Expression and functional analysis of Toll-like receptor 4 in human cervical carcinoma.

Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV-) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells.

Mesenchymal stem cells as carriers and amplifiers in CRAd delivery to tumors.

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern, kinetic delivery of adenovirus, and therapeutic efficacy of the MSC loading of E1A mutant conditionally replicative adenovirus Adv-Stat3(-) which selectively replicated and expressed high levels of anti-sense Stat3 complementary DNA in breast cancer and melanoma cells. METHODS: We assessed the release ability of conditionally replicative adenovirus (CRAd) from MSC using crystal violet staining, TCID(50) assay, and quantitative PCR. In vitro killing competence of MSCs carrying Adv-Stat3(-) toward breast cancer and melanoma was performed using co-culture system of transwell plates. We examined tumor tropism of MSC by Prussian blue staining and immunofluorescence. In vivo killing competence of MSCs carrying Adv-Stat3(-) toward breast tumor was analyzed by comparison of tumor volumes and survival periods. RESULTS: Adv-Stat3(-) amplified in MSCs and were released 4 days after infection. MSCs carrying Adv-Stat3(-) caused viral amplification, depletion of Stat3 and its downstream proteins, and led to significant apoptosis in breast cancer and melanoma cell lines. In vivo experiments confirmed the preferential localization of MSCs in the tumor periphery 24 hours after tail vein injection, and this localization was mainly detected in the tumor parenchyma after 72 hours. Intravenous injection of MSCs carrying Adv-Stat3(-) suppressed the Stat3 pathway, down-regulated Ki67 expression, and recruited CD11b-positive cells in the local tumor, inhibiting tumor growth and increasing the survival of tumor-bearing mice. CONCLUSIONS: These results indicate that MSCs migrate to the tumor site in a time-dependent manner and could be an effective platform for the targeted delivery of CRAd and the amplification of tumor killing effects.

Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer.

BACKGROUND: P21(WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. METHODS: RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. RESULTS: p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. CONCLUSIONS: Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment.

TLR9 expression and its role in chemosensitivity to DDP in human cervical cancer cells in vitro.

Inflammation and infection play an important role in the pathogenesis of many cancers. Toll-like receptors (TLRs) are a class of pattern recognition receptors that recognize conserved components of microbes and trigger the immune response against invading microorganisms. Toll-like receptor 9 (TLR9) recognizes non-methylated cytosine-phosphateguanosine (CpG) DNA sequences which are the surrogate for viral DNA. TLR9 may react to tumor development and progression during chronic inflammation that involves the tumor microenvironment. In order to study the role of TLR9 in cervical cancer, we analyzed the TLR9 expression in different types of HPV infection cervical cancer cells. Then we detected if CpG sequences influenced the TLR9 expression and the sensitivity to cisplatin (DDP) of these cervical cancer cells in vitro. The expression of TLR9 mRNA and protein in SiHa, Hela and C33A cells was detected by RT-PCR and Western blotting. Real-time PCR was used to examine the TLR9 expression changes induced by CpG. Chemosensitivity of the cervical cancer cells to cisplatin (DDP) was measured by MTT. It was observed that the expression of TLR9 mRNA and protein was increased gradually in SiHa (HPV16+), Hela (HPV18+) and C33A (HPV-) cells. Low doses of CpG increased the TLR9 expression only in C33A (HPV-) cells, but not in SiHa (HPV16+) and Hela (HPV18+) cells. Furthermore, low dose of CpG significantly increased the sensitivity of C33A (HPV-) cells, but not that of SiHa (HPV16+) and Hela (HPV18+) cells. These results indicated that TLR9 may serve as a protective agent in HPV negative cervical cancer cells. It was concluded that TLR9 could improve the sensitivity to DDP in HPV negative cervical cancer cells and might represent a potential therapeutic option in clinical practice.

Distinct Side Population Cell Subtypes Have Different Stemness Levels in Human Ovarian Cancer Cells.

The stemness of different side population (SP) cell subtypes in ovarian cancer cells was studied, and the heterogeneity of ovarian cancer stem cells was analyzed. The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342. A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP (LSP) cells, upper-SP (USP) cells, and non-SP cells (NSP) based on their sensitivity to the staining time and Hoechst dye concentration. The sphere-forming capability, expression levels of stem cell markers, resistance to high concentrations of cisplatin, and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated. The C13 cells contained SP cells with stemness characteristics, and the LSP cell subtype expressed higher levels of stem cell markers, had higher in vitro sphere-forming capability, higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells (P<0.05). NSP cells had no stemness. In conclusion, different subtypes of ovarian cancer SP cells have different stemness levels, and ovarian cancer stem cells may be heterogeneous.

Relationship between the expression of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase and survival in epithelial ovarian cancer.

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14+/-0.62 and 0.59+/-0.06 in tumor tissue, and 0.71+/-0.14 and 0.16+/-0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11+/-0.02) than in normal controls (0.38+/-0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients' prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.

A multi-shRNA vector enhances the silencing efficiency of exogenous and endogenous genes in human cells.

RNA interference (RNAi) is a powerful technology for suppressing gene function. In most studies, small interfering RNAs (siRNAs) consist of one short hairpin RNA (shRNA) and, therefore, are often unable to achieve loss-of-function of their target genes. In the current study, an RNAi vector containing three shRNAs under the control of three RNA polymerase III U6 promoters was constructed. RNAi vectors containing one or two shRNAs were generated for comparisons. A pilot study targeting exogenously expressed DsRed in the HEK293 cell line revealed promising effects and a high selectivity for the multi-shRNA RNAi vector. Akt2 is constitutively expressed in cultured SKOV3 human ovarian cancer cells, and the multi-shRNA RNAi vector showed a strong efficiency for downregulating the expression of Akt2 in these cells, with no apparent interferon response. In addition, the Akt2-3shRNA vector, containing three shRNAs targeting Akt2, showed the best effect of all the shRNA vectors in reversing paclitaxel-induced resistance in SKOV3 cells. This study developed a widely applicable resource for enhancing the efficiency of gene silencing and a novel technique for performing complex loss-of-function screens in mammalian cells.

Akt mediates metastasis-associated gene 1 (MTA1) regulating the expression of E-cadherin and promoting the invasiveness of prostate cancer cells.

Human metastasis-associated gene 1 (MTA1) is highly associated with the metastasis of prostate cancer; however, the molecular functions of MTA1 that facilitate metastasis remain unclear. In this study, we demonstrate that the silencing of MTA1 by siRNA treatment results in the upregulation of E-cadherin expression by the phosphorylation of AKT (p-AKT) and decreases the invasiveness of prostate cancer cells. We show that MTA1 is expressed in over 90% of prostate cancer tissues, especially metastatic prostate cancer tissue, comparing to non-expression in normal prostate tissue. RT-PCR analysis and Western blot assay showed that MTA1 expression is significantly higher in highly metastatic prostate cancer PC-3M-1E8 cells (1E8) than in poorly metastatic prostate cancer PC-3M-2B4 cells (2B4). Silencing MTA1 expression by siRNA treatment in 1E8 cells increased the cellular malignant characters, including the cellular adhesive ability, decreased the cellular invasive ability and changed the polarity of cellular cytoskeleton. 1E8 cells over-expressing MTA1 had a reduced expression of E-cadherin, while 1E8 cells treated with MTA1 siRNA had a higher expression of E-cadherin. The expression of phosphorylated AKT (p-AKT) or the inhibition of p-AKT by wortmannin treatment (100 nM) significantly altered the function of MTA1 in the regulation of E-cadherin expression. Alterations in E-cadherin expression changed the role of p-AKT in cellular malignant characters. All of these results demonstrate that MTA1 plays an important role in controlling the malignant transformation of prostate cancer cells through the p-AKT/E-cadherin pathway. This study also provides a new mechanistic role for MTA1 in the regulation of prostate cancer metastasis.

C4orf7 contributes to ovarian cancer metastasis by promoting cancer cell migration and invasion.

Gene C4orf7, renamed FDC-SP (follicular dendritic cell secreted protein, FDC-SP) was first isolated from human tonsils. Up to the present, the function of this gene was still poorly understood. In this study, we report the expression of gene C4orf7 in a panel of tumor types. The percentages of C4orf7 positive expression in the tumor patients with metastases were notably higher than those in the cancer without metastases. On the contrary, the expression was hardly noted in normal tissues and corresponding benign lesions. In vitro, the up-regulation of C4orf7 in ovarian cancer cell lines was associated with enhanced motility and invasiveness. Furthermore, the overexpression of C4orf7 resulted in Akt ser473 phosphorylation and decreased E-cadherin expression. The role of C4orf7 in ovarian cancer cell morphology, motility and invasion was demonstrated.

Implication of the Akt2/survivin pathway as a critical target in paclitaxel treatment in human ovarian cancer cells.

PURPOSE: Although multiple mechanisms have been implicated in paclitaxel (PTX)-induced resistance in ovarian cancer, recent evidence has suggested that Akt2 has an important role in the protection of cells from paclitaxel-induced apoptosis. In the present study, we investigated the role of the Akt2/survivin pathway in paclitaxel-induced resistance by a modified method to generate an effective shRNA vector. METHODS: We applied RNAi-mediated silencing techniques to investigate the mechanism of the Akt2/survivin pathway on PTX-induced resistance in ovarian cancer cells (A2780 and SKOV3). The expression of Akt2 and survivin mRNA and related protein levels were evaluated with semiquantitative real-time RT-PCR and western blot analysis, respectively. Inhibition of cell proliferation was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, and the induction of apoptosis was examined through flow cytometry (FACS) and Hoechst staining. RESULTS: Akt2 down-regulation sensitized ovarian cancer cells to paclitaxel-induced apoptosis, and inhibited survivin expression. We further demonstrated that suppressing the inhibition of survivin expression can induce the drug-resistance to paclitaxel. We introduced a modified vector to generate shRNA to induce RNA interference, which contained three U6 promoters to express different shRNAs; it severely reduced Akt2 gene expression and showed good specificity. CONCLUSION: Our findings will aid in understanding the molecular mechanism of paclitaxel-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.

Inhibition of Akt signaling by SN-38 induces apoptosis in cervical cancer.

Cervical cancer still remains a major health problem in women worldwide. Inhibitors of topoisomerase I have proven to be among the most promising new classes of anti-neoplastic agents introducing into the clinic in recent years. CPT-11 is one of the most widely used Camptothecin analogues and is converted to form the active metabolite SN-38. The study tried to explore the in vitro mechanisms of apoptosis induced by SN-38 in cervical cancer cell lines HeLa and SiHa. The results demonstrated here that SN-38 inhibited cell proliferation in a time- and dose-dependant manner. Western Blot showed that SN-38 down-regulated protein expression of p-Akt and increased protein expression of p53 and p21, but it had no effects on protein expression of Bax, Bcl-2 and Akt. Transfection of the full-length Akt cDNA into HeLa and SiHa cells resulted in the reduction of apoptosis induced by SN-38, and Akt kinase activity regulated the p53 pathway, indicating that inhibition of the Akt pathway played an important role in exhibition of SN-38-mediated cytotoxic effect. Our data suggested that SN-38 could induce apoptosis through a p53 pathway and that activation of p53 in response to S-38 is governed by Akt.

Reversal of the malignant phenotype of ovarian cancer A2780 cells through transfection with wild-type PTEN gene.

OBJECTIVE: PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene identified on human chromosome 10q23. Substantial studies have demonstrated that PTEN can inhibit cell proliferation, migration and invasion of many cancer cells. The purpose of this study was to determine whether upregulation of PTEN gene by transfection wild-type PTEN gene to ovarian cancer cells can inhibit growth and migration and to explore the potential for PTEN gene therapy of ovarian cancers. METHOD: Wild-type and phosphatase-inactive (C124A) PTEN plasmids were transfected into ovarian epithelial cancer A2780 cells, and their effects on cell apoptosis, cell proliferation, cell migration and cell invasion were analyzed by flow cytometry analysis, TUNEL assay, MTT assay, wound-healing assay and transwell assay. RESULTS: Both wild-type and mutant PTEN can upregulate the expression of PTEN gene dramatically; however, it is wild-type PTEN not phosphatase-inactive PTEN that can induce apoptosis and decrease cell migration, invasion and proliferation in ovarian cancer cells. CONCLUSION: These results demonstrated that PTEN had played an important role in the cell proliferation, cell migration and invasion dependent on its phosphatase activity. Enhanced expression of PTEN by gene transfer is sufficient to reverse the malignant phenotype of ovarian cancer cells and transfection of ovarian cancer cells with wild-type PTEN gene might be another novel approach for therapeutic intervention in ovarian cancer.