Parthenolide induces apoptosis and cell cycle arrest of human 5637 bladder cancer cells in vitro

Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium), has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose) polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.     

Calotropis procera extract induces apoptosis and cell cycle arrest at G2/M phase in human skin melanoma (SK-MEL-2) cells

Calotropis procera (family: Asclepiadaceae) contains cardiac glycosides which are cytotoxic to cancer cells. The extracts of C. procera have been reported to be cytotoxic to many cancer cell lines and this is the first report against the human skin melanoma cells (SK-MEL-2). The SK-MEL-2 cells treated with C. procera methanolic extract (CPME) were analysed for growth inhibition and apoptosis. The exposure of phosphatidylserine in apoptotic SK-MEL-2 was analysed by using the Annexin-V FITC flow cytometry method. In CPME-treated SK-MEL-2 cells, 19.6% of apoptotic and 58.3% dead cells were observed. The 15.97% and 15.85% of early apoptotic cells were found at 20 μg/mL of the ouabain and paclitaxel, respectively. Active caspases, nuclear degradation confirmed apoptotic SK-MEL-2 cells in time- and dose-dependent manner. The cell cycle analysis shows that CPME treated cells halt at G2/M phase. Significant cytotoxic activity of CPME against SK-MEL-2 may be attributed to its high cardenolide content.     

Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway

Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.     

Brassinin inhibits proliferation and induces cell cycle arrest and apoptosis in nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging.ObjectiveHere, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells.MethodologyThe in vitro free radical scavenging property of the brassinin was assessed by various free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1–50 µM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apoptosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits.ResultsThe brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not disturbed the Vero cell viability. The 30 µM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with ‘S’ and ‘G2/M’ phase. The Bax, caspase-3 and ?9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells.ConclusionOur findings discovered that the brassinin has the capacity to prevent the proliferation and stimulate the apoptotic cell death C666‐1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future.     

Euphorane C,an unusual C17-norabietane diterpenoid from Euphorbia dracunculoides induces cell cycle arrest and apoptosis in human leukemia K562 cells

Four new polycyclic diterpenoids euphoranes A ? D (1–4), together with 12 known terpenoids (5–16) were isolated from the whole plants of Euphorbia dracunculoides. Compound 2 represents the first example of aromatic 15,16,17-trinorabietane diterpenoid in Euphorbia species, and euphorane C (3) is an unusual 17-norabietane diterpenoid. The absolute configurations of 1, 2, and 4 were determined by single crystal X-ray diffraction, while that of 3 was established with the aid of ECD and 1D NMR quantum chemical calculation. Moreover, the absolute stereochemistry of 11 was demonstrated for the first time by crystallographic data. Compounds 1–16 were screened for antiproliferative activities against five human cancer cell lines, and 3 showed significant cytotoxicity against the human leukemia K562 cells with IC₅₀ value of 3.59 μM. Preliminary mechanistic investigation revealed that 3 could induce cell cycle arrest at G0/G1 phase and apoptosis in K562 cells.     

Synthesis,cytotoxicity, apoptosis and cell cycle arrest of a monoruthenium(II)-substituted Dawson polyoxotungstate

By 5-h reaction of cis-[Ru^IICl₂(DMSO)₄] (M2) with K₁₀[α₂-P₂W₁₇O₆₁] (M3) in ice-cooled, HCl-acidic aqueous solution, a water-soluble 1:2-type diamagnetic ruthenium(II) complex of formula K₁₈[Ru^II(DMSO)₂(P₂W₁₇O₆₁)₂]·35H₂O (M1) was unexpectedly obtained as an analytically pure, homogeneous tan-colored solid, in which two DMSO ligands are coordinated to the ruthenium(II) atom. The cytotoxic potential of the complex was tested on C33A, DLD-1, and HepG-2 cancer cells and human normal embryonic lung fibroblasts cell MRC-5; the viability of the treated cells was evaluated by MTT assay. The mode of cell death was assessed by morphological study of DNA damage and apoptosis assays. Compound M1 induced cell death in a dose-dependent manner, and the mode of cell death was essentially apoptosis though necrosis was also noticed. Cell cycle analysis by flow cytometry indicated that M1 caused cell cycle arrest and accumulated cells in S phase.     

Xanthatin mediates G2/M cell cycle arrest,autophagy and apoptosis via ROS/XIAP signaling in human colon cancer cells

Abstract

Xanthatin is a natural plant bicyclic sesquiterpene lactone extracted from Xanthium plants (Asteraceae). In the present study, we demonstrated for the first time that Xanthatin inhibited cell proliferation and mediated G₂/M phase arrest in human colon cancer cells. Xanthatin also activated caspase and mediated apoptosis in these cells. Concomitantly, Xanthatin triggered cell autophagic response. We found down-regulation of X-linked inhibitor of apoptosis protein (XIAP) contribute to the induction of apoptosis and autophagy. Moreover, reactive oxygen species (ROS) production was triggered upon exposure to Xanthatin in colon cancer cells. ROS inhibitor N-acetylcysteine (NAC) significantly reversed Xanthatin-mediated XIAP down-regulation, G₂/M phase arrest, apoptosis and autophagosome accumulation. In summary, our findings demonstrated that Xanthatin caused G₂/M phase arrest and mediated apoptosis and autophagy through ROS/XIAP in human colon cancer cells. We provided molecular bases for developing Xanthatin as a promising antitumor candidate for colon cancer therapy. Abbreviations ROS reactive oxygen species

DMSO dimethyl sulfoxide

5-FU 5-Fluorouracil

3-MA 3-Methyladenine

DCFH-DA 2’7’-dichlorfluorescein-diacetate

NAC N-acetylcysteine

XIAP X-linked inhibitor of apoptosis protein

     

Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest

Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC₅₀ value at 72 h (0.001 ± 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ± 0.037, 0.01 ± 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent.     

Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.     

Luteolin induces mitochondria-dependent apoptosis in human lung adenocarcinoma cell

The effects of luteolin on the proliferation of A549 cells were evaluated by MTT and clone formation assays. DNA ploidy and apoptotic cell percentage were calculated by flow cytometry. The expression of Bax, Bcl-xl, Bcl-2, Mcl-1, caspase-9, caspase-3, and PARP was analyzed by Western blotting. The membrane potential of mitochondria was detected by JC-1 fluorescence microscopy assay. Our results demonstrated that luteolin could inhibit the proliferation of A549 cells via induction of apoptosis, with the evidence of characteristic morphological changes of apoptosis in the nucleus. Furthermore, DNA flow cytometric analysis indicated that luteolin induced a S phase arrest of the cell cycle. The membrane potential of mitochondria was decreased. The protein levels of Bax, Bcl-xl, Bcl-2, Mcl-1, caspase-9, caspase-3, and PARP were activated after treatment with luteolin. Luteolin can inhibit the proliferation of A549 cells and trigger mitochondria- dependent apoptosis in them.     

Pre-treatment of HT-29 cells with 5-LOX inhibitor (MK-886) induces changes in cell cycle and increases apoptosis after photodynamic therapy with hypericin

It may be hypothesized that the lipoxygenase (LOX) metabolic pathway plays an important role in photodynamic therapy (PDT) of malignant tumours, and modification of this pathway may result in administration of lower doses of photodynamic active agents accompanied by reduced side effects. In this study, we examine in more detail the cytokinetic parameters of human colon adenocarcinoma HT-29 cells pre-treated for 48 or 24h with LOX inhibitor MK-886, followed by PDT induced by hypericin. Based on MTT assay the concentrations of both agents (MK-886 and hypericin) with relatively slight (non-significant) cytotoxic effects were selected. These concentrations were used for combined treatment, where MTT response, total cell number, floating cells quantification, viability, cell cycle progression and DNA synthesis were detected. Hoechst/PI staining, PARP fragmentation and mitochondrial membrane potential (MMP) were evaluated to determine the extent of apoptosis. While MK-886 alone caused mainly necrosis, 48h pre-treatment of cells with MK-886 followed by PDT with hypericin clearly shifted the type of cell death to apoptosis. PDT with hypericin alone caused apoptosis in 19% of the cell population. Some combined modalities significantly potentiated the apoptotic effect (31% of apoptotic cells; 2.5microM MK-886/0.1microM hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116kDa) cleavage to characteristic 89kDa fragments and changes in MMP. Increasing concentration of MK-886 was accompanied by massive changes in the cell cycle progression. Combined treatment with lower concentrations of MK-886 and hypericin increased accumulation of cells in the S phase, accompanied by inhibition of DNA synthesis. Increasing concentration of MK-886 in this combination caused the opposite effect, manifesting significant accumulation of cells in the G0/G1 phase. More pronounced effects were observed after the 48h pre-treatment schedule. This anti-proliferative effect was confirmed by BrdU incorporation. These results indicate that combined treatment involving PDT and LOX inhibitor MK-886 may improve the therapeutic effectiveness of PDT.     

UVA-activated 8-methoxypsoralen (PUVA) causes G2/M cell cycle arrest in Karpas 299 T-lymphoma cells

We investigated the effect of UVA-activated 8-methoxypsoralen (PUVA) on the cell line Karpas 299 derived from anaplastic large-cell lymphoma (ALCL) expressing chimeric fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM/ALK). NPM/ALK activates phosphatidylinositol 3 kinase (PI3K)/Akt pathway responsible for the cell protection from apoptosis. We found that PUVA treatment first induced G2/M cell cycle arrest resulting in a decrease in the cell proliferation rate. The mitochondrial apoptosis was triggered immediately following PUVA treatment, as we judged from the unmasking of mitochondrial membrane antigen 7A6. However, the mitochondrial membrane depolarization was not observed and caspase-3 was only slightly activated. The late apoptotic events were lacking: neither translocation of phosphatidylserine to the outer side of plasma membrane nor DNA fragmentation occurred. We revealed that PUVA enhanced the expression of peroxiredoxin, stress protein endoplasmin and galectin-3. Galectin-3 has been shown to protect mitochondrial membrane integrity and prevent cytochrome c release thereby blocking the effector stage of apoptosis. We suggest that the elevated level of this protein following PUVA treatment acts in synergy with the constitutively expressed chimeric kinase NPM/ALK to block the apoptosis.     

Methyl-beta-cyclodextrin inhibits cell growth and cell cycle arrest via a prostaglandin E(2) independent pathway

Methyl-beta-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-beta-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-beta-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.     

Cytotoxicity, cellular uptake, cell cycle arrest, apoptosis, reactive oxygen species and DNA-binding studies of ruthenium(II) complexes

Three ruthenium(II) polypyridyl complexes [Ru(dmb)₂(dadppz)]²⁺ 1, [Ru(bpy)₂(dadppz)]²⁺ 2 and [Ru(phen)₂(dadppz)]²⁺ 3 were synthesized and characterized by elemental analysis, ES-MS, ¹H NMR and ¹³C NMR. Their DNA-binding behaviors were investigated by absorption titration, fluorescence spectroscopy and viscosity measurements. Cytotoxicity in vitro, apoptosis, cell cycle arrest, cellular uptake and reactive oxygen species assays were performed. The complexes were found to show moderate DNA-binding affinities and high cytotoxicities toward A549, BEL-7402, MG-63 and SKBR-3 cell lines. These complexes can effectively induce apoptosis of BEL-7402. In cell cycle assays, the complexes induced S-phase arrest on BEL-7402 cells and G0/G1-phase arrest on SKBR-3 cells. The DNA-binding experiments showed that the three complexes interact with CT-DNA through an intercalative mode.     

Selenium speciation determines the angiogenesis effect through regulating selenoproteins to trigger ROS-mediated cell apoptosis and cell cycle arrest

Tumor angiogenesis is closely related to tumor development, immune escape, and drug resistance. Therefore, the development of effective anti-tumor angiogenesis drugs is of great research significance. Although the current clinical angiogenesis inhibitors have achieved certain efficacy, they also pose the problems of limited and short duration of efficacy, drug resistance, and intrinsic toxicity. Anti-tumor angiogenesis strategies targeting endothelial cells (ECs) have attracted widespread attention in the development of highly effective and low toxicity anti-angiogenesis inhibitors. Studies have verified that the trace element selenium (Se) can inhibit tumor growth by inhibiting tumor angiogenesis through different mechanisms. Nevertheless, it is unclear whether Se speciation has different effects on anti-tumor angiogenesis. Herein, we found that Se exhibited effective anti-angiogenic activity, and its mechanisms of activity were determined by its chemical speciation. Organic Se can significantly inhibit tumor angiogenesis by targeting thioredoxin reductase (TrxR) to trigger cell apoptosis and cell cycle arrest and by increasing reactive oxygen species (ROS) production in ECs. Inorganic Se can induce cell cycle arrest and increase ROS production in ECs, showing promising anti-angiogenic effects. Se nanoparticles (SeNPs) slightly inhibit tumor angiogenesis by inducing apoptosis and cell cycle arrest and by increasing the production of ROS. In summary, this study elucidates the anti-angiogenic activity of Se speciation control with a view to providing a scientific reference for the design and development of novel Se-based highly effective and low toxicity angiogenesis inhibitors.     

The growth suppressing effects of girinimbine on HepG2 involve induction of apoptosis and cell cycle arrest

Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G(0)/G(1) peak (hypodiploid) and caused G(0)/G(1)-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.     

Knockdown of GGCT inhibits cell proliferation and induces late apoptosis in human gastric cancer

Background

Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.

Methods

The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.

Results

We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.

Conclusion

This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.

     

Narrow-band UVB induces apoptosis in human keratinocytes

Narrow-band ultraviolet (NB-UVB) phototherapy emits mostly 311/312 nm light and is commonly used in the treatment of inflammatory skin disorders. As a source of UVB irradiation, NB-UVB causes apoptosis in T lymphocytes but its effects on keratinocytes are unknown. Herein, we have investigated the ability of NB-UVB to induce apoptosis in keratinocytes. Two types of human keratinocytes, primary and immortalized, were exposed to NB-UVB and broad-band UVB (BB-UVB; 315-280 nm) and tested for apoptosis. Both UVB light sources induced apoptosis in keratinocytes as determined by the presence of DNA ladders, although NB-UVB required approximately ten fold higher doses; NB-UVB (1000 mJ/cm2) and BB-UVB (125 mJ/cm2). By comparison, lower doses of NB-UVB (750 mJ/cm2) induced apoptosis in T lymphocytes, suggesting cell type specificity for NB-UVB induced apoptosis. Approximately, 50% or more of the cells underwent apoptosis when exposed to NB-UVB or BB-UVB as revealed by TUNEL assay. Electron micrographs showed that NB-UVB irradiated keratinocytes contained marked chromatin condensation, extensive cytoplasmic vacuolization and fragmentation of the nuclear envelope. Furthermore, Western blot analysis confirmed the presence of activated products of caspase 3 in keratinocytes that received apoptotic doses of NB-UVB. This study defines conditions by which NB-UVB irradiation causes apoptosis in keratinocytes.     

Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.