STAT3 plays a significant role in the development of cholangiocarcinoma (CCA) associated with the liver fluke (
Cholangiocarcinoma (CCA) or bile duct epithelial cancer associated with the liver fluke (
A signal transducer and activator of transcription (STATs) family of protein kinases play roles in the immune response mechanism, inflammation and cellular development (
During the past decade, the strategy for cancer prevention and treatment of the identification and characterizations of dietary phytochemicals that are capable of blocking or reversing carcinogenesis as well as possessing anticancer properties has received increased research focus (
Human CCA cells, M214 and M139 were cultured and maintained as previously described (
Antibodies for western blotting were as follows: anti-phospho-STAT3 (Cambridge, UK), anti-phospho-STAT3, phospho-Akt, total Akt (Cell Signaling Technology, Danvers, MA, USA), anti-p65 NFκB (Santa Cruz Biotechology, Santa Cruz, CA, USA), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Recombinant human IL-6 was commercially available and purchased from R&D Systems, Minneapolis, MN, USA. XN was kindly provided by Hopsteiner, Mainberge, Germany.
Western blot analysis was performed as previously described (
M214 and M139 CCA cells (2×103/100
For the XN suppressed IL-6-induced STAT3 activation experiment, cell proliferation was determined by trypan blue exclusion assay. Cells were treated with 10 ng/ml recombinant human IL-6 concomitant with the indicated concentration of XN (0, 10, 20 and 50
Six-week-old female BALB/cAJcl-nu/nu mice were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions at the animal center, Institute of Medical Science, The University of Tokyo. All animal experiments were performed according to institutional guidelines. Mice were subcutaneously injected with 2×106 cells of KKU-M214 at both flanks. One week after tumors were visible, animals were divided into two groups; the control group was provided with a vehicle (0.5% ethanol) whereas treatment groups were administrated 20 and 50
Immunostaining of Ki67, proliferation marker was performed on paraffin-embedded nude mouse tumor tissues to determine the antiproliferative effect of XN in a CCA animal model. Nude mouse tissue sections were deparaffinized in xylene followed by rehydration in a series of different ethanol concentrations. Then, the antigen was retrieved using Tris-EDTA buffer, pH 8.8 in pressure cooker and 0.3% H2O2 was used to block endogenous peroxidase activity for 30 min with agitation. Nonspecific binding was blocked by 10% skim milk in phosphate-buffered saline (PBS) for 30 min. Sections were incubated with the anti-Ki67 antibody at 4°C overnight in a moisture chamber.
Sections were then incubated with peroxidase-conjugated EnVision™ secondary antibody (Dako, Denmark) followed by washing with working PBS for 5 min, three times. After that the color was developed with 0.1% diaminobenzidine tetrahy-drochloride solution for 5 min and followed by counterstaining with Mayer's hematoxylin. Sections were observed under a light microscope (Carl Zeiss, Germany). Ki67-positive cells of each tumor section was counted in at least five of the ×200 power fields.
Histologic analysis of DNA fragmentation was used to identify apoptotic cells in the paraffin sections of CCA nude mouse tissues.
Results from cell proliferation, Ki67 staining analysis, apoptosis assay and animal experiments are represented as mean ± SD, statistical significance was addressed by independent samples t-test and a two-way ANOVA (GraphPad Prism 5 software). P-value of <0.05 was considered to indicate a statistically significant result.
The effects of XN on the growth of CCA cells were determined in human CCA cell lines established from primary tumors of Ov-associated CCA patients namely, KKUM214 and KKU-M139. The results showed that XN inhibited CCA cell growth which occurred in a dose- and time-dependent manner. A 20
We then evaluated whether inhibiting STAT3 activation leads to growth inhibition as well as apoptosis induction in CCA cells. CCA cells were exposed to XN upon stimulation with IL-6. The results showed that a low concentration of XN (10
To investigate if suppression of STAT3 activation by XN inhibited CCA growth resulted from apoptosis induction, we examined the expression of anti-apoptosis protein, Bcl-2 as well as BAX, pro-apoptotic protein. The results demonstrated that decreasing protein levels of Bcl-2 was seen in M214 and M139 CCA cells after treatment with XN, whereas BAX protein expression was increased (
To evaluate an
As shown in the
The above data revealed an inhibitory effect of XN on STAT3 activation both
Our results showed that XN also suppresses Akt activation as well as the nuclear translocation activity of p65 NFκB in both IL-6-induced CCA cells (
STAT3 is a protein kinase, which plays various roles as a signal messenger and as a transcription factor. STAT3 signaling can be triggered by inflammatory cytokines, growth factors and hormones, particularly IL-6 (
Our previous study showed that the activation of the STAT protein family occurred in both CCA cells and tissues (
XN, prenylated chalcone which can be isolated from the hop plant (
The present study showed that XN can inhibit CCA cell proliferation in a dose- and time-dependent manner. Moreover, this is the first time that an inhibitory effect of XN on STAT3 activation has been demonstrated. We revealed that XN at 20
Based on the
Our
Furthermore, we explored the molecular mechanisms by which XN inhibits STAT3 activation in CCA. Results showed that XN provided anticancer activities via the suppression of Akt and NFκB, the molecules that are involved in the proliferation, survival and angiogenesis of tumor cells. Moreover, the interconnection between Akt-NFκB and STAT3 signaling has been described (
In conclusion, we have shown that XN can inhibit STAT3 activation in human CCA cell lines as well as CCA inoculated mice. Moreover, XN can effectively suppress the growth of tumor and induce apoptosis in CCA cells and tumor inoculated mice without any noticeable side-effects. This is the first time that STAT3 has been demonstrated as a potential target of XN. Moreover, our results have shown the potential efficacy of XN for CCA treatment. The above knowledge can provide the basis to develop new therapeutic strategies for CCA using XN alone and/or combined with conventional chemotherapy drugs to improve the efficacy of CCA treatment.
We thank the research technicians (Division of Molecular Pathology, Department of Cancer Biology, Institute of Medical Science, The University of Tokyo) who kindly assisted us in the animal experiment. The present study was supported by Liver Fluke and Cholangiocarcinoma Research Center to H.D., the Research Assistantship Grant of the Faculty of Medicine, Khon Kaen University (grant no. AS57202) and the Khon Kaen University Grant (KKU59), the co-funding from Japan Science and Technology Agency (JST), Ministry of Education, Culture, Sport, Science and Technology of Japan, and grant of the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster (SHeP-GMS), KhonKaen University. We also thank Professor Ross H. Andrews for editing the initial submission via Publication Clinic KKU, Thailand.
Growth inhibitory effect of XN on CCA cells. (A) KKU-M214 CCA and (B) KKU-M139 CCA cell lines were cultured with XN at the designated concentrations ranging from 2.5 to 50
Inhibitory effects of XN on STAT3 activation and CCA cell growth. (A) Western blot analysis of STAT3 activation in M214 and M139 CCA cell lines after treatment with XN upon IL-6 stimulation compared to untreated control. (B) CCA cell proliferation after treatment with XN upon IL-6 stimulation was determined by trypan blue dye exclusion assay. (C) Western blot analysis of the cell cycle regulation proteins, cyclin D1 and CDK4 proteins expression. Data in (B) are mean ± SD of two independent experiments and analyzed by independent samples t-test. *P<0.05 and **P<0.001 compared to the control.
Effects of XN on apoptosis induction of CCA cell lines. (A) Western blot analysis showed apoptotic protein expression in CCA cell lines after treatment with XN and (B) BAX/Bcl-2 protein expression ratio. Data in B are mean ± SD of protein band intensity which were normalized to intensity of β-actin from two independent experiments.
Antitumor activity of XN on CCA-inoculated mice. (A) Tumor growth in mice that received 20 and 50
Inhibitory effects of XN on STAT3 activation and tumor development in CCA mouse model. (A) Western blot analysis presents the decrease of STAT3 activation in tumor tissues from XN-treated mice. (B) Proliferative cells were determined by IHC of Ki67 (magnification, ×200). (C) The number of proliferative cells was decreased in XN-treated mouse tumor tissues. (D) Apoptotic cells in tumor tissues (arrowheads) which were identified by TUNEL, magnification, ×200. (E) The number of apoptotic cells was increased in XN-treated mouse tumor tissues. Data in C and E, is the mean ± SD, *P<0.05 analyzed by independent samples t-test.
Molecular mechanisms by which XN inhibits STAT3 activation in CCA. (A) Western blot results represent the decreased Akt activation in IL-6-induced CCA cell lines after treatment with XN. (B) The decreased p65 NFκB nuclear translocation activity in IL-6-induced CCA cells after treatment with 50