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

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.
Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; toxicity ; Apoptosis ; drug effects ; Caspases ; genetics ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dioscoreaceae ; chemistry ; Heterografts ; drug effects ; growth & development ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Nude ; Phosphorylation ; drug effects ; Plant Tubers ; chemistry ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Saponins ; isolation & purification ; pharmacology ; toxicity

OBJECTIVE: To investigate the anti-proliferative effects of saponins prepared from Plena Clematis (PC) cultured in Fujian Province, China on 4 human tumor cell lines and its possible anti-tumor mechanism. METHODS: The growth inhibition assays of saponins on human esophageal squamous carcinoma cell line (EC9706), human hepatoma cell line (HepG-2), human oral cancer cell line (KB) and human gastric cancer cell line (BGC-823) were evaluated in vitro by thiazolyl blue (MTT) method. The inhibitory effects on EC9706 treated with different concentrations of saponins (15.62, 31.25, 62.50, 125, 250 and 500 μg/mL) were performed in vitro by MTT method. The morphology and nuclear staining with acridine orange/ethidium bromide of EC9706 cells treated with saponins were illustrated under an inverted phase fluorescence microscope. The apoptotic effects of saponins were further evaluated by annexin-V/propidium iodide dual staining experiment to examine the occurrence of phosphatidylserine externalization onto the cell surface by a flflow cytometer. RESULTS: MTT assay showed that the saponins could inhibit the proliferation of 4 tumor cell lines. Among them, the maximum inhibition rate of 73.1% was detected in EC9706 cells at the saponins concentration of 250 μg/mL for 24 h. Further investigation indicated that the saponins induced EC9706 cells apoposis. The EC9706 cells presented apoptotic characteristics when treated with saponins, including that the morphologies of EC9706 cells were appeared round-shaped with higher refraction, and the cell nuclear stained orange with EB after 250 μg/mL saponins exposure. The flow cytometry analysis results showed that the induction of cell cycle arrest in apoptotic system may participate in the anti-proliferative activity of saponins on EC9706 cells. CONCLUSION The saponins from PC exhibited significant cytotoxicity against human EC9706, KB, BGC-823, and HepG-2 cells and might be beneficial to development of ethnic pharmaceutical plant for potential anti-tumor drugs.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Clematis ; chemistry ; Humans ; Saponins ; pharmacology

Glioblastoma is a common brain tumor and the overall survival rate of the patients is very low, so it is an effective way to develop the potential chemotherapy or adjuvant chemotherapy drugs in glioblastoma treatment. As a well-known antimalarial drug, artesunate(ARTs) has clear side effects, and recently it has been reported to have antitumor effects, but rarely reported in glioblastoma. Different concentrations of ARTs were used to treat the glioblastoma cells, and then the inhibitory effect of ARTs on glioblastoma proliferation was detected by MTT assay; Ki67 immunofluorescence assay was used to detect the proliferation of cells; Soft agar experiment was used to explain the clonal formation abilities ; Flow Cytometry was used to detect the cell cycle; and Western blot assay was used to determine the expression of key cell cycle protein. MTT assay results indicated that ARTs-treated glioblastoma cell A172, U251, U87 were significantly inhibited in a time-and-dose dependent manner as compared to the control group(DMSO treatment group). Soft agar experiment showed that ARTs could significantly reduce the clonal formation ability of glioblastoma. Furthermore, Flow cytometry analysis showed that ARTs could obviously increase the cell proportion in G₀/G₁ phase and reduce the cell proportion in S phase. Western blot results showed that the expressions of cell cycle-related proteins CDK2, CDK4, cyclin D1 and cyclin B1 were all obviously down-regulated. Above all, ARTs may inhibit the proliferation of glioblastoma cells by arresting cell cycle in G₀/G₁ phase through down-regulating the expression of CDK2, CDK4, cyclin D1, cyclin B1. These results may not only provide a novel method for rediscovering and reusing ARTs but also provide a new potential drug for treating glioblastoma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Artesunate ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Glioblastoma ; drug therapy ; pathology ; Humans

OBJECTIVETo explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.

METHODSThe HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.

RESULTSThe bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.

CONCLUSIONSBufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

Apoptosis ; drug effects ; genetics ; Bufanolides ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia, Erythroblastic, Acute ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects ; genetics ; WT1 Proteins ; genetics ; metabolism

Pharmaceutical research has focused on the discovery and development of anticancer drugs. Clinical application of chemotherapy drugs is limited due to their severe side effects. In this regard, new naturally occurring anticancer drugs have gained increasing attention because of their potential effectiveness and safety. Fruits and vegetables are promising sources of anticancer remedy. Clausena (family Rutaceae) is a genus of flowering plants and includes several kinds of edible fruits and vegetables. Phytochemical and pharmacological studies show that carbazole alkaloids and coumarins from Clausena plants exhibit anticancer activity. This review summarizes research progresses made in the anticancer properties of plants belonging to Clausena; in particular, compounds with direct cytotoxicity, cell cycle arrest, apoptosis induction, and immune potentiation effects are discussed. This review reveals the potential use of plants from Clausena in preventing and treating cancer and provides a basis for development of relevant therapeutic agents.
Alkaloids ; chemistry ; pharmacology ; therapeutic use ; Antineoplastic Agents ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carbazoles ; chemistry ; pharmacology ; therapeutic use ; Cell Cycle Checkpoints ; drug effects ; Clausena ; chemistry ; Coumarins ; chemistry ; pharmacology ; therapeutic use ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Humans ; Plants, Medicinal ; chemistry

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.
Animals ; Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Survival ; drug effects ; Dogs ; Ginkgo biloba ; chemistry ; toxicity ; Lysosomes ; drug effects ; metabolism ; Madin Darby Canine Kidney Cells ; Mitochondria ; drug effects ; metabolism ; Necrosis ; drug therapy ; metabolism ; physiopathology ; Plant Extracts ; toxicity ; Salicylates ; chemistry ; toxicity

BACKGROUND/AIMS: The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. METHODS: We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. RESULTS: Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-kappaB (NF-kappaB) and beta-catenin. CONCLUSIONS: We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-kappaB and beta-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.
Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Apoptosis Regulatory Proteins/metabolism ; Benzoates/*pharmacology ; Cell Proliferation/drug effects ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*drug effects ; G1 Phase Cell Cycle Checkpoints/drug effects ; Humans ; Imatinib Mesylate/*pharmacology ; Iron Chelating Agents/*pharmacology ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Signal Transduction/drug effects ; Triazoles/*pharmacology

OBJECTIVETo provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study.

METHODSEffects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes.

RESULTSTreatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target.

CONCLUSIONPsorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2.

Caspase 3 ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Homeopathy ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Reactive Oxygen Species ; metabolism ; bcl-2-Associated X Protein ; analysis

OBJECTIVETo investigate the effects of ophiopogonin D on human breast cancer MCF-7 cells.

METHODSCell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation experiments. Cell cycle was measured with cell cycle flow cytometry and a living cell assay. Apoptosis and terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling assays were performed to detect the apoptosis of MCF-7 cells induced by ophiopogonin D. Finally, Western blotting was used to explore the mechanism.

RESULTSExposure of cells to ophiopogonin D resulted in marked decreases in viable cells and colony formation with a dose-dependent manner. Treatment of these cells with ophiopogonin D also resulted in cell cycle arrest at the G(2)/M phase, and increased apoptosis. Mechanistically, ophiopogonin D-induced G(2)/M cell cycle arrest was associated with down-regulation of cyclin B1. Furthermore, activation of caspase-8 and caspase-9 was involved in ophiopogonin D-induced apoptosis.

CONCLUSIONThe data suggested that ophiopogonin D inhibits MCF-7 cell growth via the induction of cell cycle arrest at the G(2)/M phase.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; G2 Phase Cell Cycle Checkpoints ; drug effects ; Humans ; M Phase Cell Cycle Checkpoints ; drug effects ; MCF-7 Cells ; Saponins ; pharmacology ; Spirostans ; pharmacology