Shanling Liu

The antitumoral effect of Paris Saponin I associated with the induction of apoptosis through the mitochondrial pathway

Rhizoma Paridis, a traditional Chinese medicine, has shown promise in cancer prevention and therapy. In the present study, we isolated Paris Saponin I (PSI), an active component of Rhizoma paridis, and evaluated its effects on a panel of human cell lines and in a mouse model of human ovarian cancer to explore the mechanisms of its activity. PSI had more potent and selective cytotoxic effects on tumor cell lines than etoposide had, promoting dramatic G2-M phase arrest and apoptosis in SKOV3 cells in a time- and dose-dependent manner. Furthermore, PSI treatment increased levels of Bax, cytochrome c, activated caspase-3, active caspase-9, and cleaved poly(ADP-ribose) polymerase and decreased both Bcl-2 expression levels and extracellular signal–regulated kinase-1/2 activity. We also assessed the antitumor efficacy of i.p. and p.o. PSI administration in mice bearing SKOV3 tumors; both significantly inhibited the growth of SKOV3 cells in a subcutaneous xenograft mouse model (by 66% and 52%, respectively). These results indicate that PSI mediates its effects via mitochondrial apoptosis, mitogen-activated protein kinase pathways, and G2-M cell cycle arrest. Most important, the efficacy of PSI in xenografts when administered p.o. or i.p. suggests its clinical potential. Thus, PSI is a potent antitumor compound and should be developed as a natural agent for cancer therapy.[Mol Cancer Ther 2009;8(5):1179–88]

USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma

BackgroundUbiquitination is a reversible process of posttranslational protein modification through the action of the family of deubiquitylating enzymes which contain ubiquitin-specific protease 9x (USP9X). Recent evidence indicates that USP9X is involved in the progression of various human cancers. The aim was to detect the expression of USP9X in the progression from normal epithelium to invasive esophageal squamous cell cancer (ESCC) and evaluate the relevance of USP9X expression to the tumor progression and prognosis.MethodsIn this study, USP9X immunohistochemical analysis was performed on tissues constructed from ESCC combined with either normal epithelium or adjacent precursor tissues of 102 patients. All analyses were performed by SPSS 13.0 software.ResultsWe observed that the level of high USP9X expression increased gradually in the transformation from normal epithelium (4.0%), to low grade intraepithelial neoplasia (10.5%), then to high grade intraepithelial neoplasia (28.6%), and finally to invasive ESCC (40.2%). The expression of USP9X was found to be significantly different between the normal mucosa and ESCC (P < 0.001), and between low grade intraepithelial neoplasia and high grade intraepithelial neoplasia (p = 0.012). However, no difference was observed between the high expression of USP9X in normal mucosa and low grade intraepithelial neoplasia (P = 0.369), nor between high grade intraepithelial neoplasia and ESCC (p = 0.115). Interestingly, the most intensive staining for USP9X was usually observed in the basal and lower spinous layers of the esophageal epithelium with precursor lesions which often resulted in the earliest malignant lesion. USP9X expression status was positively associated with both depth of invasion (p = 0.046) and lymph node metastasis (p = 0.032). Increased USP9X expression was significantly correlated to poorer survival rate in ESCC patients (p = 0.001). When adjusted by multivariate analysis, USP9X expression (HR 2.066, P = 0.005), together with TNM stage (HR 1.702, P = 0.042) was an independent predictor for overall survival.ConclusionsUp-regulation of USP9X plays an important role in formation and progression of precancerous lesions in ESCC and USP9X expression levels were significantly correlated with the survival of ESCC patients. Thus, USP9X could be considered as a potential biomarker and prognostic predictor for ESCC.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1945302932102737

Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

BackgroundHuman papillomavirus (HPV) infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC) and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN) and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening.MethodsExfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27), cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24) and cervical carcinoma (CA, n=21), and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH) probe and HPV-DNA with Hybrid Capture 2.From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP) and expression of human telomerase reverse transcriptase (hTERT), with immunohistochemistry (IHC). All biopsies were clinically confirmed by phathologists.ResultsAmplification of hTERC was significantly associated with the histologic diagnoses (p<0.05). The positive correlation was found between the level of hTERC amplification and histologic grading of dysplasia (CIN2/3 from CIN1 or normal, P=0.03). A profounding increase in the accumulation of HPV and hTERC positive cases was observed in the CIN3 subgroup compared with the CIN2 group, 25% versus 62.96%, respectively (p=0.007).ConclusionshTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1857134686755648.

miR-126 Suppresses the Proliferation of Cervical Cancer Cells and Alters Cell Sensitivity to the Chemotherapeutic Drug Bleomycin

In cervical cancer, one of the most common malignant tumors in women worldwide, miR-126 has been reported to exhibit decreased expression. However, its role in cervical cancer cell proliferation and drug sensitivity has remained relatively unexplored. Here, we compared the expression of miR-126 in cervical cancer tissues (n = 20) with that in normal cervical tissue (n = 20) using quantitative RT-PCR. The viability of Siha cervical cancer cells was further measured by MTT assay after transfection with miR-126 mimic (Siha-miR-126 mimic) or microRNA mimic negative control (Siha-miR mimic NC) and after treatment with various concentrations of bleomycin (BLM). IC50s were calculated, and the survival rates (SRs) of Siha cells were calculated. miR-126 expression in cervical cancer tissue was significantly decreased compared with that in normal cervical tissue (P < 0.01). The relative SRs of Siha-miR-126 mimic cells were also significantly decreased compared with those of Siha-miR mimic NC cells at 24-96 h after transfection. The IC50 of BLM in Siha-miR-126 mimic cells (50.3 ± 2.02 μg/mL) was decreased compared with that in Siha-miR mimic NC cells (70.5 ± 4.33 μg/mL) at 48 h after transfection (P < 0.05). Finally, the SRs of Siha-miR-126 mimic cells were significantly lower than those of Siha- miR mimic NC cells after cultured in medium containing 40 μg/mL BLM for 24-96 h (P < 0.05). These results suggest that miR-126 is expressed at low levels in cervical cancer. Upregulation of miR-126 inhibited cervical cancer cell proliferation and enhanced the sensitivity to BLM. Thus, miR-126 may represent a novel approach to cervical cancer treatment.

Paris Saponin II suppresses the growth of human ovarian cancer xenografts via modulating VEGF-mediated angiogenesis and tumor cell migration

PurposeParis Saponin II (PSII) is an active component of Rhizoma Paridis—an essential ingredient in traditional Chinese herbal medicines. PSII can induce cytotoxic effects in cancer cells and inhibit ovarian cancer growth. Since pathological angiogenesis (henceforth, angiogenesis) is often associated with gynecological cancers, here, we investigated whether PSII renders effects on angiogenesis and examined possible molecular mechanisms underlying the effects of PSII.MethodsThe effects of PSII on the biofunctions of endothelial cells (EC), the crucial components of blood vessels, were examined by standardized angiogenesis in vitro and ex vivo assays, Western blot analysis, ELISA, and kinase assay. Angiogenesis in a xenograft mouse model of ovarian cancer was evaluated by color Doppler ultrasound and immunohistochemistry.ResultsPSII exerted marked inhibitory effect on the growth of VEGF-stimulated human umbilical vein endothelial cells in a dose–time-dependent manner, inhibited cell’s motility, and interfered with tubulogenesis. PSII also blocked microvessel outgrowth in a rat aortic ring assay and compromised angiogenesis in a mouse model of ovarian carcinoma using either SKOV3 or HOC-7 cell lines. VEGF levels in PSII-treated EC and tumor cells were reduced. In EC, PSII blocked the activation of VEGFR2 in dose-dependent manner leading to the reduction of VEGF-induced phosphorylation on several intracellular pro-angiogenic kinase, including the extracellular signal-related kinase, Src family kinase, focal adhesion kinase, and AKT kinase.ConclusionsThe results provided the first insight into the anti-angiogenesis properties of Saponin family in solid tumors and suggested a promising therapeutic potential of PSII in the ovarian cancer treatment.

Accumulation of invariant NKT cells with increased IFN-γ production in persistent high-risk HPV-infected high-grade cervical intraepithelial neoplasia.

BackgroundPersistent high-risk human papillomavirus (HR-HPV) infection has been implicated in the development of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. Invariant natural killer T (iNKT) cells produce large amounts of cytokines to regulate immune responses. However, the role of iNKT cells in human persistent HPV-infected cervical tissues is unknown.MethodsIn our study, 201 patients with diagnoses ranging from normal ectocervical tissue to CINIII from June 2010 to May 2012 were enrolled. HPV DNA and HPV types were detected using the hybrid capture-2 HPV DNA test. Flow cytometry was used to investigate iNKT and CD3+ T cell infiltration into cervical tissues. Real-time quantitative reverse transcription-polymerase chain reaction was used to study IFN-γ expression and immunohistochemistry was used to determine CD3+ T cell distribution.ResultsA significant increase in iNKT cells was observed in HPV-positive cervical tissues (p < 0.05), especially in CINII-III (p < 0.01). IFN-γ expression was also increased in HPV-positive cervical tissues (p < 0.05). CD3+ T cells were detected among both epithelium and stromal layers in cervical tissues, and the percentage of CD3+ T cells in HPV-positive cervical tissues was similar to that in HPV-negative cervical tissues (p > 0.05).ConclusionsThe iNKT cell aggregation in cervical tissues during the progression from HPV infection to CIN indicates that iNKT cells might play an important role in suppressing immunity. IFN-γ expression could also be related to the HPV infection status. Preventing the accumulation or functioning of iNKT cells in cervical tissues may be a viable method to prevent the development of CIN.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2521874671514142

Establishment of a new representative model of human ovarian cancer in mice

BackgroundIntraperitoneal (i.p.) models that accurately mimic the feature behavior of human ovarian cancer are required to investigate the pathology and therapeutics of the disease. However, established i.p. models which are well-characterized and reliable are few. The purposes of this study are to establish a representative mice i.p. model of the disease and to analyze the consequent pathology.MethodsFresh tumor cells fiom the ascites of patient were injected into female NOD/SCID mice intraperitoneally. Histology, Cytogenetic, immunohistochemistry,tumor markers of CA125,AFP, CA-199 and CEA were used to analyze the model.ResultsThe mice developed marked abdominal distention within 6 months after inoculated with tumor cells from a patient with epithelial ovarian carcinoma. The mice developed clinically evident intraperitoneal tumors and massive ascites containing numerous tumor cells in clumps. CA125 level in our model was high in both serum and ascites supernatants, while levels of other tumor markers, such as AFP, CA-199 and CEA, were normal. Cytogenetic analysis and immunohistochemical staining confirmed its characteristics resembling human epithelial ovarian tumor.ConclusionsThe model described in this paper accurately mimics the features of ovarian tumor, which may be useful for evaluation of new therapeutics.

Noninvasive Prenatal Diagnosis of Down Syndrome in Samples from Southwest Chinese Gravidas Using Pregnant Plasma Placental RNA Allelic Ratio

OBJECTIVE The purpose of this research was to attempt a preliminary study of noninvasive prenatal diagnosis of Down syndrome in Southwest Chinese gravidas by using the plasma placental RNA allelic ratio. METHODS The genotypes of the single-nucleotide polymorphisms (SNPs) located in the transcribed regions of the gene PLAC4 were detected in population samples collected in Southwest China by using polymerase chain reaction-restriction fragment length polymorphism, and SNPs with a higher heterozygosity were selected. Mass spectrometer analysis was adopted, and cases with the heterozygous SNPs on PLAC4 mRNA in maternal plasma were selected from 29 pregnancies with a euploid fetus and from 21 pregnancies with a trisomy-21 fetus, and then their RNA-SNP allelic ratios were further determined for noninvasive prenatal diagnosis of Down syndrome. RESULTS Of all 50 singleton pregnancies, 37 gravidas were found with at least one heterozygous SNP on PLAC4 mRNA in maternal plasma. Among them, 13 pregnancies with a trisomy-21 fetus were detected by the analysis of the RNA-SNP allelic ratio. CONCLUSION The plasma placental RNA allelic ratio can be used for noninvasive prenatal diagnosis of Down syndrome, if SNPs on PLAC4 mRNA in maternal plasma are heterozygous.

The specifical inhibition of the expression of integrin alpha5/beta1 probably enhances the treatment effects and improves the prognosis of epithelial ovarian cancer

Due to subtle symptoms and the absence of effective early screening methods, most of epithelial ovarian cancer patients are diagnosed in the late stage, when current treatment strategies are not so satisfactory. To date, ovarian cancer is still the leading cause of gynecological malignancy deaths in women. The formation of massive ascites is one of the important characteristics of epithelial ovarian carcinoma in the late stage. Cancer cells, existing in ascites in the form of spheroids, play an important role in metastasis and recurrence of the malignancy. Alpha5/beta1 integrin has been found to participate in the formation of epithelial ovarian cancer multicellular spheroids in vitro. But if we want to choose alpha5- and beta1-integrin subunits as treatment targets, we must specifically block the two subunits in cancer cells, because these two subunits are very important for the physiological activities in normal cells. Based on the knowledge mentioned above, we present hypotheses that we can inhibit specifically the expression of alpha5/beta1 integrin in cancer cells with the help of complementary replication defective adenovirus. As a result, the formation of cancer cells spheroids in ascites might be interfered with and the treatment effects and prognosis of epithelial ovarian cancer might be improved.

1‑calcium phosphate‑uracil, a synthesized pyrimidine derivative agent, has anti‑proliferative, pro‑apoptotic and anti‑invasion effects on multiple tumor cell lines

1-calcium phosphate-uracil (1-CP-U), a synthetic pyrimidine derivative, has been documented to demonstrate a variety of different biological activities. However, the potency and mechanisms of this agent’s anti-cancer activity have not been elucidated to date. In the present study, the anti-cancer effects of 1-CP-U were examined in a range of in vitro assays. Different cell lines were treated with 1-CP-U at varied concentrations (0.7, 1.0, 1.4 μmol/l) for indicated durations. The cell proliferation was then examined by MTT assay. The cellular apoptotic effects were detected by Hoechst 33342 and Annexin V/propidium iodide staining, while the capacity of 1-CP-U on invasion and migration were examined by cell invasion and wound healing assays. The expression of matrix metalloproteinase proteins, as well as pro- and antiapoptotic proteins was detected by western blotting analysis. The results identified that 1-CP-U was able to inhibit the viability of SKOV3, HeLa, SMMC-7721 and A549 cell lines in a dose- and time-dependent manner, while it exerted only marginal toxic effects on non-cancerous cells. The IC50 concentration of 1-CP-U for tumor cell lines was ~1.0 μmol/l. The growth inhibition induced by 1-CP-U was accompanied by a broad spectrum of pro-apoptotic activities, in which different cell lines varied in their sensitivity to 1-CP-U. Meanwhile, the increased expression of the pro-apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl-2 levels were associated with increased 1-CP-U concentrations. Additionally, anti-migration and anti-invasion effects of 1-CP-U were evidently associated with the downregulation of matrix metalloproteinase proteins. Of note, it was observed that 1-CP-U significantly inhibited both the migration and invasion at a lower concentration, as compared with the dose required to achieve significant inhibition of apoptosis. These results indicated that 1-CP-U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis. In conclusion, the present study was the first, to the best of our knowledge, to demonstrate the function of 1-CP-U in tumor proliferation, apoptosis and invasion with specific effects against cancer cells in vitro, suggesting 1-CP-U as a potential novel anticancer agent.