Mechanisms of cell death induced by betulinic acid and dihydroartemisinin polymer-drug conjugates on pancreatic cancer cells

Abstract

Introduction: Pancreatic cancer (PC) is a lethal malignancy characterised by a poor response to standard chemotherapy. Like other cancers, PC employs apoptosis evasion mechanisms to ensure tumour survival. This makes apoptosis evasion an important cancer hallmark that can be targeted for effective treatment. Betulinic acid (BA) and Dihydroartemisinin (DHA) have shown promising anticancer properties via induction of apoptosis; however, limitations such as poor aqueous solubility and high molecular weights limit their overall therapeutic effect. Polymer drug conjugation is a promising solution. This project aimed to confirm the cytotoxic potential and determine the mechanism of cell death induced by BA and DHA polymer-drug conjugates on pancreatic cancer cells.

Methodology: The cytotoxic effect of the free drugs, BA and DHA, and the complimentary conjugates on Vero (non-cancerous cells) and MIA PaCa-2 pancreatic cancer cells was confirmed using tetrazolium salt assays. The antioxidant potential and effect of reactive oxygen species (ROS) activity were conducted using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and N, N-Dimethyl-p-phenylenediamine (DEPPD) assays. The mode of cell death and the effect of the drugs on the cycling characteristics of the cells were determined using flow cytometric assays. Their effect on the expression of proapoptotic genes was assessed using real-time polymerase chain reaction (PCR). The apoptosis evasion mechanism of the pancreatic cancer cells was determined using the human nuclear factor kappa-B p65 subunit (NF-κB/p65) enzyme-linked immunosorbent assay (ELISA) kit.

Results: The conjugates PEG-BA and PEG-DHA demonstrated lower IC50 values compared to the free compounds BA and DHA with a selectivity index of 3.00 and 1.61 for the conjugates, and 1.12 and 1.71 for BA and DHA, respectively. Antioxidant analysis showed that the free drugs, BA and DHA, had a weak antioxidant potential with IC50 values of more than 100 μM while the conjugates resulted in lower IC50. PEG-BA (4 μM) induced higher apoptosis than free BA (53.07% vs 7.68%) on the MIA PaCa-2 cells. Furthermore, PEG- DHA resulted in 50.99% of the MIA PaCa-2 cells undergoing apoptosis compared to free DHA (12.90%). Both conjugates induced low levels of necrosis (< 1%). After treatment with PEG-BA, the MIA PaCa-2 cells underwent a dose-dependent Sub-G1 arrest, which further indicated apoptosis. Similarly, PEG-DHA also induced a Sub-G1 arrest in addition to G2/M arrest on the MIA PaCa-2 cells. Compared to the free BA, PEG-BA treatment resulted in the highest overexpression of the following proapoptotic genes: TNF, BAX, CASPASE 3, CASPASE 2 and CASPASE 8. Both BA and PEG-BA induced a moderate increase in the concentration of NF-κB compared to the untreated MIA PaCa-2 cells.

Conclusion: This study confirmed that conjugation of the polymer PEG to natural compounds with anticancer properties, such as BA, further improves the apoptosis-inducing ability against pancreatic cancer cells with the overexpression of proapoptotic genes, necessitating further exploration