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  • br human breast cancer cells To confirm this


    200 human breast cancer cells. To confirm this, the AKT1 and AKT2 genes were overexpressed in
    201 SKBR3 Cucurbitacin I treated with CK. As expected, CK clearly suppressed the phosphorylation of
    202 AKT1 in SKBR3 cells transfected with AKT1 (Fig. 2D, left panel), but phosphorylation of
    203 AKT2 was not affected by overexpression (Fig. 2D, right panel). These findings indicate that
    204 Compound K increases apoptosis of cancer cells via downregulation of AKT1.
    207 To confirm the regulation of AKT1 and AKT2 by Compound K at the cellular level, cellular
    208 proliferation of AKT1- and AKT2-knockdown SKBR3 cells was examined using MTT assays.
    209 Cell viability was decreased only in shAKT2 cells treated with CK (25 mM) (Fig. 3A). These 210 results confirm that CK is involved in cancer cell death through regulation of AKT1. Because
    211 cancer metastasis is often fatal, and cancer cell migration is one of the important markers of
    212 cancer cell invasion and metastasis [21], we analyzed the suppressive effects of CK on
    213 metastasis using invasion and migration assays. In the invasion assay, CK dose-dependently
    214 reduced the invasion area of SKBR3 cells after treatment for 48 h (Fig. 3B). For the cell
    215 migration assay, SKBR3 cells were scratched and then treated with varying concentrations of
    216 CK, and the cell migration ability was observed at several time points over 24 h [22]. The
    217 cell-migration rate was inhibited with 50 mM CK (Fig. 3C). Taken together, the invasion and 218 migration assays reveal that CK reduces cancer cell invasion and metastasis. Clonogenic (or
    219 colony-formation) assays are typically used to validate cytotoxic agents or anti-cancer
    220 therapeutics [23,24]. The number and area of SKBR3 colonies were greatly decreased by CK
    225 226 Ginsenosides, the major bioactive components of ginseng, have been extensively researched, 227 in particular for their anticancer activity in angiogenesis and metastasis [25,26,27]. In
    228 addition, Compound K, which was recently discovered as an active ingredient of
    229 ginsenosides, has been studied for its pharmacological activities such as antiproliferative and
    231 antitumor effects through induction of apoptosis in leukemia and prostate cancer cells
    233 and the molecular mechanisms of this compound have not been fully elucidated. SKBR3 is a
    234 hormone-independent human breast cancer cell line. Since SKBR3 overexpresses the HER-2
    235 (human epidermal growth factor receptor-2) gene (Neu / ErbB-2), an important regulator in
    237 development of breast cancer therapies targeting Her2 [33]. Thus, we aimed to investigate the
    238 antitumor activities of CK in human breast cancer SKBR3 cells and its molecular mechanism.
    239 Apoptosis is an important biological response that prevents excessive cell growth, but
    240 defects in apoptosis cause various diseases including cancer [34]. For this reason, cancer
    241 therapy strategies inducing Cucurbitacin I apoptosis have been widely used [35]. In the present study, we
    242 first investigated whether CK induces apoptosis in breast cancer cells. Cell viability assays
    243 confirmed that the growth of SKBR3 cells treated with CK was inhibited in a dose- and time-
    244 dependent manner (Fig. 1A). In annexin V/PI fluorescent staining, early apoptotic cells are
    245 only positive for annexin V, and cells in which apoptosis has progressed are annexin V- and
    246 PI-positive [36]. Using these characteristics, the cell death of SKBR3 cells was measured
    247 using flow cytometry analysis. Treatment with CK dose-dependently increased the number of
    248 both early apoptotic and late apoptotic SKBR3 cells (Fig. 1B). When apoptosis occurs,
    249 apoptotic bodies appear along with morphological changes such as nuclear shrinkage and
    250 chromatin condensation[37]. The number of apoptotic bodies was increased in 50 mM CK- 251 treated SKBR3 cells (Fig. 1C). These results suggest that CK leads to cell death through
    253 We further identified the molecular mechanism of the apoptotic activity of CK. Apoptosis
    254 has two major signaling pathways, the extrinsic pathway and the intrinsic pathway [38], and
    256 representative caspases involved in the extrinsic and intrinsic pathways, respectively [39].
    257 Both cleaved caspase-8 and cleaved caspase-9, which are the active forms of procaspases,
    258 were increased in CK-treated SKBR3 cells. Caspase-7, a downstream executive enzyme, was
    260 expression of Bcl2, which plays a role as an apoptosis inhibitor, was suppressed by CK (Fig.
    261 2B). Taken together, these results indicate that CK induces the caspase-dependent apoptotic