PBISe, a novel selenium-containing drug for the treatment of malignant melanoma

Malignant melanoma is the most deadly form of skin cancer due to its highly metastatic nature. Untargeted therapies are ineffective for treating metastatic disease, leading to the development of agents specifically inhibiting proteins or pathways deregulated in melanoma. The deregulation of inducible nitric oxide synthase (iNOS) is one such event occurring in melanoma, and is correlated with poor survival. Current iNOS inhibitors, such as PBIT [S,S′-1,4-phenylenebis(1,2-ethanediyl)bis-isothiourea], require high concentrations for clinical efficacy causing systemic toxicity. To develop more potent agents effective at significantly lower concentrations, a novel isosteric analogue of PBIT was synthesized, called PBISe [S,S′-1,4-phenylenebis(1,2-ethanediyl)bis-isoselenourea], in which sulfur was replaced with selenium. PBISe kills melanoma cells >10-fold more effectively than PBIT, and cultured cancer cells are 2- to 5-fold more sensitive than normal cells. Like PBIT, PBISe targets iNOS but also has new inhibitory properties acting as an Akt3 pathway inhibitor and mitogen-activated protein kinase (MAPK) cascade activator, which causes decreased cancer cell proliferation and increased apoptosis. Inhibition of cellular proliferation mediated by PBISe induced a G₂-M phase cell cycle block linked to excessively high MAPK activity causing decreased cyclin D1 and increased p21 as well as p27 levels. PBISe promotes apoptosis by inhibiting Akt3 signaling, elevating cleaved caspase-3 and PARP levels. Compared with PBIT, PBISe reduced tumor development by 30% to 50% in mice inducing a 2-fold increase in apoptosis with negligible associated systemic toxicity. Collectively, these results suggest that PBISe is a potent chemotherapeutic agent with novel properties enabling the targeting of iNOS, Akt3, and MAPK signaling, thereby promoting melanoma cell apoptosis and inhibition of proliferation. [Mol Cancer Ther 2008;7(5):1297–308]

Of the three main forms of skin cancer, malignant melanoma has the most significant effect on human health and carries the highest risk of mortality from metastasis (1). Nontargeted therapeutics ranging from surgery to immunotherapy, radiotherapy, and chemotherapy have all failed to provide effective long-term treatment for patients suffering from advanced stages of this disease (2, 3). Thus, the 2007 prognosis for patients with metastatic disease remains very poor, with average survival ranging from 6 to 10 months (4). In spite of the widely appreciated magnitude of the problem, there is still a critical gap in the development of chemotherapeutics targeting proteins or signaling pathways whose deregulation contributes to melanoma development (4, 5).

Inducible nitric oxide synthase (iNOS) is a calcium-independent, cytokine-inducible enzyme involved in the production of a bioactive, pleiotropic regulatory and signaling molecule from arginine called nitric oxide (6). Recent studies have implicated iNOS as a potential therapeutic target in melanoma (7–10). Among the three isoforms of NOS, calcium-independent iNOS produces high levels of nitric oxide, promoting the development of a malignant phenotype (6–8, 10). Elevated iNOS expression/activity is also correlated with poor survival rates of patients with melanoma (7, 8). A number of small molecule iNOS inhibitors have been identified and tested in vitro and in vivo (6, 11–13). In vitro studies using iNOS inhibitors, S-methylisothiourea and aminoguanidine, have shown the inhibition of nitric oxide production and induction of apoptosis mediated by caspase-1/3 and PARP cleavage (10). PBIT [S,S′-1,4-phenylenebis(1,2-ethanediyl)bis-isothiourea] is an iNOS-selective inhibitor that is effective in preventing colon and esophageal cancer in rats following dietary administration (13–15). However, due to its low potency, poor cell permeability, and associated systemic toxicity, its use in clinical settings is questioned (13–15).

To create a more potent compound that would be an effective melanoma inhibitor at significantly lower concentrations, a novel isosteric analogue of PBIT was synthesized, called PBISe [S,S′-1,4-phenylenebis(1,2-ethanediyl)bis-isoselenourea], in which sulfur was replaced with selenium. Selenium is a trace element required for the protection of cell membranes, and is a component of various biomolecules (16, 17). Low selenium levels occur in serum isolated from patients suffering from a variety of different cancer types including melanoma (18). Furthermore, dietary selenium supplementation can reduce tumor growth as well as improve quality of life for patients (18–22).

Incorporation of selenium into the structure of PBIT has potential to provide the agent with novel tumor-inhibitory properties, due to the presence of selenium as well as structure-activity relationships associated with the compound (13, 23, 24). Specifically, selenium has inhibitory effects on phosphorylation of the survival kinase Akt in the phosphoinositide-3-kinase (PI3K) signaling pathway, thereby inhibiting its activity and that of the downstream signaling cascade (25). Akt kinase family members Akt1/PKBα, Akt2/PKBβ, or Akt3/PKBγ play important roles in the development of a large variety of cancers including melanoma (26, 27). Therefore, a novel compound inhibiting iNOS and Akt signaling might be a more effective tumor inhibitor (8, 10).

Among the three Akt family members, Akt3 is the predominantly active isoform in ∼70% of melanomas (27). Akt3 activity promotes tumorigenesis by signaling through PRAS40 to decrease the apoptotic sensitivity of melanoma cells (26–29). Reduction of Akt3 or PRAS40 activity using small interfering RNA or through PTEN expression significantly decreased the tumorigenic potential of melanoma cells by increasing cellular apoptotic sensitivity (28, 29). Therefore, the Akt3 signaling cascade is one of the major pathways important in melanoma and inhibiting it could be therapeutically important for treating this disease.

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway (Ras/Raf/Mek/Erk) through Ras mutations in 10% to 15% and B-Raf mutation in ∼60% of melanomas is a second important signaling cascade deregulated in melanomas (30). Activating B-Raf mutations are acquired, somatic, postzygotic events and are not inherited in families (31). Among different B-Raf mutations, a single-base missense substitution (T to A at nucleotide 1799) that changes valine to glutamic acid at codon 600 (V600E) in exon 15 is prevalent in ∼90% of melanoma tumors with a mutation of B-Raf (32). ^V600EB-Raf protein leads to kinase activity 10.7 times higher than occurs in normal cells and causes hyperactivity of the MAPK pathway (32).

Treatment of cells with PBIT to inhibit iNOS increases MAPK pathway activity leading to higher levels of active phosphorylated Erk1/2 and increased expression of downstream iNOS (33, 34). Abnormally high activation of the MAPK pathway can inhibit cellular growth in a wide variety of normal and cancer cells by promoting cellular senescence, including melanomas (35). Recent evidence suggests that constitutively active ^V600EB-Raf initially promotes nevi development during melanoma tumor progression resulting in high, intense activation of the MAPK pathway, which is inhibitory, and that Akt3 activity is required to phosphorylate^V600EB-Raf in order to reduce its activity and the MAPK pathway activity to levels promoting rather than inhibiting proliferation (36). Thus, increasing MAPK pathway activity following PBIT treatment or by inhibiting Akt3 could inhibit tumor cell proliferation and thereby play therapeutically important roles for the treatment of melanoma.

In this study, we show that PBISe but not PBIT effectively kills melanoma cells and that cultured cancer cells are 2- to 5-fold more sensitive to the compound than normal cells. Although PBISe targets iNOS in a manner similar to PBIT, it also has new inhibitory properties acting as an Akt3 signaling cascade inhibitor and MAPK pathway activator. These novel properties lead to decreased cell proliferation and increased apoptosis of melanoma cells. Only PBISe but not PBIT inhibits cancer cell proliferation by blocking cultured cells in the G₂-M phase of the cell cycle and decreases cyclin D1 expression with corresponding increases in p21 and p27 levels. Furthermore, PBISe but not PBIT promotes apoptosis in melanoma cells through the inhibition of Akt3 signaling leading to increased levels of cleaved caspase-3/7 and PARP. Compared with PBIT, PBISe reduced tumor development by 30% to 50% in mice with negligible associated toxicity. Mechanistically, tumors undergo 2- to 3-fold more apoptosis following treatment with PBISe compared with PBIT. Thus, PBISe is a more potent chemotherapeutic agent than PBIT.

The human metastatic melanoma cell lines UACC 903 and 1205 Lu, normal human fibroblast cells (FF2441), were maintained in DMEM (Invitrogen) and supplemented with 10% fetal bovine serum (FBS; Hyclone). The vertical growth phase melanoma cell line WM115 was maintained in Tu2% medium lacking calcium chloride, but supplemented with 2% heat-treated FBS (56°C for 30 min) and l-glutamine (Mediatech; ref. 27). Colon adenocarcinoma cell line Caco-2 was grown in Advanced DMEM supplemented with 10% heat-inactivated FBS (56°C for 30 min) and 2 mmol/L of l-glutamine.

PBIT and PBISe was synthesized as reported (13).⁷

The methyl ester of 1,4-phenylenediacetic acid was reduced with lithium aluminum hydride to obtain a diol, which upon bromination with carbon tetrabromide and triphenylphospine, resulted in the generation of a dibromo derivative. This was reacted with thiourea to generate PBIT. Similarly, PBISe was synthesized by reacting a dibromo compound with selenourea instead of thiourea.⁷ Monosubstituted derivatives S-phenylethyl isothiourea (PEIT) and S-phenylethyl isoselenourea (PEISe) were synthesized by reacting phenyl ethylbromide with thiourea or selenourea, respectively.⁷ The purity of PBIT and PBISe were confirmed by high-performance liquid chromatography and structure characterized using proton nuclear magnetic resonance (500 MHz, Bruker; Bruker Optics, Inc.) and mass spectrometry (Finnigan Mat95).⁷ High-performance liquid chromatography analysis showed that PBIT and derivatives PBISe, PEIT, and PEISe were 99.5% pure. Results were PEIT δ: 7.32 to 7.24 (m, 5H, aromatic), 3.34 (t, 2H, J = 7.0 Hz, CH₂-Ph), 2.99 (t, 2H, J = 7.0 Hz, CH2-S), m/z ion intensity −181 (M⁺, 100), 159 (10), 105 (10); PEISe: 7.29 to 7.20 (m, 5H, aromatic), 3.02 (t, 2H, J = 7.0 Hz, CH₂-Ph), 3.48 (t, 2H, J = 7.0 Hz, CH₂-Se), m/z ion intensity −229 (M+, 100), 181 (80), 159 (10), 105 (10); PBIT: δ: 7.25 (s, 4H, aromatic), 3.37 (t, 4H, J = 7.2 Hz, Ph-CH2), and 3.0 (t, 4H, J = 6.86 Hz, C-CH2), m/z ion intensity 283 (M⁺¹, 100), 266 (40), 207 (30), 142 (70), 131 (100), 104 (10), 77 (30); and PBISe: δ: 7.25 (s, 4H, aromatic), 3.37 (t, 4H, J = 7.2 Hz, Ph-CH2), and 3.21 (t, 4H, J = 6.86 Hz, C-CH2), m/z ion intensity 377 and 379 (M⁺¹, 50), 335 and 337 (80), 253 and 255 (70), 188 and 190 (100), 131 (50), 73 (10). Furthermore, electrospray-mass spectrometry showed an observed molecular weight close to the calculated mass. For example, the calculated m/z for PBISe was 378.9934 and the observed m/z was 378.9935.

Cell lysates were harvested by the addition of lysis buffers containing 50 mmol/L of HEPES (pH 7.5), 150 mmol/L of NaCl, 10 mmol/L of EDTA, 10% glycerol, 1% Triton X-100, 1 mmol/L of sodium orthovanadate, 0.1 mmol/L of sodium molybdate, 1 mmol/L of phenylmethylsulfonyl fluoride, 20 μg/mL of aprotinin, and 5 μg/mL of leupeptin. Whole cell lysates were centrifuged (≥10,000 × g) for 10 min at 4°C to remove cell debris. Protein concentrations were quantitated using the bicinchoninic acid assay (Pierce), and 30 μg of lysate loaded per lane onto NuPAGE Gels (Life Technologies). Following electrophoresis, samples were transferred to polyvinylidene difluoride membrane (Pall Corporation). Blots were probed with antibodies to PRAS40 and phosphorylated PRAS40 (Thr²⁴⁶; Invitrogen); antibodies to iNOS, cyclin D1, p27, p21, Erk2, α-enolase, and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology; and antibodies to Akt3, phosphorylated-Akt (Ser⁴⁷³), phosphorylated-Erk 1/2 (Thr²⁰²/Tyr²⁰⁴), caspase-3 and cleaved PARP were from Cell Signaling Technology. Immunoblots were developed using enhanced chemiluminescence (Pierce).

The viability and IC₅₀ of melanoma cells following treatment with PBIT, PBISe, PEIT, and PEISe were measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega). A total of 5 × 10³ cells/well in 100 μL of DMEM-10% FBS were grown in a 96-well plate for 36 or 72 h, respectively, for melanoma (UACC 903, 1205 Lu, and WM115) and human fibroblast (FF2441) cell lines, and treated with either PBS vehicle or increasing concentrations (3.5–21 μmol/L) of PBIT, PBISe, PEIT, or PEISe for 24 h. Cellular viability compared with control-treated cells was measured using the MTS assay. IC₅₀ (μmol/L) values for each compound in respective cell lines was determined from three independent experiments using GraphPad Prism version 4.01 (GraphPad).

Cellular proliferation and apoptosis rates were measured by seeding 5 × 10³ cells in 96-well plates, followed by treatment for 24 h with each respective agent. Proliferation and apoptosis rates were measured using a bromodeoxyuridine ELISA kit (Roche Applied Sciences) or Apo-ONE homogenous caspase-3/7 assay kit (Promega), respectively (29).

Cell cycle analysis was undertaken by growing 1.5 × 10⁶ melanoma cells in 100-mm culture dishes followed by treatment with PBIT or PBISe for 24 h. Cells were collected and stained using propidium iodide (100 μg/mL; Sigma), 20 μg/mL of RNase A (Roche), and 3 μg/mL of Triton X-100 dissolved in 0.1% (W/V) sodium citrate for 30 min at 4°C (37). Stained cells were analyzed using the FACScan analyzer (Becton Dickinson) and data processed using ModFit LT software (Verity Software House).

Nitrite content in the cell culture medium was determined using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Company). Caco-2 cells (1 × 10⁶) were plated in 60-mm culture dishes in advanced DMEM containing 10% heat-treated FBS for 12 h, and growing cells conditioned with phenol red–free DMEM containing 0.5% FBS for an additional 12 h. Cells were then treated with increasing concentrations of PBIT (40 and 80 μmol/L) or PBISe (2 and 4 μmol/L) dissolved in phenol red–free DMEM (2 mL) containing 0.5% FBS for 72 h. Culture supernatants were collected and total nitrite (nitrate + nitrite) measured by incubating 80 μL of the supernatant with an enzyme cofactor mixture (10 μL) and a nitrate reductase (10 μL) for 2 h. Total nitrite was measured by the addition of Griess reagent I and II (50 μL each). A nitrate standard curve (5–35 μmol/L) was simultaneously prepared. The total nitrite present in the medium alone was subtracted from all experimental values and represented as micromolar nitrite produced following each treatment.

Tumor kinetics were measured by s.c. injection of 5 × 10⁶ UACC 903 or 2.5 × 10⁶ 1205 Lu cells in 0.2 mL of DMEM containing 10% FBS above the left and right rib cages of 4- to 6-week-old female nude mice (Harlan Sprague-Dawley). Six days later, mice were randomly placed in control DMSO and experimental (PBIT and PBISe) groups (n = 5 animals; 10 tumors total) followed by i.p. treatment with PBIT (0.315 μmol/L) or PBISe (0.315 μmol/L; equivalent to 2.5 ppm selenium/20 g mouse) on Monday, Wednesday, and Friday for 3 weeks. The dimensions of the developing tumors and body weights were measured.

For mechanistic studies, 5 × 10⁶ UACC 903 cells were injected s.c. into nude mice, generating tumors of the same size developing at parallel time points. Six days later, mice were treated i.p. with DMSO vehicle, PBIT, or PBISe (0.315 μmol/L each) on alternate days up to day 15. Size- and time-matched tumors were harvested at days 9, 11, 13, and 15 to assess changes in cell proliferation and apoptosis. A small portion of tumor was flash-frozen in liquid nitrogen for caspase-3/7 activity analysis and processed by pulverizing into powder and adding protein lysis buffer [600–800 μL/50 mg powder, 50 mmol/L Tris-HCl (pH 7.5) containing 0.1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, 50 mmol/L sodium fluoride, 10 mmol/L sodium β-glycerol phosphate, 5 mmol/L sodium PPi, 1 mmol/L activated sodium orthovanadate, protease inhibitor cocktail, and 0.1% (v/v) 2-mercaptoethanol] followed by repeated centrifugation (10,000 × g). Protein concentration was quantitated using Bio-Rad protein assay reagent (Bio-Rad Laboratories) and analyzed by Western blotting. For caspase-3/7 activity determination, 100 μg of protein lysate was incubated with a rhodamine-110–conjugated caspase-3/7 substrate (Z-DEVD) for 1 to 2 h and fluorescence (485 nm excitation and 520 nm emission) measured in a SPECTRAmax M2 plate reader. Cell proliferation was measured in formalin-fixed, paraffin-embedded tumor sections using purified mouse anti-human Ki-67 from PharMingen. A minimum of six different tumors with four to six fields per tumor were analyzed and results represented as the average ± SE.

Four- to 6-week-old female nude mice (Harlan Sprague-Dawley) were injected i.p. with either control DMSO vehicle, PBIT, or PBISe (0.315 μmol/L each) for 3 weeks by following the regime used for tumorigenicity. Blood was then collected from each animal in plasma separator tubes with lithium heparin (BD Microtainer, BD) following cardiac puncture and analyzed for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, and creatinine to ascertain liver, heart, kidney, and pancreas-related toxicity. For morphologic examination of blood cells, whole blood was collected in microtainer tubes containing K₂EDTA (BD Microtainer, BD) and RBC, WBC, lymphocytes, monocytes, eosinophils, platelets, total hemoglobin, and hematocrit percentage were analyzed. Blood was also microscopically examined for segregates, polychromatin bodies, and smudge cells. A portion of the vital organs—liver, heart, kidney, intestine, pancreas, and adrenal glands—from each animal was formalin-fixed and paraffin-embedded to examine for toxicity-associated changes in cell morphology and organization following H&E staining.

Statistical analysis was undertaken using the one-way or two-way ANOVA followed by Tukey's or Bonferroni's post hoc tests. Results were considered significant at P < 0.05.

PBIT is a non–amino acid analogue of l-arginine that inhibits iNOS activity at rates 190- and 5-fold higher than eNOS or nNOS family members, respectively (6, 13). It prevents colon and esophageal tumor development in animals but has low potency and poor cell permeability, requiring high concentrations for efficacy with significant acute toxicity, reducing its clinical utility (13–15). To overcome these limitations, a novel derivative, called PBISe, was synthesized in which sulfur was replaced with selenium in order to increase the potency of the compound (Fig. 1A). Control monosubstituted sulfur and selenium compounds, PEIT and PEISe, were also synthesized to determine whether the disubstitution in the aromatic ring contributed to chemotherapeutic efficacy (Fig. 1A).

Because PBIT is a known iNOS-specific inhibitor, with demonstrated efficacy at reducing iNOS activity and colon adenocarcinoma cell growth, similar approaches to those reported in the literature were used to validate growth and iNOS-inhibitory effects mediated by PBIT on cultured Caco-2 cells and compared it to PBISe to confirm that the new compound acted in a manner similar to the original compound (14). Treatment with increasing concentrations of PBIT (40–120 μmol/L) for 72 h showed the predicted dose-response curve with a resulting IC₅₀ of ∼60 μmol/L (Fig. 1B). Using the same approach, PBISe was significantly more effective, having an IC₅₀ of 2.4 μmol/L indicating a 25-fold increase in potency compared with PBIT (Fig. 1B). Thus, PBISe is a far superior and more potent inhibitor of colon adenocarcinoma cells than PBIT.

To show that PBIT and PBISe similarly inhibited iNOS activity in terms of nitrite production, total nitrate + nitrite levels were measured in Caco-2 cells using a reported colorimetric assay (Fig. 1C; ref. 10). Results showed that 80 μmol/L of PBIT significantly reduced nitrate + nitrite levels compared with control PBS-treated cells, which was indicative of iNOS inhibition (P < 0.05; one-way ANOVA; ref. 10). Similarly, PBISe also reduced total nitrate + nitrite production in Caco-2 cells but at much lower concentrations of 2 to 4 μmol/L compared with PBIT (Fig. 1C). Unfortunately, higher concentrations could not be used because PBISe treatment killed >80% of cells at 2 to 4 μmol/L; therefore, total nitrate + nitrite levels regulated by live cells could not be measured. Thus, like PBIT, PBISe inhibited iNOS activity but also had additional inhibitory capabilities.

To compare the effectiveness of PBISe versus PBIT for killing melanoma cells, three independently derived melanoma cell lines, WM115, UACC 903, and 1205 Lu were treated with 3.5 to 21 μmol/L of PBISe or 10 to 100 μmol/L of PBIT, PEIT, and PEISe for 24 h and cell viability measured using the MTS assay. A representative example of the MTS analysis for UACC 903 is shown in Fig. 2A, demonstrating the effectiveness of PBISe for decreasing melanoma cell viability compared with PBIT. Dose-response curves for these agents were generated and IC₅₀ values determined (Table 1). PBISe decreased the viability of all three melanoma cell lines with an IC₅₀ range of 8 to 10 μmol/L (Table 1). In contrast, PBIT or control monosubstituted derivatives PEIT or PEISe had no effect at these concentrations with average IC₅₀ values >100 μmol/L (Table 1). PEISe was moderately better with an IC₅₀ of ∼80 μmol/L for the WM115 cell line; however, because a similar trend was not observed for other cell lines, this difference was considered negligible. Thus, PBISe was >10-fold more effective than PBIT, PEIT, or PEISe at killing all three melanoma cell lines.

Next, the sensitivity of melanoma cells to PBISe was compared with normal cells. Normal human fibroblast, FF2441, and UACC 903 cells were treated with 2.5 to 100 μmol/L of PBISe and IC₅₀ measured at 4, 6, 12, and 24 h (Fig. 2B). Consistently, 2- to 5-fold higher drug concentrations were required to kill fibroblasts compared with melanoma cells (Fig. 2B). Thus, cultured cancer cells are 2- to 5-fold more sensitive to PBISe than normal cells.

To determine the mechanism by which PBISe inhibited cultured cancer cells, rates of cellular proliferation and apoptosis were examined in WM115, UACC 903, and 1205 Lu melanoma cells exposed to PBISe or PBIT. PBISe more effectively decreased cellular proliferation and increased apoptosis than PBIT in all three cell lines. Figure 2C and D are representative graphs illustrating changes in proliferation and apoptosis in UACC 903 cells exposed to PBISe or PBIT. Similar results were seen for WM115 and 1205 Lu cells (data not shown). Cell cycle analysis following PBISe but not PBIT exposure showed an increased sub–G₀-G₁ population (Fig. 3A), indicating elevated apoptosis and a ∼3-fold increase in G₂-M cells (Fig. 3B and C), indicating a cell cycle arrest mediated by PBISe but not PBIT. Thus, PBISe inhibited proliferation by arresting cultured cells in the G₂-M phase of the cell cycle and induced apoptosis.

Because deregulated PI3K (through overexpressed Akt3 and PTEN loss) and MAPK (through mutant ^V600EB-Raf) signaling promotes melanoma development (26, 27, 38), the effect of PBISe on these signaling cascades was examined by Western blot analysis of cellular lysates. Compared with PBIT, PBISe treatment inhibited Akt phosphorylation and decreased total Akt protein levels (Fig. 4A). Furthermore, phosphorylation of downstream Akt3 substrate PRAS40 was significantly inhibited with associated increases in cleaved caspase-3 and PARP indicating elevated apoptosis (Fig. 4A; ref. 29).

UACC 903, WM115, and 1205 Lu cells have elevated MAPK pathway signaling due to the presence of constitutively active ^V600EB-Raf (38, 39). Both PBIT and PBISe led to increased pErk1/2 in these cell lines, indicating increased MAPK pathway activity following treatment, which can be inhibitory to melanoma cells if the activity of this pathway is overly active (representative example, Fig. 4B; ref. 36). In agreement with previous reports, increased pErk1/2 following PBIT treatment elevates iNOS expression (Fig. 4B; refs. 33, 34). However, PBIT did not alter cyclin D1 levels (Fig. 4C). In contrast, PBISe dramatically reduced the expression of cyclin-D1 and increased p27 as well as p21 levels in three cell lines (representative example, Fig. 4C). It is possible that this difference is due to PBISe-mediated inhibition of Akt3 activity, which can increase MAPK activity in melanoma cells by decreasing the phosphorylation of ^V600EB-Raf, which causes excessively high MAPK pathway activity (36). Under these circumstances, PBISe inhibits Akt3 activity making it unable to phosphorylate ^V600EB-Raf at Ser³⁶⁴ and Ser⁴²⁸ to reduce the activity of ^V600EB-Raf and downstream MAPK pathway to levels that promote rather than inhibit proliferation (36). The result is elevated MAPK activity reaching inhibitory levels, which decrease cyclin-D1 expression and increase p27 as well as p21 compared to controls (36). Thus, PBISe-mediated inhibition of Akt3 pathway signaling, increased MAPK activity to levels previously reported to be inhibitory, thereby increasing cellular apoptosis and decreasing cellular proliferation.

The effectiveness of PBISe for inhibiting melanoma tumor development was evaluated on preexisting tumors in nude mice. 1205 Lu or UACC 903 human melanoma cells were injected s.c., and after 6 days, when a fully vascularized tumor had developed, mice were exposed to PBISe, PBIT (0.315 μmol/L each), or DMSO vehicle. Treatment with PBISe but not PBIT significantly reduced 1205 Lu (Fig. 5A) and UACC 903 (Fig. 5B) tumor development by ∼30% to 50%. Thus, PBISe has the potential to appreciably reduce melanomas compared with PBIT.

The systemic toxicity associated with PBISe in animals was evaluated in nude mice. Mice were exposed to PBISe or PBIT (0.315 μmol/L each) or vehicle DMSO, thrice a week, and animals were weighed to ascertain possible toxicity. No significant difference in body weight between PBISe-treated animals and controls was observed, suggesting negligible toxicity (see insets in Fig. 5A and B). Furthermore, blood parameters (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea, glucose, and creatinine) indicative of systemic toxicity did not detect significant liver, kidney, or cardiac-related toxicity (Fig. 5C). Histologic examination of H&E-stained vital organ sections revealed that PBISe treatment did not significantly change cell morphology or organ structure (data not shown). Thus, PBISe treatment has negligible associated systemic toxicity at the concentrations examined.

Because PBISe significantly reduced tumor development, subsequent studies focused on identifying the mechanism by which inhibition occurred. Rates of tumor cell apoptosis and proliferation were compared in size-matched and time-matched tumors following PBISe, PBIT, or control DMSO vehicle treatment. The magnitude of apoptosis, assessed by caspase-3/7 activities in tumor protein lysates, were examined (Fig. 5D). UACC 903 tumors treated with PBISe had double the caspase-3/7 activity by day 15 observed in tumors treated with PBIT or DMSO vehicle (Fig. 5D, P < 0.05; one-way ANOVA). In contrast, no statistically significant difference was observed in rates of cellular proliferation, which remained between 1% and 2% for PBISe-treated and PBIT-treated tumors (data not shown). However, PBISe-treated and PBIT-treated tumors had 2- to 3-fold fewer proliferating cells than animals exposed to control DMSO. Thus, PBISe inhibited melanoma tumor development by increasing apoptosis levels compared with PBIT but no significant differences in proliferation rates were observed between the two compounds.

Melanoma is one of the most difficult cancers to treat in its advanced stages (4, 26, 40). Cells are resistant to most untargeted chemotherapeutic agents, which are driving the search for agent combinations targeting proteins or signaling cascades deregulated during development (4, 5). For the targeted approach to be effective, deregulated proteins or signaling pathways need to be identified and potent drugs developed inhibiting these targets, which are effective in preclinical as well as clinical systems (26, 40).

PBISe is more effective than PBIT at killing melanoma cells because it inhibits proliferation and survival pathways that have been shown to be important for melanoma development. The PI3K and MAPK pathways play prominent roles in melanoma by relaying extracellular signals from cell surface to nucleus, thereby regulating diverse cellular processes including cellular proliferation and survival (41). Increased activity of the PI3K pathway occurs in ∼70% of sporadic melanomas due to loss of PTEN and/or increased expression of Akt3 resulting from an increase in the gene copy number (27, 28). The highest level of Akt3 activity occurs in metastatic melanomas, deregulating apoptotic signaling and promoting chemoresistance (27). In this study, PBISe is shown to inhibit Akt3 signaling leading to increased levels of melanoma cell apoptosis in cultured cells and melanoma tumors mediated by caspase-3/7 and PARP cleavage.

PBISe also regulates the activity of the MAPK pathway, which is activated through Ras mutations in 10% to 15% of melanomas and B-Raf mutations in ∼60% of melanomas (32, 42–44). Moderate levels of MAPK pathway activation promote cell cycle progression and cancer development whereas excessively high levels inhibit cellular growth in a wide variety of normal and cancer cells, including melanomas, by promoting cellular senescence (36, 45). Senescence is linked to the induction of a variety of cyclin dependent kinase inhibitors, such as p21^Cip1, p16^Ink4a, and p27^Kip1, which functions as a putative defense mechanism of normal cells to overcome oncogene activation (46–48). PBIT has been reported to increase MAPK activity in cells, which is confirmed to occur in this study following either PBIT or PBISe treatment (34, 36).

This study shows the preclinical evaluation of a novel selenium-containing small molecule inhibitor developed from the well-characterized iNOS inhibitor PBIT (13). PBIT is a non–amino acid competitive inhibitor of iNOS (6, 13) which, this study shows, is ineffective at killing melanoma cells. However, the selenium-containing analog, PBISe, has improved chemotherapeutic inhibitory activities making it >10-fold more effective at killing melanoma cells compared with PBIT. In addition to targeting iNOS similar to PBIT, PBISe also acts as a PI3K signaling cascade inhibitor in melanoma cells, making Akt3 unable to phosphorylate ^V600EB-Raf at Ser³⁶⁴ and Ser⁴²⁸. Akt3 has been shown in melanomas to directly regulate the activity of mutant ^V600EB-Raf by phosphorylating Ser³⁶⁴ and Ser⁴²⁸ to reduce the activity of ^V600EB-Raf and the downstream MAPK pathway (36). Because PBISe inhibits Akt3 activity, Akt3 is unable to phosphorylate ^V600EB-Raf, which elevates MAPK activity to levels that inhibit rather than promote melanoma development (36). Excessively elevated MAPK activity following PBISe treatment then causes a G₂-M cell cycle block mediated by decreased cyclin D1 and increased p21 as well as p27 levels (36). This novel property of PBISe causes decreased cell proliferation and increased apoptosis of melanoma cells. Furthermore, PBISe kills cultured cancer cells at concentrations that are 2- to 5-fold less than would be required for killing normal cells. Thus, PBISe has significant potential as a melanoma therapeutic.

By targeting the PI3K and MAPK pathways in melanomas, PBISe effectively reduces the growth of established tumors by 30% to 50% with negligible associated systemic toxicity. Caspase-3/7 activity in size-matched and time-matched tumors was increased 2-fold in PBISe-treated animals compared with controls. Similarly, studies targeting Akt3 in melanomas using either small interfering RNA or small molecule inhibitors showed that decreased Akt3 activity reduced tumor volume by increasing apoptosis (27, 29). PBISe-treated and PBIT-treated tumors also had 2- to 3-fold fewer proliferating cells than animals exposed to control DMSO. Because Akt3 is a key protein deregulated in melanoma, inhibitors such as PBISe that reduce its activity will be a key part of an effective chemotherapeutic cocktail of agents for successfully treating melanoma.

Several reports have documented the beneficiary effects associated with selenium for preventing cancer development (17, 49). Dietary consumption of selenium-enriched cruciferous vegetables such as broccoli, cabbage, and cauliflower has been shown to reduce the incidence of esophageal, colorectal, lung, and prostate cancers (17, 50). Synthetic and naturally occurring organoselenium compounds such as p-XSC, methyl seleninic acid, methyl selenol, and selenocysteine significantly decrease melanoma metastasis and lung cancer development (20, 21, 24). Although the exact mechanism by which selenium prevents cancer development remains uncertain, selenium is present as a constituent of proteins, modifies the activities of various enzymes, and regulates protein transport (49, 51). It also maintains the cellular redox balance thereby protecting cell membranes and lipid messengers within cells (50). Several studies have shown that serum from patients with advanced-stage melanoma contains significantly lower levels of selenium compared with serum from control patients; and that dietary supplementation can improve the quality and duration of life (18, 22). Thus, selenium plays an important role in preventing and treating cancer. Therefore, incorporating selenium into the structure of compounds could lead to the creation of more effective therapeutic agents as shown through the development of PBISe from PBIT.

In conclusion, selenium-containing PBISe has been shown to be a more effective therapeutic agent than PBIT. It kills melanoma cells >10-fold more effectively than PBIT and cultured cancer cells are 2- to 5-fold more sensitive than normal cells. Compared with PBIT, PBISe decreases the growth of established melanomas by 30% to 50% inducing a 2-fold increase in apoptosis and 2- to 3-fold decrease in tumor cell proliferation with negligible associated systemic toxicity. Thus, PBISe is a potent chemotherapeutic agent with novel properties enabling the targeting of iNOS, Akt3, and MAPK signaling, thereby promoting melanoma cell apoptosis while inhibiting proliferation. To achieve complete melanoma tumor remission, future studies could combine PBISe with agents that would induce cellular apoptosis because decreasing Akt3 activity mediated by this compound would sensitize the cells to killing via these agents.

No potential conflicts of interest were disclosed.

We thank Melissa Tran, Arun Sharma, Krishnegowda Gowdahalli, and Raghavendragowda Chandagaludoreswamy for technical assistance.