Polymer conjugate of a microtubule destabilizer inhibits lung metastatic melanoma

Abstract

Melanoma is the most aggressive type of skin cancer. It is highly metastatic, migrating through lymph nodes to distant sites of the body, especially to lungs, liver and brain. Systemic chemotherapy remains the mainstay of treatment; however, the development of multidrug resistance (MDR) restricts the efficacy of current chemotherapeutic drugs. We synthesized a series of microtubule destabilizers, substituted methoxybenzoyl-ary-thiazole (SMART) compounds, which inhibited tubulin polymerization and effectively circumvented MDR. Due to poor water solubility of SMART compounds, co-solvent delivery is required for their systemic administration, which is usually associated with hepatotoxicity, nephrotoxicity and hemolysis. To solve this problem and also to increase circulation time, we synthesized a new SMART analogue, SMART-OH, and its polymer-drug conjugate, methoxy-poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate-graft-SMART-graft-dodecanol) (abbreviated as P-SMART), with 14.3±2.8% drug payload of SMART-OH. Similar to its parent drug, P-SMART showed significant anticancer activity against melanoma cells in cytotoxicity, colony formation, and cell invasion studies. In addition, P-SMART treatment led to cell cycle arrest at G2/M phase and cell accumulation in sub-G1 phase. We established a model of metastatic melanoma to the lung in C57/BL6 albino mice to determine in vivo efficacy of P-SMART and SMART-OH at the dose of 20mg/kg. P-SMART treatment resulted in significant inhibition of tumor growth and prolonged mouse median survival. In conclusion, P-SMART, a novel polymer-microtubule destabilizer conjugate, has the potential to treat metastatic melanoma.

Keywords: Metastatic melanoma; Microtubule destabilizer; Nanomedicine; Polymer drug conjugate.

Figure 1. Synthesis and characterization of SMART-OH and its inhibition of tubulin

A) Synthetic scheme and ¹H NMR spectrum of SMART-OH. B) Molecular docking. Docking image showed that SMART-OH bind to the colchicine binding site in tubulin. C) Tubulin polymerization assay. Tubulin (3.33 mg/ml) was exposed to 10 μM of SMART-OH, colchicine or vehicle control (5% DMSO), respectively, and incubated in general tubulin buffer. Absorbance at 340 nm was monitored at 37°C every minute for 15 min. Both SMART-OH and colchicine effectively inhibited polymerization, while robust polymerization was observed in the control group.

Figure 2. Synthesis and characterization of mPEG-b-PCC-g-SMART-g-DC (P-SMART)

A) Synthetic scheme of P-SMART. B) ¹H NMR (500 MHz, DMSO-d₆) spectrum of P-SMART. C) Particle size distribution of P-SMART by dynamic light scattering (DLS). The mean particle size of P-SMART was 71.51±0.47 nm. D) In vitro drug release profile of P-SMART. Concentration of released SMART-OH at each time point was measured by HPLC and drug release profiles were represented as the mean ± SEM (n=3). P-SMART showed a slow but sustained release of SMART-OH at neutral and acidic condition.

Figure 3. Cytotoxicity and colony formation assay

A) Cytotoxicity of SMART-OH and P-SMART was determined in A375 and B16-F10 cells for 72 h. The IC₅₀ of P-SMART was 0.75 μM in two cell lines. B) To determine colony formation of melanoma cells, 250 cells per well were seeded to 6-well culture plates. At 24 h, drug formulations were added and at 7 days, cell colonies were fixed, stained and counted. The long-term treatment of P-SMART resulted in significant anti-proliferative effect. Data represented as the mean ± SEM (n=3). *p<0.05, **p<0.01 compared to Control.

Figure 4. Cell invasion assay

B16-F10 cells were treated with either SMART-OH (0.5 μM) or P-SMART (0.5 μM, equivalent to parent drug) and allowed to invade through Matrigel for 24 h. Results were shown as mean number of invaded cells ± SEM (n=3). Treatment of SMART-OH suppressed 87% of cell invasion and P-SMART blocked 73% of cell invasion. *p<0.05, **p<0.01 compared to Control.

Figure 5. Cell cycle analysis and apoptosis of P-SMART

Cells were treated with P-SMART for 48 h, stained with propidium iodide (PI), and analyzed on a flow cytometer. A) A375. B) B16F10. Results were expressed as the mean ± SEM (n=3). The percent of cells in G2/M phase and sub-G1 phase was augmented in a dose dependent manner after treatment with P-SMART.

Figure 6. In vivo representative bioluminescent images at first day and last day of treatments

Mice (n=5) from Control (saline), SMART-OH and P-SMART groups were taken bioluminescent images every alternate day during the treatment. Images of four mice from each group were shown.

Figure 7. In vivo efficacy of SMART-OH and P-SMART in B16-F10 lung metastatic animal model

Mice received saline, 20 mg/kg SMART-OH or 20 mg/kg P-SMART intravenously once every three days for a total of five times when the radiance of tumor had reached 10⁵ (day 10 after tumor implantation). A) Radiance intensity plot of all groups was measured from day 10 to day 24. Data represented as the mean ± SEM (n=5). B) Representative tumors of each group were excised after sacrificing the mice at the end of the efficacy study. Significantly higher tumor growth inhibition and less number of lung tumor nodules was observed in the group treated with P-SMART compared to SMART-OH treated group. C) The weight of mouse lungs from each group was measured at the end of the study. D) Survival analysis of control group, SMART-OH group and P-SMART group. The median survival was 31 days in SMART-OH group and 38 days in P-SMART group while in control group median survival was 28 days.

Figure 8. Analysis of lung samples by hematoxylin and eosin (H&E), Caspase 3 stain and analysis of major organs by H&E stain

Lung samples from control, SMART-OH and P-SMART treated groups were excised, fixed and immunostained for A) H&E and B) Caspase 3. In control group the metastasis was throughout the lung lobe but in the treated groups the metastasis and proliferation of tumor cells was inhibited. Cleaved caspase-3 stain indicated significant the induction of apoptosis by P-SMART. C) Organ samples from three groups were excised, fixed and stained for H&E. No obvious histological changes were observed in the livers, spleens, kidneys and hearts from all the treated groups.