Nanoparticle-Based Celecoxib and Plumbagin for the Synergistic Treatment of Melanoma

Abstract

Using multiple drugs to kill cancer cells can decrease drug resistance development. However, this approach is frequently limited by the bioavailability and toxicity of the combined agents and delivery at ratios to specific locations that synergistically kill cancer cells. Loading the individual agents into a nanoparticle that releases the drugs at synergizing ratios at a single location is one approach to resolve this concern. Celecoxib and plumbagin are two drugs that were identified from a screen to synergistically kill melanoma cells compared with normal cells. Combined use of these agents by traditional approaches was not possible due to poor bioavailability and toxicologic concerns. This study details the development of a nanoliposomal-based agent containing celecoxib and plumbagin, called CelePlum-777, which is stable and releases these drugs at an optimal ratio for maximal synergistic killing efficacy. CelePlum-777 was more effective at killing melanoma than normal cells and inhibited xenograft melanoma tumor growth by up to 72% without apparent toxicity. Mechanistically, the drug combination in CelePlum-777 led to enhanced inhibition of melanoma cell proliferation mediated by decreasing levels of key cyclins important for cancer cell proliferation and survival, which was not observed with the individual agents. Thus, a novel nanoparticle-based drug has been developed containing celecoxib and plumbagin that lacks toxicity and delivers the agents at a synergistically killing drug ratio to kill cancer cells. Mol Cancer Ther; 16(3); 440-52. ©2016 AACR.

Figure 1. Development of synergistically acting CelePlum-777

(1A). Celecoxib and Plumbagin inhibit the viability of UACC 903 cells in a cooperative manner as measured by MTS assay. Data represent averages of at least 3 independent experiments; bars, S.E.M. (1B). Calcusyn analysis suggest synergistic killing by Celecoxib and Plumbagin and CI values range from 0.74 to 0.83. (1C). Structure of CelePlum-777 showing predicted location of Celecoxib and Plumbagin within the liposome. (1D). Size and charge of CelePlum-777 dissolved in water or saline. Size and zeta potential of empty liposomes, Celecoxib or Plumbagin liposomes alone, or CelePlum-777. Data represent averages of at least 3 independent experiments; bars, S.E.M.

Figure 2. Characterization of drug encapsulation, stability, and release kinetics of CelePlum-777

(2A). Drug encapsulation efficiency. Nanoliposomes containing Celecoxib alone (5.2 mM), Plumbagin alone (0.26 mM), or the combination (5.2 mM Celecoxib and 0.26 mM Plumbagin), Plumbagin encapsulation alone was 75.1%, Celecoxib encapsulation alone was 91.3% and the encapsulation of both agents was 67.6% and 89.3%, respectively. Data represent averages of at least 3 independent experiments; bars, S.E.M. (2B). Drug release kinetics of CelePlum-777. 71% of Celecoxib and 69% of Plumbagin was released from CelePlum-777 over 96 hours. (2C). Stability of CelePlum-777. CelePlum-777 was stored at 4°C and stability measured over weekly assessing size, charge, and cancer cell killing efficacy indicating stability for up to 5-weeks. Data represent averages of at least 2 independent experiments.

Figure 3. Efficacy of CelePlum-777 compared to nanoliposomes containing individual drugs

Normal (3A) or melanoma cell lines (3B) were treated with empty liposome, Celecoxib liposome (100 μmol/L), Plumbagin liposome (5 μmol/L), or CelePlum-777 (100 μmol/L Celecoxib + 5 μmol/L Plumbagin) for 24, 48, and 72 hours and cell survival assessed by MTS assay. Data represent averages of at least 3 independent experiments; bars, S.E.M.

Figure 4. CelePlum-777 treatment synergistically inhibited melanoma tumor growth

Vascularized xenografts of UACC 903 (A and B) or 1205 Lu (C, D, E, and F) melanoma cells were treated intravenously on alternate days with liposomes containing single agents (Celecoxib 15 mg/Kg or Plumbagin 0.75 or 1.5 mg/Kg body weight), or CelePlum-777 (Celecoxib 15 mg/Kg + Plumbagin 0.75 or 1.5 mg/Kg body weight) for 3 to 4 weeks. The line graph indicates tumor volume (mm³) and inset body weight. Data represent experiments of 3 mice per group, containing two tumors per mouse; bars, S.E.M.

Figure 5. CelePlum-777 treatment consistently led to enhanced inhibition of tumor and cultured cell proliferation

Size and time matched xenografted tumors were removed from mice on days 13 and 15, following treatment from day 6 with liposomes containing Celecoxib at 15 mg/Kg body weight, Plumbagin at 1.5 mg/Kg body weight, or CelePlum-777 containing Celecoxib 15 mg/Kg + Plumbagin 1.5 mg/Kg body weight. Tumor sections were immunostained for Ki-67 (5A) or CD31 (5B) to assess proliferation and vascular development, respectively. Images were quantified and plotted as fold difference in cells expressing Ki-67 or area occupied by blood vessel compared with controls. Data was obtained from three to four tumors, with four to five fields averaged per tumor. Data represent averages of at least 3 independent experiments; bars, S.E.M. Rates of cell proliferation in cultured cells (5C) following CelePlum-777 treatment for 72 hours reduced melanoma cell proliferation compared to liposomes containing Celecoxib or Plumbagin alone.

Figure 6. CelePlum-777 decreased STAT3 and COX-2 activity leading to an enhanced decrease in key cyclin levels

A, B, and C. UACC 903 or 1205 Lu melanoma cells were treated with empty liposome, Celecoxib liposome (100 μmol/L), Plumbagin liposome (5 μmol/L), or CelePlum-777 (100 μmol/L Celecoxib + 5 μmol/L Plumbagin) for 6 and 24 hours. Western blotting measure changes in protein expression of (6A) COX-2, (6B) STAT3 (6C) Cyclins and (6D) Apoptosis signaling pathways. Quantified pSTAT3 levels were normalized over STAT3 levels using ImageJ software.