Quantification of Nitric Oxide in Single Cells Using the Single-Probe Mass Spectrometry Technique.

Nitric oxide (NO) is a small molecule that plays important roles in biological systems and human diseases. The abundance of intracellular NO is tightly related to numerous biological processes. Due to cell heterogeneity, the intracellular NO amounts significantly vary from cell to cell, and therefore, any meaningful studies need to be conducted at the single-cell level. However, measuring NO in single cells is very challenging, primarily due to the extremely small size of single cells and reactive nature of NO. In the current studies, the quantitative reaction between NO and amlodipine, a compound containing the Hantzsch ester group, was performed in live cells. The product dehydro amlodipine was then detected by the Single-probe single-cell mass spectrometry technique to quantify NO in single cells. The experimental results indicated heterogeneous distributions of intracellular NO amounts in single cells with the existence of subpopulations.

Metabolomics studies of cell-cell interactions using single cell mass spectrometry combined with fluorescence microscopy.

Cell-cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell-cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell-cell contact) to study metabolomics of single cells affected by cell-cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations.

Single cell mass spectrometry studies reveal metabolomic features and potential mechanisms of drug-resistant cancer cell lines.

Irinotecan (Iri) is a key drug to treat metastatic colorectal cancer, but its clinical activity is often limited by de novo and acquired drug resistance. Studying the underlying mechanisms of drug resistance is necessary for developing novel therapeutic strategies. In this study, we used both regular and irinotecan-resistant (Iri-resistant) colorectal cell lines as models, and performed single cell mass spectrometry (SCMS) metabolomics studies combined with analyses from cytotoxicity assay, western blot, flow cytometry, quantitative real-time polymerase chain reaction (qPCR), and reactive oxygen species (ROS). Our SCMS results indicate that Iri-resistant cancer cells possess higher levels of unsaturated lipids compared with the regular cancer cells. In addition, multiple protein biomarkers and their corresponding mRNAs of colon cancer stem cells are overexpressed in Iri-resistance cells. Particularly, stearoyl-CoA desaturase 1 (SCD1) is upregulated with the development of drug resistance in Iri-resistant cells, whereas inhibiting the activity of SCD1 efficiently increase their sensitivity to Iri treatment. In addition, we demonstrated that SCD1 directly regulates the expression of ALDH1A1, which contributes to the cancer stemness and ROS level in Iri-resistant cell lines.

Single cell mass spectrometry analysis of drug-resistant cancer cells: Metabolomics studies of synergetic effect of combinational treatment.

Irinotecan (IRI), a topoisomerase I inhibitor blocking DNA synthesis, is a widely used chemotherapy drug for metastatic colorectal cancer. Despite being an effective chemotherapy drug, its clinical effectiveness is limited by both intrinsic and acquired drug resistance. Previous studies indicate IRI induces cancer stemness in irinotecan-resistant (IRI-resistant) cells. Metformin, an oral antidiabetic drug, was recently reported for anticancer effects, likely due to its selective killing of cancer stem cells (CSCs). Given IRI-resistant cells exhibiting high cancer stemness, we hypothesize metformin can sensitize IRI-resistant cells and rescue the therapeutic effect. In this work, we utilized the Single-probe mass spectrometry technique to analyze live IRI-resistant cells under different treatment conditions. We discovered that metformin treatment was associated with the downregulation of lipids and fatty acids, potentially through the inhibition of fatty acid synthase (FASN). Importantly, certain species can be only detected from cells in their living status. The level of synergistic effect of metformin and IRI in their co-treatment of IRI-resistant cells was evaluated using Chou-Talalay combinational index. Using enzymatic activity assay, we determined that the co-treatment exhibit the highest FASN inhibition compared with the mono-treatment of IRI or metformin. To our knowledge, this is the first single-cell MS metabolomics study demonstrating metformin-IRI synergistic effect overcoming drug resistance in IRI-resistant cells.

Schizandrin A exerts anti-tumor effects on A375 cells by down-regulating H19.

Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.

Declination of long noncoding RNA paternally expressed gene 10 inhibits A375 cells proliferation, migration, and invasion via mediating microRNA-33a.

The importance of long noncoding RNAs (lncRNAs) has been certified in malignant melanoma. Nonetheless, the functions of lncRNA paternally expressed gene 10 (PEG10) in malignant melanoma remain uninvestigated. This research discloses the influence of PEG10 in the biological actions of malignant melanoma cells. The sh-PEG10 plasmid was transfected into A375 cells; meanwhile, the effects of declined PEG10 on cell viability, apoptosis, migration, invasion, and the correlative protein levels were probed. The miR-33a expression in sh-PEG10-transfected cells was examined, and the above biological processes were studied again in miR-33a inhibitor-transfected A375 cells. Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mechanistic target of rapamycin (mTOR) pathways were delved via Western blot. We found that the enhancement of PEG10 was discovered in melanoma tissues compared to related nonmelanoma tissues. Declination of PEG10 frustrated cell viability, repressed cyclinD1 and CDK4 expression, and triggered apoptosis, as well as suppressed migration and invasion in A375 cells. A negative correction between PEG10 and miR-33a was confirmed, and repressed miR-33a inverted the functions of PEG10 repression in A375 cells. In addition, PEG10 repression discouraged the activation of PI3K/AKT and mTOR pathways via elevation of miR-33a. These results indicated that declination of PEG10 restrained A375 cell growth, migration, and invasion via adjusting miR-33a and PI3K/AKT and mTOR pathways.

Schizandrin A exerts anti-tumor effects on A375 cells by down-regulating H19

Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.

Copper-catalyzed Direct 2-Arylation of Benzoxazoles and Benzoimidazoles with Aryl Bromides and Cytotoxicity of Products.

An efficient copper-catalyzed direct 2-arylation of benzoxazoles and benzoimidazoles with aryl bromides is presented. The CuI/PPh3-based catalyst promotes the installation of various aryl and heteroaryl groups through a C-H activation process in good to excellent yields. The cytotoxicity of obtained 2-aryl benzoxazoles (benzoimidazoles) was also evaluated and 1-methyl-2-(naphthalen-1-yl)benzoimidazole showed potential cytotoxicity.

Effects of error on dose of target region and organs at risk in treating nasopharynx cancer with intensity modulated radiation therapy.

OBJECTIVE: To measure setup error of head and neck neoplasm in radiotherapy and discuss over effects of error on physical dose acting on target region and organs at risk of nasopharynx cancer (NPC) patients treated with intensity modulated radiation therapy (IMRT). METHODS: A total of 152 patients who developed head and neck neoplasm and received IMRT were randomly selected. Through comparing digital portal image and digital reconstruction image, we measured setup error, calculated expanding margin from clinical target volume (CTV) to planning target volume (PTV) and analyzed whether there was rules between setup error and treatment time. Additionally, 20 cases of NPC were selected. Three-dimensional error was simulated in planning system. Dose distribution was recalculated and a series of dose parameters of target volume and OAR were analyzed. RESULTS: Setup error in left-right, head-feet and ventral-dorsal direction was (-0.62±1.46) mm, (-0.41±1.54) mm and (-0.31±1.67) mm respectively. Regarding limit value, the maximum and minimum value in left-right direction, head-feet direction and ventral-dorsal direction was 2.70 mm and -6.00 mm; 3.00 mm and -5.00 mm, 5.00 mm and -7.50 mm. Expanding margin from CTV to PTV was 2.26 mm, 1.88 mm and 1.97 mm in left-right direction, head-feet direction and ventral-dorsal direction. CONCLUSION: During IMRT, only when setup error is controlled below 3 mm can sharply reduce the damage caused by radiation to normal tissue; therefore, quality security and control of electronic portal imaging device need (EPID) to be improved.

Recent advances in design, synthesis and bioactivity of paclitaxel-mimics.

Taxane-type anticancer drugs, including paclitaxel and its semi-synthetic derivatives docetaxel and cabazitaxel, are widely applied to chemotherapy of malignancy like breast cancer, ovarian cancer, non-small cell lung cancer and prostate cancer. However, their clinical applications are generally limited by scarce natural resources, various side effects and multidrug resistance. Therefore, it is significant to develop paclitaxel-mimics with simplified structure, fewer side effects and improved pharmaceutical properties. Based on our investigation on chemistry of paclitaxel, the current review summarized the most recent advances in the design, synthesis and biological activities of paclitaxel-mimics, which could be appealing to researchers in the field of medicinal chemistry and oncology. Meanwhile, smart design, interesting synthesis and potential bioactivities of these novel compounds may also provide valuable reference for the wider scientific communities.

Design, synthesis and biological evaluation of paclitaxel-mimics possessing only the oxetane D-ring and side chain structures.

Two spiro paclitaxel-mimics consisting only of an oxetane D-ring and a C-13 side chain were designed and synthesized on the basis of analysis of structure-activity relationships (SAR) of paclitaxel. In vitro microtubule-stabilizing and antiproliferative assays indicated a moderate weaker activity of the mimics than paclitaxel, but which still represented the first example of simplified paclitaxel analogues with significant anti-tumor biological activity.