Anti-Cancer and Immunomodulatory Activity of a Polyethylene Glycol-Betulinic Acid Conjugate on Pancreatic Cancer Cells

Abstract

Drug delivery systems involving polymer therapeutics enhance drug potency by improved solubility and specificity and may assist in circumventing chemoresistance in pancreatic cancer (PC). We compared the effectiveness of the naturally occurring drug, betulinic acid (BA), alone and in a polymer conjugate construct of polyethylene glycol (PEG), (PEG–BA), on PC cells (MIA PaCa-2), a normal cell line (Vero) and on peripheral blood mononuclear cells (PBMCs). PEG–BA, was tested for its effect on cell death, immunomodulation and chemoresistance-linked signalling pathways. The conjugate was significantly more toxic to PC cells (p < 0.001, IC50 of 1.35 ± 0.11 µM) compared to BA (IC50 of 12.70 ± 0.34 µM), with a selectivity index (SI) of 7.28 compared to 1.4 in Vero cells. Cytotoxicity was confirmed by increased apoptotic cell death. PEG–BA inhibited the production of IL-6 by 4–5.5 fold compared to BA-treated cells. Furthermore, PEG–BA treatment of MIA PaCa-2 cells resulted in the dysregulation of crucial chemoresistance genes such as WNT3A, TXNRD1, SLC2A1 and GATA3. The dysregulation of chemoresistance-associated genes and the inhibition of cytokines such as IL-6 by the model polymer construct, PEG–BA, holds promise for further exploration in PC treatment.

Polyethyleneglycol-Betulinic Acid (PEG-BA) Polymer-Drug Conjugate Induces Apoptosis and Antioxidation in a Biological Model of Pancreatic Cancer

“…The cell cycle status of the MIA PaCa-2 cells before and after treatment was performed using propidium iodide dye on 4 µM of BA and PEG-BA treated cells. This is a concentration in the IC 50 range from this ( Figure S3 ) and a previous study [ 8 ]. The untreated MIA PaCa-2 cells showed a high percentage of cells in the G 1 /G 0 (45.27 ± 4.70%) compared with cells undergoing DNA synthesis (S: 17.67 ± 1.22%) and those preparing to enter mitotic division or already in the phases of mitosis (G 2 /M: 25.07 ± 2.44%) ( Figure 2 ).…”

“…The compounds were synthesised and characterised as previously described [ 8 , 39 ]. The free compound (BA), conjugate (PEG-BA) and standard (gemcitabine) were dissolved to 20 mg/mL in tissue culture grade dimethylsulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) and stored in single-use aliquots at −20 °C until required for experiments.…”

“…Cytotoxicity assays were performed using tetrazolium dyes as previously described [ 8 ]. The IC 50 value, the half-maximal inhibitory concentration representing the concentration at which the compounds will inhibit cell viability by 50% [ 42 ], was calculated using GraphPad Prism (v8.0.2.263).…”

“…Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid), the carboxylic acid derivative of betulin, is a naturally occurring, widely distributed pentacyclic triterpene sourced from the outer bark of the Betula alba tree [ 5 ]. Betulinic acid (BA) has shown promising anticancer efficacy against several tumours, including cervical cancer, melanomas, leukaemia, blastomas and PC [ 6 , 7 , 8 ]. However, like any other natural compound, BA is associated with several limitations, including a high molecular weight, which inhibits passage through the lipid bilayer of cells, and poor solubility, which reduces biodistribution, bioavailability and overall therapeutic index [ 9 ].…”

“…Drug conjugation is known to maximise efficacy, reduce adverse side effects, prolong blood circulation, passively deliver drugs to tumours via the enhanced permeability and retention (EPR) effect and overcome tumour resistance [ 11 , 12 ]. In a recent study from our laboratory, conjugation of polyethylene glycol (PEG) to BA showed selective toxicity to PC cells compared with BA alone, resulting in increased apoptotic cell death [ 8 ]. However, the specific mechanism of apoptotic cell death and possible evasion strategies employed by the cancer cells were not investigated.…”