Exosomes biogenesis was increased in metformin-treated human ovary cancer cells; possibly to mediate resistance.

BACKGROUND: Exosomes derived from tumor cells contribute to the pathogenesis of cancers. Metformin, the most usually used drug for type 2 diabetes, has been frequently investigated for anticancer effects. Here, we examined whether metformin affects exosomes signaling in human ovary cancer cells in vitro. METHODS: Human ovary cancer cells, including A2780 and Skov3 cells, were treated with metformin for either 24-48 h. Cell viability and caspase-3 activity were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and colorimetric assays respectively. Oil-Red-O staining and in vitro, scratch assays were used to examine cellular toxicity and wound healing rate. After treatment with metformin, exosomes were isolated from cells and quantified by acetylcholinesterase (AChE) assay, Dynamic Light Scattering (DLS), and their markers. Genes related to exosomes signaling were analyzed by real-time PCR or western blotting. RESULTS: Our results showed that metformin decreased the viability of both cells dose/time-dependently (P < 0.05). Metformin increased the activity of caspase-3 (P < 0.05) as well as the number of Oil-Red-O positive cells in both cell lines. In vitro scratch assay showed that the cell migration rate of metformin-treated cells was decreased (P < 0.05), whereas AChE activity of exosomes from metformin-treated cells was increased (P < 0.05). Concurrent with an increase in CD63 protein levels, expression of Alix, CD63, CD81, Lamp-2, and Rab27b up-regulated in treated cells (P < 0.05). CONCLUSION: Results indicated that metformin had a cytotoxic effect on ovary cancer cells and enhanced exosome biogenesis and secretion.

Probing Antibacterial and Anticancer Potential of Selenicereus undatus, Pistacia vera L. and Olea europaea L. against Uropathogens, MCF-7 and A2780 Cancer Cells.

Urinary tract infection is an infectious disease that requires immediate treatment. It can occur in any age group and involves both genders equally. The present study was to check the resistance of some antibiotics and to assess the antibacterial potential of three extracts of three plants against notorious bacteria involved in urinary tract infections. Along with assessing the antibacterial activity of plant extracts, we checked for the anticancer potential of these extracts against the cancer cell lines MCF-7 and A2780. Cancer is the leading cause of mortality in developed countries. Determinations of total flavonoid content, total phenolic content, total alkaloid content, total tannin content, total carotenoid content, and total steroid content were performed. The disk diffusion method was used to analyze the antibacterial activity of plant extracts. Ethanolic extract of Selenicereus undatus showed sensitivity (25-28 mm) against bacteria, whereas chloroform and hexane extracts showed resistance against all bacteria except Staphylococcus (25 mm). Ethanolic extract of Pistacia vera L. showed sensitivity (22-25 mm) against bacteria, whereas chloroform and hexane extracts showed resistance. Ethanolic extract of Olea europaea L. showed sensitivity (8-16 mm) against all bacteria except Staphylococcus, whereas chloroform and hexane extracts showed resistance. Positive controls showed variable zones of inhibition (2-60 mm), and negative control showed 0-1 mm. The antibiotic resistance was much more prominent in the case of hexane and chloroform extracts of all plants, whereas ethanolic extract showed a sensitivity of bacteria against extracts. Both cell lines, MCF-7 and A2780, displayed decreased live cells when treated with plant extracts.

Study on the Intervention Mechanism of Cryptotanshinone on Human A2780 Ovarian Cancer Cell Line Using GC-MS-Based Cellular Metabolomics.

Cryptotanshinone (CT), an active component of the traditional Chinese medicine Salvia miltiorrhiza Bunge, exhibits a wide range of biological and pharmacological activities. Although the anticancer activity of CT is well known, the knowledge of its effect on the regulation of cancer cell metabolism is relatively new. The present study investigated the anticancer mechanism of CT in ovarian cancer with a focus on cancer metabolism. CCK8 assays, apoptosis assays, and cell cycle assays were conducted to reveal the growth-suppressive effect of CT on ovarian cancer A2780 cells. To explore the potential underlying mechanisms of CT, the changes in endogenous metabolites in A2780 cells before and after CT intervention were investigated using the gas chromatography-mass spectrometry (GC-MS) approach. A total of 28 important potential biomarkers underwent significant changes, mainly involving aminoacyl-tRNA biosynthesis, energy metabolism, and other pathways. Changes in the ATP and amino acid contents were verified with in vitro and in vivo experiments. Our results indicate that CT may exert an anti-ovarian cancer effect by inhibiting ATP production, promoting the protein catabolic process, and inhibiting protein synthesis, which may lead to cell cycle arrest and apoptosis.

Intracellular Biotransformation of Ultrasmall Iron Oxide Nanoparticles and Their Effect in Cultured Human Cells and in Drosophila Larvae In Vivo.

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.

Effect of Icaritin on Proliferation， Apoptosis， Migration and Invasion of Human Ovarian Cancer A2780 Cells / 中国实验方剂学杂志

Objective:To study the effect of icaritin on the proliferation， apoptosis， migration and invasion of human epithelial ovarian cancer A2780 cells and the inhibitory mechanism of icaritin against cell invasion and migration via the regulation of epithelial-mesenchymal transformation （EMT）-related molecule expression. Method:A2780 cells were divided into the blank control group and low-， medium-， and high-dose （5， 10， 20 μmol·L^-1） icaritin groups and received the corresponding inventions for 48 h. Cell proliferation and viability were detected using the cell counting kit-8 （CCK-8）. The cellular proliferation inhibition and apoptosis rates were assayed by flow cytometry. The cell invasion and migration were observed in Scratch test and transwell test， followed by the calculation of wound healing rate and migration rate. The protein and mRNA expression levels of EMT-related molecules including E-cadherin， N-cadherin， and Vimentin and tumor invasion and migration-related molecule matrix metalloproteinase-9 （MMP-9） were measured by Western blot and real-time polymerase chain reaction （Real-time PCR）. Result:As revealed by CCK-8 assay and flow cytometry， compared with the blank control group， the icaritin groups all exhibited elevated proliferation inhibition rate （<italic>P</italic><0.01） and apoptosis rate （<italic>P</italic><0.05）. According to the Scratch test and transwell test， compared with the blank control group， the icaritin groups displayed weakened invasion and migration ability and decreased number and rate of cell invasion and migration （<italic>P</italic><0.05）. Western blot and Real-time PCR results showed that the protein and mRNA expression levels of N-cadherin， MMP-9 and Vimentin in each icaritin group were down-regulated as compared with those in the blank control group， while the expression of E-cadherin was up-regulated. Conclusion:Icaritin inhibits the proliferation and promotes the apoptosis of human ovarian cancer A2780 cells， and it inhibits the invasion and migration of A2780 cells possibly by regulating the expression of EMT-related molecules.

Liriopesides B from Liriope spicata var. prolifera inhibits metastasis and induces apoptosis in A2780 human ovarian cancer cells.

Ovarian cancer is the most frequent cause of death among gynecological cancers. In the present study, the anti­cancer effect of liriopesides B, a steroidal saponin from Liriope spicata var. prolifera, against A2780 cells was investigated. Transwell chambers were adopted to assess its effect on cell invasion and chemotaxis abilities. Flow cytometry was used to analyze the cell cycle and apoptosis. Reverse transcription­quantitative PCR was employed to examine gene expression levels. Western blot analysis was performed to detect protein expression levels. Liriopesides B inhibited the invasion and chemotactic movement ability of A2780 cells in a dose­dependent manner. Furthermore, liriopesides B caused cell cycle arrest in A2780 cells at the G1 phase following incubation for 24, 48 and 72 h. Hoechst 33258 staining indicated that, following incubation for 48 h, liriopesides B induced cell apoptosis in a dose­dependent manner. Flow cytometry verified that liriopesides B induced apoptosis in A2780 cells and induced late apoptosis in a dose­dependent manner. Furthermore, liriopesides B significantly increased the mRNA expression levels of E­CADHERIN, p21 and p27 and decreased the gene expression levels of BCL­2, which was consistent with its protein expression levels. In conclusion, liriopesides B possess anti­cancer properties, including inhibition of metastasis­associated behaviors, cell cycle arrest and induction of apoptosis. Therefore, liriopesides B may be considered as a candidate drug against ovarian cancer.

Mitochondria-targeting zeolitic imidazole frameworks to overcome platinum-resistant ovarian cancer.

Epithelial ovarian cancer is still the leading cause of death in gynecology due to its resistance to platinum-based first-line chemotherapeutic drugs. The utilization of mitochondria-targeted drugs has become an important direction in anti-tumor drug research and development. In this work, cisplatin (DDP)-loaded ZIF-90 with mitochondrial-targeting was synthesized at room temperature with a high drug loading (11.7 %, calculated based on Pt content). The ZIF-90@DDP showed high cellular uptake and less toxicity in both non- and DDP-resistant ovarian cancer cells with effective pH- and ATP-responsive drug release. Both mitochondria-targeting and responsive drug release could increase the drug concentration in mitochondria of drug-resistant cancer cells to reverse such resistance. Conclusively, the mitochondria-targeting ZIF-90@DDP with high drug loading could trigger responsive drug release in mitochondria of epithelial ovarian cancer cells, inhibit DPP-resistant epithelial ovarian cancer cells, and reverse drug resistance.

Down-regulation of Beclin1 inhibits the resistance of ovarian cancer A2780/DDP cells for the cisplatin / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of Beclin1 knockdown on cisplatin resistance in ovarian cancer A2780 cells and its related mechanisms. Methods: The mRNA and protein expressions of Beclin1 in A2780 cells and drug resistant A2780/DDP cells were determined by qPCR and Western blotting. After transfection with Beclin1 siRNA, the sensitivity of A2780/DDP cells to cisplatin was detected by MTT assay; Cell clone formation and apoptosis were detected by the Colony formation assay and Flow cytometry assay, respectively; cell autophagy was monitored by monodansylcadaverin (MDC) staining. Furthermore, the protein levels of cell autophagy related proteins, lysosomal associated membrane protein Lamp-2 and Cathepsin B were detected by Western blotting. Results: The mRNA and protein expression levels of Beclin1 in cisplatin-resistant A2780/DDP cells were significantly higher than those in A2780 cells (all P<0.05). The expression of Beclin1 was significantly increased in A2780 cells after treated with cisplatin (P<0.05). Beclin1 knockdown promoted cisplatin induced apoptosis of A2780/DDP cells (P<0.05), inhibited autophagy and cell colony formation (all P<0.05), and increased cell sensitivity to cisplatin (P<0.05). Meanwhile, Western blotting showed that Beclin1 knockdown increased the protein levels of cleaved-caspase 3 and Cathepsin B in A2780/DDP cells, while down-regulated the protein expressions of Atg3, Atg7, LC3Ⅱ/Ⅰand Lamp-2 (all P<0.05). Conclusion: Beclin1 knockdown can improve the sensitivity of A2780/DDP cells to cisplatin, and the mechanism may be related to the inhibition of protective autophagy of cells by regulating the expressions of autophagy related proteins, and the regulation of lysosomes, thus further promoting cisplatin-induced apoptosis of drug-resistant cells.

Establishment of an miR-137-knockout cell model using CRISPR/Cas9 genome editing.

MicroRNA-137 (miR-137) has been reported to be abnormally expressed in a variety of types of cancer, including ovarian cancer. However, the roles served by miR-137 in cancer are not fully understood. In the present study, 3 single guide RNAs (sgRNAs) were designed, synthesized and inserted into pXPR001 plasmids. The pXPR001-sgRNA plasmids were verified using sequencing and integrated into the genome of the ovarian cancer cell line, A2780, through lentiviral transduction, puromycin selection and single-cell culture. PCR products amplified from single-cell cultures using primers covering miR-137 targeting sites were sequenced to identify clones with miR-137 gene disruption. Genome editing was detected in 72% of the clones derived from sgRNA2, 4% from sgRNA3 and 0% from sgRNA1. Of the clones from sgRNA2, 32% contained 1 edited miR-137 allele and 40% contained 2 edited miR-137 alleles. The expression of miR-137 in clones #2 and #3 could not be detected by reverse transcription-quantitative polymerase chain reaction. In addition, an MTT assay demonstrated that clones #2 and #3 exhibited enhanced proliferation. In conclusion, an miR-137-knockout cell model was successfully established in A2780 cells using CRISPR/Cas9 technology.

Liriopesides B inhibited cell growth and decreased CA125 level in human ovarian cancer A2780 cells.

To study the effect of liriopesides B on cell growth curve, cell doubling time, the activity of tumour marker CA125 and alkaline phosphatase (AKP) in human ovarian cancer A2780 cells. Both cell growth curve and doubling time were studied by MTT assay, CA125 level and AKP activity were determined by respective kits. Results showed that liriopesides B could shift down the A2780 cells growth curve in a dose-time-dependent manner and inhibit the proliferation in A2780 cells with the maximum inhibitory rate 94.462% at 120 h, the doubling time was prolonged too. CA125 level was decreased in a dose-dependent way as well as AKP activity. Liriopesides B exhibited potential anticancer activity against human ovarian cancer A2780 cells.

Characterisation of the antiproliferative constituents and activity of Ficus exasperata (Vahl) on ovarian cancer cells -a preliminary investigation.

Ovarian cancer is one of the most common gynaecological cancers today. This study therefore investigates the anticancer effects of Ficus exasperata extracts and fractions on ovarian cancer cells. The antiproliferative activity of the crude extracts (1 mg/mL) was assessed using the MTT assay on A2780 (ovarian cancer) cell line. Bio-activity guided fractionation was performed and preliminary identification was further achieved using high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. All crude extracts tested exhibited antiproliferative activity except for the methanol extract which interestingly showed proliferative effects. Five fatty acids were identified from the active fractions (FB1-10 and FB1-12). FB1-12 exhibited an IC50 value of 15.20 µg/mL. The least potent fraction (FB1-4 + 5) had an IC50 value of 34.51 µg/mL. H1-HEX and H1-MET exhibited 97.2 and 97.9%, respectively, compared to control. This study therefore provides proof-of-principle that fatty acids of Ficus exasperata exhibit significant antiproliferative effects on ovarian cancer cells.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology.

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 µg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Cytotoxic effects of mithramycin DIG-MSK can depend on the rise of autophagy.

DIG-MSK (demycarosil-3D-ß-D-digitoxosyl mithramycin SK; EC-8042), a novel analogue of mithramycin A, induced autophagy in HCT116 human colon carcinoma and, to a lesser extent, in A2780 human ovarian carcinoma cell lines, which was followed by apoptosis and/or necrotic cell death in a time-dependent way. The effects of DIG-MSK included changes in the expression of a set of genes involved in autophagy, the progression of cells through the different phases of cell cycle, and their halting at the checkpoints. Cells treated with the glucose analogue 2-DG (2-deoxy-D-glucose), which induces autophagy because it impairs cell metabolism, or co-treated with 2-DG plus DIG-MSK, also showed altered gene expression and autophagy. In A2780 cells, some genes involved in autophagy were down-regulated by the different treatments, yet the levels of the proteins they encode could be enough to ensure autophagic flux. In HCT116 cells, up-regulation of several pro-autophagic genes resulted in strong autophagic response. Acidic cell organelles and autophagic flux were more evident in HCT116 than in A2780 cells. DIG-MSK was still cytotoxic in cells that underwent autophagy induced by 2-DG. Therefore, we verified that autophagy resulting from a stress response did not protect cells against DIG-MSK, but, instead, autophagy promoted by either 2-DG or the novel mithralogue can enhance the antitumour activity, which depended on the cell type.

Smilax china L. rhizome extract inhibits nuclear factor-κB and induces apoptosis in ovarian cancer cells.

OBJECTIVE: To study the antitumor effects and associated mechanisms of extract of the Smilax china L. rhizome (SCR) on ovarian cancer cells. METHODS: Ovarian cancer cells A2780 were treated with different concentrations of SCR extract (SCRE), and compared with controls. Effects on cell growth were evaluated by cell counting kit-8 (CCK-8) assay; proliferation effects by EdU incorporation assay; cell cycle by propidium iodide staining; apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide; cellular distribution of nuclear factor-κB (NF-κB) by immunofluorescence; protein levels of NF-κB, caspase-3, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP), Bcl-2-associated X protein (Bax), cellular inhibitor of apoptosis (cIAP)-1, anti-X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-extra large (Bcl-XL), B-cell lymphoma-2 (Bcl-2) and AKT by Western blotting; and effects of SCRE combined with cisplatin or adriamycin on A2780 cells by CCK-8 assay. RESULTS: SCRE suppressed A2780 cell proliferation in a dose-dependent manner (P<0.05,P<0.01), arrested cells in G2/M phase and induced apoptosis by activating caspase-3, PARP and Bax. SCRE treatment also correlated with inhibition of NF-κB and downregulation of Bcl-2, Bcl-XL, cIAP-1, XIAP and AKT. SCRE can promote chemosensitivity to cisplatin and adriamycin in A2780 cells (P<0.01). CONCLUSION: SCR effectively inhibits NF-κB, induces apoptosis and reduces chemoresistance to cisplatin and adriamycin in ovarian cancer cells, which might be its molecular basis for treating ovarian cancer.

Smilax china L. rhizome extract inhibits nuclear factor-κB and induces apoptosis in ovarian cancer cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the antitumor effects and associated mechanisms of extract of the Smilax china L. rhizome (SCR) on ovarian cancer cells.</p><p><b>METHODS</b>Ovarian cancer cells A2780 were treated with different concentrations of SCR extract (SCRE), and compared with controls. Effects on cell growth were evaluated by cell counting kit-8 (CCK-8) assay; proliferation effects by EdU incorporation assay; cell cycle by propidium iodide staining; apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide; cellular distribution of nuclear factor-κB (NF-κB) by immunofluorescence; protein levels of NF-κB, caspase-3, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP), Bcl-2-associated X protein (Bax), cellular inhibitor of apoptosis (cIAP)-1, anti-X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-extra large (Bcl-XL), B-cell lymphoma-2 (Bcl-2) and AKT by Western blotting; and effects of SCRE combined with cisplatin or adriamycin on A2780 cells by CCK-8 assay.</p><p><b>RESULTS</b>SCRE suppressed A2780 cell proliferation in a dose-dependent manner (P<0.05,P<0.01), arrested cells in G2/M phase and induced apoptosis by activating caspase-3, PARP and Bax. SCRE treatment also correlated with inhibition of NF-κB and downregulation of Bcl-2, Bcl-XL, cIAP-1, XIAP and AKT. SCRE can promote chemosensitivity to cisplatin and adriamycin in A2780 cells (P<0.01).</p><p><b>CONCLUSION</b>SCR effectively inhibits NF-κB, induces apoptosis and reduces chemoresistance to cisplatin and adriamycin in ovarian cancer cells, which might be its molecular basis for treating ovarian cancer.</p>