OBJECTIVE: To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. METHODS: Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). RESULTS: Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). CONCLUSION Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.
Humans ; Antineoplastic Agents/therapeutic use* ; Apoptosis/drug effects* ; Apoptosis Regulatory Proteins/immunology* ; Autophagy/immunology* ; bcl-2-Associated X Protein/immunology* ; Bortezomib/therapeutic use* ; Burkitt Lymphoma/immunology* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Drug Therapy, Combination ; Proto-Oncogene Proteins c-akt ; Proto-Oncogene Proteins c-bcl-2 ; Receptors, CXCR/immunology* ; RNA, Messenger ; TOR Serine-Threonine Kinases ; Xanthones/therapeutic use*

Metabolic reprogramming is a common phenomenon in cancer, with aerobic glycolysis being one of its important characteristics. Hypoxia-inducible factor-1α (HIF1Α) is thought to play an important role in aerobic glycolysis. Meanwhile, naringin is a natural flavanone glycoside derived from grapefruits and many other citrus fruits. In this work, we identified glycolytic genes related to HIF1Α by analyzing the colon cancer database. The analysis of extracellular acidification rate and cell function verified the regulatory effects of HIF1Α overexpression on glycolysis, and the proliferation and migration of colon cancer cells. Moreover, naringin was used as an inhibitor of colon cancer cells to illustrate its effect on HIF1Α function. The results showed that the HIF1Α and enolase 2 (ENO2) levels in colon cancer tissues were highly correlated, and their high expression indicated a poor prognosis for colon cancer patients. Mechanistically, HIF1Α directly binds to the DNA promoter region and upregulates the transcription of ENO2; ectopic expression of ENO2 increased aerobic glycolysis in colon cancer cells. Most importantly, we found that the appropriate concentration of naringin inhibited the transcriptional activity of HIF1Α, which in turn decreased aerobic glycolysis in colon cancer cells. Generally, naringin reduces glycolysis in colon cancer cells by reducing the transcriptional activity of HIF1Α and the proliferation and invasion of colon cancer cells. This study helps to elucidate the relationship between colon cancer progression and glucose metabolism, and demonstrates the efficacy of naringin in the treatment of colon cancer.
Glycolysis ; Colonic Neoplasms/metabolism* ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Phosphopyruvate Hydratase/metabolism* ; Flavanones/pharmacology* ; Cell Line, Tumor ; Databases, Genetic ; Cell Proliferation/drug effects* ; Transfection ; Warburg Effect, Oncologic

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.
Carcinoma, Non-Small-Cell Lung/pathology* ; Cell Line, Tumor ; Cell Movement/drug effects* ; Cell Proliferation/drug effects* ; Humans ; Isoflavones/pharmacology* ; Lung Neoplasms ; MicroRNAs/metabolism* ; Ubiquitin-Protein Ligases/metabolism*

Apoptosis/drug effects* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Pentacyclic Triterpenes/pharmacology* ; Stomach Neoplasms/pathology*

Objective: Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation. Methods: Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR). Results: Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation Conclusion Antimony activated astrocytes by activating the NF-κB signaling pathway.
Animals ; Antimony/toxicity* ; Astrocytes/metabolism* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Glial Fibrillary Acidic Protein/metabolism* ; MAP Kinase Kinase Kinases ; Male ; Mice, Inbred ICR ; NF-kappa B/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Rats ; Signal Transduction/drug effects*

OBJECTIVE: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. METHODS: The cytotoxic effects of 28 Nilotinib derivatives on K562, KA, KG, HA and 32D cell lines were detected by MTT assays, and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP RESULTS: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives, which impairs leukemia cell lines, but spares normal hematopoietic cell line. Comparing with Nilotinib, Nilo 22 could induce the apoptosis of GFP CONCLUSION Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells, especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.
Animals ; Apoptosis/drug effects* ; Cell Cycle/drug effects* ; Cell Line, Tumor ; Humans ; Leukemia ; Mice ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Telomerase/metabolism* ; Telomere/metabolism*

OBJECTIVE: To investigate the inhibitory effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI) HS-10296 on the proliferation of triple-negative breast cancer (TNBC) MDA-MB-231 cells and explore the possible molecular mechanism. METHODS: MDA-MB-231 cells were treated with HS-10296 for 24, 48, or 72 h, and CCK-8 assay was used to assess the changes in the cell viability. The inhibitory effect of HS-10296 on cell proliferation was determined by clonogenic assay. JC-1 and flow cytometry were employed for analyzing the cell apoptosis, and the ultrastructure of the cells was observed under electron microscope. After pretreatment with autophagy inhibitor chloroquine (CQ), MDA-MB-231 cells were divided into control group, CQ treatment group, HS-10296 (4 and 6 μmol/L) treatment groups and combined treatment groups, and the sensitivity of the treated cells to HS-10296 was determined using CCK-8 assay. The effects of HS-10296 on EGFR pathway and apoptosis- and autophagy-related proteins in MDA-MB-231 cells were investigated using Western blotting. RESULTS: HS-10296 significantly inhibited the proliferation of MDA-MB-231 cells with IC values at 24, 48 and 72 h of 8.393, 2.777 and 2.016 μmol/L, respectively. JC-1 and flow cytometry showed that HS-10296 induced obvious apoptosis of MDA-MB-231 cells, which showed an apoptosis rate of (21.63 ± 2.97)% following treatment with 8 μmol/L HS-10296. Autophagy vesicles were observed in the cells treated with HS-10296 under electron microscope. In MDA-MB-231 cells pretreated with CQ, inhibition of autophagy significantly enhanced HS-10296-induced cell death. Western blotting showed that the apoptosis-related protein caspase-3 was activated after HS-10296 treatment to cut its substrate PARP. The expression of autophagy-related protein light chain 3B (LC3B) was significantly enhanced after HS-10296 treatment ( < 0.01), which also resulted in inhibited phosphorylation of EGFR and AKT proteins in the cells. CONCLUSIONS HS-10296 can inhibit the proliferation and induce autophagy and apoptosis of MDA-MB-231 cells by inhibiting the EGFR/PI3K/AKT signaling pathway.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Breast Neoplasms ; drug therapy ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; ErbB Receptors ; metabolism ; Humans ; Protein Kinase Inhibitors ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVE: To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects. METHODS: The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells. RESULTS: Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P < 0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P < 0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P < 0.05; 35.01%±1.24% vs. 16.54%±1.31%, P < 0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn't show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P < 0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P < 0.05). CONCLUSION Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.
Antineoplastic Agents/therapeutic use* ; Apoptosis/drug effects* ; Carcinoma, Squamous Cell/drug therapy* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Humans ; Mouth Neoplasms/drug therapy* ; Pyrans

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*