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. 2017 Jun;16(2):227-243.
doi: 10.1177/1534735416649659. Epub 2016 May 16.

Supercritical Fluid Extraction of Citrus iyo Hort. ex Tanaka Pericarp Inhibits Growth and Induces Apoptosis Through Abrogation of STAT3 Regulated Gene Products in Human Prostate Cancer Xenograft Mouse Model

Chulwon Kim  1 , Il Ho Lee  1 , Ho Bong Hyun  2 , Jong-Chan Kim  3 , Rajendra Gyawali  4 , Seok-Geun Lee  1 , Junhee Lee  1 , Sung-Hoon Kim  1 , Bum Sang Shim  1 , Somi K Cho  2 , Kwang Seok Ahn  1
Affiliations

Supercritical Fluid Extraction of Citrus iyo Hort. ex Tanaka Pericarp Inhibits Growth and Induces Apoptosis Through Abrogation of STAT3 Regulated Gene Products in Human Prostate Cancer Xenograft Mouse Model

Chulwon Kim et al. Integr Cancer Ther. 2017 Jun.

Abstract

Activation of signal transducer and activator of transcription 3 (STAT3) is well known to play a major role in the cell growth, survival, proliferation, metastasis, and angiogenesis of various cancer cells. Most of the citrus species offer large quantities of phytochemicals that have beneficial effects attributed to their chemical components. Our study was carried out to evaluate the anticancer effects of the pericarp of Iyokan ( Citrus iyo Hort. ex Tanaka), locally known as yeagam in Korea, through modulation of the STAT3 signaling pathway in both tumor cells and a nude mice model. The effect of supercritical extracts of yeagam peel (SEYG) on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was examined. The in vivo effect of SEYG on the growth of DU145 human prostate xenograft tumors in athymic nu/nu male mice was also investigated. We found SEYG exerted substantial inhibitory effect on STAT3 activation in human prostate cancer DU145 cells as compared to other tumor cells analyzed. SEYG inhibited proliferation and downregulated the expression of various STAT3-regulated gene products such as bcl-2, bcl-xL, survivin, IAP-1/2, cyclin D1, cyclin E, COX-2, VEGF, and MMP-9. This correlated with an increase in apoptosis as indicated by an increase in the expression of p53 and p21 proteins, the sub-G1 arrest, and caspase-3-induced PARP cleavage. When administered intraperitoneally, SEYG reduced the growth of DU145 human prostate xenograft tumors through downmodulation of STAT3 activation in athymic nu/nu male mice. Overall, these results suggest that SEYG extract has the potential source of STAT3 inhibitors that may have a potential in chemoprevention of human prostate cancer cells.

Keywords: Citrus iyo Hort. ex Tanaka; DU145; STAT3; apoptosis.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
SEYG suppresses p-STAT3 levels in a time- and dose-dependent manner. (A) U266, K562, DU145, MDA-MB-231, HepG2, and RWPE-1 cells (1 × 104 cells/well) were incubated at 37°C with various indicated concentrations of SEYG for 24 hours, and the viable cells were assayed using the MTT reagent. ***P < .001 compared to nontreated. (B and C) Cells (1 × 106 cells/well) were incubated with the indicated concentrations of SEYG for 6 hours. Whole-cell extracts were prepared, then equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-STAT3 and STAT3. The results shown here are representative of 3 independent experiments. Graphs represent band intensities of indicated proteins. All data were expressed as mean ± SD. ***P < .001 versus control. (D and E) DU145 cells (1 × 106 cells/well) were treated with various indicated concentrations of SEYG and time intervals. Then equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-STAT3 (Tyr705) and STAT3. The results shown here are representative of 3 independent experiments. Graphs represent band intensities of indicated proteins. All data were expressed as mean ± SD. **P < .01 versus control.
Figure 2.
Figure 2.
SEYG downregulates p-JAK1, JAK2, and Src levels in a time- and dose-dependent manner. (A-F) DU145 cells (1 × 106 cells/well) were treated with various indicated concentrations of SEYG and time intervals. Then equal amounts of lysates were analyzed by Western blot analysis using antibodies against p-JAK1 (Tyr1022/1023), JAK1, p-JAK2 (Tyr1007/1008), JAK2, p-Src (Tyr416), and Src. The results shown here are representative of 3 independent experiments. Graphs represent band intensities of indicated proteins. All data were expressed as mean ± SD. **P < .01 versus control.
Figure 3.
Figure 3.
SEYG inhibits binding of STAT3 to the DNA and expression of various gene products in human prostate cancer cells. (A) SEYG suppresses STAT3 binding activity. DU145 cells (1 × 106 cells/well) were treated with various indicated concentrations of SEYG for 6 hours, analyzed for nuclear STAT3 levels by EMSA. Oct-1 EMSA is shown as a loading control. (B) SEYG causes the inhibition of translocation of STAT3 to the nucleus. After 6 hours of SEYG treatment, the cells were fixed and permeabilized. STAT3 (green) was immunostained with rabbit anti-STAT3 followed by FITC-conjugated secondary antibodies and the nuclei (blue) were stained with DAPI. The third panels show the merged images of the first and second panels. The results shown are representative of 2 independent experiments. (C) Cell proliferation assay was performed using the Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH) as described in “Material and Methods.” After DU145 cells (5 × 103 cells/well) were seeded onto 96-well E-plates and continuously monitored using impedance technology. (D-F) DU145 cells (1 × 106 cells/well) were incubated with the indicated concentrations of SEYG for 24 hours. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by SDS-PAGE, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against bcl-2, bcl-xL, survivin, IAP1/2, cyclin D1, cyclin E, COX-2, VEGF, and MMP-9 as described in “Materials and Methods.” The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of 3 independent experiments.
Figure 4.
Figure 4.
SEYG induces apoptosis by PARP cleavage through activation of caspase-3. (A) DU145 cells (1 × 106 cells/well) were incubated with the indicated concentrations of SEYG for 24 hours. Whole-cell extracts were prepared, and 20 µg of the whole-cell lysate was resolved by SDS-PAGE, electrotransferred to nitrocellulose membrane, sliced from the membrane based on the molecular weight, and then probed with antibodies against p53 and p21 as described in “Materials and Methods.” The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of 3 independent experiments. (B) After DU145 cells (1 × 106 cells/well) were seeded onto 6-well plates, they were treated with various indicated concentrations of SEYG for 24 hours. Thereafter, equal amounts of lysates were analyzed by Western blot analysis using antibodies against caspase-3 and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) MEF cells were transiently transfected with pMXs-STAT3C or pMXs-gw (control vector) plasmid. STAT3C protein was overexpressed in pMXs-STAT3C tranfected MEF cells compared to control. Transiently transfected cells were treated with indicated concentrations of SEYG for 6 hours. Then, equal amounts of lysate were analyzed by Western blot analysis using antibodies against phospho-STAT3 (Tyr705) and STAT3. (D) MEF cells were transiently transfected with pMXs-STAT3C or pMXs-gw (control vector) plasmid. STAT3C protein was overexpressed in pMXs-STAT3C tranfected MEF cells compared to control. Transiently transfected cells were treated with indicated concentrations of SEYG for 24 hours. Equal amounts of lysate were analyzed by Western blot analysis using antibody against cyclin D1 and PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of 3 independent experiments. (E) After DU145 cells (1 × 106 cells/well) were seeded onto 6-well plates, they were incubated with the indicated concentrations of SEYG for 24 hours. Then, the cells were fixed and analyzed using flow cytometry. The results shown here are representative of 3 independent experiments. (F) Cells were treated with indicated concentrations of SEYG for 24 hours. Thereafter, they were incubated with anti-annexin V antibody conjugated with FITC plus PI and analyzed with a flow cytometer for apoptotic effects. The results shown here are representative of 3 independent experiments. (G) Cells were treated with indicated concentrations of SEYG and caspase-3 inhibitor for 24 hours. Thereafter, they were incubated with anti-annexin V antibody conjugated with FITC plus PI and analyzed with a flow cytometer for apoptotic effects. The results shown here are representative of 3 independent experiments. (H) Cells were treated with indicated concentrations of SEYG for 24 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed with a flow cytometer.
Figure 5.
Figure 5.
Effects of SEYG in human prostate cancer cells growth in nude mice induced by DU145. (A) Schematic representation of experimental protocol described in “Materials and Methods.” DU145 cells (4 × 106 cells/mice) were injected subcutaneously into the right flank of the mice. The animals were randomized after 1 week of tumor cell injection into 3 groups based on tumor volume. Group I was given PBS (200 µL, ip thrice/week), group II was given SEYG (50 mg/kg body weight, ip thrice/week), and group III was given SEYG (200 mg/kg body weight, ip thrice/week). (B) Necropsy photographs of mice bearing subcutaneously implanted prostate tumors. (C) Tumor volumes in mice measured during the course of experiment and calculated using the formula V = 4/3πr3, ** indicates P < .01. (D) Tumor volumes in mice measured on the last day of the experiment at autopsy using Vernier calipers and calculated using the formula V = 4/3πr3 (n = 11). Columns, mean; bars, SD. (E) Tumor weight was measured at the end of the experiment. Columns, mean; bars, SD.
Figure 6.
Figure 6.
SEYG exerts the effect against tumor cell proliferation and angiogenesis in prostate cancer. (A) Immunohistochemical analysis of proliferation marker Ki-67+ cell indicates the inhibition of human prostate cancer cells proliferation by SEYG dose-dependent treated groups of animals. Samples from 3 animals in each treatment group were analyzed, and representative data are shown (A, left panel). Quantification of Ki-67 proliferation index as described in “Materials and Methods.” Values are represented as mean ± SD of triplicate (A, right panel). Columns, mean of triplicate; bars, SD. (B) Immunohistochemical analysis of CD31 for microvessel density in prostate tumors indicates the inhibition of angiogenesis by SEYG dose-dependent treated groups of animals. Samples from 3 animals in each treatment group were analyzed, and representative data are shown (B, left panel). Quantification of CD31 angiogenesis index as described in “Materials and Methods.” Values are represented as mean ± SD of triplicate (B, right panel). Columns, mean of triplicate; bars, SD. (C) Immunohistochemical analysis of cleaved caspase-3 in prostate tumors. Samples from 3 animals in each treatment group were analyzed, and representative data are shown (C, left panel). Quantification of cleaved caspase-3 as described in “Materials and Methods.” Values are represented as mean ± SD of triplicate (C, right panel). Columns, mean of triplicate; bars, SD. (D) Western blot analysis showed the inhibition of p-STAT3, p-JAK1, p-JAK2, and p-Src by SEYG in whole cell extracts from animal tissue. The same blots were stripped and reprobed with STAT3, JAK1, JAK2, and Src antibody to verify equal protein loading. (E-G) Equal amounts of lysates were analyzed by Western blot analysis using antibodies against bcl-2, bcl-xL, survivin, IAP-1, IAP-2, COX-2, cyclin D1, cyclin E, VEGF, MMP-9, p53, and p21. β-Actin was used as a loading control. Western blotting samples from 3 mice in each group were analyzed and representative data are shown.
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