Objective:

2

To examine the mechanism of action of tanshinone ⅡA (Tan ⅡA) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP).

Methods:

2

The effects of different concentrations of Tan ⅡA (0–80 μmol/L) and DDP (0–2 μmol/L) on the proliferation of osteosarcoma cell lines (U2R

U2OS

143B

and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 μmol/L Tan ⅡA

0.5 μmol/L DDP alone

and a combination of 10 μmol/L Tan ⅡA and 0.25 μmol/L DDP using the transwell assay. After treating U2R and U2OS cells for 48 h with predetermined concentrations of each group of drugs

the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment

apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan ⅡA (15 mg/kg)

DDP (3 mg/kg)

Tan ⅡA (7.5 mg/kg) + DDP (1.5 mg/kg)

and normal saline

respectively. The body weight of the nude mice was weighed

and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot.

Results:

2

In vitro

studies have shown that Tan ⅡA

DDP and the combination of Tan ⅡA and DDP inhibit the proliferation

migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan ⅡA and DDP combined treatment group (

P

<

0.05 or

P

<

0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan ⅡA-DDP combination treatment (

P

<

0.05 or

P

<

0.01).

In vivo

studies demonstrated that the Tan ⅡA-DD combination treatment group significantly inhibited tumor growth compared to the Tan ⅡA and DDP single drug group (

P

<

0.01). Additionally

we found that the combination of Tan ⅡA and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38

cleaved caspase-3

and Bax and lower caspase-3

and Bcl-2 expressions with the combination of Tan ⅡA and DDP treatment compared to the single drug treatment (

P

<

0.01).

Conclusion:

2

Tan ⅡA synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions

and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions

thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.