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Cancer Prev Res (Phila). Author manuscript; available in PMC Nov 1, 2012.
Published in final edited form as:
PMCID: PMC3244047
NIHMSID: NIHMS321141
(3-Chloroacetyl)-indole, a novel allosteric AKT inhibitor suppresses colon cancer growth in vitro and in vivo
Dong Joon Kim,1 Kanamata Reddy,1 Myoung Ok Kim,1 Yan Li,1 Janos Nadas,1 Yong-Yeon Cho,1,2 Jong-Eun Kim,1 Jung-Hyun Shim,3 Nu Ry Song,4 Andria Carper,1 Ronald A. Lubet,5 Ann M Bode,1 and Zigang Dong1*
1The Hormel Institute, University of Minnesota, MN 55912, USA
2Department of Pharmacology, College of Pharmacy, The Catholic University of Korea, Bucheon 420-102, Republic of Korea
3Department of Biochemistry, College of Medicine, Soonchunhyang University, Cheonan 330-090, Republic of Korea
4Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea
5National Cancer Institute, Bethesda, MD
* Corresponding Author: Zigang Dong, The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN 55912. Tel: 1-507-437-9600; FAX: 1-507-437-9606; zgdong/at/hi.umn.edu
Indole-3-carbinol (I3C) is produced in Brassica vegetables such as broccoli and cabbage and has been shown to inhibit proliferation and induce apoptosis in various cancer cells, including breast, prostate, colon, and leukemia. However, only high doses of I3C were shown to inhibit cell proliferation (IC50 = 200~300 μM). Our goal here was to develop a more potent antitumor agent by modifying the structure of I3C. We created I3C derivatives and found that (3-chloroacetyl)-indole (3CAI) more strongly inhibited colon cancer cell growth compared to I3C. Additionally, by screening 85 kinases in a competitive kinase assay, we found that 3CAI was a specific AKT inhibitor. AKT is a serine/threonine kinase that plays a pivotal role in promoting transformation and chemoresistance by inducing proliferation and inhibiting apoptosis. Therefore, AKT is regarded as a critical target for cancer therapy. 3ICA, a derivative of I3C, is a potent and specific AKT inhibitor. This compound showed significant inhibition of AKT in an in vitro kinase assay and suppressed expression of AKT direct downstream targets such as mTOR and GSK3β as well as induced growth inhibition and apoptosis in colon cancer cells. Additionally, oral administration of this potent AKT inhibitor suppressed cancer cell growth in an in vivo xenograft mouse model.
Keywords: (3-Chloroacetyl)-indole, AKT1, AKT2, indole-3-carbinol, colon cancer
Indole-3-carbinol (I3C), a natural product found in broccoli and cabbage, has chemopreventive properties such as anti-proliferative and pro-apoptosis activities against various cancers (1, 2). I3C reportedly targets a broad range of signaling pathways involved in cell cycle regulation and survival, including those mediated by AKT, nuclear factor-κB (NF-κB), Bcl-2, mitogen activated protein kinases (MAPKs), cyclin-dependent kinase (CDK) inhibitors and cyclin D1 in vitro (3-5). It is also reported to protect against chemically-induced carcinogenesis in vivo (6-8). These previous studies suggest that many dietary plants produce unique compounds that could be a source of starting molecules from which to synthetically develop new chemotherapeutic molecules with potent anti-cancer properties.
AKT/PKB is a serine/threonine kinase that belongs to the AGC family of kinases (9). Three members, AKT1, AKT2 and AKT3, have been identified and are composed of a conserved N-terminal pleckstrin homology (PH) domain, a central catalytic domain and a C-terminal regulatory hydrophobic motif (HM). The PH domain directs AKT translocation from the cytosol to the plasma membrane by binding to the membrane lipids phosphatidylinositide-3,4-P2 and 3,4,5-P3, which are products of phsphatidylinositide-3-kinase (PI3K). The AKT kinases are activated by phosphorylation of a threonine residue (Thr308) in the activation loop and a serine residue (Ser473) in the COOH-terminal activation domain (10, 11). The PI3K/AKT pathway regulates many cellular functions through a wide range of downstream targets, including the tuberous sclerosis complex 2 (TSC2), which negatively regulates the mammalian target of rapamycin (mTOR). Phosphorylation of TSC2 by AKT releases mTOR activity, which can stimulate protein synthesis in response to nutrients as well as regulate cell growth (12, 13). Another major substrate of AKT is glycogen synthesis kinase 3β (GSK3β), which is inactivated by AKT phosphorylation leading to increased glycogen synthesis during glucose metabolism following insulin stimulation (14, 15).
The PI3K signaling pathway is genetically altered in numerous types of cancers. For example, activating mutations of PIK3CA or mutations of PTEN are found in tumors of the colon, breast, brain, prostate, stomach and many other organs (16). AKTs are good candidates for mediating PI3K-dependent cell survival responses. Indeed, AKT activation and overexpression are often associated with resistance to chemotherapy or radiotherapy and dominant-negative mutants of AKT enhance the cytotoxicity of chemotherapeutic agents (17-19). In contrast, only a few AKT inhibitors have been identified (20). Thus, development of AKT inhibitors should be useful in clinical cancer therapy. Herein we report that the I3C, (3-chloroacetyl)-indole (3CAI) is a potent allosteric and specific AKT inhibitor, which exerts efficacy in vitro and in vivo.
Reagents
I3C (purity: 95%) was purchased from Sigma-Aldrich (St Louis, MO). 3CAI (purity: 95%), 5-methoxy-3CAI (purity: 95%), 5-fluoro-3CAI (purity: 95%) and 2-(4-(2-hydroxyethyl)piperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone) (purity: 95%) were purchased from InterBioScreen (Moscow, Russia). CNBr-Sepharose 4B beads were purchased from GE Healthcare (Piscataway, NJ). The active AKTs, active MEK1, active JNK1, active ERK1 human recombinant protein, histone H2B and H2AX for kinase assays were purchased from Millipore (Temecula, CA). The active TOPK human recombinant protein for the kinase assay was purchased from SignalChem (Richmond, BC). PI3K was obtained from Upstate Biotechnology (Lake placid, NY). AKT, p-AKT (Thr308), mTOR, p-mTOR (Ser2448), GSK3β, p-GSK3β (Ser9), Bad, Bcl2 and p-ASK1 (Ser83) and CDKN1A antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to detect p53 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 was purchased from Gibco BRL (Grand Island, NY). AKT inhibitor VIII was purchased from Merck KGaA (Darmstadt, Germany).
Synthesis of 3CAI
3CAI (purity: 95%) was synthesized as described (21) and purity and structure were analyzed using HPLC and NMR.
Cell culture
All cell lines were purchased from American Type Culture Collection (ATCC) and were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 8 weeks. Enough frozen vials were available for each cell line to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less. HCT116 and HT29 human colon cancer cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.
Molecular modeling
The crystal structure of the pleckstrin homology (PH) domain of AKT1 was obtained from the RCSB Protein Data Bank, PDB entry 1UNQ (22), which is a complex structure of the AKT1 PH domain and Ins(1,3,4,5)P4 and has an atomic resolution of 0.98 Å. The crystal structure was prepared using the Protein Preparation Wizard in Maestro v.9.2. Hydrogens were added to the protein structure consistent with a pH of 7. All water molecules in the crystal structure were removed. Then the crystal structure was minimized with an RMSD cutoff value of 0.3 Å. The structure of the AKT2 PH domain used in this study was modeled with the template structure of 1UNQ using Prime v.3.0. Energy grids for docking were computed for each protein structure using default settings in Glide v.5.7. 3CAI was prepared using LigPrep v.2.5 and then was docked into the PH domains of AKT1 and 2 with Glide extra precision (XP) mode.
Anchorage independent cell growth
Cells (8 × 103 per well) suspended in complete growth medium (McCoy's 5A supplemented with 10% FBS and 1% antibiotics) were added to 0.6% agar with different doses of each compound in a base layer and a top layer of 0.3% agar. The cultures were maintained at 37°C in a 5% CO2 incubator for 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v. 4) program (Media Cybernetics).
Western blot analysis
Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 × protease inhibitor tablet). Equal amounts of protein were determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. After washing with PBS containing 0.1% Tween 20, the membrane was incubated with a horseradish peroxidase–conjugated secondary antibody at 1:5,000 dilution and the signal detected with a chemiluminescence reagent (Amersham Biosciences Corp).
In vitro pull-down assay
Recombinant human AKTs (200 ng) were incubated with 3CAI-Sepharose 4B (or Sepharose 4B only as a control) beads (50 μl, 50% slurry) in reaction buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 μg/mL bovine serum albumin). After incubation with gentle rocking overnight at 4°C, the beads were washed 5 times with buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40) and binding was visualized by Western blotting.
Cell proliferation assay
Cells were seeded (1 × 103 cells per well) in 96-well plates and incubated for 24 h and then treated with different doses of each compound. After incubation for 48 h, 20 μl of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% CO2 incubator. Absorbance was measured at 492 nm.
Apoptosis assay
Colon cancer cells were plated into 60-mm culture dishes (1 × 105 cells/dish) and incubated for 1 day in medium containing 10% FBS. The culture medium was then replaced with a 1% serum medium and cultured for 4 days with 3CAI, I3C or a commercial AKT inhibitor. The cells were collected by trypsinization and washed with phosphate buffered saline (PBS). The cells were resuspended in 200 μl of binding buffer. Annexin V staining was accomplished following the product instructions (Clontech, Palo Alto, CA). The cells were observed under a fluorescence microscope using a dual filter set for FITC and propidium iodide and then analyzed by flow cytometry.
In vitro kinase assay
The kinase assay was performed in accordance with instructions provided by Upstate Biotechnology (Billerica, MA). Briefly, the reaction was carried out in the presence of 10 μCi of [γ-32P]ATP with each compound in 40 μl of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 μl protein loading buffer and the mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relative amounts of incorporated radioactivity were assessed by autoradiography.
Hematoxylin-eosin staining and immunohistochemistry
Tumor tissues from mice were embedded in a paraffin block and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. Tumor tissues were de-paraffinized and hydrated then permeabilized with 0.5% Triton X-100/1 × PBS for 10 min, hybridized with Ki-67 (1:500) as the primary antibody and horse-radish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG antibody was used as secondary antibody. After developing with 3, 3′-diaminobenzidine, the sections were counterstained with H&E. All sections were observed by microscope and the Image-Pro Plus software (v. 4) program (Media Cybernetics).
Xenograft mouse model
Athymic mice [Cr:NIH(S), NIH Swiss nude, 6–9 wk old] were obtained from Charles River and were maintained under “specific pathogen-free” conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Mice were divided into five groups: 1) untreated vehicle group (n = 15); 2) 20 mg 3CAI/kg of body weight (n = 15), 3) 30 mg 3CAI/kg body weight (n = 15); 4) 100 mg I3C/kg of body weight (n = 15); 5) no cells and 30 mg 3CAI/kg of body weight (n = 15). HCT116 cells (3×106 cells/100 μl) were suspended in serum free McCoy's 5A medium and inoculated subcutaneously into the right flank of each mouse. 3CAI, I3C or vehicle was administered orally 5 times per week for 21 days. Tumor volume was calculated from measurements of 2 diameters of the individual tumor base using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1 cm3 total volume, at which time mice were euthanized and tumors were extracted.
Statistical analysis
All quantitative results are expressed as mean values ± S.D. Statistically significant differences were obtained using the Student's t test or by one-way ANOVA. A value of p < 0.05 was considered to be statistically significant.
An I3C derivative, 3CAI, suppresses colon cancer cell growth
We screened for I3C derivatives that could effectively inhibit proliferation and anchorage-independent cell growth. To compare the effects of I3C (Fig. 1A) and its derivatives (Fig. 1B) on cancer cell growth, HCT116 colon cancer cells were treated with various concentrations of I3C or derivatives for 48 h. Proliferation was assessed by MTS assay and results indicated that growth was significantly decreased by I3C derivatives (#1, #2 or #3), but I3C or derivative #4 had little effect (Fig. 1C). Additionally, we compared the effect of the 5 compounds on anchorage-independent cell growth. HCT116 colon cancer cells were seeded with I3C or its derivatives in 0.3% agar and incubated for 3 weeks. Data showed that only the high dose of I3C (200 μM) or I3C derivatives (#1, #2 or #3), but not derivative #4, strongly suppressed anchorage-independent cell growth (Fig. 1D). Interestingly, I3C derivatives #2 or #3 had inhibitory effects on growth similar to derivative #1. Therefore, these findings suggested that methoxy and fluoro modification of 3CAI was not important for inhibiting proliferation or anchorage-independent cell growth. Based on these results, 3CAI appears to be the most effective anti-colon cancer compound of the 4 derivatives tested and was used in further studies.
Figure 1
Figure 1
Anti-cancer activity of I3C derivatives
3CAI is a potent inhibitor of AKT kinase activity
To identify the direct molecular target of 3CAI, we screened 85 kinases against 3CAI in a high-throughput substrate-competitive assay (http://www.kinomescan.com/). Results identified 3CAI as a potential inhibitor of AKT (Supplemental Table 1). Based on these screening data, we tested the effect of 3CAI on the kinase activities of AKT1, MEK1, JNK1, ERK1 and TOPK using in vitro kinase assays. The results showed that 3CAI (1 μM) suppressed only AKT1 kinase activity and the other kinases tested were not affected by 3CAI (Fig. 2A). We also studied the affect of 3CAI on kinases upstream of AKT. PI3K activity was potently inhibited by LY294002, a well-known inhibitor of PI3K, and 3CAI inhibited PI3K by 60% at the highest concentration (10 μM; Fig. 2B). These data suggest that 3CAI is a much more potent AKT1 inhibitor than PI3K (60% inhibition at 1 vs 10 μM, respectively). Additionally, we compared the effect of I3C, 3CAI and the AKT inhibitor VIII on AKT1 and 2 activities. 3CAI, but not I3C, substantially suppressed AKT1 activity (Fig. 2C) as well as AKT2 activity (Fig. 2D) in a dose dependent manner. These data showed that 3CAI is a potent and specific AKT1 and AKT2 inhibitor.
Figure 2
Figure 2
Effect of (3-chloroacetyl)indol (3CAI) on AKT activity
3CAI directly binds with AKT1 or AKT2 in an ATP non-competitive manner
We next performed a molecular docking study with 3CAI and AKT1 and AKT2 in order to determine its binding orientation. The docking score of 3CAI with AKT1 was -2.03 Kcal/mol, which was a little less favorable than the docking score of 3CAI with AKT2 (-2.25 Kcal/mol). 3CAI forms a hydrogen bond with Glu17 in the AKT1 PH domain, whereas 3CAI forms three hydrogen bonds with Lys14, Leu52 and Arg86 in the AKT2 PH domain (Fig. 3A). The structures of AKT1 or AKT2 were aligned and superimposed to compare the docked conformation of 3CAI. 3CAI adopts a pose parallel to Ins(1,3,4,5)P4 in AKT1, whereas 3CAI adopts a pose perpendicular with Ins(1,3,4,5)P4 in AKT2 (Fig. 3B). To confirm the results of the computer docking model, we performed in vitro pull-down assays using 3CAI or I3C-conjugated Sepharose 4B beads. These results showed that 3CAI directly bound to recombinant AKT1 (Fig. 4A) and AKT2 (Fig. 4B) in an ATP non-competitive manner. I3C showed no binding. Similar results were obtained using an HCT116 colon cancer cell lysate (Fig. 4C and 4D). These results suggest that 3CAI binds to an AKT allosteric site and not the ATP pocket.
Figure 3
Figure 3
Computer modeling of 3CAI and AKT1/2
Figure 4
Figure 4
3CAI directly binds to AKT1 or 2 in an ATP non-competitive manner
3CAI inhibits down-stream targets of AKT and induces apoptosis
We investigated the effect of 3CAI on down-stream targets of AKT, including teh phosphorylation of mTOR and GSK3β. Results indicated that the AKT-mediated phosphorlyation site of mTOR (Ser2448) and GSK3β (Ser9) were substantially decreased by 3CAI in a time-dependent manner (Fig. 5A). However, phosphorylation of AKT (Thr308) was not changed. Furthermore, pro-apoptotic marker proteins p53 and p21 were also upregulated by 3CAI after 12 or 24 h of treatment. Additionally, the anti-apoptotic marker protein Bcl2 and AKT-mediated phosphorylation of ASK1 (Ser83) were significantly decreased (Fig. 4B). These findings suggested that pro- or anti-apoptotic marker proteins are regulated by 3CAI in colon cancer cells. Next, to determine whether apoptosis was induced by 3CAI, we compared the effect on cell death of this compound, I3C and an AKT inhibitor. HCT116 and HT29 colon cancer cells were seeded on 6 cm dishes in 1% FBS/McCoy's 5A (HCT116) with 3CAI, I3C or the AKT inhibitor and then incubated for 4 days. Results showed that the number of apoptotic cells was significantly increased by 3CAI in HCT116 and HT29 colon cancer cells compared with untreated control cells (Fig. 5C).
Figure 5
Figure 5
Effect of 3CAI on the AKT signaling pathway
3CAI inhibits growth of colon cancer cells in a xenograft model
To examine the antitumor activity of 3CAI in vivo, HCT116 cancer cells were injected into the right flank of individual athymic nude mice. Mice were orally administered 3CAI at 20 or 30 mg/kg, I3C at 100 mg/kg, or vehicle 5 times a week for 21 days. Treatment of mice with 30 mg/kg of 3CAI significantly suppressed HCT116 tumor growth by 50% relative to the vehicle-treated group (Fig. 6A; p < 0.05). Remarkably, mice seemed to tolerate treatment with these doses of 3CAI without overt signs of toxicity or significant loss of body weight compared with vehicle-treated group (Fig. 6B). The effects of 3CAI on a tumor proliferation marker were evaluated by immunohistochemistry and H&E staining of HCT116 tumor tissues after the 21 days of treatments. The expression of Ki67 was markedly decreased by treatment with 3CAI (Fig. 6C). Next, we examined the effect of 3CAI on down-stream targets of AKT such as phosphorylation of mTOR and GSK3β by Western blot using the in vivo tumor tissues. Expression of these AKT-target proteins was strongly suppressed by 30mg/kg of 3CAI in tumor tissues (Fig. 6D). These data suggested that HCT116 colon tumor development was suppressed by 3CAI through inhibition of AKT signaling pathway.
Figure 6
Figure 6
Effect of 3CAI on colon cancer growth in a xenograft mouse model
The natural phytochemical, I3C, has been reported to exert potent anti-proliferative activities in cell-based studies and has been implicated as a potential therapy for human cancers. However, only high concentrations of I3C can induce anti-cancer activity and appear to involve a non-specific broad range of targets. Therefore, interest in developing more potent synthetic I3C-based compounds has grown. However, only a few I3C analogues have been reported and exert only a low enhancement of potency in biological activity (23, 24). In contrast, 1-benzyl-I3C was reported as the most potent synthetic derivative of I3C with an approximate 1000-fold increased potency against breast cancer. The investigators suggested that 1-benzyl-I3C inhibited CDK2 enzymatic activity and CDK6 activity through the downregulation of CDK6 transcription and protein expression (25). However, direct targets of 1-benzyl-I3C or its specificity were not determined. We have identified a potent derivative of I3C, 3CAI, from a high throughput screening of 85 kinases (Supplemental Table 1). We found that 3CAI inhibited only AKT kinase activity (Fig 2A, B), suggesting that 3CAI is a specific AKT inhibitor.
Additionally, we determined the binding orientation between 3CAI and AKT using a computer docking model. About 20 crystal structures of AKT2 and 10 of AKT1 are available. The molecular alignment of the protein sequences using EMBOSS (26) showed that they possess about 85% and 92% identity. 3CAI was docked to the allosteric site of AKT1 and AKT2. The preference for AKT2 over AKT1 is much more difficult to explain because the crystal structure of the AKT2 PH domain has not yet been solved. The sequences of AKT1 and 2 are highly conserved, and the sequence identity between the PH domains is about 80%. Thus a reliable homology model of the AKT2 PH domain can be built based on the known structure of the AKT1 PH domain. The structure of the AKT2 PH domain used in this study was modeled with the template structure of 1UNQ. The reason as to why 3CAI prefers the allosteric site of AKT1 and AKT2 over the ATP site is puzzling. The observed preference might not lie in a classical explanation where specific ligand-protein noncovalent interactions do or do not exist, but potentially the explanation lies in the realm of statistics. 3CAI was computationally predicted to possess multiple binding orientations within the ATP binding site of both AKT1 and AKT2 (data not shown). However, 3CAI preferred to dock only to the PH-domain in AKT1 and AKT2 (Fig. 3A and 3B). Therefore, we needed to analyze a docking simulation between 3CAI and the AKT1 and 2 proteins as well as examine binding with a deletion mutant of AKT1 and 2.
Previous studies showed that HER-2 mediated AKT activation to induce translocation of MDM2 from the cytoplasm to the nucleus. MDM2 directly binds to p53 and induces ubiquitination (27, 28). In other reports, an effect of AKT on MDM2 subcellular localization from the cytoplasm to the nucleus was not detected, but AKT was shown to facilitate the function of MDM2 to promote p53 ubiquitination by phosphorylation of Ser186 (29). In our study, the protein level of p53 was substantially increased by 3CAI in a time-dependent manner, as was the abundance of p21, a target of p53 (Fig. 5B). We examined whether inhibition of AKT kinase activity by 3CAI could induce stability of p53 by suppressing phosphorylation of MDM2 (Ser166). We confirmed the phosphorylation of MDM2 and p53 protein level using an immunofluoresence assay and Western blot analysis of cytoplasmic and nuclear protein fractions. However, we did not observe any significant translocation of MDM2 (data not shown). Importantly, 3CAI suppressed colon cancer cell growth and induced apoptosis more potently than I3C or a commercially available AKT inhibitor (Fig. 5). Results of a xenograft mouse model showed that oral administration of 3CAI at 30 mg/kg B.W. for 21 days significantly inhibited colon cancer cell growth and was not toxic (Fig. 6A, 6B).
In conclusion, we found that the I3C derivative, 3CAI, is a potent and specific AKT inhibitor and suppressed cell growth and induced apoptosis both in vitro and in vivo. These findings should be useful for developing drugs targeting AKT and 3CAI seems to be a promising lead compound.
Supplementary Material
Acknowledgments
We greatly appreciate the help from Dr. Margarita Malakhova for the RSK2 protein An external file that holds a picture, illustration, etc.
Object name is nihms321141ig1.jpgRSK2?? I used a RSK2 protein for EKR2 kinase assay substrate and Todd Schuster for apoptosis analysis. This work was supported by The Hormel Foundation and National Institutes of Health grants R37 CA081064, CA120388, ES016548, CA0227501 and National Cancer Institute Contract No. HHSN-261200533001C-NO1-CN-53301.
Footnotes
Conflict of Interest: The authors state no conflict of interest.
1. Chinni SR, Li Y, Upadhyay S, Koppolu PK, Sarkar FH. Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene. 2001;20:2927–36. [PubMed]
2. Zhang J, Hsu BAJ, Kinseth BAM, Bjeldanes LF, Firestone GL. Indole-3-carbinol induces a G1 cell cycle arrest and inhibits prostate-specific antigen production in human LNCaP prostate carcinoma cells. Cancer. 2003;98:2511–20. [PubMed]
3. Sarkar FH, Li Y. Indole-3-carbinol and prostate cancer. J Nutr. 2004;134:3493S–8S. [PubMed]
4. Aggarwal BB, Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle. 2005;4:1201–15. [PubMed]
5. Wang TT, Milner MJ, Milner JA, Kim YS. Estrogen receptor alpha as a target for indole-3-carbinol. J Nutr Biochem. 2006;17:659–64. [PubMed]
6. Grubbs CJ, Steele VE, Casebolt T, Juliana MM, Eto I, Whitaker LM, et al. Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Res. 1995;15:709–16. [PubMed]
7. Jin L, Qi M, Chen DZ, Anderson A, Yang GY, Arbeit JM, et al. Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice. Cancer Res. 1999;59:3991–7. [PubMed]
8. Oganesian A, Hendricks JD, Williams DE. Long term dietary indole-3-carbinol inhibits diethylnitrosamine-initiated hepatocarcinogenesis in the infant mouse model. Cancer Lett. 1997;118:87–94. [PubMed]
9. Bellacosa A, Kumar CC, Di Cristofano A, Testa JR. Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res. 2005;94:29–86. [PubMed]
10. Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT--a major therapeutic target. Biochim Biophys Acta. 2004;1697:3–16. [PubMed]
11. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999;13:2905–27. [PubMed]
12. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002;10:151–62. [PubMed]
13. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4:648–57. [PubMed]
14. van Weeren PC, de Bruyn KM, de Vries-Smits AM, van Lint J, Burgering BM. Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem. 1998;273:13150–6. [PubMed]
15. Summers SA, Kao AW, Kohn AD, Backus GS, Roth RA, Pessin JE, et al. The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. J Biol Chem. 1999;274:17934–40. [PubMed]
16. Samuels Y, Velculescu VE. Oncogenic mutations of PIK3CA in human cancers. Cell Cycle. 2004;3:1221–4. [PubMed]
17. Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH. AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway. Cancer Res. 2007;67:6325–32. [PubMed]
18. Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001;61:3986–97. [PubMed]
19. Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther. 2002;1:707–17. [PubMed]
20. Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones RE, et al. Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J. 2005;385:399–408. [PubMed]
21. Sudipta Roy SH, Gordon W. Gribble. Synthesis of Novel Oxazolyl-indoles. Synthesis. 2006;23:3948–54.
22. Milburn CC, Deak M, Kelly SM, Price NC, Alessi DR, Van Aalten DM. Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change. Biochem J. 2003;375:531–8. [PubMed]
23. Brandi G, Paiardini M, Cervasi B, Fiorucci C, Filippone P, De Marco C, et al. A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines. Cancer Res. 2003;63:4028–36. [PubMed]
24. Chao WR, Yean D, Amin K, Green C, Jong L. Computer-aided rational drug design: a novel agent (SR13668) designed to mimic the unique anticancer mechanisms of dietary indole-3-carbinol to block Akt signaling. J Med Chem. 2007;50:3412–5. [PubMed]
25. Nguyen HH, Lavrenov SN, Sundar SN, Nguyen DH, Tseng M, Marconett CN, et al. 1-Benzyl-indole-3-carbinol is a novel indole-3-carbinol derivative with significantly enhanced potency of anti-proliferative and anti-estrogenic properties in human breast cancer cells. Chem Biol Interact. 2010;186:255–66. [PMC free article] [PubMed]
26. Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16:276–7. [PubMed]
27. Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A. 2001;98:11598–603. [PubMed]
28. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol. 2001;3:973–82. [PubMed]
29. Ogawara Y, Kishishita S, Obata T, Isazawa Y, Suzuki T, Tanaka K, et al. Akt enhances Mdm2-mediated ubiquitination and degradation of p53. J Biol Chem. 2002;277:21843–50. [PubMed]