![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
3,
*
North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030 and Department of Otolaryngology, Long Island Jewish Medical Center, The Long Island Campus of Albert Einstein College of Medicine, New Hyde Park, NY 11030; and
Department of Biological Sciences, St. John’s University, Jamaica, NY 11439
3To whom correspondence should be addressed. E-mail: carterth{at}aol.com.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • cervical cancer • indole-3-carbinol • diindolylmethane • apoptosis • mice
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). A diet rich in cruciferous vegetables (2
) or supplements of I3C (3
) causes regression of tumors or decreases their rate of growth or recurrence in two thirds of patients with recurrent laryngeal papillomatosis. Clinical trials are planned to test the efficacy of I3C as a preventive treatment for breast cancer (4
). Laboratory studies suggest that this phytochemical can act in several different ways to prevent transformation and/or tumor progression, as well as to kill transformed cells selectively.
I3C is rapidly converted in the stomach to a variety of condensation products, chiefly diindolylmethane (DIM) (5
). Plasma from humans and rats fed I3C contains no detectable I3C, but large amounts of DIM, as well as other metabolites, some of which remain uncharacterized (6
; L. Bjeldanes, University of California at Berkeley, personal communication). Thus DIM, rather than I3C, is probably the major compound initially available to cells after ingestion of I3C. I3C is also converted slowly to DIM at neutral pH (5
), with the result that either compound is active in vitro. For example, both I3C and DIM induce apoptosis in MCF-7 breast carcinoma cells growing in culture (7
,8
).
We showed previously that dietary I3C prevents the appearance of cervical cancer after chronic estrogen exposure in transgenic mice expressing the type 16 human papillomavirus (HPV) oncogenes (9
). This effect is accompanied by a shift in estrogen metabolism to favor the production of 2-OH estrone rather than the 16
-OH metabolite, which is associated with prolonged estrogenic activity and carcinogenesis (our unpublished data)(10
,11
). Thus, it is likely that one of the major pathways by which I3C and its derivatives prevent the onset of cervical cancer involves alteration of estrogen metabolism by inducing specific cytochrome P450 isoforms via the aryl hydrocarbon receptor (9
,10
) for which DIM is a weak ligand (12
,13
).
However, we observed that estradiol and I3C act in opposing ways on the balance of proliferation of cervical cancer cells (unpublished data), and others have reported that the inhibitory effect of I3C on MCF-7 breast cancer cells is independent of estrogen signaling (14
). Therefore, at least in vitro, there must be an additional, estrogen-independent pathway by which I3C interferes with the establishment and progression of malignancy. Both I3C and DIM induce apoptotic changes in breast cancer cells in vitro (7
,8
). However, the published clinical data on therapeutic use of I3C involve laryngeal papillomas and cervical cancer, which are both HPV-associated diseases. It is therefore important to determine whether I3C and DIM can induce apoptosis in a variety of cervical cancer cell lines, including those with and without HPV genes, and whether apoptotic changes occur in target tissues in vivo. We looked for apoptotic cells in dysplastic cervical epithelium of HPV16 transgenic mice fed I3C under conditions that would otherwise lead to the development of cervical neoplasia. Furthermore, we asked whether normal cells in culture, as well as normal cervical tissue, are affected by I3C/DIM. Our results indicate that the ability of I3C and DIM to induce apoptosis extends to a variety of cervical cancer cell lines and to preneoplastic cervical epithelium. This effect appears to be specific for transformed cells, raising the possibility that these dietary phytochemicals may be generally useful as therapeutic agents.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
17ß-Estradiol (E2) and I3C were purchased from Sigma (St. Louis, MO). Diindolylmethane was a gift from Dr. M. Zeligs, BioResponse, Boulder, CO.
Cell lines and cell culture.
The cervical cancer cell lines CaSki (containing multiple copies of integrated HPV16 DNA) and C33A (HPV negative, mutant p53) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). C33AE6 cells (15
) were stably transfected with the HPV16 E6 gene (pLXSN16E6 from D. Galloway, Fred Hutchinson Cancer Research Center, Seattle, WA) and express low levels of E6 transcripts (15
). All cells were maintained as monolayer cultures at 37°C, 7% CO2. Cervical cancer cells and 3T3 fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose and bicarbonate (GIBCO-BRL, Gaithersburg, MD), supplemented with 110 mg/L sodium pyruvate, 200 mmol/L glutamine, 100 mL/L fetal bovine serum, and 1 x 105 U/L each of penicillin and streptomycin. Normal human foreskin keratinocytes were grown in F12-DMEM on feeder layers by the method of Rheinwald and Green (16
) as described previously (16
,17
).
Mice.
K14-HPV16 transgenic mice were derived and described by Arbeit et al. (18
), and were characterized and maintained by us as described previously (9
,17
). Control and experimental groups were as described previously (9
). Virgin normal and transgenic mice (4–5 wk old) were implanted subcutaneously with 0.25 mg/d release pellets of E2 and fed diets with or without I3C as described below. Implants were repeated every 60 d until the end of the study. Mice were housed in groups of 5/cage. All experiments involving mice were done in strict adherence to IACUC-approved procedures.
Diet.
Mice consumed ad libitum the AIN76a diet or AIN76a diet (19
) enriched with 0.1 g/kg I3C. Diets were prepared by Ziegler (Gardner, PA). The AIN76a diet contains 5% corn oil and supplies a total of 18.5 MJ/kg, with 22% of energy from protein, 11% from fat and 67% from carbohydrates.
Cell viability.
C33A Cells were trypsinized, seeded at 104 cells/well in 96-well plates containing 100 µL medium/well and incubated over night. The next day, the medium was changed to 200 µL containing either DIM or dimethyl sulfoxide (DMSO) as solvent control, employing a minimum of four replicate wells per condition. Viability was determined at indicated times by a mitochondrial function assay [reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)] using the Cell Titer Aqueous One kit (Promega, Madison, WI) according to the manufacturer’s instructions. Absorbance at 595 nm of the solution in individual wells was determined with a multiwell plate reader. Data were analyzed by plotting the mean and SD of cell viability vs. DIM concentration. Protein concentration was measured with the MicroBCA kit (Pierce, Rockford, IL) using a bovine serum albumin (BSA) standard.
Nucleosomal leakage apoptosis assay.
Nucleosomal leakage was monitored with a Cell Death Detection ELISAPLUS kit from Roche Molecular Biochemicals (Mannheim, Germany) that detects histones and DNA in cytoplasmic extracts. Cells were grown in 96-well plates as described above in replicates of 6 wells/condition. Cells in duplicate wells were lysed and the postnuclear supernatant solution was analyzed for nucleosomal leakage according to manufacturer’s instructions. Results were determined by measuring absorbance at 405 nm with a microwell plate reader as above. The second set of 4 wells from the same plate was analyzed for viability using the MTS assay by absorbance at 595 nm, and the ELISA results were normalized to this value (A405/A595) to correct for reduction in viable cell number after treatment with I3C and DIM. The mean and SD of the normalized data were plotted vs. DIM concentration.
Western blotting.
Cells treated with I3C, or vehicle controls, each with or without estradiol, were lysed at room temperature in buffer containing 10 mmol/L NaH2PO4, 20 g/L triton X-100, 12g/L SDS, 10g/L sodium deoxycholate supplemented just before use with 2 µmol/L aprotinin, 100 µmol/L phenylmethylsulfonyl fluoride and 0.1 mmol/L EDTA, boiled for 2 min, and centrifuged for 10 min at 12,000 x g at 4°C. Supernatant solutions were stored at -80°C until use. Extract protein (100 µg) in sample buffer (125 mmol/L Tris-HCl, pH6.8, 10 g/LSDS, 20 g/L ß-mercaptoethanol and 0.1g/L bromophenol blue) was loaded onto a 12% SDS-polyacrylamide gel. After electrophoresis at 32 V for 3 h at room temperature, protein bands were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) by electroblotting overnight in transfer buffer (192 mmol/L glycine, 25 mmol/L Tris and 200 g/L methanol). Before incubation with antibodies, the membrane was blocked with Tris buffered saline with Tween (TBST)/milk (20mmol/L Tris-HCl, 137mmol/L NaCl, 15g/L nonfat dry milk and 1g/L Tween20, pH7.6) for 1 h. BCL-2, BAX and -tubulin were detected with specific mouse monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) by incubating dilutions (1:500–1:1000) for 1 h. After washing in TBST/milk, the filters were incubated with horesradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G antibody (Santa Cruz) at 1:2000 dilution for 1 h at room temperature. Antibody bound to protein was detected using the enhanced chemiluminescence system (Amersham Life Science, Piscataway, NJ).
TdT-mediated dUTP nick-end labeling (TUNEL) assay.
The assay used the in situ cell death detection kit, peroxidase (POD), from Boehringer Mannheim (Indianapolis, IN). Cells were grown for 24 h in 8-well chamber slides seeded with 105 cells/well, treated with 200 µmol/L I3C and incubated at 37°C for 48h. The slides were washed in PBS and fixed with 40 g/L paraformaldehyde for 30 min at room temperature. Fixed cells were washed in PBS, permeabilized with sodium citrate buffer containing 1 g/L Triton X-100 for 2 min on ice, and then incubated with terminal deoxynucleotidyl transferase for 1 h at 37°C. After being rinsed with PBS, slides were treated with converter-POD (conjugated with HRP) at 37°C for 30 min and mounted with a glass cover slip. At least 200 cells/well were evaluated for staining.
Assessment of DNA fragmentation by gel electrophoresis.
Cells were grown for 48 h in the presence of various concentrations of I3C or DIM. After treatment, the cells were harvested, centrifuged at 500 x g for 5 min and washed with PBS. The cell pellet was lysed in 200 µL of lysis buffer containing 50 mmol/L Tris-HCl, 20 mmol/L EDTA and 10 g/L NP-40, and centrifuged at 1600 x g for 5 min. The supernatant solution was incubated with (5 mg/L) RNase A for 2 h at 56°C and then digested with proteinase K (2.5 mg/L) at 37°C for 16 h. DNA was precipitated with an equal volume of 10 mol/L ammonium acetate and 2.5 volumes of ethanol. The precipitates were rinsed with 700 g/L ethanol, air dried, dissolved in Tris/EDTA buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA), electrophoresed through a 1.5% agarose gel, stained with ethidium bromide and photographed on a UV transilluminator.
Immunohistochemistry.
Tissues were procured, fixed and processed for immunostaining as described (9
). For detection of activated caspase 3, both cells in culture and tissue slices were fixed in 10 g/L paraformaldehyde for 60 min at room temperature, followed by three washes in PBS. After being blocked overnight with 150 g/L BSA in PBS, the cells or tissue slices were incubated with a polyclonal antibody specific for the activate form of caspase 3 (Promega) overnight at 4°C. After three washes in PBS, samples were incubated with peroxidase-conjugated goat anti-rabbit second antibody (Santa Cruz Biochemicals); the signal was developed according to the manufacturer’s instructions. For TUNEL staining of tissue sections, paraffin-embedded tissues were sectioned at a thickness of 5 µm and processed using the Complete ApopTag in situ hybridization kit (Intergen, Purchase, NY). Hematoxylin and eosin (H&E) staining was accomplished as described (9
).
Cytologic detection of apoptosis.
Two groups of mice (n = 50 transgenic and 50 nontransgenic) were treated continuously with E2 starting at 4–5 wk of age. Half of each group was fed a diet containing 2 g/kg I3C and the other half a control diet without I3C. At 6 mo, when the majority of E2-treated transgenic mice fed the control diet were found to have cervical tumors (9
), all mice were killed by CO2 inhalation and their cervical tissue examined by H&E staining as described (9
). Samples were coded and the determination of apoptotic cells was carried out by a single individual unaware of treatment groups (M.Q.).
Fluorescence staining of nuclei.
C33A cells and normal human keratinocytes growing in monolayer were fixed in paraformaldehyde as above, followed by 10 µg/L 4,6-diamidino-2-phenylindole (DAPI) in methanol (Boehringer Mannheim, Germany) for 30 min at 37°C. Stained cells were mounted with Aqueous Mounting Medium (Biomedia, Loomis, CA) before fluorescence microscopy.
Fluorescence-activated cell sorting analysis.
To determine altered permeability, C33A cells were treated with or without 200 µmol/L I3C for 48 h and trypsinized. After washing with PBS, the cells were incubated in PBS containing 8 mg/L of 7-amino actinomycin D (7-AAD)-fluorescein isothiocyanate (Sigma) in the dark at 4°C for 20 min and then washed in PBS + 150 g/L BSA + 0.2 g/L NaN3 containing 20 mg/L of nonfluorescent actinomycin D (AD; Sigma). The resulting cell suspension was then analyzed cytofluorometrically on a Coulter Elite flow cytometer, set for single color, ungated fluorescence intensity.
Statistical analysis of data.
Standard deviations were calculated for all quantitative data as indicated in the figures and figure legends. Significant differences (P < 0.05) were determined using Student’s t test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
After treatment with either I3C or DIM, DNA from I3C-treated C33A cervical cancer cells accumulated double-strand breaks that generated the characteristic apoptotic pattern of nucleosomal "laddering" (Fig. 1A
, lanes 1–5), and the effect was dose dependent because an equal amount of DNA from cells treated with increasing concentrations of I3C gave progressively more intense patterns of nucleosomal repeat-sized DNA fragments. A similar apoptotic pattern was obtained when cells were treated with DIM (Fig. 1B
). In contrast, nontransformed cells such as 3T3 fibroblasts, did not accumulate double-strand DNA breaks after DIM treatment (Fig. 1A
, lanes 6–10).
|
). C33AE6, which express theHPV16 E6 oncogene (15
), showed an intermediate response (Fig. 2)
. Thus, it appeared that the induction of DNA strand breaks by I3C was a general phenomenon for cervical cancer cell lines and was independent of viral gene expression because C33A cells do not express detectable viral gene products (20
), whereas both CaSki and C33AE6 do (15
,21
).
|
If the DNA fragmentation caused by I3C were the result of late-stage apoptosis, then it should be possible to detect early apoptotic changes in these cells. We next determined whether I3C could cause the cytoplasmic membrane changes characteristic of this process. Extroversion of phosphatidylserine during apoptosis (22
) is accompanied by altered membrane permeability such that a fluorescent derivative of AD, namely, 7-AAD, can enter apoptotic cells and bind to DNA in the nucleus (23
,24
). Fluorescence-activated cell sorting (FACS) analysis of 7-AAD–stained C33A cells that had been treated with 200 µmol/L I3C confirmed that the compound increased the percentage of stained cells from 9.8% of the total (Fig. 3A
) to 55% of the total (Fig. 3B
) after 48 h of exposure to I3C.
|
If the DIM, the dimeric adduct of I3C, were the active molecular form, or more proximate to the active form, then DIM would be expected to act faster and at a lower concentration than I3C because the latter is converted only slowly to DIM in cell culture. Figure 4A
shows the effect of increasing concentrations of DIM and I3C on C33A cells after a 48-h exposure, using a mitochondrial function assay (MTS assay) as an indirect measure of cell viability. We observed a reduction in the number of viable cells in cultures treated with as little as 40 µmol/L DIM, whereas I3C had no observable effect at concentrations <100 µmol/L (Fig. 4A
). The dose at which the number of viable cells is 50% of that in untreated cultures (LD50) for DIM was 
60 µmol/L, compared with 200 µmol/L for I3C. We next compared the rate of cell killing by DIM and I3C; 100 µmol/L DIM began to have an observable effect between 16 and 20 h after addition to growing cells, whereas 300 µmol/L I3C had no observable effect until 36 h (Fig. 4B
). To confirm that DIM was in fact inducing apoptosis, the dose-response experiment in Figure 4A
was repeated, and ELISA was used to detect nucleosomal leakage into the cytoplasm. Histones and DNA were detected in the cytoplasm of cells treated with 75 µmol/L DIM, whereas release in I3C-treated cells required concentrations several fold higher (Fig. 4C
).
|
shows an immunofluorescence photomicrograph of DAPI-stained C33A cells (panels A and B) and normal human keratinocytes (panels C and D) treated with DIM. Because DAPI stains only DNA and chromatin, the outline of the stained region indicates the relative size and shape of the nucleus, as well as the distribution of DNA within it. At concentrations as low as 30 µmol/L, DIM caused observable changes in the nuclear morphology of virtually all of the C33A cells (data not shown); at 60 µmol/L DIM, condensed, fragmented late-stage apoptotic nuclei were observed in C33A cells and virtually all of the nuclei were contracted compared with those in untreated cells (panel B, white arrows). At 100 µmol/L, few C33A cells remained on the cover slip after staining (not shown). In contrast, normal keratinocytes did not exhibit these changes even at 100 µmol/L DIM (compare Fig. 5C
and D
). When duplicate cultures of C33A cells were stained for activated caspase 3 by immunohistochemistry, a similar dose response was observed (Fig. 6
).
|
|
One of the points at which several pathways of apoptotic induction converge is the breakdown of mitochondrial membrane integrity. This process is thought to be controlled in part by the relative abundance of various members of the Bcl-2 family of proteins, notably BCL-2 itself and BAX, the former acting as an antiapoptotic agent and the latter as an apoptotic inducer [reviewed in (15
)]. I3C caused a time- and dose-dependent reduction in the amount of Bcl-2 protein detected by Western blot (Fig. 7A
and B), whereas BAX was not affected by treatment with 300 µmol/L I3C for 72 h. DIM reduced the amount of BCL-2 to undetectable levels by 72 h.
|
The observation that I3C and its major condensation product DIM can induce apoptotic changes in cervical cancer cells in vitro led us to ask whether I3C had the same effect in vivo. Transgenic mice expressing the human HPV E6 and E7 oncogenes under control of the keratin 14 promoter all develop cervical cancer when exposed chronically to estradiol (9
,18
), but a diet supplemented with I3C protects nearly all of these mice (9
). We examined sections of cervical epithelium from normal and transgenic mice by H&E staining in a double-blind protocol, scoring for apoptotic cell nuclei (Fig. 8A
and B
). A small number of apoptotic nuclei were observed throughout the cervical epithelium of normal mice, but this number did not change significantly in mice treated with E2 (Fig. 8C)
, although the E2-treated mice showed increased cervical dysplasia (9
). Transgenic mice exposed to estradiol, on the other hand, had significantly fewer apoptotic cells (Fig. 8C
). When these estradiol-treated transgenic mice were also fed I3C, the number of apoptotic cells detected in H&E stained sections returned to normal.
|
shows clear evidence of stained cells in cervical epithelium only from transgenic mice chronically exposed to estrogen, i.e., those expected to develop cervical cancer but also fed I3C. We did not observe TUNEL staining cells in the cervical epithelium of normal mice or in transgenic mice exposed to amounts of estrogen that induce epithelial hypertrophy and dysplasia.
|
, individual cells stained positively for active caspase 3 in the suprabasal epithelium of the transgenic mice fed I3C (Fig. 10B
), but not of placebo-fed controls (Fig. 10D
).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) are present in I3C- and DIM-treated cells growing in culture, i.e., altered nuclear morphology (Fig. 5)
, DNA fragmentation (Figs. 1
, 2)
and nucleosomal leakage (Fig. 4C)
, changes in membrane permeability (Fig. 3)
and activation of caspase 3 (Fig. 6)
. The quantitative data from the various assays used to detect cell killing and apoptosis were in good agreement. For example, exposure to 300 µmol/L I3C for 48 h caused 35% of the cells to stain positively for DNA strand breaks in the TUNEL assay (a late apoptotic event) and induced membrane permeability changes (an early event) in 55%. Similarly, 100 µmol/L DIM for 48 h killed between 80 and 90% of the cells (Fig. 4A
and B
) and active caspase 3 was visible by immunostaining in roughly half of the surviving cells (Fig. 6)
. In contrast to cervical cancer cells, normal human foreskin keratinocytes, cells that are similar (although clearly not identical) to those in the cervix that most commonly undergo malignant transformation, did not exhibit major changes in nuclear morphology associated with apoptosis upon exposure to DIM (Fig. 5
, panels C and D).
The dose-response curves for I3C and DIM (Fig. 4)
are consistent with slow conversion of I3C to DIM in cell culture medium, assuming that DIM is the active compound or is further metabolically converted to the active compound(s). However, serum levels of DIM in rats fed I3C were only 10% of those required to induce apoptosis in vitro in our experiments (6
). The fact that relatively high concentrations of DIM (and I3C), pharmacologic as opposed to dietary levels, were required to obtain biologic effects in vitro is consistent with a requirement for metabolic conversion of DIM to a secondary active compound. However, it is now widely recognized that serum levels of bioactive compounds are often uninformative because other factors such as carrier proteins, intracellular accumulation and localized concentration in specific tissues may all come into play. It is therefore not surprising that the concentrations of DIM required to produce effects in vitro are higher than those normally attainable in vivo. Similar observations have been made for other bioactive natural products, for example isoflavones from soy (26
,27
). In any case, the fact that dietary I3C appeared to induce apoptosis in the cervical epithelium of HPV16 transgenic mice suggests that this compound or its active metabolites do indeed reach effective intracellular levels in vivo.
Detection of apoptotic cells in cervical epithelia was undoubtedly facilitated by our experimental model system. In the HPV transgenic mouse, just as in human cervical cancer, nearly every cell is expressing HPV oncogenes and is thus initiated for transformation. If, as our results suggest, transformed cells or cells undergoing preneoplastic conversion are differentially sensitive to I3C/DIM, then the enrichment for these cells in cervical epithelium as a result of VP6 and VP7 gene expression would explain our ability to detect what in other tissues or experimental systems would be a rare event. In cervical epithelium from HPV16 mice, chronic exposure to elevated E2 is required for expression of the transformed phenotype (9
,18
). The observed reduction in late-stage apoptotic cells identifiable by routine histopatholgy in samples from HPV16 mice exposed to E2 (Fig. 8C
) is therefore consistent with the generally observed phenomenon that apoptosis is inhibited in neoplastic and preneoplastic cells [reviewed in (25
)]. By contrast, when E2-treated HPV transgenic mice were also fed I3C, we did not observe the E2-related reduction in apoptotic cells. This suggests that I3C either prevented transformation or killed the cells as they became transformed. Our in vitro data are consistent with the latter explanation.
We do not yet know either the mechanism by which these phytochemicals induce apoptosis or what determines their apparent specificity for transformed cells. Our data and published reports from other laboratories rule out both HPV and estrogen effects as requirements for induction of apoptosis by I3C/DIM, although both mechanisms may play ancillary roles in specific instances. Apart from sex, two differences of potential relevance between cervical cancer cells, which are sensitive to DIM, and foreskin keratinocytes, which are not, are the presence of HPV oncogenes and estrogen receptors in the former. However C33A cells, unlike C33AE6 and CaSki cells, do not express HPV oncogenes or any other viral genes at detectable levels (15
); therefore, it is unlikely that viral gene products account for the sensitivity of cervical cancer cells to I3C and DIM. The reduced sensitivity of C33AE6 cells to killing by I3C compared with the parental C33A cell line (Fig. 2)
may have been caused by the reported antiapoptotic effect of E6 (28
–30
). However, even the protection afforded by expression of this viral oncogene was not complete.
Cell killing by DIM or I3C similarly does not seem to require estrogen; in fact, I3C and E2 have opposing effects on cell survival. Although C33A cells possess estrogen receptors (our unpublished data), and estrogen induces proliferation of epithelial cells in the cervix, estradiol interferes with induction of apoptosis caused by a number of agents, including I3C (our unpublished observations). Conversely, I3C interferes with signaling from the estrogen receptor (31
). The cytotoxic specificity of I3C/DIM for transformed cells also appears to be reflected in cervical epithelium in vivo, regardless of whether mice were exposed chronically to elevated estrogen (Figs. 8
9
10)
. During the course of multiple studies spanning >2 y, we have never seen apoptotic cells in cervical epithelium or any evidence of cervical histopathology in normal mice fed a diet containing DIM. We conclude that induction of apoptosis in cervical cancer cells exposed to I3C or DIM both in vitro and in vivo occurs by a pathway separate from, and independent of estrogen-responsive mechanisms, and that it is specific for transformed cells.
How does I3C/DIM induce apoptosis of cancer cells? Several possibilities are suggested by published work from our laboratory and others. One mechanism that could account for the sensitivity of transformed cells is cell cycle inhibition. I3C inhibits cdk6 expression in MCF-7 cells (32
) by interfering with transcription (33
). Thus it might be that proliferating cells are more sensitive to I3C and DIM due to a need for cdk6 activation, which these agents prevent. Another possibility involves the induction of proapoptotic genes, for example, via the aryl hydrocarbon receptor (12
,13
) or perhaps the sensitization of cells to the cytotoxic effects of cytokines or other factors in the cellular microenvironment.
Our results provide further mechanistic underpinning for the epidemiologic data supporting the efficacy of a diet rich in cruciferous vegetables for reducing the incidence of cervical cancer. However, this is also one of the first reports of apoptotic changes induced by chemotherapeutic agents in vivo, particularly involving solid tumors. The fact that normal keratinocytes did not show evidence of apoptotic morphologic changes after prolonged exposure to high DIM concentrations, and that normal cervical epithelium never showed any indication of apoptosis extensive enough to score positively in TUNEL staining, suggests that I3C/DIM may have a utility beyond dietary prevention, as a relatively innocuous chemotherapeutic agent in the treatment of cervical cancer and, potentially, other malignancies.
|
FOOTNOTES |
|---|
pathway. p. 122 (abs.)].
2 Supported by National Institutes of Health, National Cancer Institute grant CA73385 (to K.J.A.).
4 Abbreviations used: 7-AAD, 7-amino actinomycin D; AD, actinomycin D; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole; DIM, diindolylmethane; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; E2, 17ß-estradiol; H&E, hematoxylin and eosin; HPV, human papillomavirus; HRP, horseradish peroxidase; I3C, indole-3-carbinol; LD50, dose at which the number of viable cells is 50% of that in untreated cultures; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; POD, peroxidase; TUNEL, TdT-mediated dUTP nick-end labeling.
Manuscript received June 6, 2001. Initial review completed July 12, 2001. Revision accepted September 19, 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Bell, M. C., Crowley-Nowick, P., Bradlow, H. L., Sepkovic, D. W., Schmidt-Grimminger, D., Howell, P., Mayeaux, E. J., Tucker, A., Turbat-Herrera, E. A. & Mathis, J. M. (2000) Placebo-controlled trial of indole-3-carbinol in the treatment of CIN. Gynecol. Oncol. 78:123-129.[Medline]
2. Auborn, K., Abramson, A., Bradlow, H. L., Sepkovic, D. & Mullooly, V. (1998) Estrogen metabolism and laryngeal papillomatosis: a pilot study on dietary prevention. Anticancer Res 18:4569-4574.[Medline]
3. Rosen, C. A., Woodson, G. E., Thompson, J. W., Hengesteg, A. P. & Bradlow, H. L. (1998) Preliminary results of the use of indole-3-carbinol for recurrent respiratory papillomatosis. Otolaryngol. Head Neck Surg. 118:810-815.[Medline]
4. Anon, A. (1996) A clinical development plan: indole-3-carbinol. J. Cell. Biochem. 26:127-136.
5. Grose, K. R. & Bjeldanes, L. F. (1992) Oligomerization of indole-3-carbinol in aqueous acid. Chem. Res. Toxicol. 5:188-193.[Medline]
6. Stresser, D. M., Williams, D. E., Griffen, D. A. & Bailey, G. S. (1995) Mechanisms of tumor modulation by indole-3-carbinol. disposition and excretion in male Fisher. 344 rats. Drug Metab. Disp. 23:966-975.
7. Ge, X., Yannai, S., Rennert, G., Gruener, N. & Farres, F. A. (1996) 3,3'-Diindolylmethane induces apoptosis in human cancer cells. Biochem. Biophys. Res. Commun. 228:153-158.[Medline]
8. Ge, X., Fares, F. A. & Yannai, S. (1999) Induction of apoptosis in MCF-7 cells by indol-3-carbinol is independent of p53 and bax. Anticancer Res 19:3199-3203.[Medline]
9. Jin, L., Qi, M., Chen, D.-Z., Arbeit, J. M., Yang, G.-Y. & Auborn, K. J. (1999) Indole-3-carbinol prevents cervical cancer in HPV16 transgenic mice. Cancer Res 59:1693-1699.
10. Auborn, K. J., Woodworth, C., DiPaolo, J. & Bradlow, H. L. (1991) The interaction between HPV infection and estrogen metabolism in cervical carcinogenesis. Int. J. Cancer 49:867-869.[Medline]
11. Yuan, F., Chen, D.-Z., Liu, K., Sepkovic, D. W., Bradlow, H. L. & Auborn, K. (1999) Anti-estrogenic activities of indole-3-carbinol in cervical cancer cells: implication for cervical cancer. Anticancer Res 19:1673-1680.[Medline]
12.
Bjeldanes, L. F., Kim, J.-Y., Grose, K. R., Bartholomew, J. C. & Bradfield, C. A. (1991) Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. U.S.A. 88:9543-9547.
13.
Chen, I., McDougal, A., Wang, F. & Safe, S. (1998) Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19:1631-1639.
14.
Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F. & Firestone, G. L. (1998) Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J. Biol. Chem. 273:3838-3847.
15. Etscheid, B. G., Foster, S. A. & Galloway, D. A. (1994) The E6 protein of human papillomavirus type 16 functions as a transcriptional repressor in a mechanism independent of the tumor suppressor protein, p53. Virology 205:583-585.[Medline]
16. Rheinwald, J. G. & Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-343.[Medline]
17.
Chen, D.-Z. & Auborn, K. (1999) Fish oil constituent docosahexaenic acid selectively inhibits growth of human papillomavirus immortalized keratinocytes. Carcinogenesis 20:249-254.
18.
Arbeit, J. M., Munger, K., Howley, P. M. & Hanahan, D. (1994) Progressive squamous epithelial neoplasia in K-14 human papillomavirus type 16 transgenic mice. J. Virol. 68:4358-4368.
19. Dillehay, D. L., Webb, S. K., Schmelz, E. M. & Merrill, A. H., Jr (1994) Dietary sphingomyelin inhibits 1,2-dimethylhydrazine-induced colon cancer in CF1 mice. J. Nutr. 124:615-620.
20. Crook, T., Wrede, D. & Vousden, K. H. (1991) p53 point mutation in HPV negative human cervical carcinoma cell lines. Oncogene 6:873-875.[Medline]
21.
Smotkin, D. & Wettstein, F. O. (1986) Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. U.S.A. 83:4680-4684.
22. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L. & Henson, P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148:2207-2216.[Abstract]
23. Schmid, I., Uittenbogaart, C. H. & Giorgi, J. V. (1994) Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15:12-20.[Medline]
24. Lecoeur, H., Ledru, E., Prevost, M. C. & Gougeon, M. L. (1997) Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods. J. Immunol. Methods 209:111-123.[Medline]
25. Strasser, A., O’Connor, L. & Dixit, V. M. (2000) Apoptosis signaling. Annu. Rev. Biochem. 69:217-245.[Medline]
26. Frey, R. S., Li, J. & Singletary, K. W. (2001) Effects of genistein on cell proliferation and cell cycle arrest in non-neoplastic human mammary epithelial cells: involvement of Cdc2, p21(waf/cip1), p27(kip1), and Cdc25C expression. Biochem. Pharmacol. 61:979-989.[Medline]
27. Constantinou, A. I., Kamath, N. & Murley, J. S. (1998) Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinoma MCF-7 cells. Eur. J. Cancer 34:1927-1934.
28. Park, J. S., Kim, E. J., Lee, J. Y., Sin, H. S., Namkoong, S. E. & Um, S. J. (2001) Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins. Int. J. Cancer. 91:822-827.[Medline]
29.
Butz, K., Denk, C., Ullmann, A., Scheffner, M. & Hoppe-Seyler, F. (2000) Induction of apoptosis in human papillomavirus positive cancer cells by peptide aptamers targeting the viral E6 oncoprotein. Proc. Natl. Acad. Sci. U.S.A. 97:6693-6697.
30. Kamradt, M. C., Mohideen, N., Krueger, E., Walter, S. & Vaughan, A. T. (2000) Inhibition of radiation-induced apoptosis by dexamethasone in cervical carcinoma cell lines depends upon increased HPV E6/E7. Br. J. Cancer 82:1709-1716.[Medline]
31.
Meng, Q., Yuan, F., Goldberg, I. D., Rosen, E. M., Auborn, K. & Fan, S. (2000) Indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells. J. Nutr. 130:2927-2931.
32. Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F. & Firestone, G. L. (1998) Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J. Biol. Chem. 273:3838-3847.
33.
Cram, E. J., Liu, B. D., Bjeldanes, L. F. & Firestone, G. L. (2001) Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. J Biol. Chem. 276:22332-22340.
This article has been cited by other articles:
|
|
 |
|
  K. Kunimasa, T. Kobayashi, K. Kaji, and T. Ohta Antiangiogenic Effects of Indole-3-Carbinol and 3,3'-Diindolylmethane Are Associated with Their Differential Regulation of ERK1/2 and Akt in Tube-Forming HUVEC J. Nutr., January 1, 2010; 140(1): 1 - 6. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Swanick, K. Windstar-Hamlin, and H. Zwickey An Alternative Treatment for Cervical Intraepithelial Neoplasia II, III Integr Cancer Ther, June 1, 2009; 8(2): 164 - 167. [Abstract] [PDF]
|
|
|
|
 |
|
 H. H. Nguyen, I. Aronchik, G. A. Brar, D. H. H. Nguyen, L. F. Bjeldanes, and G. L. Firestone The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing PNAS, December 16, 2008; 105(50): 19750 - 19755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Kassie, I. Matise, M. Negia, P. Upadhyaya, and S. S. Hecht Dose-Dependent Inhibition of Tobacco Smoke Carcinogen-Induced Lung Tumorigenesis in A/J Mice by Indole-3-Carbinol Cancer Prevention Research, December 1, 2008; 1(7): 568 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Moiseeva, R. Heukers, and M. M. Manson EGFR and Src are involved in indole-3-carbinol-induced death and cell cycle arrest of human breast cancer cells Carcinogenesis, February 1, 2007; 28(2): 435 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Auborn Can Indole-3-Carbinol-Induced Changes in Cervical Intraepithelial Neoplasia Be Extrapolated to Other Food Components? J. Nutr., October 1, 2006; 136(10): 2676S - 2678S. [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Savino III, J. F. Evans, D. Rabinowitz, K. J. Auborn, and T. H. Carter Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane. Mol. Cancer Ther., March 1, 2006; 5(3): 556 - 563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. McCarty and K. I. Block Multifocal Angiostatic Therapy: An Update Integr Cancer Ther, December 1, 2005; 4(4): 301 - 314. [Abstract] [PDF]
|
|
|
|
 |
|
 K. W. Rahman and F. H. Sarkar Inhibition of Nuclear Translocation of Nuclear Factor-{kappa}B Contributes to 3,3'-Diindolylmethane-Induced Apoptosis in Breast Cancer Cells Cancer Res., January 1, 2005; 65(1): 364 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. H. Sarkar and Y. Li Indole-3-Carbinol and Prostate Cancer J. Nutr., December 1, 2004; 134(12): 3493S - 3498S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Anderton, M. M. Manson, R. Verschoyle, A. Gescher, W. P. Steward, M. L. Williams, and D. E. Mager PHYSIOLOGICAL MODELING OF FORMULATED AND CRYSTALLINE 3,3'-DIINDOLYLMETHANE PHARMACOKINETICS FOLLOWING ORAL ADMINISTRATION IN MICE Drug Metab. Dispos., June 1, 2004; 32(6): 632 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Leibelt, O. R. Hedstrom, K. A. Fischer, C. B. Pereira, and D. E. Williams Evaluation of Chronic Dietary Exposure to Indole-3-Carbinol and Absorption-Enhanced 3,3'-Diindolylmethane in Sprague-Dawley Rats Toxicol. Sci., July 1, 2003; 74(1): 10 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Firestone and L. F. Bjeldanes Indole-3-Carbinol and 3-3'-Diindolylmethane Antiproliferative Signaling Pathways Control Cell-Cycle Gene Transcription in Human Breast Cancer Cells by Regulating Promoter-Sp1 Transcription Factor Interactions J. Nutr., July 1, 2003; 133(7): 2448S - 2455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Auborn, S. Fan, E. M. Rosen, L. Goodwin, A. Chandraskaren, D. E. Williams, D. Chen, and T. H. Carter Indole-3-Carbinol Is a Negative Regulator of Estrogen J. Nutr., July 1, 2003; 133(7): 2470S - 2475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Carter, K. Liu, W. Ralph Jr., D. Chen, M. Qi, S. Fan, F. Yuan, E. M. Rosen, and K. J. Auborn Diindolylmethane Alters Gene Expression in Human Keratinocytes In Vitro J. Nutr., November 1, 2002; 132(11): 3314 - 3324. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
