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#28543190   2017/05/25 Save this To Up

Effects of shRNA-Mediated Silencing of PKM2 Gene on Aerobic Glycolysis, Cell Migration, Cell Invasion, and Apoptosis in Colorectal Cancer Cells.

This study aims to explore the effects of shRNA-mediated silencing on Pyruvate kinase type M2 (PKM2) gene during aerobic glycolysis in colorectal cancer (CRC) cells. CRC tissues and adjacent normal tissues were obtained from 136 patients diagnosed with qRT-PCR, Western blotting, and immunohistochemistry (IHC) were performed to detect mRNA and protein expressions of PKM2. CRC cells were divided into a blank, vector, and PKM2-shRNA groups. Hexokinase (HK) and PKM2 activity were both determined by glucose-6-phosphate dehydrogenase (G-6-PD) coupled colorimetric assay and enzyme coupling rate method. The extracellular lactate concentration was measured by ultraviolet spectrophotometer and caspase activity was measured using spectrophotometry. The proliferation, cell cycle, apoptosis, invasion, and migration of CRC cells were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, transwell assay, and scratch test. Three groups of nude mice were injected with 0.2 mL single-cell suspension from the blank, vector, and PKM2-shRNA groups, respectively. PKM2 protein content in CRC tissues was higher than that in adjacent normal tissues. Results showed that the PKM2-shRNA group exhibited significantly lower mRNA and protein expressions of PKM2, decreased PKM2 activity, reduced lactate metabolism level, increased cell apoptosis rate, elevated caspase-3 and caspase-9 activity, weakened proliferation, and a reduction in cell invasion and migration ability compared to the vector and blank groups. The optical density (OD) value was lower in the PKM2-shRNA group than in the blank and vector groups. These findings indicate that shRNA-mediated silencing of PKM2 gene promotes apoptosis and inhibits aerobic glycolysis, proliferation, migration, and invasion in CRC cells. J. Cell. Biochem. 118: 4792-4803, 2017. © 2017 Wiley Periodicals, Inc.

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#28147711   2017/02/02 Save this To Up

Pharmacokinetics and in vitro and in vivo delivery of sulforaphane by PCL-PEG-PCL copolymeric-based micelles.

A reliable and efficient drug delivery system using PCL-PEG-PCL copolymers was established for the anti-cancer compound sulforaphane (SF) in this study. Encapsulated SF by PCL-PEG-PCL nanoparticles led to formation of SF-loaded PCL-PEG-PCL micelles. Micelles characterization and stability, the particle size and their morphology were determined by DLS and AFM. The loading efficiency of SF was 19.33 ± 1.28%. The results of AFM showed that the micelles had spherical shapes with the size of 107 nm. In vitro release of SF from SF-entrapped micelles was remarkably sustained. The cytotoxicity of free SF, PCL-PEG-PCL and SF/PCL-PEG-PCL micelles was analysis by MTT colorimetric assay on MCF-7, 4T1 and MCF10A cell lines. Expression levels of BCL-2, PARP, COX-2, Caspase-9 and ACTB genes were quantified by real-time PCR. Flow cytometry analysis was performed using the Annexin V-FITC Apoptosis Detection Kit to evaluate the apoptotic effects of free SF compared with SF/PCL-PEG-PCL micelles. Study of the in vivo pharmacokinetics of the SF-loaded micelles was carried out on SF-loaded PCL-PEG-PCL micelles in comparison with free SF. The results of in vivo experiments indicated that the SF loaded micelles significantly reduced the tumor size. In vivo results showed that the multiple injections of SF-loaded micelles could prolong the circulation period and increase the therapeutic efficacy of SF. Also, in comparison with the free-SF solution, encapsulation of the SF in micelles increased the mean residence time from 0.5 to 4 h and the area under the concentration-time curve up to 50 folds.

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#25344211   2014/12/24 Save this To Up

ALDH1A1 mediates resistance of diffuse large B cell lymphoma to the CHOP regimen.

Although there have been substantial advances in our knowledge of the resistance of diffuse large B cell lymphoma (DLBCL) to chemotherapy, there are few efficient treatment strategies for recurrent/refractory DLBCL. The aim of this study was to investigate the role of aldehyde dehydrogenase (ALDH) 1A1 in the resistance of diffuse large B cell lymphoma to the chemotherapeutic mixture consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The involvement of ALDH1A1 in DLBCL was elucidated by knockdown and pharmacologic inhibition; Cell Counting Kit-8 (CCK-8) and clone formation assays were used to determine its role in CHOP sensitivity and clone formation ability. Caspase colorimetric assay was used to measure the extent of apoptosis. Western blot analysis was used to measure signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa B (NF-κB) signaling proteins, and quantitative real-time PCR (RT-PCR) was used to measure the differential expression of ALDH1A1 of DLBCL patients and healthy donors. ALDH1A1 showed a 5.64-fold higher expression in malignant B cells than in normal B cells. Diethylaminobenzaldehyde (DEAB) decreased the half maximal inhibitory concentration (IC50) of the CHOP regimen in Farage cells from 344.78 ± 65.75 to 183.88 ± 49.75 ng/ml (P = 0.004). Both knockdown and inhibition of ALDH1A1 reduced clonogenicity, increased caspase-3/caspase-9 activity, and attenuated the phosphorylation status of STAT3/NF-κB. The prognosis of patients with a high level of ALDH1A1 expression was poor compared with that of patients with low levels of expression (P = 0.044). ALDH1A1 is a new mediator for resistance of DLBCL to CHOP; it is a predictor of clinical prognosis and may serve as a potential target to improve chemotherapy responsiveness of human DLBCL.

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#24250574   2013/11/19 Save this To Up

Apoptosis Induction of Salvia chorassanica Root Extract on Human Cervical Cancer Cell Line.

Salvia chorassanica Bunge is one of the Iranian endemic species of Salvia. There is not any reported literature on S. chorassanica. This study was designed to examine the in-vitro anti-proliferative and proapoptotic effects of the methanol extract of S. chorassanica and its fractions on HeLa cell line. Cells were cultured in EX-CELL®, an animal free medium specially designed for HeLa cell line and incubated with different concentrations of plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry (sub-G1 peak). Activity of caspase -3, -8 and -9 was measured by the caspase colorimetric kit assay. S. chorassanica inhibited the growth of malignant cells and the CH2Cl2 fraction was determined as the most cytotoxic fraction in comparison with other fractions. The calculated IC50 values for methanol extract, n-hexane, CH2Cl2 and EtOAc fractions were 8.841, 5.45, 2.38, and 58.03 μg/mL, respectively. S. chorassanica induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that the cytotoxic mechanism is characterized by apoptosis induction. The activity of caspase-3 and 8 proteins in treated HeLa cells was significantly higher than that of the control while caspase-9 activity did not change significantly. Based on the result obtained from our study, the apoptosis pathway involved in S. chorassanica-induced cell death may be through the extrinsic pathway and it can be a novel promising candidate in the treatment of cancer.

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#24091880   2014/01/08 Save this To Up

Daphnoretin-induced apoptosis in HeLa cells: a possible mitochondria-dependent pathway.

Daphnoretin is a bicoumarin compound isolated from a natural product, Wikstroemia indica, which has been used to treat many diseases. It has strong antiviral and anti-tumor activities. Taking the anti-tumor activity of daphnoretin as a starting point, the present study aimed to test the pro-apoptotic effect of daphnoretin and its underlying mechanism in HeLa cells. The inhibitory effects of daphnoretin on viability and proliferation of HeLa cells were determined by the MTT assay. Daphnoretin-induced apoptotic morphological changes were analyzed by mitochondrial membrane potential and Hoechst staining. The number and stage of apoptotic HeLa cells were determined by flow cytometry. Gene expression was determined by reverse-transcription polymerase chain reaction. Protein expression was determined by western blot. The caspase activity of HeLa cells was detected by a caspase-3 and caspase-9 colorimetric assay kit. We found that daphnoretin significantly inhibited HeLa cells' viability by the MTT assay and flow cytometry. The nuclei of the apoptotic cells exhibited strong, blue fluorescence in Hoechst staining. Bax mRNA and protein levels were increased while bcl-2 mRNA levels were decreased after daphnoretin treatment. Daphnoretin also activated both caspase-3 and caspase-9. These findings suggest that daphnoretin promotes apoptosis of HeLa cells in a mitochondria-mediated way. Daphnoretin therefore has potential to be a promising drug to treat uterine cervix cancer.

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#22876686   2012/08/10 Save this To Up

[Study on anti-proliferation activity and the mechanisms of alkaloid monomers from Gelsemium elegans on HepG2 cell in vitro].

To investigate the effects of alkaloid monomers from Gelsemium elegans on proliferation of HepG2 cell in vitro and the possible mechanism.

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#22457823   2012/03/29 Save this To Up

Puerarin induces mitochondria-dependent apoptosis in hypoxic human pulmonary arterial smooth muscle cells.

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs), and to determine the possible underlying mechanisms.

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#21725610   2011/08/29 Save this To Up

Induction of apoptosis by casticin in cervical cancer cells through reactive oxygen species-mediated mitochondrial signaling pathways.

Casticin, one of the main components from Fructus Viticis, has been reported to inhibit the growth of various cancer cells, including the human cervical cancer cell line HeLa. The purpose of this study was to examine the apoptotic activity and molecular mechanism of casticin action on human cervical cancer cells. The apoptotic activity of casticin on human cervical cancer HeLa, CasKi, SiHa and peripheral blood mononuclear cells (PBMCs) was measured using a histone/DNA ELISA assay, flow cytometry with propidium iodide (PI) staining and DNA agarose gel electrophoresis. The mitochondrial membrane potential and reactive oxygen species (ROS) production were evaluated by flow cytometry analysis. Caspase activities were assayed using a caspase colorimetric activity assay kit. Protein expression levels of cytochrome c, Bax, Bcl-2, Bcl-xL and XIAP were analyzed by Western blotting. Casticin caused accumulation of the Sub-G1 cells and increased reactive oxygen species (ROS) production in HeLa, CasKi, SiHa cell lines, but not in PBMCs. Apoptosis of HeLa cells was induced by casticin via mitochondrial release of cytochrome c due to the reduction of mitochondrial trans-membrane potential, activation of caspase-3 and -9, and the production of reactive oxygen species. The pan caspase inhibitor zVAD-FMK, the caspase-9 inhibitor zLEHD-fmk and N-acetylcysteine suppressed casticin-induced apoptosis. Bax was upregulated, while expression levels of Bcl-xL and XIAP were downregulated. However, there was no change in the expression of Bcl-2 under the same treatment. Our results indicate that casticin-induced apoptosis of cervical cancer cells is mediated by ROS generation and mitochondrial signaling pathways.

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#21542969   2011/05/05 Save this To Up

Silencing of high mobility group A1 enhances gemcitabine chemosensitivity of lung adenocarcinoma cells.

The high mobility group A1 (HMGA1) proteins are architectural transcription factors found to be overexpressed in lung adenocarcinoma. Lentivirus-mediated RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in human cancer cells. Our preliminary study shows that gemcitabine inhibits growth of the human lung cancer cell line SPCA-1 and induces apoptosis, and this effect might link with down-regulation of HMGA1 expression. This study aimed to investigate the chemosensitivity change of the lung adenocarcinoma cells SPCA-1 after HMGA1 inhibition by lentivirus-mediated RNAi.

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#21473903   2011/05/23 Save this To Up

Apoptogenic activity of 2α,3α-dihydroxyurs-12-ene-28-oic acid from Prunella vulgaris var. lilacina is mediated via mitochondria-dependent activation of caspase cascade regulated by Bcl-2 in human acute leukemia Jurkat T cells.

The dried spikes of Prunella vulgaris var. lilacina (Labiatae) have been used for traditional herbal medicine to treat fever, inflammation, dropsy, gonorrhea and cancer in Korea, Japan and China. The present study evaluated the apoptotic effect of 2α,3α-dihydroxyurs-12-en-28-oic acid (DHURS), purified from the dried spikes on human acute leukemia Jurkat T cells.

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