(143f) Targeted and Controlled Combination Therapy Using siRNA and Resveratrol for Inducing Leukemic Cell Apoptosis

Abstract:

Introduction: Approximately every 3 minutes, a person is diagnosed with blood cancer accounts for almost 10.2% of new cancer cases in the US. Almost 171,550 people are diagnosed with leukemia, lymphoma, and myeloma in 2016. Many approaches have been explored including small interfering RNA (siRNA) and small chemicals. siRNA has been proposed with 84% protein suppression level, however, cell death levels have not been shown. On the other hand, Resveratrol, a polyphenol found in wine and grapes, induces ~60% apoptosis via inhibition of the sphingosine kinase 1 enzyme (SPHK1). Generally, leukemia treatment with small molecules, suggests that despite inhibiting certain pathways with high percentage, cells can still survive by alternative pathways, hence low cell apoptosis level are obtained. In addition, small molecule cancer therapeutics are also hindered by issues with pharmacokinetics. For example, resveratrol has a very short half live (3-5 hours) and its bioavailability is very low. In this study, we questioned whether a combination of siRNA with controlled release of resveratrol can induce higher cell death level. First, we explored the effect of combination of resveratrol and siRNA on K562 cell line derived from chronic myelogenous leukemia (CML). Using cell concentration of 2 x 10⁵cells/ml of solution, and 40 µM Resveratrol, we explored forming coaxial hybrid fibers from hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic gelatin (GT) in two different configurations, and the release of resveratrol at 37°C over five days.

Selected siRNA was a custom-synthesized BCR-ABL siRNA (5′-GCAGAGUUCAAAAGCCCTT-3′). First, 2 x 10⁵K562 cells/mL of solution were exposed to a range of resveratrol concentrations (10, 20, 40, 80, and 160 µM) and a range of siRNA concentrations (12, 24, 36, 48, and 60 nM). Both conditions were analyzed for cell viability after incubation for 72 hours using Annexin V, PI staining and flow cytometry. Apoptotic cells level of 48% was achieved with resveratrol concentration of 40 µM. Apoptotic cell level of 47% was achieved with 36 nM siRNA concentration. A combination of both drugs was then investigated following the same methodology. From these studies, 40 µM resveratrol concentration and 36 nM siRNA concentration was selected in combination therapy. Also, similar concentration was used in forming the electrospun fibers. In addition, a factorial design of experiments was used to develop a surface plot using a mid-point concentration of 20 µM resveratrol and 18 nM siRNA. Results showed that addition of both molecules had an additive effect on apoptosis; nearly 72(±2.9)% cells and 25(±1.8)% (mid-point) were apoptotic under these conditions. Next, PCL and GT were dissolved separately in trifluoroethanol (TFE) and mixed together in 1:1 ratio for the hybrid layers. Fabricated fibers at the same settings and same solvent were characterized for their structural morphology, various components, viability and apoptosis of K562 cell over 72 hr. Resveratrol release from the fibers were also characterized by incubating samples in a buffer, similar to our previously publication [1]. Amount of resveratrol released at various times was assessed using HPLC. These results show a promising potential of the approach.

[1] Khalf A, Madihally SV. Modeling the Permeability of Multiaxial Electrospun poly(ε-caprolactone)- Gelatin Hybrid Fibers for Controlled Doxycycline Release. Materials Science & Engineering C: Materials for Biological Applications. 76:161-170, 2017.