Stat3 activation in human endometrial and cervical cancers - PubMed (original) (raw)

Stat3 activation in human endometrial and cervical cancers

C-L Chen et al. Br J Cancer. 2007.

Abstract

The activation of signal transducer and activator of transcription 3 (Stat3) has been implicated in the oncogenesis of cancer and is regarded as a novel target for cancer therapy. Stat3 is classified as a proto-oncogene, because an activated form of Stat3 can mediate oncogenic transformation in cultured cells and tumour formation in nude mice. The constitutive activation of Stat3 has been frequently detected in various types of human cancers. However, the constitutive activation of Stat3 in endometrial and cervical cancers has not been studied. We examined tyrosine phosphorylation of Stat3 (activated form of Stat3) in multiple endometrial and cervical cancer tissues using tissue microarray slides as well as cancer cell lines to explore the possible activation of Stat3. Our results indicated that elevated phosphorylation of Stat3 was detected in cervical and endometrial cancer cell lines. Our results also showed that elevated levels of phosphorylation of Stat3 protein were detected in the endometrial and cervical cancer specimens. This is the first study to demonstrate that Stat3 is activated in human endometrial and cervical cancer tissues. Immunohistochemical staining showed that activated Stat3 is associated with increased expression of downstream antiapoptotic genes, Bcl-xL, survivin, and Mcl-1 in these tissues. Expression of a dominant-negative Stat3 mutant using adenovirus-mediated gene transfer inhibited cell growth and induced apoptosis in HeLa and SiHa cervical cancer cell lines expressing elevated levels of Stat3 phosphorylation. Further, a JAK/Stat3 small molecular inhibitor, JSI-124, induced apoptosis more selectively in HeLa and SiHa cancer cell lines than Ishikawa cell line without elevated levels of Stat3 phosphorylation. These results indicate that Stat3 is activated in human endometrial and cervical cancers and the inhibition of constitutive Stat3 signaling may be an effective target for cancer intervention in these two cancers.

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Figures

Figure 1

Figure 1

Expression of Stat3 phosphorylation (Tyr705) in (A) endometrial cancer tissues and (B) cervical cancer tissues. Endometrial and cervical cancer tissue microarray slides were stained using IHC methods and an IHC-validated phospho-specific Stat3 antibody (Tyr705). The representative examples of phosphorylated Stat3 staining in normal tissues and positive staining of endometrial cancer tissues (grades I, II and III) are shown. The representative examples of phosphorylated Stat3 staining in normal tissues and positive staining of cervical cancer tissues (different stages) are shown.

Figure 2

Figure 2

(A) Expression of Stat3, Stat1 and Stat5 phosphorylation in human immortalised cervical cell line (Ect1/E6E7) and cervical and endometrial cancer cell lines. A total of 100 _μ_g of total protein of cell lysates from various cell lines were resolved on 8% SDS–PAGE and subjected to Western blot analysis using antibodies that recognise phospho-specific Stat3 (Tyr705), Stat3 (Ser727), Stat1 (Tyr701), Stat5 (Tyr694), and GAPDH, respectively. (B) Densitometric quantitation of phospho-Stat3 (Tyr705) and Stat3 (Ser727) expressions. The phospho-Stat3 expressions are normalised to GAPDH expression in each cell line and shown in percentage of phospho-Stat3 (Tyr705) in SiHa cells and phospho-Stat3 (Ser727) in RL95-2 cells, respectively.

Figure 3

Figure 3

Transduction of dnStat3 inhibits cell growth and viability in cervical cancer cell lines. (A) Expression of dnStat3 mediated by rAd vector in a dose-dependent manner in HeLa and SiHa cervical cancer cell lines. One hundred micrograms of cell lysates were analysed on 10% PAGE analysis and then subjected to immunoblots probed with anti-FLAG, -Stat3 and – GAPDH antibodies. The dose-dependent expression of FLAG-tagged dnStat3 is according to the increased expression levels of total Stat3 protein. There was no detectable FLAG-tagged dnStat3 in cell lysates of untransduced or cells transduced with rAd/eGFP. (B) and (C) dnStat3 inhibits HeLa and SiHa cell growth. HeLa and SiHa cells were transduced with either rAd/dnStat3 or rAd/eGFP (MOI=10–400). Representative phase-contrast images of HeLa and SiHa cells 48 h post-transduction were shown at magnification × 100. Only representative data (MOI=400) are shown for cells transduced with rAd/eGFP for cells transduced with MOI lower than 400 showed no adverse effects on cell growth. Cells in five random individual microscopic fields (× 100) were scored on day 2 post-transduction of rAd/eGFP or rAd/dnStat3. The cell growth is shown in cell density/control (%). The averages and standard deviations are based on triplicate independent experiments. (D) dnStat3 reduces cell viability of cancer cells with elevated phospho-Stat3 at 48 h post-transduction. HeLa and Ishikawa cells were transduced with rAd/eGFP and rAd/dnStat3 (MOI=250). Cell viability was analysed using MTT assay.

Figure 4

Figure 4

Transduction of dnStat3 induces apoptosis through caspase 3 pathway in (A) HeLa and (B) SiHa cervical cancer cell lines day 2 post-transduction. Cells were transduced with rAd/dnStat3 or rAd/eGFP (MOI=400). Cells incubated with JSI-124 (5 and 10 μ

M

), a Stat3 inhibitor, and DMSO served as a positive and negative control, respectively. Cells were fixed in methanol/acetone (v : v=1 : 1) and then immunostained with anti-cleaved caspase-3 antibody after 48 h. Cleaved caspase-3 immunoreactivies were observed in cells transduced with rAd/dnStat3, but much less in cells transduced with rAd/eGFP or the untransduced controls. Magnifications of all images were × 100. The percentages of cleaved caspase-3 positive cells were scored in five microscopic fields. The averages and standard deviations are based on three independent experiments. (C) dnStat3 induces cleavage of PARP in HeLa and SiHa cell lines. HeLa and SiHa cells were transduced with rAd/dnStat3 and rAd/eGFP (MOI:10, 100 and 400). After 48 h, cell lysates (100 _μ_g) were fractionated using SDS–PAGE and subject to Western blots probed with anti-cleaved PARP and -GAPDH antibodies. Cleaved caspase 3: anticleaved-caspase-3 antibody immunofluorescent staining; DAPI: nuclear staining with DAPI; none: untransduced cells.

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