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    r> Apoptosis is a programmed cell death which can be triggered by external or internal stimuli. In fact, majority of chemotherapeutic agents eradicate malignant tumor 2-NBDG by inducing apoptosis [26]. In the present study, it was found that bufothionine increased nuclear chromatin density, which reflected the condensation, coagulation, and fragmentation of the nuclear chromatin. Flow cytometric assay also showed that bufothionine dramatically promoted GC cell apoptosis. Activation of caspase cascade is the hallmark for apoptotic cell death [27]. Among the caspases, activation of casapse-8 and casapse-9 leads to apoptosis through extrinsic and intrinsic pathways, respectively [28]. The results indicated that bufothionine markedly elevated the in-tracellular level of cleaved caspase-3, cleaved caspase-8 and cleaved caspase9, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. Moreover, caspase inhibitor blocked the bu-fothionine-induced apoptosis, suggesting that bufothionine triggered caspase-dependent apoptosis. The apoptotic process is also tightly regulated by a series of pro-apoptotic and anti-apoptotic proteins, in-cluding Bax, Bak, Bid, Bcl-2, and Bcl-xL. Particularly, the interaction between Bax and Bcl-2 triggers mitochondrial membrane disruption and subsequent release of mitochondrial cytochrome c into the cyto-plasm, which lead to the activation of caspase-cascade [29]. Here, it was observed that bufothionine upregulated pro-apoptotic protein Bax and downregulated anti-apoptotic protein Bcl-2, implying that mi-tochondrial dysfunction was involved in the bufothionine-induced apoptosis. These findings are in agreement with the report by Zhou which showed that bufothionine promoted cell apoptosis in mouse liver tumor cells by activating mitochondrial apoptosis signaling [18].
    The pim family members possess serine/threonine kinase activity [30]. Recently, three kinases have been added to this family, termed as PIM1, PIM2, and PIM3 [31]. PIM1, as a proviral insertion site in Mo-loney murine leukemia virus T cell lymphomas, positively contributes  Life Sciences 232 (2019) 116615
    to lymphomagenesis. On the other hand, PIM2 was initially identified as a proviral integration site [32,33] whereas PIM3 was initially iden-tified as an oncogenic gene [34]. It was later found that PIM3 is ex-pressed in normal brain and heart tissue [4,7,8]. Interestingly, PIM3 is unregulated in cancerous tissues, especially endoderm-derived tumors including the colon, liver, pancreas, and stomach [4,7,8]. Since then, small molecule Pim kinase inhibitors were developed [35,36]. An ear-lier study showed that a stemonamide synthetic intermediate, named T-18, exhibited anticancer activities against GC by functioning as a PIM3 inhibitor [37]. Our results showed that PIM3 is a potential therapeutic target for GC treatment. The experimental data in this study revealed that the anti-growth and pro-apoptotic activities of bufothionine were attributed to downregulation of PIM3. Moreover, the results provided compelling evidence that PIM3 is a key mediator of bufothionine-in-duced apoptosis of GC cells, which is consistent with studies in color-ectal cancer, glioma and pancreatic cancer [6,38,39]. Several studies have shown that PIM3 positively contributes to metastasis [6], che-moresistance [40,41], and radio resistance [42]. Therefore, it is rea-sonable to believe that bufothionine might exhibit anti-metastatic ac-tivities or enhance the sensitivity to chemotherapy and radiotherapy by downregulating PIM3.
    In summary, the findings in this study show that bufothionine suppresses GC growth in vitro and in vivo. Moreover, PIM3 may act as the primary molecular target that mediates the anti-cancer activities of bufothionine against GC. These findings provide novel insights into the chemotherapeutic potential of bufothionine for GC treatment.
    Author contribution
    Guojun Wang designed the research, provided the fund and wrote the paper; Guanghui Liu, Yanwei Ye and Yang Fu performed experi-ments; Xiefu Zhang analyzed the data.
    Declaration of Competing Interest
    [8] Y.Y. Li, B.K. Popivanova, Y. Nagai, et al., Pim-3, a proto-oncogene with serine/ threonine kinase activity, is aberrantly expressed in human pancreatic cancer and phosphorylates bad to block bad-mediated apoptosis in human pancreatic cancer cell lines, Cancer Res. 66 (13) (2006) 6741–6747.
    [10] Q. Guo, P. Lan, X. Yu, et al., Immunotherapy for hepatoma using a dual-function vector with both immunostimulatory and pim-3-silencing effects, Mol. Cancer Ther.
    [16] W. Xu, H. Luo, Y. Zhang, et al., Simultaneous determination of five main active bufadienolides of Chan Su in rat plasma by liquid chromatography tandem mass spectrometry, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 859 (2) (2007)