Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo.
BACKGROUND
Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis.


OBJECTIVE
We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo.


METHODS
We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo.


RESULTS
Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo.


CONCLUSIONS
Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma.
Katagiri
Yoshiyuki
Y
Department of Dermatology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, 990-9585 Yamagata, Japan. ykatagir@med.id.yamagata-u.ac.jp
Hozumi
Yutaka
Y
Kondo
Shigeo
S
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000187:drug effects* / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007976:Leupeptins; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D008546:Melanoma, Experimental; D051379:Mice; D008819:Mice, Nude; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology / Q000627:therapeutic use*; D044786:S-Phase Kinase-Associated Proteins / Q000187:drug effects* / Q000378:metabolism
D007976:Leupeptins; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde