Annals of Clinical & Laboratory Science 36:455-460 (2006)
© 2006 Association of Clinical Scientists
Antitumor and Normal Cell Protective Effect of PKC412 in the Athymic Mouse Model of Ovarian Cancer
Myungshin Kim1,
In-Yang Park2,
Jihyang Lim1,
Yonggoo Kim1,
Ku Taek Han2,
Won Heui Chung1 and
Kyungja Han1
1 Department of Laboratory Medicine and 2 Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul, Korea
Address correspondence to Kyungja Han, M.D., Department of Laboratory Medicine, St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 62 Youido-dong, Youngdeungpo-gu, Seoul 150-713, Korea; tel 82 2 3779 1310; fax 82 2 3779 2285; e-mail microkim{at}catholic.ac.kr.
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Abstract
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N-benzoyl-staurosporine (PKC412) is a selective inhibitor of protein kinase C, and it inhibits the growth of human cancer cells. In this study, we examined the antitumor effect of PKC412, given singly and in combination with paclitaxel, on tumor regression and chemotherapeutic side effects by assessing tumor burden and cytokine production responses in vivo. Twenty-six nude mice intraperitoneally inoculated with SKOV3 cells were treated differently in 4 treatment groups: PKC412 plus paclitaxel (n = 7), paclitaxel-only (n = 6), PKC412-only (n = 6), and controls (n = 7). At autopsy, we found that PKC412 itself slightly reduced the mass of tumor but did not fully inhibit tumor formation. The incidence of evident disease was decreased when PKC412 was combined with paclitaxel (43%). From the body weight of the tumor-bearing mice, we observed that PKC412 plus paclitaxel treated mice were less wasted than paclitaxel-only treated mice (18.1 g vs 22.4 g, p = 0.001). We measured intracellular TNF
, IFN
, IL-4, and IL-10 in stimulated mouse splenocytes using flow cytometry to determine if PKC412 inhibited cytokine production in T cells. TNF, IFN, and IL-10 production were all significantly inhibited in the paclitaxel-treated mice. The inhibitory effects on cytokine production by paclitaxel were compensated with PKC412 combination (p = 0.008, 0.035, 0.014, respectively). From this study, we deduce that PKC412 may have clinical applications in promoting tumor regression in ovarian cancer when combined with paclitaxel. Moreover, PKC412 is able to prevent weight loss and immunosuppression induced by paclitaxel because it rescues normal proliferating cells from cytotoxic effects.
Keywords: protein kinase C inhibitor, ovarian cancer, paclitaxel, PKC412, cytokine
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Introduction
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Ovarian cancer patients are likely to be diagnosed late, at an advanced stage, because of the rapid growth and spread of this intraperitoneal tumor, sometimes with a large volume of ascites. Despite improvements in surgery and chemotherapy, the high mortality rates in women with advanced, recurrent, or persistent ovarian cancer have been largely unchanged for the past 4 decades [1]. Paclitaxel is a widely used antineoplastic agents that promotes assembly of microtubules, inhibits tubulin disassembly, and blocks cell cycling at the G2/M stage. It also inhibits DNA synthesis, releases tumor necrosis factor- (TNF), and causes apoptotic cell death in a variety of cancer cell types, including ovarian cancer [2]. A major limiting factor in cancer chemotherapy is the serious cellular toxicity affecting proliferating normal cells, such as hematopoietic precursors, intestinal epithelial cells, and hair follicle cells [3]. To diminish the severity of these side effects, decreased doses of chemotherapy agents may be administered, allowing some cancer cells to survive and develop drug resistance. The patients ultimately relapse.
In an effort to develop more effective forms of therapy for ovarian carcinoma, we have investigated N-benzoyl-staurosporine (PKC412)-based combination therapy. PKC412 is a selective inhibitor of protein kinase C (PKC), and it inhibits the growth of human cancer cells in vitro and in vivo [4]. The rationale for testing PKC412 as an antitumor agent is based on the hypothesis that by inhibiting the deregulated cellular signaling pathways in malignant cells, as well as blocking tumor-induced angiogenesis, we may inhibit tumor growth and encounter fewer adverse side effects. Preliminary experiments with PKC412, in combination with conventional anticancer agents (including paclitaxel) against human NCI H-460 large cell lung carcinoma, provided evidence for agonistic effects. There was no exacerbation of the toxic effects of conventional agents [5].
Therefore, we examined the interactive impact of PKC412 given with paclitaxel on tumor regression and chemotherapeutic side effects by assessing tumor burden and cytokine production responses in an in vivo model.
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Materials and Methods
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Materials.
PKC412 and its placebo were provided by Novartis Pharma Inc. (Basel, Switzerland). The cancer cell line, SKOV-3, was purchased from ATCC (Rockville, MD). The cells were grown in RPMI1640 medium, supplemented with 20% (v/v) fetal bovine serum (GIBCO BRL, Grand Island, NY), 2 mmol/L L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin. The cell cultures were kept in a humidified incubator at 37°C and atmosphere of 95% air and 5% CO2.
In vivo study.
Five- to 7-wk-old BALB/c nude mice (20–25 g body wt, n = 26) were purchased from Atsugi Breeding Center (Charles River Japan Inc., Tokyo, Japan). The mice were acclimatized for 1 wk prior to the experiments in a room with an air-conditioned barrier-system and specific pathogen-free conditions. The mice were housed in polysulfone cages (4–5 mice/cage; Daejeong E&C, Seoul, Korea) with autoclaved wood-shavings; they were fed -ray irradiated laboratory mouse food (A04-10, UAR Quality Assurance, Orge, France) and had access to water containing minocycline ad libitum. The study protocols were approved by the Animal Care and Use Committee of The Catholic Univerity of Korea.
The cultured tumor cells were suspended (2x107 cells/ml) in phosphate-buffered saline (PBS). Cell viability was >95% by the trypan blue dye exclusion test. One ml of tumor cell suspension (2x107 cells) was injected ip into each mouse. At 4 wk after inoculation, the mice were divided into 4 treatment groups (n = 6 to 7 mice/group). One group (n = 7) was treated with PKC412 plus paclitaxel (Sigma Chemical Co., St. Louis, MO) for 6 wk. The second group of mice (paclitaxel-only treated, n = 6) was treated with paclitaxel and PKC412 placebo. The third group of mice (PKC412-only treated, n = 6) was treated with PKC412 and paclitaxel vehicle (PBS). The control group (n = 7) was treated with PBS and PKC412 placebo. Paclitaxel and PBS were injected ip twice weekly and the dose of paclitaxel (20 µg/g body wt) was based on previous studies [2]. The PKC412 (100 mg/kg) was given orally every other day. Body wt was measured twice weekly. When the mice died, autopsies were performed. Two mice in the PKC412-only treated group died during the treatment period. At autopsy, there were no specific findings except small tumor masses on the abdominal wall. All surviving mice were killed 1 wk after the end of treatment. The size and weight of the solid ip lesions were recorded.
Flow cytometric assay of cytokine production.
Splenocytes were isolated from mice and filtered through a 53 µm pore-size nylon mesh. The cells were stimulated with 10 ng/ml of phytohemagglutinin (M Form, lyophilized, GIBCO BRL) in the presence of BD GolgiPlug (1 µl/ml) for 5 hr. The cells (1x106 cells/50 µl of PBS) were stained with 20 µl of fluorescein isothiocyanate-conjugated anti-mouse-CD4 (BD Biosciences, San Jose, CA) (30 min, 4°C). After being washed, the cells were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences), according to the manufacturer’s directions. Ten µl of phycoerythrin-conjugated monoclonal antibodies to mouse TNF, IFN, IL-4, and IL-10 (BD Biosciences) were added to each tube. The tubes were incubated for 30 min at 4°C. After being washed with Perm/Wash solution supplied with the kit, 20,000 cells were analyzed by flow cytometry (FACSCalibur, BectonDickinson Immunocytometry System, San Jose, CA) using a CellQuest program. The experiments were repeated two or more times.
Statistics.
Results are presented as means ± SE. Data were analyzed using one-way analysis of variance, followed by the unpaired t-test for comparison between groups. Differences between groups were considered significant at p <0.05.
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Results
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We examined the potential interactions occurring between PKC412 and paclitaxel, given singly and in combination, upon the control of ovarian tumor growth and upon the cellular immunity of mouse splenocytes in order to assess whether these interactions (a) increase the therapeutic effects of each agent individually, and (b) reduce the side effects of paclitaxel.
The first signs of tumor growth in the mice were observed in the abdominal wall needle track at 2 wk after cancer cell inoculation. At postmortem examination, all mice in the control group had tumors on the surface of the peritoneum, intestines, and mesentery. All mice of the PKC4l2-only treated group had detectable tumors on the abdominal wall. The incidence of disease in mice of the paclitaxel-only treated group was decreased 50% (3 of 6 mice). Three of 7 (43%) mice receiving PKC412 plus paclitaxel had small subcutaneous tumors at the injection site. Three of 7 (43%) control mice exhibited abdominal swelling with ascites formation, but no detectable swelling or ascites developed in the other groups.
Body wt change during drug administration was used to evaluate the toxicity of the treatment modality. Fig. 1
summarizes the body wt changes during and after treatment. The paclitaxel-only treated mice showed more severe wt loss and weakness during the treatment period than mice in the other treatment groups. All of the other treatment groups showed body wt loss of <10%. There was a significant difference in weight at the end of treatment between the paclitaxel-only treated group and the PKC412 plus paclitaxel treated group (18.1 g vs 22.4 g, p = 0.001).
To determine if PKC412 inhibited cytokine production in T cells when given singly and in combination with paclitaxel, we measured intracellular TNF, IFN, IL-4, and IL-10 in mouse splenocytes stimulated with PHA [6]. Cytokine production in the different treatment groups is shown in Fig. 2. TNF, IFN, IL-4, and IL-10 production were all significantly inhibited in the paclitaxel-only treated mice compared to controls. Otherwise, cytokine production was not reduced in the PKC4l2-only treated and PKC412 plus paclitaxel treated mice compared to controls. The inhibitory effects on cytokine production (TNF, IFN, and IL-10) by paclitaxel were compensated when it was combined with PKC412 (p = 0.008, 0.035, 0.014, respectively).
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Discussion
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PKC412 has a broad spectrum of antitumor activity as a single agent, but its use in ovarian carcinoma has not been studied. In this study, we examined the antitumor effect of PKC412, given singly and in combination with paclitaxel, in a model of intraperitoneal ovarian carcinoma in athymic mice inoculated with SKOV3 cells. Because of the nature of tumor dissemination within the abdomen, tumor mass could not be quantified accurately during treatment. At autopsy, we found that PKC412 itself slightly reduced the mass of the tumor but did not fully inhibit tumor formation. The incidence of evident tumor was decreased when PKC412 was combined with paclitaxel (43%).
Attempts to potentiate cytotoxic cancer therapies by use of monoclonal antibody (mAb) to inhibit human vascular endothelial growth factor (VEGF), or by use of PKCß inhibitor, have been reported in previous studies. It is believed that angiogenesis may play a major role in the progression of most solid tumors, including ovarian carcinoma [7,8]. VEGF mAb and PKCß inhibitor were not very effective against SKOV-3 cells, but exposure to combinations with paclitaxel resulted in a marked reduction of tumor growth and ascites, and promoted long-term survival. VEGF promotes tumor angiogenesis and ascites production through its stimulation of vascular permeability [7]- PKC412 was originally identified and developed as an inhibitor of PKC, but was subsequently found to inhibit kinase insert-domain receptor (KDR), which is the major receptor for VEGF [9]. Neutralization of VEGF activity by PKC412 may have clinical applications in tumor regression and in inhibiting malignant ascites formation in ovarian cancer, when combined with paclitaxel.
From the body wt of the tumor-bearing mice, we observed that PKC412 plus paclitaxel treated mice were less wasted than paclitaxel-only treated mice. Paclitaxel is an effective chemotherapeutic agent for the treatment of ovarian carcinoma, but it affects normal bone marrow and gastrointestinal epithelial cells, which can lead to diarrhea, wt loss, and general weakness. The paclitaxel-only treated mice in this study also suffered from diarrhea. We cannot describe the definite mechanism by which PKC412 mitigates the wt loss caused by paclitaxel, but we suspect that normal proliferating cells are protected from paclitaxel by the presence of PKC412. Normal cells are tightly controlled by regulation of cell cycle checkpoints, but tumor cells have deregulated cell cycles, which allow for their continued, unabated proliferation [10,11]. The staurosporine pretreatment of normal cells inhibits their proliferation, but does not affect the proliferation of breast and ovarian cancer cells. Staurosporin-induced arrest of normal cells in the G0/G1 phase is reversible, whereas tumor cells, which are insensitive to staurosporine, are killed by chemotherapeutic agents [9,11].
PKC412 itself appears to have a minor effect on TNF, IFN, and IL-10 production. TNF, IFN, and IL-10 production were not inhibited when PKC412 was used combined with paclitaxel, compared to the paclitaxel-only treated group. These results suggest that PKC412 protects the inhibition of cytokine production that occurs with paclitaxel. In a previous report, PKC412 inhibited the production of TNF and IL-6 in patients receiving it in higher doses (15–300 mg/day); this inhibition was reversible 2–6 mo after finishing treatment [12]. Another study showed that PKC412 inhibited TNF production by T cells with an IC50 of 0.5 µM, and did not significantly inhibit the production of IL-2 or IFN [13]. We suspect that the concentrations we used were not able to reach the cytokine inhibiting level. It is also possible that the inhibition effect of PKC412 is reversible [14], so that the cytokines became normalized after the treatment cycle. Since the paclitaxel-only treated mice showed a marked inhibition of cytokine production, including TNF, IFN, IL-4, and IL-10, at the end of treatment, it seems reasonable to assume that PKC412 prevents the severe side effects of paclitaxel.
From this study, we believe that PKC412 may have clinical applications in promoting tumor regression in ovarian cancer when combined with paclitaxel. Moreover, PKC412 is able to prevent weight loss and immunosuppression induced by paclitaxel because it rescues normal proliferating cells from cytotoxic treatment. More studies are necessary to determine the optimal dosage and treatment protocol.
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Acknowledgments
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This study was supported by grant # 0320170-2 from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea.
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Abstract
Introduction
