Fig 1.
WYE-687 is cytotoxic to cultured human RCC cells.
Established human RCC cell lines (786-O and A498), primary human RCC cells, or HK-2 tubular epithelial cells were treated with indicated concentration of WYE-687, rapamycin or RAD001 for applied time, cell viability was tested by MTT assay (A, C and D, n = 5). 786-O cells were treated with 100 nM of WYE-687, rapamycin or RAD001 for 10 days, the number surviving colonies was recorded (B, n = 5). *p< 0.05 vs. untreated control group (“C”). #p< 0.05 vs. WYE-687 group (B).Vehicle control (0.1% of DMSO) failed to affect survival and proliferation of above cells.
Fig 2.
WYE-687 induces apoptosis in cultured human RCC cells.
RCC cell lines (786-O and A498), primary human RCC cells (“Primary”), or HK-2 normal tubular epithelial cells were treated with described WYE-687 (“WYE”) for applied time, cell apoptosis was tested by listed assays (A, B, E, n = 3). For C and D, 786-O cells were pre-treated for 1 hour with Ac-DEVD-cho (“+DEVD”, 50 μM) or Ac-VAD-cho (“+ VAD”, 50 μM) before applied WYE-687 treatment, cell apoptosis (Annexin V assay, C, n = 3) and survival (MTT assay, D, n = 5) were tested. *p< 0.05 vs. untreated control group (“C”). #p< 0.05 vs. WYE-687treatment only (C and D).
Fig 3.
WYE-687 inhibits human RCC cell proliferation.
RCC cell lines (786-O and A498), primary human RCC cells, or HK-2 tubular epithelial cells were treated with indicated concentrations of WYE-687, rapamycin or RAD001 for applied time, cell proliferation was tested by listed assays (A-D, n = 5). *p< 0.05 vs. untreated control group (“C”). #p< 0.05 vs. WYE-687 group (C).
Fig 4.
WYE-687 blocks mTORC1 and mTORC2 activation in RCC cells.
786-O cells (A and D), primary human RCC cells (B and E), or HK-2 tubular epithelial cells (C) were treated withWYE-687 (100 nM) for applied time, expression of listed proteins was tested by Western blot assay.
Fig 5.
WYE-687 oral administration inhibits 786-O RCC tumor growth in nude mice.
The growth curve of 786-O xenografts in nude beige mice with daily administration ofWYE-687 (oral gavage, 25 mg/kg body weight) or vehicle control (“Vehicle”) was presented (A). Each treatment group comprised 9 mice, mean estimated tumor volume (A) and mice body weight (C) were recorded every 5 days. Estimated daily tumor growth was also presented (B). To test signaling changes, at treatment day-2, one mice per group was sacrificed, and tumor xenografts were excised; Expressions of indicated proteins in xenograft tissues were analyzed by Western blot assay (D and E) and IHC staining assay (F, bar = 50 μm). *p< 0.05.