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RESEARCH ARTICLE

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Role of Reactive Oxygen Species in the Cytotoxicity of Arsenic Trioxide and Pamidronate for Human Prostate Cancer Cells

James H. Doroshow1–3 and Shikha Gaur3 

1Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; 2Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; 3City of Hope National Medical Center, Duarte, CA 91010, USA 

Correspondence: doroshoj@mail.nih.gov (J.H.D.) 

Doroshow JH and Gaur S. Reactive Oxygen Species 9(26):81–94, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.811

(Received: November 20, 2019; Accepted: December 9, 2019) 

ABSTRACT | To examine whether combining arsenic trioxide (ARS) and pamidronate (PAM), anticancer drugs that generate reactive oxygen species (ROS), enhanced targeting of redox sensitive growth signals, we studied cloning efficiency, protein tyrosine phosphatase (PTPase) activity, and epidermal growth factor receptor (EGFR) phosphorylation in DU-145 and PC-3 human prostate cancer cells in response to treatment with ARS and/or PAM for 24 h. IC50 concentrations in a clonogenic assay for ARS and PAM were 9 and 20 μM, respectively, in DU-145 cells; and 2 and 12 μM, in PC-3 cells. When combined, ARS and PAM demonstrated additive cytotoxicity in the DU-145 line (combination index [CI] of 1.10) and synergy for PC-3 cells (CI of 0.86). ARS (20 μM for 24 h) inhibited PTPase activity by 36 ± 7 %, p < 0.05 vs. untreated controls, in DU-145 cells; and by 58 ± 8%, p< 0.05, in the PC-3 line. PAM (20 μM for 24 h) decreased PTPase activity by 24 ± 9%, p= 0.06, and 8 ± 1%, p < 0.01, in DU-145 and PC-3 cells, respectively. Combining ARS and PAM significantly inhibited PTPase activity in both cell lines at lower concentrations of each drug. Pretreatment with N-acetyl-L-cysteine reversed ARS- and PAM-induced inhibition of PTPase activity. PTPase inhibition by ARS and/or PAM treatment in both DU-145 and PC-3 cells was associated with prolonged EGFR activation. These experiments demonstrate additive or synergistic cell killing by the ARS/PAM combination in DU-145 or PC-3 cells and suggest that enhanced antitumor activity may be related to alterations in receptor tyrosine kinase signaling that occur, in part, due to ROS-mediated PTPase inhibition. 

KEYWORDS | Arsenic trioxide; Bisphosphonates; Epidermal growth factor receptor; Pamidronate; Prostate cancer; Protein tyrosine phosphatase; Reactive oxygen species; Signal transduction 

ABBREVIATIONS | ARS, arsenic trioxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; NAC, N-acetyl-L-cysteine; PAM, pamidronate; pNPP,p-nitrophenyl phosphate; PTPase, protein tyrosine phosphatase; ROS, reactive oxygen species 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Materials

2.2. Cell Culture

2.3. Detection of ROS

2.4. Colony Formation Assay

2.5. Measurement of Cellular Protein Tyrosine Phosphatase Activity

2.6. Measurement of Specific Activity of PTP1B

2.7. Western Blotting of PTP1B

2.8. Analysis of Protein Tyrosine Phosphorylation

2.9. Statistical Analysis

  1. Results

3.1. Sensitivity of Human Prostate Carcinoma Cell Lines to ARS and/or PAM

3.2. Drug Dependent ROS Production by ARS and PAM

3.3. Effect of ARS and/or PAM on Protein Tyrosine Phosphatase Activity

3.4. PTP1B Activity and Protein Expression Are Downregulated in Response to ARS and/or PAM

3.5. ARS and/or PAM Prolong the Duration of EGF-Related Tyrosine Phosphorylation of EGFR

  1. Discussion

1. INTRODUCTION 

Prostate cancer is the most common solid cancer in men and the second most frequent cause of death for men in the United States [1]. Most prostate cancers are initially androgen dependent, but the response to androgen ablation therapy is transient [2]. Although various therapeutic approaches are available, new treatment options for patients in the androgen-independent stage of prostate cancer remain an investigative priority [3]. 

As2O3 (ARS) is a chemotherapeutic agent that is part of standard therapy for patients with acute promyelocytic leukemia [4]. The antineoplastic mechanism(s) of action for ARS, however, are pleiotropic and include activation of the apoptotic cascade, alterations in DNA repair and signal transduction pathways, changes in mitochondrial permeability, and enhancement of oxidative stress [5–8]. ARS has been shown to generate reactive oxygen species (ROS) and induce apoptosis in PC-3 and DU-145 human prostate cancer cell lines, and to produce growth inhibition in vivo for a PC-3 orthotopic model of androgen-independent prostate cancer [9]. ARS treatment has also been demonstrated to significantly diminish proliferation in several other human tumor cell models [10, 11]. 

Because ARS has been shown to enhance the apoptotic effect of other drugs, such as resveratrol, that increase tumor cell oxidative stress [11], we evaluated whether other agents that inhibit prostate cancer cell growth, in part through the generation of ROS, might be appropriate to combine with ARS. One such class of drugs is the bisphosphonates (including pamidronate, PAM) which play a critical role in controlling the function of osteoclasts and improving outcomes for patients with osteoporosis as well as tumors that often metastasize to bone [12–14]. PAM and related bisphosphonates inhibit the growth and invasion of prostate cancer cells in cell culture [15, 16]; these drugs also generate ROS in a wide variety of model cell systems [17–19]. 

Several studies have provided evidence that PAM exerts its effects on bone, at least in part, by inhibition of protein tyrosine phosphatases (PTPase) [20, 21]. The cytoplasmic PTPase, PTP1B, has also been shown to be inhibited by the bisphosphonate alendronate in an H2O2-dependent fashion [20]. Although phosphorylation-dependent signaling is generally understood to be the result of kinase activation, inhibition of PTPases also activates intracellular signaling by permitting the accumulation of phosphotyrosines produced by basal levels of kinase activity [22]. Because PTPases are known to be regulated post-transcriptionally through oxidation of critical cysteine groups [23], we hypothesized that ROS generated by ARS and/or PAM might contribute to PTPase inhibition and the control of prostate cancer cell growth by these drugs. 

To examine whether combining ARS and PAM could provide enhanced targeting of redox sensitive signals critical for tumor cell growth control, we studied cloning efficiency, PTPase activity, and EGFR phosphorylation in DU-145 and PC-3 cells in response to ARS and/or PAM exposure. We demonstrate herein the synergistic cytotoxicity of the ARS/PAM combination for human prostate cancer cells that is associated with enhanced ROS production and prolonged inhibition of PTPase activity.

2. MATERIALS AND METHODS 

2.1. Materials 

Human prostate cancer cell lines DU-145 and PC-3 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture media and serum were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). p-Nitrophenyl phosphate (pNPP), arsenic trioxide, and epidermal growth factor (EGF) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pamidronate disodium (Bedford laboratories, OH, USA) was obtained from the City of Hope Pharmacy (Duarte, CA, USA). Mouse monoclonal antibody against PTP1B, rabbit polyclonal anti-phosphotyrosine (pY99) antibody, antibody against EGFR, HRP-labeled goat anti-mouse IgG, and protein A/G plus beads were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). 5,6-Chloromethyl-2′7′-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA) was purchased from Molecular Probes (Eugene, OR, USA). HRP-labeled anti-rabbit IgG and the enhanced chemiluminescence plus Western blotting detection system were purchased from Amersham Bioscience (Arlington Heights, IL, USA). All other reagents were obtained from Fischer Scientific (Pittsburg, PA, USA). 

2.2. Cell Culture 

The human prostate cancer cell lines PC-3 and DU-145 were maintained as adherent cells routinely cultured in a humidified atmosphere of 5% CO2 in air at 37 oC in RPMI-1640 and MEM with 10% heat-inactivated fetal calf serum, respectively. The cells were treated overnight in medium alone or medium containing ARS (0–100 μM) and/or PAM (0–100 μM). The cells were serum starved for experiments that employed growth factor stimulation. Where indicated, 25 mM N-acetyl-L-cysteine (NAC) was added to the cells 1 h prior to the treatment with ARS or PAM. 

2.3. Detection of ROS 

Intracellular ROS accumulation was monitored using CM-H2DCF-DA, which passively diffuses into cells and is then deacetylated by intracellular esterases. Hydrolyzed, oxidized CM-H2DCF-DA emits green fluorescence at 529 nm. Briefly, after treatment with the drugs, control and treated cells were collected and washed once with Ringer’s solution. The cell pellets were then resuspended in 1 ml of Ringer’s, and 1 μM CM-H2DCF-DA was added to the cells for 1 h. The cells were visualized at an excitation wavelength of 488 nm and emission was measured at 515–540 nm with a MoFlo flow cytometer (Cytomation, Fort Collins, CO, USA). 

2.4. Colony Formation Assay 

The cells were washed, trypsinized, and counted after treatment with various concentrations of the drugs. A total of 1000 cells were plated on 1.5-cm tissue culture dishes in triplicate and incubated at 37oC for 4 to 5 days to form colonies. The number of colonies (more than 40 cells each) was then counted and dose-response curves and combination indices [CI] were derived using Calcusyn® software (Biosoft, Cambridge, UK). 

2.5. Measurement of Cellular Protein Tyrosine Phosphatase Activity 

Cells were collected after treatment with the drugs and washed once with phosphate-buffered saline (PBS). The cells were sonicated in ice-cold homogenization buffer (150 mM NaCl, 5 mM EDTA, 5 mM EGTA in 50 mM Hepes, pH 7.5, containing a protease inhibitor cocktail). Approximately 200 μl homogenate (cell extract, CE) was removed into different tubes, and the remainder was subjected to centrifugation at 10,000 g for 30 min. The supernatant was taken as the soluble fraction (SF). Protein was measured in both fractions using the method of Bradford. PTPase activity was determined in the cell fractions containing approximately 20 μg protein in a final volume of 100 μl at 37oC for 30 min in a reaction mixture containing 10 mM pNPP, 2 mM EDTA, and 20 mM MES at pH 6.0. The reaction was stopped by the addition of 50 μl 1N NaOH, and the absorption was determined at 410 nm. 

2.6. Measurement of Specific Activity of PTP1B 

The cells were collected after the treatment and washed once with PBS. Lysates were prepared by homogenizing cells in a lysis buffer containing 10 mM Tris (pH 8.0), 140 mM NaCl, 0.025% NaN3, 1 mM EDTA, 1 mM phenylmethylsufonylfluoride (PMSF), 1% triton X-100, and 50 U/ml of protease inhibitor cocktail. The lysates were left on ice before the centrifugation at 10,000 g for 30 min. Protein was measured as described earlier. PTP1B was immunoprecipitated from cell lysates with a monoclonal antibody directed at a C-terminal epitope that preserves its enzymatic activity following adsorption to Trisacryl protein G. PTPase activity was measured by the hydrolysis of pNPP in the washed immunoprecipitates using the same method as described above. 

2.7. Western Blotting of PTP1B 

Whole cell lysates (20 μg protein per lane) were denatured by boiling in Laemmli sample buffer and resolved by 12.5% SDS-PAGE. The proteins were transferred to PVDF membranes (Amersham) by electroblotting using Tris buffer containing 10% methanol. The blots were blocked with 20% horse serum and probed with anti-PTP1B. Blots were then incubated with HRP-linked secondary antibody followed by ECL detection. Actin was measured as the protein for the loading control. The blots were quantified using ImageQuant® software (Molecular Dynamics/GE Health Care, Chicago, IL, USA). 

2.8. Analysis of Protein Tyrosine Phosphorylation 

After drug treatment, tumor cells (2 × 106 cells for each treatment group) were washed with PBS and exposed to 10 ng epidermal growth factor (EGF) for different times (1 to 360 min). The cells were then washed again with ice-cold PBS, harvested, and lysates were prepared as described earlier. EGFR was immunoprecipitated from the cell lysates with anti-EGFR antibody followed by adsorption to protein A/G+ beads. The beads were washed, and the samples were subjected to 5% SDS-PAGE. The proteins were transferred to PVDF and subjected to Western blot analysis using a monoclonal anti-phosphotyrosine antibody (pY99). The same blots were stripped and reblotted with anti-EGFR as a loading control. The images were quantified using ImageQuant® (Molecular Dynamics/GE Health Care). 

2.9. Statistical Analysis 

Results are expressed as mean ± SE of at least three independent experiments. Statistical analyses were performed with Student’s two-tailed t test. Values of p < 0.05 were considered statistically significant.

3. RESULTS 

3.1. Sensitivity of Human Prostate Carcinoma Cell Lines to ARS and/or PAM 

Two hormone resistant prostate carcinoma lines (DU-145 and PC-3) were selected for this study because previous studies had demonstrated that both ARS and PAM are individually cytotoxic for these cells [10, 15]. Cells were treated with a range of concentrations (0–100 μM) of the two drugs separately. Cell viability was determined by clonogenic assay after treatment with ARS or PAM after 6 h, 24 h, or 48 h of drug exposure. At the 6 h time point, neither of the two drugs inhibited clonogenic survival at the 100 µM concentration. However, inhibition of clonogenic growth was demonstrable at clinically achievable concentrations of ARS or PAM after incubation for 24 h. Treatment for 48 h with either drug alone killed more than 80% of the cells. Therefore, we selected 24 h of drug exposure for several of the experiments in this study. The IC50’s for ARS and PAM were 9 and 20 μM, respectively, for DU-145 cells; and 2 and 12 μM for PC-3 cells over 24 h of drug treatment. When the drugs were used in combination, ARS and PAM demonstrated an additive effect in DU-145 cells with a combination index [CI] of 1.10. Treatment of PC-3 cells with both agents revealed therapeutic synergy with a combination index of 0.86 (Figure 1). Pretreatment of the cells for 1 h with 25 mM NAC, an antioxidant thiol, prior to anticancer drug exposure completely reversed the cytotoxic effect of PAM on both DU-145 and PC-3 cells. However, PC-3 cells demonstrated only partial reversal of the ARS effect after treatment with NAC in contrast to complete reversal in DU-145 cells (Figure 2). NAC pretreatment also blocked the cytotoxic effect of the ARS/PAM combination in the DU-145 line; significant but partial inhibition of the cytotoxicity of the combination was observed for PC-3 cells (Figure 2).

 

FIGURE 1. Dose-effect, isobologram, and combination index [CI] curves for ARS and/or PAM following 24 h of drug treatment in DU-145 and PC-3 cells. CI’s were calculated by using Calcusyn® software. DU-145 cells showed an additive effect at lower concentrations and synergistic effects at higher concentrations with a CI of 1.10. PC-3 cells demonstrated synergistic effects at much lower concentrations compared to DU-145 cells with a CI of 0.86. ED50, 75, or 90 represent the concentrations (μM) required for 50, 75, or 90% inhibition of clonogenic survival. Data are representative of three independent experiments.

 

FIGURE 2. Effect of ARS and/or PAM at their IC50’s for a 24h exposure on the formation of colonies in DU-145 (A) and PC-3 (B) cells. Each bar represents the mean ± SE. Both ARS and PAM demonstrate significant inhibition of clonogenic survival compared to untreated cells; the drug combination further enhances tumor cell cytotoxicity versus the single agents. Prior treatment for 1 h with 25 mM NAC blocks the effect of ARS and PAM as well as the ARS/PAM combination completely in DU-145 cells and partially in PC-3 cells (*, p 0.001; n = 6). 

3.2. Drug Dependent ROS Production by ARS and PAM 

To confirm previous studies demonstrating that ARS or PAM exposure led to ROS formation in tumor cells [18], we investigated the effects of ARS and/or PAM on cellular ROS levels in the DU-145 and PC-3 prostate cancer lines. Exposure of DU-145 cells to ARS (20 µM) or PAM (20 µM) for 24 h elevated the cellular ROS levels from 327.7 to 422.4 or 349.7 mean log fluorescence units; and from 692.7 to 810.6 or 804.4 mean log fluorescence units in PC-3 cells. These increases were more substantial when either cell line was treated with the two-drug combination (Figure 3).

 

FIGURE 3. Effect of ARS and/or PAM on reactive oxygen production in DU-145 and PC-3 cells which was measured by flow cytometry. ARS and PAM each independently increase the production of ROS. The two drugs in combination further increase ROS formation. The numbers in parentheses show mean fluorescence values. Data are representative of three identical experiments. 

3.3. Effect of ARS and/or PAM on Protein Tyrosine Phosphatase Activity 

Protein tyrosine phosphatases (PTPase) play key roles in modulating cellular metabolism; many possess catalytic cysteines that are potential sites of interaction with ARS [5, 24]. Studies have also provided evidence that PAM may exert certain of its biochemical effects through inhibition of protein tyrosine phosphatase activity [25]. To explore the effects of these two drugs on tyrosine phosphatases, we measured protein tyrosine phosphatase activity in the whole cell lysates and soluble fractions of DU-145 and PC-3 cells prepared after treatment of the cells with either ARS or PAM or the two-drug mixture. In the soluble fraction, ARS (20 µM for 24 h) decreased PTPase activity by 56 ± 2%, p< 0.01 compared to untreated control DU-145 cells; and by55±11%, p<0.01 in PC-3 cells. PAM (20 µM for 24 h) decreased the PTPase activity in the soluble cell fraction by 39 ± 0.5%, p<0.01 and by 18 ± 4%, p<0.05 in DU-145 and PC-3 cells, respectively (Figure 4). Whole cell lysates also showed similar decreases in the PTPase activities of both cell lines treated with ARS or PAM. Combining the two drugs further decreased PTPase activities in both cell lines at lower concentrations of each drug in cell extracts as well as soluble fractions. The particulate fractions (the residual cell pellet following centrifugation) of ARS- or PAM-treated cells did not demonstrate any differences in PTPase activity compared to untreated controls (data not shown). As demonstrated in Figure 4C and 4F, we did not observe significant recovery of PTPase activity for at least 6 h in either DU-145 or PC-3 cell lines following removal of ARS or PAM after the two drugs were replaced with fresh media. Pretreatment of the cells for 1 h with 25 mM NAC reversed ARS- and PAM-induced inhibition of PTPase activity (Figure 5).

 

FIGURE 4. Effect of 20 µM ARS and/or 20 µM PAM (24 h exposure) on PTPase activity in DU-145 and PC-3 cells. The x-axis is a multiplier for 20 µM ARS or PAM or the combination of 20 µM of each drug. The PTPase activity (calculated as % of untreated control) decreased significantly (+, p 0.05 and *, p 0.01) in cell extracts and the soluble fractions of treated cells for both drugs (A, B, D, E). The combination of the two drugs decreased PTPase activity further. Cells were washed after 24 h of drug treatment, and the recovery of PTPase activity was measured at different times in both the cell extracts (CE) and soluble fractions (SF). The arrows indicate the end of drug exposure. Enzyme activity did not recover significantly for the 6 h after the end of drug treatment (C, F). Each point represents the mean ± SE of at least three experiments.

 

FIGURE 5. Effect of 25 mM NAC on the activity of PTPase in cells treated with 20 µM ARS and 20 µM PAM for 24 h (n = 3). Each error bar represents the mean ± SE. ARS and PAM significantly (*, p < 0.01) decreased PTPase activity compared to untreated controls in both cell extract and soluble fractions of DU-145 (A, B) and PC-3 cells (C, D). NAC, added 1 h prior to the addition of ARS or PAM, prevented the drug-induced inhibition of PTPase in all fractions of both cell lines. 

3.4. PTP1B Activity and Protein Expression Are Downregulated in Response to ARS and/or PAM 

Previous studies have demonstrated that inhibition of the protein tyrosine phosphatase PTP1B in vitro by alendronate, a bisphosphonate that has, like PAM, been employed for treatment of osteoporosis, depends upon oxidation of its active site thiol [20]. We measured the activity of PTP1B in washed immunoprecipitates from ARS- and/or PAM-treated DU-145 and PC-3 cells. Following a 24 h exposure, ARS inhibited PTP1B activity by 30 ± 7%, p < 0.01, and 37 ± 9%, p < 0.01 in DU-145 and PC-3 cells, respectively, compared to untreated control cells (Figure 6A and 6B). PAM decreased the specific activity of PTP1B by 33 ± 5%, p < 0.01 and 31 ± 7%, p < 0.01 in DU-145 and PC-3 cells, respectively. Combining ARS and PAM further decreased the activity of PTP1B to 57 ± 3%, p < 0.01, in DU-145 and 63 ± 3%, p < 0.01, in PC-3 cells. NAC blocked the effects of ARS and/or PAM on PTP1B activity for both cell lines. To further investigate these findings, we examined the effect of our drug treatment conditions on PTP1B protein expression. PTP1B expression was decreased in response to ARS as well as PAM; protein expression decreased further when the two drugs were used in combination in both DU-145 and PC-3 cells (Figure 6C and 6D). NAC pre-treatment partially prevented the drug-induced decrease in PTP1B expression in both cell lines.

 

FIGURE 6. Blockade of the effect of ARS and/or PAM on the activity and expression of PTP1B by 25 mM NAC in DU-145 (A) and PC-3 (B) cells. The treated cells demonstrate significantly decreased enzyme activity (*, p < 0.01; n = 6) compared to untreated cells. Each error bar represents the mean ± SE. Combination of the two drugs further decreased the activity of PTP1B. Panels (C) and (D) demonstrate that incubation of DU-145 and PC-3 cells with 20 µM ARS and/or PAM for 24 h downregulates PTP1B protein expression. Pretreatment with NAC (25 mM) 1 h prior to drug treatment partially blocked the effect of ARS and PAM on PTP1B expression in PC-3 cells to a greater extent than in the DU-145 line. The upper panels in Figure 6C and 6D demonstrate the Western blots; the lower panels show the ratios of the densities of PTP1B to actin calculated using ImageQuant® software. The Western blots are representative of three separate experiments. 

3.5. ARS and/or PAM Prolong the Duration of EGF-Related Tyrosine Phosphorylation of EGFR 

Cellular redox reactions related to the control of PTPase function have been implicated in growth factor receptor signaling [26–28]. Furthermore, H2O2 has also been shown to mimic ligand-mediated signaling by EGF and plate-derived growth factor (PDGF) [23]. To evaluate the effect of drug-induced alterations in PTPase function on receptor tyrosine kinase signaling, we studied EGF-related EGF receptor phosphorylation in response to treatment of DU-145 and PC-3 cells with ARS and/or PAM. For these experiments, tumor cells were exposed to EGF for 10 min after treatment with ARS or PAM for 24 h, and then washed with PBS. Tyrosine phosphorylation of EGFR was then measured at time points from 0 to 360 min in treated cells and compared to untreated cells. ARS, PAM, and the ARS/PAM combination markedly prolonged the duration and degree of activation of overall cellular tyrosine phosphorylation of the EGFR for up to 6 h in both DU-145 and PC-3 cells, compared to approximately 30–60 min in untreated cells (Figure 7A and 7B).

 

FIGURE 7. Incubation of DU-145 and PC-3 cells with 20 µM ARS and/or 20 µM PAM for 24 h prolongs EGF-related protein tyrosine phosphorylation of the EGFR. Cells were treated with the drugs for 24 h, washed with PBS, and incubated with 10 ng/ml EGF for 10 min. After washing again, the cells were collected at time points from 0 to 360 min, and cell lysates were prepared. The protein was measured using the Bradford method. Cell lysates (1 mg protein) were subjected to immunoprecipitation with anti-EGFR (1mg/ml) antibody. The immunoprecipitates were subjected to Western blot, transferred to nitrocellulose, and probed with an anti-phosphotyrosine antibody. The bar diagrams show the calculated ratios (using ImageQuant®) of phosphorylated EGFR to EGFR after re-probing the membranes with anti-EGFR antibody. These figures are representative of three identical experiments.

4. DISCUSSION 

In these experiments, we found that the anticancer agents ARS and PAM, demonstrated previously as individual drugs to decrease the proliferative rate of human prostate cancer cells [10, 15], produce synergistic and/or greater than additive inhibition of clonogenic cell growth for the PC-3 and DU-145 lines when the drugs are delivered together (Figure 1). The combination of ARS and PAM also enhanced the production of ROS, the degree of PTPase inhibition, the inhibition of the activity and protein expression of the phosphatase PTP1B, and the duration of EGF-mediated phosphorylation of the EGF receptor in human prostate cancer cells. These combination effects are consistent with at least additive mechanistic interactions between the two drugs that promote growth inhibition. 

PTPases play critical roles in cancer and tumor cell signal transduction, including the development of breast cancer and the promotion of migration and invasion of prostate cancer in vitro [29]. Small molecule inhibitors of PTPases significantly diminish the growth of hepatocellular carcinoma cells, in part by producing sustained phosphorylation along the MAPK cascade [30]. In the current experiments, we demonstrated that ARS and PAM, individually and in combination, inhibit PTPase and PTP1B activities in a fashion that is significantly decreased by pretreatment with the antioxidant thiol NAC (Figures 4, 5, and 6). Bisphosphonates have been demonstrated previously to inhibit PTPase activity in an H2O2-dependent fashion [20]; similar effects have also been elucidated for ARS [24]. As shown in Figure 3, treatment with either ARS or PAM, and to a greater extent the combination, enhanced the production of ROS. Many PTPases, and specifically PTP1B, are well known to be regulated by oxidative modification of a critical cysteine at their active site [24]. And thus, the inhibition of phosphatase activity observed in these experiments, and their prevention by NAC, might be explained by the enhanced level of intracellular oxidant production by ARS and/or PAM. 

However, we also found that PTPase inhibition by ARS/PAM was not reversible for at least 6 h following removal of the drugs from the cell culture (Figure 4). These experiments suggest the possibility that, in addition to oxidative stress, covalent interactions of ARS or PAM at the PTPase cysteine could have occurred, producing prolonged (possibly irreversible) inactivation of the phosphatase [22], facilitating cytotoxicity. In the absence of measurements of such covalent by-products or of the redox state of the PTPase cysteine, a definitive explanation for the dose-related inhibition of PTPase activity by ARS/PAM is not possible based on the data presented herein. 

As demonstrated in Figure 6, we observed that ARS and PAM in PC-3 cells, and the combination in DU-145 cells, also decreased the protein expression of PTP1B, which was partially prevented by NAC pretreatment. Drug-induced changes in PTP1B expression at the protein level, which could contribute to decreased PTPase activity, have not been clearly described previously as a result of anticancer drug exposure. However, regulation of PTP1B expression at the transcriptional level has been demonstrated for the oxidant-sensitive transcription factors Sp-1 and NF-κB, as well as for a variety of non-oxidant-related post-translational modifications [22]. Partial protection of PTP1B protein expression in these studies by NAC was not expected; hence, further experiments will be required to understand the mechanism, oxidant or not, by which ARS/PAM exposure alters PTP1B protein levels. 

To more clearly understand potential mechanisms that might explain the greater than additive cytotoxicity of ARS/PAM, we examined growth factor-induced phosphorylation of EGFR in both DU-145 and PC-3 cells. Previous studies have demonstrated that hyperactivation of the EGFR (due to high levels of EGFR receptor density or high levels of EGF) inhibits growth (rather than stimulating proliferation) across both breast and lung cancer cell lines, due, in part, to a block in cell cycle progression at the late S/G2 interface [31–33]. Prolonged inhibition of PTPase function by small molecule phosphatase inhibitors produces prolonged, unchecked phosphorylation of the mitogen-activated protein kinase (MAPK) pathway, leading to unbalanced tyrosine kinase activity that is closely correlated with growth inhibition in human hepatoma cells [33]. We found (Figure 7) that the ARS/PAM combination, particularly in PC-3 cells, led to pronounced EGFR phosphorylation after a short exposure to EGF that continued for up to 6 h (rather than the 30–60 min observed in the absence of either drug). While the cytotoxicity of ARS/PAM that we observed could have been due to other direct effects of these drugs on, for example, mitochondrial respiration or endoplasmic reticulum (ER) stress, or from secondary pathological changes produced by a drug-enhanced reactive oxygen cascade per se, it seems likely that the profound alterations in growth factor signaling produced by drug-related inhibition of PTPase activity contributed in a substantive fashion to the cytotoxic effects of the ARS/PAM combination. 

In summary, we observed that the combination of ARS/PAM enhanced cytotoxicity and inhibition of protein tyrosine phosphatase function in human prostate cancer cells in a thiol-dependent fashion which was accompanied by prolonged phosphorylation of EGFR after EGF stimulation. These experiments suggest that therapeutic strategies focusing on altering PTPase activity to induce a drug-related imbalance in tyrosine kinase/phosphatase function may be a fruitful approach for treatment of androgen-independent prostate cancer. 

ACKNOWLEDGMENTS 

This work was supported by the U.S. National Cancer Institute grant ZIA BC 011078 and the City of Hope National Medical Center (Duarte, CA, USA). The authors declare no conflicts of interest. 

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