NUTRITION AND CANCER, 58(2), 188196

C 2007, Lawrence Erlbaum Associates, Inc.

Copyright

Evaluation of the Antiproliferative Effects of EssiacTM on In Vitro

and In Vivo Models of Prostate Cancer Compared to Paclitaxel
Andy Eberding, Candice Madera, Sherwin Xie, Catherine A. Wood, Paula N. Brown,
and Emma S. Guns
Abstract: EssiacTM , a widely consumed, sparsely tested
herbal tea, was evaluated for preparation consistency
and antiproliferative effects on prostate cancer cells
and xenografts. High performance liquid chromatography
(HPLC) was used to compare different lots of Essiac and
evaluate extraction consistency by comparing peak areas
in concentrated preparations. Repeated analysis of one lot
showed <2% RSD between corresponding peaks. Absolute
peak areas varied widely between lots, but similarity in relative size of corresponding peaks was observed. Cytotoxic
effects of Essiac were tested in vitro by crystal violet assay
and analysis of cell cycle distribution by flow cytometry, but
no differences between control and treatment groups was observed. Paclitaxel was used as a positive control in cell cycle
analysis and was the only treatment which showed significant effects on cell cycle distribution. Toxicity in nude mice
was tested, and efficacy in inhibiting PC-3 xenograft growth.
No toxicity or tumour size difference was observed dosing
up to 240 mg/kg QD, over 28 days , excepting the positive
control group treated with paclitaxel. Ki-67 and PCNA expression was analyzed in treated tumors, but no difference in
expression of either marker was observed. These evaluations
suggest Essiac has no marked antiproliferative effect on the
models tested.

Introduction
Prostate cancer is one of the most prevalent new cancer
diagnoses in men (1). Epidemiological studies of prostate
cancer have revealed that both genetic and environmental
factors contribute to the incidence of the disease (1,2). Often
patients attempt to inhibit the progression of their disease
by altering their diet-for instance, adjusting their intake of
vitamins, minerals, red meat, or vegetables. Another practice

common among patients is the introduction of complementary or alternative medicines (CAM) into their daily regimens
(3). Despite the known popularity of CAM treatments with
prostate cancer patients, there is a lack of rigorous scientific
research proving their efficacy.
Among the many CAM treatments currently in
widespread use is the botanical formulation EssiacTM. A survey of cancer patients with advanced malignancies found
that of patients who used CAM, 9.5% of these used Essiac
(4). Essiac is a proprietary mixture of four herbs: Burdock
root (Arctium lappa), Indian Rhubarb root (Rheum Palmatum), Slippery Elm bark (Ulmus fulva or Ulmus rubra), and
Sheep Sorrel (Rumex acetosella). Many active compounds
have been previously identified in the component plants of the
mixture. Burdock contains several flavonesnamely, genistein, biochanin A, nobiletin, and tangeretinthat possess
an ability to initiate apoptosis in stomach cancer cells in
vivo. (5). Burdock also contains arctegenin, which has been
shown to slow progression of M1 mouse leukemia cells (6).
Rhubarb extracts used to treat sarcoma 37 cells in vivo led
to increased tumor necrosis (7,8). Emodin, an anthraquinone
found in Rhubarb and Sheep Sorrel, has demonstrated mutagenic and cytotoxic properties in breast FM3A and C3H cells
(9). Emodin has also been shown to inhibit in vivo mammary
and Ehrlich-ascites carcinomas (10).
This blend has a long history, first being formulated in
1922. The Canadian Breast Cancer Research Initiative compiled the most complete review of the modest research that
has been conducted on Essiac (11). This review concluded
that Essiac shows Weak evidence of effectiveness. Little evidence of harm. This is a widely used agent which has been
incompletely studied. Health Canada issued a disparaging
statement in 1989 entitled EssiacTM : An ineffective cancer treatment. This review was based on the results of a
clinical study that Health Canada authorized in 1978 (12).
Other authors, however, countered this finding, contending
that the information on which the statement was based was

A. Eberding, C. Madera, S. Xie, C. A. Wood, and E. S. Guns are affiliated with the Prostate Centre at Vancouver General Hospital, Vancouver, British
Columbia, Canada V6H 3Z6. E. S. Guns is also affiliated with the University of British Columbia, Faculty of Medicine, Department of Urologic Sciences,
Division of Urology, Vancouver, British Columbia, Canada V5Z 1M9. P. N. Brown is affiliated with the British Columbia Institute of Technology, Technology
Centre & School of Health Sciences, Natural Health Products Research Group, Burnaby, British Columbia, Canada, V5G 3H2.

incomplete, as the study was cancelled prior to completion.

Despite these results, cancer patients continue to consume
the product.
After a long period of seeming disinterest by the research
community, there has recently been an increase in the number
of publications pertaining to Essiac. Positive data has been
reported relating to Essiacs antioxidant and free radical scavenging abilities (13,14). It has also been reported that Essiac
reduced proliferation in prostate cancer cells metastasized to
a lymph node (LNCaP) and Chinese hamster ovary (CHO)
cells, with a greater degree of inhibition in LNCaP cells,
and did not reduce proliferation in nontransformed cells(15).
However, in vitro studies using breast cancer cell lines indicate that Essiac actually stimulates human breast cancer
cell growth in both estrogen receptor positive and negative
cell lines (16). Recently, another publication reported a single case study describing an apparent remission of hormone
refractory prostate cancer in a 64-yr-old man which was ascribed to Essiac use (17). The experiments described herein
were carried out to assess the preclinical efficacy of the blend
to inhibit the growth of prostate cancer in models used to
study the disease.
Patients with advanced disease have been reported to be
more likely to use CAM (18), so to more accurately recreate
the conditions in which Essiac would be taken, the androgen
independent PC-3 model was chosen as a more representative model of late stage prostate cancer(19). This cell line
was derived from a bone metastasis of an androgen independent prostate cancer (19). Also, because herbal preparations
are complex mixtures and may exhibit variation between
production lots, a high-performance liquid chromatography
(HPLC) validation of the aqueous extract was performed to
ensure consistency between lots.

Materials and Methods

Chemicals and Reagents
Essiac herbal powder (Lots 17694, 17903, and 18146)
was provided as a donation from Essiac Canada International (Ottawa, Ontario, Canada). This product is available
on the Internet, and all lots were available worldwide. Micellar paclitaxel used in animal studies was provided as a kind
donation from Dr Helen Burt, Faculty of Pharmaceutical
Sciences, University of British Canada (Vancouver, British
Columbia, Canada). Ki-67 antibodies were obtained from
DAKO Cytomation (Mississauga, Ontario, Canada). Proliferating cell nuclear antigen (PCNA) antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).

in the barrier containment unit at the Jack Bell Research

Centre for the duration of the study.
Essiac Preparation
Essiac extraction was performed as per manufacturers
directions. In short, Essiac was added to water, covered and
boiled, then simmered for 10 min. The mixture was cooled
for 4 h before being boiled again and then simmered for another 5 min. The Essiac was then removed from heat and
refrigerated for 16 h before decanting. Normal dose ratio
is 1.5 oz (43 g) Essiac in 88 oz (2.5 l) water (17.2 g/l),
although smaller volumes were prepared for dosing. Animals were dosed based on the manufacturers recommendation for humans converted to an approximate mouse dosage
based on the body mass differential. The recommended human dose will be considered a 1X dose. Increasing amounts
of Essiac were added to fixed volumes of water to produce
more concentrated extracts. The manufacturers suggested
dose (recommended human dose) was prepared as well as 3
more concentrated extracts: 2X, 5X, and 10X. All samples
were refrigerated throughout the study as recommended by
the manufacturer. This method was utilized to mimic consumer use as closely as possible and to compensate for any
changes that may occur in the extract during storage time,
which might not be represented by freshly prepared samples
at each time point. HPLC analysis required the evaluation of
all available commercial lots of Essiac: 17694, 17903, and
18146. Lot 17903 was used exclusively throughout the rest
of the study including all animal studies.

HPLC Analysis
Triplicate samples were prepared for each sample. Each
sample was centrifuged to remove particulate and filtered (40
m). The HPLC was fitted with a Zorbax SB-C18 column
(4.6 150 mm, 5 m) maintained at 25 C. HPLC conditions
were as follows: Solvent A, 1% formic acid in water and Solvent B, acetonitrile. Gradient started at 100% A, decreasing
to 79% over 2.5 min, holding for 5.5 min (8 min, then ramping down to 65% for 13 min (21 min). Gradient continued to
0% A over 2 min (23 min), holding for 2 minutes (25 min),
and then returning to 100% A over the last 2 min of the 27min run. Flow rate was 0.75 ml/min, and injection volume
was 5 l of sample. Detection of peaks was by absorbance
at 330 nm.

Cytotoxicity Analysis of Essiac With Crystal

Violet Assay
Animals
Male athymic nude mice were purchased from Harlan
Sprague Dawley, Inc. (Indianapolis, IN) at 68 wk of age
with a body weight of 2025 g. These animals were housed
Vol. 58, No. 2

Cultured prostate cancer cells metastasized to a lymph

node (LNCaP) or PC-3 cells were plated in 80 l media [RPMI 1640 and Dulbeccos Modified Eagles Medium
(DMEM), respectively] with 5% fetal bovine serum (FBS)
per well (96-well plate) at 5 104 cells/ml. After 24 h, the
189

Table 1. Immunohistochemical Staining of Ki-67 and PCNA in PC-3 Control and Treated Tumorsa
Animal No.

Ki-67
n (cores)
Average
Control
SD
100 mg/kg bid
n (cores)
Average
SD
PCNA
n (cores)
Average
Control
SD
100 mg/kg bid
n (cores)
Average
SD

7
37.0

5
39.7

7
54.8

5
54.8

5
43.5

4
38.5

6
44.5

7.0

9.5

7.0

7.0

10.2

12.9

10.8

4
37.1
12.0

6
41.1
9.2

7
37.0
10.5

4
57.1
3.9

5
59.0
12.8

5
52.6
7.8

7
2.4

6
5.4

5
10.1

4
5.5

7
4.9

7
9.0

6
7.1

1.3

4.0

5.1

3.3

2.7

5.4

4.7

5
1.5
0.6

6
3.7
2.9

5
8.7
1.6

4
15.7
3.2

6
4.7
2.8

6
7.9
3.5

5
49.0
13.9

5
11.3
2.6

Overall

7
46.8
12.9

7
8.1
5.2

positive immunohistochemical staining of control and EssiacTM treated PC-3 tumors with Ki-67 and proliferating cell nuclear antigen antibodies.
The values shown reflect the percentage of positively stained cells. The animal number refers to the identifier of the individual mouse from which the tumor
was harvested. Although sections were taken from 11 tumors, many lacked sufficient tissue to allow quantification of the proliferation markers of interest. The
number of cores used to determine the average staining levels has been indicated. The overall averages of the treated and control sections have no statistically
significant difference (P < 0.05) determined using a Student t-test.

a Percent

cells were treated in octuplicate with 20 l Essiac extract

diluted in media with 5% FBS and 25% deionized water
(dH2 O) to final concentrations of 0, 6, 60, and 600 g/ml of
Essiac (concentration indicates the amount of dry material
initially extracted in a volume of water). The cells were incubated at 37 C in 5% CO2 for 48 h and then fixed with 25
l of 4% glutaraldehyde in dH2 O. The media was aspirated
and the cells rinsed with 200 l dH2 O and allowed to air dry.
Cells were stained with 100 l of 0.5% crystal violet solution
(0.5 g crystal violet dissolved in 100 ml of 20% ethanol) for
5 min. Excess crystal violet solution was aspirated and the
cells rinsed with 200 l dH2 O, aspirated, and air dried. The
fixed dye was dissolved in 100 l of Sorensens solution (9
mg trisodium citrate, 195 ml 0.1 N HCl, 500 ml 90% ethanol,
and 305 ml dH2 O) added to each well and incubated at room
temperature for 5 min. The absorbance at 592 nm (A592)
was recorded. The recorded absorbance for each treatment
group was normalized by subtracting the average absorbance
for the blank wells. The resulting average relative absorbance
for the dH2 O treated controls was then used to calculate the
percentage of cytotoxicity resulting from each treatment.

media with 5% FBS. The cells were incubated at 37 C in

5% CO2 to allow adhesion to plates. After 24 h, media and
nonadherent cells were aspirated from the plates. Prepared
Essiac was determined to have an initial concentration of 17
mg Essiac per ml of water, which was then diluted for cell
treatment. Cells were treated with 0, 6, 60, and 600 g/ml
Essiac or 7 M paclitaxel in DMEM with 5% FBS, 5%
dH2 O, and 0.1% dimethylsulfoxide. After a 48-hr incubation
at 37 C in 5% CO2, media was collected and replaced
with 10 mM ethylenediamine tetraacetic acid (EDTA),
20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), 0.1% bovine serum albumin at pH 7.4 in
phosphate buffered saline (PBS). The cells were incubated
for 5 min at 37 C in 5% CO2 and gently rinsed off the
plate. The cells were pelleted with the previously collected
media and 35 l 100 mM CaCl2 , 100 mM MgCl2 . Each
pellet was rinsed with 1 ml 0.1% glucose in PBS. Cells
were then pelleted and resuspended in propidium iodide
staining solution (10 l10 mg/ml RNase A, 5 l10 mg/ml
propidium iodide per 1ml of PBS, 0.1% glucose). After 30
min, the cells were analyzed using a Coulter Epics XL-MCL
flow cytometer (Beckman Coulter, Inc., Fullerton, CA).

Cell Cycle Analysis

Toxicity in Athymic Nude Mice

Cells were prepared for analysis by plating 5 105 PC-3

cells onto 100 mm diameter tissue culture plates in DMEM

A total of 8 groups of 3 male athymic nude mice were administered Essiac at doses of 24, 48, 120, and 240 mg/kg daily

190

Nutrition and Cancer 2007

by oral gavage using the 1X, 2X, 5X, and 10X extracts. Four
groups were dosed once daily and 4 groups were dosed at 12h intervals with half the daily dose. The control group was
administered vehicle only (water). Formulations were prepared such that a 30 g mouse would be dosed with 100 l to
receive the correct dosage. The mice were dosed for 28 days.
Effect of Essiac on Tumor Growth in Athymic
Nude Mice
Sixty athymic nude mice with 2 106 PC-3 cells. The
mice were divided into 5 treatment groups (100 mg/kg bid
orally, 200 mg/kg qd orally, 10 mg/kg bid orally, micellular
paclitaxel intravenous and control). After 14 days, weight
and tumor sizes were recorded, and treatment was initiated.
For each treatment group, tumors were measured linearly
in 3 dimensions semiweekly. All tumor volumes specified
represent total tumor burden. On euthanization, all tumors
were fixed by immediate immersion in 10% formalin for 24
h followed by long-term storage in 80% ethanol for studying
proliferation markers in tissue micro arrays.
Tissue Micro Array
Seven 0.6 mm diameter core samples from each paraffin embedded tumor were transferred to a recipient paraffin
block into two 7 11 grids of tissue core samples (the 100
mg/kg bid treated samples and the control tumors from the
efficacy study). Sections of the block were cut to a thickness
of 4 m and mounted on glass microscope slides.
Immunohistochemical Staining and Analysis
Tissue micro array sections were deparaffinized in xylene
and rehydrated with graded ethanol. Slides were steamed in
citrate buffer for 30 min. The sections were cooled to room
temperature and washed 3 times with PBS before blocking
with 3% bovine serum albumin (BSA) to reduce background
staining. Prior to staining, 3% hydrogen peroxide in PBS
was applied. The tissue micro array sections were incubated
overnight at room temperature in either Ki-67 antibodies
(DAKO Cytomation, Mississauga, Ontario, Canada) diluted
at 1:100 in 1% BSA solution or PCNA antibodies (Santa
Cruz Biotechnology, Santa Cruz, CA) diluted with 1% BSA
solution at 1:6400. Biotinylated secondary antibody (DAKO
Cytomation) and streptavidin-peroxidase conjugate (DAKO
Cytomation) incubations followed. The section was then
stained with Nova Red (Vector Laboratories, Burlingame,
CA) and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA).
Each core section was digitally photographed using
an Olympus BX51 microscope (Olympus Canada, Inc.,
Markham, ON, Canada) coupled with a Diagnostics Instruments RT 2.2.1 color digital camera (Diagnostic Instruments,
Inc., St. Sterling Heights, MI) controlled by SPOT software,
version 3.5.5. The extent of positive staining was then analyzed using Image Pro PLUS software, version 4.5.1.22
Vol. 58, No. 2

(Media Cybernetics, Inc., Silver Springs, MD). The number

of Ki-67 positively stained cells and the total number of cells
was counted in each core section. The result was expressed as
the average percentage of Ki-67 positively stained cells per
core section. The extent of PCNA positive staining was analyzed using Image Pro PLUS software, version 4.5.1.22. The
percentage area of PCNA positive staining was determined
for each core section.
Statistical Analysis
All statistical analyses were performed using Sigma Stat
for Windows, version 2.03 (SPSS, Inc., Chicago, IL). Cytotoxicity assays and proliferation marker expression levels
were assessed using a student t-test with the P value set at
0.05. The results are expressed as means SD. The comparison of the treatment groups in the in vivo efficacy study
was made using a 2-way analysis of variance (ANOVA). A
post hoc analysis using a Tukey test was used to indicate
which group was significantly different. A 1-way ANOVA
was performed on the cell cycle data followed by an analysis
of the groups using 2 post hoc tests, the Tukey test, and the
Student-Newman-Keuls test. The P value was set at 0.05 for
all tests, and the results are expressed as means SD.
Results
Essiac Extract Within-Lot and Between-Lot Variability
Operating conditions were optimized for the development
of the method including flow rate, column temperature, solvent system, and gradient. The separation method consisted
of an acetonitrile and acidified water gradient as indicated
in Materials and Methods. The final method provided a
large number of peaks with sufficient resolution to allow for
semiqualitative analysis (Fig. 1).
Twenty-three peaks (A-W) were quantified in each lot
tested. These peak areas were compared, and the average
value and SD determined for each. The variation in peak
areas within lots is predictably small, typically >2% relative SD (RSD); however, 1 peak (Peak V) varied between
preparations of the same lot, producing RSDs near 25%. Homogeneity between lots was determined by calculating the
mean percent variation in peak area between all lots. The average percent variation was calculated to be 16.7%, ranging
from 5.0% to 29.3%. This calculation of SD relative to the
average of all values for each peak (9 in all, 3 replicates of 3
lots) resulted in the percent variation for each peak. The percent variations of all peaks were then averaged to determine
mean percent variation. Using the same calculation applied
to peak areas relative to total peak area, the mean percent
variation was determined to be 10.3%, ranging from 3.3% to
35.7%, which indicates that plant sources were similar. The
objective of the final HPLC analysis was to ensure that an
increase in the quantity of material extracted would actually
produce comparable increases in corresponding peak areas.
In calculating the ratio of each peak area in the concentrated
preparations to the corresponding 1X peak areas, peak areas
191

Figure 1. A sample high-performance liquid chromatography (HPLC) chromatograph of EssiacTM extract acquired using the method developed specifically
for this purpose and outlined in Materials and Methods. The chromatograph has been labeled to identify the specific peaks that were quantified for the
HPLC-based consistency analysis. The specific chromatograph shown was acquired from a run of Lot 17903-60 mg/ml, measuring absorbance at 330 nm.

in the 2X preparation were determined to be an average of 1.9

times greater than the corresponding 1X preparation peak areas. Likewise, the peaks in the 5X and 10X preparations were
on average 4.2 and 8.3 times greater than the corresponding
1X preparation peaks, respectively. Relative abundance of
each individual peak was expressed as a proportion of the
total area under the curve (AUC) for each concentration. The
deviations in these ratios were small across the entire set
of peaks analyzed with the exception of Peak V, which was
substantially lower at 3.0 and 6.1 times the area of the 1X
preparation in the 5X and 10X preparations, respectively.
The RSD of each peaks relative increase in area compared
to the 1X preparation was highly stable, remaining at >4%
for most peaks, with the noted exception of Peak V with a
resulting mean variation in peak area of 3.2%, ranging from
0.8 to 19.3%.
In Vitro Effects of Essiac on PC-3 and LNCaP Cell Lines
The cytotoxicity assay was performed on both LNCaP
and PC-3 cell lines treated with Essiac. As shown in Fig. 2
the effect of 6, 60, and 600 g/ml Essiac on proliferation in
LNCaP cells based on the absorbance of the crystal violet
dye was 98.6 6.4%, 87.3 4.6%, and 92.3% 6.8%
of the proliferation seen in cells treated with an equivalent
volume of dH2 O control, respectively. Figure 2 also shows
that the same concentrations applied to PC-3 cells produced
a marginal but not statistically significant enhancement in
proliferation of 113.7 6.7%, 126.3 12%, and 113.1
6.1%, respectively, relative to the dH2 O controls.
Cell Cycle Analysis of Essiac Treated PC-3 Cells
PC-3 cells treated with Essiac had a cell cycle distribution
that typically resembled the distribution seen in the control
192

cells. For example, the control cells containing a diploid

quantity of DNA, or cells in the G1/G0 phase, represented
49.4 0.7% of the all the control cells, while those treated
with 0.6, 0.06, and 0.006 mg/ml Essiac had 49.1 1.6%,
52.5 1.0%, and 51.7 1.7%, respectively, of G1/G0 cells
(illustrated in Fig. 3. This similarity was observed across all
the cell cycle phases. Alternatively, treatment of PC-3 cells
with 7 M paclitaxel resulted in a dramatically different
distribution; for instance, the G1/G0 phase contained 18.6
1.1% of the cells. There was no significant difference
between the distribution of the control cells and those treated
with Essiac.

Subacute Toxicity Testing of Essiac in Athymic Mice

All treatment groups displayed an increase in average
body weight over the treatment period, ranging from 5.4%
to 19.1% of the initial average body weight. No animals
decreased in body weight, behaved abnormally, or exhibited
gross symptoms of toxicity. Overall body weight increase
had no relationship to Essiac dosage.

Efficacy of Essiac to Inhibit Tumor Growth in Athymic

Nude Mice
The only group that showed a significant difference from
the control group was the micellar paclitaxel treated group,
which showed a final average tumor growth of 32% 11% of
initial tumor size. Average tumor growth for each treatment
group over the duration of the study is shown in Figure 4.
Final average tumor growth for untreated and Essiac treated
groups ranged from 490% to 608% of the initial tumor size.
Nutrition and Cancer 2007

Figure 2. Results of cytotoxicity evaluation of EssiacTM extract over a 48-h incubation period using both LNCaP (A) and PC-3 (B) prostate cancer cell lines
with an exposure level ranging from 0.006 to 0.6 mg/ml. The results are expressed as a percentage of the controls absorbance.

Figure 3. Cell cycle analysis results of PC-3 cells treated with EssiacTM extract (0.6, 0.06, and 0.006 mg/ml) and Paclitaxel (0.007 mM) for 48 h. Following
incubation, the cells were fixed with ethanol, and the DNA was stained with propidium iodide. Each cell then had its DNA content measured using flow
cytometry. Each sample analyzed contained 10,000 cells. The histogram shows means SD of triplicate samples. A 1-way analysis of variance was performed
on the cell cycle data followed by an analysis of the groups using 2 post hoc tests, the Tukey test and the StudentNewmanKeuls test; P < .05 was considered
statistically significant.

Vol. 58, No. 2

193

Figure 4. Efficacy study average tumor growth chart of all treatment groups expressed in relation to average group tumor size at initiation of treatment.
Volumes were calculated using V = xyz( /6). Average of the 100 mg/kg bid treatment group is shown with error bars indicating SD. All treatments
were administered via oral gavage except micellar paclitaxel, the positive control, which was administered via tail vein injection. For all groups, n= 11. The
comparison of the treatment groups was performed with a 2-way analysis of variance. A post hoc analysis using a Tukey test was used to indicate which group
was significantly different. A P value of .05 was considered statistically significant.

Essiac treatments did not result in a significant difference in

tumor growth at either of the tested concentrations using the
dosing regimens applied.

Tissue Micro Array Analysis

Ki-67 Immunohistochemical Staining: Table 1 summarizes the average percent of tumor core sections positively
stained with Ki-67, which ranged from 37.0 10.5% to 59.0
12.8% to produce an overall average of 46.8 12.9%.
The average positive staining in the control tumor core sections analyzed ranged from 37.0 7.0% to 54.8 7.0% to
produce an overall average of 44.5 10.8%. There is no
significant difference between these values. Approximately
50% of cores showed evidence of necrosis on examination.
PCNA Immunohistochemical Staining: Table 1 summarizes the average percent of tumor core sections positively
stained for PCNA, which spanned from 1.5 0.6% to 12.3
3.5%, while the area of positive staining in the control tumor
core sections spanned from 2.4 1.2% to 11.5 4.1%. The
overall average of the positively stained areas in the treated
tumor core sections was 8.1 5.2%, while the corresponding
average for the control tumor core sections was 7.1 4.7%.
194

Discussion
The results of the in vitro crystal violet assay of Essiac
on LNCaP and PC-3 prostate cancer cell lines indicate Essiac had no significant antiproliferative effect compared to
cells treated with an equivalent volume of water. While the
quantified A592 values from the LNCaP assay suggested a
marginal cytotoxic effect, results from the PC-3 assay were
more indicative of a proliferative effect. However, none of
the findings were statistically significant or dose dependent.
Flow cytometry results showed that cell cycle phase distribution of the PC-3 cells treated with Essiac was not significantly different from the control treated cells and corroborate
cytotoxicity results. The combination of the 2 assay results
provided strong evidence that Essiac does not produce a detectable effect on PC-3 cells in vitro. The paclitaxel treatment
represented a positive control; the known mechanisms of action for paclitaxel support the effects seen in this treatment
(20, 21).
Increases in body weight in all study groups indicated
that these dosing concentrations were not toxic. Although
only monitored subjectively, no physiological indicators of
toxicity, such as shakiness, lethargy, loss of coordination, or
change in body temperature, were apparent at any time. At the
2 highest dosing levels evaluated, 240 mg/kg qd and bid, two
of the largest average body weight increases were observed;
Nutrition and Cancer 2007

however, it is difficult to form a definitive conclusion due

to the small group size in this study (n = 3). In all, results
support that the treatment would not induce toxicity after
repeated dosing.
Treatment of mice with Essiac led to neither tumor growth
inhibition nor reduced growth rates. Only the positive control
group treated with micellar paclitaxel demonstrated a significant treatment effect. There were no statistically significant
differences in the average tumor growth of any groups treated
with Essiac when compared to the control group.
One of the first considerations when a study results in
negative findings with oral administration of a treatment is
the bioavailability of the dose. As with any herbal product,
bioavailability is difficult to address due to the large number
of components present in each dose and the lack of knowledge regarding their possible activity. Each constituent of
the extract potentially has its own bioavailability; and without complete knowledge of their identities and activities,
each would have to be isolated, characterized, and tested.
This type of evaluation is not feasible on a blend that has not
previously been shown to be efficacious.
The positive control group that was dosed intravenously
with micellar paclitaxel had a statistically significant decrease in tumor growth rate when compared to the control
group. Until now, this result has not been previously published in a PC-3 tumor model. Similar results were reported
with mean LNCaP tumor volumes falling 91% after 3 cycles of treatment (5 mg/day administered on the first 5 days
of a 21-day cycle) (22). In this study, a 68% decrease was
observed after 2 cycles of treatment.
A second pharmacokinetic consideration is distribution.
In some instances, drugs can be stored in reservoirs such as
adipose tissue or bone, as is the case with tetracycline and
bisphosphonates (23). It is unlikely that compounds from
an aqueous extract would be sequestered into a lipid-rich
environment such as adipose tissue. The bone, however, represents a more interesting environment. Prostate and breast
cancers consistently metastasize to skeletal sites, and Essiac
is alleged to be a successful treatment for late-stage cancer.
If any active compound present in Essiac is sequestered to
bone, it may be more effective in treating metastases than in
treating primary tumors. In addition, a longer term treatment
than was employed here would likely be required to achieve
therapeutic levels systemically. There is, however, no experimental evidence to indicate that this process is occurring.
Unfortunately, the use of subcutaneous PC-3 tumors in
nude mice inherently limited conclusions that could be drawn
from this work. The cell line was chosen because it is a widely
accepted model of advanced human prostate cancer. This is
an aggressive tumor system and has a restricted dosing period. Also, it is a human tumor model, which requires immune
compromised carrier animals that can support a xenograft,
but this may also play a role in the effectiveness of treatment.
Two proliferation markers were quantified in this study,
PCNA and Ki-67 (24,25). In both instances, Essiac-treated
tumors were actually shown to have slightly higher amount
of proliferation markers; however, these results are not staVol. 58, No. 2

tistically significant. It would have been useful to have also

included a positive control treatment group in the tissue micro
array. Unfortunately, there was only a small amount of micellar paclitaxel treated tumor tissue available due to tumor regression. The tumor tissue harvested, although not histologically examined, probably contained a low density of striving
cancerous cells. This low density would have been reflected
in the staining with a low number of quantifiable sections.
The recognized need to execute quality control (QC) tests
when dealing with herbal products of unknown integrity
prompted development of the analytical HPLC method described. In performing this QC step, we have been assured of
the test extracts uniformity of composition. No attempt was
made to determine the identity of the compounds giving rise
to any of the peaks.
Analysis of repeated extractions from a single lot revealed that the manufacturers preparation protocol generates
a product that produces reproducible HPLC chromatographs
with a RSD less than 5% at 330 nm. This wavelength was
chosen, as it produced clean chromatograms, and fluorescent
detection can be more sensitive and selective than UV detection. Using this wavelength may limit the detection of other
compounds that may be bioactive but do not absorb well
at 330 nm. The analytical comparison of different lots was
less supportive of the extraction technique. One lot (18146)
contained peak areas that are substantially lower than the
equivalent peaks of the other 2 lots. It appears that Lot 18146
less readily allows for extraction of its components. This
lot, because the blend consists of only raw plant material,
might have a lower amount of extractable components per
net weight. This may be a reflection of variation in the growing season, time, and conditions of harvest of raw materials,
postharvesting processing differences, not necessarily suggestive of noncompliance to good manufacturing practices.
Despite the fact that the peak areas are of substantially different size, the relative area of each individual peak with respect to the cumulative peak area remained quite consistent
throughout the analysis (Fig. 3). Although there are active
compounds in the herbs of this product, the analysis performed in this study did not attempt to reveal which HPLC
peak(s) might represent them. The objective of this study was
to establish if Essiac had antiproliferative properties when
taken in the form that is supplied by the manufacturer. The
findings of the in vivo efficacy study did not suggest that
pharmacokinetic research was justified.

Conclusion
The initial results of this work showed that the aqueous
extract of the herbal product Essiac, despite having a difference in absolute quantities of components between lots,
maintained a stable ratio of its component parts. The in vitro
results indicated that 48-h treatments of PC-3 and LNCaP
prostate cancer cells with Essiac at concentrations up to 6
g/ml have no statistically significant difference in proliferation rate from the control group. Treatment with Essiac was
195

not found to result in toxicity in nude mice dosed with the

product for 4 wk, as would have been evidenced by weight
loss or observed distress of the animals, but at the same time
showed no efficacy against a prostate cancer tumor growth.
As a confirmation of the results recorded during the efficacy
study, the tumors harvested were probed with antibodies for
Ki-67 and PCNA proliferation markers. The quantification
of the immunohistochemical staining showed no difference
in proliferation marker levels between treated and untreated
tumours.
The findings in this project did not identify any significant difference between proliferation levels and markers in
Essiac treated and untreated prostate cancer cells. Throughout the study, there were only marginal differences observed
between the treatment groups. This study indicates that variation in detected HPLC peaks was fairly small, and Essiac
had no significant effect on prostate tumor cell growth in
vitro or in vivo.

Acknowledgments and Notes

We thank EssiacTM Products Inc. for donating the Essiac and Dr. Helen
Burt for supplying the micellar paclitaxel used in this study. This research
was supported by a private donation from John Scrymgeour and the CPCRI
Large Centre Training Award. Address correspondence to Dr. Emma S.
Guns, The Prostate Centre at Vancouver General Hospital, 2660 Oak Street,
Vancouver, BC, Canada V6H 3Z6. Phone: 604-875-4111, ext. 63430. FAX:
604-875-5654. E-mail: Emma.Guns@vch.ca.
Submitted June 2006; accepted in final form January 2007.

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