Delicaflavone Represses Lung Cancer Growth by Activating Antitumor Immune Response through N6-Methyladenosine Transferases and Oxidative Stress.

Our previous studies have shown that delicaflavone (DLL), a biocomponent extracted from Selaginella doederleinii Hieron, has antitumor activity. However, the role of DLL in the antitumor immune response is unknown. In this study, we tested the potential roles of DLL in antitumor immune response. An animal tumor model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established to determine whether DLL induced the tumor-bearing host's antitumor immune response. m6A-MeRIP-qPCR, western blot, and flow cytometry were performed to explore the underlying mechanisms. DLL inhibited the proliferation of 3LL lung cancer cells in vitro and in vivo and induced tumor cell oxidative stress. DLL significantly inhibited tumor growth in immunocompetent mice compared with nude mice. DLL treatment significantly increased Th1 cytokine production and CD8+ T cell infiltration into tumor tissues in tumor-bearing mice. DLL-mediated antitumor immune effects were reversed by overexpression of the N6-methyladenosine (m6A) transferase Mettl3/Mettl14. Mechanistically, DLL upregulated the expression of Stat1 and Irf1 and the secretion of cytokines by inhibiting Mettl3 and Mettl14 in lung cancer cells. In conclusion, DLL inhibited lung cancer cell growth by suppressing Mettl3/Mettl14 to activate antitumor immunity. These findings provided an opportunity to enhance lung cancer immunotherapy.

Study on the Biosensor Based on Biomimetic PDA Vesicles Fluorescence Resonance Energy Transfer for the Determination of Ovarian Cancer Marker miRNA-21.

In recent years, more and more research is being conducted on microRNAs and their involvement in the regulation of autophagy phagocytosis which is closely related to tumor growth. MicroRNA-21 is a kind of small RNA that can regulate gene expression and plays a significant role in autophagy of tumor cells. But the detection of microRNAs had always been a problem in the field of biological analysis. In this study, we designed a new fluorescent sensor for the detection of miRNA-21. The sensor was based on the successful signal reporting by E36-encapsulated vesicles and the specific interaction between E36 and miRNA-21. In the presence of miRNA, the E36/miRNA-21 complex formed and served as a donor molecule inside the acceptor PDA vesicles to amplify the fluorescence through FRET. Additionally, the sensor was applied to detect miRNA-21 in complex biological samples with satisfactory results.

Delicaflavone Reverses Cisplatin Resistance via Endoplasmic Reticulum Stress Signaling Pathway in Non-Small Cell Lung Cancer Cells.

BACKGROUND: The incidence and mortality of lung cancer continue to increase around the world; in 2018, new lung cancer cases accounted for 11.6% of all cancer cases, and lung cancer deaths accounted for 18.4% of cancer deaths. Cisplatin (DDP) is a first-line chemotherapy drug for lung cancer; however, DDP resistance can lead to a poor prognosis in patients with lung cancer. Therefore, reversing DDP resistance is a treatment goal. MATERIALS AND METHODS: Cell counting kit-8 (CCK8) assays, wound healing analyses, Transwell assays, in vitro tumor xenografts, and flow cytometry were used to detect the proliferation, migration, invasion, and apoptosis of multidrug resistant A549/DDP and PC9/DDP cells, respectively. Western blot was performed to detect protein levels of cleaved caspase-3, CHOP, and GRP78. RESULTS: Delicaflavone inhibited DDP resistance of lung cancer cells and decreased proliferation in a dose- and time-dependent manner. It also decreased migration and invasion and enhanced apoptosis. Western blots showed that delicaflavone overcame DDP resistance by increasing the expression of GRP78 and CHOP and the apoptosis-related protein cleaved caspase-3. CONCLUSION: Delicaflavone can reverse DDP resistance in A549/DDP and PC9/DDP cells by inhibiting cell proliferation and migration and enhancing apoptosis and cleaved caspase-3 levels by increasing the expression of CHOP and GRP78 protein via the endoplasmic reticular stress pathway. It could be a useful therapeutic adjunct to treat DDP-resistant lung cancer.

CYP2D6*10 pharmacogenetic-guided SERM could be a cost-effective strategy in Chinese patients with hormone receptor-positive breast cancer.

Aim: To assess the cost-effectiveness of CYP2D6*10 genetic testing for the management of Chinese women with hormone receptor-positive (HR+) breast cancer treated with selective estrogen receptor modulator. Methods: A Markov model was developed to evaluate a total expected cost and an incremental cost-effectiveness ratio (ICER). Robustness of the model was addressed in one-way analyses and probabilistic sensitivity analysis. Results: The cost of strategies of tamoxifen, toremifene without genotyping and the strategy base on CYP2D6*10 genotype were $63,879.19, $90,156.60 and $95,021.41, and the quality-adjusted life years gained are 8.1588, 12.89687 and 13.85911, respectively. The incremental cost-effectiveness ratio of the CYP2D6*10 testing versus toremifene were 5,055.74221/quality-adjusted life year, respectively. Conclusion:CYP2D6*10 pharmacogenetic-guided selective estrogen receptor modulator can be a cost-effective strategy in the Chinese patients with hormone receptor-positive breast cancer.

Delicaflavone induces ROS-mediated apoptosis and inhibits PI3K/AKT/mTOR and Ras/MEK/Erk signaling pathways in colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and tends to have drug resistance. Delicaflavone (DLF), a novel anticancer agent of biflavonoid from Selaginella doederleinii Hieron, showed strong anti-CRC activities, which has not yet been reported. In this study, we investigated the effects and possible anti-CRC mechanism of DLF in vitro and in vivo. It was shown that DLF significantly inhibited the cells viability and induced G2/M phase arrest, apoptosis, the loss of mitochondrial membrane potential (Δψm), generation of ROS and increase of intracellular Ca2+ in HT29 and HCT116 cells by MTT assay, TEM, flow cytometry and inverted fluorescence microscope. Western blot and qPCR assays results further confirmed DLF induced caspase-dependent apoptosis and inhibited PI3K/AKT/mTOR and Ras/MEK/Erk signaling pathways in CRC cells. Meanwhile, DLF significantly suppressed the tumor growth via activation of Caspase-9 and Caspase-3 protein and decrease of ki67 and CD34 protein without apparent side effects in vivo. In summary, these results indicated DLF induced ROS-mediated cell cycle arrest and apoptosis through ER stress and mitochondrial pathway accompanying with the inhibition of PI3K/AKT/mTOR and Ras/MEK/Erk signaling cascade. Thus DLF could be a potential therapeutic agent for CRC.

Delicaflavone induces apoptosis via mitochondrial pathway accompanying G2/M cycle arrest and inhibition of MAPK signaling cascades in cervical cancer HeLa cells.

BACKGROUND: Cervical cancer (CCa) represents the fourth most common cause of cancer-related death in women worldwide. CCa therapy is still a major clinical challenge worldwide. Finding and developing new anti-CCa chemotherapeutic drugs is a very significant issue. Delicaflavone is a rare biflavonoid from Selaginella doederleinii Hieron, which has shown strong anti-cancer activities in our preliminary screening. PURPOSE: The present study aimed to investigate the apoptotic effect and mechanism of delicaflavone against CCa. METHODS: In this study, the effect and potential mechanism of delicaflavone against CCa were investigated in vitro and in vivo by MTT assay, TEM, flow cytometry, western blot assay, qPCR assay, immunofluorescence assay and the mouse xenograft tumor model. RESULTS: It was confirmed that delicaflavone inhibited the proliferation of human CCa HeLa cells, and induced morphological changes, G2/M phase arrest and apoptosis in a dose- and time-dependent manner. HeLa cells treated with delicaflavone showed the loss of mitochondrial membrane potential, release of Cytochrome c, activation of caspases, alteration of Bax/Bcl-2 balance, and the inhibition of MAPK signaling cascades. Furthermore, delicaflavone significantly decreased tumor growth in a dose-dependent manner without apparent side effects in a xenograft tumor model of HeLa cells. Immunohistochemistry analysis confirmed the up-regulation of Caspase-9, Caspase-3, Bax protein and down-regulation of Bcl-2 protein in the xenografts tumors, which was consistent with the results in vitro. CONCLUSION: The results of the current study show that apoptosis is induced by the mitochondrial pathway accompanying with G2/M cycle arrest and inhibition of MAPK signaling cascades in human CCa HeLa cells, which can be used as a promising therapeutic drug for CCa.

Association of SENPs single-nucleotide polymorphism and breast cancer in Chinese population.

SUMO-specific Cysteine Proteases (SENPs) have involvement in the initiation and progression of human cancers. In the present study, we evaluated the association of SENPs polymorphism with susceptibility as well as clinicopathologic features and patients' response of breast cancer (BC) in a Chinese population.We genotyped SENP1 (rs61918808), SENP2 (rs6762208), SENP7 (rs61697963) by sequencing in a case-control study including 210 BC patients and 225 healthy volunteers. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assume the association strength.No significant association was found between polymorphism of the 3 SENPs and BC susceptibility. However, SENP1 rs61918808 (C>T) and SENP7 rs61697963 (A>C) was associated with HER-2 expression (P < .05). SENP2 rs6762208(C>A) was correlated with increasing risk of lymph node metastases (P < .05). Among the patients who received neoadjuvant chemotherapy, T allele and TT genotype of SENP1 rs61918808 were less likely to achieve pCR (P < .05).We first reported SENPs variants were not associated with BC risk in Chinese population, but presented specific effect on clinicopathological features of BC. Moreover, SENP1 rs61918808 may be a predictor for the clinical response in local advanced BC patients who received neoadjuvant chemotherapy.

Cost-effectiveness analysis of UGT1A1*6/*28 genotyping for preventing FOLFIRI-induced severe neutropenia in Chinese colorectal cancer patients.

AIM: To assess the cost-effectiveness of UGT1A1*6/*28 genotyping compared with no genotyping or no dose adjustment before irinotecan administration in China. MATERIALS & METHODS: A decision tree model was developed to evaluate costs and health outcomes represented as quality-adjusted life years gained. Model inputs for the frequency of genotypes, the probability of neutropenia under FOLFIRI chemotherapy and direct costs and utilities were obtained from published sources. One-way sensitivity analyses were performed. RESULTS: The strategy of genotyping with dose reduction dominated all remaining strategies. Compared with the strategies of no genotyping and genotyping with unchanged dose, it resulted in only marginal quality-adjusted life year increases (0.0011 and 0.0012) but a cost reduction of $651.12 and $805.22 per patient, respectively. One-way sensitivity analyses revealed that the model was relatively robust. CONCLUSION: UGT1A1*6/*28 genotyping was cost saving for Chinese colorectal cancer patients.

[Expression of Apoptosis-related Protein Livin and Caspase-3 in Diffuse Large B Cell Lymphoma and Its Clinical Significance].

OBJECTIVE: To detect the expression levels of Livin and Caspase-3 proteins in diffuse large B cell lymphoma (DLBCL), and to explore its correlation with clinicopathological characteristics, so as to provide a reference for clinical treatment and evaluation prognosis of DLBCL. METHODS: Immunohisto-chemical staining was performed to detect the expression of Livin and Caspase-3 in the samples from 51 DLBCL patients and 20 patients with reactive hyperplasia of lymph node (RHL). The relation between Livin and Caspase-3 with the factors influencing the prognosis of DLBCL was analyzed. RESULTS: Livin+ was not expressed in the tissues of RHL, but its expression rates reached to 58.82%(30/51) in 51 cases of DLBCL(P<0.05). The expression rats of Caspase-3 in DLBCL and RHL were 25.49%(13/51) and 85.0%(17/20) respectively, and the difference was statistically significant(P<0.05). The expression level of Livin in DLBCL tissues was related with clinical stage and pathological type(P<0.01, P<0.01). The expression of Livin was not obviously related with sex, age and extranodal involvement. The expression level of Caspase-3 in DLBCL tissues was related with clinical stage (P<0.05). The expression of Caspase-3 was not obviously related with sex, age, pathological type and extranodal involvement. There was a negative correlation between Livin and Caspase-3 in DLBCL(γs=-0.333,P=0.017). Kaplan-meier survival analysis revealed that the expressions of Livin and Caspase-3 were related stalistically significantly with the patients prognosis (P<0.01, P<0.05). CONCLUSION: Over-expression of Livin in DLBCL and DLBCL of non-GCB subtypes, and low-expression of Caspase-3 in DLBCL may play a significant role in clinical prognosis of DLBCL.

Circadian Gene CLOCK Affects Drug-Resistant Gene Expression and Cell Proliferation in Ovarian Cancer SKOV3/DDP Cell Lines Through Autophagy.

Abnormal autophagy regulation affects the chemoresistance of ovarian cancer, during which the circadian gene clock may play a major role. In this study, RNA interference plasmid pSUPER-Clock and overexpression plasmid pcDNA3.1-Clock of CLOCK were used to stably transfect the SKOV3/DDP cells by lipofection. Upon screening, the in vitro transfected cell lines with pSUPER-Clock, the autophagy level, and G0/G1 phase cells were significantly reduced, and the expression levels of Clock, LC3, P-gp, and MRP2 were inhibited. In contrast, the autophagy level and G0/G1 phase cells in cell lines transfected with pcDNA3.1-Clock were significantly increased, and the expressions of Clock, LC3, P-gp, and MRP2 were enhanced. In comparison with the untransfected control group showed the percentage of apoptotic cells in SKOV3/DDP cell lines of Clock interfering expression group after cisplatin treatment was significantly increased while the survival was substantially reduced. These results indicated that inhibiting the circadian gene Clock expression can reverse the cisplatin resistance of ovarian cancer SKOV3/DDP cell lines by affecting the protein expression of drug resistance genes during which autophagy plays an important role. The CLOCK gene may be designated as a novel candidate for targeted gene therapy in drug-resistant ovarian cancer.

Primary mediastinal clear cell sarcoma: a case report and review of the literature.

BACKGROUND: Clear cell sarcoma (CCS) is a rare malignant soft-tissue neoplasm that displays melanocytic markers and exhibits striking histopathological features. The tumour has a predilection for the lower extremities and rarely presents in the mediastinum. CASE PRESENTATION: We present a case of primary mediastinal CCS in a 57-year-old man. Computer tomography (CT) revealed a 12 × 12 × 7.5 cm mass in the anterior mediastinum. Microscopically, the tumour mainly consisted of epithelioid cells with oval vesicular nuclei and eosinophilic cytoplasm. Immunohistochemically, the tumour was positive for human melanoma black 45 (HMB-45) and vimentin but negative for S-100 and Melan-A. Fluorescence in situ hybridisation (FISH) showed a translocation involving the EWSR1 gene region. CONCLUSION: This report will illustrate that the mediastinum is a potential site for primary CCS and FISH plays an important role in making a conclusive diagnosis.

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. KEY MESSAGES: Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.

Interfering EZH2 Expression Reverses the Cisplatin Resistance in Human Ovarian Cancer by Inhibiting Autophagy.

We aimed to determine the effects of the inhibition of enhancer of zeste homolog 2 (EZH2) gene expression on the cisplatin resistance of the human ovarian cancer cell line, SKOV3/DDP, and to identify the underlying mechanisms. SKOV3/DDP cells were stably transfected with pSUPER-EZH2 (EZH2 RNA interference plasmid) or pcDNA3.1-EZH2 (EZH2 gene overexpression plasmid) using the lipofection method. Real-time fluorescence quantitative reverse transcription polymerase chain reaction and western blotting confirmed that EZH2 expression was downregulated in pSUPER-EZH2-transfected cells. Flow cytometry revealed that EZH2 inhibition did not induce apoptosis, but significantly inhibited autophagy. In addition, it significantly increased the expression of the cellular senescence-signaling proteins p14(ARF), p16(INK4a), p53, pRb, and p21, and significantly decreased the expression of cyclin-dependent kinase (CDK)1, CDK2, and H3K27me3. Cellular senescence was characterized by a significant increase in the G0/G1 ratio and the restoration of sensitivity to cisplatin in the drug-resistant cells. These findings suggest that interfering with EZH2 expression can inhibit SKOV3/DDP cell autophagy and reverse resistance to cisplatin. The underlying mechanisms could be associated with the regulation of the cellular senescence-signaling pathway.

Ethyl acetate extract from Selaginella doederleinii Hieron inhibits the growth of human lung cancer cells A549 via caspase-dependent apoptosis pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Selaginella doederleinii Hieron has been used as a folk medicine for the treatment of different cancers, especially for nasopharyngeal carcinoma, lung cancer and trophoblastic tumor in China. Previously, the ethyl acetate extract from S. doederleinii (SDEA extract) showed favorable anti-cancer potentials. However, the main chemical composition and anticancer mechanism of the SDEA extract were still not very clear. Until now, there are no reports available about the oral toxicity of the extract. AIM OF STUDY: The present study was to further elucidate the chemical composition and anti-lung cancer mechanism of the SDEA extract, and evaluate the acute oral toxicity of the extract. MATERIALS AND METHODS: The SDEA extract was separated and analysed by HPLC to disclose its main chemicals. The effects of the extract were then investigated in vitro on cell viability, apoptosis and cell cycle using fluorescence microscopy and flow cytometry, and the molecular mechanism against human lung cancer cells A549 was further studied by western blot assays. The in vivo anti-cancer effect of the extract was evaluated in A549 xenograft mice model by histochemical assay, and tumor growth, microvascular density (MVD) and Ki67 expression were also measured. In addition, acute oral toxicity test of the extract was executed in mice. RESULTS: SDEA extract mainly contained eight biflavonoids. The extract caused the loss of mitochondrial membrane potential and induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating caspase-9 and caspase-3 and blocked the cell cycle in S phase. The extract reduced expression of antigen Ki67, decreased MVD, and significantly inhibited the tumor growth. The extract did not show apparent oral acute toxicity in healthy mice. CONCLUSION: The SDEA extract exerted anti-tumor effect through activating mitochondrial pathways and reducing Ki67 expression and MVD. Low oral acute toxicity suggested it a promising chemotherapy agent.

Proteomic analysis of Eimeria acervulina sporozoite proteins interaction with duodenal epithelial cells by shotgun LC-MS/MS.

Although it has been known for many years that Eimeria acervulina (E. acervulina) initiates infection by invading the duodenal epithelial cells of chicken, the key protein molecules and the mechanisms of the parasite in invading are unknow. In this study, we found that 85 proteins of E. acervulina could bind with the chicken duodenal epithelial cells from Eimeria protein database. Among them, sixteen were identified only in Eimeria spp. correlation with invasion and evasion and 69 proteins were found in Eimeria spp. with more than 2 unique pep count. Nine out of the 16 proteins and 41 out of the 69 proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Most of the 9 annotated proteins occurred in binding, catalytic activity and cellular process whereas, 29 (70.73%) out of the 41 proteins had binding activity and 20 proteins (48.78%) had catalytic activity. The findings provided an insight into the interactive relationship between E. acervulina and host cells and will shed new lights on the understanding of molecular mechanisms of E. acervulina invasion and pathogenesis.

Effect of autophagy-related beclin1 on sensitivity of cisplatin-resistant ovarian cancer cells to chemotherapeutic agents.

The purpose of the study was to determine the effects of autophagy related gene Beclin1 at different levels of expression on the sensitivity of cisplatin-resistant ovarian cancer cells (SKOV3/DDP) to different chemotherapeutics. In pSUPER-Beclin1 transfected cells, real-time fluorescence quantitative RT-PCR and Western blot analysis showed that expression was significantly inhibited. Flow cytometry revealed that the mean fluorescence intensity (MDC), reflecting autophagy, and cells in the G0/G1 phase were markedly reduced. When compared with the blank control group, inhibition of Beclin1 expression in SKOV3/DDP cells not only increased the rate of apoptosis following treatment with chemotherapeutics, but also increased the sensitivity. These findings suggest that Beclin1 expression plays an important role in chemotherapeutic agent-induced death of SKOV3/DDP cells. Inhibition of autophagy related gene Beclin1 expression in SKOV3/DDP cells may increase the rate of apoptosis and elevate the sensitivity to chemotherapeutics.

Identification and molecular characterization of microneme 5 of Eimeria acervulina.

In the present study, the microneme 5 gene of Eimeria acervulina (E. acervulina) (EaMIC5) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, GenBank Accession No. EH386430.1) to amplify the 3'- and 5'-ends of EaMIC5. The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. Sequence analysis revealed that the open reading frame (ORF) of EaMIC5 was 336 bp and encoded a protein of 111 amino acids with 12.18 kDa. The ORF was inserted into pET-32a (+) to produce recombinant EaMIC5. Using western blotting assay, the recombinant protein was successfully recognized by the sera of chicks experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC5. Immunofluorescence analysis using antibody against recombinant protein EaMIC5 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC5 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens, and presented anti-coccidial index (ACI) more than 160. All the above results suggested that the EaMIC5 was a novel E. acervulina antigen and could be an effective candidate for the development of a new vaccine against this parasite.

Inhibition of Beclin 1 expression enhances cisplatin-induced apoptosis through a mitochondrial-dependent pathway in human ovarian cancer SKOV3/DDP cells.

The purpose of this study was to determine the influence of autophagy on cisplatin-induced ovarian cancer SKOV3/DDP cell line death through regulation of the expression of the autophagy gene, Beclin 1, and to explore the potential mechanism underlying the relationship between autophagy and apoptosis. When compared with a blank control group, the proportion of apoptotic cells undergoing Beclin 1 interfering increased significantly after cisplatin treatment, accompanied by reduction in mitochondrial membrane potential, increase in activities of caspase-9/3 and cytoplasmic cytochrome C, elevation of Bax expression, and reduction in Bcl-2 expression. However, the proportion of apoptotic cells with Beclin 1 overexpression reduced. These findings suggest that Beclin 1 plays an important role in the regulation of potent antitumor activity through a mitochondrial-dependent pathway in SKOV3/DDP cell line, and inhibition of Beclin 1 expression may become a new target for the sensitization therapy of ovarian cancer with cisplatin.

Preparative isolation of six anti-tumour biflavonoids from Selaginella doederleinii Hieron by high-speed counter-current chromatography.

INTRODUCTION: Biflavonoids are the primary constituents of Selaginella doederleinii Hieron, to which different bioactivities have been attributed, including anti-cancer, anti-inflammatory, anti-oxidant, anti-fungal and anti-virus activity. However, effective methods for separation of these compounds are not currently available. OBJECTIVE: To develop a high performance and bioassay-guided method for preparative isolation of biflavonoids from S. doederleini via high-speed counter-current chromatography (HSCCC). METHODS: The anti-proliferation effects of four fractions (70% ethanol, petroleum ether, dichloromethane and acetic ether extracts) of S. doederleinii on five human cancer cells were monitored. The dichloromethane and acetic ether extracts showed good cytotoxicities to the studied cancer cell lines, guiding the subsequent separation. Two solvent systems composed of n-hexane:ethyl acetate:methanol:water (1:2:1.5:1.5, v/v) and n-hexane:ethyl acetate:methanol:water (3:2:3:2, v/v) were developed for separation of the active fractions, respectively. Identification of the biflavonoids was performed by EI-MS(n) , (1) H- and (13) C-NMR.' RESULTS: Under the optimised conditions, 12.6 mg amentoflavone (91.4%), 6.6 mg robustaflavone (90.4%), 7.5 mg 2'', 3''-dihydro-3', 3'''-biapigenin (98.2%) and 7.3 mg 3', 3'''-binaringenin (90.3%) from acetic ether extract (500 mg) and 6.3 mg heveaflavone (93.5%) and 5.3 mg 7, 4', 7'', 4'''-tetra-O-methyl-amentoflavone (94.5%) from dichloromethane extract (200 mg) were obtained, respectively. The anti-proliferation effects of the six biflavonoids on the five human cancer cells were further verified. CONCLUSION: The study provides methodological references for simultaneously preparative isolation of several bioactive biflavones from the herbal family of Selaginella. It is the first report discovering 2'', 3''-dihydro-3', 3'''-biapigenin and 3', 3'''-binaringenin from this herb and describing their cytotoxicities to human cancer cell lines.

Beclin 1 influences cisplatin-induced apoptosis in cervical cancer CaSki cells by mitochondrial dependent pathway.

PURPOSE: To investigate the role of Beclin 1 expression on the cisplatin-induced apoptosis in cervical cancer CaSki cells and to explore the potential mechanism underlying this effect. MATERIALS AND METHODS: After overexpression or partial silencing of Beclin 1 in cervical cancer CaSki cells, the transfected group and the control group were treated with cisplatin for 24 hours. The percentage of apoptotic cells were assessed by flow cytometry. The mitochondrial membrane potential and activities of caspase-8/9/3 were detected by JC-1 fluorescence staining and colorimetry. The expression of cytochrome c was measured using a Western blot. The messenger RNA expression of Bax and Bcl-2 were detected by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Expression of Beclin 1 protein was up-regulated in overexpressed transfectants of CaSki cells. After treatment with cisplatin, the Beclin 1 overexpression group led to the decrease of mitochondrial membrane potential and increase of activities of caspase-9 and caspase-3, and showed a greater increase in apoptosis than did the nontransfected group. Furthermore, Beclin 1 overexpression resulted in increased cytoplasmic cytochrome c and Bax expression and decreased mitochondrial cytochrome c and Bcl-2 expression. CONCLUSION: Overexpression of Beclin 1 in CaSki cells may influence cisplatin-induced apoptosis by mitochondrial dependent pathway.