Induction of apoptosis by hydroxydibenzoylmethane through coordinative modulation of cyclin D3, Bcl-X(L), and Bax,release of cytochrome c,and sequential activation of caspases in human colorectal carcinoma cells

DBM (dibenzoylmethane) is a minor constituent of licorice that has antimutagenic activity. However, its other biological activities are not well-known. The structurally related beta-diketones hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) were able to induce apoptosis in colorectal carcinoma COLO 205 cells. Thus, the effect of structurally related beta-diketones on cell viability, DNA fragmentation, and caspase activity was assessed. The potency of these compounds on these features of apoptosis were in the order of HDB > HMDB > DBM in colorectal carcinoma COLO 205 cells. Here, we found that HDB-induced apoptotic cell death was accompanied by upregulation of cyclin D3, Bax, and p21 and down-regulation of Bcl-X(L), while HDB had no effect on the levels of Bcl-2 and Bad protein. These results indicate that HDB allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 degradation. It is suggested that HDB-induced apoptosis is triggered by the release of cytochrome c into cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by HDB may provide a pivotal mechanism for its cancer chemopreventive action.     

Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells

Garcinol, a polyisoprenylated benzophenone, was purified from Garcinia indica fruit rind. The effects of garcinol and curcumin on cell viability in human leukemia HL-60 cells were investigated. Garcinol and curcumin displayed strong growth inhibitory effects against human leukemia HL-60 cells, with estimated IC(50) values of 9.42 and 19.5 microM, respectively. Garcinol was able to induce apoptosis in a concentration- and time-dependent manner; however, curcumin was less effective. Treatment with garcinol caused induction of caspase-3/CPP32 activity in a dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly(ADP-ribose) polymerase (PARP). Pretreatment with caspase-3 inhibitor inhibited garcinol-induced DNA fragmentation. Treatment with garcinol (20 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. The cleavage of D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. Of these, Bcl-2, Bad, and Bax were studied. The level of expression of Bcl-2 slightly decreased, while the levels of Bad and Bax were dramatically increased in cells treated with garcinol. These results indicate that garcinol allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. It is suggested that garcinol-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by garcinol may provide a pivotal mechanism for its cancer chemopreventive action.     

Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells

This study examined the growth inhibitory effects of theasinensin A (from oolong tea) and black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate (TF-2a) and theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) in human cancer cells. Theasinensin A, TF-1, and TF-2 displayed strong growth inhibitory effects against human histolytic lymphoma U937, with estimated IC50 values of 12 microM, but were less effective against human acute T cell leukemia Jurkat, whereas TF-3 and (-)-epigallocatechin-3-gallate (EGCG) had lower activities. The molecular mechanisms of tea polyphenol-induced apoptosis as determined by annexin V apoptosis assay, DNA fragmentation, and caspase activation were further investigated. Loss of membrane potential and reactive oxygen species (ROS) generation were also detected by flow cytometry. Treatment with tea polyphenols caused rapid induction of caspase-3, but not caspase-1, activity and stimulated proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Pretreatment with a potent caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone, inhibited theasinensin A induced DNA fragmentation. Furthermore, it was found that theasinensin A induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of caspase-9 activity. These results indicate that theasinensin A allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. The results suggest that induction of apoptosis by theasinensin A may provide a pivotal mechanism for their cancer chemopreventive function.     

Induction of apoptosis by 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione through reactive oxygen species production, GADD153 expression, and caspases activation in human epidermoid carcinoma cells

This study examined the growth inhibitory effects of the structurally related beta-diketones compounds in human cancer cells. Here, we report that 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB) induces growth inhibition of human cancer cells and induction of apoptosis in A431 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of HMDB-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that HMDB induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bad and p21; down-regulation of Bcl-2, Bcl-XL, Bid, p53, and fatty acid synthase; and cleavage of Bax were found in HMDB-treated A431 cells. Glutathione and N-acetylcysteine (NAC) suppress HMDB-induced apoptosis. HMDB markedly enhanced growth arrest DNA damage inducible gene 153 (GADD153) mRNA and protein in a time- and concentration-dependent manner. NAC prevented up-regulation of GADD153 mRNA expression caused by HMDB. These findings suggest that HMDB creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in A431 cells.     

Induction of apoptosis by the Anthocyanidins through regulation of Bcl-2 gene and activation of c-Jun N-terminal kinase cascade in hepatoma cells

Anthocyanidins that are reddish pigments widely distributed in fruit and vegetables have been reported to possess antioxidant and anticancer activities. To understand the molecular basis of the putative anticancer activity of anthocyanidins, we investigated the antiproliferation effects of anthocyanidins in human hepatoma cell lines. Delphinidin, cyanidin, and malvidin exhibited strong growth inhibitory effects against human hepatoma HepG(2), but were less effective against Hep3B. According to the appearance of the caspase-3 fragments and stimulated proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) in time-dependent studies, delphinidin induced apoptotic cell death characterized by internucleosomal DNA fragmentation and caused a rapid induction of caspase-3 activity. RT-PCR and Western blot data revealed that delphinidin stimulated an increase in the c-Jun and JNK phosphorylation expression at mRNA and protein levels, respectively. Moreover, delphinidin-induced apoptotic cell death was accompanied by up-regulation of Bax and down-regulation of Bcl-2 protein. Dephinidin-induced DNA fragmentation was blocked by N-acetyl-l-cysteine and catalase, suggesting that the death signaling was triggered by oxidative stress. Our experiments provide evidence that delphinidin is an effective apoptosis inducer in HepG(2) cells through regulation of Bcl-2 family moleculars and activation of c-Jun N-terminal kinase cascade. The results suggest that induction of apoptosis by anthocyanidins is a pivotal mechanism of their cancer chemopreventive functions.     

4-acetylantroquinonol B isolated from Antrodia cinnamomea arrests proliferation of human hepatocellular carcinoma HepG2 cell by affecting p53, p21 and p27 levels

The 4-acetylantroquinonol B isolated from the mycelium of Antrodia cinnamomea could inhibit proliferation of hepatocellular carcinoma cells HepG2 with IC(50) 0.1 μg/mL. When the HepG2 cells were treated with 4-acetylantroquinonol B for 72 h, the proportion of cells in the G1 phase of the cell cycle increased and that in the S phase decreased significantly, and the proportion of G2/M phase cells were not obviously changed. In addition, the 4-acetylantroquinonol B treatment resulted in the decreases of CDK2 and CDK4, and an increase of p27 in a dose-dependent manner. The protein levels of p53 and p21 proteins were also increased when the cells were treated with low dosage (0.1 μg/mL) of 4-acetylantroquinonol B. Higher dosages, however, decreased the expression of p53 and p21 proteins. Assay of RT-PCR indicated that, corresponding to the increases of p53 and p21 proteins at the dosage of 0.1 μg/mL, the mRNAs of p53 and p21 showed 1.66- and 1.61-fold upregulations, respectively. Corresponding to the decreases of CDK2 and CDK4 proteins, the mRNAs of CDK2 and CDK4 showed -1.02- and -1.13-fold downregulations, respectively. However, level of p27 mRNA showed -1.2-fold downregulation in spite of the increase in p27 protein. This observation, again, confirms the fact that the p27 gene rarely undergoes homozygous inactivation in cancer cells. Our finding suggested that the 4-acetylantroquinonol B inhibits proliferation of HepG2 cells via affecting p53, p21 and p27 proteins, and can be considered as a potential cancer drug.     

Induction of apoptosis by vitamin D2, ergocalciferol, via reactive oxygen species generation, glutathione depletion, and caspase activation in human leukemia Cells

This study demonstrated that ergocalciferol was able to inhibit leukemia cell growth in a concentration-dependent manner. Exploration of the acting mechanisms involved this event revealed that ergocalciferol induced DNA fragmentation and increased sub-G1 DNA contents in HL-60 cells, both of which are hallmarks of apoptosis. Analysis of the integrity of mitochondria demonstrated that ergocalciferol caused loss of mitochondrial membrane potential with release cytochrome c to cytosol, generation of reactive oxygen species (ROS), and depletion of glutathione (GSH), suggesting that ergocalciferol may induce apoptosis in HL-60 cells through a ROS-dependent pathway. Further results show that caspases-2, -3, -6, and -9 were all activated by ergocalciferol, together with cleavage of the downstream caspase-3 targets, DNA fragmentation factor (DFF-45), and poly(ADP-ribose) polymerase. In addition, ergocalciferol led to the increase in pro-apoptotic factor Bax accompanied with the decrease in anti-apoptotic member Mcl-1, and the reduced Mcl-1 to Bax ratio may be a critical event concerning mitochondrial decay by ergocalciferol. Furthermore, ergocalciferol also led to induction of Fas death receptor closely linked to caspase-2 activation, suggesting the involvement of a Fas-mediated pathway in ergocalciferol-induced apoptosis. Totally, these findings suggest that ergocalciferol causes HL-60 apoptosis via a modulation of mitochondria involving ROS production, GSH depletion, caspase activation, and Fas induction. On the basis of anticancer activity of ergocalciferol, it may be feasible to develop chemopreventive agents from edible mushrooms or hop.     

Acacetin induces apoptosis in human gastric carcinoma cells accompanied by activation of caspase cascades and production of reactive oxygen species

Acacetin (5,7-dihydrocy-4'-methoxy flavone), which is a flavonoid compound, possesses anti-peroxidative and anti-inflammatory effects. The effects of acacetin on cell viability in human gastric carcinoma AGS cells were investigated. This study demonstrated that acacetin was able to inhibit cell proliferation and induce apoptosis in a concentration- and time-dependent manner. Acacetin-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation, and cell morphology. The molecular mechanism of acacetin-induced apoptosis was also investigated. Treatment with acacetin caused induction of caspase-3 activity in a time-dependent manner, but not caspase-1 activity, and induced the degradation of DNA fragmentation factor (DFF-45) and poly(ADP-riobse) polymerase. Cell death was completely prevented by a pancaspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone. Furthermore, treatment with acacetin caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Antioxidants such as N-acetylcysteine and catalase, but not superoxide dismutase, allopurinol, or pyrrolidine dithiocarbamate, significantly inhibited acacetin-induced cell death. In addition, it was found that acacetin promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in acacetin-induced apoptosis. On the other hand, the results showed that acacetin-induced apoptosis was accompanied by up-regulation of Bax and p53, down-regulation of Bcl-2, and cleavage of Bad. Taken together, these results suggest that ROS production and a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to acacetin-induced apoptosis in AGS cells. The induction of apoptosis by acacetin may provide a pivotal mechanism for its cancer chemopreventive action.     

Goniothalamin inhibits growth of human lung cancer cells through DNA damage, apoptosis, and reduced migration ability

We evaluated the possible anticancer performance of a natural compound, goniothalamin (GTN), against human lung cancer using as a non-small cell lung cancer (NSCLC) cell line, H1299, as the model system. Cellular proliferation was significantly inhibited by GTN. Using an improved alkaline comet-nuclear extract (comet-NE) assay, GTN was found to induce a significant increase in the tail DNA. Wound healing and zymography assays showed that GTN attenuated cell migration and caused a reduction in the activity level of two major migration-associated matrix metalloproteinases, MMP-2 and MMP-9. It can be concluded that the DNA-damaging effect of GTN against lung cancer cells leads to growth inhibition as well as a depression in migration ability. Therefore, GTN has potential as a chemotherapeutic agent against lung cancer.     

A mixture of trans, trans conjugated linoleic acid induces apoptosis in MCF-7 human breast cancer cells with reciprocal expression of Bax and Bcl-2

The growth inhibitory effect of a mixture of trans, trans conjugated linoleic acid isomers (t, t CLA) was investigated in a human breast cancer cell line, MCF-7, with references to c9, t11 CLA, t10, c12 CLA, and linoleic acid. The t, t CLA treatment effectively induced a cytotoxic effect in a time-dependent (0-6 days) and concentration-dependent (0-40 microM) manner, as compared to the reference and control treatments. The apoptotic parameters were measured on cells treated with 40 microM t, t CLA for 4 days. The occurrence of the characteristic morphological changes and DNA fragmentation confirmed apoptosis. The t, t CLA treatment led to an increase in the level of p53 tumor suppressor protein and Bax protein, but suppressed the expression of Bcl-2 protein. In addition, cytochrome c was released from the mitochondria into the cytosol, and the activation of caspase-3 led to the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, the composition of the linoleic and arachidonic acids was decreased in cellular membranes. These findings suggest that incorporation of t, t CLA in the membrane induces a mitochondria-mediated apoptosis that can enhance the antiproliferative effect of t, t CLA in MCF-7 cells.     

The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway

Chalcones have been described to represent cancer chemopreventive food components that are rich in fruits and vegetables. In this study, we examined the anti-oral cancer effect of flavokawain B (FKB), a naturally occurring chalcone isolated from Alpinia pricei (shell gingers), and revealed its molecular mechanism of action. Treatment of human oral carcinoma (HSC-3) cells with FKB (1.25-10 μg/mL; 4.4-35.2 μM) inhibited cell viability and caused G(2)/M arrest through reductions in cyclin A/B1, Cdc2, and Cdc25C levels. Moreover, FKB treatment resulted in the induction of apoptosis, which was associated with DNA fragmentation, mitochondria dysfunction, cytochrome c and AIF release, caspase-3 and caspase-9 activation, and Bcl-2/Bax dysregulation. Furthermore, increased Fas activity and procaspase-8, procaspase-4, and procaspase-12 cleavages were accompanied by death receptor and ER-stress, indicating the involvement of mitochondria, death-receptor, and ER-stress signaling pathways. FKB induces apoptosis through ROS generation as evidenced by the upregulation of oxidative-stress markers HO-1/Nrf2. This mechanism was further confirmed by the finding that the antioxidant N-acetylcysteine (NAC) significantly blocked ROS generation and consequently inhibited FKB-induced apoptosis. Moreover, FKB downregulated the phosphorylation of Akt and p38 MAPK, while their inhibitors LY294002 and SB203580, respectively, induced G(2)/M arrest and apoptosis. The profound reduction in cell number was observed in combination treatment with FKB and Akt/p38 MAPK inhibitors, indicating that the disruption of Akt and p38 MAPK cascades plays a functional role in FKB-induced G(2)/M arrest and apoptosis in HSC-3 cells.     

5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone induces apoptosis through reactive oxygen species production, growth arrest and DNA damage-inducible gene 153 expression, and caspase activation in human leukemia cells

This study examined the growth inhibitory effects of structurally related polymethoxylated flavones in human cancer cells. Here, we report that 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) induces growth inhibition of human cancer cells and induction of apoptosis in HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 5-OH-HxMF-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that 5-OH-HxMF induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bax was found in 5-OH-HxMF-treated HL-60 cells. In addition, a caspase-independent pathway indicated by endonuclease G also contributed to apoptosis caused by 5-OH-HxMF. Antioxidants suppress 5-OH-HxMF-induced apoptosis. 5-OH-HxMF markedly enhanced growth arrest DNA damage-inducible gene 153 (GADD153) protein in a time-dependent manner. N-acetylcysteine (NAC) and catalase prevented up-regulation of GADD153 expression caused by 5-OH-HxMF. These findings suggest that 5-OH-HxMF creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in HL-60 cells. Meanwhile, ROS were proven an important inducer in this apoptotic process. The C-5 hydroxyl on the ring of 5-OH-HxMF was found to be essential for the antiproliferative and apoptosis-inducing activity. Our study identified the novel mechanisms of 5-OH-HxMF-induced apoptosis and indicated that these results have significant applications as potential chemopreventive and chemotherapeutic agents.