Bemis, PhD, Jillian L. Capodice, LAc, MS, Manisha Desai, PhD, Aaron E. Katz, MD, Ralph Buttyan, PhD
Bark extracts from the Amazonian rain forest tree Geissospermum vellosii (pao pereira), enriched in ]-carboline alkaloids have signifi-
cant anticancer activities in certain preclinical models. Because of the predominance of prostate cancer as a cause of cancer-related
morbidity and mortality for men of Western countries, we preclinically tested the in vitro and in vivo effects of a pao pereira extract
against a prototypical human prostate cancer cell line, LNCaP. When added to cultured LNCaP cells, pao pereira extract significantly
suppressed cell growth in a dose-dependent fashion and induced apoptosis. Immunodeficient mice heterotopically xenografted with
LNCaP cells were gavaged daily with pao pereira extract or vehicle control over 6 weeks. Tumor growth was suppressed by up to
80% in some groups compared with tumors in vehicle-treated mice. However, we observed a striking U-shaped dose-response curve
in which the highest dose tested (50 mg/kg/d) was much less effective in inducing tumor cell apoptosis and in reducing tumor cell
proliferation and xenograft growth compared with lower doses (10 or 20 mg/kg/d). Although this study supports the idea that a pao
pereira bark extract has activity against human prostate cancer, our in vivo results suggest that its potential effectiveness in prostate
cancer treatment may be limited to a narrow dose range.
pao pereira, preclinical, prostate cancerKey words:
Debra L. Bemis, Jillian L. Capodice, and Aaron E. Katz: Department of
Urology, College of Physicians and Surgeons, Columbia University Medical
Center, New York, NY; Manisha Desai: Department of Biostatistics, Mailman
School of Public Health, Columbia University Medical Center, New York, NY;
Ralph Buttyan: The Ordway Research Institute, Albany, NY.
Debra L. Bemis, PhD, Reprint requests: Ludwig Institute for Cancer Research,
605 Third Avenue, New York, NY 10158; e-mail: dbemis@licr.org.
DOI 10.2310/7200.2009.0009
059-065_JSIO_2009_0009.indd 59 3/29/09 1:42:55 AM

60 Journal of the Society for Integrative Oncology, Spring 2009, Volume 7, Number 2
condition). WST-1 labeling solution (WST-1 Cell Proliferation
Assay Kit, Roche Diagnostics, Indianapolis, IN) was added
at 24, 48, or 72 hours following the addition of the extract.
Three hours later, formazan product was detected at 420 nm
in a spectrophotometric plate reader (Tecan, Männedorf,
Switzerland). Each experiment was repeated in triplicate.
Cell Cycle Analysis
Cultured LNCaP prostate cancer cells were exposed to
various concentrations of pao pereira extract for 24 hours.
Adherent cells were trypsinized and pooled with the cells
in suspension, centrifuged, and washed twice with ice-
cold phosphate-buffered saline. A fraction of washed cells
was stained with trypan blue and counted. The cells were
adjusted to 1 million cells per milliliter and fixed in a 2:1
ratio (vol/vol) of chilled ethanol overnight before staining
with propidium iodide in the presence of ribonuclease. Cell
cycle distribution was analyzed on a Becton Dickinson Flow
Cytometer (BD Biosciences, San Jose, CA), and 10,000 cells
were analyzed for each experimental condition. Data analy-
sis was performed using the CellQuest cell cycle analysis
software (BD Biosciences).
Generation of Tumor Xenografts and Oral Gavage with
Pao Pereira Extract
LNCaP tumor xenografts were generated in 4- to 5-week-old
male athymic nude mice (nu/nu; Harlan Inc., Indianapolis,
IN). Mice were randomized into four groups of eight each.
Following 3-day acclimation, freshly harvested LNCaP cells
were xenografted subcutaneously over the right flank of the
mice as previously described.8 Oral gavage (0.5 mL) was ini-
tiated 48 hours following xenografting, as follows: (1) vehicle
control (0.12% DMSO); (2) 10 mg/kg pao pereira extract;
(3) 20 mg/kg pao pereira extract; or (4) 50 mg/kg pao pereira
extract. Gavage was performed 6 days per week at the same
time per day for 6 weeks. Tumor volumes were calculated
one to two times per week using caliper measurements
of length, width, and depth [volume = (Pxh) × (h2 + 3a2)/6,
a = (L + W)/4].9 Mice were injected subcutaneously with a
10 mM solution of bromodeoxyuridine (BrdU) at a dose of
15 ML/g body weight 4.5 hours prior to euthanasia. Tumors
were then removed, fixed for 24 hours in 10% formalin solu-
tion, paraffin embedded, and sectioned onto glass slides for
immunohistochemical analyses.
Immunohistochemical Analysis of Tumor Xenografts
Tumor sections were stained for BrdU incorporation using
the In Situ Cell Proliferation Kit II POD (Roche Applied
the hormone-refractory stage, few chemotherapeutic agents
are known to affect patient survival. Recently, it was shown
that a combination of docetaxel, a paclitaxel (Taxol) deriva-
tive, with estramustine does offer some therapeutic advan-
tage for hormone-refractory prostate cancer patients, but the
survival gained is limited to a few months at most.7 Therefore,
there is good reason to consider whether natural agents
that act by mechanisms different from the chemotherapeu-
tics now in use might help prostate cancer patients. In this
regard, we had previously used LNCaP cells to identify the
anti–prostate cancer activity of a B-carboline alkaloid–rich
extract of another medicinal plant, Rauwolfia vomitoria.8 In
contrast to our findings with Rauwolfia extracts, however, we
report herein results showing a striking discrepancy involv-
ing the effectiveness of escalated doses of pao pereira extract
for in vivo tumor growth inhibition. Our results suggest that
the pao pereira extract might differ fundamentally from the
B-carboline-rich extracts of other medicinal plants.
Methods and Materials
Pao Pereira Extracts
All experiments were conducted with a proprietary extract of
pao pereira bark enriched in B-carboline alkaloids (Natural
Source International, Ltd., New York, NY). A single batch
of the extract, which was determined by high-performance
liquid chromatrography analysis to contain 54% B-carboline
alkaloids, was used. The pao pereira extract was dissolved in
6% dimethyl sulfoxide (DMSO) in water and was filtered
through a 0.2 µm membrane. The final concentration of
DMSO following dilution in cell culture was 0.12%. A stock
solution of 6% DMSO was used for vehicle controls.
Cell Culture
The androgen-sensitive human prostate cancer LNCaP cells
were obtained from the American Type Culture Collection
(Manassas, VA) and were grown in RPMI-1640 medium
with L-glutamine (GIBCO Invitrogen Corp., Carlsbad, CA),
supplemented with 10% fetal bovine serum and 100 mg/L
erythromycin (Sigma-Aldrich, St. Louis, MO). The cells were
maintained at 37°C in a humidified atmosphere of 95% air
and 5% CO2.
Cell Growth Assays
LNCaP cells were seeded in 96-well plates at a density of
5,000 cells per well in a final volume of 100 µL. Twenty-four
hours later, the medium was replaced with 100 µL of fresh
medium containing different concentrations of pao pereira
extract (100, 250, and 500 µg/mL) or vehicle (n = 8 for each
059-065_JSIO_2009_0009.indd 60 3/29/09 1:42:55 AM

Bemis et al, Pao Pereira Suppresses Prostate Cancer 61
Sciences, Indianapolis, IN) and were counterstained with
methyl green. TUNEL immunostaining was conducted on
sequential sections using the In Situ Cell Death Detection Kit
POD (Roche Applied Sciences, Indianapolis, IN). Sections
were counterstained with Harris hematoxylin. Slides were
imaged under a light microscope (×200 magnification),
and images were captured using a SPOT insight color digi-
tal camera and analysis software (Diagnostic Instruments).
Positive nuclei were counted to determine the apoptotic and
proliferation indices, as previously described.8
Statistical Evaluation of Data
Statistical methods used to analyze the data consisted of the
Student t-test, analysis of variance (ANOVA) techniques,
and the Kruskal-Wallis test. All tests were two-sided and
conducted at the .05 level of significance.
For the in vitro studies, the outcomes of interest were cell
growth, cell cycle progression, and cell death. The Student
t-test was used to determine if the pao pereira extract induced
significant effects on these outcomes in comparison with
the control conditions. For the in vivo study, outcomes of
interest were rate of change in log tumor volume over time,
change in final log tumor volume from baseline, baseline
and final tumor volume, and percent positive staining nuclei
per total cells for the BrdU and TUNEL assays. Owing to the
skewed nature of some of the tumor volume–related out-
comes, the Kruskal-Wallis test was applied to assess differ-
ences among the dose groups where appropriate. Log tumor
volume measurements over time for the different treatment
groups were also presented graphically. Differences in per-
cent positive staining nuclei per total cells between control
and pao pereira–treated mice for both BrdU and TUNEL
assays were assessed using a Student t-test.
Results
Effects of Pao Pereira Extract on LNCaP Cell Growth
and Apoptosis In Vitro
In vitro growth of LNCaP cells was significantly reduced fol-
lowing treatment with 100 or 500 µg/mL pao pereira extract
(p < .001, Student t-test) compared with vehicle-treated cells
(Figure 1). There was a striking dose-dependent difference in
growth inhibition between the 100 and 500 µg/mL doses, with
a greater than 90% suppression of growth at the 500 µg/mL
dose. Fluorescence-activated cell sorter (FACS) analysis
showed that there was no significant effect on the overall
cell cycle distribution of cells treated at 100 µg/mL; how-
ever, at 500 µg/mL, there was a significant reduction in the
percentage of cells in the S and G2/M phases of the cell cycle
and increased accumulation in the sub-G1/G0 population
(cells containing subgenomic levels of deoxyribonucleic acid
[DNA]) (Figure 2). The increased number of cells in sub-
G1/G0 indicated that the 500 µg/mL dose induced cell death that
was not observed at 100 µg/mL. To determine if the cell death
was the consequence of apoptosis, protein lysates from pao
pereira–treated (500 µg/mL) or control cells were analyzed for
the presence of cleaved poly (adenosine diphosphate [ADP]-
ribose) polymerase (PARP) by Western blot analysis. Extensive
PARP cleavage was observed in cells treated for 24 hours with
pao pereira but not in control cells (data not shown).
In Vivo Effects of Pao Pereira on LNCaP Xenograft
Volume and Tumor Cell Proliferation or Apoptosis
To determine if the pao pereira extract could suppress the
in vivo growth of LNCaP tumor xenografts, male immuno-
deficient mice were subcutaneously xenografted with 1 × 106
LNCaP cells and the mice were randomly divided into groups,
80
100
Percent of Control
0
20
40
60
0 24 48 72
*
*
Time (hr)
100 µg/mL
500 µg/mL
1.Figure Pao pereira extract suppresses in vitro cell growth
in the human prostate cancer cell line LNCaP. *p < .001,
Student t-test.
70
80
20
30
40
50
60 Control
100 µg/mL Pao
500 µg/mL Pao
0
10
Sub GO G1 S G2/M
**
% of Total Cell Population
Cell Cycle Phase
2.Figure Analysis of LNCaP cell cycle progression following
24-hour treatment with pao pereira extract. *p < .01, Student
t-test.
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62 Journal of the Society for Integrative Oncology, Spring 2009, Volume 7, Number 2
each of which was gavaged daily with vehicle or with 10, 20,
or 50 mg/kg/d of the pao pereira extract. The extract did not
appear to affect the overall health of the mice as no signifi-
cant difference in weight was observed over the study period
across the treatment or control groups (ANOVA-based
F-test, p = .3852) (Figure 3). However, the tumor volumes
at the first measurement were already significantly lower
in the 10 and 20 mg/kg treated groups (Figure 4; Kruskal-
Wallis test, p = .025). Also, by the end of the experiment,
median tumor volumes in the 10 and 20 mg/kg treatment
groups were decreased by 80% and 75%, respectively, com-
pared with the vehicle control group (Kruskal-Wallis test,
p = .021). The tumor-suppressing effect of the pao pereira
extract was apparently lost at the highest dose (50 mg/kg/d)
as the distribution of tumor volume at both baseline and the
end of the study in this group was not statistically different
from that of vehicle-treated controls.
Change in tumor volume over time was evaluated by
considering both the rate of change in log tumor volume and
the difference in final log tumor volume from baseline (see
Figure 4B). There was no evidence of differences in tumor
volume behavior over time across the dosing groups for
these two outcomes (p = .792 and p = .682, respectively).
These results then suggest that the 10 mg/kg and 20 mg/kg
doses slowed the ability of the tumor cells to establish them-
selves and grow within the subcutaneous environment of the
mice compared with the control or the 50 mg/kg treatment
group.
The effect of the extract on tumor cell proliferation was
analyzed by immunohistochemical detection of BrdU incor-
poration into tumor cell nuclei. A quantitative assessment of
35
*
15
20
25
30
Mouse Weight (g)
0
5
10
0 10 20 30 40
Time (days)
0
10 mg/kg
20 mg/kg
50 mg/kg
3.Figure Average mouse weights of the pao pereira treatment
and control groups. *p = .3852, ANOVA-based F-test.
30
40
Tumor Volume (mm3)
0
20
10
Dose Group (pao pereira mg/kg)
0 10 20
A
50
4.Figure Effect of pao pereira extract (10, 20, and 50 mg/kg) on
LNCaP tumor xenograft growth in immunodeficient mice A, Box
plot of baseline tumor volumes for control and pao pereira treat-
ment groups. A statistically significant difference was observed
across all dose groups (p = .025, Kruskal-Wallis test). B, Overall
tumor volume in mouse xenografts was reduced in 10 mg/kg and
20 mg/kg pao pereira–treated mice. *p = .005, ANOVA (median
log tumor volume).
B
Control
50 mg/kg
* 10 mg/kg
Time (days following treatment initiation)
* 20 mg/kg
7
0
2
4
15 23 32 41
Log Tumor Volume
6
the average number of BrdU-positive nuclei revealed that
all treatment groups induced statistically significant reduc-
tions in overall tumor cell BrdU incorporation, as shown
in Figure 5 (Student t-test, p < .001). The 10 mg/kg and
20 mg/kg extract reduced tumor cell proliferation by 52%
and 73%, respectively. Although the 50 mg/kg dose inhib-
ited tumor cell proliferation by 46% compared with the
control group (Student t-test, p < .001), the effect was
not different from that caused by the 10 mg/kg dose, sup-
portive of a biphasic dose response. Additionally, tumor
sections from all mice were analyzed by TUNEL staining
to detect cells undergoing apoptosis (Figure 6A). As illus-
trated in Figure 6B, TUNEL-positive nuclei were signifi-
cantly increased by 1.6-fold and 2.6-fold in the 10 mg/kg
and 20 mg/kg groups, respectively, compared with con-
trol levels (Student t-test, p < .05). The 50 mg/kg dose
did not induce tumor cell apoptosis over control levels
(p > .05). Again, a trend toward a biphasic dose response
059-065_JSIO_2009_0009.indd 62 3/29/09 1:42:58 AM

Bemis et al, Pao Pereira Suppresses Prostate Cancer 63
in vitro and in vivo, and it supports the idea that other plants
rich in B-carbolines might also be natural sources of prostate
cancer preventive or therapeutic agents.
Although we do not understand the mechanism(s)
through which the pao pereira extract affects prostate can-
cer cell growth and survival, we presume that the effects
of this agent might be related to the B-carboline alkaloids.
B-Carboline alkaloids suppress CDK activity5 that is needed
to drive normal and cancerous cells through the cell cycle,
but they also bind to DNA and may cause other cellular
anomalies specific to cancer cells that suppress cell division
or induce cell death. In our previous work with R. vomitoria
extract, we used a targeted microarray analysis approach to
evaluate the effects of the extract on gene expression patterns
in LNCaP cells, especially on genes involved in DNA dam-
age repair.8 We reported that numerous genes involved in
DNA damage and repair were modulated by this extract. We
did perform a similar but preliminary (unvalidated) analysis
using the same DNA damage and repair microarray to com-
pare gene expression in pao pereira–treated (100 µg/mL)
LNCaP cells with that in control (vehicle treated) cells, but
much fewer and different genes were affected in this path-
way compared with treatment with the Rauwolfia extract.
In our assay using pao pereira–treated cell complementary
DNA, we observed particularly strong upregulation of the
DNA repair response genes BRCA1 (24.67-fold) and DDIT3
was evident, suggesting that the 50 mg/kg pao pereira was
beyond the optimal dose required to induce maximal tumor
cell apoptosis.
Discussion
Prostate cancer is a problematic human disease whose bur-
den to health in the United States could be decreased by a
suitable preventive agent or treatment, especially one that
might be used as a nutritional supplement. Here we tested
whether a B-carboline-rich extract of pao pereira bark might
have any efficacy in suppressing in vitro or in vivo growth of
a prototypic human prostate cancer cell line, LNCaP. Our
results showed that this extract, when added to the medium of
cultured LNCaP cells, suppressed their growth and induced
apoptosis in a dose-dependent manner. Furthermore, when
pao pereira extract was gavaged into mice that had been
xenografted with LNCaP cells, tumor size was significantly
smaller in two of three groups of treated mice compared with
the tumor size in vehicle-treated mice over the experimen-
tal period. To some extent, this response was similar to our
previous finding that a B-carboline-rich extract of another
medicinal plant, R. vomitoria, also suppressed LNCaP growth
A
B
10 mg/kg 20 mg/kg 50 mg/kg
Pao Pereira Extract (50 mg/kg)
% TUNEL Positive/Field (Avg.)
*
*
00 10 20 30
5
10
15
6.Figure Six-week oral dosing of pao pereira extract–induced
apoptosis in LNCaP tumor xenografts. A, Cells undergoing apop-
tosis stained brown (TUNEL staining; ×200 magnification). B,
Quantification of apoptotic cells in LCNaP tumor xenografts from
each treatment group. Error bars indicate ± SEM; *p < .05, Student
t-test.
Control 50 mg/kg
*
*
*
Pao Pereira Extract (mg/kg)
% Brdu Positive/Field (Avg.)
0
0
2
4
6
8
10
10 20 50
B
A
5.Figure Inhibition of proliferation in LNCaP tumor xeno-
grafts following 6-week gavage of pao pereira extract. A, Cells
stained dark blue represent proliferating cells (BrdU incorpo-
ration; ×200 magnification). B, Quantification of proliferating
cells in LNCaP tumor xenografts. Errors bars indicate ± SEM;
*p < .05, Student t-test.
059-065_JSIO_2009_0009.indd 63 3/29/09 1:43:00 AM

64 Journal of the Society for Integrative Oncology, Spring 2009, Volume 7, Number 2
agent(s) present and that the tumor cell–protective effect is
manifest at the higher doses of the extract.
According to the Mertz model, a U-shaped dose-response
relationship exists between essential nutrients and their bio-
logic impact; therefore, the optimal concentration of these
agents required to achieve their desired biologic effects is in
the midrange of the dose-response curve.10 Several studies
have reported that this relationship also applies to anticancer
nutritional agents,11–13 and our data using pao pereira extract
also appear to validate this idea. Regardless, our data dem-
onstrate the anti–prostate cancer activities of the pao pereira
extract in both in vitro and in vivo prostate cancer cell model
systems, and this work indicates the need for continued
exploration into its use in the prevention and/or treatment
of prostate cancer.
Acknowledgments
Financial disclosure of authors: This research was supported
by The Center for Holistic Urology at Columbia University
Medical Center, The Charles Royce Foundation, and Natural
Source International, Ltd.
Financial disclosure of reviewers: None reported.
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unusual treatment effect. All groups were gavaged daily at
the same times by the same investigators, and the doses were
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organ systems, unlike single cells in cell culture. Finally, we
must consider the possibility that the complex nature of pao
pereira extract allows for the potential presence of ingre-
dients that oppose the actions of any cancer-suppressing
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Bemis et al, Pao Pereira Suppresses Prostate Cancer 65
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