Effects of CTS on cell cycle status in SiHa cervical cancer cells.

<p>(A) Cell cycle profiles of CTS treated SiHa cells. (B) The proportion of cells in the sub-G1 phase. Results of **<i>p</i> < 0.005 were considered statistically significant. CTS-treated cells were compared to untreated cells.</p

The HPLC analyses of composition in <i>Cudrania tricuspidata</i> stem (CTS) extract.

<p>The seventeen kinds of the phenolic acid composition of the sample were analyzed by comparing the spectrum of the sample and standards components matched to create a standard curve from Peak area per component to quantify the amount of change. (A) The seventeen kinds of the reference phenolic acid compounds. (B) <i>Cudrania tricuspidata</i> stem (CTS) extract. The HPLC analysis showed the presence of eight compounds corresponding to 8 among 17 standard compounds. The 5, 7, 11 peaks represented chlorogenic acid, caffeic acid, and veratric acid, respectively.</p

Effects of CTS on SiHa cervical cancer cell morphological changes and apoptosis.

<p>(A) Microscopic images of SiHa cells treated with CTS for 24 h. The photographs were taken by phase-contrast microscopy at 100× magnification. (B) Fluorescence microscopic images of SiHa cells treated with CTS for 24 h. Nuclear condensation and chromatin shrinkage were observed. (C) The data on apoptotic nuclei of whole DAPI stained cells were summarized as bar graphs. Results of *<i>p</i> < 0.05 and **<i>p</i> < 0.005 were considered statistically significant. (D) After treatment with the indicated concentration of CTS for 24 h, SiHa and HaCaT cells were stained with annexin V-FITC/PI.</p

Effects of CTS treatment on oncoprotein E6/E7 mRNA levels and protein levels of E6/E7 targeting p53, pRb, and p-pRb.

<p>(A) mRNA levels of oncoproteins E6 and E7 as detected by qRT-PCR. (B) Western blot analyses of pRb, p-pRb, p53, p21, and p27. SiHa cells were treated with the indicated concentration of CTS for 24 h.</p

<i>Cudrania tricuspidata</i> Stem Extract Induces Apoptosis via the Extrinsic Pathway in SiHa Cervical Cancer Cells

<div><p>The focus of this study is the anti-cancer effects of <i>Cudrania tricuspidata</i> stem (CTS) extract on cervical cancer cells. The effect of CTS on cell viability was investigated in HPV-positive cervical cancer cells and HaCaT human normal keratinocytes. CTS showed significant dose-dependent cytotoxic effects in cervical cancer cells. However, there was no cytotoxic effect of CTS on HaCaT keratinocytes at concentrations of 0.125–0.5 mg/mL. Based on this cytotoxic effect, we demonstrated that CTS induced apoptosis by down-regulating the E6 and E7 viral oncogenes. Apoptosis was detected by DAPI staining, annexin V-FITC/PI staining, cell cycle analysis, western blotting, RT-PCR, and JC-1 staining in SiHa cervical cancer cells. The mRNA expression levels of extrinsic pathway molecules such as Fas, death receptor 5 (DR5), and TNF-related apoptosis-inducing ligand (TRAIL) were increased by CTS. Furthermore, CTS treatment activated caspase-3/caspase-8 and cleavage of poly (ADP-ribose) polymerase (PARP). However, the mitochondrial membrane potential and expression levels of intrinsic pathway molecules such as Bcl-2, Bcl-xL, Bax, and cytochrome C were not modulated by CTS. Taken together, these results indicate that CTS induced apoptosis by activating the extrinsic pathway, but not the intrinsic pathway, in SiHa cervical cancer cells. These results suggest that CTS can be used as a modulating agent in cervical cancer.</p></div

Effects of CTS on extrinsic pathway-related factors in SiHa cervical cancer cells.

<p>(A) mRNA levels of TRAIL, DR5 and Fas as detected by qRT-PCR. (B) Western blot analysis of extrinsic pathway-related factors. SiHa cells were treated with the indicated concentration of CTS for 24 h. (C) Western blot analysis of the effects of CTS following pretreatment with general caspase inhibitor Z-VAD-FMK or caspase-8 inhibitor Z-IETD-FMK in SiHa cells.</p

Cytotoxic effects of CTS extract on cervical cancer cells and normal keratinocytes.

<p>(A) HaCaT, (B) SiHa and CaSki cells were treated for 24–48 h with various concentrations of CTS extract, after which cell viability was investigated using the MTS assay. Results of *p < 0.05 and **p < 0.005 were considered statistically significant. The CTS-treated cells were compared to the control cells.</p

Volatile compounds identified in <i>Cudrania tricuspidata</i> stem (CTS) extract.

<p>Volatile compounds identified in <i>Cudrania tricuspidata</i> stem (CTS) extract.</p

Quantitative HPLC analyses of phenolic compounds in the <i>Cudrania tricuspidata</i> stem (CTS) extract.

<p>Quantitative HPLC analyses of phenolic compounds in the <i>Cudrania tricuspidata</i> stem (CTS) extract.</p