Akt Inactivation Is a Key Event in Indole-3-carbinol-induced Apoptosis in PC-3 Cells¹ Free

I3C³ is a bioactive compound generated from the hydrolysis of glucobrasscin, which is a major glucosinolate in cruciferous vegetables. When cruciferous vegetables are crushed, the plant enzyme myrosinase comes into contact with the glucosinolates and hydrolyzes the glucosinolates, generating a mixture of products containing I3C (1). Several animal studies have shown that consumption of brassica vegetables, such as cabbage, cauliflower, and broccoli, or purified I3C have protective effects against carcinogen-induced tumors. The protective effects of I3C compound appear to be mediated by selective alterations in Phase I and II carcinogen-metabolizing enzymes (2). I3C induces the Phase I enzyme estradiol 2-hydroxylase leading to an altered metabolic ratio of 2-hydroxyestrone:16α-hydroxyestrone. Human studies with consumption of either I3C or brassica vegetables also showed an increase in estradiol 2-hydroxylation, thereby implicating the protective effect of I3C against hormone-related carcinogenesis (3, 4, 5). In addition, I3C and its oligomeric compound, indolo [3,2-β]carbazole, have been shown to have weak binding affinity for estrogen receptor (6). In vitro data with breast (7, 8) and PCa cells (9) showed I3C-induced cell growth inhibition, reversible G₁ cell cycle arrest, and apoptosis. The physiological concentrations of I3C in the populations, who consume high brassica vegetables, are not known precisely. I3C-induced apoptosis was accompanied by the alterations in several molecular targets, such as altered ratio of Bax:Bcl-2 and translocation of Bax to mitochondria (7).

These investigations have suggested that I3C could be an effective agent against PCa; however, further molecular dissection of signaling pathway by which I3C elicits its biological effects on PCa requires additional studies. PCa is the most common cancer and second leading cause of death in American men in the United States. An estimated 198,100 new cases of PCa are expected to be diagnosed in the United States in 2001, and 31,500 men are expected to die from this disease during 2001 (10). Recent studies involving detailed histological analysis of prostate glands from autopsy specimens have shown that the PCa has a long latency period (11). Recent dietary and epidemiological studies have suggested the benefit of dietary intake of fruits and vegetables, against the incidence of many types of cancer, including hormone-related cancers (12, 13). Although mechanism(s) involved in the progression of PCa are unclear, growing evidence in the literature point toward the role of autocrine and paracrine factors in the development of prostate tumorigenesis. EGF and transforming growth factor α-mediated activation of EGFR signaling events have been identified in PCa cells (14). Furthermore, studies with EGFR tyrosine kinase inhibitors (15) and anti-EGFR antibodies with PCa cells (16) reveal inhibition of cell growth and tumor formation in mice, indicating the importance of EGFR signaling events in PCa. EGFR family is composed of EGFR, ErbB-2, ErbB-3, and ErbB-4, and receptor activation is mediated by various ligand-induced dimerization events and autophosphorylation (17, 18, 19). The PI3K and MAP kinase signaling pathways are the major downstream signaling events of EGFR.

Akt is a serine/threonine protein kinase functioning downstream of PI3K in response to mitogen or growth factor stimulation. Three isoforms of Akt are present in mammalian cells, Akt1/PKBα and Akt2/PKBβ, which are ubiquitously expressed, and Akt3/PKBγ, which shows tissue specificity (20, 21, 22). Threonine 308 and Serine 473 are phosphorylated by PDK1 and PDK2, respectively (23, 24). PI3K products phosphatidyl inositol 3,4,5-trisphosphates have been shown to be the activators of PDK isoforms, in addition to targeting Akt to membranes by interacting with the plextrin homology domain of Akt (24). The tumor suppressor gene PTEN or MMAC1 acts as a lipid phosphatase for phosphatidyl inositol at 3′ (25, 26), and SHIP is another phosphatase for phosphatidyl inositol at the 5′ position (27, 28, 29). Both of these proteins negatively regulate the activity of Akt/PKB by depleting the pool of phosphatidyl inositol 3,4,5-trisphosphates. In addition to metabolic and transcriptional modulatory effects of Akt, an antiapoptotic role of Akt is gaining prominence by virtue of its critical role in growth factor, and mitogen-induced cell survival pathways as observed in several cell types, including cancer cells (23). Akt has been shown to affect the apoptotic processes by multiple mechanisms involving phosphorylation of several components of the apoptotic machinery, including BAD (30) and caspases 9 (31), and also modulate apoptosis indirectly by influencing the activities of several families of transcription factors, including fork head transcription factor, NF-κB, and cyclic AMP-responsive element binding protein (23).

In the present study, we show that treatment of the PCa cell line PC-3 with I3C induces apoptosis in a dose- and time-dependent manner, and the induction of apoptosis was partly because of the inhibition of Akt activation and down-regulation of BAD and Bcl-x_L. In addition, I3C inhibited EGF-mediated activation of EGFR by decreasing its autophosphorylation and, in turn, inhibiting phosphorylation of Akt and PI3K in PC-3 cells. These results suggest an important role of the Akt signaling pathway in mediating the effects of I3C in PCa cells. Molecular targeting of the Akt pathway by I3C alone or followed by treatment with cytotoxic agent(s) may be a useful and novel strategy for the prevention and/or treatment of PCa, particularly for the treatment of hormone refractory disease, for which there is no effective therapy.

The human PCa cell line, PC-3, was obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37°C. The cells were seeded at a density of 1 × 10⁶/100-mm culture dish. After 24 h, the cells were treated with indicated concentrations of I3C (Sigma Chemical Co., St. Louis, MO) and stimulated with EGF (Invitrogen) as mentioned in the figure legends.

7-AAD staining method was used to detect cells undergoing apoptosis as reported previously (32). Briefly, 5 × 10⁵ cells were seeded in 60-mm plates and subsequently treated with 30, 60, and 100 μm I3C (dissolved in DMSO and added to cultures at a final concentration of 0.1% DMSO) for 24, 48, and 72 h, and control cells received 0.1% DMSO in culture medium. After the indicated incubation periods, the cells were trypsinized, and the cell pellet was resuspended in 1 ml of PBS. The cells were treated with 100 μm 7-AAD for 30 min. in the dark and analyzed by flow cytometry for 7-AAD staining.

PC-3 cells were plated and cultured in complete medium and allowed to attach for 24 h followed by the addition of 30, 60, and 100 μm I3C prepared as stated earlier. Incubation was carried out for 24, 48, and 72 h. Control cells were incubated in the medium with 0.1% DMSO for the same time period. After the indicated incubation period, the cells were harvested by scraping the cells from culture dishes and collected by centrifugation. Cells were resuspended in Tris-HCl (pH 6.8) buffer, sonicated two times for 10 s, and lysed using an equal volume of 4% SDS. Protein concentration was then measured using bicinchoninic acid protein assay reagents (Pierce, Rockford, IL). Cell extracts were boiled for 10 min and chilled on ice, briefly centrifuged, and subjected to 10% SDS-PAGE, unless otherwise indicated, and electrophoretically transferred to a nitrocellulose membrane. Each membrane was incubated with primary antibodies: Akt (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Akt (serine 473 and threonine 308; Cell Signaling Technology, Inc., Beverly, MA), β-actin, BAD, Bcl-x_L (Santa Cruz Biotechnology), p85 of PI3K (Upstate Biotechnology, Lake Placid, NY), and phospho-Tyrosine antibodies (Transduction Laboratories, San Diego, CA). The membranes were washed with Tris-buffered saline and incubated with secondary antibodies conjugated with peroxidase, and the signal was detected by the chemiluminescent detection system (Pierce).

Autoradiograms of the Western blots were scanned with the Gel Doc 1000 image scanner (Bio-Rad, Hercules, CA), and band intensities were quantified and analyzed with the Molecular Analyst software program (Bio-Rad).

PC-3 cells (1 × 10⁶) were lysed in NP-40 lysis buffer containing 50 mm Tris (pH 8.0), 0.5% NP-40, 0.5% sodium deoxycholate, 150 mm NaCl, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 100 mm sodium fluoride, 2 mm sodium orthovanadate, and 10 mm sodium PP_i. Protein concentration was determined using Protein A assay kit from Pierce, and equal amounts of proteins from I3C-treated and untreated cell lysates were incubated with appropriate primary antibodies for overnight at 4°C, followed by the addition of Protein-A agarose, and incubated at 4°C for 1 h. Agarose beads were washed for five times with NP-40 lysis buffer, resuspended in 2 × SDS sample buffer, and subjected to Western immunoblot analysis with antiphosphotyrosine antibodies.

Akt kinase assay was performed using reagents supplied as a kit obtained from Cell Signaling Technology, Inc. Briefly, 1 × 10⁶ PC-3 cells were lysed in cell lysis buffer containing 20 mm Tris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium PP_i, 1 mm β-glycerophosphate, 1 mm Na₃VO₄, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Equal amounts of lysed proteins were immunoprecipitated with agarose-coupled anti-Akt antibodies for overnight at 4°C. Immunocomplexes were washed four times with lysis buffer and twice with kinase buffer composed of 25 mm Tris (pH 7.5), 5 mm β-glycerophosphate, 2 mm DTT, 0.1 mm Na₃VO₄, and 10 mm MgCl₂. Immune complexes were resuspended in 40 μl of kinase buffer, supplemented with 200 μm ATP and 1 μg of GSK-3 fusion protein, and incubated at 30°C for 30 min. Kinase reaction was terminated with the addition of 20 μl of 3 × SDS sample buffer. Sample (20 μl) was analyzed by Western immunoblotting with antiphospho-GSK-3α/β antibody.

We have shown previously that I3C inhibits growth of cultured PC-3 cells, and this growth inhibition was partly because of the induction of cell cycle arrest at the G₁ phase of the cell cycle and also because of the induction of apoptotic cell death; however, the degree of cell death and its molecular mechanism have not been investigated. Although the attainable serum levels of I3C in animal or humans after consumption of I3C or cruciferous vegetables are currently unknown, the concentrations used in our study are comparable with other published results with cell culture system (7, 8, 9, 33, 34, 35). And it is reasonable to assume that these concentrations fall under pharmacological range rather than physiological circulating levels. Here we show that I3C-treated PC-3 cells undergo apoptosis in a dose- and time-dependent manner as measured by 7-AAD staining of PC-3 cells (Fig. 1). A significant number of cells (29.78%) started to undergo apoptosis as early as 48 h after treatment with 60 μm I3C. Similar degree of apoptosis (24.91%) was also observed with lower concentration of I3C (30 μm) treatment for 72 h. The maximum number of cells (84.82%) underwent apoptosis with 100 μm I3C treatment for 72 h. The concentrations of I3C used in this study are comparable with other studies reported in the literature, and similar induction of apoptosis by I3C was also observed in breast cancer cells as reported in our earlier study (7). To further understand the molecular mechanism of I3C-induced apoptosis in PC-3 cells, gene expression levels of the cell survival and apoptotic machinery were investigated.

A common survival pathway operates in cells in response to growth factor stimuli, and the proto-oncogene product Akt kinase plays a major role in cell survival pathway by regulating cell survival and apoptotic processes. Activation of Akt signaling is known to initiate from membrane-bound receptors via PI3k products and subsequent phosphorylation of downstream effector molecules, such as proapoptotic protein BAD, causing its inactivation and, in turn, inhibiting apoptotic processes in response to growth factor stimuli. Here, we investigated whether activation of Akt protein was altered during I3C-induced apoptosis in PC-3 cells. PC-3 cells treated with I3C have no effect on steady-state levels of total Akt protein, whereas phosphorylation at serine 473 and threonine 308 were inhibited significantly in a dose- and time-dependent manner (Fig. 2). The inhibition of phosphorylation at threonine 308 appears to be more sensitive to I3C treatment in PC-3 cells. Interestingly, the basal level of phosphorylation at these two residues were predominant in the PC-3 cell line, which could be because of the mutation and inactivation of PTEN tumor suppressor gene (36, 37), which acts as phosphatase for PI3K product and, thereby, increases the basal activation of Akt and subsequent inhibition of the apoptotic processes. DU145 PCa cells have an intact PTEN gene and, therefore, do not show any detectable basal phosphorylation at serine 473 (data not shown). The Western blot analysis of PI3K, an upstream activator of Akt, shows no effect on treatment with I3C in PC-3 cells; hence, further investigation was conducted to understand the molecular mechanism of I3C action as detailed in the following paragraphs. The inhibition of Akt phosphorylation could be attributed to the inactivation of cell survival pathways, resulting in the subsequent induction of apoptosis in PC-3 cells treated with I3C.

Apoptosis is an ordered cascade of enzymatic activities leading to genome disintegration and cell death. Several lines of evidence suggest that activated Akt, in response to growth factor stimuli, phosphorylates the proapoptotic protein BAD at serine 136, inhibiting its membrane interaction with Bcl-x_L. Because I3C inhibits the phosphorylation of Akt at both serine and threonine residues, we investigated whether the effect of I3C is mediated by the alterations in the BAD and Bcl-x_L proteins in PC-3 cells. We found that both BAD and Bcl-x_L protein levels were down-regulated on I3C treatment (Fig. 3). Down-regulation of Bcl-x_L expression was more pronounced with 100 μm I3C treatment beginning at 48 h. These results suggest that down-regulation of Bcl-x_L protein and BAD are more distal effects of I3C action in PC-3 cells, promoting apoptotic processes. Therefore, we investigated the effect of I3C on signaling molecules that are more proximal to Akt signaling pathway.

In PCa, EGF acts as an autocrine growth factor, transducing signals through EGFR (14, 38, 39). Constitutive expression and activation of EGFR has been observed in a variety of cancers, including PCa (39). EGFR signaling includes activation of PI3K/Akt molecules, which leads to antiapoptotic and cell survival signals, accompanied by the activation of many intracellular signaling pathways. Here we investigated whether I3C has any effect on the expression of EGFR and its associated signaling events leading to activation of Akt. Western blot analysis of I3C-treated PC-3 cells showed down-regulation of EGFR protein levels in a concentration- and time-dependent manner (Fig. 4). The maximum effect of I3C was observed at 60–100 μm I3C treatments starting at 48 h. In addition, EGFR autophosphorylation in response to EGF treatment was also inhibited in I3C-treated PC-3 cells. These results suggest that the effect of I3C could be because of either down-regulation of EGFR protein expression and/or inhibition of EGFR autophosphorylation (Fig. 5). PI3K and Akt kinase protein levels were unaltered by the I3C treatment; however, the phosphorylation of p85 subunit of PI3K and Akt were inhibited in response to I3C treatment. In addition, I3C pretreatment of cells inhibited EGF-mediated phosphorylation of p85 subunit of PI3K (Fig. 6). Total Akt protein levels were also unaltered in response to I3C treatment or I3C treatment followed by EGF stimulation in PC-3 cells; however, phosphorylation at serine 473 and threonine 308 were inhibited by I3C. EGF treatment of PC-3 cells enhanced the phosphorylation at serine 473, whereas treatment of cells with LY294002, an inhibitor of PI3K, inhibited the serine phosphorylation, indicating that serine 473 phosphorylation is PI3K dependent. Pretreatment of PC-3 cells with I3C followed by EGF treatment fails to activate phosphorylation at serine 473. In addition, the PC-3 cells showed higher basal levels of Akt kinase activity, and it was further induced by 2-fold on EGF treatment. I3C treatment decreased both basal- and EGF-induced Akt kinase activity in PC-3 cells (Fig. 7). Collectively, these results indicate that I3C treatment of PC-3 cells not only down-regulates EGFR protein levels but also regulates the EGFR-induced PI3K/Akt signaling pathway by inhibiting phosphorylation of critical serine/threonine/tyrosine residues and, in turn, inhibiting PI3K and Akt activity.

We have shown previously that I3C induces cell growth inhibition of PC-3 cells accompanied by the induction of apoptotic processes as evidenced by a change in Bax:Bcl-2 ratio and intranucleosomal breakdown of DNA (9). Here, we investigated the signaling molecules that may be involved during the induction of apoptotic processes induced by I3C in PCa PC-3 cells. We found that the components of cell survival pathway are affected in I3C-treated PC-3 cells. Akt/PKB plays an important role in inhibiting apoptosis in response to growth factor and mitogen stimuli. Many signaling molecules in the apoptotic pathway have been shown to be either direct or indirect targets of Akt. Direct targets of Akt include the proapoptotic protein BAD, caspases 9, and forkhead family of transcription factors, and Akt can indirectly inhibit the NF-κB transcription factor activity and Fas-mediated apoptosis. We found that I3C inhibits the phosphosphorylation at serine 473 and threonine 308 without significantly affecting Akt protein levels, but Akt kinase activity was inhibited in I3C-treated PC-3 cells. Similar inhibition of Akt phosphorylation has been observed recently in gemcitabine-treated pancreatic cancer cells (40), topotecan-treated lung cancer cells (41), and several kinds of stress-inducing agents, including hyperosmosis, γ-irradiation, and UV irradiation (42). Conversely, oxidative stress induces Akt phosphorylation through an EGFR-dependent PI3K/Akt pathway (43). Given the cytotoxicity and significant side effects of chemotherapeutic drugs, a new interest in cancer chemoprevention is gaining prominence. I3C is one such reagent that has been shown to be very effective in in vivo animal studies against carcinogen-induced tumors. The molecular basis of the chemopreventive action of I3C could be attributed to its effect on cell growth inhibition, cell cycle arrest, inhibition of tumor invasion and metastasis (35), and induction of apoptosis (7) in a variety of cancer cells. Here we show that I3C induces apoptosis through inhibition of the PI3K/Akt pathway. Additional studies with either dominant negative constructs of p85 subunit of PI3K or Akt need to be used to understand the precise mechanism of I3C-mediated inhibition of Akt activation in PC-3 cells, and such studies are ongoing in our laboratory.

Aberrant expression of EGFR family members has been documented in a variety of cancers, including PCa. Signaling events associated with receptor tyrosine kinase play a prominent role in the regulation of cell growth and differentiation (44). In addition, overexpression of EGFR and autocrine activation by EGF and transforming growth factor-α has been observed in advanced androgen-independent PCa (16, 45, 46). We observed a dose- and time-dependent down-regulation of EGFR levels in I3C-treated PC-3 cells (Fig. 4), an androgen-independent PCa cell line, which lacks androgen receptor expression. The down-regulation of EGFR could be attributed to either transcriptional inhibition by I3C or inhibition of recycling of internalized EGFR to the plasma membrane. Transcriptional down-regulation of EGFR by retinoic acid has been observed in human papilloma virus 16-immortalized human ectocervical epithelial cells (47) and human epidermoid carcinoma ME180 cells (48), nerve growth factor in PC12 cells (49), and a leucine-rich proteoglycan decorin in A431 squamous carcinoma cells (50). In vitro studies have shown that EGFR is a good substrate for Phase II caspases, particularly caspases 3 and 7, and its degradation occurs during tumor necrosis factor-α-induced apoptosis in the presence of cycloheximide in A431 cells (51). Hence, the activation of apoptosis induced by I3C could lead to the degradation of many cellular proteins, including EGFR by caspases. On ligand engaging with EGFR, the ligand-EGFR complex undergoes internalization by recruitment into clatherin-coated pits on the plasma membrane. These newly formed sorting endosomes can be either targeted to late endosomes and lysosomes for degradation or on ligand dissociation in sorting endosomes because of moderately acidic pH, EGFR can recycle back to plasma membrane. Proto-oncogenes, c-abl ubiquitin ligase plays a role in targeting EGFR to lysosomal degradation by elevating the ubiquitination of EGFR (52), and protein kinase C has been shown to phosphorylate threonine 654 at EGFR, which diverts internalized EGFR to plasma membrane (53). Similar mechanism(s) may exist during I3C-induced inactivation of EGFR. Thus, our results on the down-regulation of EGFR by I3C may open new avenues for the prevention and/or treatment of PCa. However, the precise role of I3C in the down-regulation of EGFR remains to be further elucidated to fully understand the molecular mechanism of I3C action in PCa cells.

Acute treatment of cells with EGF is known to induce phosphorylation of many intracellular proteins involving PI3K/Akt and MAP kinase pathway. Akt phosphorylation at serine 473 and its corresponding biological activity through EGFR signaling is mediated by PI3K, and these signaling pathways were found to be down-regulated by I3C in PC-3 PCa cells. In the current investigation, we focused our efforts on the I3C-mediated alterations in PI3K/Akt pathway. However, we recognize that MAP kinase pathway may also participate in I3C-induced effects on PC-3 PCa cells, and, thus, it is premature to conclude whether I3C-mediated effects are specific to Akt cell survival pathway. Activated Akt has been shown to phosphorylate pro-apoptotic protein BAD at serine 136, which leads to its dissociation from Bcl-x_L and, in turn, promotes survival. However, this molecular pathway was found to be compromised severely in I3C-treated PCa cells as demonstrated by the down-regulation of Bcl-x_L at the protein levels. In addition, we observed decreased association of Bcl-x_L with BAD protein on I3C treatment, and this effect was not altered on EGF treatment of PC-3 cells pretreated with I3C. However, we were unable to demonstrate BAD interaction with 14-3-3ς even in untreated PC-3 cells despite using antibodies from several sources. This could be because of the specificity of antibodies, or BAD could be interacting with a different isoform of 14-3-3 proteins other than 14-3-3ς in PC-3 cells. Nevertheless, we are now actively investigating the molecular role of BAD phosphorylation in resting PC-3 cells using overexpression of BAD and 14-3-3ς isoform to understand the downstream functional consequences of constitutively active Akt in PC-3 cells. In keratinocytes, the basal expression of Bcl-x_L was dependent on EGFR-mediated MEK activity. Inhibition of MEK activity by either MEK inhibitor PD98059 or expression of dominant negative MEK construct led to down-regulation of Bcl-x_L expression (54). I3C induced down-regulation of EGFR protein levels, and the inhibition of its associated tyrosine kinase autophosphorylation activity could be attributed to down-regulation of Bcl-x_L expression. In addition, Bcl-x_L expression was shown to be down-regulated during etoposide-induced apoptosis in myeloid leukemia K562 cells (55), supporting our results indicating that the down-regulation of Bcl-x_L expression is an important molecular event during I3C-induced apoptosis in PC-3 cells.

In conclusion, the results of our studies provide mechanistic evidence, for the first time, that I3C induces apoptosis by inhibiting EGF-induced EGFR autophosphorylation and, in turn, inhibiting PI3K and Akt phosphorylation. The apoptosis-inducing ability of I3C, in conjunction with its nontoxic nature, could make it a potentially effective preventive and/or therapeutic agent against PCa. However, additional in vivo studies are needed to establish the role of I3C as a chemopreventive and/or therapeutic agent for PCa.

We thank the George Puschelberg Foundation for its support.