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  • br AKT is a primary kinase involved in cell su

    2020-07-06


    263 AKT is a primary kinase involved in cell survival, and it is important in cancer because it
    264 plays a role in the balance between cell proliferation and apoptosis [40,41]. AKT is activated
    265 by phosphorylation and promotes cell growth through inhibition of apoptosis mechanisms
    266 [42]. Thus, suppression of AKT could be an effective strategy for cancer treatment. AKT
    267 isoforms, including AKT1, AKT2, and AKT3, are highly homologous, but they have different
    268 expression patterns and functions [43,44]. The knockout of AKT1 is defective in growth,
    269 whereas the knockout of AKT2 is reported to have a problem in glucose homeostasis. In
    270 addition, it has been reported that AKT1 regulates local tumor growth and AKT2 regulates
    271 distant tumor dissemination in breast cancer progression [45]. As shown in Figs. 2C and 2D,
    272 CK specifically blocked the activation of AKT1, an isoform of AKT. In particular, cell
    273 growth was clearly reduced in AKT2-knockdown SKBR3 Lycopene treated with CK, but there
    275 proliferation and induces apoptosis by targeting AKT1. Since the AKT signaling pathway is
    276 involved in cancer metastasis, we investigated the antimetastatic activity of CK using
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    277 migration and invasion assays. CK significantly inhibited the invasion and migration of
    278 SKBR3 cells in a dose-dependent manner (Figs. 3B and 3C). Since metastasis determines the
    279 prognosis of cancer patients, suppression of metastasis is crucial to cure cancer. In this study,
    280 we ascertained inhibitory effect of CK only in vitro not in vivo. Lee et al. reported that that
    281 CK has in vivo anti-cancer activity in triple-negative breast cancer [16]. CK significantly
    282 reduced the tumor mass and metastasis of colon cancer cells [46]. Based on previous studies,
    283 CK has in vivo anticancer activities, so we could expect that CK inhibit the cancer growth
    284 and metastasis in HER2-positive breast cancer.
    285 In conclusion, we demonstrated that CK strongly inhibits the proliferation and metastasis of
    286 human breast cancer SKBR3 cells by upregulating apoptosis, as summarized in Fig. 4. The
    287 pro-apoptotic activities of CK are related to the caspase-dependent apoptotic pathway and
    288 AKT1-mediated cell survival signaling. CK also suppresses the metastasis of SKBR3 cells
    289 through inhibition of AKT1 signaling. Thus, our findings suggest that Compound K has the
    290 potential to be an effective chemotherapeutic drug for breast cancer treatment.
    291 292 Acknowledgements
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    296 References
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    416 In vitro anti-cancer effects of CK on SKBR3 cells. (A) Viability of SKBR3 cells treated with
    417 the indicated dose of CK (0-50 mM) for 3-24 h was measured using MTT assay. (B) The anti- 418 apoptotic effects of CK were determined by measuring the numbers of apoptotic cells after
    424 The effect of CK on activation of apoptosis-related proteins in SKBR3 cells. (A) Caspase
    425 levels of CK-treated SKBR3 cells were analyzed using Western blotting with antibodies
    426 against cleaved or full-length proteins. (B) SKBR3 cells were treated with CK (0-50 mM) for 427 6 h, and the mRNA level of Bcl2 was measured using semi-quantitative RT-PCR. (C) The
    428 protein levels of phospho- and total-AKT1 and -AKT2 in whole cell lysates were determined
    429 using immunoblotting after treating SKBR3 cells with CK (0-50 mM) for 6 h. (D) SKBR3 430 cells overexpressing HA-AKT1 (left panel) or HA-AKT2 (right panel) were treated with CK
    431 (0-50 mM) for 6 h. The protein levels of phospho-AKT1/2, HA, and b-actin (internal control) 432 were identified using immunoblotting. Quantification of band intensity was measured by
    436 The effects of CK on AKT activation and metastasis. (A) SKBR3 cells were treated with
    439 treated with various dosages of CK (0-25 mM) for 24 h was analyzed using hematoxylin and 440 eosin staining. Quantification of invasion capacity was performed using Image J software. (C)
    441 The effects of CK on cell migration were evaluated using wound healing assay. A wound was
    442 created by scraping the cell monolayer with a pipette tip. Photographs were captured with a
    443 digital camera after 24 h of treatment with CK (0-25 mM). (D) In the clonogenic assay, 444 SKBR3 cells were seeded on plates and incubated until the colonies formed. The colonies
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