Search

Objective: To compare the effects of three different fixation solutions on immunofluorescence staining of Beclin1 and LC3 proteins in two hypoxic cell models. Methods: In this study, primary myocardial fibroblasts and MCF-7 breast cancer cell lines of hypoxic cell models were fixed with acetone/methanol (1:1), methanol and 4% paraformaldehyde, respectively, and then double immunofluorescence staining were carried out to compare the staining effects of the three fixatives on the key autophagy regulatory proteins Beclin1 and LC3. Results: There were significant differences in the results of immunofluorescence staining of Beclin1 and LC3 proteins in two hypoxic cell models with three kinds of fixed solutions. The best effect of immunofluorescence staining was fixed by acetone/methanol (1:1). The localization and expression of the two proteins could be seen clearly, while methanol, and 4% paraformaldehyde appeared poor effect. Conclusions: In the autophagy-associated protein immunofluorescence double staining experiment of cardiac fibroblasts and MCF-7 breast cancer cells, when other fixatives are used and the staining effect is worse, acetone/methanol (1:1) fixation solution is a good choice before immunofluorescence staining. Moreover, choosing a more suitable fixation solution according to different experimental requirements could help to achieve the best fluorescence staining results. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Modern Laboratory Medicine is the property of Journal of Modern Laboratory Medicine Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To detect the effects of LCL161 combined with docetaxel on the proliferation and apoptosis of human breast cancer MCF-7 cells and to explore its mechanism. Methods CCK-8 ( Cell counting kit-8 ) was used to de­tect the proliferation inhibition effect of various concentrations of LCL161 (1-5 jjimol/L) on the human breast cancer MCF- 7 cells. MCF-7 cells were divided into four groups ： the LCL161 group which was added with 2 jjimol/L LCL161，docetaxel group with 0. 6 mg/L docetaxel, LCL161 combined with docetaxel group with 0. 6 mg/L docetaxel and 2 |jLmol/L LCL161， and the control group with a drug-free medium. CCK-8 was used to detect the cell viability. The colony cloning ability of ach group was detected by cell colony cloning assay. The apoptosis of each group was observed by Hoechst33258 staining. The apoptosis rate of each group was measured by flow cytometry. Western blotting was used to detect the changes of apop- tosis-related protein cellular inhibitor of apoptosis protein 1/2 ( cIAPl/2) and necroptosis protein RIP1. Results The rel­ative cell viability rates of the LCL161 group, docetaxel group, LCL161 combined with docetaxel group, and control group were 86. 84% ±3. 83%，81. 29% ±6.71%，46. 91% ±4. 76%，and 92. 77% ±2. 18%，and the number of colonies was 117 ±14，95 ±9，61 ±12，and 132 ±7，respectivelyï¼› no significant difference was found between the LCL161 group and the control group ( P > 0. 05 ) , and significant difference was found between the rest groups ( all P < 0. 05 ) . In the LCL161 group, the cell membrane permeability increased, with chromatin condensation and some existed nuclear fragmentation. The nuclei of the docetaxel group and control group were lightly stained，the nucleus morphology was elliptical，and the cell membrane was relatively intact. The number of cells in the LCL161 combined with docetaxel group was significantly de­creased ,with fragmented nucleus, and some nuclear disintegration. The apoptosis rates of the LCL161 group, docetaxel group, LCL161 combined with docetaxel group, and control group were 15. 16% ±2.26%，21.42% 土 1. 17%，34. 38% ±3.27%，and 5. 18% ±0. 89%，respectivelyï¼› no significant difference was found between the LCL161 group and the control group ( P > 0. 05 )，and significant difference was found between the rest groups ( all P <0. 05). The relative ex­pression levels of cIAPl protein in the LCL161 group, docetaxel group, LCL161 combined with docetaxel group, and con­trol group were 0.41 ± 0. 09，0. 29 ± 0. 11，0. 11 ± 0. 04，and 0. 62 ±0. 17. The relative expression levels of cIAP2 protein in the LCL161 group, docetaxel group, LCL161 combined with docetaxel group, and control group were 0. 86 ±0. 11，0. 77 ± 0. 15，0. 51 ±0. 12，and 0. 98 ± 0. 01，respectively; no significant difference was found between the LCL161 group and the control group ( P > 0. 05 )，and significant difference was found between the rest groups ( all P < 0. 05 ) . The relative expression levels of RIP1 protein in the LCL161 group, docetaxel group, LCL161 combined with docetaxel group and con­trol group were 1.97 ±0.22，1.84 ±0. 19，3. 17 ±0.26，and 1.00 ±0.07; no significant difference was found between the LCL161 group and the control group ( P > 0. 05 )，and significant difference was found between the rest groups ( all P <0.05). Conclusion LCL161 combined with docetaxel can inhibit the proliferation and promote apoptosis of breast cancer cells ï¼› LCL161 combined with docetaxel can induce the apoptosis of breast cancer cells by down-regulating apoptosis inhibi tory protein and up-regulating the expression of necroptosis protein to exert its anti-tumor effect. [ABSTRACT FROM AUTHOR]

The effects of oenothein B(OEB) on the proliferation, apoptosis, invasion, and migration of breast cancer MCF-7 and MDA-MB-231 cells were investigated by cell culture in vitro, network pharmacology, and molecular docking. In vitro cell experiments revealed that OEB inhibited the proliferation and colony formation ability, and promoted the apoptosis and formation of apoptotic bodies in breast cancer cells, as well as inhibited the invasion and migration of breast cancer cells. The targets of OEB were obtained using SwissTargetPrediction database and breast cancer targets were obtained from GeneCards. The targets of OEB and breast cancer were entered separately in Venny 2.1 software to obtain the Venn diagram of common targets of OEB and breast cancer. The common targets of OEB and breast cancer were input into STRING database to construct a protein-protein interaction(PPI) network, which was entered into Cytoscape 3.7.2 software for network topology analysis. Key targets were screened according to protein association strength, and analyzed for KEGG pathway enrichment. Molecular docking of OEB to key targets using AutoDock software revealed that OEB stably bound to the active pocket of P53, while OEB promoted the expression of P53 protein. MCF-7 and MDA-MB-231 cell viability and migration ability increased after silencing P53, and this change was reversed after treatment with OEB. Therefore, this study showed that OEB inhibited the proliferation, migration, and invasion of breast cancer MCF-7 and MDA-MB-231 cells, and promoted the apoptosis of breast cancer MCF-7 and MDA-MB-231 cells, which may be related to the targeted regulation of P53.

Objective To investigate the effect of metallothionein 2A (MT2A) silencing on cytochrome C oxidase 6B2 subunit (Cpy6B2) expression of breast cancer cells. Methods The breast cancer wild cell line MCF-7 (hereinafter referred as the MCF-7) was divided into the blank control group and MT 2A interference group, and each group had 12 holes. The blank control group did not transfect any genetic materials, and the MT2A interference group transfected the shRNA-MT2A vector. Using semi-quantitative PCR to detect the MT2A mRNA expression levels of the two groups. The MT2A interference group successfully obtained the MT 2A gene silencing. The real-time quantitative PCR was used to analyze the expression of Cpy6B2 in the two groups. Results The results showed that the expression level of MCF-7 Cpy6B2 was increased under the MT2A gene silencing. Conclusion MT2A can inhibit the Cpy6B2 expression of breast cancer cells. [ABSTRACT FROM AUTHOR]

Orthogonal experiments were used to optimize the process parameters of curcumin TPP-PEG-PCL nanomicelles; the particle size, electric potential and morphology under the electron microscope were systematically detected for the curcumin TPP-PEG-PCL nanomicelles; and the stability and in vitro release of the curcumin TPP-PEG-PCL nanomicelles were investigated. With DID fluorescent dye as the fluorescent probe, flow cytometry was used to study the uptake of nanomicelles by breast cancer cells, and laser confocal microscopy was used to study the mitochondrial targeting and lysosomal escape functions of nanomicelles. Under the same dosage conditions, the effect of curcumin TPP-PEG-PCL nanomicelles on promoting the apoptosis of breast cancer cells was evaluated. The optimal particle size of curcumin TPP-PEG-PCL nanomicelle was(17.3±0.3) nm, and the Zeta potential was(14.6±2.6) mV in orthogonal test. Under such conditions, the micelle appeared as regular spheres under the transmission electron microscope. Fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote drug uptake by tumor cells, escape from lysosomal phagocytosis, and target the mitochondria. The cell survival rate and Hoechst staining positive test results showed that curcumin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of breast cancer cells. The curcumin TPP-PEG-PCL micelles can significantly reduce the mitochondrial membrane potential of breast cancer cells, increase the release of cytochrome C, significantly increase the expression of pro-apoptotic protein Bcl-2 and reduce the expression of anti-apoptotic Bax protein. These test results were significantly better than those of curcumin PEG-PCL nanomicelles and curcumin, with statistically significant differences. The results revealed that curcumin TPP-PEG-PCL nanomicelles can well target breast cancer cell mitochondria and escape from the lysosomal capture, thereby enhancing the drug's role in promoting tumor cell apoptosis.