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OBJECTIVE To study in vitro inhibitory effects of realgar nanoparticles on breast cancer stem cells. METHODS Human breast cancer MCF- 7 parent cells were selected as subjects and cultured by serum-free culture to obtain breast cancer stem cells. Using adriamycin （1 mg/L）as positive control ，same concentration of water-processed realgar as reference ，the effects of realgar nanoparticles on the proliferation of MCF- 7 parent cells and stem cells were detected by CCK- 8 method. The effects of realgar nanoparticles on the formation of mammosphere ，the ability of differentiation ，migration and invasion ，the proportion of CD44+/CD24－ subgroup in breast cancer stem cells were detected by mammosphere formation and differentiation experiment ， scratch experiment ，Transwell invasion experiment and flow cytometry. Western blot assay was used to detect the expression of proteins related to epithelial mesenchymal transformation pathway （E-cadherin and vimentin ） in breast cancer stem cells. RESULTS The survival rates of MCF- 7 parent cells and stem cells （except for breast cancer stem cells in both 1 mg/mL groups ）in 1，5，10，40，60，80 mg/L groups of water-processed realgar and realgar nanoparticles were significantly lower than blank control group（P＜0.01）. The number of mammosphere （＞20 stem cells ）in 1，2.5，5，10 mg/L groups of water-processed realgar and realgar nanoparticles was significantly lower than blank control group （P＜0.01）；the volume of mammosphere decreased and the differentiated adherent cells decreased ；the healing rate of wound ，relative invasion rate （except for water-processed realgar 1 mg/L group）and the proportion of CD 44+/CD24－ subgroup were significantly lower than blank control group （P＜0.01）. The expressions of E-cadherin in 2.5，10 mg/L groups of water-processed realgar and realgar nanoparticles was significantly higher than blank control group ，and the expressions of vimentin was significantly lower than those in blank control group （P＜0.01）. The above effects of realgar nanoparticles were generally better than those of water-processed realgar with the same mass concentration （P＜ 0.01）. CONCLUSIONS Compared with water-processed realgar with the same mass concentration ，realgar nanoparti cles can significantly inhibit the proliferation of breast cancer stem cells， the formulation and differential ability of mammo- sphere，and reduce the proportion of CD 44+/CD24－ subgroup. The effect may be associated with the inhibition of migration and invasion of breast cancer stem cells by inhibiting the expression of proteins related to epithelial mesenchymal transformation pathway.
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Objective::To observe the effect of realgar nanoparticles (a representative drug in toxin eliminating therapeutics) targeting hypoxia-inducible factors (HIF), which act as effector molecules on metabolic reprogramming of lung cancer stem cells, and to explore the effect mechanism of lung cancer stem cells and metabolic reprogramming in the process of lung cancer metastasis, so as to verify the effectiveness of toxin eliminating therapeutics in the prevention and treatment of lung cancer metastasis. Method::Lung cancer A549 cells were cultured in vitro, and lung cancer stem cells were then identified and selected. The stem cells were divided into blank control group, cisplatin group (5 mg·L-1), realgar nanoparticles low, medium and high dose groups (100, 200, 400 mg·L-1). After intervention, glucose oxidase method was used to detect the effect of realgar nanoparticles on the glucose metabolism of lung cancer stem cells, real-time polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression levels of hypoxia-inducible factors-1α (HIF-1α), C-myc and p53, while Western blot was used to detect the expression of related proteins HIF-1α, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR), and enzyme linked immunosorbent assay (ELISA) was used to detect the glucose transporter 1 (GLUT1), pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M (PKM), phosphofructokinase(PFK), pyruvate dehydrogenase (PDH) and lactic dehydrogenase (LDH) expression. Result::As compared with the blank control group, realgar nanoparticles can reduce the glucose consumption of lung cancer stem cells, and the glucose consumption was reduced with the increase of dose in a time-and dose-dependent manner (P<0.01). Realgar nanoparticles can inhibit the mRNA expression of HIF-1α, a key factor in metabolic reprogramming of lung cancer stem cells (P<0.05, P<0.01), down-regulated C-myc mRNA and up-regulated the p53 mRNA expression (P<0.05, P<0.01), down-regulated protein expressions of PI3K, Akt, mTOR(P<0.05, P<0.01), and inhibited the expression of related enzymes GLUT1, PDK1, PFK, PKM, PDH, and LDH levels (P<0.05, P<0.01). With the increase of dose, the regulation and control ability of realgar nanoparticles gradually increased. Conclusion::Toxin eliminating therapeutics can drive the metabolic reprogramming of lung cancer stem cells by targeting HIF effector molecule, and then inhibit the invasion and metastasis of lung cancer.
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Objective Quantitative analysis of four arsenic species As (III), As (V), monomethyl arsenate (MMA), dimethyl arsenate (DMA) in rat serum, liver, kidney, and spleen was performed to compare their differences between realgar and realgar nanoparticles (NPs) groups. Methods SD rats were ig treated with blank solvent, realgar, and realgar NPs (800 mg/kg) respectively. After 28 d of continuous administration, serum and tissues were collected and four arsenic species were determined by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Results Four arsenic species were detected in serum and kidney of rats, three were detected in the liver and two in the spleen. The content of arsenic species in the realgar NPs group was significantly higher than that in the realgar group. Conclusion Nanotechnology enhanced the bioavailability of realgar, and more arsenic was absorbed into the body and underwent metabolic transformation, which might lead to increased toxicity of realgar NPs.
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Aim To observe the effects of realgar nano-particles on B cell non-Hodgkin's lymphoma Raji cells in vitro. Methods Realgar nanoparticles and crude realgar particles were characterized with a laser particle size analyzer, a transmission electron microscopy (TEM)and an atomic force microscopy(AFM). The morphological changes of proliferation of Raji cells brought about by the use of realgar naoparticles and crude realgar particles were observed with a light mi-croscope. The membrane changes of Raji cells treated with realgar naoparticles and crude realgar particles were observed with AFM. The ultrastructures of Raji cells were observed with TEM. The inhibitory effects of Raji cells treated with realgar naoparticles and crude realgar particles were measured with MTT. The nuclear apoptosis morphologies of Raji cells were observed with fluorescence microscopy. The apoptosis rates and the cell cycle distributions of Raji cells treated with real-gars were measured with flow cytometry. Results The size of realgar nanoparticles and crude realgar particles was (79 ± 8)nm and (1. 89 ± 0. 2)μm,respectively. Light microscopy showed that realgar nanoparticles could inhibit the aggregation growth of Raji cells. AFM showed that Raji cells treated with realgar nanoparticle became shrank, had smaller volume and lost the growth state of stretching out. Raji cells treated with crude realgars did not change significantly. TEM showed Raji cells treated with realgar nanoparticle had damaged subcellular organelles and mitochondria with increased vacuoles. The Raji cells treated with crude realgar did not change significantly. MTT assay showed that when treated with the final concentration of 50 mg ·L - 1 of realgar nanoparticle for 24 h,the cell survival rate of Raji cells was (40 ± 2)% . When treated with the same concentration of crude realgar,its survival rate was (65 ± 3)% . When treated with 50 mg·L - 1 of realgar nanoparticle for 48 h,its survival rate was only 10 % ,and when treated with crude realgar ,its survival rate was (42 ± 2 )% . Fluorescence micro-scope indicated that the Raji cells treated with realgar nanoparticle had obvious nuclear apoptosis,which was not obvious in crude realgar group. Flow cytometry showed that the total apoptosis rate of Raji cells in-duced by realgar nanoparticles and by crude realgar was 11. 14%,15. 9%,respectively. Compared with those treated with crude realgar,the Raji cells treated with realgar nanoparticles presented a significantly higher ratio cell distribution in G1 phase and an obvious decreased ratio in S phase. Conclusion Compared with crude realgar particles,the same dose of realgar nanoparticles can significantly inhibit the proliferation of Raji cells,destroy their sub-cellular structure,and induce the cell apoptosis of Raji cells.
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The effect of realgar nanoparticles (NPs) on endogenous small molecules in rat kidney was analyzed by mass spectrometry-based metabolomics.The relationship between the changes of metabolites and the nephrotoxicity of realgar NPs was also discussed to provide a basis for the further toxicity study and the clinical application of realgar NPs.SD rats were randomly divided into seven groups,including control group,three doses (40,200,1 000 mg/kg) of relegar and realgar NPs groups,respectly.After 28 days of continuous intragastric administration,all rats were sacrificed and their serum and kidney samples were collected.The toxic effect of realgar NPs on kidney tissues were examined by biochemical analysis and histopathologic examination,which revealed a dosedependent nephrotoxicity induced by realgar NPs.The LC-MS and GC-MS analysis were performed for the subsequent metabolomics study.A series of 32 metabolites were found to be altered significandy in the kindey of realgar NPs treated rats,and might serve as potential nephrotoxicity biomarkers.The results of metabolic pathway analysis indicated that the nephrotoxicity of realgar NPs might be associated with the disorders of the amino acids and phosphatidic acid metabolism.
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Realgar nanoparticles (NPs) are increasingly used as therapeutic agents for their enhanced anti-proliferation effect and cytotoxicity on cancer cells. However, the alteration of particle size may enhance biological reactivity as well as toxicity. A LC/MS and GC/MS based metabolomics approach was employed to explore the mechanism of realgar NPs-induced hepatotoxicity and identify potential biomarkers. Male Sprague-Dawley rats were administrated intragastrically with realgar or realgar NPs at a dose of 1.0 g·kg·d for 28 days and toxic effects of realgar NPs on liver tissues were examined by biochemical indicator analysis and histopathologic examination. Increased levels of serum enzymes and high hepatic steatosis were discovered in the realgar NPs treated group. Multivariate data analysis revealed that rats with realgar NPs-induced hepatotoxicity could be distinctively differentiated from the animals in the control and realgar treated groups. In addition, 21 and 32 endogenous metabolites were apparently changed in the serum and live extracts, respectively. Realgar NPs might induce free fatty acid and triglyceride accumulation, resulting in hepatotoxicity. In conclusion, the present study represents the first comprehensive LC/MS- and GC/MS-based metabolomics analysis of realgar NPs-induced hepatotoxicity, which may help further research of nanotoxicity.