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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey (G.E.A., X.Z., X.X.C., A.D.R., A.H., R.L.C., Y.P.L., Y.R.L., A.H.C.); Cancer Institute of New Jersey, New Brunswick, New Jersey (A.B.R., W.J.S., A.H.C.); Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School and Rutgers, The State University of New Jersey, Piscataway, New Jersey (J.S., A.B.R.); Division of Biometrics, School of Public Health, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey (W.J.S., Y.L.); and Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana (P.C.)
Received April 8, 2005; accepted June 14, 2005.
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Abstract |
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; Rovera et al., 1979; Zheng et al., 2001), and TPA was also shown to stimulate differentiation in peripheral leukemia cells obtained from patients with acute myeloid leukemia when the cells were cultured in vitro (Koeffler et al., 1980; Pegoraro et al., 1980).
In China, we administered TPA intravenously over a 1-h interval, one to three times per week alone or in combination with vitamin D3 or cytosine arabinoside to patients with acute myeloid leukemia or chronic myeloid leukemia with blast crisis who were refractory to cytosine arabinoside, all-trans retinoic acid (ATRA), and several other chemotherapeutic drugs. This study, which was the first to describe the administration of TPA to humans, demonstrated that administration of TPA decreased the number of myeloblasts in the bone marrow and in the peripheral blood (Han et al., 1998). The side effects associated with intravenous TPA administration, namely, dyspnea, fever, chills, venous irritation, and hematuria, were reversible. More recently, a phase I clinical trial at the Cancer Institute of New Jersey in New Brunswick also indicated an acceptable toxicity profile for TPA in patients with hematological malignancies (Strair et al., 2002).
In studies with cultured cells from solid tumors, TPA was shown to inhibit the growth, stimulate apoptosis, or enhance differentiation in human tumor cell lines derived from patients with melanoma, prostate, breast, colon, or lung cancer (Guilbaud et al., 1990; Salge et al., 1990; Arita et al., 1994; Garzotto et al., 1998; Rickard et al., 1999). Treatment of prostate cancer LNCaP cells with clinically achievable concentrations of TPA (1–1.6 nM) resulted in growth inhibition (Konno et al., 1996; Powell et al., 1996; Garzotto et al., 1998; Fujii et al., 2000), and treatment of these cells with a severalfold higher concentration of TPA caused apoptosis (Konno et al., 1996; Powell et al., 1996; Garzotto et al., 1998; Fujii et al., 2000). Treatment of LNCaP cells with a combination of TPA and gamma radiation resulted in a synergistic increase in ceramide synthesis and apoptosis (Garzotto et al., 1999). In other studies, treatment of prostate cancer cells with ATRA or 9-cis retinoic acid inhibited growth and induced apoptosis (Blutt et al., 1997; Pasquali et al., 1999). Our studies with TPA in prostate cancer cells demonstrated a synergistic inhibitory effect of TPA and ATRA on the growth of cultured LNCaP cells, and we also found an inhibitory effect of TPA or TPA and ATRA administration on the growth of well established LNCaP tumors in immunodeficient mice (Zheng et al., 2004).
Carcinoma of the pancreas is the fifth and fourth leading cause of cancer-related death in women and men, respectively, in the United States (Silverman et al., 1999), and the survival rate for this cancer is less than 4% (Greenlee et al., 2000). Currently, the front-line treatment for pancreatic cancer is the pyrimidine antimetabolite gemcitabine. As a relatively effective inhibitor of DNA synthesis and repair, gemcitabine has shown promise as a chemotherapeutic drug; however, the majority of patients with cancer of the pancreas show a poor response to chemotherapy. Accordingly, there is a need to develop better treatment regimens for pancreatic cancer. In the present report, we provide data on the inhibitory effect of TPA on the growth of PANC-1, MIA PaCa-2, and BxPC-3 human pancreatic cancer cell lines as well as data on the inhibitory effect of TPA on the growth of well established PANC-1 and BxPC-3 pancreatic tumor xenografts in immunodeficient mice.
The properties of PANC-1, MIA PaCa-2, and BxPC-3 cells are described at the web site http://pathology2.jhu.edu/pancreas/geneticsweb/Profiles.htm. All three cell lines have a mutation in p53, whereas only PANC-1 and MIA PaCa-2 also have a ras mutation. All three cell lines have 9p, 17p, and 18q loss of heterozygosity as well as a p16 homozygous deletion. PANC-1 and BxPC-3 cells have a 13q loss of heterozygosity.
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Materials and Methods |
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,25-dihydroxyvitamin D3, propylene glycol, polysorbate 80, benzyl alcohol, and ethanol were obtained from Sigma-Aldrich (St. Louis, MO), and gemcitabine hydrochloride was from Eli Lilly & Co. (Indianapolis, IN). Matrigel was obtained from BD Biosciences (Bedford, MA). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, McCoy's 5A medium, penicillin-streptomycin, L-glutamine, and fetal bovine serum were from Invitrogen (Carlsbad, CA). PANC-1 and MIA PaCa-2 cells were both maintained in DMEM, and BxPC-3 cells were maintained in RPMI 1640 medium as described previously. All media were supplemented with 10% fetal bovine serum, penicillin (100 units/ml)-streptomycin (100 µg/ml), and L-glutamine (300 µg/ml). The three cell lines were grown at 37°C in a humidified incubator with a 5% CO2 in air atmosphere, and stock cultures were passed two to three times per week.
Determination of the Number of Viable Cells. Cells were seeded at a density of 0.02 x 106 cells/ml in 35-mm plastic tissue culture dishes (Corning Glassworks, Corning, NY) and incubated for 24 h before treatment with vehicle or with TPA alone or in combination with other agents in vehicle for 96 h. The number of viable cells after a 96-h incubation was determined using a trypan blue exclusion assay (Zheng et al., 2004).
Flow Cytometry. For cell cycle analysis, cells were seeded at a density of 0.02 x 106 cells/ml in 100-mm plastic tissue culture dishes (Corning Glassworks) 24 h before treatment, and 1 x 106 cells were used for flow cytometry analysis. Cells were washed once with phosphate-buffered saline (PBS) and resuspended in 500 µl of stain solution [20 mg/ml polyethylene glycol 8000 (Fisher Scientific Co., Pittsburgh, PA), 50 µg/ml propidium iodide, 2.22 mg/ml RNase A, 0.1% Triton X-100, and 4 mM sodium citrate (Sigma-Aldrich)] for 1 h at room temperature. Five hundred microliters of salt solution (20 mg/ml polyethylene glycol 8000, 50 µg/ml propidium iodide, 0.1% Triton X-100, and 0.4 M sodium chloride) was added, and the mixture (1 ml) was incubated overnight at 4°C in the dark before being analyzed on a Beckman-Coulter Cytomics FC500 flow cytometer (Beckman Coulter, Fullerton, CA). The proportion of cells in each phase of the cell cycle was calculated by the cytologic software from Beckman Coulter (Fullerton, CA), and pre-G0/G1 cells were considered apoptotic cells.
Immunoblotting. Cells (1.5 x 106) were lysed in 500 µl of ice-cold RIPA buffer [10 mM Tris-HCl, pH 8, 10 mM EDTA (Sigma-Aldrich) at pH 8, 150 mM NaCl, 1% Nonidet P-40 (Sigma-Aldrich), and 0.5% SDS (Sigma-Aldrich)] containing a protease inhibitor cocktail [a 50x stock solution was prepared by dissolving one Complete tablet (Hoffman-La Roche, Nutley, NJ) in 1 ml of deionized water]. Twenty microliters of this stock solution was added to each milliliter of RIPA buffer. Lysates were passed through a 25-gauge needle four times to homogenize the sample and to shear the DNA. Samples (20 µl) containing 20 µg of protein were solubilized for 5 min at 100°C in Laemmli's SDS-PAGE sample buffer (Bio-Rad, Hercules, CA) containing 10% 
-mercaptoethanol (50 µl of -mercaptoethanol per 950 µl of sample buffer) (Sigma-Aldrich), loaded onto a 4 to 15% SDS-polyacrylamide gradient gel (SDS-PAGE) (Bio-Rad, Hercules, CA) or a 12% SDS-PAGE gel, and electrophoresed at 84 V for 2 h. Protein concentrations were determined by the DC protein assay method (Bio-Rad), a proprietary modification of the Lowry method. Protein was transferred to a polyvinylidene difluoride membrane (GE Healthcare, Piscataway, NJ) at 100 V for 60 min. The membrane was incubated in blocking buffer (PBS containing 0.1% Tween 20, 5% nonfat dry milk, and 1:1000 normal serum) for 1 h at room temperature or overnight at 4°C, followed by incubation in a 1:3000 dilution of the relevant primary antibody for 1 h at room temperature, and then incubated in a 1:10,000 dilution of horseradish-peroxidase conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 h at room temperature. The antibody to p21 (catalog no. 556430) was obtained from BD Biosciences Phar-Mingen (San Diego, CA), and the antibody to phospho-STAT1 (catalog no. sc-8394) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to phospho-Rb (catalog no. 9301S) and phospho-p44/42 mitogen-activated protein kinase (MAPK) (phospho-ERK) (catalog no. 9101) were obtained from Cell Signaling Technology Inc. (Beverly, MA). Bands were visualized by enhanced chemiluminescence (GE Healthcare). The extent of protein loading was determined by blotting for -actin. The membrane was incubated in stripping buffer (100 mM -mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl at pH 6.7) at 50°C for 30 min with occasional agitation before incubating in blocking buffer and reprobing using anti--actin (catalog no. sc-8432; Santa Cruz Biotechnology, Inc.).
Immunocytochemistry. PANC-1, MIA PaCa-2, and BxPC-3 cells were seeded at a density of 0.02 x 106 cells/ml in 60-mm plastic culture dishes (5 ml of medium/dish) and incubated for 24 h. The cells were then left untreated, treated with ethanol (solvent control), or treated with 10 nM TPA for 0.5, 2, 24, or 96 h. At appropriate time intervals, the culture medium was removed, and the cells were washed twice with PBS and fixed in a 1:1 methanol/acetone solution. Cells were incubated for 20 min with normal goat serum (1:1000) (Jackson ImmunoResearch Laboratories Inc.), washed twice for 5 min in PBS, and then incubated with mouse monoclonal antibodies to PKC (catalog no. sc-8393) or PKC (catalog no. sc-1681), or rabbit polyclonal antibodies to PKCI (catalog no. sc-209), PKCII (catalog no. sc-210), PKC
(catalog no. sc-213), or PKC
(catalog no. sc-216) for 1 h at room temperature. All PKC antibodies were used at 1:50 dilutions and were obtained from Santa Cruz Biotechnology Inc. Cells were washed twice for 5 min in PBS and then incubated with biotin-conjugated goat anti-mouse secondary antibody (1:200) (Jackson ImmunoResearch Laboratories Inc.) for 30 min at room temperature. Cells were washed and incubated in avidin-biotin complex (ABC; Vector Laboratories, Burlingame, CA) for 30 min and then in 1x 3,3'-diaminobenzidine tetrahydrochloride substrate for 5 min. The PKC antibodies used recognize human, mouse, and rat PKCs.
Growth of Pancreatic Tumor Xenografts in Immunodeficient Mice. Male NCr-immunodeficient mice (6–7 weeks old) were obtained from Taconic Farms (Germantown, NY). The animals were maintained in sterile microisolator cages. PANC-1 cells (1.5, 3.5, or 5 x 106 cells/0.1 ml/mouse), MIA PaCa-2 cells (2 x 106 cells/0.1 ml/mouse), or BxPC-3 cells (3.5 x 106 cells/0.1 ml/mouse) suspended in a 1:1 mixture of Matrigel and tissue culture medium (DMEM for PANC-1 and MIA PaCa-2 cells and RPMI 1640 medium for BxPC-3 cells) were injected subcutaneously into the right flank. Mice with well established palpable tumors measuring 0.4 to 1.0 cm in length and 0.4 to 1.0 cm in width were used for studies on the effects of TPA on tumor growth. All animals assigned to the experimental groups received daily injections of a vehicle (5 µl/g body weight) composed of propylene glycol, polysorbate 80, benzyl alcohol, ethanol, and water (40:0.5:1:10:48.5) or drug dissolved in vehicle. Tumor size was measured using calipers and areas were calculated (length x width). Both tumor size and body weight were recorded daily. Animals were sacrificed by cervical dislocation, after which the tumors were excised and fixed in 10% phosphate-buffered formalin at room temperature for 2 days before being transferred to 70% ethanol.
Statistical Analyses. The Student's t test was used for simple comparisons of two groups. The analysis of variance (ANOVA) model with Dunnett's adjustment (Hsu, 1996) was used for the comparison of multiple treatment groups with a common control. The analyses of the percentage of initial tumor size were based on the mixed effect regression (repeated measurement) model (Lindsey, 1993). The effects of the treatments also were assessed by comparing the rates of change over time between treatment groups (i.e., comparing the slopes between treatment groups). Pearson correlation coefficients together with 95% confidence intervals were calculated to assess the strength of correlations between changes in tumor size and body weight.
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Results |
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Effect of TPA on the Cell Cycle Distribution of Cultured PANC-1, MIA PaCa-2, and BxPC-3 Human Pancreatic Cancer Cells
Treatment of PANC-1 cells with 0.5 to 10 nM TPA for 96 h resulted in a dose-dependent decrease in the proportion of cells in the G0/G1 phase, an increase in the proportion of cells in the G2/M phase, and a small increase in the proportion of pre-G0/G1 cells (Table 2). Treatment of MIA PaCa-2 cells with 0.5 to 10 nM TPA for 96 h resulted in a dose-dependent increase in the proportion of cells in the G0/G1 phase, a decrease in the proportion of cells in the S phase, and a decrease in the proportion of cells in the G2/M phase. There was little or no change in the proportion of cells in the pre-G0/G1 phase (Table 2). In BxPC-3 cells, treatment with 0.5 to 10 nM TPA for 96 h inhibited cell growth but had no effect on the percentage of cells in G0/G1, S, or G2/M phases of the cell cycle (Table 2). An increased number of dead cells was observed after 24 or 48 h of TPA treatment (about 4-fold higher than in non-TPA-treated cells), but the number of dead cells was not appreciably increased after 96 h of TPA treatment. These results suggest that TPA-resistant BxPC-3 cells were the predominant cell type present after 96 h of TPA treatment, and it is likely that this is why TPA did not influence the cell cycle when measured after 96 h of TPA treatment.
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Effects of TPA on the Level and Intracellular Distribution of PKC Isoforms in Cultured PANC-1, MIA PaCa-2, and BxPC-3 Human Pancreatic Cancer Cells as Measured by Immunohistochemistry
Treatment of PANC-1 cells with 10 nM TPA for 96 h decreased the level of PKC in the cytoplasm, increased the level of PKC in the nucleus and cytoplasm, increased the level of PKCII in the perinuclear region and cytoplasm, increased the level of PKC in the perinuclear region, nucleus, and cytoplasm, and increased the level of PKC in the nucleus. Treatment of MIA PaCa-2 cells with 10 nM TPA for 96 h increased the level of PKC in the cytoplasm and nucleus, increased the level of PKCII in the cytoplasm and nucleus, caused translocation of PKC into the nucleus, and increased the level of PKC in the cytoplasm and nucleus. Treatment of BxPC-3 cells with 10 nM TPA for 96 h decreased the level of PKC in the cytoplasm and perinuclear area and increased the level of PKC and PKC in the cytoplasm and nucleus.
In another study, BxPC-3 cells were treated with 10 nM TPA, and the cells were fixed and stained with antibody for PKC, PKC, PKCI, PKCII, PKC, or PKC at 0.5, 2, 24, and 96 h. Treatment of the cells with TPA increased perinuclear staining for PKCII at 2 and 24 h and cytoplasmic staining at 24 and 96 h. Cytoplasmic and perinuclear PKC staining was increased from 0.5 to 96 h, and cytoplasmic and perinuclear PKC staining was increased from 24 to 96 h.
The results of our studies indicate that treatment of PANC-1, MIA PaCa-2, and BxPC-3 cells with TPA caused characteristic changes in the levels and intracellular distribution of the PKC isozymes in each cell line. The results of our studies on the effect of TPA on the level and intracellular distribution of PKC isozymes suggest that TPA-induced modulation of the PKC isozymes may be responsible for the inhibitory effect of TPA on the growth of PANC-1, MIA PaCa-2, and BxPC-3 cells.
Effect of TPA on the Level of p21 in Cultured PANC-1, MIA PaCa-2, and BxPC-3 Human Pancreatic Cancer Cells
Previous studies indicated that p21 is an important regulator of cell proliferation. Therefore, we investigated the effect of TPA on the level of p21 in PANC-1, MIA PaCa-2, and BxPC-3 cells. PANC-1, MIA PaCa-2, and BxPC-3 cells were seeded at a density of 0.1 x 106 cells/ml in 100-mm dishes and incubated for 24 h to allow the cells time to attach. The cells were then treated with 10 nM TPA for 2, 4, 6, 8, 10, 12, 16, and 24 h, and the cells were harvested at each time interval. The cells were then lysed in RIPA buffer and analyzed for p21 by immunoblot analysis. No p21 was detected in control untreated or ethanol-treated PANC-1 cells at any time interval, but a measurable level was observed at 2 h after TPA treatment, and a maximum level was observed at 4 h after treatment with TPA (Fig. 1A). The level of p21 decreased somewhat at 6 h, and at later time points, the level of p21 was less than that seen at 6 h but remained approximately constant up to 24 h, as indicated by optical density measurements of the bands at each time interval (normalized for actin) (Fig. 1A). p21 protein levels were undetectable in untreated, ethanol-treated, or TPA-treated MIA PaCa-2 and BxPC-3 cells at all time points (data not shown).
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B, Sp1, AP2, and STAT1.
Since TPA increased the level of PKC in the nucleus and cytoplasm and increased the perinuclear localization of PKCII and PKC in PANC-1 cells, we investigated the effects of four inhibitors of PKC, [bisindolylmaleimide I (Gö6850; GF109203X), Gö6976, Gö6983, and rottlerin] on the TPA-induced increase in the level of p21 in PANC-1 cells. Bisindolylmaleimide I and Gö6983 were proposed as selective inhibitors of the conventional and novel PKCs (Toullec et al., 1991; Gschwendt et al., 1996). Bisindolylmaleimide I (8 nM–6 µM) was shown to inhibit PKC, PKC1, PKC2, PKC, PKC, PKC, and PKC
(Toullec et al., 1991; Martiny-Baron et al., 1993), and Gö6983 (7–60 nM) was effective as an inhibitor of PKC, PKC, PKC, PKC, and PKC (Gschwendt et al., 1996). Gö6976 (2–20 nM) was effective as an inhibitor of PKC, PKC1, and PKCµ (Martiny-Baron et al., 1993; Gschwendt et al., 1996), and rottlerin (3–6 µM) was proposed as a selective inhibitor of PKC (Gschwendt et al., 1994).
PANC-1 cells were pretreated with vehicle or a PKC inhibitor in vehicle for 1 h before the addition of 10 nM TPA. Concentrations of inhibitors were selected that were previously shown to inhibit PKC. The cells were harvested 4 h after treatment with TPA and lysed, and the level of p21 was determined by immunoblot analysis. Treatment of PANC-1 cells with bisindolylmaleimide I (2 µM) or rottlerin (5 µM) completely inhibited the TPA-induced increase in the level of p21 in PANC-1 cells at 4 h after TPA (Fig. 1B). Pretreatment of PANC-1 cells with Gö6976 (100 nM) or Gö6983 (100 nM), however, had no inhibitory effect (Fig. 1B). The strong inhibitory effect of rottlerin and bisindolylmaleimide on TPA-induced increase of p21 suggests that the TPA effect was mediated by PKC. Electrophoretic mobility shift assays showed that treatment of PANC-1 cells with TPA (10 nM) for 15 min, 30 min, 1 h, or 2 h did not increase the level of NF-B (data not shown), indicating that the TPA-induced increase in the level of p21 is not associated with activation of NF-B.
Since phosphorylation of STAT1 and extracellular signal-related kinase [(ERK)1/2; p44/42 MAPK] in PANC-1 cells are potential downstream targets of PKC that can modulate p21 levels, we studied the effect of TPA on these pathways by immunoblot analysis using antibodies for phosphorylated STAT1 (Tyr701) and ERK1/2. Treatment of PANC-1 cells with 10 nM TPA for 2, 4, 6, 8, 10, 12, 16, or 24 h had no effect on the level of phosphorylated STAT1 (Tyr701) or ERK1/2 (data not shown). The results of our studies indicate that the TPA-induced increase in the level of p21 in PANC-1 cells is mediated by TPA-induced modulation of specific PKC isoforms but is not associated with activation of ERK, NF-B, or STAT1.
Effect of TPA on the Phosphorylation of Retinoblastoma Protein in Cultured PANC-1 Cells
p21 is an important regulator of the G1 cyclin-dependent kinases that in turn exert effects on the cell cycle through regulation of the phosphorylation of the retinoblastoma (Rb) protein. We therefore examined the status of Rb phosphorylation in TPA-treated PANC-1 cells (Fig. 1C). Lysates of ethanol- or TPA-treated PANC-1 cells were subjected to Western blot analysis using an antibody that reacts with nonphosphorylated or phosphorylated Rb. In ethanol-treated cells, only a single strongly reactive band was observed at 16 and 48 h corresponding to Rb phosphorylated at Ser795. In contrast, cells treated with TPA for 16 or 48 h showed the appearance of a more weakly reactive, more rapidly migrating Rb band indicative of hypophosphorylated Rb. Since phosphorylation of Rb at Ser795 is critical for inactivation of Rb transcriptional repressor function (Connell-Crowley et al., 1997), TPA-induced increase in the level of dephospho-Rb should result in the inhibition of transcription.
Effect of BAY 11-7082 Alone and in Combination with TPA on the Proliferation of Cultured PANC-1, MIA PaCa-2, and BxPC-3 Human Pancreatic Cancer Cells
Studies on the effects of TPA in combination with other drugs were initiated to determine whether enhanced inhibition of proliferation or enhanced cell killing could be achieved. BAY 11-7082 was tested together with TPA because BAY 11-7082 is a potent inhibitor of NF-B, and PANC-1, MIA PaCa-2, and BxPC-3 were previously shown to have high levels of NF-B (our unpublished data and studies by other investigators) (Liptay et al., 2003; Li et al., 2004). BxPC-3 cells were either left untreated, treated with DMSO (vehicle control), or treated with varying concentrations of BAY 11-7082. Concentrations of 0.2, 0.5, 1, and 5 µM BAY 11-7082 (dissolved in DMSO) were added, and the total incubation time was 96 h for all treatments. Although BAY 11-7082 inhibited the proliferation of all three cell lines in a dose-dependent manner as determined by the trypan blue exclusion assay, dead cells were not observed. Treatment of PANC-1, MIA PaCa-2, or BxPC-3 cells with 1 µM BAY 11-7082 for 96 h decreased the number of viable cells by 41, 67, and 25%, respectively, compared with vehicle-treated control cells (data not shown), and treatment of these cell lines with 5 µM BAY 11-7082 for 96 h decreased the number of viable cells by 88, 92, and 71%, respectively, compared with the vehicle-treated control cells (data not shown). The possibility that differences in the sensitivity of the three cell lines to BAY 11-7082 may be due to differences in the level of NF-B was explored by electrophoretic mobility shift assays. The basal level of expression of NF-B was similar in all three cell lines, and treatment of PANC-1 cells with TPA (10 nM) did not alter the level of NF-B at 15, 30, 60, or 120 min after TPA treatment (data not shown).
Treatment of PANC-1 cells with TPA (0.5 nM), BAY 11-7082 (0.2 µM), or TPA (0.5 nM) and BAY 11-7082 (0.2 µM) for 96 h decreased the number of viable cells by 38, 3, and 51% compared with the vehicle-treated control (Fig. 2A). Treatment of MIA PaCa-2 cells with TPA (0.5 nM), BAY 11-7082 (0.2 µM), or TPA (0.5 nM) and BAY 11-7082 (0.2 µM) for 96 h decreased the number of viable cells by 23, 36, and 75%, respectively, compared with vehicle-treated control cells, indicating that treatment of these cells with a combination of TPA and BAY 11-7082 inhibited the growth of MIA PaCa-2 cells to a greater extent than treatment with the individual compounds alone (Fig. 2B). No dead cells were observed. Treatment of BxPC-3 cells with a combination of TPA (0.5 nM) and BAY 11-7082 (0.2 µM) did not have a greater inhibitory effect on proliferation than TPA (0.5 nM) or BAY 11-7082 (0.2 µM) alone (data not shown). The results indicate that TPA in combination with BAY 11-7082 has a greater effect on the growth of MIA PaCa-2 cells (and to a lesser extent in PANC-1 cells) than either compound alone, but this was not observed in BxPC-3 cells.
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Effect of Sulindac Alone and in Combination with TPA on the Proliferation of Cultured PANC-1, MIA PaCa-2, and BxPC-3 Human Pancreatic Cancer Cells
Sulindac is a nonsteroidal anti-inflammatory drug that has previously been shown to inhibit NF-B (Yamamoto et al., 1999) and may be of potential clinical utility when combined with TPA. Treatment of PANC-1 cells with TPA (0.5 nM), sulindac (0.2 mM), or TPA (0.5 nM) and sulindac (0.2 mM) decreased the number of viable cells by 28, 15, and 72%, respectively, compared with the vehicle-treated control cells (Fig. 2C). The percentage of dead cells in vehicle-treated, TPA-treated, sulindac-treated, and sulindac plus TPA-treated PANC-1 cells was 3, 4, 0, and 9%, respectively (Fig. 2C). Treatment of PANC-1 cells with 0.5 nM TPA and 0.5 mM sulindac killed all of the cells (Fig. 2C).
Treatment of MIA PaCa-2 cells with TPA (0.5 nM), sulindac (0.2 mM), or TPA (0.5 nM) and sulindac (0.2 mM) decreased the number of viable cells by 67, 31, and 77%, respectively, compared with vehicle-treated control cells (data not shown). In this study, the percentage of dead cells in vehicle-treated, TPA-treated, sulindac-treated, and sulindac plus TPA-treated MIA PaCa-2 cells was 9, 24, 4, and 26%, respectively (data not shown).
Treatment of BxPC-3 cells with TPA (0.5 nM), sulindac (0.2 mM), or TPA (0.5 nM) and sulindac (0.2 mM) decreased the number of viable cells by 1, 62, and 69%, respectively (data not shown). None of the treatments increased the number of dead cells. In summary, the results indicate that TPA in combination with sulindac has a greater effect on the growth of PANC-1 cells than either compound alone, but this was not observed in MIA PaCa-2 or BxPC-3 cells.
Effects of ATRA Alone and in Combination with TPA on the Growth of Cultured BxPC-3 Human Pancreatic Cancer Cells
Since combinations of TPA and ATRA were more effective inhibitors of proliferation in HL-60 myeloid leukemia cells and in LNCaP prostate cancer cells (Zheng et al., 2001, 2004), we evaluated the effect of this combination on the growth of BxPC-3 pancreas cancer cells. In three experiments, TPA alone (0.5 nM) or ATRA alone (0.5–2 µM) had a modest inhibitory effect on the growth of BxPC-3 cells (Table 3), and the combination of TPA (0.5 nM) and ATRA (0.5–2 µM) had a somewhat greater inhibitory effect on the growth of BxPC-3 cells than TPA or ATRA alone (Table 3). Although growth inhibition occurred in these studies, there was little or no effect of the TPA/ATRA combination on cell killing (data not presented). Treatment of PANC-1 or MIA PaCa-2 cells with combinations of TPA (0.5 nM) and ATRA (0.5–2 µM) did not markedly increase growth inhibition compared with either compound alone (data not presented).
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Effects of Paclitaxel (Taxol) Alone and in Combination with TPA on the Proliferation of Cultured BxPC-3 Human Pancreatic Cancer Cells
In three experiments with BxPC-3 cells, the combination of Taxol and TPA had a greater inhibitory effect on proliferation than was obtained using TPA or Taxol alone (Table 3). In additional experiments, pretreatment of BxPC-3 cells with 0.5 nM TPA for 48 h before treatment with Taxol (0.5–5 ng/ml) for 48 h also enhanced the growth inhibitory effect of Taxol (data not presented). In PANC-1 cells, treatment with 0.5–5 ng/ml Taxol and 0.2 nM TPA caused effects comparable with that obtained with TPA alone or less than that obtained with either compound alone, depending on the concentration of Taxol used (data not presented). In MIA PaCa-2 cells, treatment with Taxol and TPA caused approximately additive effects compared with the effects obtained with either compound alone (data not presented).
Blood Levels of TPA after Its Intraperitoneal Injection in NCr-Immunodeficient Mice
Since low concentrations of TPA inhibited the growth of three cultured pancreatic cancer cell lines in our studies, we wanted to determine whether these concentrations could be achieved in vivo in mice before determining whether administration of TPA could inhibit the growth of pancreatic tumors in these animals. An i.p. injection of 50 or 100 ng of TPA/g body weight in a vehicle that consisted of propylene glycol, polysorbate 80, benzyl alcohol, ethanol, and water (40:0.5:1:10:48.5) resulted in peak blood levels at 30 to 60 min (Fig. 3). The peak blood level after a 100-ng/g body weight injection was about 1.5 nM (0.94 ng/ml) and after a 50-ng/g injection, the peak blood level was about 0.7 nM (0.44 ng/ml). Blood level data are expressed as nanomolar equivalents of TPA in blood as described in a recently developed bioassay (Cui et al., 2002). The half-life of TPA in these mice (measured between 6 and 12 h after the dose) was about 6 to 8 h. The concentrations of TPA observed were within the range that inhibited the growth of cultured pancreas cancer cells.
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Effects of Daily Intraperitoneal Injections of TPA on the Growth of PANC-1 Human Pancreatic Tumor Xenografts in NCr-Immunodeficient Mice
Because we were able to achieve blood levels of TPA in mice that inhibited the growth of cultured pancreas cancer cells in vitro, we evaluated the in vivo effect of TPA on the growth of well established pancreas tumors in mice. Male NCr mice were injected subcutaneously with 1.5 to 5 x 106 PANC-1, MIA PaCa-2, or BxPC-3 cells in Matrigel, and the mice were observed for the formation of tumors for 8 weeks. Tumor formation was slow and was observed in only 38% of the animals injected with PANC-1 cells at 6 to 8 weeks after the injection. Although no tumors were observed in animals injected with MIA PaCa-2 cells, even at 8 weeks after the injection, all animals injected with BxPC-3 cells had tumors by 2 weeks.
Effects of TPA on Tumor Growth and Body Weight. Initial in vivo experiments with TPA were done with PANC-1 tumors. Mice with well established and palpable PANC-1 tumors measuring 0.4 to 1.0 cm in length and 0.4 to 1.0 cm in width were injected i.p. with vehicle (5 µl/g body weight), 50 ng of TPA/g body weight, or with 100 ng TPA/g body weight in vehicle (5 µl/g body weight) once a day for 33 days (13 mice in the control group, five mice in the 50 ng/g TPA group, and four mice in the 100 ng/g TPA group). One additional mouse treated with 100 ng/g TPA died after eight daily injections and was not included in the analysis of data. Tumor growth was measured daily and expressed as a percentage of the initial tumor size on day 0 (Fig. 4A). On day 33, the mean ± S.E. for the percentage of initial tumor size was 188 ± 33 for the control group, 96 ± 11 for the 50 ng/g TPA group, and 62 ± 13 for the 100 ng/g TPA group (Fig. 4A). Statistical analysis of the data in Fig. 4 by the Kruskal-Wallis nonparametric ANOVA using Dunnett's multiple comparisons test (used because the data are not normally distributed) showed that the differences in percentage of initial tumor size at the termination of the study between the control group and the TPA (50 ng/g body weight/day) group and between the control group and the TPA (100 ng/g body weight/day) group were statistically significant (P < 0.05 and P < 0.01, respectively). Differences between the two TPA groups were not significant. Statistical analysis of the data in Fig. 4A showed that the linear time trends were significantly different between the vehicle control and TPA 50 and 100 ng/g-treated groups (P = 0.0092).
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The effects of vehicle and TPA in vehicle on the body weight of the mice with PANC-1 tumors was evaluated as a measure of drug toxicity. Expressing body weights at the end of the study as a percentage of the initial body weight on day 0, the mean ± S.E. was 98 ± 1% for the control group, 85 ± 4% for the 50 ng/g TPA group, and 91 ± 5% for the 100 ng/g TPA group. The mice treated with TPA initially showed a moderate drop in body weight with a subsequent increase and plateau in body weight with continued treatment. Statistical analysis of the data by the Kruskal-Wallis nonparametric ANOVA using Dunnett's multiple comparisons test showed that the differences in percentage of initial body weight at the termination of the study between the control group and the TPA (50 ng/g body weight/day) were statistically significant (P < 0.01). Differences between the control group and the TPA (100 ng/g body weight/day) group and between the two TPA groups were not statistically significant (P > 0.05).
The effects of vehicle and TPA treatments on the growth of PANC-1 tumors in individual mice are shown in Fig. 4B. None of the mice treated with vehicle showed tumor regression, and tumor size on day 33 ranged from 119 to 574% of the initial size. All mice treated with 100 ng of TPA/g body weight had some tumor regression, and tumor size on day 33 ranged from 34 to 95% of the initial tumor size (Fig. 4B). A substantial but somewhat smaller effect on tumor growth was seen in the mice treated with daily injections of 50 ng of TPA/g body weight (Fig. 4B). A comparison of data relating changes in body weight to changes in tumor size at the end of the experiment in individual mice from all of the 22 mice treated for 33 days indicated a relationship between these two parameters (r = 0.464) that is statistically different from zero (P = 0.03). There was no statistically significant relationship between these two parameters in the nine mice treated with TPA alone (r = –0.144). These results indicate that among mice treated with TPA, effects on body weight in individual mice were not related to changes in tumor size.
In a preliminary study, four vehicle-treated control mice and two TPA-treated mice with PANC-1 tumors were continued in the study for an additional 33 days. The average tumor size in the four mice injected i.p. with vehicle every day for 66 days was 314 ± 158% (S.E.) of the initial size (n = 4). The average tumor size in the two mice injected i.p. with TPA (100 ng/g body weight) every day for 66 days was 241% (287, 195%) of the initial size (n = 2). The average body weight of the four control mice at the end of the study was 105 ± 1% (S.E.) of the initial body weight, and the body weight of the two TPA-treated mice was 84.9% (81.6, 88.1%) of the initial body weight.
Effects of TPA on Mitosis and Apoptosis in Tumors. Mitotic cells were identified in H&E-stained section under a light microscope as described previously (Ergun et al., 1999). The percentage of mitotic cells in PANC-1 tumors from mice treated with vehicle or with 100 ng of TPA/g body weight i.p. daily for 66 days was determined as an indicator of proliferation. As shown in Table 4, the average percentage of mitotic cells ± S.E. in tumors was 0.60 ± 0.05 for the four mice in the control group and 0.36 (0.43 and 0.29) for the two mice in the 100 ng/g TPA group. The percentage of caspase-3-positive cells in tumors from these animals was determined as an indicator of apoptosis. The average percentage of caspase-3-positive cells ± S.E. was 0.45 ± 0.06 for the control group, and this was increased to 1.60 (1.66 and 1.53) for the 100 ng/g TPA group (Table 4). The ratio of percentage of mitotic cells/percentage of caspase-3-positive cells ± S.E. was 1.52 ± 0.40 for the control group and 0.22 (individual values of 0.26 and 0.19) for the 100 ng/g TPA group (Table 4). Similar but somewhat smaller effects were observed in PANC-1 tumor-bearing mice treated with daily i.p. injections of 50 ng of TPA/g body weight for 33 days (Table 4). The average ratio of the percentage of mitotic cells/percentage of caspase-3-positive cells in the tumors was decreased from 1.39 in the control animals to 0.45 in the TPA-treated animals (Table 4).
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Effects of Daily Intraperitoneal Injections of TPA Alone or Together with All-Trans Tetinoic Acid on the Growth of BxPC-3 Human Pancreatic Tumor Xenografts in NCr-Immunodeficient Mice
Effects of TPA and ATRA on Tumor Growth. Because BxPC-3 cells formed tumors with 100% efficacy in NCr mice, we repeated the in vivo experiments using this cell line. BxPC-3 cells (3.5 x 106) were injected subcutaneously into male NCr mice. Animals with well established tumors measuring 0.4 to 1.0 cm in length and 0.4 to 1.0 cm in width were injected i.p. with vehicle (5 µl/g body weight), TPA (30 or 50 ng/g body weight; 5 µl of vehicle/g body weight), ATRA (150 ng/g body weight; 5 µl vehicle/g body weight), or a combination of TPA and all-trans retinoic acid (50 and 150 ng/g body weight, respectively; 5 µl of vehicle/g body weight) once a day for 22 days (seven mice per group). Tumor growth was measured daily and expressed as a percentage of the initial tumor size on day 0 (Fig. 5A). It can be seen that treatment of the mice with daily injections of TPA (30 or 50 ng/g) or ATRA (150 ng/g) inhibited tumor growth for about 10 days, but at later time intervals, the rate of tumor growth was no longer inhibited (Fig. 5A). Tumor growth was inhibited to a greater extent and for longer time intervals when the mice were injected with a combination of TPA (50 ng/g body weight) and ATRA (150 ng/g body weight) (Fig. 5A). After 22 days of treatment, the mean ± S.E. for the percentage of initial tumor size was 191 ± 12 for the control group, 167 ± 14 for the 30 ng/g TPA group, 160 ± 14 for the 50 ng/g TPA group, 158 ± 18 for the ATRA group, and 115 ± 4 for the TPA/ATRA combination group (Fig. 5A). Although only two animals treated with the TPA/ATRA combination showed tumor regression at day 22 (Fig. 5B), it was observed that most animals experienced an initial decrease in tumor size with the treatment until day 17, at which time the tumors began to grow (Fig. 5A). Statistical analysis of the data by one-way ANOVA using the Student-Newman-Keuls multiple comparisons test showed that differences in percentage of initial tumor size at the termination of the study on day 22 were statistically significant between the control group and the TPA/ATRA group (P < 0.001), between the 30 ng/g TPA group and the TPA/ATRA group (P < 0.01), between the ATRA group and the TPA/ATRA group (P < 0.05), and between the 50 ng/g TPA group and the TPA/ATRA group (P < 0.05). Differences were not statistically significant between the control group and the 50 ng/g TPA group, between the control group and the 30 ng/g TPA group, between the control group and the ATRA group, between the 30 ng/g TPA group and the 50 ng/g TPA group, between the 30 ng/g TPA group and the ATRA group, and between the 50 ng/g TPA group and the ATRA group (P > 0.05). Statistical analysis was performed using the repeated measurement (mixed effect) models evaluating the change in percentage of tumor volume from baseline over time. Pairwise comparisons of the regression slopes (rate of change in percent of tumor size) of the treatment groups showed that the rate of percentage of increase in tumor size from baseline for the vehicle-treated group was significantly greater than for any other group (P 
0.0311), the rate for the TPA plus ATRA-treated group was significantly smaller than for any other group (P < 0.0001) and the rates for the TPA- and ATRA-treated groups were not statistically different from each other (P 
0.1556). This analysis showed that the percent increase in tumor size from baseline was increased by 4.4, 3.5, 2.9, 2.9, and 0.4% per day for the vehicle-treated group, the TPA 30 ng/g-treated group, the TPA 50 ng/g-treated group, the ATRA-treated group, and the TPA plus ATRA-treated group, respectively. In summary, the data indicate that daily injections of TPA (30 or 50 ng/g body weight) or ATRA (150 ng/g body weight) inhibited the growth of well established BxPC-3 tumors and a combination of TPA and ATRA was more effective.
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Effects of TPA and ATRA on Body Weight. The effects of the treatments on the body weight of the mice were expressed as a percentage of the initial body weight at day 0. The percentage of the initial body weight ± S.E. on day 23 was 101 ± 1% for the control group, 94 ± 2% for the 30 ng/g body weight TPA group, 93 ± 3% for the 50 ng/g body weight TPA group, 92 ± 3% for the ATRA group, and 91 ± 2% for the TPA/ATRA combination group (Fig. 5C). Statistical analysis of the data by one-way ANOVA using the Student-Newman-Keuls multiple comparisons test showed that the percentages of initial body weight at the termination of the study were statistically different between the control group and the TPA/ATRA group (P < 0.01). Differences between all other groups were not statistically significant (P > 0.05). The effects of the treatments on the tumor size and body weight in individual mice are shown in Fig. 5, B and C. A comparison of data relating changes in body weight to changes in tumor size at the end of the experiment in individual mice from all of the 35 mice treated for 23 days indicated a relationship between these two parameters (r = 0.598) that is statistically different from zero (P = 0.0001). However, there was no statistically significant relationship between these two parameters in the 14 mice treated with TPA alone (r = 0.131), indicating that among TPA-treated mice TPA-induced changes in tumor size were not related to TPA-induced changes in body weight. In addition, treatment of the mice with TPA (50 ng/g body weight), ATRA (150 ng/g body weight), or with TPA and ATRA had about the same inhibitory effect on body weight (Fig. 5C), but the combined treatment with TPA and ATRA was more effective at inhibiting tumor growth than TPA or ATRA alone (Fig. 5, A and B).
Effects of TPA and ATRA on Mitosis and Apoptosis in Tumors. The percentage of mitotic cells in tumors from animals from all the treatment groups was determined as an indicator of proliferation. The average percentage of mitotic cells ± S.E. was 0.51 ± 0.02% for the control group, 0.41 ± 0.02% for the 30 ng/g TPA group, 0.41 ± 0.01% for the 50 ng/g TPA group, 0.37 ± 0.02% for the ATRA group, and 0.34 ± 0.02% for the TPA/ATRA combination group (Table 4). Statistical analysis by one-way ANOVA, using the Tukey-Kramer multiple comparisons test, showed that the average percentage of mitotic cells of tumors from mice treated with the vehicle was significantly different from that of tumors from mice treated with the TPA/ATRA combination (P = 0.0153) but not from that of tumors from mice treated with 30 ng/g TPA, 50 ng/g TPA, or ATRA alone. The percentage of caspase-3-positive cells in tumors from animals from all the treatment groups was determined as an indicator of apoptosis. The average percentage of caspase-3-positive cells ± S.E. was 0.34 ± 0.03% for the control group, 0.49 ± 0.09% for the 30 ng/g TPA group, 0.42 ± 0.09% for the 50 ng/g TPA group, 0.39 ± 0.04% for the ATRA group, and 0.94 ± 0.17% for the TPA/ATRA combination group (Table 4). Statistical analysis using the Tukey-Kramer multiple comparisons test indicated that the percentage of caspase-3-positive cells in tumors from animals treated with the TPA/ATRA combination was significantly different from that of animals treated with vehicle (P < 0.01), animals treated with 30 ng/g TPA (P < 0.05), animals treated with 50 ng/g TPA (P < 0.01), and animals treated with ATRA (P < 0.05). The ratio of percentage of mitotic cells to percentage of caspase-3-positive cells ± S.E. was 1.57 ± 0.15% for the control group, 1.08 ± 0.22% for the 30 ng/g TPA group, 1.14 ± 0.22% for the 50 ng/g TPA group, 0.93 ± 0.07% for the ATRA group, and 0.42 ± 0.10% for the TPA/ATRA combination group (Table 4). Statistical analysis by the Tukey-Kramer multiple comparisons test showed that the ratio of percentage of mitotic cells to percentage of caspase-3-positive cells seen in tumors from animals treated with the TPA/ATRA combination was significantly different from that of tumors from vehicle-treated animals (P < 0.01) but not from that of tumors from animals in any of the other groups. For all parameters measured, only the TPA/ATRA group was statistically different from the vehicle control (Table 4).
In a second study using low dose levels of TPA and ATRA, animals with BxPC-3 tumors were injected i.p. with vehicle (5 µl/g body weight) (n = 6), TPA (25 ng/g body weight; 5 µl/g body weight) (n = 6), ATRA (100 ng/g body weight; 5 µl/g body weight) (n = 7), or a combination of TPA and all-trans retinoic acid (25 and 100 ng/g body weight, respectively; 5 µl/g body weight) (n = 5) once a day for 25 days. As in the previous experiments, tumor growth was measured daily and expressed as a percentage of the initial tumor size on day 0. On day 25, the mean percentage of the initial tumor size ± S.E. was 266 ± 16% for the control group, 211 ± 24% for the TPA group, 209 ± 15% for the ATRA group, and 185 ± 15% for the TPA/ATRA combination group. Animals treated with the TPA/ATRA combination experienced an initial strong suppression in tumor growth, but after about 15 days, tumors started to grow at a rate comparable with the vehicle-treated control group. Statistical analysis of the data by one-way ANOVA using the Tukey-Kramer multiple comparisons test showed that the differences in percentage of initial tumor size at termination of the study on day 25 were statistically significant only between the control group and the TPA/ATRA combination group (P < 0.05). For all other groups, the differences from the control group were not statistically significant (P > 0.05).
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Effects on Tumor Growth. Tumor growth was measured daily and expressed as a percentage of the initial tumor size on day 0 (Fig. 6, A and C). The effects of the different treatments on tumor growth were similar to those described earlier in Fig. 5A. Animals treated with the TPA/ATRA combination experienced a substantial inhibition of tumor growth compared with animals treated with vehicle or to animals treated with TPA or ATRA alone. Animals that were treated with vehicle and were diet-restricted experienced a rate of tumor growth similar to that observed in animals treated with vehicle but with free access to food (Fig. 6, A and C). After 17 days of treatment, the mean percentage of initial tumor size ± S.E. was 147 ± 7 for the control group, 135 ± 11 for the diet-restricted group, 132 ± 7 for the TPA group, 139 ± 6 for the ATRA group, and 79 ± 2 for the TPA/ATRA combination group (Fig. 6, A and C). The effect of the treatments on tumor size in individual mice is shown in Fig. 6C.
Effects on Body Weight. On day 17, the mean percentage of initial body weight was 100 ± 1 for the control group, 78 ± 4 for the diet-restricted group, 85 ± 1 for the TPA group, 98 ± 1 for the ATRA group, and 90 ± 3 for the TPA/ATRA combination group (Fig. 6B). The effect of the treatments on the body weight of individual mice is shown in Fig. 6D. The results of this study indicate that decreasing body weight by restricting food intake is not associated with an inhibition of tumor growth.
Effects on Mitosis and Apoptosis in Tumors. Tumors from diet-restricted animals or animals treated with TPA, ATRA, or TPA + ATRA for 17 days as described in Fig. 6 were examined for mitoses and caspase-3-positive cells as indices of proliferation and apoptosis. We found that diet restriction did not influence mitoses or caspase-3-positive cells in the tumors (Table 4). Treatment of the mice with TPA (50 ng/g) or ATRA (150 ng/g) daily for 17 days caused modest decreases in the ratio of the percentage of mitotic cells/percentage of caspase-3-positive cells, whereas treatment of the mice with a combination of TPA and ATRA caused a substantial (79%) decrease in the ratio of percentage of mitotic cells/percentage of caspase-3-positive cells (Table 4).
Effects of Daily Intraperitoneal Injections of TPA and Taxol on the Growth of BxPC-3 Human Pancreatic Tumor Xenografts in NCr-Immunodeficient Mice
NCr mice with BxPC-3 tumors measuring 0.4 to 1.0 cm in length and 0.4 to 1.0 cm in width were injected i.p. once a day with vehicle (5 µl/g body weight), TPA (50 ng/g body weight; 5 µl of vehicle/g body weight), Taxol (10 ng/g body weight; 5 µl of vehicle/g body weight), or a combination of TPA and Taxol (50 and 10 ng/g body weight, respectively; 5 µl of vehicle/g body weight) once a day for 28 days (seven to eight mice per group). Tumor growth was measured daily and expressed as a percentage of the initial tumor size at the start of the study on day 0 (Fig. 7, A and B). After 28 days of daily injections, the mean percentage of initial tumor size ± S.E. was 259 ± 25% for the control group, 222 ± 11% for the TPA group, 166 ± 13% for the Taxol group, and 211 ± 14% for the TPA/Taxol group (Fig. 7, A and B). Statistical analysis of the data by one-way ANOVA using the Tukey-Kramer multiple comparisons test showed that the differences in percentage of initial tumor size at the termination of the study on day 28 were statistically significant between the control group and the Taxol group (P < 0.01) but not between the control and any of the other groups (P > 0.05). A pairwise comparison of slopes indicated that the difference in slopes between the vehicle control and Taxol group (P < 0.0001) or between the vehicle control and TPA + Taxol group (P = 0.01) was significant. The difference in slopes between the Taxol group and the Taxol + TPA group was also significant (P = 0.01).
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In a repeat of the above-mentioned TPA/Taxol study, the difference in slopes for tumor growth between the vehicle control and TPA group (P = 0.002), between the vehicle and Taxol group (P < 0.0001), and between the vehicle and TPA + Taxol group (P < 0.0001) was significant (data not presented). In this repeat experiment, the inhibitory effect of TPA alone on tumor growth was greater than in the first experiment. Although the effect of TPA + Taxol on tumor growth was somewhat less than that for Taxol alone, the difference between slopes was not significant. The overall conclusion from the two studies was that treatment of NCr mice with TPA did not increase the effectiveness of Taxol for suppressing BxPC-3 tumor growth and that TPA antagonized the effect of Taxol in one of the two studies.
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). TPA-induced inhibition of growth was different in each cell line. PANC-1 and MIA PaCa-2 cells were more sensitive to growth inhibition by TPA than BxPC-3 cells, and TPA had different effects on the cell cycle in the three cell lines. In PANC-1 cells, TPA decreased the percentage of cells in the G0/G1 phase of the cell cycle and increased the percentage of cells in the G2/M phase. This effect of TPA on the cell cycle in PANC-1 cells is not consistent with the effect of TPA to increase the level of p21 and indicates that the TPA-induced up-regulation of p21 is not translated into an expected accumulation of cells in G0/G1. The reason for this unexpected result is unknown. In MIA PaCa-2 cells, TPA increased the percentage of cells in the G0/G1 phase of the cell cycle while decreasing the percentage of cells in the S and G2/M phase. In contrast to these results, there was no effect of TPA treatment on the cell cycle distribution in BxPC-3 cells, although there was substantial growth inhibition.
Although treatment of PANC-1 cells with TPA markedly increased the level of p21 protein, this effect was not observed in MIA PaCa-2 or BxPC-3 cells. The TPA-induced increase in the level of p21 in PANC-1 cells was paralleled by a TPA-induced increase in the level of Ser795-dephosphorylated Rb. The stimulatory effect of TPA on the level of p21 in PANC-1 cells was blocked by bisindolylmaleimide, an inhibitor of conventional and novel PKCs (PKC, PKC, PKCµ, and PKC) and by rottlerin, a selective inhibitor of PKC. In human Jurkat T-cells, TPA activation of p21 expression occurred by a PKC-dependent mechanism involving an Sp1 transcription factor, and the TPA-induced increase in the level of p21 was mediated by the second most upstream Sp1 site in the p21 promoter (Schavinsky-Khrapunsky et al., 2003). It is of interest that several reports have suggested that an increased level of p21 is associated with an antiapoptotic effect (cited in Schavinsky-Khrapunsky et al., 2003).
In another study, the growth inhibitory effect of TPA (1 µM) in DanG pancreas cancer cells was associated with an increased level of p21 and hypophosphorylated Rb, but as in our study, apoptosis was not increased (Detjen et al., 2000). Pretreatment of DanG pancreas cancer cells with GF109203X (bisindolylmaleimide I) prevented the growth inhibitory effect of TPA, and evidence was presented that TPA inhibited the growth of DanG cells by activation of PKC (Detjen et al., 2000). In a more recent study, treatment of MIA PaCa-2 cells with TPA enhanced MAPK, and this treatment enhanced anchorage-independent growth of these cells growing in suspension, but these effects were not observed in attached cells (Ishino et al., 2002). The stimulatory effect of TPA on anchorage-independent growth of MIA PaCa-2 cells was inhibited by inhibitors of PKC and mitogen-activated protein kinase kinase, and overexpression of PKC, PKC, or PKC increased the phosphorylation of MAPK in suspended cells (Ishino et al., 2002). Overexpression of PKC or PKC in MIA PaCa-2 cells also enhanced anchorage-independent growth by 30 to 50%, and overexpression of PKC lowered the concentration of TPA that was needed to stimulate anchorage-independent growth (Ishino et al., 2002).
Several cancer cell lines, including PANC-1, LNCaP, and PC-3 cells, have an elevated level of phosphorylated Akt (protein kinase B) that is thought to enhance proliferation and/or inhibit apoptosis (Yang et al., 2004). Treatment of PANC-1 cells with triciribine (API-2), an inhibitor of phospho-Akt formation, inhibited cell growth, caused a 6-fold increase in apoptosis, and inhibited the growth of PANC-1 tumors in immunodeficient mice (Yang et al., 2004). Similar inhibitory effects of API-2 on the growth of cultured cells or tumor xenografts were observed in other tumor cell lines with high levels of phospho-Akt but not in tumor cell lines with low levels of phospho-Akt (Yang et al., 2004). These results indicate that API-2 inhibits cell growth and induces apoptosis preferentially in cells that overexpress phospho-Akt. It will be of interest to study the effect of combinations of TPA and API-2 on the growth of PANC-1 and other pancreas tumors. In other studies, it was shown that Akt interacts directly with Smad3 to regulate the sensitivity of cells to transforming growth factor--induced apoptosis (Conery et al., 2004; Remy et al., 2004) so that modulation of Akt may affect apoptosis by influencing transforming growth factor- signaling.
The results of our studies indicated that TPA in combination with BAY 11-7082 (an inhibitor of NF-B) had a greater inhibitory effect on the growth of MIA PaCa-2 cells than either compound alone, and combinations of TPA and sulindac had a greater inhibitory effect on the growth of PANC-1 cells than either compound alone. The results of our investigations indicated that the three cultured pancreas cancer cell lines studied each had its own characteristic response to inhibitors of cell growth and that clinically relevant concentrations of TPA inhibited the growth of all three cultured cell lines.
Although combinations of Taxol and TPA resulted in a greater inhibitory effect on the growth of cultured BxPC-3 cells than Taxol or TPA alone (Table 3), administration of TPA and Taxol to BxPC-3 tumor-bearing mice may be less effective than Taxol alone for inhibiting tumor growth (Fig. 7). The reason why TPA enhanced the inhibitory effect of Taxol on the growth of BxPC-3 cultured cells but antagonized or had no effect on the inhibitory effect of Taxol on the growth of these tumors in NCr mice is not known.
Daily i.p. injections of 50 or 100 ng of TPA/g body weight inhibited the growth or caused tumor regressions in well established PANC-1 tumors in NCr immunodeficient mice (Fig. 4). Treatment of the mice with TPA (50 or 100 ng/g body weight/day) caused a decrease in the mitotic index in PANC-1 tumors and an increase in apoptosis as measured by activated caspase-3 staining in the tumors (Table 4). The ratio of the percentage of mitotic cells/percentage of apoptotic cells in PANC-1 tumors was decreased by 68% in mice treated with 50 ng of TPA per g body weight/day and by 86% in mice treated with 100 ng of TPA/g body weight/day (Table 4). This finding indicates that TPA caused a strong inhibitory effect on the proliferation of PANC-1 tumors in vivo.
Daily i.p. injections of TPA (50 ng/g body weight/day) or ATRA (150 ng/g body weight/day) in NCr mice with well established BxPC-3 tumors had only a modest inhibitory effect on tumor growth but administration of a combination of TPA and ATRA at these dose levels caused tumor regressions or substantial inhibition of tumor growth (Figs. 5 and 6), and the ratio of the percentage of mitotic cells/percentage of apoptotic cells in the tumors was substantially decreased (Table 4). The results described here for BxPC-3 pancreatic tumors are similar to our previous observations indicating that treatment of mice with a combination of TPA and ATRA caused regressions in human LNCaP prostate tumors growing in NCr immunodeficient mice and decreased the ratio of the percentage of mitotic cells/percentage of apoptotic cells in the tumors (Zheng et al., 2004). It is of interest that the inhibitory effect of the TPA/ATRA combination on the growth of BxPC-3 pancreas tumors in NCr mice was predicted from the effect of TPA/ATRA on the growth of cultured BxPC-3 cells (Table 3). Although growth inhibition of cultured BxPC-3 cells treated with TPA + ATRA was not associated with an increase in apoptosis, treatment of mice bearing BxPC-3 tumors with TPA + ATRA caused an increase in apoptosis in the tumors. TPA is known to stimulate the production of cytokines such as tumor necrosis factor-, interleukin-1, and interleukin-6 in a variety of cells. It is possible that the levels of these cytokines were elevated in the tumors of mice treated with TPA. The combination of TPA, ATRA, and these cytokines may have induced apoptosis in BxPC-3 tumors growing in these mice.
The peak blood level of TPA after a 50 or 100 ng of TPA/g body weight i.p. injection was approximately 0.7 or 1.5 nM, respectively (Fig. 3). These concentrations of TPA, which are associated with inhibition of PANC-1 and BxPC-3 pancreas tumor growth and with LNCaP prostate tumor growth in NCr immunodeficient mice, are achievable in patients treated with TPA (Cui et al., 2002). The peak blood level of ATRA ± S.D. after treatment of patients with ATRA (15 mg/m2) was 1.13 ± 0.68 µM (Chen et al., 1996). Although we do not know the concentration of ATRA in mice injected with ATRA in our studies, assuming equal distribution of ATRA throughout the mouse and the lack of metabolism, the maximum achievable concentration of ATRA would be 0.5 µM, and it is likely that the concentration of ATRA is considerably less.
Our studies indicating that combined treatment with TPA and ATRA decreased growth, increased apoptosis, and caused regressions of prostate and pancreas tumors in immunodeficient mice at blood levels of these drugs that are clinically achievable, provide encouragement for a clinical trial to evaluate the effectiveness of combined therapy with TPA and ATRA in patients with prostate and pancreas cancer.
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ABBREVIATIONS: TPA, 12-O-tetradecanoylphorbol-13-acetate; ATRA, all-trans retinoic acid; BAY 11-7082, (E)-3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; ANOVA, analysis of variance; NF-B, nuclear factor-B; STAT, signal transducer and activator of transcription; Rb, retinoblastoma; DMSO, dimethyl sulfoxide; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole; Gö6983, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide; API-2, triciribine.
1 Current address: Department of Medical Oncology and Medicine, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA. 
Address correspondence to: Dr. Allan H. Conney, Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854. E-mail: aconney{at}rci.rutgers.edu
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