br The cell lysates from CK treated SKBR cells

116 The cell lysates from CK-treated SKBR3 Z-Guggulsterone were prepared using lysis buffer (20 mM
121 according to the manufacturer’s instructions. Western blotting assays were conducted as
123 124 2.6. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
125 To determine the mRNA expression of Bcl2, total RNA was extracted from CK-treated
126 SKBR3 cells using TRIzol reagent (Gibco) according to the manufacturer’s instructions. The
127 cDNA synthesis and RT-PCR were performed as previously described [19], and the primer
131 The short hairpin RNA (shRNA) coding sequences against AKT1- or AKT2-containing
132 constructs were cloned following the Addgene protocol (www.addgene.org). Non-targeting
133 scrambled shRNA sequences and AKT1 or AKT2 shRNA sequences were cloned into the
134 pLKO.1 vector. Lentivirus was produced using transient HEK293T cell transfection. SKBR3
135 cells were infected with the virus, and the infected cells were selected through puromycin
136 treatment. The knockdown levels of AKT1 and AKT2 were confirmed using immunoblotting.
140 previously reported [20]. Briefly, Matrigel was diluted with serum-free DMEM (1:3) and
141 used to coat the upper surface of the transwell chamber. The lower chamber was filled with
142 culture medium containing 10% FBS, SKBR3 cells, and various concentration of CK and
143 was incubated at 37 °C for 48 h. The cells were fixed in 4% formaldehyde and then stained 144 with hematoxylin and eosin. Images of the cell invasion were produced using a microscope,
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145 and the invasion area was measured using Image J software.
150 colony formation. The media was discarded, and the cells washed once with PBS. The cells
151 were then fixed in fixation buffer (1:7 acetic acid:methanol) for 5 min. The fixation buffer
152 was removed via suction, and the cells were incubated with 0.5% crystal violet solution for 2
153 h. The stained cells were washed with tap water and dried. Images of cells were captured, and
154 the areas of colonies were measured using Image J software.
163 All data presented in this study are expressed as mean±SD of experiments performed with
164 three replicates. For statistical comparisons, the results were analyzed using either ANOVA
165 with the Scheffe post hoc test (for normally-distributed data) or the Kruskal–Wallis or Mann–
167 statistically significant.
172 To investigate the anti-cancer activities of compound K, we first evaluated the cell growth
173 of SKBR3 human breast cancer cells using cell viability assay. SKBR3 cells were treated
176 death, we further identified which mechanism of cell death was involved. To confirm the
177 apoptotic effects of CK, we analyzed cell death using PI and annexin V staining (Fig. 1B).
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179 concentration (0-50 mM). In contrast, the proportion of live cells decreased from 90.7% to 180 63.06%. Consequently, the CK-induced cell death was the result of apoptosis. To confirm the
181 apoptotic effects of CK, we further examined the morphological changes in CK-treated
182 SKBR3 cells. Actin cytoskeleton-mediated morphological changes, including apoptotic
183 bodies and nuclear condensation, were seen after treatment with CK for 24 h (Fig 1C). These
184 results suggest that Compound K suppresses the proliferation of cancer cells by increasing
188 pathways in SKBR3 cells
189 To determine the molecular mechanism underlying the pro-apoptotic activity of Compound
190 K, we identified the active forms of caspases using western blot analysis. The levels of
191 cleaved caspase-7, -8, and -9, which are the active forms of pro-caspases, were increased in
192 CK-treated SKBR3 cells, and the pro- forms were reduced in a dose-dependent manner (Fig.
193 2A). Moreover, the mRNA expression of Bcl2, an anti-apoptotic protein, was suppressed by
194 CK treatment (Fig. 2B). These results indicate that CK promotes apoptotic signaling cascades
195 via both intrinsic and extrinsic pathways. To examine which molecules involved in activation
196 of the apoptotic pathway are regulated by CK, we further analyzed the expression of AKT,
197 which is a primary regulator of apoptosis. Phosphorylation of the AKT isoform AKT1 was
198 significantly reduced in the presence of CK, but phosphorylation of AKT2 was not inhibited
199 by CK (Fig. 2C). The western blotting results suggest that AKT1 is a specific target of CK in