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Clinical Cancer Research 13, 6827, November 15, 2007. doi: 10.1158/1078-0432.CCR-07-0454
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Inhibition of Protein Kinase Cß by Enzastaurin Enhances Radiation Cytotoxicity in Pancreatic Cancer

Aaron C. Spalding1, Richard Watson1, Mary E. Davis1, Alex C. Kim2, Theodore S. Lawrence1 and Edgar Ben-Josef1

Authors' Affiliations: 1 Department of Radiation Oncology and 2 Cellular and Molecular Biology Training Program, The University of Michigan Medical School, Ann Arbor, Michigan

Requests for reprints: Aaron C. Spalding, Department of Radiation Oncology, The University of Michigan Medical School, UH B2C490, Box 0010, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0010. Phone: 734-936-8207; Fax: 734-763-7370; E-mail: spalda{at}med.umich.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Aberrant activation of protein kinase Cß (PKCß) by pancreatic cancer cells facilitates angiogenesis and tumor cell survival. Targeting PKCß with enzastaurin, a well-tolerated drug in clinical trials, would be expected to radiosensitize pancreatic tumors through direct antitumor and antivascular effects.

Experimental Design: We tested the hypothesis that enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo through inhibition of PKCß. We analyzed pancreatic cancer xenografts for growth delay and microvessel density after treatment with enzastaurin, radiation, or both. We determined the effect of radiation and enzastaurin on glycogen synthase kinase 3ß, a mediator of cell death in culture and in vivo.

Results: At concentrations attained in patients, enzastaurin reduced levels of active PKCß measured by phosphorylation at Thr500 in culture and in xenografts. Enzastaurin alone did not affect pancreatic cancer cell survival, proliferation, or xenograft growth. However, enzastaurin radiosensitized pancreatic cancer cells in culture by colony formation assay. Enzastaurin alone decreased microvessel density of pancreatic cancer xenografts without appreciable effects on tumor size. When combined with radiation, enzastaurin increased radiation-induced tumor growth delay with a corresponding decrease in microvessel density. Enzastaurin inhibited radiation-induced phosphorylation of glycogen synthase kinase 3ß at Ser9 in pancreatic cancer cells in culture and in tumor xenografts, suggesting a possible mechanism for the observed radiosensitization.

Conclusions: Enzastaurin inhibits PKCß in pancreatic cancer cells in culture, enhancing radiation cytotoxicity. Additional antivascular effects of enzastaurin were observed in vivo, resulting in greater radiosensitization. These results provide the rationale for a clinical trial in locally advanced pancreatic cancer combining enzastaurin with radiation.

Aberrant activation of protein kinase Cß (PKCß), an intracellular serine/threonine kinase, promotes neoplastic transformation, tumor cell survival, and angiogenesis. With respect to tumorigenesis, growth factor receptor activation of PKCß promotes colon carcinogenesis (1) and is necessary for invasiveness of colon carcinoma (2) and melanoma (3) cells. PKCß promotes colon (4) and breast (5) cancer cell cycle progression. Targeted silencing of PKCß or pharmacologic inhibition has been shown to inhibit proliferation in culture of colon, glioma, breast, and ovarian cancer cells. PKCß overexpression correlates with poor prognosis in diffuse large B-cell lymphoma (6) and is overexpressed in colon cancer as well. PKCß inhibition inhibits survival cascades in cancer cells (7, 8). Therefore, PKCß is an attractive target for radiosensitization of tumor cells.

In addition to its direct tumor effects, PKCß activation has been associated with pathologic angiogenesis in cancer (9), inflammation (10), and diabetes (11). Vascular endothelial growth factor (VEGF) receptor activation leads to PKCß-dependent endothelial cell proliferation. Tumor-induced angiogenesis requires activation of PKCß (9). Thus, PKCß inhibition could prevent tumor recruitment of endothelial cells in addition to direct antineoplastic activity.

Enzastaurin is a potent and selective inhibitor of PKCß that has antitumor and anti–endothelial cell activities. Enzastaurin has an additive effect with paclitaxel and carboplatin on inhibiting proliferation of breast and ovarian cancer cells in culture (12). Enzastaurin has activity against multiple myeloma in culture and in vivo (13). Further studies show that enzastaurin suppresses VEGF-induced angiogenesis in the rat corneal micropocket assay (14), decreases microvessel density, and prevents VEGF secretion from human tumor cell xenografts in nude mice (15). Prolonged courses of enzastaurin increase chemotherapy or radiation tumor growth delay of glioma, breast, and small-cell lung cancer xenografts (14, 16). Enzastaurin has activity in vitro and in animal models against a spectrum of malignancies, consistent with antivascular and antitumor effects.

There are data to suggest that enzastaurin would be a promising drug for pancreatic cancer. PKCß is overexpressed in pancreatic cancer relative to surrounding stroma and normal pancreatic tissue (17). In addition, angiogenesis is thought to be an important driving force in pancreatic cancer pathogenesis. Combining PKCß inhibition with radiation could result in improved tumor response for two reasons. First, PKCß promotes glycogen synthase kinase 3ß (GSK3ß) suppression in tumor cells, potentially providing resistance to radiation. Second, PKCß activation promotes angiogenesis in endothelial cells surrounding tumor cells; the antivascular effects may potentiate the effects of radiation through normalization of tumor perfusion, restoring normoxia and increasing delivery of systemic agents. Therefore, we tested the hypothesis that inhibition of PKCß with enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo.

PKCß modulates tumor cell survival, in part, by inhibiting GSK3ß. Growth factor–induced PKC activation (18) or radiation (19) can result in GSK3ß inactivation by phosphorylation at Ser9, stabilizing the mitochondrial membrane to prevent apoptosis. Graff et al. (8) showed that enzastaurin caused a time-dependent reduction of GSK3ß Ser9 phosphorylation in the colon cancer cell line HCT116 in culture and nude mouse xenografts. Enzastaurin also suppresses GSK3ß Ser9 phosphorylation in glioma cell lines both in culture and in intracranial glioma xenografts (16). Therefore, we also conducted experiments to determine if radiosensitization is mediated through changes in GSK3ß Ser9 phosphorylation.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. Panc1 and BxPC3 human pancreatic cancer cells were obtained from the American Type Culture Collection and were maintained in RPMI medium containing 10% bovine serum in an atmosphere of 93% air and 7% carbon dioxide.

Colony formation assays. After enzastaurin treatment or irradiation, cells were trypsinized, counted, and plated at clonal densities. Fourteen days later, cells were fixed and stained with crystal violet. Colony counting was done using an automated counter. The median effective radiation dose (20) was calculated for control and each dose of enzastaurin, and the enhancement ratio was calculated as the median effective radiation dose in the control curve divided by the median effective radiation dose in the enzastaurin curve.

Antibodies and immunoblotting. Antibodies to PKCß (Santa Cruz Biotechnology), phospho-Thr500 PKCß (Abcam), GSK3ß (Cell Signaling), phospho-Ser9 GSK3ß (Cell Signaling), and ß-actin (Sigma) were used at dilutions per manufacturer's instruction. Cell lysate production with radioimmunoprecipitation assay buffer and immunoblotting were done using detailed protocols from Cell Signaling. Xenografts were collected immediately following treatment and frozen in a dry ice-ethanol bath. The samples were ground at –80°C with a mortar and pestle and suspended in radioimmunoprecipitation assay buffer for immunoblotting.

Immunohistochemistry and immunofluorescence. Xenografts were collected on the last day of treatment for each group and half was placed in formalin overnight, followed by 70% ethanol. Samples were embedded in paraffin, and immunohistochemistry was done with anti-CD31 for microvessel density by the Immunohistochemistry Core at the University of Michigan. To determine microvessel density, the slides were blinded to the counter, and the number of patent vessels was scored in 10 high-power fields (400x).

Immunofluorescence was done with phospho-Ser9 GSK3ß antibody from Cell Signaling according to the manufacturer's protocol. A secondary antirabbit immunoglobulin G conjugated to FITC was used for antigen visualization. Nuclei were stained with propidium iodide to help determine subcellular localization and verify the number of cells per field.

Xenografts. Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the U.S. Department of Agriculture and Department of Health and Human Services. Under an institutionally approved protocol, 4-week-old female athymic nude mice were implanted with 5 x 107 BxPC3 or Panc1 cells s.c. Tumor volume was calculated as follows: tumor volume = {pi}/6 x a x b2, where a and b are the longer and shorter dimensions of the tumor, respectively.

When the average tumor volume achieved 100 mm3, mice were randomized into treatment groups of vehicle alone, enzastaurin (100 mg/kg gavage twice daily, separated by 8 h) alone, radiation alone, or enzastaurin with radiation. This schedule and dose of enzastaurin produces a 1 µmol/L serum concentration, similar to those achieved in patients from phase I trials (8). This schedule also suppresses GSK3ß dephosphorylation in mouse xenografts from 3 to 8 h after enzastaurin administration (8). Radiation was given as shown in Supplementary Fig. S1.

Data are expressed as the ratio of tumor volume at varying times after treatment compared with the day of irradiation (day 0). The absolute or normalized growth delay was calculated to compare the efficacy of each regimen. Absolute growth delay is defined as the time in days for tumors in the treatment arms to quadruple their volume minus the time in days for the tumors in the untreated control group to reach the same size. Normalized growth delay is defined as the time for tumors in groups treated with a combined regimen to quadruple their volume minus the time to reach the same size in mice treated with enzastaurin alone. Radiation enhancement ratio is the ratio of normalized growth delay (for each combined regimen) to absolute growth delay (for radiation alone; ref. 21).

Irradiation. Cells or xenografts were irradiated using a Phillips 250 orthovoltage unit at ~2 Gy/min for cells or 1.4 Gy/min for mice in the Irradiation Core of the University of Michigan Cancer Center. Dosimetry is carried out using an ionization chamber connected to an electrometer system, which is directly traceable to a National Institute of Standards and Technology calibration. Mice were anesthetized with a mixture of ketamine 60 mg/kg and xylazine 3 mg/kg, and positioned such that the apex of each flank tumor was at the center of a 2.4-cm aperture in the secondary collimator and irradiated with the rest of the mouse being shielded from radiation.

Statistical analysis. Differences between groups in microvessel density or mean growth delay were tested for significance with two-tailed Student's t test. The xenograft studies were powered at 80% to detect a 30% difference in absolute growth delay between the 12 tumors per group.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
We began by showing that both Panc1 and BxPC3 cells express active PKCß (Fig. 1 ). Treatment with enzastaurin leads to inactivation of PKCß in these pancreatic cancer lines as evidenced by Thr500 PKCß phosphorylation. Radiation also reduced the levels of Thr500 PKCß phosphorylation to the limit of detection by 120 min. Concentrations of enzastaurin above 100 µmol/L were required to inhibit survival as assessed by colony formation of both BxPC3 and Panc1 cells (Fig. 2A ). Achievable enzastaurin serum concentrations in patients are of the order of 1 µmol/L; at this concentration, enzastaurin inhibited PKCß activation but did not affect BxPC3 or Panc1 cell survival or proliferation when grown in 10% FCS (Supplementary Fig. S2). We therefore used enzastaurin at a concentration of ≤1 µmol/L for the remaining experiments described therein.


Figure 1
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  		Fig. 1. The pancreatic cancer cell lines BxPC3 and Panc1 express PKCß, and enzastaurin decreases PKCß phosphorylation. BxPC3 or Panc1 cells were treated with vehicle or 1 µmol/L enzastaurin for 24 h. A postradiation time course was conducted, and Western blotting for phospho-Thr500 or total PKCß and ß-actin was done.

 

Figure 2
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  		Fig. 2. Enzastaurin radiosensitizes BxPC3 and Panc1 cells at nontoxic concentrations obtained in patients. A, Panc1 or BxPC3 cells were incubated with semi-log incremental concentrations of enzastaurin for 24 h and assayed for clonogenic survival. Points, mean of three independent experiments done in triplicate; bars, SE. At 1 µmol/L, the serum concentration achieved in phase I trials, enzastaurin did not decrease clonogenic survival. B, BxPC3 or Panc1 cells were incubated with vehicle (solid line) or 1 µmol/L enzastaurin (dashed line) for 24 h before irradiation as indicated on the x-axis. The surviving fraction was assessed by colony formation at 14 d. A representative experiment done in triplicate is plotted with the SE bars too small to be visible. Similar results were obtained in at least three independent experiments.

 
After characterizing the effect of enzastaurin alone, we studied whether enzastaurin could radiosensitize pancreatic cancer cells. Incubation of BxPC3 or Panc1 cells for 24 h with 1 µmol/L enzastaurin led to radiosensitization by colony formation assay (Fig. 2B). Similar results were obtained with 0.3 µmol/L enzastaurin (data not shown). Enzastaurin produced modest radiosensitization in BxPC3 cells (enhancement ratio, 1.2 ± 0.03; n = 4) and Panc1 cells (enhancement ratio, 1.3 ± 0.03; n = 3). We conducted additional experiments and determined that pretreatment with 1 µmol/L enzastaurin for 16 h, but not 2 or 8 h, produced similar radiosensitization (Supplementary Fig. S3).

We next conducted nude mouse xenograft experiments to determine the in vivo effect of concurrent enzastaurin with radiation. We used ten 2-Gy daily fractions for BxPC3 xenografts and five 2-Gy daily fractions for Panc1 xenografts, as previous experiments had determined these regimens to cause 20 ± 5 day absolute growth delay for each cell type. Both BxPC3 (Fig. 3A ) and Panc1 (Fig. 3B) xenografts were treated with vehicle alone, enzastaurin alone, radiation alone, or enzastaurin with radiation. The combination of concurrent enzastaurin with radiation induced a greater tumor growth delay than the sum of the individual treatments, resulting in a radiation enhancement factor of 1.8 in BxPC3 cells and 2.1 for Panc1 cells (Supplementary Table S1). We also conducted experiments with 3-Gy fractions and obtained longer tumor growth delays with radiation alone. However, enzastaurin still enhanced radiation tumor growth delay by 1.5 in BxPC3 and by 1.8 in Panc1. Despite the enhancement of radiation-induced tumor growth delay, enzastaurin treatment did not increase toxicity because there was no difference in animal weight between the treatment groups (Supplementary Fig. S4). Radiation induced PKCß phosphorylation after five fractions in both Panc1 and BxPC3 xenografts in contrast to the inhibition seen with one fraction in culture. We confirmed that enzastaurin prevented Thr500 PKCß phosphorylation in vivo by Western blotting (Fig. 3C). The magnitude of radiosensitization by enzastaurin in vivo was greater than that seen in culture, leading to investigations to explain this observation.


Figure 3
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  		Fig. 3. Enzastaurin radiosensitizes BxPC3 and Panc1 xenografts. A, female athymic nude mice were implanted with BxPC3 and randomized: control, enzastaurin alone, 2 Gy daily x 10, or 2 Gy daily x 10 with enzastaurin. B, Panc1 cells were treated with control, enzastaurin alone, 2 Gy daily x 5, or 2 Gy daily x 5 with enzastaurin. Points, mean tumor volumes; bars, SE. Solid lines indicate treatment periods. P < 0.05, between radiation alone arm and radiation with enzastaurin arm. C, tumor lysates from each group were collected at the end of the treatment period and assayed for Thr500 PKCß phosphorylation, total PKCß, and ß-actin.

 
Because enzastaurin resulted in greater radiosensitization in vivo, we hypothesized that enzastaurin had both direct antitumor effects and antivascular effects not shown in culture. To investigate antivascular effects, we stained tumors from each treatment group for CD31 to determine the effect on blood vessel density of each treatment regimen (Fig. 4 ). After 5 days of treatment, radiation, enzastaurin, and the combination all resulted in decreased microvessel density compared with vehicle alone in Panc1 xenografts. Radiation alone, but not enzastaurin alone, resulted in decreased microvessel density in BxPC3 xenografts. For both BxPC3 and Panc1 xenografts, the combination of concurrent enzastaurin and radiation treatment resulted in the lowest microvessel density of all (n = 3; P < 0.05). Further decreases in microvessel density were not seen with a 10-day course of enzastaurin compared with a 5-day regimen.


Figure 4
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  		Fig. 4. Effects of radiation, enzastaurin, and the combination on vascular density of BxPC3 and Panc1 xenografts. Female athymic nude mice bearing either BxPC3 or Panc1 xenografts were randomized into treatment groups: control, enzastaurin alone for 5 d, 2 Gy x 5, or 2 Gy x 5 with enzastaurin. Tumors were harvested following the last treatment and anti-CD31 staining was done. *, P < 0.05, enzastaurin combined with radiation arm versus control, enzastaurin alone, and radiation alone.

 
Because GSK3ß has been implicated in determining the response of endothelial cells to radiation (19), and PKCß can modulate GSK3ß activity, we next conducted experiments to determine the level of GSK3ß phosphorylation under radiosensitizing conditions. As shown in Fig. 5A , radiation alone induces phosphorylation of GSK3ß at Ser9 in a time-dependent fashion in BxPC3 or Panc1 cells grown in culture, with the phosphorylation peaking at 15 min for BxPC3 and 60 min for Panc1. Enzastaurin pretreatment prevented the phosphorylation at Ser9. In vivo, a similar pattern occurs as shown by Western blotting in Fig. 5B. Tumors from animals treated with radiation alone show detectable phospho-Ser9 GSK3ß, in contrast to animals treated with concurrent enzastaurin and radiation.


Figure 5
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  		Fig. 5. Enzastaurin inhibits radiation-induced GSK3ß phosphorylation in pancreatic cancer cells in culture and in vivo. A, BxPC3 or Panc1 cells were treated with vehicle or 1 µmol/L enzastaurin for 24 h. A postradiation time course was conducted, and Western blotting for phospho-Ser9 or total GSK3ß and ß-actin was done. B, female athymic nude mice bearing either BxPC3 or Panc1 xenografts were randomized into treatment groups: control, enzastaurin alone for 5 d, 2 Gy x 5, or 2 Gy x 5 with enzastaurin. Tumors were harvested following the last treatment and subjected to immunoblotting for phospho-Ser9 and total GSK3ß. ß-Actin is shown as a loading control.

 
To confirm these findings and help determine the subcellular localization of radiation-induced phospho-Ser9 GSK3ß, we carried out immunofluorescence analysis of paraffin-embedded tumor sections (Fig. 6 ). In both BxPC3 and Panc1 cells, radiation treatment resulted in phosphorylation of GSK3ß at Ser9, with both nuclear and cytoplasmic localization. There was no detectable difference in total GSK3ß amount or localization by immunofluorescence (data not shown). These findings show that radiation leads to phosphorylation of GSK3ß in pancreatic cancer cells as well as in tumor-associated endothelial cells and that enzastaurin prevents radiation-induced GSK3ß phosphorylation.


Figure 6
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  		Fig. 6. Enzastaurin inhibits radiation-induced GSK3ß phosphorylation and subcellular distribution in pancreatic cancer cells in vivo. Female athymic nude mice bearing either BxPC3 or Panc1 xenografts were randomized into treatment groups: control, enzastaurin alone for 5 d, 2 Gy x 5, or 2 Gy x 5 with enzastaurin. Tumors were harvested following the last treatment and subjected to immunofluorescence for phosphorylated Ser9, detected by antirabbit immunoglobulin G-FITC (green). Propidium iodine staining (red) shows cell nuclei.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have found that inhibition of PKCß with enzastaurin radiosensitizes pancreatic cancer cells in vitro and lengthens the duration of radiation-induced tumor growth delay in vivo. We found that human pancreatic cancer cell lines express active PKCß, and that treating these cells with enzastaurin at noncytotoxic concentrations achievable in patients produces modest radiosensitization in culture. Although a brief course of enzastaurin treatment alone did not cause a tumor growth delay, microvessel density was reduced. When mice were treated with concurrent radiation and enzastaurin, the microvessel density and tumor growth delay were greater than the sum of either agent alone. These xenograft experiments suggest that the antivascular effects of enzastaurin account, at least in part, for the tumor radiosensitization seen in vivo. We also found that radiation induces Ser9 phosphorylation of GSK3ß in human pancreatic cancer cells. Enzastaurin effectively blocked this radiation-induced survival signal both in culture and in xenografts. Taken together, these data suggest that enzastaurin provides a dual mechanism for radiosensitization through anti–cancer cell and antivascular actions.

There is evidence that PKCß plays a role in neoplastic progression and tumor aggressiveness in patients, and strategies targeting PKCß could improve clinical outcomes. PKCß overexpression has been shown to correlate with relapse and death after cyclophosphamide-Adriamycin (hydroxydaunorubicin)-vincristine (Oncovin)-prednisone chemotherapy for diffuse large B-cell lymphoma (22). High-grade glioma has also shown increased PKC activity, which is potentially responsible for the rapid proliferation found in this malignancy (23). The data on human pancreatic adenocarcinomas suggest overexpression of PKCß relative to surrounding stroma (17). Our work shows that human pancreatic cell lines express active PKCß, and enzastaurin does inhibit PKCß. By effectively blocking PKCß signaling, enzastaurin was able to potentiate radiation cytotoxicity of human pancreatic cancer cells. Others have shown that pharmacologic agents that inhibit PKC isoforms can radiosensitize cancer cells in culture and in vivo (7, 24, 25).

Although we have shown the antivascular effects of enzastaurin in vivo, the direct radiosensitization of human pancreatic cancer cells could distinguish this compound from other agents that target endothelial cells more exclusively. Human pancreatic cancer cells do not universally express receptors for VEGF, whereas most do secrete VEGF (26). Therefore, pharmacologic strategies that target VEGF receptor would not likely directly radiosensitize pancreatic cancer cells. However, PKCß lies downstream of VEGF receptor (in endothelial cells) and of other growth factor receptors (27) that are overactive in pancreatic cancer, potentially accounting for enzastaurin-mediated radiosensitization of both tumor and endothelial cells. We showed a modest, reproducible radiosensitization with nontoxic concentrations of enzastaurin in pancreatic cancer lines. The magnitude of the direct antitumor effect in vivo is uncertain. However, because these pancreatic cancer cells express active PKCß, we would hypothesize that direct antitumor effects augment xenograft radiosensitization.

Radiation can induce tumor regression through antivascular effects. Tumor xenografts in asmase-deficient mice have been shown to be resistant to high-dose single-fraction radiation compared with the same tumors in wild-type mice; these differences have been attributed to endothelial cell radioresistance (28). The disordered tumor-associated blood vessels have increased permeability and interstitial pressure compared with normal vasculature (29), leading to decreased perfusion. Our data show that radiation reduced microvessel density in both pancreatic cancer cell lines, but the combination of enzastaurin with radiation resulted in greater-than-additive gains. Enzastaurin could potentially improve the effects of radiation by promoting normalization of tumor vasculature to restore blood flow and delivery of oxygen (30), although we need to confirm this with additional experiments.

There is rationale for combining antivascular therapy with radiation in the treatment of pancreatic cancer. Pancreatic cancers have high microvessel density, which correlates with shorter overall survival time (31). Increased pancreatic tumor microvessel density also predicts for higher rates of liver metastasis (32). However, the microvessel density does not lead to higher perfusion because the pathologic angiogenesis leads to increased vascular permeability. Pancreatic tumors have high interstitial pressure and are hypoxic (33). Interrupting tumor-mediated recruitment of blood vessels with enzastaurin could reverse the hyperpermeable state of pancreatic tumor blood supply. The resulting restoration of normoxia could enhance the cytotoxic effects of radiation in pancreatic tumors.

Our data suggest that enzastaurin can enhance radiation effects with very little toxicity of its own. When used alone at the concentrations achievable in patients, enzastaurin did not blunt proliferation or decrease colony formation. Previous reports have shown enzastaurin to have antiproliferative activity in vitro in 1% serum. It is possible that the 10% serum used in our experiments blunted the antiproliferative effect of enzastaurin. In our xenograft experiments, enzastaurin treatment alone for up to 10 days did not have an effect on tumor growth. Previous reports had shown enzastaurin to have activity alone after 21 consecutive days of treatment. Our experiments show that enzastaurin did not require efficacy or toxicity alone to potentiate radiation in vivo. Enzastaurin treatment did not cause a decrease in animal weight and has minimal reported side effects in clinical trials (34–36). Traditional cytotoxic systemic agents, in contrast, have not only intrinsic antitumor activity but also additional toxicity in rapidly proliferating normal tissues such as bowel mucosa. Given the close proximity of small intestine and other normal noninvolved organs to the pancreatic tumor, we expect that enzastaurin would improve the therapeutic index when combined with radiation in the treatment of pancreatic cancer patients.

In summary, we have found that enzastaurin has both radiosensitizing effects on pancreatic cancer cells in vitro and antivascular effects in pancreatic cancer xenografts. We provide data to support a clinical trial using enzastaurin with radiation for the treatment of locally advanced pancreatic cancer. These data suggest that this strategy might improve radiation effectiveness and clinical outcomes. Our data suggest that both direct anti–cancer cell and antivascular effects are involved. We are currently studying the interactions of enzastaurin with chemotherapy and chemoradiation regimens to determine if additional gains can be realized. Further studies are necessary to more definitively define the role of GSK3ß in enzastaurin-induced radiosensitization.


   Acknowledgments
 
We thank Steven Kronenberg for his graphical expertise.


   Footnotes
 
Grant support: American Society for Therapeutic Radiology and Oncology Resident Seed Grant (A.C. Spalding), Lilly Pharmaceuticals (E. Ben-Josef), and NIH grant 1 R03 CA127050-01 (E. Ben-Josef).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

A. Spalding has been designated a B. Leonard Holman Pathway Fellow by the American Board of Radiology. This work was presented in part at the 2007 Gastrointestinal Malignancies Symposium, January 19-21 2007, Orlando, Florida.

Received 2/27/07; revised 8/ 4/07; accepted 8/14/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gokmen-Polar Y, Murray NR, Velasco MA, Gatalica Z, Fields AP. Elevated protein kinase CßII is an early promotive event in colon carcinogenesis. Cancer Res 2001;61:1375–81.[Abstract/Free Full Text]
  2. Zhang J, Anastasiadis PZ, Liu Y, Thompson EA, Fields AP. Protein kinase C (PKC)ßII induces cell invasion through a Ras/Mek-, PKC{iota}/Rac1-dependent signaling pathway. J Biol Chem 2004;279:22118–23.[Abstract/Free Full Text]
  3. Khatib AM, Nip J, Fallavollita L, Lehmann M, Jensen G, Brodt P. Regulation of urokinase plasminogen activator/plasmin-mediated invasion of melanoma cells by the integrin vitronectin receptor {alpha}Vß3. Int J Cancer 2001;91:300–8.[CrossRef][Medline]
  4. Wang Q, Zhou Y, Evers BM. Neurotensin phosphorylates GSK-3{alpha}/ß through the activation of PKC in human colon cancer cells. Neoplasia 2006;8:781–7.[CrossRef][Medline]
  5. Li H, Weinstein IB. Protein kinase Cß enhances growth and expression of cyclin D1 in human breast cancer cells. Cancer Res 2006;66:11399–408.[Abstract/Free Full Text]
  6. Hans CP, Weisenburger DD, Greiner TC, et al. Expression of PKC-ß or cyclin D2 predicts for inferior survival in diffuse large B-cell lymphoma. Mod Pathol 2005;18:1377–84.[CrossRef][Medline]
  7. Chmura SJ, Mauceri HJ, Advani S, et al. Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation. Cancer Res 1997;57:4340–7.[Abstract/Free Full Text]
  8. Graff JR, McNulty AM, Hanna KR, et al. The protein kinase Cß-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res 2005;65:7462–9.[Abstract/Free Full Text]
  9. Yoshiji H, Kuriyama S, Ways DK, et al. Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res 1999;59:4413–8.[Abstract/Free Full Text]
  10. Andrassy M, Belov D, Harja E, et al. Central role of PKCß in neointimal expansion triggered by acute arterial injury. Circ Res 2005;96:476–83.[Abstract/Free Full Text]
  11. Curtis TM, Scholfield CN. The role of lipids and protein kinase Cs in the pathogenesis of diabetic retinopathy. Diabetes Metab Res Rev 2004;20:28–43.[CrossRef][Medline]
  12. Teicher BA, Menon K, Alvarez E, Shih C, Faul MM. Antiangiogenic and antitumor effects of a protein kinase Cß inhibitor in human breast cancer and ovarian cancer xenografts. Invest New Drugs 2002;20:241–51.[CrossRef][Medline]
  13. Podar K, Raab MS, Zhang J, 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 2007;109:1669–77.[Abstract/Free Full Text]
  14. Teicher BA, Alvarez E, Menon K, et al. Antiangiogenic effects of a protein kinase Cß-selective small molecule. Cancer Chemother Pharmacol 2002;49:69–77.[CrossRef][Medline]
  15. Keyes KA, Mann L, Sherman M, et al. LY317615 decreases plasma VEGF levels in human tumor xenograft-bearing mice. Cancer Chemother Pharmacol 2004;53:133–40.[CrossRef][Medline]
  16. Tabatabai G, Frank B, Wick A, et al. Synergistic antiglioma activity of radiotherapy and enzastaurin. Ann Neurol 2007;61:153–61.[CrossRef][Medline]
  17. Evans JD, Cornford PA, Dodson A, Neoptolemos JP, Foster CS. Expression patterns of protein kinase C isoenzymes are characteristically modulated in chronic pancreatitis and pancreatic cancer. Am J Clin Pathol 2003;119:392–402.[CrossRef][Medline]
  18. Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR, Mills GB. Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway. Mol Cell Biol 2002;22:2099–110.[Abstract/Free Full Text]
  19. Tan J, Geng L, Yazlovitskaya EM, Hallahan DE. Protein kinase B/Akt-dependent phosphorylation of glycogen synthase kinase-3ß in irradiated vascular endothelium. Cancer Res 2006;66:2320–7.[Abstract/Free Full Text]
  20. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55.[CrossRef][Medline]
  21. Milas L, Fujii T, Hunter N, et al. Enhancement of tumor radioresponse in vivo by gemcitabine. Cancer Res 1999;59:107–14.[Abstract/Free Full Text]
  22. Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med 2002;8:68–74.[CrossRef][Medline]
  23. Baltuch GH, Dooley NP, Villemure JG, Yong VW. Protein kinase C and growth regulation of malignant gliomas. Can J Neurol Sci 1995;22:264–71.[Medline]
  24. Akinaga S, Nomura K, Gomi K, Okabe M. Effect of UCN-01, a selective inhibitor of protein kinase C, on the cell-cycle distribution of human epidermoid carcinoma, A431 cells. Cancer Chemother Pharmacol 1994;33:273–80.[Medline]
  25. Tenzer A, Zingg D, Rocha S, et al. The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C. Cancer Res 2001;61:8203–10.[Abstract/Free Full Text]
  26. Wey JS, Fan F, Gray MJ, et al. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer 2005;104:427–38.[CrossRef][Medline]
  27. Oliva JL, Griner EM, Kazanietz MG. PKC isozymes and diacylglycerol-regulated proteins as effectors of growth factor receptors. Growth Factors 2005;23:245–52.[CrossRef][Medline]
  28. Garcia-Barros M, Paris F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 2003;300:1155–9.[Abstract/Free Full Text]
  29. Hagendoorn J, Tong R, Fukumura D, et al. Onset of abnormal blood and lymphatic vessel function and interstitial hypertension in early stages of carcinogenesis. Cancer Res 2006;66:3360–4.[Abstract/Free Full Text]
  30. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005;307:58–62.[Abstract/Free Full Text]
  31. Ikeda N, Adachi M, Taki T, et al. Prognostic significance of angiogenesis in human pancreatic cancer. Br J Cancer 1999;79:1553–63.[CrossRef][Medline]
  32. Fujioka S, Yoshida K, Yanagisawa S, Kawakami M, Aoki T, Yamazaki Y. Angiogenesis in pancreatic carcinoma: thymidine phosphorylase expression in stromal cells and intratumoral microvessel density as independent predictors of overall and relapse-free survival. Cancer 2001;92:1788–97.[CrossRef][Medline]
  33. Koong AC, Mehta VK, Le QT, et al. Pancreatic tumors show high levels of hypoxia. Int J Radiat Oncol Biol Phys 2000;48:919–22.[CrossRef][Medline]
  34. Rademaker-Lakhai J, Beereport L, Witteveen EO, et al. Phase I and pharmacologic study of enzastaurin HCl, gemcitabine and cisplatin. 2004 ASCO Annual Meeting; 2004.
  35. Fine HA, Kim L, Royce C, et al. Results from phase II trial of Enzastaurin (LY317615) in patients with recurrent high grade gliomas. 2005 ASCO Annual Meeting; 2005.
  36. Carducci MA, Musib L, Kies MS, et al. Phase I dose escalation and pharmacokinetic study of enzastaurin, an oral protein kinase Cß inhibitor, in patients with advanced cancer. J Clin Oncol 2006;24:4092–9.[Abstract/Free Full Text]



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B. A. Teicher
In Vivo/Ex Vivo and In Situ Assays Used in Cancer Research: A Brief Review
Toxicol Pathol, January 1, 2009; 37(1): 114 - 122.
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