Myristicin from nutmeg induces apoptosis via the mitochondrial pathway and down regulates genes of the DNA damage response pathways in human leukaemia K562 cells (original) (raw)
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Journal of Acupuncture and Meridian Studies, 2013
Extract of Myrica cerifera bark has long been fruitfully used as a hepato-protective and anticancer drug in various complementary and alternative systems of medicine. Myricanone, its principal bioactive compound, had also been reported to have apoptosis-promoting ability. We evaluated its anti-cancer potential in vitro in HepG2 liver cancer cells and tried to understand the signal cascades involved in accomplishing apoptosis. Further, we ascertained by using a (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT) assay if it had cytotoxic effects on normal noncancerous liver cells (WRL-68). We deployed various tools and protocols, like phase contrast, scanning electron and fluorescence microscopies, performed an annexinV-FITC/PI assay and cell cycle analysis, and estimated the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization through flow cytometry. Further, analyses of cytochrome-c translocation and of HSP70 and caspase expressions were also done by using immunoblota and Enzyme linked immunosorbent assay (ELISA). Results revealed that myricanone induced apoptosis in HepG2 cells through generation of ROS, depolarization of the mitochondrial membrane, early release of cytochrome-c, down-regulation of HSP70 and activation of a caspase cascade; it had no, or insignificant, cytotoxic effects in WRL-68 cells in vitro and in mice in vivo. Thus, myricanone has great potential for use in formulating an effective drug against both hepatotoxicity and hepatocellular cancer.
Myristicin: a potential cancer chemopreventive agent from parsley leaf oil
Journal of Agricultural and Food Chemistry, 1992
Parsley (Petroselinum sativum Hoffm.) is used extensively as a culinary herb for garnishing and seasoning. Bioassay-directed fractionation of parsley leaf oil has resulted in the isolation of an active compound named myristicin. Myristicin and other fractions were tested for their ability to induce increased activity of the detoxifying enzyme system glutathione S-transferase in several mouse target tissues. Myristicin showed high activity as a GST inducer in the liver and small intestinal mucosa. To establish preliminary structure-activity relationship, the olefinic bond of myristicin was reduced by catalytic hydrogenation to yield dihydromyristicin. The latter compound retained high activity in the liver and small intestinal mucosa. Thus, the isolated double bond is not required for the enzyme-inducing activity. Since the ability to induce an increase in the detoxifying enzyme activity by anticarcinogenic natural products has been found to correlate with their activity in the inhibition of tumorigenesis, myristicin may be considered a potential chemopreventive agent.
Biochemical Pharmacology, 2005
Abrogation of mitochondrial permeability and induction of reactive oxygen species (ROS) production have been observed in chemicalinduced apoptosis; however, the relationship between the mitochondria and intracellular ROS levels in apoptosis is still unclear. In the present study, myricetin (ME) but not its respective glycoside, myricitrin (MI; myricetin-3-O-rhamnose) reduced the viability of human leukemia HL-60 cells via apoptosis, characterized by the occurrence of DNA ladders and hypodiploid cells. Results of Western blotting and caspase activity assays showed that activation of caspases 3 and 9 but not caspases 1, 6 or 8 with cleavage of PARP and D4-GDI proteins is involved in ME-induced apoptosis. A reduction in mitochondrial functions characterized by a decrease in the Bcl-2/Bax protein ratio and translocation of cytochrome c (cyt c) from the mitochondria to the cytosol in accordance with a decrease in mitochondrial membrane potential were observed in ME-treated HL-60 cells. No significant induction of intracellular ROS levels by ME was observed by the DCHF-DA assay, DPPH assay or plasmid digestion assay, and antioxidants including N-acetyl-cysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and tiron (TIR) showed no protective effects on ME-induced apoptosis. A PKC activator, 12-Otetradecaoylphorbol-13-acetate (TPA) significantly attenuated ME-induced apoptosis via preventing cytochrome c release to the cytosol and maintaining the mitochondrial membrane potential by inhibiting the decrease in the Bcl-2/Bax protein ratio; these effects were blocked by protein kinase C (PKC) inhibitors including GF-109203X, H7, and staurosporin. Removing mitochondria by ethidium bromide (EtBr) treatment reduced the apoptotic effect of ME. Results of SAR studies showed that the presence of OH at C3 0 , C4 0 , and C5 0 is important for the apoptosis-inducing activities of ME, and that ME induces apoptosis in another leukemia cell line, Jurkat cells, but not in primary human polymorphonuclear (PMN) cells or in murine peritoneal macrophages (PMs). The results of the present study suggest that apoptosis induced by ME occurs through a novel mitochondrion-dependent, ROS-independent pathway; TPA protects cells from MEinduced apoptosis via PKC activation which prevents the occurrence of mitochondrial destruction during apoptosis.
Apoptosis, 2007
Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Dw mt ) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases -9, -3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-jB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.
Biochemical Pharmacology, 2010
Artemisinin and its derivatives (ARTs) are effective antimalarial drugs and also possess profound anticancer activity. However, the mechanism accounted for its distinctive activity in tumor cells remains unelucidated. We computed Pair wise Pearson correlation coefficients to identify genes that show significant correlation with ARTs activity in NCI-55 cell lines using data obtained from studies with HG-U133A Affymetrix chip. We found c-myc is one of the genes that showed the highest positive correlation coefficients among the probe sets analyzed (r = 0.585, P < 0.001). Dihydroartemisinin (DHA), the main active metabolite of ARTs, induced significant apoptosis in HL-60 and HCT116 cells that express high levels of c-MYC. Stable knockdown of c-myc abrogated DHA-induced apoptosis in HCT116 cells. Conversely, forced expression of c-myc in NIH3T3 cells sensitized these cells to DHA-induced apoptosis. Interestingly, DHA irreversibly down-regulated the protein level of c-MYC in DHA-sensitive HCT116 cells, which is consistent to persistent G1 phase arrest induced by DHA. Further studies demonstrated that DHA accelerated the degradation of c-MYC protein and this process was blocked by pretreatment with the proteasome inhibitor MG-132 or GSK 3b inhibitor LiCl in HCT116 cells. Taken together, ARTs might be useful in the treatment of c-MYC-overexpressing tumors. We also suggest that c-MYC may potentially be a biomarker candidate for prediction of the antitumor efficacies of ARTs. ß
Food and Chemical Toxicology, 2011
Some food flavourings, such as safrole and methyleugenol, are known for their genotoxic and hepatocarcinogenic properties whereas for others, such as myristicin, there is less data. Myristicin and eugenol are both alkenylbenzenes, and we compared their direct genotoxicity in repair proficient (AA8) and repair deficient XRCC À (EM9) Chinese hamster ovary cells. Cell viability was assessed by the MTT assay. The comet assay was used to evaluate DNA breaks, and the c-H2AX assay to evaluate induction of double strand breaks. We assessed apoptosis by measuring caspases activation, and the TUNEL assay. Reduction of cell viability was similar in AA8 and EM9 cells, for both compounds. After 1 h eugenol produced DNA strand breaks in the comet assay and induced double strand breaks in the c-H2AX assay in AA8 cells, while myristicin was not genotoxic in both the comet and the c-H2AX assays. Both flavourings were negative in EM9 cells. After 24 h eugenol and myristicin induced DNA fragmentation detected by TUNEL in both cell lines, but only myristicin activated caspases. Myristicin was more apoptotic than eugenol, in both cell lines. The XRCC1 protein does not influence the apoptotic activity of either compound.
Quantitative Proteomic Analysis of Myc-induced Apoptosis
Journal of Biological Chemistry, 2005
Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an isotopecoded affinity tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.
Journal of Biological Chemistry, 2004
c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin-induced cell death. We have investigated the molecular mechanisms involved in executing doxorubicin-induced apoptosis and show caspase-3 activation by both mitochondria-dependent and-independent pathways. Moreover, serine proteases participate in doxorubicin-induced apoptosis partly by contributing to caspase-3 activation. Finally, a complete rescue from doxorubicininduced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore support a model in which doxorubicin simultaneously triggers multiple c-Myc-dependent apoptosis pathways.
Molecular medicine reports, 2016
The mechanisms underlying myricetin-induced cancer cell apoptosis remain to be elucidated. Certain previous studies have shown that myricetin induces apoptosis through the mitochondrial pathway. Apoptosis, however, can also be induced by other classical pathways, including endoplasmic reticulum (ER) stress and DNA double‑strand breaks (DSBs). The aim of the present study was to assess whether these two apoptotic pathways are involved in myricetin‑induced cell death in SKOV3 ovarian cancer cells. The results revealed that treatment with myricetin inhibited viability of SKOV3 cells in a dose‑dependent manner. Myricetin induced nuclear chromatin condensation and fragmentation, and also upregulated the protein levels of active caspase 3 in a time‑dependent manner. In addition, myricetin upregulated ER stress‑associated proteins, glucose‑regulated protein‑78 and C/EBP homologous protein in SKOV3 cells. Phosphorylation of H2AX, a marker of DNA DSBs, was revealed to be upregulated in myric...