Cytotoxic effects of walnut hull extracts against human osteosarcoma cell line (U2OS)

Abstract

This study aimed to evaluate the total phenolic, total flavonoid content and the antioxidant activity of walnut (Juglans regia L.) hull (JRH) extracts and investigate the cytotoxic effect of different walnut hull extracts on osteosarcoma U2OS cell line and study their impact on apoptosis, autophagy and heat shock protein 90. For this purpose, the dried walnut hulls were extracted by soxhlet apparatus using methanol, ethanol, ethyl acetate and hexane as extraction solvents. The total phenolic content, total flavonoid content and antioxidant activity of each extract were determined by three colorimetric methods: Folin-Ciocalteu method, AlCl3 method and DPPH radical scavenging assay, respectively. In order to investigate the osteosarcoma U2OS cells viability in response to treatment with each JRH extracts, MTT assays were performed. qRT-PCR was used to investigate the cytotoxic effect of the extracts. Caspase-3 and Bcl-2 were checked to determine their role in apoptosis, Beclin 1 for autophagy and HSP90 for viability/proliferation. Overall, the highest extraction yield (41.9 %.), total phenolic content (85.16 ± 5.43 mg GAE/g), total flavonoid content (105.49 ± 6.44 mg RE/g), the antioxidant activity (IC50 = 15.484 μg/mL) and cytotoxic effect on osteosarcoma cell line (IC50 = 180.20 ± 27 μg/mL) were determined in JRH methanol, ethanol, ethyl acetate, ethanol, methanol extracts respectively. The assessments of the present investigation demonstrated that all JRH extracts reduced the viability of osteosarcoma U2OS cells in a dose dependent manner and methanol extract showed the highest anticancer activity by upregulating the gene expression of Caspase-3 and downregulating the gene expressions of Beclin-1, Bcl-2 and HSP-90.