Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937

Background

Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.

Methods

Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.

Results

Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.

Conclusions

These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.

General significance

This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.     

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs–SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.     

Tetrazolium violet inhibits cell growth and induces cell death in C127 mouse breast tumor cells

Tetrazolium violet (TV), a tetrazolium salt, has been applied in several fields, including estimating respiration rate, as a redox indicator of microbial growth, and for estimating the number of viable animal cells. It has recently been found that TV is capable of inducing apoptosis in rat glioblastoma cells by way of an elusive mechanism. In this study, we demonstrated that TV also induced apoptosis in mouse breast tumor C127 cells as evidenced by nucleus condensation and nucleus fragmentation. Our data showed that TV caused activation of caspase-3 and caspase-8, but not caspase-9. An enhancement in Fas and its two ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by TV. Also, the results first reported that TV not only inhibited C127 cells proliferation but also blocked cell cycle progression in the G1 and G2 phase, determined by MTT assay and flow cytometry analysis. Immunofluorescence assay demonstrated that TV significantly increased the expression of p53 protein, which caused cell cycle arrest. Taken together, p53, Fas/FasL, and the caspase apoptotic system may participate in the antiproliferative activity of TV in C127 cells.     

Role of the mitochondrial pathway in serum deprivation-induced apoptosis of rat endplate cells

The apoptosis of cartilage endplates (CEPs), acting as an initiating factor, plays a vital role in the pathogenesis of intervertebral disc degenerative diseases, the underlying molecular mechanism of the apoptotic process in CEPs is still not clear. The present study aimed to investigate the mechanism of CEP cell apoptosis. We found that low levels of fetal bovine serum (FBS) can induce cell apoptosis. Serum deprivation led to high expression levels of caspase-9, caspase-3, PARP, cytochrome-c and Bax. Flow cytometric analysis showed that inhibition of the intrinsic pathway by a caspase-9 inhibitor (z-LEHD-fmk) significantly suppressed serum deprivation-induced apoptosis. However, a caspase-8 inhibitor (z-IETD-fmk) did not reduce apoptotic cell death. These data suggest that serum deprivation induces apoptosis in rat CEP cells via the activation of the intrinsic apoptotic pathway. The efficacy of a caspase-9 inhibitor in attenuating or preventing apoptosis of serum deprivation-induced disc cell apoptosis suggests that targeting the intrinsic apoptotic pathway may be used as a potential therapy for the treatment of disc degeneration.     

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.     

MST1 activation by curcumin mediates JNK activation,Foxo3a nuclear translocation and apoptosis in melanoma cells

Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.     

Cadmium activates CaMK-II and initiates CaMK-II-dependent apoptosis in mesangial cells

Cadmium is a toxic metal that initiates both mitogenic responses and cell death. We show that Cd(2+) increases phosphorylation and activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) in mesangial cells, in a concentration-dependent manner. Activation is biphasic with peaks at 1-5 min and 4-6 h. Cadmium also activates Erk, but this appears to be independent of CaMK-II. At 10-20 microM, Cd(2+) initiates apoptosis in 25-55% of mesangial cells by 6h. Inhibition of CaMK-II, but not of Erk, suppresses Cd(2+)-induced apoptosis. We conclude that activation of CaMK-II by Cd(2+) contributes to apoptotic cell death, independent of Erk activation.     

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Kaempferol induces apoptosis in human HCT116 colon cancer cells via the Ataxia-Telangiectasia Mutated-p53 pathway with the involvement of p53 Upregulated Modulator of Apoptosis

Dietary flavonols have been found to possess preventive and therapeutic potential against several kinds of cancers. This study is conducted to investigate the anti-proliferation effects of kaempferol, a major component of food flavonols, against colon cancer cells. In the human HCT116 colon cancer cell line, kaempferol induced p53-dependent growth inhibition and apoptosis. Furthermore, kaempferol was found to induce cytochrome c release from mitochondria and activate caspase-3 cleavage. The Bcl-2 family proteins including PUMA were involved in this process. Kaempferol also induced ATM and H2AX phosphorylation in HCT116 cells, inhibition of ATM by a chemical inhibitor resulted in abrogation of the downstream apoptotic cascades. These findings suggest kaempferol could be a potent candidate for colorectal cancer management.     

Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, have serious gastrointestinal side effects. Since their direct cytotoxicity was suggested to be involved in this side effect, we here tried to identify NSAID-resistant genes. We screened for Saccharomyces cerevisiae genes whose overexpression causes indomethacin resistance and identified the TPO1 gene, which encodes a major facilitator superfamily transporter. Its overexpression or deletion made yeast cells resistant or sensitive, respectively, to some NSAIDs. A BLAST search identified the possible human orthologue of Tpo1p, tetracycline transporter-like protein (TETRAN), whose overexpression in cultured human cells caused resistance to some NSAIDs, suggesting that TETRAN is an efflux pump for some NSAIDs.     

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

Background

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.

Methods

MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.

Results

BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α₁, α₆ and β₁ integrin subunit expression, and increases α₅ subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.

Conclusion

BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.

General significance

Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.     

7,8-Dihydroxy-4-methylcoumarin induces apoptosis of human lung adenocarcinoma cells by ROS-independent mitochondrial pathway through partial inhibition of ERK/MAPK signaling

Coumarins have attracted intense interest in recent years because they have been identified from natural sources, especially green plants and have diverse pharmacological properties. In this study, we investigated whether 7,8-dihydroxy-4-methylcoumarin (DHMC) caused apoptosis in A549 human non-small cell lung carcinoma cells (NSCLC) and, if so, by what mechanisms. Here, we show that, in A549 human NSCLC cells, DHMC induces apoptosis through mitochondria-mediated caspase-dependent pathway. Although an increase in the levels of reactive oxygen species (ROS) was observed, pre-treatment with antioxidant showed no protective effect against DHMC-induced apoptosis. In addition, our immunoblot data revealed that DHMC treatment led to down-regulation of Bcl-xl, Bax, p21, Cox-2, p53 and upregulation of c-Myc. Results in the present study for the first time suggest that DHMC induces apoptosis in human lung A549 cells through partial inhibition of ERK/MAPK signaling.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22 kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200 μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100–400 μg/ml for 24 h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP.     

Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3

Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10-60 microg/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 microg/ml at 24 h and 31.5 microg/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 microg/ml GLA for 48 h and with 40 microg/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pan-caspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation.     

p38 MAPK is involved in CB2 receptor-induced apoptosis of human leukaemia cells

Cannabinoids have been shown to inhibit the growth of a broad spectrum of tumour cells. However, the molecular mechanisms involved in that effect have not been completely elucidated. Here, we investigated the possible involvement of mitogen-activated protein kinases (MAPKs) in CB2 receptor-induced apoptosis of human leukaemia cells. Results show that stimulation of the CB2 receptor leads to p38 MAPK activation and that inhibition of this kinase attenuates CB2 receptor-induced caspase activation and apoptosis. These findings support a role for p38 MAPK in CB2 receptor-induced apoptosis of human leukaemia cells.     

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.     

Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.