6‐Dehydrogingerdione,an active constituent of dietary ginger,induces cell cycle arrest and apoptosis through reactive oxygen species/c‐Jun N‐terminal kinase pathways in human breast cancer cells

This study is the first to investigate the anticancer effect of 6‐dehydrogingerdione (DGE), an active constituent of dietary ginger, in human breast cancer MDA‐MB‐231 and MCF‐7 cells. DGE exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2 and Cdc25C. DGE also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. DGE triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl‐2 ratios, resulting in caspase‐9 activation. We also found the generation of reactive oxygen species is a critical mediator in DGE‐induced cell growth inhibition. DGE clearly increased the activation of apoptosis signal‐regulating kinase 1 and c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38. In addition, antioxidants vitamin C and catalase significantly decreased DGE‐mediated JNK activation and apoptosis. Moreover, blocking JNK by specific inhibitors suppressed DGE‐triggered mitochondrial apoptotic pathway. Taken together, these findings suggest that a critical role for reactive oxygen species and JNK in DGE‐mediated apoptosis of human breast cancer.     

Xanthohumol induces apoptosis in cultured 40-16 human colon cancer cells by activation of the death receptor- and mitochondrial pathway

Xanthohumol (XN) is one of the major prenylflavonoids found in hop cones (Humulus lupulus L.). In this study, we investigated the cell growth inhibitory potential of XN on cultured human colon cancer cells. Cell proliferation was measured by sulforhodamine B staining. Poly(ADP-ribose)polymerase (PARP) cleavage, activation of caspases-3, -7, -8, and -9, and Bcl-2 family protein expression were detected by Western blot analyses. XN significantly reduced proliferation of the HCT 116-derived colon cancer cell line 40--16. Half-maximal inhibitory concentrations decreased from 4.1 microM after 24 h treatment to 3.6 and 2.6 microM after 48 and 72 h incubation, respectively. Treatment with 15 microM XN for 48 h and with 5 microM for 72 h led to the detection of the cleaved 89 kDa fragment of 116 kDa PARP as an indication of apoptosis induction. Concomitantly, we observed activation and cleavage of the effector caspases-3 and -7, induced by activation of the initiator caspases -8 and -9. Expression of anti-apoptotic Bcl-2 was down regulated when the cells were treated with XN for 48--72 h. We conclude that induction of apoptosis by downregulation of Bcl-2 and activation of the caspase cascade may contribute to the chemopreventive or therapeutic potential of XN.     

Some natural flavonoids are competitive inhibitors of Caspase-1, -3 and -7 despite their cellular toxicity

A common feature of both apoptosis and inflammation is the activation of caspases. Caspases are aspartate-directed cysteine proteases that have numerous cellular targets. It has been discovered that several flavonoids are inhibitors of caspases. Flavonoids are members of a family of polyphenolic compounds from plants that have many biological properties, one of which is the ability to induce cell death. Some flavonoids are selective inhibitors of particular caspases. Since some of the inhibitory flavonoids are nevertheless cytotoxic, these results suggest that flavonoid-induced cell death may be occurring through a non-classical apoptosis pathway that is not dependent on caspase activity.     

The production of hydrogen peroxide is not a common mechanism by which olive oil phenols induce apoptosis on HL60 cells

We have recently demonstrated that hydroxytyrosol (3,4-DHPEA), the most representative olive oil phenol, induces apoptosis on HL60 cells through the production of considerable amount of extracellular hydrogen peroxide (H₂O₂). The aims of the present investigation were first to assess the ability of different phenolic compounds to both produce extracellular H₂O₂ and induce apoptosis on HL60 cells, and second to elucidate whether the pro-apoptotic activity was mediated by the production of H₂O₂ in the cell culture medium. Based on the results phenols can be classified as follows: (1) those which were not able to induce both apoptosis and H₂O₂ accumulation (tyrosol, homovanillic alcohol and protocatechuic, o-coumaric, vanillic, homovanillic, ferulic and syringic acids); (2) those which showed a pro-apoptotic activity mediated, at least in part, by the production of H₂O₂, as evidenced by the ability of catalase to inhibit apoptosis (3,4-DHPEA, dopamine, 3,4-dihydroxyphenylacetic, 3,4-dihydroxy-hydrocinnamic, caffeic and gallic acids); and (3) those which induced apoptosis without the involvement of H₂O₂ (the secoiridoid derivatives of both hydroxytyrosol and tyrosol). Oleuropein showed a peculiar behaviour since, and although it caused an abundant production of H₂O₂ in the cell culture medium, it exerted a weak pro-apoptotic effect. From these results we may conclude that the cathecol moiety of the phenol molecule is necessary for the H₂O₂ producing activity, and that the 3,4-DHPEA metabolism to homovanillic alcohol and homovanillic acid may significantly reduce its pro-apoptotic potential. The real in vivo meaning of the phenol-induced H₂O₂ production remains to be investigated.     

Antiproliferative,proapoptotic and morphogenic effects of the flavonoid rutin on human glioblastoma cells

In this study, we investigated the effects of the flavonoid rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rutinoside) on glioma cells, using the highly proliferative human cell line GL-15 as a model. We observed that rutin (50–100 μM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100 μM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50 μM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with overexpression of GFAP. Because of its capacity to induce differentiation and apoptosis in cultured human glioblastoma cells, rutin could be considered as a potential candidate for malignant gliomas treatment.     

Protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells

The protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVECs), as compared with Cordyceps sinensis, was examined. The cytotoxicity of HUVECs induced by 40 mM glucose was ameliorated by water extracts of C. militaris (CME) and water extracts of C. sinensis (CSE). CME and CSE inhibited the increase in ROS and NO in HUVECs induced by 40 mM high glucose. Moreover, CME increased the Bcl-2/Bax ratio, modulated the mitochondrial membrane potential and reduced the caspase-3 activity in high glucose-induced HUVECs. In addition, cordycepin, a component of CME and CSE, displayed protective effects against oxidative stress, which was partly responsible for the cytoprotective effects of CME and CSE against high glucose-induced oxidative stress in HUVECs. Overall, the obtained results show C. militaris helps preventing diabetic endothelial dysfunction and related complications.     

Effects of camptothecin, etoposide and Ca2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing

Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca(2+) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca(2+) was more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.     

Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells

Hydroxytyrosol [3,4‐dihydroxyphenylethanol (3,4‐DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth‐suppressive and pro‐apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro‐apoptotic activity of 3,4‐DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4‐DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V‐FITC kit. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation‐xylenol orange method. The pro‐apoptotic effect of 3,4‐DHPEA (100 μM) was prevented by N‐acetyl‐cysteine, ascorbate, and α‐tocopherol. Catalase suppressed the 3,4‐DHPEA‐induced apoptosis, while the Fe(II)‐chelating reagent o‐phenantroline showed no effect, suggesting the involvement of H₂O₂ but not of OH^?. Indeed, 3,4‐DHPEA caused accumulation of H₂O₂ in the culture medium. Tyrosol (p‐hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4‐DHPEA but not able to generate H₂O₂, did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4‐DHPEA is mediated by the extracellular production of H₂O₂.