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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Lilly Research Labs, Eli Lilly and Company, Indianapolis, Indiana
Requests for reprints: Jeremy R. Graff, Principal Research Scientist, Cancer Growth and Translational Genetics, Oncology Division, Lilly Research Labs, Lilly Corporate Center, DC0546, Indianapolis, IN 46285. Phone: 317-277-0220; Fax: 317-277-3652; E-mail: graff_jeremy{at}lilly.com.
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Abstract |
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Introduction |
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PKC activation also contributes to tumor cell survival and proliferation and has been repeatedly implicated in the malignant progression of human cancers, notably B cell lymphomas (4), malignant gliomas (5), and colorectal carcinomas (6). PKCß expression is specifically increased in patients with fatal/refractory diffuse large B cell lymphoma (DLBCL), linking increased PKCß expression to decreased patient survival (4). Further supporting a role for PKCß in DLBCL, cultured DLBCL cells with PKCß overexpression undergo apoptosis when treated with the PKCß-selective inhibitor LY379196 (7). In intestinal epithelia, expression of a PKCß transgene elicits hyperproliferation of the colonic epithelium and increases susceptibility to carcinogen-induced colon carcinogenesis indicating that PKCßII can promote colon carcinogenesis (6, 8). Furthermore, PKCßII expression in rat intestinal epithelial cells can induce an invasive phenotype (9).
Although PKC activation has been implicated in tumor formation and progression, the signaling pathways affected by PKC activation that contribute to malignancy are unclear. PKC activation can trigger signaling through the ras/extracellular signal-regulated kinase (ERK) pathway, which may be involved in the controlling cellular proliferation and apoptosis (1) as well as the induction of intestinal cell invasiveness (9). PKCßII activity may also play a role in reducing the sensitivity of intestinal epithelia to the growth suppressive effects of transforming growth factor ß (8). Recent work has now shown a link between PKC activity and activity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, a prominent regulatory pathway governing the apoptotic response. PKC activation requires phosphorylation of the T-loop, a process triggered by the activity of phosphatidylinositol-dependent kinase-1 (PDK-1), a key effector kinase-activated immediately downstream of PI3K (10). PKC
, PKCß, and PKC
can also directly phosphorylate AKT at Ser473, which is essential for AKT activity (11–13). Moreover, both PKC (14, 15) and AKT (16) can phosphorylate glycogen synthase kinase 3ß at Ser9 (GSK3ß), further supporting the notion that these signaling pathways overlap. Crosstalk between PKC and the PI3K/AKT pathway may be an attractive mechanism by which PKCs influence the apoptotic response.
Collectively, these data have implicated PKCs in tumor progression and have prompted the development of novel anticancer therapeutics targeting PKC. Enzastaurin (LY317615.HCl) was developed as a selective PKCß inhibitor (17). Based upon the role established for PKCß in angiogenic signaling (1, 3), enzastaurin was initially evaluated in preclinical tumor models for antiangiogenic activity. Enzastaurin treatment dramatically suppressed the growth of new vasculature towards a VEGF-impregnated disc implanted in the rat corneal micropocket (18). Enzastaurin also decreased microvessel density and VEGF expression in human tumor xenografts (19). The striking antiangiogenic effects of enzastaurin prompted the clinical development of enzastaurin.
In addition to the antiangiogenic effects of enzastaurin, we now show that enzastaurin directly suppresses proliferation and induces apoptosis of tumor cells in culture and suppresses phosphorylation of GSK3ß, ribosomal protein S6, and AKT. Oral dosing of enzastaurin to achieve plasma concentrations of drug comparable with those achieved in clinical trials significantly suppresses the growth of human colon and glioblastoma xenografts. As in cell culture, GSK3ß phosphorylation was suppressed in these tumor tissues. Moreover, GSK3ß phosphorylation was suppressed to a similar extent and with a similar time course in peripheral blood mononuclear cells (PBMCs) from these xenograft-bearing mice. These data support the notion that enzastaurin elicits an antitumor effect by suppressing signaling through the AKT pathway, directly inducing tumor cell death and suppressing tumor cell proliferation.
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Materials and Methods |
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, PKC
, or PKC
activity by enzastaurin was determined using a filter plate assay format measuring 33P incorporation into myelin basic protein substrate. Reactions were done in 100-µL reaction volumes in 96-well polystyrene plates with final conditions as follows: 90 mmol/L HEPES (pH 7.5), 0.001% Triton X-100, 4% DMSO, 5 mmol/L MgCl2, 100 µmol/L CaCl2, 0.1 mg/mL phosphatidylserine (Avanti Polar Lipids, Alabaster, AL), 5 µg/mL diacetyl glyerol (Avanti Polar Lipids), 30 µmol/L ATP, 0.005 µCi/µL 33ATP (NEN, Boston, MA), 0.25 mg/mL myelin basic protein (Sigma, St.Louis, MO), serial dilutions of enzastaurin (1-2,000 nmol/L), and recombinant human PKCßII, PKC, PKC, or PKC enzymes (390, 169, 719, or 128 pmol/L, respectively; PanVera, Madison, WI). Reactions were started with enzyme addition, incubated at room temperature for 60 minutes, quenched with 10% H3PO4, transferred to multiscreen anionic phosphocellulose 96-well filter plates (Millipore, Billerica, MA), incubated 30 to 90 minutes, filtered, and washed with 4 volumes of 0.5% H3PO4 on a vacuum manifold (Millipore). Scintillation cocktail was added and plates were read on a Microbeta scintillation counter (Wallac, Turku, Finland). IC50 values were determined by fitting a three-variable logistic equation to the 10-point dose-response data using ActivityBase 4.0 (ID Business Solutions, Ltd., Cambridge, MA). Upstate Kinase Profiler data were derived as per the provider (Upstate, Charlottesville, VA). Data are presented as the percent of kinase activity without enzastaurin.
Cell culture. HCT116 human colorectal cancer, U87MG human glioblastoma, and PC3 human prostatic adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in media with fetal bovine serum (FBS; Hyclone, Logan, UT) without antibiotics, as prescribed (ATCC).
Xenograft tumor studies. Five million HCT116 human colon cancer cells or U87MG human glioblastoma cells were injected s.c. in the flank of female, 6 to 8 weeks old, athymic nude mice (Harlan, Indianapolis, IN) in a 1:1 mixture of serum-free growth media and matrigel (Becton Dickinson, Bedford, MA). Mice were monitored daily for palpable tumors. Enzastaurin treatment was initiated when the tumors reached a group mean of 100 mm3. Enzastaurin was suspended in 10% acacia (Fisher Scientific, Fair Lawn, NJ) in water and dosed by gavage twice daily at 75 mg/kg based upon weekly body measurements for each treated group. Control groups were treated only with vehicle.
Protein lysates from cells and in vivo tissues. Lysates for western blot experiments and ELISA assays were prepared using Biosource Cell Extraction Buffer (Biosource International, Camarillo, CA) plus protease inhibitor cocktails (Sigma #P8340, #P2850, #P5726) at 10 µL of each per mL of cell extraction buffer (complete lysis buffer). Extracted tumors from in vivo studies were placed in 1 mL of ice-cold complete lysis buffer in Bio-101 tubes (QBiogene, Irvine, CA) for immediate homogenization. The Fast Prep FP-120 instrument (QBiogene) homogenized the tissues during two 30-second blasts. The homogenate was then transferred to Eppendorf tubes and centrifuged 10 minutes at 12,000 rpm. The supernatant (protein lysate) was then saved for western blotting or ELISA. The Bio-Rad DC-Protein assay kit (Bio-Rad, Hercules, CA) was used to determine protein concentrations.
PBMCs from the HCT116 tumor-bearing mice treated with enzastaurin were isolated as per the manufacturer's protocol included with the BD Vacutainer CPT tubes (BD, Franklin Lakes, NJ). Briefly, blood from five identically treated mice was pooled into a single CPT tube and centrifuged at 1,800 RCF for 30 minutes to separate blood components. The PBMC layer was collected and washed with 5 mL of ice-cold D-PBS, centrifuged 10 minutes at 200 rpm, and resuspended in 250 µL of complete lysis buffer. After centrifugation, the supernatant was collected for protein analysis.
Western blot and ELISA assays. The following antibodies used for western blotting were phosphoGSK3ßSer9, S6 ribosomal protein, phosphoS6Ser240/244, and phosphoAKTThr308 (Cell Signaling Technologies, Beverly, MA). Antibodies for total GSK3ß and total AKT were purchased from BD Biosciences (San Jose, CA). Western blots were done as described (20).
Proliferation assays. Proliferation was assessed for all cell lines over a 6-day time course in media supplemented with 1% FBS (7 days total). Briefly, 1,000 cells were plated per well in a 96-well plate and changed to fresh media (1% FBS) with or without enzastaurin on days 1 and 4. On day 7, the media were removed and 100 µL propidium iodide (PI) solution (10 µg/mL in D-PBS) were added to each well. An initial reading for PI staining (excitation at 500 nmol/L, absorbance at 615 nmol/L) was done using the WallacVictor plate reader following a 30-minute incubation to determine the nonviable cell fraction. The plate was then frozen at –80 °C for 2 hours, thawed, and reread. The proliferative index was scored by subtracting the prefreeze data (nonviable cells) from the postfreeze data (all cells).
Apoptosis assays. Apoptosis induction by enzastaurin was measured by nucleosomal fragmentation (Cell Death Detection ELISAplus, Roche Applied Science, Indianapolis, IN) and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining for HCT116 and U87MG cell lines. Briefly, 5,000 cells were plated per well in 96-well plates (1% FBS-supplemented media conditions), incubated with or without enzastaurin for 48 to 72 hours (as indicated) and run as per the manufacturer's protocol (Roche Applied Science). The absorbance values were normalized to those from control-treated cells to derive a nucleosomal enrichment factor at all concentrations as per the manufacturer's protocol (Roche Applied Science). The concentrations studied ranged from 0.1 to 10 µmol/L. In situ TUNEL staining was assayed with the In situ Cell Death Detection, Fluorescein kit (Roche Applied Science). Cells (75,000) were plated per well in 6-well plates and incubated 72 hours in 1% FBS-supplemented media ± enzastaurin. Fluorescein-labeled DNA strand breaks were detected with the BD epics flow cytometer. Ten thousand, single-cell, FITC-staining events were collected for each test.
PhosphoGSK3ßSer9 ELISA. Lysates prepared from HCT116 and U87MG tumors or mouse PBMCs were prepared as described above. PhosphoGSK3ßSer9 was quantitated using the Assay Design, Inc. (Ann Arbor, MI) immunometric assay kit. Briefly, 15 µL of tumor lysate (400-600 µg protein per well) or 25 µL PBMC lysate (50-100 µg protein per well) were added to all test wells. Absolute phosphoGSK3ßSer9 values are reported in pg phosphoGSK3ßSer9/mg lysate.
Statistical analyses. Tumor volume data are transformed to a log scale to equalize variance across time and treatment groups. The log volume data are analyzed with a two-way repeated-measures ANOVA by time and treatment using SAS PROC MIXED software (SAS Institute, Inc., Cary, NC). Treatment groups are compared with the control group at each time point. The data are plotted as means and SEs for each treatment group versus time. Statistical significance for the effect of enzastaurin treatment on GSK3ß phosphorylation in xenograft tumors was assessed by Dunnett's method, one-way ANOVA (JMP Statistical Discovery Software, SAS Institute).
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Results |
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We next sought to determine whether enzastaurin might induce apoptosis in tumor cells. As measured by oligonucleosomal fragmentation, enzastaurin induced apoptosis in both HCT116 colon carcinoma cells and U87MG glioblastoma cells in the low micromolar range (Fig. 2A). To confirm these results, we also evaluated apoptosis by TUNEL staining. Enzastaurin treatment of HCT116 colon carcinoma cells induced apoptosis in a dose-dependent manner with the percentage of TUNEL positive cells increasing from a basal level of roughly 2% to >50% in HCT116 cells treated with 4 µmol/L enzastaurin (Fig. 2B). Apoptosis was also evident by examining the sub-G0-G1 fraction after fluorescence activated cell sorting in HCT116 cells (data not shown). These analyses show that enzastaurin induces apoptosis in cultured human cancer cell lines in the low micromolar range (1-4 µmol/L).
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, PKC
, PKC
, PKC
, and PKC by 
90% or more but did not show substantial inhibition of PKCµ and PKC
. At 1 µmol/L, enzastaurin also suppressed the activity of p70S6 kinase, PKC
, and PKC by nearly 50% but failed to show substantial inhibition of other serine-threonine kinases (IKK and IKKß, c-jun NH2-terminal kinases, MKKs, stress-activated protein kinases, mitogen-activated protein kinases, AMPK, PRK2, PKB, PKBß, or PDK1) or tyrosine kinases (epidermal growth factor receptor, platelet-derived growth factor receptor, fibroblast growth factor receptor, BTK, SRC, Abl; Table 2). These data reaffirm that enzastaurin selectively inhibits PKCß at low concentrations and inhibits the activity of other PKC isozymes at higher concentrations, concentrations that are reached or surpassed in clinical trials.
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Oral dosing of enzastaurin suppresses human tumor xenograft growth and GSK3ßSer9 phosphorylation. The phase I clinical trials for enzastaurin have shown that oral administration at 525 mg/d yields 2 µmol/L mean steady-state plasma exposure of enzastaurin and its analytes (21). To examine more fully the effects of orally given enzastaurin on the growth of human tumor xenografts, we sought to identify a dose that would yield levels of enzastaurin and its metabolites comparable with what is reached in clinical trials. We chose a dose of 75 mg/kg given orally by gavage twice daily. Xenograft-bearing mice were treated for 21 consecutive days starting when the mean tumor volume reached 100 mm3. Enzastaurin treatment significantly suppressed the growth of both HCT116 colon carcinoma (Fig. 4A) and U87MG glioblastoma (Fig. 5A) xenografts (P < 0.01 for both studies). In the HCT116 xenograft-bearing mice, plasma exposure for enzastaurin was 2 µmol/L at 30 minutes, increased to nearly 3 µmol/L at 1 hour, and dropped to 1.5 µmol/L by 2 hours. Between 8 and 12 hours after dosing, the plasma concentrations of enzastaurin were nearly undetectable (Fig. 4B). The time course for enzastaurin plasma exposure was similar on day 1 and on day 21 after dosing (Fig. 4B). The time course for plasma exposure of enzastaurin dosed orally in mouse models consistently reflects the data in Fig. 4B, with nearly undetectable levels of drug in plasma between 8 and 12 hours after dosing. Consequently, 12-hour time points were not included in further studies.
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In both cultured tumor cells and in xenograft tumor tissues from the same cell lines, enzastaurin treatment suppressed GSK3ßSer9 phosphorylation, indicating that GSK3ßSer9 phosphorylation may serve as a reliable marker for enzastaurin activity. To do higher throughput analyses for GSK3ßSer9 phosphorylation, we chose a commercially available ELISA detection method (Assay Designs). ELISA analyses of U87MG xenograft tissues revealed >50% reduction in GSK3ßSer9 phosphorylation evident at the 2-hour time point and persisting through 8 hours (Fig. 5B; P < 0.0001 for all time points). In HCT116 xenograft tissues, ELISA analyses showed that GSK3ßSer9 phosphorylation was reduced by 50% after 30 minutes and by 80% at 2 hours. Consistent with the western blotting data (Fig. 4C), the reduction in GSK3ßSer9 phosphorylation persisted through 8 hours. Results for GSK3ßSer9 phosphorylation were comparable by western blotting and by ELISA (Fig. 4C versus Fig. 6), although the extent of reduction may be more pronounced by western blotting (compare 8-hour time points in Fig. 4C and Fig. 6). The differences in the extent to which GSK3ßSer9 phosphorylation was reduced in U87MG glioblastoma and HCT116 colon carcinoma xenografts may reflect an inherent difference in the biology of these tumors (for instance, U87MG lacks PTEN expression). Alternately, these data may simply reflect differences in these xenografted tumors related to tumor content, host tissue content, etc. In any case, both xenograft models show a significant, reproducible reduction in GSK3ßSer9 phosphorylation evident no later than 2 hours and persisting through 8 hours after oral dosing with enzastaurin.
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Discussion |
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We now show that enzastaurin also exhibits a direct effect on human tumor cells, inducing apoptosis in, and suppressing proliferation of, a wide array of cultured human tumor cells. Enzastaurin treatment interferes with signaling through the AKT pathway, suppressing the phosphorylation of GSK3ßSer9, ribosomal protein S6Ser240/244, and AKTThr308. The suppression of GSK3ßSer9 phosphorylation by enzastaurin was also evident in human xenograft tissues. Oral dosing of xenograft-bearing mice with enzastaurin (to achieve plasma exposure levels similar to that reached in human clinical trials) suppressed GSK3ßSer9 phosphorylation by >50% in both colon cancer and glioblastoma xenografts. GSK3ßSer9 phosphorylation was reduced in these tissues in a time-dependent manner, coinciding with plasma exposure levels of enzastaurin. Phosphorylation of GSK3ßSer9 was suppressed in the PBMCs of these xenograft-bearing mice to a similar extent and with a similar time course as in the xenograft tumor tissue, suggesting that GSK3ßSer9 phosphorylation in PBMCs may serve as a reliable pharmacodynamic marker for enzastaurin activity.
The direct, proapoptotic effects of enzastaurin treatment on human tumor cells were achieved at drug concentrations similar to those achieved in the plasma of clinical trials patients (1-4 µmol/L). At these drug concentrations, enzastaurin treatment interferes with signaling through the PI3K/AKT pathway, suppressing phosphorylation of GSK3ßSer9, ribosomal protein S6Serine 240/244, and AKTThr308. The PI3K/AKT pathway is a key regulator of cellular survival and is frequently activated in many human cancers, most notably glioblastomas, melanomas, and prostate, ovarian, and endometrial carcinomas (23). Interestingly, previous work had indicated that enzastaurin treatment suppressed VEGF expression by tumor xenografts (19). VEGF expression is regulated at both the transcriptional and posttranscriptional levels through activation of the AKT pathway (24–26).
It is unclear how enzastaurin may interfere with signaling through the PI3K/AKT pathway. In in vitro kinase assays, enzastaurin shows minimal inhibitory activity against p70S6 kinase and virtually no inhibition of AKT-1 or PDK-1, suggesting that the kinases responsible for phosphorylation of GSK3ßSer9, ribosomal protein S6Ser240/244, and AKTThr308 may not be directly inhibited by enzastaurin (LY317615.HCl). Furthermore, the time course for these signaling changes is different. Decreased phosphorylation of GSK3ßSer9 is evident within 30 minutes, whereas decreased phosphorylation of ribosomal protein S6Ser240/244 and AKTThr308 is evident at 2 and 4 hours post-treatment, respectively. Collectively, these data suggest that enzastaurin may indirectly suppress signaling through the AKT pathway. The PKC inhibitor PKC412 has also been shown to suppress AKT pathway signaling, upstream or at the level of AKT, although no direct mechanism was clear in these studies either (22).
Recent evidence has now shown that various PKC family members can regulate AKT activity. PKC can regulate the activity of AKT by directly stimulating phosphorylation of Ser473 in endothelial cells (11). PKCßII can also directly phosphorylate and activate AKT in mast cells (12). Activation of PKC can also activate AKT in glioblastoma cells, supporting glioblastoma proliferation (13). Our data show that the AKT pathway is suppressed in cells treated with 1 to 4 µmol/L enzastaurin. At these concentrations, enzastaurin can directly suppress the kinase activity of multiple PKC isoforms. It is therefore conceivable that interference with the AKT signaling pathway may be related to the effect of enzastaurin on multiple PKC family members.
Enzastaurin has now successfully advanced to phase II clinical trials for the treatment of refractory glioblastoma and DLBCL. With this report, we now show that enzastaurin exhibits direct antitumor activity, inducing tumor cell apoptosis and suppressing tumor cell proliferation. Moreover, enzastaurin interferes with signaling through the AKT pathway, a pathway frequently activated in a variety of human cancers. Finally, we show that enzastaurin profoundly suppresses the phosphorylation of GSK3ßSer9 both in human tumor xenograft tissue and in PBMCs harvested from xenograft-bearing mice, suggesting that GSK3ßSer9 phosphorylation may serve as a reliable pharmacodynamic marker of enzastaurin activity. With previous data, these data show that enzastaurin suppresses tumor growth through multiple mechanisms- the direct induction of tumor cell death and the suppression of tumor cell proliferation coupled to the indirect effect of suppressing tumor-induced angiogenesis.
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Acknowledgments |
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We thank Drs. Richard Gaynor, Kerry Blanchard, Jake Starling, John McDonald, Homer Pearce, Christopher Slapak, and Alan Hatfield for supporting this work and Cecile Gonzalez-Cerimele for all of her assistance.
Received 1/11/05. Revised 3/31/05. Accepted 5/27/05.
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J. M. Rademaker-Lakhai, L. V. Beerepoot, N. Mehra, S. A. Radema, R. van Maanen, J. S. Vermaat, E. O. Witteveen, C. M. Visseren-Grul, L. Musib, N. Enas, et al. Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral Protein Kinase C {beta}-Inhibitor Enzastaurin in Combination with Gemcitabine and Cisplatin in Patients with Advanced Cancer Clin. Cancer Res., August 1, 2007; 13(15): 4474 - 4481. [Abstract] [Full Text] [PDF]
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R. S. Herbst, Y. Oh, A. Wagle, and M. Lahn Enzastaurin, a Protein Kinase C{beta} Selective Inhibitor, and Its Potential Application as an Anticancer Agent in Lung Cancer Clin. Cancer Res., August 1, 2007; 13(15): 4641s - 4646s. [Abstract] [Full Text] [PDF]
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A. M.P. Omuro, S. Faivre, and E. Raymond Lessons learned in the development of targeted therapy for malignant gliomas Mol. Cancer Ther., July 1, 2007; 6(7): 1909 - 1919. [Abstract] [Full Text] [PDF]
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H. Younes, X. Leleu, E. Hatjiharissi, A.-S. Moreau, T. Hideshima, P. Richardson, K. C. Anderson, and I. M. Ghobrial Targeting the Phosphatidylinositol 3-Kinase Pathway in Multiple Myeloma Clin. Cancer Res., July 1, 2007; 13(13): 3771 - 3775. [Abstract] [Full Text] [PDF]
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R. W. Robey, S. Shukla, K. Steadman, T. Obrzut, E. M. Finley, S. V. Ambudkar, and S. E. Bates Inhibition of ABCG2-mediated transport by protein kinase inhibitors with a bisindolylmaleimide or indolocarbazole structure Mol. Cancer Ther., June 1, 2007; 6(6): 1877 - 1885. [Abstract] [Full Text] [PDF]
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A.-S. Moreau, X. Jia, H. T. Ngo, X. Leleu, G. O'Sullivan, Y. Alsayed, A. Leontovich, K. Podar, J. Kutok, J. Daley, et al. Protein kinase C inhibitor enzastaurin induces in vitro and in vivo antitumor activity in Waldenstrom macroglobulinemia Blood, June 1, 2007; 109(11): 4964 - 4972. [Abstract] [Full Text] [PDF]
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M. J. Robertson, B. S. Kahl, J. M. Vose, S. de Vos, M. Laughlin, P. J. Flynn, K. Rowland, J. C. Cruz, S. L. Goldberg, L. Musib, et al. Phase II Study of Enzastaurin, a Protein Kinase C Beta Inhibitor, in Patients With Relapsed or Refractory Diffuse Large B-Cell Lymphoma J. Clin. Oncol., May 1, 2007; 25(13): 1741 - 1746. [Abstract] [Full Text] [PDF]
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K. Podar, M. S. Raab, J. Zhang, D. McMillin, I. Breitkreutz, Y.-T. Tai, B. K. Lin, N. Munshi, T. Hideshima, D. Chauhan, et al. Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl) Blood, February 15, 2007; 109(4): 1669 - 1677. [Abstract] [Full Text] [PDF]
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E. I. Fomchenko and E. C. Holland Mouse models of brain tumors and their applications in preclinical trials. Clin. Cancer Res., September 15, 2006; 12(18): 5288 - 5297. [Abstract] [Full Text] [PDF]
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M. A. Carducci, L. Musib, M. S. Kies, R. Pili, M. Truong, J. R. Brahmer, P. Cole, R. Sullivan, J. Riddle, J. Schmidt, et al. Phase I Dose Escalation and Pharmacokinetic Study of Enzastaurin, an Oral Protein Kinase C Beta Inhibitor, in Patients With Advanced Cancer J. Clin. Oncol., September 1, 2006; 24(25): 4092 - 4099. [Abstract] [Full Text] [PDF]
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M. A. Rizvi, K. Ghias, K. M. Davies, C. Ma, F. Weinberg, H. G. Munshi, N. L. Krett, and S. T. Rosen Enzastaurin (LY317615), a protein kinase C{beta} inhibitor, inhibits the AKT pathway and induces apoptosis in multiple myeloma cell lines. Mol. Cancer Ther., July 1, 2006; 5(7): 1783 - 1789. [Abstract] [Full Text] [PDF]
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L. J. Green, P. Marder, C. Ray, C. A. Cook, S. Jaken, L. C. Musib, R. S. Herbst, M. Carducci, C. D. Britten, M. Basche, et al. Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-{beta} Inhibitor. Clin. Cancer Res., June 1, 2006; 12(11): 3408 - 3415. [Abstract] [Full Text] [PDF]
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) and day 21 (
) of dosing at 0.5, 1, 2, 4, 8, and 12 hours after dose. Plasma concentrations for enzastaurin were assessed by liquid chromatography tandem mass spectrometry. C, on day 21 of dosing, mice were killed and tumors harvested at 0.5, 1, 2, 4, 8, and 12 hours after dose. GSK3ßSer9 phosphorylation was assessed from these protein lysates by western blotting.
