Iris Nertschinskia Ethanol Extract Differentially Induces Cytotoxicity in Human Breast Cancer Cells Depending on AKT 1 / 2 Activity

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.


Introduction
Iris species have activities that are beneficial for the treatment of diverse human diseases, such as cancer, inflammation, and viral infections (Han, 1988;Steinegger et al., 1988).Several Iris species, including Iris tectorum, Iris germanica and Iris missouriensis, have attracted the attention of researchers in the oncology field (Wong et al., 1986;Tan et al., 2001;Wollenweber et al., 2003;Rui et al., 2008).However, there are no studies on the anticancer activities of Iris nertschinskia, traditionally used in East Asian medicine for the treatment of inflammation-related diseases.
The anticancer effects of plant extracts are typically assessed by evaluating their cytotoxic effects on panels of human tumor-derived cell lines.This approach has also revealed relationships between cancer-specific signaling molecules and sensitivity of cancer cells to plant extracts.Protein kinases in cancer cells are key molecules that control intracellular signal transduction pathways regulating cell proliferation, survival, and differentiation (Sachsenmaier, 2001).In particular, AKT, a serine/ threonine protein kinase, plays a crucial role in cancer cell survival (Hers et al., 2001).AKT is a major downstream target of growth factor receptor tyrosine kinases that signal through phosphatidylinositol 3-kinase (PI3K) (Soung et al., 2006).To date, three mammalian isoforms of AKT have been identified: AKT1/PKB-α, AKT2/ PKB-β, and AKT3/PKB-γ (Datta et al., 1999).Activated AKT promotes survival and suppresses apoptosis in response to various stimuli through inhibition of PTEN (phosphatase and tensin homolog), known as tumor suppressor gene (Simpson et al., 2001).AKT activity is present in primary carcinomas of a variety of cancers,
We previously reported that an ethanol extract of I. nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line (Shin et al., 2011).Here, we investigated the inhibitory effects and mechanisms of action of an ethanol extract of I. nertschinskia on the Hs578T and MDA-MB231 human breast carcinoma cell lines, which differ in their levels of AKT1/2 activity.Our results demonstrated an association between AKT activity and cellular sensitivity to the cytotoxic effects of an ethanol extract of I. nertschinskia.

Cell cytotoxicity
Cell viability was determined using trypan blue exclusion by counting at least 500 cells in each culture.Cells were treated with 10, 20, 30, 40, or 50 μg/ml of I. nertschinskia ethanol extract for 24 h, and live and dead cells were counted.

Preparation of ethanol extracts of I.nertschinskia
I. nertschinskia (50 g) was extracted with ethanol (100%, 500 ml) for 18 h at room temperature to produce 24 g of solid extract.The ethanol extract was condensed by decompression concentration and suspended in distilled water.The suspended extract was frozen at -70°C and dried.The resulting powder, a yellow solid, was dissolved in dimethyl sulfoxide.
Following incubation with horseradish peroxidaseconjugated goat anti-mouse or goat anti-rabbit secondary antibody, as appropriate, blots were developed using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK).

Human breast cancer cells are differentially sensitive to I. nertschinskia ethanol extract
Recently, we reported that breast cancer cells with varying degrees of malignancy are differentially sensitivity to drug-induced apoptotic effects (Shin et al., 2011).Here, using this experimental system, we attempted to demonstrate a dependence of I.nertschinskia cytotoxicity on the degree of cancer cell malignancy.We first measured the viability of two breast cancer cell lines, Hs578T and MDA-MB231,following exposure to different concentrations (0, 10, 20, 30, 40, and 50 μg/ml) of I. nertschinskia ethanol extract.The viability of MDA-MB231 cells was lower in than that of Hs578T cells (Figure 1A).Consistent with this, time-course studies showed that I.nertschinskia induced cell death in MDA-MB231 cells at earlier time points than was observed in Hs578T cells (Figure 1B).Collectively, these results indicate that MDA-MB231 cells are more sensitive to I.nertschinskia-induced cell death than Hs578T cells.
To further examine the effect of I.nertschinskia ethanol extract in the two human breast cancer cell lines, we performed flow cytometry analyses (Figure 2).The ethanol extract of I. nertschinskia induced a dramatic concentration-dependent increase in the number of cells in the sub-G1 (apoptotic) phase in MDA-MB231 cells but not in Hs578T cells (Figure 2A and B).This specific increase in sub-G1 phase MDA-MB231 cells was also clearly time dependent (Figure 2C).Taken together, these results, which paralleled those of cellular viability studies shown in Figure 1, suggest that breast cancer cells are differentially sensitive to I.nertschinskia depending on their degree of malignancy.
These findings prompted us to investigate whether the differential sensitivity of the breast cancer cell lines, Hs578T and MDA-MB231, to Iris nertschinskia ethanol extract was dependent upon the activity of AKT1 or AKT2 in each cell line.To analyze the activity of AKT1 andAKT2 following exposure to the ethanol extract of I.nertschinskia, we performed Western blotting analysis using antibodies specific for phosphorylated AKT1 (p-AKT1) or p-AKT2.These experiments revealed that the phosphorylation of AKT1 was dramatically increased after I. Nertschinskia treatment in both breast cancer cell lines, whereas phosphorylated AKT2 was only increased in Hs578T cells (Figure 3).Neither AKT1 nor AKT 2 protein levels were affected by I. nertschinskia treatment (data not shown).Taken together, these results imply that the sensitivity to Iris nertschinskia-induced cell death may be regulated by the activity of AKT1, but not by AKT2.Treatment with extract more dramatically reduced phosphorylation of AKTs in MDA-MB231 cells than in Hs578T cells.Notably, no change in AKT2 phosphorylation was observed in MDA-MB231 cells after treatment with the extract, suggesting that Hs578T cells are more resistant to extract-induced cell death owing to their unsuppressed AKT activity.

The activity of AKT1, but not AKT2, controls the cellular sensitivity to I.nertschinskia
On the basis of the above data, we examined whether knockdown of endogenous AKTs by siRNA sensitizes cells to I.nertschinskia-induced cell death.We focused first on the effect of AKT1-siRNA on the sensitivity to I.nertschinskia.Two different siRNAs targeting human AKT1 were tested for AKT1-suppressive activity.AKT1-siRNA I clearly decreased I. nertschinskia-induced Hs578T andMDA-MB231 cells were treated with an ethanol extract ofI.nertschinskia(30μg/ml).Cell lysates were prepared at the indicated time points and analyzed byWestern blottingusing anti-phospho-AKT1, anti-phospho-AKT2, anti-AKT1, and anti-AKT2 antibodies.γ-tubulin levels were used as loading controls.Next, we investigated the effect of I. nertschinskia treatment following siRNA-mediated AKT2 knockdown.Of the two siRNA that targeted human AKT2 tested, AKT2-siRNA II clearly decreased I. nertschinskiainduced phosphorylation of AKT2 (Figure 5A).Knockdown of AKT2 by siRNA II sensitized both Hs578T and MDA-MB231 cells to the effects of I.nertschinskia treatment compared to cells transfected with scrambled RNA or treated with I. nertschinskia only (Figure 5B).Consistent with this, the number of cells in the sub-G1 phase after I. nertschinskia treatment was significantly increased following AKT2 knockdown by siRNA II in each cell line (Figure 5C), indicating that AKT2 repression also increases sensitivity to I. nertschinskia-induced cell death.

Discussion
Recently, we reported that breast cancer cells with varying degrees of malignancy were differentially sensitive to drug-induced apoptotic effects (Seo et al., 2009).To generalize this effect in breast cancer cells, we examined I. nertschinskia-induced cell death in two cell lines-Hs578T and MDA-MB231-with differing degrees of malignancy.As predicted, these cells were differentially sensitive to an ethanol extract of I.nertschinskia: the extract induced a dramatic, concentration-and time-dependent cell death in highly malignant MDA-MB231 cells.In contrast, the extract induced cell death to a lesser extent in the Hs578T line.Mechanistically, I.nertschinskia acted through the regulation of AKT activity, decreasing the phosphorylation of AKTs, especially in MDA-MB231 cells.The inhibitory effect of I. nertschinskiaon breast cancer cell growth was enhanced by knocking down AKT1 activity using siRNA, but not byAKT2 knockdown.These results imply that the AKT signaling pathway plays a crucial role in I.nertschinskia-induced cell death in MDA-MB231 cells.
The AKT pathway is among several signaling pathways that are important for cell survival.AKT (also known as protein kinase B or PKB), a proto-oncogenic serine/threonine kinase, has received considerable attention because of its critical regulatory role in diverse cellular processes, including cancer progression and insulin metabolism.There are three highly related isoforms of AKT-AKT1, AKT2 and AKT3-which   DOI:http://dx.doi.org/10.7314/APJCP.2012.13.12.6511AKT1 Activity Affects Cytotoxicity of an Iris Nertschinskia Ethanol Extract constitute major elements in the PI3Ksignaling pathway.AKT regulates cell growth through its effects on the mTOR and p70 S6 kinase pathways, and modulates the cell cycle and cell proliferation through direct actions on the CDK inhibitors p21 and p27 and indirect effects on the levels of cyclin D1 and p53.AKT is a major mediator of cell survival through direct inhibition of pro-apoptotic factors, such as the Bcl-2 family member Bad and members of the Forkhead family of transcription factors.These findings make AKT/PKB an important therapeutic target for the treatment of cancer.
Small organic molecules derived from higher plants have been among the mainstays of cancer chemotherapy for the past half-century.Several selected, single chemical entity natural products of plant origin and their semi-synthetic derivatives are currently featured in clinical trials as new cancer chemotherapeutic drug candidates (Lee et al., 2010).These compounds obtained from plants show promising in vivo biological activity, exhibiting potential as anticancer agents.Because extracts of only a relatively small proportion of the approximately 300,000 higher plants on earth have been screened biologically to date, bioactive compounds from plants are likely to play an important role in future anticancer drug discovery efforts.
Previously we reported that I. nertschinskia, an ornamental plant utilized in traditional East Asian medicine for the treatment of skin diseases, showed anti-tumor effects on MCF7 human breast cancer cells (Shin et al., 2011).An ethanol extract of I. nertschinskia triggered p53-dependent cell death in these cells by inducing the Bax protein, a key regulator of p53dependent apoptotic cell death, and promoting caspase-7 cleavage.Cells treated with p53-specific siRNA or a caspase inhibitor were resistant to I. nertschinskiainduced apoptotic cell death.Mechanistically, these results suggest that p53 sensitizes tumor cells to the ethanol extract of I. nertschinskia.Activation of the AKT signaling pathway induces phosphorylation of Mdm2, which ubiquitinates the p53 tumor suppressor, targeting it for proteasome-mediated degradation.The role of AKT activity in I. nertschinskia-induced cytotoxicity revealed in the current study is consistent with this p53-dependent mechanism.
Future studies will further elucidate the novel mechanism underlying the cell growth-inhibitory effects of I. nertschinskia extracts and validate this plants of the source of a potential chemotherapeutic candidate.These data suggest that treatment of an ethanol extract of I. nertschinskia differentially induces cell death through Akt1/2 expression in breast cancer cells.

Figure 1 .
Figure 1.Ethanol extract of I. nertschinskia induces cytotoxicity in human breast cancer cells.(A) Hs578T and MDA-MB231 cells were treated with the indicated concentrations of an ethanol extract of I. nertschinskia, and their cell numbers were monitored at 24 h.(B) Both cell lines were treated with an ethanol extract of I. nertschinskia (30 μg/ml), and cell numbers were counted at the indicated times.The percentage of dead cells was measured by trypan blue staining.The results represent mean values and standard deviations of three independent experiments.

Figure 2 .Figure 3 .
Figure 2. Differential Sensitivity of Hs578T and MDA-MB231 Cells to I.nertschinskia Ethanol Extract-induced Cell Death.(A and B) Each cell type, treated with an ethanol extract of I. nertschinskia as in Figure 1A,was analyzed by flow cytometry after staining with PI. (C).Cells were prepared as in Figure 1B and then stained with PI for flow cytometric analysis.
, whereas AKT1-siRNA II did not (Figure4A).Transfection with AKT1-siRNA I enhanced the sensitivity of both Hs578T and MDA-MB231 cells to I.nertschinskia-induced cytotoxicity compared to cells transfected with scrambled RNA or treated with I.nertschinskia only (Figure4B).Consistent with this, the number of cells in the sub-G1 phase after I.nertschinskia treatment was significantly increased following transfection of AKT1-siRNA I in each cell line (Figure4C), indicating that AKT1 repression increases sensitivity to I.nertschinskia-induced cell death.

Figure 4 .
Figure 4. AKT1 Activity is Necessary for Resistance to an Ethanol Extract of I. nertschinskia.(A) Cells were transfected with scrambled siRNA (control), AKT1-siRNAI or AKT1-siRNAII, and then the efficiency of AKT1 knockdown was analyzed by Western blotting.The level of phospho-AKT1 after treatment with an ethanol extract of I. nertschinskia alone was used as a positive control.γ-tubulin levels were used as loading controls.(B) Hs578T and MDA-MB231 cells were transfected with AKT1-siRNA I and then incubated with or without an ethanol extract of I. nertschinskia (30μg/ml).Cell death was determined by counting the number of trypan blue-stained cells at 24h.The data represent means and standard deviations of three independent experiments.(C) Cells prepared as in Figure 4B were stained with PI for flow cytometric analysis.

Figure 5 .
Figure 5. AKT2 activity is partially required for resistance to an ethanol extract of I. nertschinskia.(A)Cells were transfected with scrambled siRNA, AKT2-siRNAI or AKT2-siRNAII, and then the expression of AKT2 was analyzed by Western blotting.(B) Hs578T and MDA-MB-231 cells were transfected with AKT2-siRNAII and then incubated with or without an ethanol extract of I. nertschinskia (30μg/ml) for 24h.Cell death was determined using the trypan blue exclusion method.The data represent means and standard deviations of three independent experiments.(C) Cells prepared as in Figure5Bwere stained with PI for FACS analysis.