SKP2 siRNA inhibits the degradation of P27<sup>kip1</sup> and down-regulates the expression of MRP in HL-60/A cells

Abstract

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitinmediated degradation of P27 kip1 . SKP2 and P27 kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27 kip1 , and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27 kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27 kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G 0 /G 1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

siRNA KNOCKDOWN SKP2 GENE IN BREAST CANCER

“…This observation is in agreement with the reports suggesting that the inhibition of SKP2induced apoptosis is characterized by an elevated level of p27, and a decrease in the G1 phase [13,16,29]. The findings of a high level of p27 and low G1 cell population resulting from inhibited SKP2 are also reliable with the function of p27 as a key regulator of G1 progression [30,31]. Thus, SKP2-siRNA could inhibit tumor growth by controlling the level of SKP2, which could in turn activate the p27 pathway that leads to apoptosis, in the present study, we found that SKP2 reduction induced increased levels of p27, followed by depletion of expression of cyclin E and CDK2 in breast cancers [6].…”

p53, SKP2, and DKK3 as MYCN Target Genes and Their Potential Therapeutic Significance

“…Overexpression of SKP2 can drive quiescent cells to enter the cell cycle (Sutterluty et al, 1999), and promote adhesion-independent growth of cancer cells (Carrano and Pagano, 2001; Signoretti et al, 2002). Downregulation or inhibition of SKP2 expression leads to growth arrest and/or apoptosis, as well as reduced cell migration, invasion, and metastasis (Koga et al, 2003; Yokoi et al, 2003; Jiang et al, 2005; Lee and McCormick, 2005; Shibahara et al, 2005; Katagiri et al, 2006; Kitagawa et al, 2008; Xiao et al, 2009; Chan et al, 2010a; Bretones et al, 2011). In transgenic mouse models, SKP2 cooperates with N-Ras to drive lymphomagenesis (Latres et al, 2001), and tissue targeted expression of SKP2 results in hyperplasia, dysplasia, and low-grade carcinoma of the prostate gland (Shim et al, 2003).…”

Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma

“…Thus, Skp2 is a tumor-promoting factor. A number of studies have revealed that the overexpression of Skp2 is associated with the progression of a variety of types of human cancer (22)(23)(24)(25). Functional deletion of Skp2 leads to stabilization of CDK inhibitors, which can subsequently induce cell-cycle delay or arrest (26,27 The aim of the study was to explore the role of Skp2 in colon carcinoma and to identify whether depletion of Skp2 by Skp2 RNA interference attenuates the proliferation and migration of colon carcinoma.…”