Yang JD, Roberts LR. HCC was also tested. For example, He activity of icaritin against HCC has not been extensively tested Existing evidences have suggested that sphingolipid metabolites are key molecule in regulating a number of cancerous behaviors . In which, sphingosine-1-phosphate (S1P) promotes malignancy cell survival and proliferation . On the other hand, ceramide and sphingosine build up could promote cell apoptosis and/or growth arrest [16, 18]. The key protein kinase that regulates the balance of these sphingolipid metabolites is definitely sphingosine kinase 1 (SphK1) . SphK1 catalyses the phosphorylation of ceramide or sphingosine to S1P, therefore reducing pro-apoptotic ceramide/sphingosine level, while increasing pro-survival S1P level [16, 19]. SphK1 activation positively regulates malignancy cell survival, proliferation, transformation, as well as apoptosis prevention and chemo-resistance [16, 19]. Reversely, inhibition, mutation or silence of SphK1 will lead to tumor cell apoptosis and tumor repression [16, 19]. Clinical studies have shown that SphK1 is definitely often over-expressed in a number of solid tumors including HCC [16, 19]. In the current study, we display that icaritin exerts significant anti-HCC activity both and possibly through inhibiting SphK1. RESULTS Icaritin is definitely cytotoxic and pro-apoptotic against human being HCC cells We here explored the potential effect of icaritin against HCC cells. As demonstrated in Number ?Number1A,1A, icaritin treatment inhibited survival of HepG2 HCC cells inside a dose-dependent manner. Icaritin was highly effective, with an IC-50 less than 5 M (Number ?(Figure1A).1A). Further, as demonstrated in Number ?Number1B,1B, the activity of icaritin was also time-dependent. It required at least 48 hours for icaritin (10 M) to exert a significant anti-survival effect (Number ?(Figure1B).1B). Colony formation in icaritin-treated HepG2 cells was also inhibited (Number ?(Number1C).1C). The potential effect of icaritin on HepG2 cell apoptosis was also tested. Results from the Histone DNA ELISA assay (Number ?(Figure1D)1D) and Annexin V FACS assay (Figure ?(Figure1E)1E) proven that icaritin at 2.5C25 M induced significant HepG2 cell apoptosis. Notably, icaritin was also cytotoxic to two additional human being HCC cell lines: Huh-7 and KYN-2 (Number ?(Figure1F).1F). Further, in the primary human being HCC cells (Patient-1 derived, or P1), icaritin (1C25 M) also decreased cell Delphinidin chloride viability (Number ?(Number1G).1G). The experiments were also repeated in main cancer cells derived from two additional HCC individuals (Patient-2/3 derived, or P2/3), and related results were acquired (Supplementary Number S1A). Note that icaritin exerted related pro-apoptotic activity in main (Supplementary Number S1B) and Huh-7/KYN-2 (Supplementary Number S1C) HCC cells. Collectively, these results demonstrate that icaritin is definitely cytotoxic and pro-apoptotic against human being HCC cells. Open in a Delphinidin chloride separate windowpane Number 1 Icaritin is definitely cytotoxic and pro-apoptotic against human being HCC cellsHepG2 (ACE), KYN-2 (F), Huh-7 (f), or main human being HCC cells (G, patient 1, or P1) were either left untreated (C, for those numbers), or treated with applied concentrations of icaritin (0.1C25 M) for indicated time, cell survival was tested by MTT assay (A, B, F and G) or clonogenicity assay (C, for HepG2 cells); HepG2 cell apoptosis was analyzed by Histone DNA ELISA assay (D) or Annexin V FACS assay (E). IC stands for icaritin (10 M, 72 h) (G). Mouse monoclonal to RUNX1 Experiments in this and all following figures were repeated three times, with related results acquired. =5 for each repeat (Same for those numbers). *< 0.05 vs. group C. Veh stands for 0.1% DMSO vehicle control (Same for those figures). Icaritin inhibits SphK1 activity, but raises cellular ceramide production in HCC cells Next, the possible involvement of SphK1 in icaritin-mediated anti-HCC activity was tested. Thus, we tested the potential effect of icaritin on SphK1 activity in HCC cells. As demonstrated in Number ?Number2A,2A, icaritin treatment significantly reduced SphK1 activity in HepG2 cells. Importantly, SphK1 protein or mRNA manifestation was not affected by the same icaritin treatment (Number ?(Figure2B).2B). On the other hand, the level of intracellular ceramide was improved in icaritin-treated HepG2 cells (Number ?(Figure2C).2C). Similarly in KYN-2 cells and main human being HCC cells, the SphK1 activity, but not SphK1 manifestation, was decreased following icaritin treatment (Number ?(Number2D2D and ?and2E).2E). As a result, the cellular ceramide level in these cells was improved (Number ?(Figure2F).2F). Collectively, icaritin inhibits SphK1 activity, but raises cellular ceramide production in HCC cells. Open in a separate window Number 2 Icaritin inhibits Delphinidin chloride SphK1 activity, but raises cellular ceramide production in HCC cellsHepG2 (ACC), KYN-2 (DCF), or main human being HCC cells (DCF) were either left untreated, or stimulated with icaritin (10 M) for indicated.
- All animal protocols were authorized and performed in accordance with the Vanderbilt University or college Medical Center Animal Care and Use Program
- Indeed, we found that S1P induced fast and transient phosphorylation of ERK1/2 inside a time-dependent way (Figure 5(b)), indicating the activation from the ERK1/2 pathway