INDOLE 3 CARBINOL

CANCER
    Cancer is a group of more than 100 diseases characterized by uncontrolled cellular growth, local tissue invasion,
and distant metastasis. Cells that have undergone neoplastic transformation usually express cell surface antigens that
may be of normal fetal type, may display other signs of apparent immaturity and may exhibit qualitative and
quantitative chromosomal abnormalities, including various translocations and appearance of amplified gene
sequence. A small subpopulation of cells within tumor can be described as tumor stem cells. Such cells can express
clonogenic or colony forming capability. Tumor stem cells often have chromosome abnormalities. The invasive and
metastatic processes as well as a series of metabolic abnormalities resulting from the cancer cause illness and
eventual death of the patient unless the neoplasm can be eradicated with treatment.

 
    The role of the pharmacist in the management of the cancer patient can be very diverse. Through knowledge of
antineoplastic drug pharmacology and pharmacokinetics is essential to prevent and manage many drug-induced
toxicities. Provision of drug information is another critical role for oncology pharmacist. Experienced pharmacists
are able to fulfill those roles to make valuable contribution to patient care in the oncology setting.

 
ETIOLOGY OF CANCER

 
Carcinogenesis:

 
    The mechanism by which cancer occur is incompletely understood. Current evidence supports the concept of
carcinogenesis as a multistage process that is genetically regulated.

 
    The first step in the process is initiation, which require exposure of normal cells to carcinogenic substances. The
carcinogens produce genetic damage, that if not repaired, result in irreversible cellular mutations. Chemical
carcinogens, (particularly those in tobacco smoke) as well as azo dyes, aflatoxins, asbestos and benzene have been
implicated in cancer induction in humans and animals. Certain herpes and papilloma group DNA virus have also
been implicated as causative agents in animal cancers.

 
    During the second phase known as promotion, carcinogens or other factors alter the environment to favour the
growth of the mutated cell population. The primary difference between promotion and initiation is that promotion is
a reversible process. In fact because it is reversible, promotion phase may be the target of future chemo prevention
strategies.

 
    The final stage of neoplastic growth called progression involves further genetic changes leading to increased cell
proliferation. The critical elements of this phase include tumor invasion into local tissues and development of
metastases.
 
    Substances that may act as carcinogens include chemical, physical, and biologic agents. Exposure to chemicals
may occur by virtue of occupational and environmental means as well as life style habits the association of aniline
dye exposure and bladder cancer is one such example. physical agents that act as carcinogens include ionizing
radiation and ultraviolet light. These radiations include mutations by forming free radical that damage DNA and
other cellular components. Viruses are biological agents that are associated with certain cancers. The Epstein-Barr
virus is believed to be an important factor in the initiation of African Burkitt's lymphoma. All the previously
mentioned carcinogens, as well as age, gender, diet, growth factors and chronic irritation, are among the factors
considered to be promoters of carcinogenesis.

 
Genetic Basis:

 
    There are two major classes of genes involved in carcinogenesis: oncogenesis and tumor suppressor genes.
Oncogenes develop from normal genes called proto-oncogenes and may have important roles in all phases of
carcinogenesis. Genetic alteration of proto-oncogenes through point mutation, chromosomal rearrangement activates
oncogene.

 
    Tumor-suppressor genes regulate and inhibit cellular growth and proliferation. Gene loss or mutation results in
loss of control over normal cell growth. Two common examples of tumor-suppressor genes are retinoblastoma (Rb)
and p53 genes.

 
DIAGNOSIS

 
The presenting signs and symptoms of vary widely and depend on type of cancer. The presentation in adult may
include:
 Change in bowel or bladder habits
 A sore that doesn't heal
 Unusual bleeding or discharge
 Thickening or lump in breast or elsewhere
 Indigestion or difficulty in swallowing
 Obvious change in wart or mole
 Nagging cough or hoarseness

 
    The definitive diagnosis of cancer relies on procurement of a sample of tissue and its pathological assessment.

 
PATHOLOGY
Tumor characteristics

 
    Tumor may be either benign or malignant. Benign tumors are non-cancerous growths that are often encapsulated,
localized. The benign tumor resembles cells from which they develop. These seldom metastasize and once removed,
they rarely occur. In contrast malignant tumor invade and destroy the surrounding tissue. The cells of malignant
tumor are genetically unstable and loss of normal cell architecture result in cells that are atypical of their tissues or
cells of origin. These loose the ability to perform their usual function. This loss of structure and function is defined
as anaplasia. Recurrences are common after removal or destruction of primary tumor.

 
Tumor Origin
    Tumor may arise from any of the four basic tissue types:
 Epithelial tissue
 Connective tissue
 Lymphoid tissue
 Nerve tissue

 
TREATMENT

 
Modalities of cancer treatment

 
    Four primary modalities employed are:
 surgery
 Radiation
 Chemotherapy
 Biological therapy

 
    Oldest is surgery. It is the treatment of choice for most solid tumors diagnosed in early stages. Although effective,
surgery and radiation are local treatments. Because most patients with cancer have metastatic diseases, localized
therapies often fail to completely eliminate the cancer. In addition systemic diseases such as leukaemia cancer
treatment with a localized modality. Chemotherapy (including hormonal therapy) assesses the systemic circulation
and can theoretically treat primary as well as any metastatic disease. Biological therapy also known as
immunotherapy involves stimulating the host immune system to fight the cancer. Many of the agents of this
category are naturally occurring cytokines, which have been produced with recombinant DNA technology. Eg:
interferons (INFs) , interleukins (ILs)
    Management of most types of cancer involves the use of combined modalities.

 
PRINCIPLES OF CHEMOTHERAPY

 
    The modern era of cancer chemotherapy was born in 1941, when Goodman and Gilman first administered
nitrogen mustard to patients with lymphoma. Since that time, numerous antineoplastic agents have been developed.

 
Response: The response to chemotherapy and other treatment modalities may be described as :
 Cure
 Complete response
 Partial response
 Stable disease
 Progression

 
Basic pharmacology of cancer chemotherapeutic drugs

 
    Agents used in cancer chemotherapy are commonly categorized by their mechanism of action or by their origin.

 
 Polyfunctional alkylating agents:
Cyclophosphamide, mechlorethamine
 
    These exert cytotoxic effects via transfer of their alkyl groups to various cellular constituents. These drugs react
with chemically with sulph hydril, amino, hydroxyl, carboxyl and phosphate groups of other cellular nucleophiles as
well. The general mechanism of action of these drugs involve intramolecular cyclization to form an ethylenimonium
ion.

 
    Active alkylating agents have direct vesicant effects and can damage tissues at the sites of injection as well as
produce systemic toxicity.

 
    Oral administration of alkylating agents agents have been developed using relatively less reactive alkylating
drugs.

 
    All alkylating agent are cytotoxic, mutagenic, teratogenic, carcinogenic and myelosupressive. Resistance to these
sgents can occur from increased DNA repair capabilities from decreased entry into or accelerated exit from cells
from increased inactivation of the agents inside cells or from lack of cellular mechanism to result in cell death
following DNA damage

 
 Antimetabolites:

 
    Neoplastic cells have a number of quantitative differences in metabolism from normal cells that render them more
susceptible to a number of antimetabolites or structural analogues.

 
    The metabolic pathways that have this far proved to be most vulnerable to antimetabolites have been those related
to nucleic acid and nucleotide synthesis in a number of instances where an enzyme is known to have a major effect
on pathways leading to cell replication, inhibitors of the reaction it catalyzes have proved to be useful anticancer
drugs. The principal drugs are: methotrexate, fluorouracil.

 Plant alkaloids:

 
    Vinblastine derived from Vinca rosea. Its mechanism of action involves depolymerisation of microtubules which
are important part of cytoskeleton and mitotic spindle which result in mitotic arrest at metaphase, dissolution of
mitotic spindle, and interference with chromosome segregation.

 
    Vincristine is also an alkaloid derivative of Vinca rosea. It has same mechanism of action as that of vinblastine
and has a strikingly different spectrum of vinblastine and has a strikingly different spectrum of clinical activity and
qualitatively different toxicities. Others of this group are vinorelbine, paclitaxel.

 
 Anti tumor antibiotics:

 
    Screening of microbial products has led to the discovery of a number of a number of growth inhibiting hormones
that have proved to be clinically useful in cancer therapy. Many of these antibiotics bind to DNA through
intercalation between specific bases and block the synthesis of RNA, DNA or both, cause DNA strand scission and
interfere with cell replication. All of the anticancer antibiotics now being used in clinical practice are products of
various strains of the soil microbe streptomycetes. These include the anthracyclines, dactinomycin, bleomycin and
mitomycin.

 
    Anthracyclines isolated from Streptimyces peucetius var caesius, are among the most widely used cytotoxic
anticancer anticancer drugs. Several other anthracycline analogs have entered clinical practice including idarubicin,
epirubicin and mitoxantrone.

 
 Hormonal agents:

 
Steroidal hormones and antisteroidal drugs

 
    Sex hormones and several other hormones are employed in the management of several other types of cancer.
Since sex hormones are actively employed in stimulation and the control of proliferation and function of certain
tissues, including the mammary and prostate glands, cancer arising from other tissues may be inhibited or stimulated
by appropriate changes in hormonal balance. Cancer of breast and of prostate can be effectively treated with sex
hormone therapy or ablation of appropriate endocrine organs.

 
    The mechanism of action of steroid hormones have been partially clarified. Most steroid sensitive cancers express
specific cell surface receptors

 
    Estrogen sensitive breast cancers, prednisone sensitive lymphomas express receptors for estrogen and
corticosteroids. It is now possible to assay tumor specimens for steroid receptors content and to identify which
individual patients are likely to benefit from hormonal therapy. High dose of estrogen is useful therapeutically in
metastatic breast cancer,but is replaced by anti-estrogen therapy.
Estrogen and androgen inhibitors-tamoxifen (antiestrogen)

 
 Biologic response modifiers

 
    The interferons are a family of proteins produced by nucleated cells and by recombinant DNA technology.
    The mechanism of IFN α's anti tumor action are complex. IFN increases the activity of cytotoxic cells within the
immune system,but direct antiproliferative effects also play a role. IFN prolong the cell cycle which result in
cytostasis,an increase in cell size and apoptosis. They can inhibit new blood vessel formation in tumor and can
increase expression of antigen on tumor cell surface making the cancerous cell more easily recognized by thew cell
of immune system. They can also inhibit or block certain oncogenes that can direct an unregulated cell growth that
is characteristic of cancerous cells.

 
    Interleukin-2(IL-2)is a lymphokine produced recombinanatly that has diverse immunologic effects.IL-2 promote
B and T cell proliferation and differentiation and initiate a cytokine cascade with multiple interacting immunologic
effects. Anti tumor effects depends on proliferation of a variety of cytotoxic cells that can recognize and destroy
tumor cells without damaging normal cells. Some of these cytotoxic cells are natural killer (NK)cells,lymphokine-
activated killer(LAT)cells,tumor infiltrating lymphocytes(TIL).

 
 Monoclonal antibodies

 
    The monoclonal antibodies have become important biologic response modifiers used in treatment of cancer. There
are currently four agents approved for use as anti cancer agents within US – trastuzumab,rituximab,gemtuzumab and
alemtuzumab. These agents consist of specific immunoglobulin sequences that are known to recognize a specific
antigen or protein on the surface of cells. There are several mechanisms by which monoclonal antibodies may
induce death of cancer cells. Direct mechanism include induction of apoptosis,blockage of a growth factor
receptor,or induction anti idiotype antibodies. Important indirect mechanism include antibody dependant cellular
toxicity (ADCC)and compliment mediated cellular toxicity.

 
 Heavy metal compounds

 
Cisplatin : it is a platinum complex with broad spectrum of anti tumor activity and remarkable in cancer treatment.

 
    Cisplatin's cytotoxicity depends on platinum binding to DNA and formation of intrastranded cross links or
adducts between neighbouring guanines. These intra strand links cause a major bending of DNA. They may cause
cellular damage by distorting normal DNA conformation and preventing bases that are normally paired from lining
up with each other. Inter strand cross links also occur.

 
    Cisplatin is a highly toxic antineoplastic agent with potential for serious nephrotoxicity and anaemia.

 
    Carboplatin is a structural analogue of cisplatin. It shares same mechanism of action of cisplatin.

 
 Topoisomerase – targetting drugs
    Etoposide and teniposide: etoposide and teniposide are semisynthetic podophyllotoxin derivatives.

 
    DNA topoisomerase enzyme relieve torsional strain during DNA unwinding and by producing strand breaks.
They cleave DNA strand , producing a gap through which DNA strand can pass , then they reseal the strand break.
Topoisomerase 1 form single stranded breake and topoisomerase2 form double stranded break. Etoposide and
teniposide , both form complex with topoisomerase2 and DNA that inhibit strand resealing.

 
    Irinotecan and topotecan poison the action of topoisomerase 1 enzyme.

 
 

INDOLE 3 CARBINOL

 
Alternative name: Indol-3-methanol, 3-indolmethanol
MV= 147,17; SMP= 96-99oC

 
Figure 6

 
Description:
    Indole-3-carbinol or I3C is a breakdown product of the glucosinolate glucobrassicin, also known as indole-3-
glucosinolate. Glucosinolates are beta-thioglucoside N-hydroxysulfates, which are primarily found in cruciferous
vegetables (cabbage, broccoli sprouts, brussels sprouts, cauliflower, bok choy and kale).
    Indole-3-carbinol may have cancer chemopreventive activity. Glucosinolates themselves have minimal anticancer
activity. Indole-3-carbinol is produced from indole-3-glucosinolate via the action of the enzyme myrosinase
(thioglucoside glucohydrolase), an enzyme which is present in cruciferous vegetables and activated upon maceration
of the vegetables.
    The possible anticancer activity of substances such as I3C was recognized by the Roman statesman, Cato the
Elder (234-149 BC), who in his treatise on medicine wrote: "If a cancerous ulcer appears upon the breasts, apply a
crushed cabbage leaf and it will make it well." Crushing a cabbage leaf would convert indole-3-glucosinolate to I3C,
among other reactions.
What is it?
    Indole-3-carbinol is one of the major anticancer substances found in cruciferous (cabbage family) vegetables. It is
a member of the class of sulfur-containing chemicals called glucosinolates.1 It is formed from parent compounds
whenever cruciferous vegetables are crushed or cooked.2
3

    Indole-3-carbinol and other glucosinolates (e.g., other indoles and isothiocyanates such as sulforaphane)
are antioxidants and potent stimulators of natural detoxifying enzymes in the body.4
5
 Indole-3-carbinol and other glucosinolates are believed to be responsible for the lowered risk of cancer in humans
that is associated with the consumption of broccoli and other cruciferous vegetables like cauliflower, cabbage,
and kale.6
7

8
9

10
11
12

    Feeding indole-3-carbinol or broccoli extracts rich in indole-3-carbinol has dramatically reduced the frequency,
size, and number of tumors in laboratory rats exposed to a carcinogen. It appears to be especially protective
against breast13
14

15
16
 and cervical17
18
 cancers because of a number of actions, including an ability to increase the breakdown of estrogen. However,
while most animal studies report protective effects, a few indicate that indole-3-carbinol may actually promote
cancer formation in certain situations, depending upon the chemical initiator of cancer, method of exposure, and
species of animal studied.19

    Until there is further research and more human clinical data to determine if indole-3-carbinol actually inhibits
rather than stimulates cancer formation, some researchers have recommended proceeding with caution when using
isolated indole-3-carbinol as a dietary supplement.21 The areas where its use has currently been documented in
humans are only preliminary, but the results are promising. Indole-3-carbinol reduced or halted the formation of
precancerous lesions (papillomas) in 12 out of 18 people with recurrent respiratory tract papillomas.22 In addition, in
a small double-blind trial, supplementation with 200 or 400 mg of indole-3-carbinol per day for 12 weeks reversed
early-stage cervical cancer in 8 of 17 women.23 Preliminary studies have also shown indole-3-carbinol has
significantly increased the conversion of estrogen from cancer-producing forms to nontoxic breakdown products.24
25

Where is it found?

    Indole-3-carbinol is found in highest concentrations in broccoli, but is also found in other cruciferous vegetables,
such as cauliflower, cabbage, and kale.
Who is likely to be deficient?
    As indole-3-carbinol is not an essential nutrient, no deficiency state exists.
Actions and Pharmacology
Actions

    Indole-3-carbinol may modulate estrogen metabolism. It may also have anticarcinogenic, antioxidant and anti-
atherogenic activities.
Mechanism of Action

    The estrogen metabolites 16 alpha-hydroxyestrone and 4-hydroxyestrone have been demonstrated to be

carcinogens and are thought to be responsible for the possible carcinogenic effects of estrogen. On the other hand,
the estrogen metabolite 2-hydroxyestrone has been found to be protective against several types of cancer, including
breast cancer. Indole-3-carbinol has been shown to increase the ratio of 2-hydroxyestrone to 16 alpha-
hydroxyestrone and also to inhibit the 4-hydroxylation of estradiol. Indole-3-carbinol increases 2-hydroxylation of
estrogens via induction of cytochrome P4501A1 (CYP1A1). Indole-3-carbinol is converted by stomach acid to
diindolymethane (DIM) and indole carbazole (ICZ). DIM and ICZ have similar activities regarding estrogen
metabolism.
    Regarding its possible anticarcinogenic effects, indole-3-carbinol has been shown to modulate the activities of
both Phase I enzymes, such as cytochrome P4501A1, -1A2, -2B1, -2B2, -3A1 and -3A2, and Phase II enzymes, such
as glutathione S-transferase (GST), quinone reductase and uridine glucuronide transferase. Indole-3-carbinol
modulates the metabolism of carcinogens, such as benzo(a)pyrene, aflatoxin B1 and 4-(methylnitrosoamino)-1-(3-
pyridyl)-1-butanone (NNK). Indole-3-carbinol has also been shown to upregulate apoptosis in some cancer cell
lines.
    As mentioned above, indole-3-carbinol induces the synthesis of 2-hydroxyestrone. 2-hydroxyestrone has been
found to inhibit the oxidation of low-density lipoprotein. This indicates that indole-3-carbinol has indirect
antioxidant activity. 2-hydroxyestrone also appears to inhibit smooth muscle proliferation. Inhibition of smooth
muscle proliferation and inhibition of the oxidation of LDL could account for the possible anti-atherogenic activity
of indole-3-carbinol.
    A number of mechanisms exist (that are not mutually exclusive)
whereby I3C (or DIM) can diminish the effects of estrogen on
tumor growth. First, I3C and DIM induce enzymes such as CYP1A1,
which converts estrone to 2-hydroxyestrone and ultimately
results in metabolites that are antiproliferative and proapoptotic. Alternative metabolism (16-hydroxylation) of
estradiol
results in compounds that increase proliferation and anchorage
independent growth. Second, in the case of genes driven
by the estrogen receptor (ER)-, I3C acts as a negative regulator. The tumor suppressor breast cancer 1 (BRCA-1),
whose
expression is upregulated by I3C/DIM, also inhibits the
expression of genes driven by ER-. Moreover, I3C and BRCA-1
work together to abrogate ER-–driven expression.
Using subtractive hybridization, Chen et al. determined
that expression of a battery of genes driven by estrogen was
abrogated by DIM. Speculation is that I3C/DIM and estradiol
modulate the ER and the aryl hydrocarbon receptor. Thus,
estrogen could modulate the activity of I3C/DIM as well. Finally,
in the absence of estrogen, I3C and DIM induce many genes that
have the potential to induce growth arrest and apoptosis and
therefore might counteract the effects of estradiol.

 
Biological Activities

Effects on Biotransformation Enzymes Involved in Carcinogen Metabolism

    Biotransformation enzymes play major roles in the metabolism and elimination of many biologically active
compounds, including steroid hormones, carcinogens toxins and drugs. In general, phase I biotransformation
enzymes, including the cytochrome P450 (CYP) family, catalyze reactions that increase the reactivity of
hydrophobic (fat-soluble) compounds, preparing them for reactions catalyzed by phase II biotransformation
enzymes. Reactions catalyzed by phase II enzymes generally increase water-solubility and promote the elimination
of these compounds .
    Acid-condensation products of I3C, particularly DIM and indole[3,2-b]carbazole (ICZ), can bind to a protein in
the cytoplasm of cells called the aryl hydrocarbon receptor (AhR) . Binding allows the AhR to enter the nucleus
where it forms a complex with the Ahr nuclear translocator (Arnt) protein. This Ahr/Arnt complex binds to specific
DNA sequences in genes known as xenobiotic response elements (XRE) and enhances their transcription. Genes for
a number of CYP enzymes and several phase II enzymes are known to contain XREs. Thus, oral consumption of
I3C results in the formation of acid condensation products that can increase the activity of certain phase I and phase
II enzymes. Increasing the activity of biotransformation enzymes is generally considered a beneficial effect because
the elimination of potential carcinogens or toxins is enhanced. However, there is a potential for adverse effects
because some procarcinogens require biotransformation by phase I enzymes to become active carcinogens.
Alterations in Estrogen Activity and Metabolism

    Endogenous estrogens, including 17beta-estradiol, exert their estrogenic effects by binding to estrogen receptors
(ERs). Within the nucleus, the estrogen-ER complex can bind to DNA sequences in genes known as estrogen
response elements (EREs), recruit coactivator molecules and enhance the transcription of estrogen-responsive genes.
Some ER-mediated effects, such as those that promote cellular proliferation in the breast and uterus, can increase the
risk of developing estrogen-sensitive cancers.
Effects on Estrogen Receptor Activity

    When added to breast cancer cells in culture, I3C has been found to inhibit the transcription of estrogen-
responsive genes stimulated by 17beta-estradiol. Acid condensation products of I3C that bind and activate AhR may
also inhibit the transcription of estrogen-responsive genes by competing for coactivators or increasing ER
degradation. In contrast, some studies in cell culture and animal models have found that acid-condensation products
of I3C enhance the transcription of estrogen-responsive genes. Further research is needed to determine the nature of
the stimulatory and inhibitory effects of I3C and its acid-condensation products on estrogen-responsive gene
transcription under conditions that are relevant to human cancer risk.
Effects on Estrogen Metabolism
    The endogenous estrogen 17beta-estradiol can be irreversibly metabolized to 16alpha-hydroxyestrone (16OHE1)
or 2-hydroxyestrone (2OHE1). In contrast to 2OHE1, 16OHE1 is highly estrogenic and has been found to stimulate
the proliferation of several estrogen-sensitive cancer cell lines. It has been hypothesized that shifting the metabolism
of 17beta-estradiol toward 2OHE1 and away from 16OHE1 could decrease the risk of estrogen sensitive cancers,
such as breast cancer. In controlled clinical trials, oral supplementation with 300-400 mg/day of I3C has consistently
increased urinary 2OHE1 levels or urinary 2OHE1:16OHE1 ratios in women. Supplementation with 108 mg/day of
DIM also increased urinary 2OHE1 levels in postmenopausal women. However, the relationship between urinary
2OHE1:16OHE1 ratios and breast cancer risk is not clear. Although women with breast cancer had lower urinary
ratios of 2OHE1:16OHE1 in several small case-control studies , larger case-control and prospective cohort studies
have not found significant associations between urinary 2OHE1:16OHE1 ratios and breast cancer risk.
Induction of Cell Cycle Arrest

    Once a cell divides, it passes through a sequence of stages collectively known as the cell cycle before it divides
again. Following DNA damage, the cell cycle can be transiently arrested at damage checkpoints which allow for
DNA repair or activation of pathways leading to cell death (apoptosis) if the damage is irreparable. Defective cell
cycle regulation may result in the propagation of mutations that contribute to the development of cancer. The
addition of I3C to prostate and breast cancer cells in culture has been found to induce cell cycle arrest. However, the
physiological relevance of these cell culture studies is unclear since little or no I3C is available to the tissue after
oral administration.
Induction of Apoptosis

    Unlike normal cells, cancerous cells lose their ability to respond to death signals by undergoing apoptosis. I3C
and DIM have been found to induce apoptosis when added to cultured prostate, breast and cervical cancer cells.
Inhibition of Tumor Invasion and Angiogenesis

    Limited evidence in cell culture experiments suggests that I3C and DIM can inhibit the invasion of normal tissue
by cancer cells and inhibit the development of new blood vessels (angiogenesis) required by tumors to fuel their
rapid growth.
Disease Prevention

Cancer

Epidemiological Studies

    Epidemiological studies provide some support for the hypothesis that higher intakes of cruciferous vegetables are
associated with lower risk for some types of cancer. However, cruciferous vegetables are relatively good sources of
other phytonutrients that may have protective effects against cancer, including vitamin C, folate, selenium,
carotenoids and fiber. Moreover, cruciferous vegetables provide a variety of glucosinolates that may be hydrolyzed
to a variety of potentially protective isothiocyanates, in addition to indole-3-carbinol. Consequently, evidence for an
inverse association between cruciferous vegetable intake and cancer risk provides relatively little information about
the specific effects of indole-3-carbinol on cancer risk.
Animal Studies

    In most animal models, exposure to a chemical carcinogen is required to cause cancer. When administered before
or at the same time as the carcinogen, oral I3C has been found to inhibit the development of cancer in a variety of
animal models and tissues, including cancers of the mammary gland (breast), stomach, colon, lung and liver.
However, a number of studies have found that I3C actually promoted or enhanced the development of cancer when
administered chronically after the carcinogen (post initiation). The cancer promoting effects of I3C were first
reported in a trout model of liver cancer. However, I3C has also been found to promote cancer of the liver, thyroid,
colon and uterus in rats. Although the long-term effects of I3C supplementation on cancer risk in humans are not
known, the contradictory results of animal studies have led several experts to caution against the widespread use of
I3C and DIM supplements in humans until their potential risks and benefits are better understood.
Disease Treatment
Diseases Related to Human Papilloma Virus Infection

Cervical Intraepithelial Neoplasia

    Infection with certain strains of human papilloma virus (HPV) is an important risk factor for cervical cancer.
Transgenic mice that express cancer-promoting HPV genes develop cervical cancer with chronic 17beta-estradiol
administration. In this model, feeding I3C markedly reduced the number of mice that developed cervical cancer. A
small placebo-controlled trial in women examined the effect of oral I3C supplementation on the progression of
precancerous cervical lesions classified as cervical intraepithelial neoplasia (CIN) 2 or CIN 3. After 12 weeks, four
out of the eight women who took 200 mg/day had complete regression of CIN and four out of the nine who took 400
mg/day had complete regression, while none of the ten who took a placebo had complete regression. Although these
preliminary results are encouraging, larger controlled clinical trials are needed to determine the efficacy of I3C
supplementation for preventing the progression of precancerous lesions of the cervix.

Recurrent Respiratory Papillomatosis

    Recurrent respiratory papillomatosis (RRP) is a rare disease of children and adults, characterized by generally
benign growths (papillomas) in the respiratory tract caused by HPV infection. These papillomas occur most
commonly on or around the vocal cords in the larynx (voice box), but they may also affect the trachea, bronchi and
lungs. The most common treatment for RRP is surgical removal of the papillomas. Since papillomas often recur,
adjunct treatments may be used to help prevent or reduce recurrences. In immune-compromised mice transplanted
with HPV-infected laryngeal tissue, only 25% of the mice fed I3C developed laryngeal papillomas, compared to
100% of the control mice. In a small observational study of RRP patients, increased ratios of urinary
2OHE1:16OHE1 ratios resulting from increased cruciferous vegetable consumption were associated with less severe
RRP. Most recently, an uncontrolled pilot study examined the effect of daily I3C supplementation (400 mg for
adults and 10 mg/kg daily for children) on papilloma recurrence in RRP patients. Over a five-year follow-up period,
11 of the original 49 patients experienced no recurrence, 10 experienced a reduction in the rate of recurrence, 12
experienced no improvement, and 12 were lost to follow-up. Although the low toxicity of I3C makes it an attractive
adjunct therapy for RRP, controlled clinical trials are needed to determine whether I3C is effective in preventing or
reducing the recurrence of respiratory papillomas.

 
Systemic Lupus Erythematosus

    Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by chronic inflammation that may
result in damage to the joints, skin, kidneys, heart, lungs, blood vessels or brain. Estrogen is thought to play a role in
the pathology of SLE because the disorder is much more common in women than men, and its onset is most
common during the reproductive years when endogenous estrogen levels are highest. The potential for I3C
supplementation to shift endogenous estrogen metabolism toward the less estrogenic metabolite 2OHE1 and away
from the highly estrogenic metabolite 16OHE1 led to interest in its use in SLE (25). In an animal model of SLE, I3C
feeding decreased the severity of renal (kidney) disease and prolonged survival. A small uncontrolled trial of I3C
supplementation (375 mg/day) in female SLE patients found that I3C supplementation increased urinary
2OHE1:16OHE1 ratios, but found no significant change in SLE symptoms after 3 months. Controlled clinical trials
are needed to determine whether 13C supplementation will have beneficial effects in SLE patients.

Pharmacokinetics
    There is much unknown about the pharmacokinetics of indole-3-carbinol in humans. It is converted to DIM and
ICZ by stomach acid, and DIM and ICZ are absorbed from the gastrointestinal tract. The extent of absorption of
I3C, DIM and ICZ, as well as their distribution, metabolism and excretion, are currently being studied.

Metabolism and Bioavailability

    A number of commonly consumed cruciferous vegetables, including broccoli, Brussels sprouts and cabbage, are
good sources of glucobrassicin, the glucosinolate precursor of I3C. Myrosinase, an enzyme that catalyzes the
hydrolysis of glucosinolates, is physically separated from glucosinolates in intact plant cells (3). When plant cells
are damaged, as when cruciferous vegetables are chopped or chewed, the interaction of myrosinase and
glucobrassicin results in the formation of I3C. In the acidic environment of the stomach, I3C molecules can combine
with each other to form a complex mixture of biologically active compounds, known collectively as acid
condensation products. Although numerous acid condensation products of I3C have been identified, some of the
most prominent include the dimer 3,3'-diindolylmethane (DIM) and a cyclic trimer (CT). The biological activities of
individual acid condensation products differ from those of I3C and are responsible for the biological effects
attributed to I3C. When plant myrosinase is inactivated (e.g., by boiling), glucosinolate hydrolysis still occurs to a
lesser degree, due to the myrosinase activity of human intestinal bacteria . Thus, when cruciferous vegetables are
cooked in a manner that inactivates myrosinase, glucobrassicin hydrolysis by intestinal bacteria still results in some
I3C formation. However, acid condensation products would be less likely to form in the more alkaline environment
of the intestine.

Safety

Adverse effects
    Slight increase in serum concentration of a liver enzyme (alanine aminotransferase; ALT) were observed in two
women who took unspecified doses of 13 C supplements for four weeks. One person reported a skin rash while
taking 375mg/kg of 13C. High doses of 13C were associated with symptoms of disequilibrium and tremor, which
resolved when the dose decreased. The effects of 13C or DIM supplementation on cancer risk in humans are not
known.

Pregnancy and lactation

The safety of 13C or DIM supplements during pregnancy or lactation has not been established.

Drug interaction
    No drug interaction in humans have been reported.
    Antacids, H2 blocker , proton pump inhibitors: The conversion of 13C to DIM or ICZrequire stomach acid. It is
unclear if indole 3 carbinol itself would have all the possible activities mentioned above if it were not converted to
DIM or ICZ.
    Tamoxiphen : indole 3 carbinol may be synergestic with tamoxiphen in protecting against breast cancer.

Contraindications

    Indole-3-carbinol is contraindicated in those hypersensitive to this substance or to any component of an indole-3-

carbinol-containing product.

Precautions
    Pregnant women and nursing mothers should avoid indole-3-carbinol supplements pending long-term safety
studies. Those with cancer should confer with their physician before deciding to use indole-3-carbinol.

Dosage and Administration

    Indole-3-carbinol is available as a stand-alone supplement and in combination products. Dosage ranges from 200
mg to 800 mg daily.
    Indole-3-carbinol, as well as diindolylmethane, are available in combination formulas used by some body
builders.

Clinical Trials
Indole-3-Carbinol in Preventing Cancer in Healthy Participants

This study is currently recruiting patients

 
Purpose
    RATIONALE: Chemoprevention is the use of certain drugs to keep cancer from forming, growing, or coming
back. The use of indole-3-carbinol may prevent cancer.
    PURPOSE: This randomized phase I trial is studying the side effects and best dose of indole-3-carbinol and to see
how well it works compared to placebo in preventing cancer in healthy participants.
Condition   Intervention  Phase 
 Drug: indole-3-carbinol
 Procedure: biologically
based therapies
 Procedure: cancer
prevention intervention
 Procedure: chemopreven
unspecified adult solid tumor, protocol specific  tion of cancer Phase I
 Procedure: complementa
ry and alternative therapy
 Procedure: dietary
intervention
 Procedure: nutritional
supplementation

 
Study Type: Interventiona
Study Design: Prevention
Official Title: Phase I Randomized Chemoprevention Study of Indole-3-Carbinol in Healthy Participants
Further Study Details: 

Objectives
Primary
 Determine the maximum tolerated dose of indole-3-carbinol in healthy participants.
 Determine the safety and tolerability of this drug in these participants.
 Determine the pharmacokinetics of this drug in these participants.
 Secondary
  Determine the effects of this drug on selected markers of sexual function in these participants.
 Determine the effects of this drug on markers of susceptibility to cancer in these participants.

Outline

This is a randomized, double-blind, placebo-controlled, dose-escalation study. Participants at each dose level are
randomized to 1 of 2 treatment arms.
 Arm I: Participants receive a single dose of oral indole-3-carbinol on day 1.
 Arm II: Participants receive a single dose of oral placebo on day 1. Cohorts of 3 participants
receive escalating doses of indole-3-carbinol until the maximum tolerated dose (MTD) is determined.
The MTD is defined as the dose preceding that at which 1 of 3 participants experiences dose-limiting
toxicity. An additional cohort of 3 participants is treated at the MTD.
Participants are followed on days 2, 3, and 6.
PROJECTED ACCRUAL: A total of 24 participants (18 in arm I and 6 in arm II) will be accrued for this study.

Eligibility
Ages Eligible for Study:  18 Years   - 70 Years,  Genders Eligible for Study:  Both
Accepts Healthy Volunteers
Criteria

Disease characteristics
 Healthy participants
 Non-smoker
 No drug abuse, as determined by urine cotinine and baseline drug screen

Patient characteristics
Age
 18 to 70
Performance status
 Not specified
Life expectancy
 At least 12 months
Hematopoietic
 Absolute granulocyte count > 1,500/mm^3
 Hemoglobin > 10 g/dL

 
Hepatic
 Bilirubin < 1.8 mg/dL
 AST and ALT < 110 U/L
 Alkaline phosphatase < 300 U/L
Renal
 Creatinine < 2.0 mg/dL
  Albumin > 3.0 g/dL
Pulmonary
 No asthma
Other
 Not pregnant or nursing
 Negative pregnancy test
 Weight within 20% of ideal body weight by the Metropolitan Life table
 No serious drug allergies
 No arthritis
 No acute, unstable, chronic, or recurring medical condition
 No strict vegetarians
 No diabetes
 No evidence of an active malignancy
 No other serious intolerance or allergies
 Mild seasonal allergies allowed
 No other serious acute or chronic illness
 None of the following chronic conditions:
 Headaches
 Dysphoria
 Fatigue
 Dizziness
 Blurred vision
 Insomnia
 Rhinorrhea
 Nausea
 Vomiting
 Abdominal pain
 Diarrhea
 Constipation
 Premenstrual syndrome
 Cessation of menses within the past 10 days (menstruating women only)

Prior concurrent therapy

Biologic therapy
 Not specified
Chemotherapy
 No prior chemotherapy
Endocrine therapy
 Concurrent oral contraceptives allowed
Radiotherapy
 Not specified
Surgery
 Not specified
 
Other
 More than 21 days since prior medications, herbal products, dietary supplements, or high-dose
vitamins
 More than 3 months since prior investigational drugs
 At least 14 days since prior and no concurrent ingestion of cruciferous vegetables, including any
of the following:
 Broccoli
 Cabbage, including coleslaw
 Cauliflower
 Bok-choy
 Brussels sprouts
 Collards
 Kale
 Kohlrabi
 Mustard greens
 Rutabaga
 Turnip
 Watercress
 At least 7 days since prior and no concurrent alcohol consumption
 At least 48 hours since prior ingestion of grapefruit-containing foods and beverages
 No concurrent chronic drug therapy
 No other concurrent supplements, including dietary supplements, vitamins, herbal products, or
over-the-counter medications

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells
    The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation
therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The
naturally occurring chemical indole-3- carbinol (I3C), found in vegetables of the Brassica genus, is a promising
anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines,
independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit
the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone.
This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage- independent growth,
reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also
caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2- specific enzymatic activity than either
compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen
ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs,
whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence
suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for
estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate
that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast
cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.

Inhibitory effects of Indole-3-carbinol on invasion and migration in human breast cancer cells
    Indole-3-carbinol (I3C) is a promising phytochemical agent in chemoprevention of breast cancer. Our present
study is the first description of I3C that significantly inhibits the cell adhesion, spreading and invasion associated
with an up-regulation of PTEN (a tumor suppressor gene) and E-cadherin (a regulator of cell-cell adhesion)
expression in T47-D human breast cancer cells. Therefore, I3C exhibits anti-cancer activities by suppressing breast
tumor cell growth and metastatic spread. Metastatic breast cancer is a devastating problem, clinical application of
I3C as a potent chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis.

Indole 3 carbinol is a negative regulator of Estrogen

Abstract
    Studies increasingly indicate that dietary indole-3-carbinol
(I3C) prevents the development of estrogen-enhanced cancers
including breast, endometrial and cervical cancers. Epidemiological,
laboratory, animal and translational studies support the efficacy
of I3C. Whereas estrogen increases the growth and survival of
tumors, I3C causes growth arrest and increased apoptosis and
ameliorates the effects of estrogen. Our long-range goal is
to best use I3C together with other nutrients to achieve maximum
benefits for cancer prevention. This study examines the possibility
that induction of growth arrest in response to DNA damage (GADD)
in genes by diindolylmethane (DIM), which is the acid-catalyzed
condensation product of I3C, promotes metabolically stressed
cancer cells to undergo apoptosis. We evaluated whether genistein,
which is the major isoflavonoid in soy, would alter the ability
of I3C/DIM to cause apoptosis and decrease expression driven
by the estrogen receptor (ER)-. Expression of GADD was evaluated
by real-time reverse transcription–polymerase chain reaction.
Proliferation and apoptosis were measured by a mitochondrial
function assay and by fluorescence-activated cell sorting analysis.
The luciferase reporter assay was used to specifically evaluate
expression driven by ER-. The estrogen-sensitive MCF-7 breast
cancer cell line was used for these studies. We show a synergistic
effect of I3C and genistein for induction of GADD expression,
thus increasing apoptosis, and for decrease of expression driven
by ER-. Because of the synergistic effect of I3C and genistein,
the potential exists for prophylactic or therapeutic efficacy
of lower concentrations of each phytochemical when used in combination.

Results
    It was hypothesized that certain genes may be instrumental in determining
how I3C/DIM ameliorates estrogen induction of tumor growth and
eventually causes growth arrest and apoptosis of these cells
(Fig. 1). We considered the involvement of GADD because of
our recent information that their expression is robustly upregulated
by DIM. GADD are a group of proteins (GADD-153, GADD-45,
-ß and - and GADD-34) that induce growth arrest and
apoptosis by different pathways. Moreover, BRCA-1, which is
induced by I3C/DIM, not only induces expression to GADD-45
but also inhibits estrogen signaling that is dependent on ER-. Finally, as shown below, genistein also can induce
expression
of GADD and can affect ER signaling.

 
FIGURE 1  Model for a subset of genes induced by indole-3-carbinol/diindolylmethane (I3C/DIM) that may
counteract estrogen-enhanced tumors.

 
DIM and genistein synergistically induce GADD

 
    Genistein, an isoflavonoid from soy that is considered to be
an anticancer phytochemical, inhibits glucose-regulated
protein. This activity would counteract the protective
response to ER stress, and raises the possibility that genistein
could be an adjunct to I3C/DIM by modulation of the endoplasmic
reticulum stress response in the direction of increased growth
arrest and apoptosis. We first asked how genistein might affect
the expression of GADD. We evaluated effects of DIM and genistein
separately and together on the expression of GADD and used real-time
RT-PCR to measure the response in estrogen-sensitive MCF-7 cells.
As shown in Figure 2A, a short-time (6-h) treatment with either
DIM (100 or 50 µmol/L) or genistein (5 or 25 µmol/L)
increases expression of GADD-34. Very little increase is detected
using 25 µmol DIM/L. However the combination of 5 µmol
genistein/L and 25 µmol DIM/L results in a synergistic
increase in the expression of this GADD. Results also are shown
for GADD-153 ( Fig. 2B) and GADD-45 ( Fig. 2C). Similar results
occur with the other isoforms of GADD-45 and are virtually identical
in C33A cells (unpublished data).

 
FIGURE 2  DIM and genistein synergistically induce growth arrest in response to DNA damage (GADD). MCF-7
cells were treated with dimethyl sulfoxide (DMSO, a solvent control), genistein, DIM or both DIM and genistein
for 16 h. The cDNA made from mRNA was used as a template to amplify GADD-34 (A), GADD-153 (B), GADD-
45 (C) and ß-actin by real-time reverse transcription–polymerase chain reaction (RT-PCR) as described in Methods
and Materials. Treatment of cells was DMSO (1), 100 µmol DIM/L (2), 50 µmol DIM/L (3), 25 µmol DIM/L (4),
25 µmol genistein/L (5), 5 µmol genistein/L (6) and the combination of 25 µmol DIM/L + 5 µmol genistein/L (7).
The amount of estrogen in media (10% fetal calf serum) was 10-15 mol/L.
 

 
DIM and genistein synergistically increase apoptosis

    If DIM and genistein synergistically induce GADD, then growth

arrest and apoptosis should be a consequence of this induction.
We used two methods [a mitochondrial function assay and fluorescence-activated
cell sorting (FACS) analysis] to evaluate growth arrest and
apoptosis. In the mitochondrial function assay ( Fig. 3), DIM
and genistein work together to decrease cell viability. When
increasing concentrations of DIM are used together with 5 µmol
genistein/L (a concentration of genistein that enhances growth
in MCF-7 cells when used alone), increased killing of cells
occurs at concentrations of DIM as low as 20 µmol/L ( Fig.
3A) compared to the requirement for DIM concentrations to be
>50–60 µmol/L when cells are exposed to DIM alone.
DIM (50 µmol/L) counteracts the proliferative effect of
genistein, and genistein (used at increasing concentrations)
potentiates cell killing by DIM ( Fig. 3B). Using FACS analysis
( Fig. 4A), the profiles of subdiploid (putative apoptotic cells,
M1), G1 (M2), S (M3) and G2 (M4) are identical for cells treated
for 24 h with DMSO (solvent control), 25 µmol DIM/L or
5 µmol genistein/L. However, the fraction of putatively
apoptotic cells is dramatically increased when cells are treated
with the combination of both DIM and genistein ( Fig. 4, A and
B). Results with C33A cells (both assays) are identical to those
of MCF-7 cells.

 
FIGURE 3  DIM and genistein work together to decrease viability. MCF-7 cells were treated for 72 h with DMSO
(solvent controls), or 5 µmol genistein/L and increasing amounts of DIM from 0 to 100 µmol/L (A) or 50 µmol
DIM/L and increasing amounts of genistein from 0 to 50 µmol/L (B). A minimum of six replicate wells was
employed per condition. Viability was determined by a mitochondrial function assay [3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)]. The amount of estrogen in media (10%
fetal calf serum) was 10-15 mol/L.

 
FIGURE 4  DIM and genistein synergistically increase apoptotic cells. MCF-7 cells were treated with DMSO,
25 µmol DIM/L, 5 µmol genistein/L or 25 µmol DIM/L + 5 µmol genistein/L. Cells were treated for 48 h, stained
with propidium iodide and sorted by fluorescence (abscissa). (top and middle panels) M1, subN2 cells; M2, G1;
M3, S; M4, G2/M. (bottom panel) The gated percent values of M1 cells for the four conditions were 6.5, 4.8, 4.4
and 42.3%.

 
DIM and genistein synergistically decrease estrogen signaling driven by ER-

 
    Because genistein is a weak estrogen but able to compete
with estradiol for the ER, we wanted to know what the
effect of I3C and genistein together have on the expression
of genes driven by the ER- ( Fig. 5). Using MCF-7 cells treated
with I3C (50 µmol/L), genistein (25 µmol/L) or the
combination of I3C and genistein, we evaluated the amount of
luciferase that genes produce when driven by an estrogen-responsive
enhancer as described previously. I3C decreases estrogen-driven
luciferase activity. Treatment with genistein results in an
even greater decrease. Treatment with the two phytochemicals
reduces expression significantly more than would have been predicted
if the effect of the two phytochemicals were additive, which
indicates a clearly synergistic effect.

 
FIGURE 5  DIM and genistein synergistically inhibit estrogen signaling. MCF-7 monolayers were transfected with
a plasmid that expresses estrogen receptor- and a plasmid for luciferase expression driven by the estrogen response
element (ERE). The cells were then treated with ±1 µmol estradiol/L, ±25 µmol/L genistein and ±50 µmol I3C/L
and were assayed for luciferase activity 24 h later as described in Methods and Materials. The final bar is not
experimental data but the theoretical value if the effects of genistein and I3C had been additive.

 
Discussion
    We provide insight into additional mechanisms whereby I3C/DIM
can counteract the growth and survival of tumors in estrogen-sensitive
cells. We performed additional analysis that confirms that not
only does DIM induce GADD, but also that DIM and genistein synergistically
induce expression of GADD. Consistent with their effects on
the induction of GADD proteins, genistein and DIM work better
together than alone to increase apoptosis. Another way by which
I3C/DIM can lead to the growth arrest of estrogen-sensitive
cancer cells is by interfering with estrogen signaling. Here,
too, genistein and DIM are synergistic in inhibiting estrogen
signaling by ER-.

    Our discovery that DIM induces GADD and other proteins involved
in the endoplasmic reticulum stress response not only
supports the possibility that GADD contribute to the growth
arrest and apoptosis associated with I3C/DIM, but also may answer
(at least in part) why I3C/DIM seems to specifically target
tumor cells opposed to normal cells. The importance of the tumor
microenvironment in malignant progression has received much
less attention in the literature than the cellular events that
trigger oncogenesis. Tumor cells protect themselves from changes
in the microenvironment such as decreased availability of oxygen
and nutrients by engaging a biochemical pathway called the metabolic
stress response. In vivo cancer cells are likely to be chronically
stressed. The cellular response to hypoxia, hypoglycemia and
nutrient starvation includes the synthesis of protective proteins
and cell cycle arrest, which can lead to apoptosis or survival
and also can involve induction of genes that promote angiogenesis
and tissue remodeling. In other words, the fate of the stressed
cell is survival by adaptation to the stressful conditions or
elimination by programmed cell death. Obviously, the desired
outcome for cancer prevention is growth arrest and apoptosis.

    The fact that genistein works synergistically with I3C/DIM has

a number of implications. Importantly (at least for induction
of GADD, apopotosis and inhibition of estrogen-increased gene
expression), the concentrations of these phytochemicals used
in vitro to achieve these activities are more in line with concentrations
that people acquire from eating the relevant foods. Additionally,
people are exposed to combinations of foods and their bioactive
constituents. Although sorting out how diets ultimately may
affect a cell necessarily involves evaluating individual nutrients,
the study of interactions between nutrients (especially well-studied
nutrients) is the next step. Clearly, the Asian diet, which
is considered protective against breast and some other cancers,
must involve many bioactive compounds and their interactions.

Dose-ranging study of indole-3-carbinol for breast cancer prevention

    Sixty women at increased risk for breast cancer were enrolled in a placebo-controlled, double-blind dose-ranging
chemoprevention study of indole-3-carbinol (I3C). Fifty-seven of these women with a mean age of 47 years (range
22-74) completed the study. Each woman took a placebo capsule or an I3C capsule daily for a total of 4 weeks; none
of the women experienced any significant toxicity effects. The urinary estrogen metabolite ratio of 2-hydroxyestrone
to 16 alpha- hydroxyestrone, as determined by an ELISA assay, served as the surrogate endpoint biomarker (SEB).
Perturbation in the levels of SEB from baseline was comparable among women in the control (C) group and the 50,
100, and 200 mg low-dose (LD) group. Similarly, it was comparable among women in the 300 and 400 mg high-
dose (HD) group. Regression analysis showed that peak relative change of SEB for women in the HD group was
significantly greater than that for women in the C and LD groups by an amount that was inversely related to baseline
ratio; the difference at the median baseline ratio was 0.48 with 95% confidence interval (0.30, 0.67). No other
factors, such as age and menopausal status, were found to be significant in the regression analysis. The results in this
study suggest that I3C at a minimum effective dose schedule of 300 mg per day is a promising chemopreventive
agent for breast cancer prevention. A larger study to validate these results and to identify an optimal effective dose
schedule of I3C for long-term breast cancer chemoprevention will be necessary.

 
Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer
cells

     Prostate cancer is one of the most common cancers in men and it is the second leading cause of cancer related
death in men in the United States. Recent dietary and epidemiological studies have suggested the benefit of dietary
intake of fruits and vegetables in lowering the incidence of prostate cancer. A diet rich in fruits and vegetables
provides phytochemicals, particularly indole-3-carbinol (I3C), which may be responsible for the prevention of many
types of cancer, including hormone-related cancers such as prostate. Studies to elucidate the role and the molecular
mechanism(s) of action of I3C in prostate cancer, however, have not been conducted. In the current study, we
investigated whether I3C had any effect against prostate cancer cells and, if so, attempts were made to identify the
potential molecular mechanism(s) by which I3C elicits its biological effects on prostate cancer cells. Here we report
for the first time that I3C inhibits the growth of PC-3 prostate cancer cells. Induction of G1 cell cycle arrest was also
observed in PC-3 cells treated with I3C, which may be due to the observed effects of I3C in the up-regulation of
p21(WAF1) and p27(Kip1) CDK inhibitors, followed by their association with cyclin D1 and E and down-
regulation of CDK6 protein kinase levels and activity. The induction of p21(WAF1) appears to be transcriptionally
upregulated and independent of the p53 responsive element. In addition, I3C inhibited the hyperpohosphorylation of
the Retinoblastoma (Rb) protein in PC-3 cells. Induction of apoptosis was also observed in this cell line when
treated with I3C, as measured by DNA laddering and poly (ADP-ribose) polymersae (PARP) cleavage. We also
found an up-regulation of Bax, and down-regulation of Bcl-2 in I3C-treated cells. These effects may also be
mediated by the down-regulation of NF-kappaB observed in I3C treated PC-3 cells. From these results, we conclude
that I3C inhibits the growth of PC-3 prostate cancer cells by inducing G1 cell cycle arrest leading to apoptosis, and
regulates the expression of apoptosis-related genes. These findings suggest that I3C may be an effective
chemopreventive or therapeutic agent against prostate cancer.

 
Indole-3-Carbinol with Standardized Broccoli 200 mg/150 mg, 60 capsules

    Indole-3-carbinol is a plant compound from cruciferous vegetables, such as broccoli and cauliflower, that has
shown impressive results in maintaining healthy cell growth.49-52* Broccoli contains several compounds that help
maintain healthy DNA function.* The broad-spectrum benefits of cruciferous vegetables have now been combined
into one capsule. The proprietary 3:1 blend of broccoli sprout concentrate provides sulforaphane for immediate
absorption and glucosinolates, which must be broken down in the body to provide a broad spectrum of
isothiocyanates.
    Unlike other products on the market that contain only the isothiocyanate sulforaphane, I3C with Standarized
Broccoli contains standardized levels of glucosinolates. Glucosinolates are precursors or the "parent" compound that
include a natural spectrum of isothiocyanates. Glucosinolates are sulfur-containing phytonutrients, which are part of
the genus Brassicaceae. It has been estimated that there are over 120 different glucosinolates known to exist in
nature. Epidemiological (population) studies have demonstrated that glucosinolates found in these vegetables have
health benefits and show supporting effects in maintaining healthy cells in the stomach, lung, and colon.

 
Figure 7

 
Supplement Facts

Serving Size 1 capsule

Servings Per Container 60
Amount Per Serving

Indole-3-Carbinol
200 mg
Broccoli sprout concentrate (Brassica oleracea) [standardized for 0.3% sulforaphane (0.45 mg), 1% total
glucosinolates (1.5 mg)]
150 mg
Other ingredients: rice flour, gelatin, magnesium stearate, titanium dioxide.

 
Dosage and Use

 One capsule per day for individuals weighing up to 179 pounds.

 Those weighing over 239 pounds, take two capsules per day.
 Indole-3-Carbinol with Standardized Broccoli also comes in a 300 mg dose for those weighing
180-239 pounds and only requires one capsule per day.
 This product can be taken with or without food.
 
Warning

 
 Keep out of reach of children
 Do not exceed recommended dose.
 Do not purchase if outer seal is broken or damaged.
 If you have a bad reaction to product discontinue use immediately
 When using nutritional supplements, please inform your physician if you are undergoing treatment
for a medical condition or if you are pregnant or lactating.

 
CONCLUSION

 
Metastatic breast cancer is a devastating problem. Clinical application of indole 3 carbinol as a potent
chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis. Many evidences suggest
that intake of cruciferous vegetables which are good source of indole 3 carbinol will help in lowering the risk of
some types of cancers.

 
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