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Antiproliferative and Proapoptotic Effect of Ascorbyl Stearate in Human Pancreatic Cancer Cells
Association with Decreased Expression of Insulin-Like Growth Factor 1 Receptor
K. AKHILENDER NAIDU, MD,* RICHARD C. KARL, MD, KAMATHAM A. NAIDU, MD, and DOMENICO COPPOLA, MD

Pancreatic cancer is an aggressive tumor with short median survival and high mortality rate. Alternative therapeutic modalities are currently being evaluated for pancreatic cancer. Here we studied the effects of ascorbyl stearate (Asc-S), a nontoxic, lipophilic derivative of ascorbic acid, on pancreatic cancer. Treatment of human pancreatic carcinoma cells with Asc-S (50200 M) resulted in a dose-dependent inhibition of their proliferation. Asc-S slowed down the cell cycle, accumulating, PANC-1 cells in late G2 -M phase. Furthermore, Asc-S treatment (150 M) markedly inhibited growth in soft agar and facilitated apoptosis of PANC-1 cells but not of Capan-2 cells. These effects were accompanied by a signicant reduction in insulin-like growth factor 1 receptor (IGF1-R) expression, as compared to untreated controls. Interestingly, Capan-2 cells, the least responsive to Asc-S treatment, did not overexpress the IGF1-R. The results demonstrate the efcacy of Asc-S in inhibing growth of pancreatic cancer cells and warrant additional studies to explore the potential utility of this compound as an alternative and/or adjuvant therapeutic modality for pancreatic cancer.
KEY WORDS: ascorbyl stearate; insulin-like growth factor 1 receptor; pancreatic cancer; cell proliferation; apoptosis; signal transduction.

Pancreatic cancer (ductal type) is the fourth most common cause of cancer death in Western society, and one of the leading causes of cancer death worldwide (eighth leading cause of death among women, and seventh among men)
Manuscript received May 26, 2002; revised manuscript received September 13, 2002; accepted September 20, 2002. From the *Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570013, India; and Departments of Surgery, Neurology and Pathology; Moftt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA. This study was supported in part by grant RPG0023401-CNE (to D.C.) from the American Cancer Society including a generous direction from Edward Bakewell, Jr. Trust. Address for requests Dr. Domenico Coppola, Department of Pathology; Moftt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive Tampa, Florida, 33612-9497, USA.

(1). Its incidence is increasing, particularly in women (2). This highly aggressive tumor affects patients usually >50 years old and has no obvious variation in risk related to social class (3). Its incidence and mortality rates are almost identical, with short median survival time after diagnosis (46 months) (4). Perhaps the reason for this gloomy prognosis is that pancreatic carcinomas usually present late in their development and are locally unresectable at diagnosis in approximately 30% of cases (5). The American Cancer Society estimated that 30,300 new cases of pancreatic cancer, and 29,700 pancreatic cancer deaths are expected to occur in the United States in the year 2002 (6). These observations attest to the lack of efcacy of current treatment modalities for this form of human cancer and our limited knowledge of the pathogenetic mechanisms
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leading to pancreatic cancer. These unsatisfactory results warrant a search for alternative treatments. The benecial effects of ascorbate and ascorbate derivatives in preventing and/or delaying the spontaneous occurrence of breast tumors and of ultraviolet light induced skin tumors in mice have been observed (7). Lipophilic synthetic derivatives of ascorbate (eg, ascorbyl esters) are being tested in vitro and in vivo as antiproliferative compounds with promising results. Ascorbyl esters have been found to inhibit cell proliferation and DNA synthesis of breast, skin, and brain tumors (711). Smart et al have shown that the dietary intake of ascorbyl palmitate is effective in inhibiting 12-O-tetradecanoylphorbol13-acetate (TPA) -induced tumor promotion (12). Similarly, in vivo antitumor activity of ascorbic acid compounds has been observed in human leukemia (13) and in human mammary tumor xenografts (14). Ascorbyl stearate (Asc-S) is a lipophilic, ascorbyl ester that can easily enter the intracellular compartment (15) and can act as free radical scavengers, reducing the production of reactive oxygen species (16, 17). In this study, we describe that the antitumoral activity of Asc-S is mediated via interference with cell cycle progression and induction of apoptosis in human pancreatic carcinoma cells. MATERIALS AND METHODS
Cell Culture. Human pancreatic cancer cells PANC-1, BxPC-3, HS 766T, SU. 86.86, MiaPaca-2, Capan-1, and Capan-2 were obtained from the American Type Culture Collection (Rockville, Maryland, USA). PANC-1, MiaPaca-2 and HS 766T cells were cultured for 24 hr in Dulbeccos modication of Eagles (DMEM); SU.86.86, Capan-1, and Capan-2 were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, Utah, USA) penicillin (50 units/ ml), streptomycin (50 g/ml), and 1.0 mM L-glutamine. P6 cells [3T3 broblast cells, transfected with a CVN plasmid constitutively expressing human insulin-like growth factor [IGF1-R], and Receptor cells (mouse embryo broblasts homozygous for the targeted disruption of the IGF1-R gene) were kindly provided by Dr. R. Baserga, Jefferson Cancer Institute, Philadelphia, Pennsylvania, USA). Cell Growth Assay. The effect of Asc-S on cell growth and viability of a panel of pancreatic primary and metastatic carcinoma cells (PANC-1, MIA PaCa-2, BxPC-3, HS 766T, AsPC-1, SU.86.86, Capan-1, and Capan-2) as well as P6 and R- cells were evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5diphenyl-tetrazoliumbromide (MTT) (Sigma Chemical Co., St. Louis, Missoni, USA) assay. The details of the procedure are as described earlier (10). The above cells were seeded at 2.5 104 viable cells/well in 0.1 ml DMEM/RPMI supplemented with 10% FBS or serum in 96-well tissue culture plates. The following day when the cells were nearly conuent, the medium was replaced with fresh medium with 0, 50, 75, 100, 125, 150, 175, and 200, M Asc-S and incubated in a CO2 incubator at 37 C for 24 hr with respective untreated controls.
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After 24 hr, MTT dye was added to the cells and the plates were incubated for 3 hr at 37 C. The solubilization reagent DMSO was added, and absorbance at 570 nm was determined using a Dynatech MR 580 reader, with a reference wavelength of 630 nm. The relationship of the absorbance of MTT to cell number was veried in a separate experiment and showed a linear relationship between cell number and absorbance with a correlation coefcient of 0.98. The decrease in absorbance is considered as the loss of viability of cells. Values were calculated as mean SD of three experiments comprising 18 replicates and expressed as absorbance of MTT at 570 nm. Cell Cycle Analysis by Flow Cytometry. PANC-1 cells grown in DMEM with 10% FBS were treated with 0, 50, 100, 150, and 200 M of Asc-S for 24 hr for doseresponse studies. For time course studies, PANC-1 cells were treated with 150 M of Asc-S and at time intervals of O (untreated control), 3, 6, 9, 12, 18, and 24 hr. The cells were washed twice with PBS, centrifuged, resuspended, and counted. Treated and untreated PANC-1 cells were adjusted to 106 cells/ml and xed in 1 ml of ice cold 70% ethanol overnight at 4 C. The cells were then stained with propidium iodide (50 g/ml) containing RN/Ase (100/ml). Samples were run on a FAC Scan ow cytometer and analyzed using cell Fit software. Values were calculated as mean SD of three experiments, expressed as percent of cells per cycle phase. Baseline Expression of IGF1-R Measured by Western Blotting. BxPC-3, PANC-1, MiaPaca-2, Capan-1, Capan-2, as well as P6 and R- cells were cultured for 24 hr in DMEM/RPMI supplemented with 10% FBS. Cells were disrupted in cold lysis buffer (10 mM tetrasodium pyrophosphate, 20 mM HEPES, 1% Triton X-100, 100 mM NaCl, 2 g/ml aprotinin, 2 g/ml leupectin, and 100 g/ml of PMSF). The crude lysate was centrifuged and protein concentration was determined using a Pierce BCA protein assay kit (Pierce Laboratory). Equal amounts of protein (25 g) were placed in 2 sample buffer [0.125 M Tris HC1 (pH 6.8), 20% glycerol, 0.2 mg/ml bromophenol blue dye, 2% SDS, and 10% -mercaptoethanol] and electrophoresed on a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) apparatus (Novex, San Diego, California, USA). The proteins were then transferred to a nitrocellulose membrane by using an electrobinding technique. Membranes were blocked for 30 min at room temperature in Tris buffer saline with Tween (TBST) and 5% nonfat milk and incubated with mouse anti-human IGF1-R antibodies to identify the 85 kDa -subunit of the IGF1-R (Pharmingen, San Diego, California, USA). The blots were then washed and incubated with the appropriate horseradish peroxidase conjugated secondary antibody (Oncogene Science, Inc., Uniondale, New York, USA.) Antigen bound to nitrocellulose membrane were detected with the ECL system (Amerscam Corporation, Arlington Heights, Illinois, USA). P6 cells were used as the positive control and R cells as the negative control for IGF1-R. Effect of Asc-S on IGF1-R and PDGF-R Expression by Western Blotting. PANC-1 cells cultured in DMEM with 10% FBS were treated with different concentrations of Asc-S (eg, 0, 50, 100, 150, and 200 M) for 24hr or with 150 M Asc-S at different time points (3, 9, 12, and 24hr). For the evaluation of Asc-S effect on plateled-derived growth factor receptor (PDGFR) expression, PANC-1 cells were treated with 150 M Asc-S for 24 hr. The Western blot was performed as reported above. To detect IGF1-R, we used the same primary antibody as above (anti-

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NAIDU ET AL human IGF1-R; Pharmingen). To detect PDGF-R we used a rabbit anti-human polyclonal antibody toward the PDGF type A receptor (Upstate Biotechnology Inc.). 3T3 cells were used as the positive control for PDGF-R. Soft Agar Experiments. The growth of Asc-S treated (150 M of Asc-S for 24 hr) and untreated PANC-1 cells in soft agar was assessed as previously described (18). In brief, the cells were seeded at a density of 3 103 /35-mm plate in 10% serum on a bottom layer of 1% agar, with a top layer of 0.3% agar. Colonies >125 m in diameter (or 10 cells) were counted at weekly intervals for 1421 days. The clonogenicity was determined in three independent experiments. Detection of Apoptosis by In Situ Hybridization Using Tunel Assay. Apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) with an in situ cell death detection kit (Boehringer Mannheim, Indianapolis, Indiana, USA). PANC-1 and Capan2 cells were split at a density of 3 104 cells/well in 6-well plates. The cells were treated with 150 M Asc-S for 24 hr. Following treatment the cells were trypsinized and cytospin preparations were obtained. Cells were washed with PBS and xed with freshly prepared paraformaldehyde (4% in PBS, pH 7.4) for 30 min. For the TUNEL assay, the cells were rinsed with PBS and incubated in permeabilization solution for 5 min. Slides were rinsed and incubated with the TUNEL reaction mixture for 60 min at 37 C in a humidied chamber. Slides were rinsed and cross-reacted with converteralkaline phosphatase solution for 30 min at 37 C in a humidied chamber. Slides were thoroughly rinsed and reacted with alkaline phosphatase substrate solution for 510 min (Vector Laboratories, Burlington, California, USA). After an additional rinse, the slides were mounted under a cover slip and analyzed under a light microscope at time 0, 6, 12, and 24 hr. Apoptotic cells were identied by a dark-brown nuclear stain. The percentage of apoptotic cells was dened as the ratio of apoptotic cells to total cells counted 100, and a minimum of 400 cells were counted for each experiment. These experiments were performed in triplicate. Statistical analysis of the data was carried out using Students t test (19).

RESULTS Asc-S Inhibits Proliferation of Pancreatic Carcinoma Cells. The MTT assay data show that increasing doses of Asc-S (50200 M) for 24 hr induced signicant inhibition of pancreatic cancer cell proliferation in a dose-dependent manner, compared to the control group (Figure 1). Among the pancreatic carcinoma cells tested, BxPC-3, Capan-1, Panc-1, and SU.86.86 were the most sensitive, their growth being inhibited by about 7080%

Fig 1. Dose-dependent inhibition of pancreatic cancer cell proliferation following Asc-S treatment for 24 hr. The cells grown in the appropriate culture media in presence of 10% FBS were exposed to different concentrations of Asc-S (0, 50, 75, 100, 125, 150, 175, 200 M) for 24 hr. P6 cells (3T3 broblasts transfected with a CVN plasmid constitutively expressing human IGF1-R) and R-cells (mouse embryo broblasts homozygous for the targeted disruption of the IGF1-R gene), were included. The values are mean SD of three experiments. The response to Asc-S of PANC-1, BxPc-3, P6, and Capan-1( ) is statistically different at P < 0.001 compared to IGF1-R negative cells (R-).

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at 175 M concentration of Asc-S as compared to the untreated cells MiaNPaca-2 and Capan-2 cells, on the other hand, were the most resistant to Asc-S treatment. The inhibitory effect of Asc.S between IGF1-R expressing PANC-1, BxPC-3, P6, and Capan-1 cells and IGF1-R negative cells (IGF-I R- ) was found to be highly signicant at P < 0.001. Asc-S Treatment Affects Cell Cycle Progression. Treatment of PANC-1 cells with increasing doses of Asc-S resulted in signicant accumulation of cells in the G2 M phase of the cell cycle, compared to untreated controls (Figure 2). The accumulation of tumor cells in the G2 M phase of the cell cycle was dose-(>50% of cells arrested in G2 M following treatment with 200 M of Asc-S) and time-dependent (gradual increase in percentage of cells in late S/G2 M phase after 18 hr of exposure to 150 M Asc-S) (Figure 3). The inhibition of the cell cycle at the G2 M phase was found to be statistically signicant at P < 0.05. Cells Sensitive to Asc-S Treatment Express High Levels of IGF1-R. When the baseline IGF1-R cellular expression of the pancreatic cell lines was measured by Western blot, it became evident that the tumor cell lines usually more sensitive to Asc-S treatment (BxPC-3, Capan-1, and PANC-1) were also those expressing higher levels of IGF1-R, as compared to the less Asc-S sensitive lines (Capan-2 and MiaPaca-2) (Figure 4).

Fig 3. Time dependent effect of Asc-S (150 M) on cell cycle distribution of PANC-1 cells. The cells grown in DMEM with 10% FBS were treated with 150 M of Asc-S and at time intervals (C = untreated control, 3, 6, 9, 12, 18, 24 hr), before being analyzed by ow cytometry. The values are means SD of three experiments, expressed as percent of cells per cycle phase (*P < 0.05).

Interestingly, the IGF1-R-positive (P6 cells) and -negative (IGF1-R knockout mouse broblasts, R- cells) controls followed a similar pattern of Asc-S sensitivity (see also Figure 1). PANC-1 tumor cells were those expressing the highest level of IGF1-R, and therefore we chose them as the representative cell line for the following additional experiments. Asc-S Treatment Reduces Expression of IGF1-R but Not of PDGF-R. To further establish a link between the antiproliferative effect of Asc-S and IGF1-R, changes in the expression of this protein were assessed in Asc-Streated and in untreated PANC-1 cells. The exposure of

Fig 2. Dose-dependent effect of Asc-S on cell cycle distribution of PANC-1 cells. The cells grown in DMEM with 10% FBS were treated with different concentrations of Asc-S (0, 50, 100, 150, 200 M) for 24 hr before being analyzed by ow cytometry. The values are means SD of three experiments, expressed as percent of cells per cycle phase (*P < 0.05). Digestive Diseases and Sciences, Vol. 48, No. 1 (January 2003)

Fig 4. IGF1-R baseline expression in pancreatic cancer cells lines. IGF1R baseline expression of MiaPaca-2, BxPC-3, PANC-1, Capan-1, and Capan-2 cells by Western blot. The cells were cultured in appropriate medium and 10% FBS before testing. P6 cells and R- cells (mouse embryo broblasts homozygous for the targeted disruption of the IGF1R gene), were included as positive and negative controls for IGF1-R, respectively.

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TABLE 1. ASC-S (150 M) INHIBITS CLONOGENICITY OF PANC-1 CELLS IN SOFT AGAR* Category Control Asc-S treated Week 1 39 2 0 Week 2 136 9 15 1 Week 3 252 10 50 4

*Number of colonies formation in soft agar. Only colonies greater than >125 m in diameter were counted. Values are mean SD of three independent experiments.

Fig 5. Dose-dependent effect of Asc-S on IGF1-R expression in PANC1 cells. IGF1-R Western blot of PANC-1 cells cultured in DMEM with 10% FBS and treated with increasing concentrations of Asc-S (C = untreated control, 50, 100, 150, 200 M) for 24 hr Untreated P6 cells were included as positive control for IGF1-R.

PANC-1 cells to increasing concentrations of Asc-S (0, 50, 100, 150, and 200 M) for 24 hr, (induced a dosedependent decrease in IGF1-R expression) that was evident at a dose of 150 M of Asc-S, and became maximal at a dose of 200 M (IGF1-R expression almost absent) (Figure 5A). When time was considered, IGFI-R expression decreased after 9 hr of exposure to Asc-S (150 M) and became minimal after 12 hr of exposure (Figure 6).

Conversely, the expression of another tyrosine kinase receptor, the PDGF-R competent factor, was not affected by Asc-S treatment (150 M for 24 hr) (Figure 5B). Asc-S Decreases Clonogenicity of PANC-1 Cells in Soft Agar. The effect of Asc-S on clonogenicity was determined by colony formation in soft agar. Table 1 shows that while the growth of untreated PANC-1 cells was exponential during the entire length (three weeks) of the experiment (39 2, 136 9, 252 10 colonies), PANC-1 cells treated with 150 M Asc-S for 24 hr and subsequently seeded in soft agar, grew at a much slower pace (0, 15 1, 50 4 colonies). In addition to a reduction in the number of colonies, the Asc-Streated cells also produced smaller colonies (data not shown) as compared to the control group. A sample of treated PANC-1 cells was subjected to trypan blue staining, before their seeding in soft agar, to conrm their viability. Asc-S Facilitates Apoptosis of PANC-1 Cells but Not of Capan-2 Cells. Using the in situ TUNEL cell death assay, we observed that PANC-1 cells treated with 150 M Asc-S for 24 hr had a signicant increase in apoptosis (approximately 2030% positively stained cells: +90/400; +107/400; +126/400), as compared to untreated PANC-1 cells (about 15% positively stained cells: +5/100; +2/100; +1/100), and to the Asc-S tested low/IGFI-R expressor Capan-2 cells (510% positively stained cells: 7/100; 5/100; 10/100).

DISCUSSION The inhibitory effect of vitamin C and vitamic C derivatives (ascorbyl esters) on cell proliferation and DNA synthesis, as well as their adjuvant action in combination with other antitumoral agents, has been observed in several tumor systems (2022). Liehr reported a statistically signicant reduction in the incidence and severity of renal tumors induced by estradiol and diethylstilbestrol in hamsters (20). It has also been observed that ascorbic acid signicantly reduces the mutagenic effects produced by
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Fig 6. Time-dependent effect of Asc-S (150 M) on IGF1-R expression in PANC-1 cells. IGF1-R Western blot of PANC-1 cells cultured in DMEM with 10% FBS and treated with 150 M of Asc-S at different time intervals (C = untreated control, 3, 9, 12, 24 hr). Untreated P6 cells were included as positive control for IGF1-R.

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alkylating agents in vivo (22) and protects from chemically induced hepatocarcinogenesis in rats (21). The antitumor activity of ascorbic acid compounds has been reported in human leukemia (13) and in human mammary tumor xenografts (14). However, these experimental in vivo and in vitro studies have sometimes yielded controversial results (23, 24). It is possible that the disparity between studies reects the nonoptimal solubility of ascorbic acid. It has been shown that the esterication of ascorbic acid with fatty acids makes it lipophilic (25) and allows it to easily cross biological barriers (15). Therefore, the antiproliferative, antitumorigenic, and antioxidative effects of ascorbic acid are much enhanced. Using these synthetic compounds, Naidu et al have reported that ascorbyl stearate was more potent in inhibiting the cell growth of glioblastoma multiforme cells as compared to water-soluble ascorbic acid (10, 11, 26) and that Asc-S treatment reduces the IGF1-R expression in these cells (26). In this study, we report that Asc-S also has potent antiproliferative and proapoptotic effects on human pancreatic carcinoma cells. Asc-S treatment is effective in slowing down the proliferation of a subset of pancreatic cancer cell lines, causing their accumulation at the late S/G2 M phase of cell cycle. Thus, Asc-S-induced cell cycle arrest at the G2 M phase checkpoint may explain the inhibition of cell growth and facilitation of programmed cell death. The demonstration that dehydroascorbic acid, the oxidized form of ascorbic acid, is also able to arrest the cell cycle in the late G2 M phase provides further support to our hypothesis (27). Furthermore, the block of these cells in the G2 M phase of the cell cycle raised the possibility, among others, that the Asc-S-induced slowing down of the cell multiplication cycle may be due to a modication in the expression of progression growth factors or of their receptors. One of the growth factor receptors known to affect the G2 M transition and to be overexpressed in pancreatic cancer is the insulin-like growth factor 1 receptor (IGF1-R) (2830). This is a cell receptor with tyrosine kinase activity that has recently emerged as a new critical player in transformation and tumorigenicity (31). Numerous studies have shown that the overexpression and activation of the IGF1-R is associated with malignant transformation, increased tumor aggressiveness, and protection from apoptosis, in a variety of tumor cell types (3236). Analysis of our results reveals that the cell lines most responsive to Asc-S treatment are those with the highest expression of the IGF1-R. Furthermore, when focusing on PANC-1 cells, expressing the highest level of IGF1-R among the pancreatic cancer cell lines studied, we noted
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that their exposure to concentrations of 150200 M of Asc-S induced a remarkable reduction in the expression of IGF1-R. We also observed that PDGF-R, another tyrosine kinase receptor, was not affected by similar treatment. Reduction in IGF1-R expression was followed by a partial reversal of the transformed phenotype, measured as number of colonies formed in soft agar (Table 1). The difference in clonogenic capability between Asc-S-treated and untreated PANC-1 cells, included a consistently larger size of the colonies formed by the untreated PANC-1 cells. These ndings are in agreement with the hypothesis that the autocrine interaction between the IGF1-R and its ligand regulates both cell proliferation and anchorage independent growth of pancreatic carcinoma cells (37). Thus, Asc-S-induced down regulation of IGF1-R may be responsible for the observed inhibition of cell proliferation and for the partial reversal of the transformed phenotype in PANC-1 cells. Our experiments also show that Asc-S treatment facilitates apoptosis of PANC-1 cells but not of Capan2 cells. This nding is consistent with the recently postulated antiapoptotic role of the IGF1-R (35, 36). It has been shown that the antiapoptotic effect of the IGF1-R is independent of its mitogenic effect (38, 39). Since Capan2 cells express much lower levels of IGF1-R compared to PANC-1 cells, the lack of apoptosis following Asc-S treatment of Capan-2 cells may indicate that the proapototic effect of Asc-S depends on the down-regulation of IGF1R. A similar conclusion can be drawn by analyzing the proliferation data. There, Capan-2 and MiaPaca-2 cells, expressing minimal levels of IGF1-R, as well as R-cells (mouse embryo broblasts homozygous for the targeted disruption of the IGF1-R gene), were among the cells least affected in their proliferative capabilities by Asc-S treatment. Ascorbyl esters are virtually nontoxic (LD50 = 25 g/kg of Asc-S for mice), and ascorbate and stearic acid, the breakdown products of Asc-S, are also nontoxic to biological systems and have been reported to have anticarcinogenic properties in different tumor types (40). Ascorbyl radicals are reported to induce apoptosis, while stearic acid, being a precursor for the synthesis of ceramide, might play a critical role in cell growth, differentiation, and apoptosis (41, 42). In conclusion, we show here that there is a correlation between Asc-S-induced modications of cell proliferation, transformation, and apoptosis and the expression level of IGF1-R. Thus, Asc-S may have multiple modes of action and should be considered as a vitamin C-based compound with antitumorigenic properties, having potential application in human pancreatic cancer.

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ACKNOWLEDGMENTS
We thank the Molecular Biology, Molecular Imaging, Flow cytometry and Pathology core facilities of Moftt Cancer Center and Research Institute.

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