Secondary Metabolites from the Cultures of Medicinal Mushroom Vanderbylia robiniophila and Their Tyrosinase Inhibitory Activities.

Vanderbylia robiniophila (Huaier in Chinese) has been used as a traditional herbal medicine in China for over 1600 years. However, the secondary metabolites of V. robiniophila have not been systematically examined. Corresponding chemical investigation in this study led to the discovery of two new compounds, (22E, 24R)-6ß, 7α-dimethoxyergosta-8(14), 22-diene-3ß, 5α-diol (1) and vanderbyliolide A (8), along with eight known ones (2-7, 9-10). Their structures were determined by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. The tyrosinase inhibitory activity of all isolated compounds was evaluated, and compound 10 showed a potential tyrosinase inhibitory effect with an IC50 value of 60.47 ± 2.63 µM. Kinetic studies of the inhibition reactions suggested that 10 provides the inhibitory ability on tyrosinase in an uncompetitive way.

Fluorescent TPE Macrocycle Relayed Light-Harvesting System for Bright Customized-Color Circularly Polarized Luminescence.

Artificial systems for sequential chirality transmission/amplification and energy relay are perpetual topics that entail learning from nature. However, engineering chiral light-harvesting supramolecular systems remains a challenge. Here, we developed new chiral light-harvesting systems with a sequential Förster resonance energy transfer process where a designed blue-violet-emitting BINOL (1,1'-Bi-2-naphthol) compound, BINOL-di-octadecylamide (BDA), functions as an initiator of chirality and light absorbance, a new green-emitting hexagonal tetraphenylethene-based macrocycle (TPEM) with aggregation-induced emission serves as a conveyor, and Nile red (NiR) or/and a near-infrared dye, tetraphenylethene (TPE)-based benzoselenodiazole (TPESe), are the terminal acceptors. Benefiting from the close contact and large optical overlap between donors and acceptors at each level, triad and tetrad relaying systems sequentially and efficiently furnish chirality transmission/amplification and energy transfer along the cascaded line BDA-TPEM-NiR (or/and TPESe), leading to bright customized-color circularly polarized luminescence (CPL) and bright white-light-emitting CPL (CIE coordinates: 0.33, 0.34) with an amplified dissymmetry factor (glum) of 3.5 × 10-2 over a wide wavelength range. This work provides a new direction for the construction of chiral light-harvesting systems for a broad range of applications in chiroptical physics and chemistry.

BNIP3 contributes to cisplatin-induced apoptosis in ovarian cancer cells.

BNIP3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of BNIP3 in cisplatin-induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of BNIP3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and BNIP3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. BNIP3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in BNIP3 levels in A2780 and OVCAR4. BNIP3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin-induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between BNIP3 level and overall survival and progression-free survival in patients with ovarian cancer. Pooled analyses showed that higher BNIP3 level was correlated with poorer overall survival (95% confidence intervals; hazard ratio = 1.18, 1.04-1.34; P = 0.013) and progression-free survival (95% confidence intervals; hazard ratio = 1.26, 1.10-1.43; P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d'Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin-induced apoptosis is dependent on BNIP3 level in ovarian cancer cell lines. Targeting BNIP3 may therefore be a potential way of restoring cisplatin sensitivity.

Let-7e sensitizes epithelial ovarian cancer to cisplatin through repressing DNA double strand break repair.

BACKGROUND: Resistance to platinum-based chemotherapy remains a great challenge for ovarian cancer treatment. The human let-7 family contains 13 members located on nine different chromosomes, and most members have been implicated in the modulation of drug sensitivity in cancers. Our previous study showed that deregulation of let-7e in epithelial ovarian cancer (EOC) promoted the development of resistance to cisplatin. In the present study, we aimed to investigate the underlying mechanism and further evaluate the clinical value of let-7e in predicting chemo-response and prognosis in EOC. RESULTS: In situ hybridization assays revealed a significantly decreased expression of let-7e in chemo-resistant EOC tissues compared with chemo-sensitive cases. Transfection with let-7e agomir sensitized EOC cells to cisplatin, down-regulated BRCA1 and Rad51 expression, and repressed the repair of cisplatin-induced DNA double strand break, while let-7e inhibitor exerted the opposite effects. In human EOC tissues, BRCA1 and Rad51 levels were increased in the chemo-resistant group compared with the sensitive group and were negatively correlated with let-7e. Low let-7e and high Rad51 were significantly associated with poor progression-free survival and overall survival and multivariate regression analyses showed that let-7e was an independent predictor for overall survival and chemotherapy response in EOC. Receiver operating characteristic analysis indicated that let-7e level was highly predictive of resistance to platinum-taxane chemotherapy with an area under the curve of 0.826. CONCLUSIONS: In EOC, low let-7e leads to activation of BRCA1 and Rad51 expression and subsequent enhancement of DSB repair, which in turn results in cisplatin-resistance. Let-7e is a potential predictor for survival and chemo-response in EOC and re-expression of let-7e might be an effective strategy for overcoming chemo-resistance.

Comparison of different sample preparation methods for platinum determination in cultured cells by graphite furnace atomic absorption spectrometry.

BACKGROUND: Platinum-based agents are widely used in chemotherapy against solid tumors and insufficient intracellular drug accumulation is one of the leading causes of platinum resistance which is associated with poor survival of tumor patients. Thus, the detection of intracellular platinum is pivotal for studies aiming to overcome platinum resistance. In the present study, we aimed to establish a reliable graphite furnace atomic absorption spectrometry (GFAAS)-based assay to quantify the intracellular platinum content for cultured cells. METHODS: Several most commonly applied cell preparation methods, including 0.2% HNO3, 0.2% Triton X-100, concentrated nitric acid, RIPA combined with concentrated nitric acid and hydroxide, followed by GFAAS for platinum detection were compared in ovarian, cervical and liver cancer cell lines to obtain the optimal one, and parameters regarding linearity, accuracy, precision and sensitivity were evaluated. Influence of other metals on platinum detection and the storage conditions of samples were also determined. RESULTS: The treatment of cells with 0.2% HNO3 was superior to other approaches with fewer platinum loss and better repeatability. The recovery rate and precision of this method were 97.3%-103.0% and 1.4%-3.8%, respectively. The average recoveries in the presence of other metals were 95.1%-103.1%. The detection limit was 13.23 ug/L. The recovery rate of platinum remained acceptable even in cell samples stored in -20 °C or -80 °C for two months. DISCUSSION: After comparison, we found that 0.2% HNO3 was optimal for intracellular platinum quantification based on GFAAS, which presented values compatible with that of inductively-coupled plasma mass-spectrometry (ICP-MS), and this is partially attributed to the simplicity of this method. Moreover, the assay was proved to be accurate, sensitive, cost-effective and suitable for the research of platinum-based antitumor therapy.

PMS2 expression in epithelial ovarian cancer is posttranslationally regulated by Akt and essential for platinum-induced apoptosis.

Epithelial ovarian cancer (EOC) is the most lethal of the gynecologic malignancies, mainly due to the advanced stage at diagnosis and development of cisplatin resistance. The sensitivity of tumor cells to cisplatin is frequently affected by defect in DNA mismatch repair (MMR), which repairs mispaired DNA sequences and regulates DNA-damage-induced apoptosis. However, the role of postmeiotic segregation increased 2 (PMS2), a member of MMR protein family, in cisplatin resistance remains elusive. In the present study, we demonstrated the frequent deficiency of PMS2 and phosphorylation of Akt in EOC cell lines and tissues. Results of complex immunoprecipitation (co-IP) and protein stability assay indicated that activated Akt could directly bind to PMS2 and cause degradation of PMS2 in EOC cells. In addition, functional experiments revealed that PMS2 was required for cisplatin-induced apoptosis and cell cycle arrest in G2/M phase. These findings provide a novel insight into molecular mechanisms linking MMR with chemoresistance and suggest that stabilization of PMS2 expression may be useful in overcoming the cisplatin resistance in EOC.

Tie-1: A potential target for anti-angiogenesis therapy.

The tyrosine kinase system angiopoietin (Ang)/Tie interacts with vascular endothelial growth factor pathway and regulates vessel quiescence in adults as well as later steps of the angiogenic cascade related to vessel maturation. Since all Angs are able to bind to Tie-2 but none binds to Tie-1, the function of Tie-2 and its ligands have captured attention. However, emerging evidence indicates unique roles of the orphan receptor Tie-1 in angiogenesis under physiological and pathological conditions. It is required for maintaining vascular endothelial cell integrity and survival during murine embryo development and in adult and may be involved in modulating differentiation of hematopoietic cells in adult. Tie-1 exhibits poor tyrosine kinase activity and signals via forming heterodimers with Tie-2, inhibiting Tie-2 signaling mediated by Angs. This inhibition can be relieved by Tie-1 ectodomain cleavage mediated by tumor- and inflammatory-related factors, which causes destabilization of vessels and initiates vessel remodeling. Up-regulated Tie-1 expression has been found not only in some leukemia cells and tumor related endothelial cells but also in cytoplasm of carcinoma cells of a variety of human solid tumors, which is associated with tumor progression. In addition, it has pro-inflammatory functions in endothelial cells and is involved in some inflammatory diseases associated with angiogenesis. Recent research indicated that Tie-1 gene ablation exhibited significant effects on tumor blood- and lymph-angiogenesis and improved anti-Ang therapy, suggesting Tie-1 may be a potential target for tumor anti-angiogenesis treatment.

Tumor-associated macrophages induce lymphangiogenesis in cervical cancer via interaction with tumor cells.

Our studies were conducted to investigate the clinical and functional significance of tumor-associated macrophages (TAMs) in cervical tumor lymphatic metastasis. We found that the increase in macrophages in tumor stroma is significantly associated with lymphatic metastasis (p = 0.017), through performing immunohistochemical staining in 111 cervical samples (55 invasive squamous carcinomas of uterine cervix, 27 cervical intraepithelial neoplasms III, and 29 normal cervix). The human lymphatic endothelial cells (HLEC), which were cultured in conditioned medium of cervical cancer cell-macrophage coculture, formed more tube-like structures in vitro, when compared with those in conditioned mediums of LEC, normal cervical epithelium, single macrophage, and single cervical cancer cell (all p < 0.001). The mRNA expressions of IL-1ß and IL-8 in cervical cancer cells cocultured with macrophages were increased, compared with those in cervical cancer cell cultured alone (pIL-1ß  < 0.05 and pIL-8  < 0.01). Meanwhile, the mRNA expression of VEGF-C and VEGF-A was increased in macrophages cocultured with cervical cancer cells, compared with the expression in those macrophages cultured alone (both p < 0.05). Taken together, the results suggest that TAMs promote lymphangiogenesis mainly through interaction with surrounding cervical cancer cells.

Proteomic identification of target proteins following Drosha knockdown in cervical cancer.

The nuclear microRNA (miRNA) processing enzyme Drosha is upregulated in cervical cancer, and its overexpression is related to an invasive tumour phenotype. However, the mechanisms that underlie this effect remain poorly understood. The aim of this study was to identify the potential targets of Drosha in cervical cancer. Here, we demonstrated that Drosha knockdown (Drosha-KD) inhibited proliferation, colony formation and the migration of cervical cancer cells in vitro. A global upregulation of proteins in Drosha-KD cells was revealed by two-dimensional gel electrophoresis (2-DE). Eighteen proteins were identified by liquid chromatography and tandem mass spectrometry technology (LC-MS/MS) from 21 selected protein spots that exhibited significant alterations in Drosha-KD cells. The majority of the identified proteins have been previously associated with tumour formation. The downregulation of tubulin 5ß in Drosha-KD cervical cancer cells was further confirmed by western blotting. Our results suggest that Drosha affects the biological activity of cervical cancer cells by regulating the expression of numerous tumour-associated proteins.

The role of EZH2 and DNA methylation in hMLH1 silencing in epithelial ovarian cancer.

Enhancer of zeste homolog 2 (EZH2) is overexpressed in various malignancies and associated with poor prognosis and drug-resistance. A recent study suggested that there is a link between EZH2 expression and the mediation of gene silencing in association with aberrant DNA methylation. In the present study, we showed an inverse correlation between EZH2 and human mutL homolog 1 gene (hMLH1) expression in 30 epithelial ovarian cancer (EOC) tissues. Moreover, we found that EZH2 downregulation could induce the re-expression of the unmethylated, basally expressed hMLH1 gene without affecting DNA methylation in the hMLH1 promoter. These results suggest that EZH2 can modulate the transcription of basally expressed hMLH1 via a non-DNA-methylation-dependent pathway, but it has no effect on hMLH1 silencing that is mediated by DNA hypermethylation.

A novel function of protein kinase B as an inducer of the mismatch repair gene hPMS2 degradation.

Human DNA mismatch repair (MMR) proteins correct DNA errors, which normally occur during DNA replication. Defects of MMR genes result in genomic instability and carcinogenesis. However, the mechanism of MMR proteins regulation has not yet been clearly explored, especially for the member of MutL-related protein, human post meiotic segregation increased 2 (hPMS2). In this study, an inverse correlation between hPMS2 level and activated Akt was detected in nine tumor cell lines by western blot. The negative regulation of hPMS2 expression by activated Akt was further verified by functional experiments manipulating Akt activity using siRNA targeting Akt, Akt Inhibitor I, Akt/PKB Signaling Inhibitor-2 (API-2) and Insulin-like Growth Factor-I (IGF-1). In addition, protein complex immunoprecipitation assays and protein stability assays using cycloheximide revealed that activated Akt (P-Akt1 S473) could bind to hPMS2 directly and induce hPMS2 degradation. Moreover, results of immunofluorescence assays showed blocking Akt activity resulted in accumulation of hPMS2 protein in nucleus. These observations indicate that activated Akt is the upstream signaling regulating hPMS2 expression, stability and nuclear localization, providing a novel insight into the regulation of hPMS2 in cancer cells.

[Association of functional polymorphisms on MMP-12 and MMP-13 gene promoter region with epithelial ovarian carcinoma].

OBJECTIVE: To investigate whether the functional polymorphisms in the promoter region of MMP-12 (-82A/G) and MMP-13(-77A/G) are associated with epithelial ovarian carcinoma (EOC). METHODS: The MMP-12 -82A/G and MMP-13 -77A/G were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 300 epithelial ovarian carcinoma patients and 300 control women. RESULTS: The A/G genotype frequency of the MMP-12 gene was significantly higher in the patients than in the controls (P= 0.003); similarly, the frequency of MMP-12 -82G allele was higher in the patient group (P= 0.004). Compared with the A/A genotype, the A/G genotype carriers significantly increased the risk of EOC development (OR= 2.81, 95%CI: 1.38-5.74). No overall association between the MMP-13 -77A/G polymorphism and EOC(P= 0.15) was observed. However, the A/A genotype carriers in the MMP-13 -77A/G locus had significantly higher risk of developing serous-papillary and mucinous ovarian cancer (OR= 1.93, 95% CI: 1.05-3.53; OR= 5.16, 95% CI: 1.62-16.44, respectively), comparing with the G/G genotype carriers. Combining the two SNPs, the haplotype distributions in patients were not significantly different from that in control women (P= 0.06). CONCLUSION: These results suggested that individuals with MMP-12 -82A/G and MMP-13 -77A/A might have higher risk of overall or special histological type of EOC development.

Expressions of SE-1, CD31 and CD105 in the vascular endothelial cells and serum of rat with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. In order to investigate the molecular biologic mechanism of HCC's development, we studied the expressions of SE-1, CD105 and CD31 in tumor endothelial cells (TECs) of HCC and in the serum of rats. METHODS: We analyzed the expressions of SE-1, CD31 and CD105 in rat HCC tumor tissues using immunohistochemistry (IHC). Twenty HCC bearing rats and eighteen normal rats were examined for the expressions of SE-1, CD31 and CD105 antigens in serum by enzyme-linked immunosorbent assay (ELISA). RESULTS: SE-1, CD31 and CD105 antigens were detected both in HCC tissue and in normal liver tissue with higher expressions of CD31 and CD105 in HCC while the SE-1 antigen expression was higher in normal liver. Similarly, serum CD31 and CD105 in rats with HCC were significantly increased compared with normal rats (t = 2.8628, P = 0.0086; t = 4.4922, P < 0.0001, respectively). In contrast, SE-1 antigen in HCC rat serum was significantly decreased compared with normal rats (t = 3.4983, P = 0.0011). CONCLUSION: SE-1, CD31 and CD105 are closely related with liver tumor angiogenesis, which is similar to their performances in terms of their expressions in the serum.

Identification of proteins differentially expressed between capillary endothelial cells of hepatocellular carcinoma and normal liver in an orthotopic rat tumor model using 2-D DIGE.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well-formed rat HCC using 2-D DIGE coupled with MALDI-TOF/TOF MS. Thirty-eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain-containing protein 3 and galectin-3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain-containing protein 3 is significantly down-regulated in TECs, but galectin-3 is up-regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.

The functional polymorphisms on promoter region of matrix metalloproteinase-12, -13 genes may alter the risk of epithelial ovarian carcinoma in Chinese.

BACKGROUNDS AND AIMS: Growing evidences indicate that single nucleotide polymorphisms (SNPs) of matrix metalloproteinases (MMPs) gene promoter may alter MMPs protein expression levels to influence malignant tumors developing and progressing. Our study was to assess the effects of the SNPs in the promoter region of MMP-12 and MMP-13 on the risk of epithelial ovarian carcinoma (EOC) developing and progressing. METHODS: MMP-12 A-82G and MMP-13 A-77G SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism in 256 EOC patients and 329 controls. RESULTS: The A/G genotype frequency of MMP-12 was significantly higher in patients than in controls (7.0% vs 3.3%, P = 0.04); similarly, the frequency of MMP-12 82G allele was higher in patients too (P = 0.04). Compared with A/A genotype, A/G genotype significantly increased the risk of EOC (odds ratio, 2.19; 95% confidence interval, 1.01-4.72). Age-stratified analysis showed that individuals with A/G genotype had a higher risk in the final diagnosis aged younger than 50 years. We observed no overall association between MMP-13-77A/G polymorphism and EOC. However, an elevated positive association was observed for A/A versus G/G + A/G genotypes in mucinous ovarian cancer. Combining the analyzed 2 SNPs, the haplotype distributions in patients were not significantly different from that in controls. CONCLUSION: These results suggested that the G allele of the MMP-12 82A/G polymorphism might be a risk factor for the development and progression of EOC and that the A/A genotype of MMP-13-77A/G polymorphism was associated with special pathological subtype and clinical stage in EOC at least in Chinese women.

[Study on the association of SNPs of MMP-2 and TIMP-2 genes with the risk of endometriosis and adenomyosis].

OBJECTIVE: To investigate the association of single nucleotide polymorphisms (SNPs) in matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) with the risk of endometriosis and adenomyosis. METHODS: Genotypes of MMP-2 and TIMP-2 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method among 298 endometriosis patients, 180 adenomyosis patients and 324 matched control women. RESULTS: No significant difference was found in allele frequencies and genotype distributions of MMP-2 -1306C/T polymorphism between endometriosis patients and control women (P> 0.05). However, there were significant differences in genotype and allele distributions of MMP-2 -1306C/T polymorphism between adenomyosis patients and control women (P< 0.05). Compared with CT+TT genotypes, CC genotype significantly increases the risk of adenomyosis, with an odds ratio of 1.83 (95% CI was 1.13-2.96). No significant difference was shown in allele frequencies and genotype distributions of the MMP-2 -735C/T polymorphism among the three groups (P>0.05). MMP-2 -1306C/T and -735C/T polymorphisms displayed linkage disequilibrium (D'=0.74). There was no significant difference in haplotype distributions of the two MMP-2 SNPs among the three groups ( P> 0.05). No significant difference was found in allele frequencies of TIMP-2 -418G/C polymorphism among the three groups (P> 0.05). However, the frequency of TIMP-2 CC genotype in endometriosis patients (0.7%) was significantly lower than that in the control women (3.7%) (P< 0.05). CONCLUSION: The C allele of MMP-2 -1306C/T polymorphism did not modify the risk of developing endometriosis but significantly increase the risk of developing adenomyosis. The MMP-2 -735C/T and TIMP-2 -418G/C polymorphisms were not associated with the risk of developing endometriosis or adenomyosis.

[Association of SNPs in the promoter of MMP-2 and TIMP-2 genes with epithelial ovarian cancer].

The association between single nucleotide polymorphisms (SNPs) in the promoter region of MMP-2 and TIMP-2 genes and the risk of epithelial ovarian cancer was investigated. MMP-2 C-1306T, C-735T and TIMP-2 G-418C SNPs were genotyped by polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) analysis in 246 patients with epithelial ovarian cancer and 324 healthy women as control. Results showed no significant difference between the patient and control groups in allele or genotype distributions of MMP-2 C-1306T (P=0.55 and P=0.42). However, the frequencies of the C allele and the C/C genotype of the MMP-2 C-735T were significantly higher in ovarian cancer patients (80.7% and 66.7%) than those in healthy controls (75.5% and 55.9%). Compared with the T/T+C/T genotypes, the C/C genotype significantly increased the risk of ovarian cancer (OR=1.58, 95%CI=1.12-2.23). Stratification analysis showed that subjects carrying C/C genotype were significantly associated with the risk of endometrioid ovarian cancer and with ovarian cancer in subjects that were 50 or older, with odds ratio at 1.69 (95%CI=1.03-2.79) and 1.71 (95%CI=1.14-2.57), respectively. Haplotype analysis showed that the frequencies of four haplotypes (T(-1306)-T(-735), T(-1306)-C(-735), C(-1306)-T(-735) and C(-1306)-C(-735)) of MMP-2 C-1306T and C-735T were not significantly different between the patient and control groups (P=0.24). The allele and genotype frequencies of TIMP-2 G-418C were not significantly different between the patient and control groups (P=0.33 and P=0.47). But TIMP-2 -418G/G genotype was associated with a trend for endometrioid ovarian cancer by stratification analysis according to histological subtypes (OR=1.62, 95%CI=0.94-2.78). Thus, the study suggested that the C/C genotype of the C-735T SNP in the promoter region of MMP-2 gene may be a potential risk factor for epithelial ovarian cancer, but the C-1306T SNP may have no association with the risk of epithelial ovarian cancer. The TIMP-2 G-418C SNP may be associated with the risk of different histological subtypes of epithelial ovarian cancer.