Roles of vimentin and 14-3-3 zeta/delta in the inhibitory effects of heparin on PC-3M cell proliferation and B16-F10-luc-G5 cells metastasis.
OBJECTIVE
To investigate the inhibitory effects of heparin on PC-3M cells proliferation in vitro and B16-F10-luc-G5 cells metastasis in Balb/c nude mice and identify the protein expression patterns to elucidate the action mechanism of heparin.


METHODS
Human prostate cancer PC-3M cells were incubated with heparin 0.5 to 125 μg/mL for 24 h. The proliferation of PC-3M cells was assessed by MTS assay. BrdU incoporation and Ki67 expression were detected using a high content screening (HCS) assay. The cell cycle and apoptosis of PC-3M cells were tested by flow cytometry. B16-F10-luc-G5 cardinoma cells were injected into the lateral tail vein of 6-week old male Balb/c nude mice and heparin 30 mg/kg was administered iv 30 min before and 24 h after injection. The metasis of B16-F10-luc-G5 cells was detected by bioluminescence assay. Activated partial thromboplastin time (APTT) and hemorheological parameters were measured on d 14 after injection of B16-F10-luc-G5 carcinoma cells in Balb/c mice. The global protein changes in PC-3M cells and frozen lung tissues from mice burdened with B16-F10-luc-G5 cells were determined by 2-dimensional gel electrophoresis and image analysis. The protein expression of vimentin and 14-3-3 zeta/delta was measured by Western blot. The mRNA transcription of vimentin, transforming growth factor (TGF)-β, E-cadherin, and α(v)-integrin was measured by RT-PCR.


RESULTS
Heparin 25 and 125 μg/mL significantly inhibited the proliferation, arrested the cells in G(1) phase, and suppressed BrdU incorporation and Ki67 expression in PC-3M cells compared with the model group. But it had no significant effect on apoptosis of PC-3M cells. Heparin 30 mg/kg markedly inhibits the metastasis of B16-F10-luc-G5 cells on day 8. Additionally, heparin administration maintained relatively normal red blood hematocrit but had no influence on APTT in nude mice burdened with B16-F10-luc-G5 cells. Thirty of down-regulated protein spots were identified after heparin treatment, many of which are related to tumor development, extracellular signaling, energy metabolism, and cellular proliferation. Vimentin and 14-3-3 zeta/delta were identified in common in PC-3M cells and the lungs of mice bearing B16-F10-luc-G5 carcinoma cells. Heparin 25 and 125 μg/mL decreased the protein expression of vimentin and 14-3-3 zeta/delta and the mRNA expression of α(v)-integrin. Heparin 125 μg/mL decreased vimentin and E-cadherin mRNA transcription while increased TGF-β mRNA transcription in the PC-3M cells, but the differences were not significant. Transfection of vimentin-targeted siRNA for 48 h significantly decreased the BrdU incoporation and Ki67 expression in PC-3M cells.


CONCLUSIONS
Heparin inhibited PC-3M cell proliferation in vitro and B16-F10-luc-G5 cells metastasis in nude mice by inhibition of vimentin, 14-3-3 zeta/delta, and α(v)-integrin expression.
State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Peking University, Beijing, China.
Yan
Y
Pan
Li-jun
LJ
Zhong
Hong
H
Zhou
Xin
X
Wang
Kui
K
Chen
Hao-peng
HP
Yang
Yilixiati
Y
Xiaokaiti
Aikebaier
A
Maimaiti
Ling
L
Jiang
Xue-jun
XJ
Li
D048948:14-3-3 Proteins / Q000235:genetics*; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D015820:Cadherins / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006493:Heparin / Q000494:pharmacology / Q000627:therapeutic use*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009362:Neoplasm Metastasis / Q000188:drug therapy / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics; D011467:Prostate / Q000187:drug effects / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics; D014746:Vimentin / Q000235:genetics*
D048948:14-3-3 Proteins; D000925:Anticoagulants; D000970:Antineoplastic Agents; D015820:Cadherins; D016212:Transforming Growth Factor beta; D014746:Vimentin; D006493:Heparin