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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (2): 234-238. doi: 10.19723/j.issn.1671-167X.2019.02.006

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Rasfonin inhibits proliferation and migration of osteosarcoma 143B cells

Fan ZHANG1,2,Tai-qiang YAN1,∆(),Wei GUO1   

  1. 1. Musculoskeletal Tumor Center, Peking University People’s Hospital, Beijing 100044, China;
    2. Department of Bone and Soft Tissue, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450008, China
  • Received:2017-04-12 Online:2019-04-18 Published:2019-04-26
  • Contact: Tai-qiang YAN E-mail:yantqzh@163.com
  • Supported by:
    the National Natural Science Foundation of China(81272381)

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Abstract:

Objective: To investigate the effects of rasfonin,a fungal secondary metabolite, on the proliferation and migration of osteosarcoma 143B cells.Methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to exa-mine 143B cell viability following treatment of rasfonin. Using dimethyl sulfoxide (DMSO) group as control, cell viability was detected when 143B cells were treated with rasfonin (3 μmol/L and 6 μmol/L) for 12 or 24 hours. The effect of rasfonin on colony forming ability was detected by clone formation assay.143B cells treated with DMSO or rasfonin (3 μmol/L) for one week, and the number of clones formed in the two groups was counted. Wound healing and transwell assay were employed to analyze cell invasion and migration upon rasfonin challenge. The DMSO group was used as control while rasfonin (3 μmol/L) was used for 24 hours. The wound healing rate and the number of invasive cells were compared between the two groups. The intracellular autophagosomes were monitored by transmission electron microscopy when 143B cells were treated with DMSO or rasfonin (3 μmol/L) for 4 hours. The expression of p62, microtubule-associated protein 1 light chain 3 fusion protein (LC3) and poly (ADP-ribose) polymerase-1(PARP-1) in response to rasfonin were detected by immunoblotting assay.Results: Rasfonin reduced the viability of 143B cells in a dose-dependent manner (12 h: F=31.36, P<0.01; 24 h: F=67.07, P<0.01). Rasfonin (3 μmol/L) completely inhibited the clonal formation of 143B cells (P<0.01). The wound healing result revealed that rasfonin significantly decreased migratory ability of 143B cells (33.91%±0.83% vs. 65.11%±0.94%, P<0.01), whereas its treatment significantly reduced the number of 143B cells penetrating through Matrigel-containing basement membrane (21.33±1.45 vs. 49.33±2.40, P<0.01). Compared with the control group, rasfonin markedly increased the number of autophagic vacuoles. The immunoblotting results revealed that rasfonin increased LC3-Ⅱ accumulation and decreased p62 levels. Choloroquine (CQ), an often used autophagic inhibitor, further accumulated rasfonin-induced LC3-Ⅱ. In addition, rasfonin appeared to cause the cleavage of PARP-1.Conclusion: Rasfonin induced autophagy and activated caspase-dependent apoptosis in 143B cells concurring with suppressing the proliferation and migration of the cells; these results provide an experimental basis for rasfonin as a potential therapeutic agent for osteosarcoma.

Key words: Osteosarcoma, Proliferation, Migration, Autophagy

CLC Number: 

  • R738.1

Figure 1

Rasfonin inhibits cell viability of 143B cells A,143B cells were treated with rasfonin (3 μmol/L and 6 μmol/L) for up to 24 h, cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)assay;B,colony growth assays were performed in 143B cells with rasfonin (3 μmol/L).*P<0.01, vs.DMSO group.DMSO, dimethyl sulfoxide."

Figure 2

Rasfonin inhibits migratory and invasive abilities of 143B cells A and B, wound healing assay and transwell assay were performed in143B cells in the present of DMSO or rasfonin (3 μmol/L); B, stained with crystal violet, ×100.DMSO, dimethyl sulfoxide."

Figure 3

Rasfonin increases autophagy and apoptosis of 143B cells A,electron microscopy was performed in 143B cells after exposed to rasfonin (3 μmol/L) for 4 h,×9 900; B and C, following treatment of rasfonin (B: 3 μmol/L and 6 μmol/L, C:3 μmol/L) for 4 h with or without Choloroquine (CQ), 143B cells were lysed and analyzed by immunoblotting using the indicated antibodies.Actin was used as a loading control; D, expression of poly (ADP-ribose) polymerase-1(PARP-1) was analyzed by immunoblotting after treated by rasfoninfor 4 h. Actin was used as a loading control.DMSO, dimethyl sulfoxide; CQ,choloroquine;LC3, microtubule associated protein 1 light chain 3 fusion protein; PARP, poly (ADP-ribose) polymerase."

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