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, 102 (11), 1592-9

STAT3 Inhibitor WP1066 as a Novel Therapeutic Agent for Renal Cell Carcinoma

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STAT3 Inhibitor WP1066 as a Novel Therapeutic Agent for Renal Cell Carcinoma

A Horiguchi et al. Br J Cancer.

Abstract

Background: Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes that mediate cell survival, proliferation, and angiogenesis and is aberrantly activated in various types of malignancies, including renal cell carcinoma (RCC). We examined whether it could be a novel therapeutic target for RCC by using the STAT3 inhibitor WP1066.

Methods: The antitumour activities and related mechanisms of WP1066 were investigated in vitro on renal cancer cell lines and in vivo on murine xenografts.

Results: In Caki-1 and 786-O renal cancer cells, 5 muM WP1066 prevented the phosphorylation of STAT3, and 2.5 muM WP1066 significantly (P<0.01) inhibited cell survival and proliferation. WP1066 suppressed the expression of Bcl-2, induced apoptosis, and inhibited the basal and hypoxia-induced expression of HIF1alpha and HIF2alpha, as well as vascular endothelial growth factor secretion into cell culture medium. Human umbilical vascular endothelial cells cocultured with media from WP1066-treated cells showed significantly reduced tubulogenesis (P<0.05). Systemic oral administration of WP1066 to mice for 19 days significantly inhibited the growth of Caki-1 xenograft tumours (P<0.05), and pathological analysis of xenografts of WP1066-treated mice showed decreased immunostaining of phosphorylated STAT3 and reduced length of CD34-positive vessels (P<0.05).

Conclusion: Our results suggest that using WP1066 to inhibit the STAT3 signalling pathway could be a novel therapeutic strategy against RCC.

Figures

Figure 1
Figure 1
WP1066 inactivates STAT3 and suppresses the growth and viability of renal cancer cells. (A) WP1066 inhibited STAT3 phosphorylation, while stimulating ERK, in Caki-1 and 786-O cells. Caki-1 and 786-O cells were treated with the indicated concentration of WP1066 for 24 h and cell lysates were subjected to western blotting. (B) MTS-assay-determined viability of Caki-1 and 786-O cells treated with the indicated concentration of WP1066 for 24 h. Note the significantly lower viability of WP1066-treated cells (#, P<0.01; ##, P<0.05). The results are expressed as the mean ± s.e. of the three sets for each group. (C) Proliferation of Caki-1 and 786-O cells in six-well plates treated with the indicated concentration of WP1066. Total cell numbers were counted at 24 and 48 h. Note the significantly reduced proliferation of WP1066-treated cells (##, P<0.01). The results are expressed as the mean ± s.e. of the three sets for each group.
Figure 2
Figure 2
WP1066 downregulates antiapoptotic protein Bcl-2 and induces apoptosis in renal cancer cells. (A) Annexin V staining for apoptosis assay in Caki-1 and 786-O cells incubated for 24 h with the indicated concentration of WP1066. The percentages of annexin-V-positive- and propidium-iodide-negative (early apoptosis) fractions are indicated. (B) Nuclear morphologies observed by fluorescence microscopy after staining with Hoechst 33258 after Caki-1 and 786-O cells had been incubated for 24 h with the indicated concentration of WP1066. Note the fragmented and condensed nuclei characteristic of apoptosis. (C) Western blot analysis of apoptosis-related proteins. Cells were treated with the indicated concentration of WP1066 for 24 h and subjected to western blotting. Actin was used as an internal control.
Figure 3
Figure 3
WP1066 downregulates HIF1α and HIF2α expression and reduces VEGF production in renal cancer cells. (A) Caki-1 and 786-O cells were incubated with the indicated concentration of WP1066 under normoxic (norm) or hypoxic (hypo, 1% O2) conditions for 24 h, and the VEGF levels in the cell culture media were measured by ELISA. Hypoxic conditions stimulated VEGF production in Caki-1 cells but not in 786-O cells (#, P<0.05). VEGF production under both normoxic and hypoxic conditions was significantly suppressed by treatment with WP1066 (##, P<0.01). The results are expressed as the mean ± s.e. of the three sets for each group. (B) Caki-1 and 786-O cells were incubated with the indicated concentration of WP1066 under normoxic or hypoxic (1% O2) conditions for 6 h, and cell lysates were subjected to western blotting. Hypoxic conditions stimulated STAT3 phosphorylation and upregulated HIF1α and HIF2α expression in Caki-1 cells, and these effects were suppressed by treatment with WP1066. Hypoxic conditions had no effect on STAT3 phosphorylation or HIF2α expression in 786-O cells, both of which were suppressed by treatment with WP1066.
Figure 4
Figure 4
WP1066 inhibited in vitro angiogenesis. The HUVECs were incubated in a cell-conditioned medium with 5 μM WP1066 under normoxic (norm) or hypoxic (hypo, 1% O2) conditions. After incubation for 11 days, HUVECs were fixed and stained with an anti-human CD31 antibody. (A) Representative photomicrographs ( × 40) of HUVEC tubular formations. (B) Tubular formation length was quantified in five randomly selected fields. (#, P<0.05; ##, P< 0.01 vs cells cultured without WP1066 under normoxic conditions). The results are expressed as the mean ± s.e. of the three sets for each group.
Figure 5
Figure 5
WP1066 inhibits tumour growth in the murine xenograft model of Caki-1 cells. (A) Established Caki-1 tumour xenografts in athymic male nude mice were treated with WP1066 (40 mg kg−1, n=8) or vehicle (20% DMSO-80% polyethylene glycol 300, n=8) by oral gavage once per day for 5 days (followed by a rest for 2 days) for a total of 19 days. Tumour volume in WP1066-treated mice on day 19 after the first administration was significantly smaller than that in control vehicle-treated mice (#, P<0.05). (B) Representative photographs of WP1066-treated mice and vehicle-treated control mice. (C) Immunohistochemical analysis of representative samples of xenograft tumours. Formalin-fixed xenograft tumours were immunostained with anti-p-STAT3, STAT3, or CD34 antibodies. Note decreased level of nuclear p-STAT3 immunostaining (upper right) and decreased tubular length of CD34-positive vessels (lower right) in WP1066-treated tumours compared with vehicle-treated control tumours. (D) Length of CD34-positive vessels was quantified in selected fields. (##, P<0.05).

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