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Placebo-Controlled Trial of Indole-3-Carbinol in the Treatment of CIN

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Most precancerous lesions of the cervix are treated with surgery or ablative therapy. Chemoprevention, using natural and synthetic compounds, may intervene in the early precancerous stages of carcinogenesis and prevent the development of invasive disease. Our trial used indole-3-carbinol (I-3-C) administered orally to treat women with CIN as a therapeutic for cervical CIN. Thirty patients with biopsy proven CIN II-III were randomized to receive placebo or 200, or 400 mg/day I-3-C administered orally for 12 weeks. If persistent CIN was diagnosed by cervical biopsy at the end of the trial, loop electrocautery excision procedure of the transformation zone was performed. HPV status was assessed in all patients. None (0 of 10) of the patients in the placebo group had complete regression of CIN. In contrast 4 of 8 patients in the 200 mg/day arm and 4 of 9 patients in the 400 mg/day arm had complete regression based on their 12-week biopsy. This protective effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0. 25 to 0.99) P = 0.023) for the 200 mg/day group and a RR of 0.55 ((95% CI, 0.31 to 0.99) P = 0.032) for the 400 mg/day group. HPV was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day group, and in 8 of 9 in the 400 mg/day group. There was a statistically significant regression of CIN in patients treated with I-3-C orally compared with placebo. The 2/16 alpha-hydroxyestrone ratio changed in a dose-dependent fashion.
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Placebo-Controlled Trial of Indole-3-Carbinol in the Treatment of CIN
Maria C. Bell,* Peg Crowley-Nowick,† H. Leon Bradlow,‡ Daniel W. Sepkovic,‡ Delf Schmidt-Grimminger,*
Patti Howell,* E. J. Mayeaux,* Angela Tucker,* Elba A. Turbat-Herrera,* and J. Michael Mathis*
*Department of Obstetrics and Gynecology, Louisiana State University Medical Center–Shreveport, 1501 Kings Highway, Shreveport,
Louisiana 71130–3932; Department of Obstetrics and Gynecology, Harvard Medical School, Fearing Laboratory, P.O. Box 701, Boston,
Massachusetts 02081; and Strang Cancer Research Laboratory, Murray Rayburn Laboratory for Biochemical Endocrinology,
Box 231, The Rockefeller University, 1239 York Avenue, New York, New York 10021
Received September 14, 1999
Objective. Most precancerous lesions of the cervix are treated
with surgery or ablative therapy. Chemoprevention, using natural
and synthetic compounds, may intervene in the early precancerous
stages of carcinogenesis and prevent the development of invasive
disease. Our trial used indole-3-carbinol (I-3-C) administered
orally to treat women with CIN as a therapeutic for cervical CIN.
Methods. Thirty patients with biopsy proven CIN II–III were
randomized to receive placebo or 200, or 400 mg/day I-3-C ad-
ministered orally for 12 weeks. If persistent CIN was diagnosed by
cervical biopsy at the end of the trial, loop electrocautery excision
procedure of the transformation zone was performed. HPV status
was assessed in all patients.
Results. None (0 of 10) of the patients in the placebo group had
complete regression of CIN. In contrast 4 of 8 patients in the 200
mg/day arm and 4 of 9 patients in the 400 mg/day arm had
complete regression based on their 12-week biopsy. This protective
effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI,
0.25 to 0.99) P0.023) for the 200 mg/day group and a RR of 0.55
((95% CI, 0.31 to 0.99) P0.032) for the 400 mg/day group. HPV
was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day
group, and in 8 of 9 in the 400 mg/day group.
Conclusions. There was a statistically significant regression of
CIN in patients treated with I-3-C orally compared with placebo.
The 2/16
-hydroxyestrone ratio changed in a dose-dependent
fashion. © 2000 Academic Press
Key Words: placebo; indoles; dysplasia.
BACKGROUND
Chemoprevention is the attempt to use natural and synthetic
compounds to intervene in the early precancerous stages of
carcinogenesis, before invasive disease begins [1–4]. Cervical
cancer is ideally suited to chemopreventive efforts given its
well-characterized preinvasive state, its multistep progression
to invasive disease, and the clinical ease with which the clini-
cian can follow the lesion without significantly invasive tech-
niques.
Many families of fruits and vegetables contain relatively
high concentrations of unique phytochemicals with potential
cancer-preventing properties. Diets high in cruciferous vege-
tables (e.g., broccoli, cabbage, cauliflower, bok choy, and
brussels sprouts) can retard cancer growth in animals and
increased consumption of these vegetables by humans is asso-
ciated with decreased cancers of the colon, lung, and breast [5].
A specific compound that has been identified in crucifers,
indole-3-carbinol (I-3-C), may be able to prevent or halt car-
cinogenesis [6].
Clinical success has been achieved in the treatment of hu-
man-papillomavirus-induced lesions of the larynx using in-
dole-3-carbinol [7, 8], and I-3-C has been shown to prevent the
development of cervical and vaginal cancers in HPV 16 trans-
genic mice [9]. Although extrapolation of data found in trans-
genic mice has its limitations, these data provide additional
information for the rationale of using I-3-C in the treatment of
cervical dysplasia. Indole-3-carbinol has been shown to
strongly induce the activity of an enzyme found in the liver in
humans and other mammals, called P450 1A1/1A2 (CYP1A1/
1A2) and, to a lesser extent, other p450s [10–12]. This group
of enzymes is known to detoxify and/or metabolize a variety of
chemical substances known to be carcinogenic, and this mech-
anism may explain its cancer-preventive activity in animals as
well as humans [13].
Indole-3-carbinol, through its action on cytochrome P-450
1A1/1A2, is known to alter the pathway of estrogen metabo-
lism in human males and females in a manner that decreases
the risk of certain tumors [13–15]. Metabolic degradation of
estradiol by liver cells results primarily in either 2-hy-
droxyestrone or 16
-hydroxyestrone, and to a lesser extent
4-hydroxyestrone—a potent carcinogen. It is known that 16-
-hydroxyestrone causes proliferation of some breast tumor
cell lines [12], while the alternative metabolite, 2-hy-
droxyestrone, is anti-estrogenic and anti-proliferative [13, 16].
Presented at the 30th Annual Meeting of the Society of Gynecologic
Oncologists, San Francisco, CA, March 20–24, 1999.
Gynecologic Oncology 78, 123–129 (2000)
doi:10.1006/gyno.2000.5847, available online at http://www.idealibrary.com on
123 0090-8258/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
Likewise, it has been shown that I-3-C abrogates the prolifer-
ative effect of estradiol on human laryngeal cells in culture and
decreases the development of papillomas in HPV-infected
grafts in immunocompromised mice [7]. Women with
CIN II/III have lower estrogen metabolite ratios than normal
women [17].
Estradiol is normally metabolized to 16
-hydroxyestrone,
2-hydroxyestrone, and, to a lesser extent, 4-hydroxyestrone.
The decreased ratio of these metabolites (i.e., elevated 16
-
hydroxyestrone) is thought to be tumorigenic and, importantly,
16
-hydroxyestrone is elevated in breast cancer [12]. 16
-
Hydroxyestrone also stimulates cell proliferation and anchor-
age-independent growth [13]. 2-Hydroxyestrone, however,
has less activity than estradiol in inducing anchorage-
independent growth and seems to have an anti-estrogenic
effect. 4-Hydroxyestrone is a known carcinogen of consid-
erable potency [16].
While tumor-associated viruses can profoundly affect the
2-hydroxyestrone to 16
-hydroxyestrone ratio, attempts to di-
rectly decrease 16
-hydroxylation have not proved effective.
However, by upregulating 2-hydroxylation, estradiol is metab-
olized to a benign product at the expense of 16
-hydroxyla-
tion. Although several compounds are capable of upregulating
2-hydroxylation, Niwa et al. found that I-3-C was the most
potent of these compounds [18]. I-3-C also suppresses 4-hy-
droxylation [19].
Since laryngeal papillomatosis (an HPV-induced disease)
has been effectively treated with indole-3-carbinol, we hypoth-
esized that indole-3-carbinol may be effective in the treatment
of cervical dysplasia—another HPV-induced disease. In this
study, our goal was to use indole-3-carbinol in capsule form to
treat women with CIN, most of which are human-papilloma-
virus-induced.
MATERIALS AND METHODS
Patient Eligibility
Thirty subjects with histologically confirmed CIN (Table 1)
were enrolled. The study protocol was reviewed and approved
for human research subjects by the Institutional Research
Board for Human Research of LSU Medical Center–Shreve-
port. Because indole-3-carbinol was available in health food
stores prior to October 1994, a FDA IND number was not
required. Eligibility requirements were as follows: adequate
colposcopy, negative ECC, no suspicion of invasion, nonpreg-
nant, and HIV. There was not a “size” requirment for the
dysplastic lesion prior to entry.
Evaluating colposcopists and patients were blinded to the
treatment. All residents and staff involved in the study were
instructed regarding the protocol. Typically second, third, and
fourth year ob/gyn residents staff the colposcopy clinic with
four different attending physicians supervising. The patients
were randomized to placebo or 200 or 400 mg/day treatment of
indole-3-carbinol administered orally for 12 weeks. Random-
ization was performed by successively assigning patients from
a computer-generated randomization table. Ten patients were
randomized to each group; however, 2 patients in the 200
mg/day treatment arm and 1 patient in the 400 mg/day treat-
ment arm did not complete the study. These patients were
randomized, but did not return for their appointment to receive
the medication and were excluded from analysis.
Patient Evaluation
All patients underwent colposcopy at the initial visit as well
as their 4-, 8-, and 12-week visits. Photodocumentation of each
colposcopic examination was obtained. At the end of the trial,
each patient was reexamined, and if the patient had persistent
CIN on 12-week biopsy or if the colposcopic impression was
persistent CIN, she underwent loop electrocautery excision
procedure of the transformation zone. Patients were asked at
each visit about side effects and compliance. Patients returned
remaining pills at the end of the trial for assessment of remain-
ing unused medication.
Pathology
The dysplasia was classified by the pathologists in the usual
manner and defined as follows: mild dysplasia meaning the
presence of immature (dysplastic) cells present in the lower
third of the squamous mucosa, moderate dysplasia in the lower
two-thirds, severe dysplasia the lower three-fourths of the
thickness of the mucosa, and carcinoma in situ full-thickness
immaturity. The cases were originally diagnosed by a sign-out
pathologist. The diagnosis of all cases was verified by the same
pathologist (ETH) for uniformity. In all cases, the pathologist
(ETH) was in agreement with the pathologist that had signed
out the case.
Indole-3-Carbinol Assay
In order to assess the purity of the indole-3-carbinol used in
this study, high-performance liquid chromatography was per-
formed on the bottles labeled “product X,” which was the
indole-3-carbinol, and on the bottles labeled “product Y,”
which was the placebo. Each of the product X capsules con-
tained slightly more than 100 mg of the indole-3-carbinol per
capsule, and no indole-3-carbinol was present in the product Y
capsule. Design Nutritional Products (Orem, UT), provided the
indole-3-carbinol capsules and placebo capsules.
HPV Analysis
HPV status was assessed in all patients by Hybrid capture
assay tests (Digene Diagnostics, Beltsville, MD) for low risk
(HPV types 6, 11, 42, 43, 44) and high risk (HPV types 16, 18,
124 BELL ET AL.
31, 33, 35, 45, 51, 52, 56). If the Hybrid capture assay was
negative, a follow-up polymerase chain reaction (PCR) analy-
sis was performed using consensus primers shown below for
the L-1 region of HPV to confirm HPV positivity.
Primer 1 (MY09) sequence: 5-CGTCCMARRGGAWAC-
TGATC-3
Primer 2 (MY11) sequence: 5-GCMCAGGGWCATA-
AYAATGG-3
The PCR amplification conditions were optimized using DNA
isolated from paraffin-embedded control HPV-positive cervi-
cal cancer specimens prior to analysis of the study patient’s
samples.
Estradiol Metabolism
Clean catch urine specimens were obtained from each pa-
tient at the initial visit and again at their 4-week visit. 2-Hy-
droxyestrone to 16-
-hydrosytestrone ratios were assessed in
each urine sample. Ascorbic acid was added to the urine
specimens to aid in preservation for accurate analysis. ELISA
assays were performed to assess the quantity of 2-hy-
droxyestrone and 16-
-hydroxyestrone in each urine sample
and were reported in nanograms per milliliter [20–22]. All
specimens were shipped to the Strang Cancer Research Labo-
ratory for analysis in the laboratory of Dr. H. Leon Bradlow.
Statistical Analysis
The placebo group was compared to each treatment group
using Fisher’s exact test with two-tailed Pvalues. Statistical
significance was defined as P0.05. Taylor 95% confidence
intervals were computed for relative risks. The statistical pack-
age used was Trus Epistat (Richardson, TX).
TABLE 1
Dose, Diagnosis, and HPV Status
Patient (No.)
dose I-3-C
(mg/day) Diagnosisa
(week 1) Diagnosisa
(week 12) Low-riskbHPV High-riskbHPV HPV PCR
1. Placebo CIN II CIN I Pos Pos
2. Placebo CIN II CIN I Neg Neg Neg
3. Placebo CIN II CIN I Neg Pos
4. Placebo CIN III CIN I Neg Pos
5. Placebo CIN III CIN I Neg Neg Pos
6. Placebo CIN II CIN I Neg Neg
7. Placebo CIN II CIN I Neg Neg
8. Placebo CIN III CIN III Neg Pos
9. Placebo CIN III CIN I Neg Pos
10. Placebo CIN III CIN III Neg Pos
11. 400 CIN II CIN I Neg Neg Pos
12. 400 CIN II CIN I Neg Pos Pos
13. 400 CIN III CIN I Neg Pos
14. 400 CIN II CIN I Neg Neg Neg
15. 400 CIN II Normal Neg Neg Pos
16. 400 CIN III CIN I Neg Pos Pos
17. 400 CIN II Normal Neg Neg Pos
18. 400 CIN III Normal Neg Neg Pos
19. 400 CIN II Normal Neg Pos
20. 200 CIN III CIN I Pos Pos
21. 200 CIN II Normal Neg Pos
22. 200 CIN III CIN III Neg Neg Neg
23. 200 CIN II Normal Neg Pos
24. 200 CIN III Normal Pos Pos
25. 200 CIN II CIN II Pos Pos
26. 200 CIN II Normal Neg Neg Pos
27. 200 CIN II CIN II Neg Pos
Note. mg/day, milligrams per day; HPV, human papillomavirus; LEEP, loop electrocautery excision procedure; CIN, cervical intraepithelial neoplasia; neg,
negative; pos, positive; PCR, polymerase chain reaction.
aDiagnosis by biopsy.
bHPV types 6, 11, 42, 43, 44.
cHPV types 16, 18, 31, 33, 35, 45, 51, 52, 56.
125INDOLE-3-CARBINOL IN THE TREATMENT OF CIN
RESULTS
In this study, we evaluated the efficacy of indole-3-carbinol
in 27 histologically confirmed cervical dysplasia patients (Ta-
ble 1). Routine pathologic evaluation of the study patients
showed that none (0 of 10) of the patients in the placebo group
had complete regression of their dysplasia as summarized in Table 1. In contrast, 50.0% (4 of 8) of patients in the 200
mg/day treatment arm and 44.4% (4 of 9) of patients in the 400
mg/day treatment arm had complete regression based on their
12-week biopsy.
In order to evaluate the degree of regression between the
treatment groups, the mean grade of CIN regression was com-
pared as shown in Fig. 1. A linear dose–response relationship
was noted among the placebo and the 200 and 400 mg/day
treatment groups. The mean grade change of CIN in the pla-
cebo group was 1.22, in the 200 mg/day treatment group 1.38,
and in the 400 mg/day treatment group 1.78. Fisher’s exact test
was used to make comparisons between groups and the signif-
icance level was defined as P0.05. The protective effect of
I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0.25
to 0.99) P0.023) for the 200 mg/day group and a RR of
0.55 ((95% CI, 0.31 to 0.99) P0.032) for the 400 mg/day
group.
The urinary 2-hydroxyestrone to 16-
-hydroxyestrone ratios
were evaluated in each patient at the initiation of the trial and
at 4 weeks in order to assess the physiologic response to
indole-3-carbinol (Table 2, Fig. 2). Although there was some
variability in the individual ratios, a general trend in increase in
the ratios was found in the 200 and 400 mg/day treatment
groups before and after therapy (Table 2). In contrast, the
placebo treatment group showed a decrease in the ratio before
and after therapy (Fig. 3).
To determine whether HPV infection alters the response to
indole-3-carbinol, HPV status was determined on all patients
(Table 1). In the placebo group, 7 of 10 patients had HPV
detected. Of these 7 placebo patients, 6 had high-risk HPV
TABLE 2
2-Hydroxyestrone/16
-Hydroxyestrone Ratio versus Dose
Patient (No.) and
dose (mg/day) Ratio
pretherapy Ratio after
I-3-C Ratio
% change
1. Placebo 0.83 1.56 88
2. Placebo 1.09 1.38 26
3. Placebo 1.26 1.91 52
4. Placebo 0.65 0.79 21
5. Placebo 0.67 0.55 18
6. Placebo 0.57 0.64 12
7. Placebo 1.18 1.43 21
8. Placebo 3.48 0.36 89
9. Placebo 2.80 1.58 44
10. Placebo ND ND ND
11. 400 1.52 1.42 0.07
12. 400 1.31 0.67 48
13. 400 0.48 0.87 81
14. 400 2.44 2.85 17
15. 400 0.61 1.10 80
16. 400 1.18 3.43 190
17. 400 1.42 1.45 0.02
18. 400 1.13 1.51 33
19. 400 0.89 1.06 19
20. 200 0.92 1.08 17
21. 200 0.69 0.69 0
22. 200 0.29 0.47 62
23. 200 1.08 0.66 39
24. 200 0.55 1.37 149
25. 200 0.70 0.91 30
26. 200 0.90 1.30 44
27. 200 1.32 1.66 26
FIG. 1. Regression of CIN by dose.
FIG. 2. Change in 2-hydroxyestrone/16
-hydroxyestrone ratio.
126 BELL ET AL.
subtypes (HPV 16 or 18) and 1 patient had HPV present by
PCR, but was not typed. In the 200 mg/day treatment group, 7
of 8 patients had detectable HPV and, of these, 6 had high-risk
subtypes and 1 patient had HPV present by PCR, but was not
typed. In the 400 mg/day treatment group, 8 of 9 patients had
detectable HPV, of which 4 were high-risk subtypes and 4
patients had HPV present by PCR, but were not typed. These
results show that the incidence of HPV was similar between all
FIG. 3. H&E staining of cervical biopsies before and after therapy. Representative sections of cervical biopsies before and after treatment with
indole-3-carbinol are shown. Note the persistent dysplasia in the placebo section at 12 weeks, but regression of CIN in the 200 and 400 mg/day sections.
127INDOLE-3-CARBINOL IN THE TREATMENT OF CIN
treatment groups and historical control, and suggest that pres-
ence of HPV does not correlate with response to indole-3-
carbinol.
DISCUSSION
HPV infection of the lower genital tract is the most common
sexually transmitted viral disease in the United States [23].
Most of these viral infections remain dormant and never result
in clinically evident disease. However, in some cases, the virus
may propagate and cause clinically recognizable HPV-associ-
ated changes including condylomata (genital warts), precan-
cerous lesions of the cervix, and invasive cervical cancer. The
association between HPV infection and genital cancer is well
documented [24, 25]. The progression of HPV infection to
genital cancer is not absolute, and other factors (e.g., smoking,
diet, and immunosuppression) probably contribute to the pro-
gression. In our study we found that the majority of patients
had HPV present in their cervix. Typing of HPV also demon-
strated a similar proportion of high-risk HPV subtypes in each
of our three treatment groups. However, it is unclear from our
data whether the presence of high-risk HPV is more or less
likely to respond to I-3-C since we did not type all of the PCR
patients.
The primary mechanism by which I-3-C is hypothesized to
work is by altering the 2-hydroxyestrone/16
-hydroxyestrone
ratio. We were able to demonstrate an increasing change in this
ratio among placebo and the 200 and 400 mg/day groups in a
dose-response fashion. Indole-3-carbinol, normally present in
cruciferous vegetables, has been administered to healthy hu-
man volunteers in short courses (3 months) in doses of 5–7
mg/kg (350–500 mg), which is equivalent to approximately
300–500 g of raw cabbage or brussels sprouts per day [11, 26].
Bradlow et al. assessed the effects of I-3-C administered to
healthy subjects versus placebo. In this study, multiple organ
systems were monitored including hematologic, renal, and
hepatic, and no adverse effects were noted in any of these
subjects at this dosage. In this study, the highest dose (400
mg/day) was chosen because it is equivalent to 1/3 of a head of
cabbage, an amount thought to be what most that people would
reasonably consume daily [27]. Anecdotal reports about chil-
dren taking I-3-C for suppression of their laryngeal papillomas
indicate that I-3-C can be administered for several years with-
out any toxicity. In our study no toxicity was observed in either
the 200 or the 400 mg/day group.
When looking at individual ratios and determining whether
a positive change in ratio would predict whether the patient
would have regression of her lesion, our data showed that six
of the eight patients who had regression demonstrated a posi-
tive change in their urinary 2-hydroxyestrone to 16
-hy-
droxyestrone ratio. In contrast, five of the six patients who had
a negative change in their urinary 2-hydroxyestrone/16
-hy-
droxyestrone ratio demonstrated persistent CIN. The fact that
not all the responders had a significant (50%) increase in
their 2/16 ratio may be explained by the other ways in which
I-3-C has been reported to work. These alternate pathways
include induction of apoptosis [28], inhibition of microtubule
assembly [29], and decreased effect of NNK—a known to-
bacco carcinogen [30, 31]. In light of the fact that five of six of
our responders had a positive shift in their urinary 2-hy-
droxyestrone/16
-hydroxyestrone ratio, the urinary 2-hy-
droxyestrone/16
-hydroxyestrone ratio was considered a fea-
sible and simple method for monitoring of potential responses.
However, further studies are necessary to confirm this result.
Indole-3-carbinol may not induce a shift in estrogen metab-
olism in a subset of the general population. Some patients have
a CYP 1A1 genotype (MSP-1 polymorph), in which case the
administration of I-3-C would not be expected to shift the
2/16-hydroxyestrone ratio. We did not assay for the CYP 1A1
genotype in our patients. The CYP 1A1 genotype is present in
1–16% of the population, depending on their ethnic back-
ground [32]. Further studies using I-3-C for cervical dysplasia
should incorporate CYP 1A1 genotyping to assess efficacy of
I-3-C in different CYP 1A1 genotypes.
Not all dysplastic lesions will go on to develop into cancer.
Mildly dysplastic lesions will spontaneously regress without
therapy in the majority of patients, and only a small subset of
cases actually posses cancerous potential. Less than one-half of
more severely dysplastic lesions in patients will eventually
progress to invasive disease [33]. There is currently no avail-
able test that can accurately predict the clinical behavior of a
cervical lesion. Therefore, most high-grade lesions are treated
with surgical excision. Chemoprevention is the attempt to use
natural and synthetic compounds to intervene in the early
precancerous stages of carcinogenesis, before invasive disease
begins. Cervical cancer is ideally suited to chemopreventive
efforts given its well-characterized preinvasive state and its
multistep progression to invasive disease and the clinical ease
with which the clinician can follow the lesion without signif-
icantly invasive techniques (i.e., colposcopy).
In conclusion, this placebo-controlled trial of indole-3-carbi-
nol in the treatment of CIN does show promise for I-3-C as a
therapeutic. We were able to demonstrate a physiologic effect
of the drug, which was seen in a dose-dependent fashion. The
strengths of this study are that there was placebo control, which
is important because of the high rate of spontaneous regression
of CIN, and that indole-3-carbinol was well tolerated with no
adverse events noted. Although the response rate in this study
is encouraging, these data need to be confirmed with a large
placebo-controlled multicenter trial. If such a large trial sup-
ports the efficacy of I-3-C for the treatment of cervical dys-
plasia, it would give patients and physicians a nonsurgical
option for the treatment of CIN.
128 BELL ET AL.
ACKNOWLEDGMENTS
The authors thank Dr. Steve London (Department of Obstetrics and Gyne-
cology, LSU Medical Center–Shreveport) for his generous support of this
project, as well as Murray and Isabella Rayburn.
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129INDOLE-3-CARBINOL IN THE TREATMENT OF CIN
... We found that the histomorphology, vascularization and proliferative activity of these reproductive organs are not affected by the treatment with I3C. This result is in line with several clinical trials assessing the therapeutic efficacy of I3C in the treatment of cervical dysplasia as well as breast and prostate cancer [39,[61][62][63]. They showed that supplementation with I3C up to 400 mg/day only elicits few, if any, adverse effects [64]. ...
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Endometriosis represents an estrogen-dependent disorder with a complex pathophysiology. Phytochemicals are promising candidates for endometriosis therapy, because they simultaneously target different cellular processes involved in the pathogenesis of endometriosis. Herein, we analyzed whether indole-3-carbinol (I3C) suppresses the development of endometriotic lesions, which were surgically induced by fixation of uterine tissue samples (diameter: 2 mm) from female BALB/c donor mice to the peritoneum of recipient animals. The mice received either I3C or vehicle (control) by peroral administration once per day. Growth, cyst formation, cell proliferation, microvascularization and protein expression of the lesions were assessed by high-resolution ultrasound imaging, caliper measurements, histology, immunohistochemistry and Western blotting. I3C inhibited the vascularization and growth of endometriotic lesions without inducing anti-angiogenic and anti-proliferative side effects on reproductive organs. This was associated with a significantly reduced number of proliferating stromal and endothelial cells and a lower expression of the pro-angiogenic signaling molecules vascular endothelial growth factor receptor-2 (VEGFR2), phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (pERK) within I3C-treated lesions when compared to controls. These findings indicate that I3C effectively inhibits endometriotic lesion formation in mice. Thus, further studies should clarify whether I3C may be also beneficial for the prevention and therapy of the human disease.
... W trakcie tych badań zaobserwowano regresję dysplazji śródbłonkowej szyjki macicy (CIN) u 50% pacjentek, którym podawano 200 mg/dzień i u 44% u pacjentek, którym podawano 400 mg/dzień. Wykazano również niewielki wzrost poziomu 2-hydroksyestronu w stosunku do 16α-hydroksyestronu, który był zależny od dawki [67]. ...
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Pochodne indolu to aromatyczne, heterocykliczne związki organiczne, które mają duże znaczenie przy poszukiwaniu nowych substancji leczniczych. Indolo-3-karbinol (I3C) i 3,3´-diindolilometan (DIM) występują naturalnie w roślinach krzyżowych, ale ich pochodne są także syntetyzowane w laboratorium. Wyniki badań in vitro wskazują, że I3C oraz DIM hamują proliferację komórek nowotworowych w fazie G1 i wywołują ich apoptozę. Dowiedziono, że mają wpływ na metabolizm estrogenów. Mikromacierzowe profilowanie ekspresji genów wskazuje, że pochodne indolu regulują ekspresję wielu genów, które mają istotne znaczenie dla kontroli cyklu komórkowego, apoptozy komórek, transdukcji sygnałowej, w angiogenezie oraz inwazji komórek. W badaniach na modelach zwierzęcych stwierdzono, że pochodne indolu zapobiegają powstawaniu nowotworów piersi i szyjki macicy. W badaniach klinicznych (faza II i III) oceniono I3C i DIM jako potencjalne środki chemoprewencyjne do stosowania w profilaktyce i wczesnych etapach rozwoju nowotworów piersi, jajnika oraz śródnabłonkowej neoplazji sromu. Dokładny mechanizm molekularny działania przeciwnowotworowego pochodnych indolu wciąż jest przedmiotem badań. Omawiane związki (I3C i DIM) są składnikami suplementów diety, zalecanych do prewencyjnego stosowania przy chorobach nowotworowych.
... Indole-3-carbinol (I3C), a phytochemical found in Brassica vegetables, has long been recognized as a chemopreventive agent to intervene in the early precancerous changes in carcinogenesis [1,2]. As expected, 3,3 -diindolylmethane (DIM), the main natural occurring substance produced following digestion of indole-3-carbinol, has been extensively investigated as an anticancer agent in vivo and in vitro, and has undergone clinical trials for multiple forms of cancer, including prostate cancer (Phase II) (ClinicalTrials.gov ...
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The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3’-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.
... In addition, a placebo-controlled trial involving 27 patients diagnosed with CIN II-CIN III, resulted in a statistically significant complete regression in four out of eight patients orally supplied with I-3-C 200 mg/day and in four out of nine patients on I-3-C 400 mg/day. A change of the 2/16a-hydroxyestrone ratio was found (Supplementary Table S1, indole-3-carbinol-based formulation no.2) [125]. ...
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Human papillomavirus (HPV) still represents an important threat to health worldwide. Better therapy in terms of further improvement of outcomes and attenuation of related side-effects is desirable. The pharmaceutical industry has always targeted natural substances—phytochemicals in particular—to identify lead compounds to be clinically validated and industrially produced as antiviral and anticancer drugs. In the field of HPV, numerous naturally occurring bioactives and dietary phytochemicals have been investigated as potentially valuable in vitro and in vivo. Interference with several pathways and improvement of the efficacy of chemotherapeutic agents have been demonstrated. Notably, some clinical trials have been conducted. Despite being endowed with general safety, these natural substances are in urgent need of further assessment to foresee their clinical exploitation. This review summarizes the basic research efforts conducted so far in the study of anti-HPV properties of bio-actives with insights into their mechanisms of action and highlights the variety of their natural origin in order to provide comprehensive mapping throughout the different sources. The clinical studies available are reported, as well, to highlight the need of uniformity and consistency of studies in the future to select those natural compounds that may be suited to clinical application.
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An efficient one-pot synthesis of an indole-xanthydrol hybrid is described in the presence of catalytic combinations of Fe(NO3)3/FeCl3. This strategy involves a series of reactions such as allylic oxidation, isomerisation, cyclisation and hydroxylation reactions in a tandem manner. This protocol offers several advantages including mild reaction conditions, operational simplicity, high selectivity, good yields and easily accessible starting materials. The synthetic utility of this protocol was further demonstrated by the one-pot synthesis of the highly substituted xanthene containing bis-indolylmethane derivative. The preliminary mechanistic studies reveal that the reaction is initiated by the generation of radicals in the presence of catalytic iron(III)-salts.
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The incidence of candidiasis is significant, especially in the group of people predisposed to the development of fungal infections due to conditions related to the underlying diseases, clinical conditions and prolonged hospitalization. The most common etiological factor causing fungal diseases are species of the genus Candida spp. Undoubtedly, it is related to the dichotomy they present. Candida albicans, as well as NAC species (Nonalbicans Candida), are commonly found in the natural environment, and are also part of the human microbiome. However, some species, especially in conditions of dysbiosis, can pose a serious pathogenic threat. Fungal infections constitute a significant diagnostic and therapeutic problem due to a number of Candida spp. Adaptations to tissue colonization, as well as their phenotypic and genotypic variability. Effective fight against this type of infections may be possible thanks to the understanding of all structural, functional and molecular mechanisms, including the study of the relationship between Candida species and the analysis of changes within the genome of a given species. The full success of mycological diagnostics can be achieved by supplementing conventional methods with molecular methods. Genotypic analysis allows for the assessment of the degree and possible routes of spread of Candida spp species. The study of the relationship between Candida spp. Species and the analysis of changes within a given species enable the identification of their diversity, determination of the spread of species with different sensitivity to antimycotics, thus facilitating the selection of appropriate treatment . Thus, the essence of molecular methods concerns the analysis of fungal DNA polymorphism. Genotyping methods provide a lot of information on the organization of all genetic material in different Candida spp species. The aim of the study is to analyze the techniques most often used to differentiate individual Candida strains: RFLP (Restriction Fragment Length Polymorphism) techniques, RAPD (Random Amplification Polymorphism DNA), RT-PCR (Reverse Transcription), Real-Time PCR, AFLP (Amplified Fragment Length Polymorphism) ) and PCR MP (Melting Profile). It is also important to demonstrate the relationship between the results of molecular tests and modern microscopic methods, such as atomic force microscopy (AFM). The test results obtained with the use of these tools enable an in-depth analysis of the phenomena underlying the colonization ability of Candida spp. The assessment of nanomechanical properties, the analysis of elasticity, stiffness and the ability to adhere to the surface (adhesion) can determine the ability of fungi to colonize tissues or elements of the environment. Determining changes in nanomechanical properties of living cells can be a source of information about changes taking place in their structures under the influence of environmental, immunological and chemical factors. These data may be of importance in assessing the susceptibility of Candida spp. To drugs used in the elimination of such infections.
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Herein we report crystallographic comparison of some geometrical and structural features for a series of biologically relevant bis-indole derivatives. Selected bond distances and bond angles of interest in a series of bis-indole derivatives have been discussed in detail. The biological activity of the substances has been correlated with based the structure-activity relationships (SAR) base which provides the different possibility of activity (Pa) and possibility of inactivity (Pi). For a better understanding of the packing interactions existing among these derivatives, an overview of crystal structure analysis with emphasis on the intramolecular hydrogen bonding in some bis-indole derivatives is presented. The role of hydrogen bonding in the crystal structure assembly of bis-indole derivatives has been found to be predominant and this observation reveals significant impact of hydrogen bonding in high value of drug-likeness of these bio-molecules.
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Cruciferous vegetables, such as cabbage, cauliflower, and broccoli, have been cultivated since antiquity as medicinal plants. Once ingested, the Cruciferae release unique phytochemical constituents able to modify the activity of cellular enzymes effecting carcinogen clearance and estrogen metabolism. The most active of these phytochemicals with regard to estrogen is the dietary indole, diindolylmethane. Understanding the dietary influences of cruciferous phytochemicals on estrogen status provides a basis for nutritional approaches to estrogen-related concerns that accompany aging in women and men.
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Intraepithelial neoplasia is of critical importance to the cancer chemoprevention field because it is a target condition for which drugs must be sought that will prevent its development or stop its progression. The term "dysplasia" refers to the morphological alterations that characterize intraepithelial neoplasia and according to many authors consists of seven basic morphological changes that occur in the majority of human epithelia, as well as in the epithelium of mouse skin papillomas induced by 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate: increased nuclear size; altered nuclear shape; increased nuclear stain uptake; nuclear pleomorphism (increased variation in nuclear size, shape, and stain uptake); increased mitoses; abnormal mitoses; and disordered or absent maturation. Clonal evolution appears to begin early in the neoplastic process during intraepithelial neoplasia. Aneuploidy has been found during intraepithelial neoplasia in many human epithelia, and, in association with other forms of genetic instability, may provide the increase in genetically variant cells required for clonal evolution to occur. It is postulated that two major factors affecting the rate of progression of intraepithelial neoplasia are the cellular mutation rate, which is enhanced by environmental carcinogens, and the cellular proliferation rate, which is enhanced by agents that include sex hormones, inducers of chronic inflammation, and irritant chemicals which stimulate reactive hyperproliferation. A preferred chemoprevention strategy should consist of the development of drugs and drug combinations which will block mutagenic carcinogens or prevent epithelial hyperproliferation or its causes. Two examples of the induction of regression of intraepithelial neoplasia by chemopreventive drugs are the regression of oral leukoplakia produced by beta-carotene and the regression of colorectal polyps in patients with familial polyposis produced by sulindac. It is evident that there is a strong need for more research on the induction of regression of intraepithelial neoplasia with chemopreventive agents. There is also a critical need to identify and develop biomarkers that correlate with the appearance and regression of intraepithelial neoplasia.
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Dietary indoles in cruciferous vegetables induce cytochrome P450 enzymes and have prevented tumors in various animal models. Because estradiol metabolism is also cytochrome P450 mediated and linked to breast cancer risk, indoles may similarly reduce estrogen-responsive tumors in humans. We examined several indoles in female Sprague-Dawley rats for induction of hepatic estradiol 2-hydroxylation. The most potent inducer, indole-3-carbinol, was administered to humans (500 mg daily for 1 wk). It significantly increased the extent (mean ± SEM) of estradiol 2-hydroxylation from 29.3% ± 2.1% to 45.6% ± 2.1% ( P <.001). These results indicate that indole-3-carbinol strongly influences estradiol metabolism in humans and may provide a new chemopreventive approach to estrogen-dependent diseases. [J Natl Cancer Inst 82:947–949, 1990]
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Alterations in the metabolism of estrogen have been implicated as an important factor in the etiology of diseases such as gynecological cancers and lupus erythematosus. The major metabolites of estradiol are hydroxylated at the C-2 or C-16α position yielding products with estrogen antagonist and agonist activities, respectively. A sensitive and specific immunodiagnostic assay to determine the balance between these competing pathways might serve as a routine biomarker for management of estrogen-related diseases. We describe here the generation of high affinity, specific murine monoclonal antibodies to 2-hydroxyesterone and 16α-hydroxyesterone by high efficiency fusion protocols. With these antibodies, we have developed a rapid and simple anzyme immunoassay (EIA) kit for the simultaneous quantitation of 2- and 16α-hydroxyestrone in unextracted urine. Initial validation studies established that urinary metabolite 2- and 16α-hydroxyestrone concentrations found by the EIA correlate well with values found by gas chromatography-mass spectroscopy. Preliminary studies with the EIA kit found total recovery of metabolites from spiked urine samples. The EIA inter- and intra-assay coefficients of variation for 2-hydroxyestrone and 16α-hydroxyestrone and the ratio of 2-hydroxyestrone to 16α-hydroxyestrone with the current EIA kit were consistently less than 9%. This kit, designated ESTRAMET™ 2/16 may provide an important new tool for research in estrogen-related diseases.
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Background: Experimental studies have provided strong evidence that human papillomavirus (HPV) is the long-sought venereal cause of cervical neoplasia, but the epidemiologic evidence has been inconsistent. Purpose : Given improvements in HPV testing that have revealed a strong link between sexual activity history and cervical HPV infection, we conducted a large case-control study of HPV and cervical intraepithelial neoplasia (CIN) to evaluate whether sexual behavior and the other established risk factors for CIN influence risk primarily via HPV infection. Methods : We studied 500 women with CIN and 500 control subjects receiving cytologic screening at Kaiser Permanente, a large prepaid health plan, in Portland, Ore. The established epidemiologic risk factors for CIN were assessed by telephone interview. We performed HPV testing of cervicovaginal lavage specimens by gene amplification using polymerase chain reaction with a consensus primer to target the LI gene region of HPV. Unconditional logistic regression analysis was used to estimate relative risk of CIN and to adjust the epidemiologic associations for HPV test results to demonstrate whether the associations were mediated by HPV. Results : The case subjects demonstrated the typical epidemiologic profile of CIN: They had more sex partners, more cigarette smoking, earlier ages at first sexual intercourse, and lower socioeconomic status. Statistical adjustment for HPV infection substantially reduced the size of each of these case-control differences. Seventy-six percent of cases could be attributed to HPV infection; the results of cytologic review suggested that the true percentage was even higher. Once HPV infection was taken into account, an association of parity with risk of CIN was observed in both HPV-negative and HPV-positive women. Conclusion : The data show that the great majority of all grades of CIN can be attributed to HPV infection, particularly with the cancer-associated types of HPV. Implications : In light of this conclusion, the investigation of the natural history of HPV has preventive as well as etiologic importance. [J Natl Cancer Inst 85:958–964, 1993]
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Indole-3-carbinol, 3,3'-diindolylmethane, and indole-3-acetonitrile, three indoles occurring in edible cruciferous vegetables, have been studied for their effects on 7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in female Sprague-Dawley rats and on benzo(a)pyrene-induced neoplasia of the forestomach in female ICR/Ha mice. When given by p.o. intubation 20 hr prior to 7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and 3,3'-diindolylmethane had an inhibitory effect on mammary tumor formation, but indole-3-acetonitrile was inactive. Indole-3-carbinol when added to the diet for 8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibited mammary tumor formation, whereas indole-3-acetonitrile did not. Dietary administration of all three indoles inhibited benzo(a)pyrene-induced neoplasia of the forestomach in ICR/Ha mice. The identification of dietary constituents that can inhibit chemical carcinogens ultimately may be of value in understanding the balance of factors that determines the neoplastic response to these cancer-producing agents in the environment.
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Estrogens are potent mammary tumor promoters influencing post-initiation events via epigenetic mechanisms. The upregulation (i.e., induction) of the C16 alpha-hydroxylation pathway during 17 beta-estradiol (E2) biotransformation has been associated with mammary cell transformation. The action of E2 metabolites on tumorigenic transformation, however, is poorly understood. The newly established mammary epithelial cell line C57/MG, derived from the C57BL mouse strain, was used to examine whether E2 or its metabolites, 16-hydroxyestrone (16 alpha-OHE1) and estriol (E3), function as initiators of mammary cell transformation. DNA repair (hydroxyurea-insensitive thymidine uptake), estrogen metabolism (3H exchange to form 3H2O), hyperproliferation (increased cell number), and acquisition of anchorage-independent growth (soft-agar colonies) were used as quantitative end points to measure the relative extent of transformation. Treatment of cells with 200 ng/mL 16 alpha-OHE1 resulted in a 55.2% increase in DNA repair synthesis, a 23.09% increase in proliferative activity, and a 18-fold increase in the number of soft-agar colonies, relative to the solvent controls (P less than .0001). The extent of upregulation of the three end points was similar to that induced by the genotoxic mammary carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA, positive control). DMBA treatment also upregulated the ratio of 16 alpha/C2 hydroxylation of E2 leading to increased formation of 16 alpha-OHE1. E2 and E3 were not effective in upregulating these markers for transformation. These results demonstrate that in nontransformed C57/MG cells, 16 alpha-OHE1 may function as an initiator, perturbing the intermediate biomarkers for preneoplastic transformation.
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Research studies have demonstrated a strong association between estrogen metabolism and the incidence of breast cancer, and we have therefore sought pharmacological means of favorably altering both metabolism and subsequent risk. Indole-3-carbinol (I3C), obtained from cruciferous vegetables (e.g., cabbage, broccoli, etc.), is a known inducer of oxidative P-450 metabolism in animals. We investigated the effects in humans of short-term oral exposure to this compound (6-7 mg/kg/day over 7 days). We used an in vivo radiometric test, which provided a highly specific and reproducible measure of estradiol 2-hydroxylation before and after exposure to I3C. In a group of 12 healthy volunteers, the average extent of reaction increased by approximately 50% during this short exposure (p less than 0.01), affecting men and women equally. We also measured the urinary excretion of two key estrogen metabolites, 2-hydroxyestrone (2OHE1) and estriol (E3). We found that the excretion of 2OHE1 relative to that of E3 was significantly increased by I3C, further confirming the ongoing induction of 2-hydroxylation. These results indicate that I3C predictably alters endogenous estrogen metabolism toward increased catechol estrogen production and may thereby provide a novel "dietary" means for reducing cancer risk.