Piperine improves the sensitivity of osteosarcoma cells to doxorubicin by inducing apoptosis and inhibiting the PI3K/AKT/GSK-3β pathway

Abstract

Background: Osteosarcoma is a primary bone malignancy associated with the highest incidence rate. Chemotherapy for osteosarcoma has not substantially changed, and survival of patients with metastatic tumours has reached a plateau. Doxorubicin (DOX) is a broad-spectrum anti-osteosarcoma drug; however, its application is limited due to its high cardiotoxicity. Piperine (PIP) has been verified to drive certain cancer cell death and increases chemosensitivity of DOX. However, the effects of PIP in promoting the chemosensitivity of osteosarcoma to DOX have not been studied.

Methods: We examined the combined effect of PIP and DOX on U2OS and 143B osteosarcoma cells. CCK-8 assays, scratch assays, flow cytometry analysis, and western blotting were performed. Furthermore, the effect of PIP combined with DOX on osteosarcoma tumours was observed in vivo using nude mice.

Results: PIP can increase the chemosensitivity of U2OS and 143B cells to DOX. Both in vitro and in vivo results showed the dramatic inhibition of cell proliferation and tumour growth by the combined therapy group compared to monotherapy groups. Apoptosis analysis revealed that PIP augments DOX-induced cell apoptosis by upregulating BAX and P53 expression, as well as reducing Bcl-2 expression. Furthermore, PIP also attenuated the initiation of the PI3K/AKT/GSK-3β signaling pathway in osteosarcoma cells by altering the expression levels of P-AKT, P-PI3K and P-GSK3β.

Conclusions: This study revealed for the first time that PIP can potentiate the sensitivity and cytotoxicity of DOX during osteosarcoma therapy in vitro and in vivo, which probably achieved by inhibiting the PI3K/AKT/GSK-3β signalling pathway.

Keywords: Doxorubicin; Osteosarcoma; PI3K/AKT/GSK3β; Piperine.

Fig. 1

Effect of combination of PIP and DOX on proliferative activity of osteosarcoma cells. A, B Cytotoxicity analysis of PIP and DOX against U2OS and 143B cell. Varying concentrations of PIP combined DOX were observed to be potent in downregulating the cell viability of U2OS and 143B for 24 h and 48 h via the CCK8 assay. C, D Varying concentrations of PIP combined DOX could effectively decrease the cell viability of U2OS and 143B for 24 h and 48 h via the CCK8 assay. E, F CDI of different PIP combined with DOX for 24 h and 48 h in U2OS and 143B cells. G The PIP + DOX group was more efficient in inhibiting cell viability compared to the DOX group for 24 h and 48 h. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Effect of PIP combined with DOX on scratch healing in osteosarcoma cells. Cells were scratched using a 100 μL tip and then treated with PBS, PIP, DOX, and PIP + DOX. Scratch healing was observed at 0, 12 and 24 h after scratching. A Wound healing area of U2OS and 143B cells in each treatment group. B Quantification of migration rates. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

PIP combined with DOX promote apoptosis in osteosarcoma cells. A U2OS and 143B cells were treated with PIP, DOX, and PIP + DOX for 48 h. Apoptosis was determined using the Annexin V-FITC Flow Cytometry. B Quantification of apoptotic cells. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Effect of PIP combined with DOX on the PI3K/AKT/GSK3β signalling pathway. U2OS and 143B cells were treated with PIP, DOX, and PIP + DOX for 48 h. The protein was extracted, and the target protein expression was observed using western blot. A Relative expression of Bax, Bcl-2, and P53 in different groups. B Relative expression of AKT, p-AKT, p-PI3K, p-GSK3β. *P < 0.05, **P < 0.01

Fig. 5

Anti-osteosarcoma effects of PIP, DOX, and PIP + DOX in vivo using mice models. Balb/c mice were randomly grouped and injected intraperitoneally with saline, PIP (30 mg/kg), DOX (3 mg/kg), and PIP + DOX (30 mg/ kg and 3 mg/kg, respectively) for 18 days. A Images of various groups of tumours. B Variations in body weight of mice in different groups per three days after administration. C Tumour volume measured every three days. D Tumour weight in each group after isolation. E AST and BUN in serum for each group. *P < 0.05, **P < 0.01, ***P < 0.001