5-Hydroxy-7-Methoxyflavone Triggers Mitochondrial-Associated Cell Death via Reactive Oxygen Species Signaling in Human Colon Carcinoma Cells

Plant-derived compounds are an important source of clinically useful anti-cancer agents. Chrysin, a biologically active flavone found in many plants, has limited usage for cancer chemotherapeutics due to its poor oral bioavailability. 5-Hydroxy-7-methoxyflavone (HMF), an active natural chrysin derivative found in various plant sources, is known to modulate several biological activities. However, the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma cells in vitro is still unknown. Herein, HMF was shown to be capable of inducing cytotoxicity in HCT-116 cells and induced cell death in a dose-dependent manner. Treatment of HCT-116 cells with HMF caused DNA damage and triggered mitochondrial membrane perturbation accompanied by Cyt c release, down-regulation of Bcl-2, activation of BID and Bax, and caspase-3-mediated apoptosis. These results show that ROS generation by HMF was the crucial mediator behind ER stress induction, resulting in intracellular Ca²⁺ release, JNK phosphorylation, and activation of the mitochondrial apoptosis pathway. Furthermore, time course study also reveals that HMF treatment leads to increase in mitochondrial and cytosolic ROS generation and decrease in antioxidant enzymes expression. Temporal upregulation of IRE1-α expression and JNK phosphorylation was noticed after HMF treatment. These results were further confirmed by pre-treatment with the ROS scavenger N-acetyl-l-cysteine (NAC), which completely reversed the effects of HMF treatment by preventing lipid peroxidation, followed by abolishment of JNK phosphorylation and attenuation of apoptogenic marker proteins. These results emphasize that ROS generation by HMF treatment regulates the mitochondrial-mediated apoptotic signaling pathway in HCT-116 cells, demonstrating HMF as a promising pro-oxidant therapeutic candidate for targeting colorectal cancer.     

Caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis: Protective role of the nitric oxide signaling module

Hydrophobic bile acids such as deoxycholate are known tumor promoters in the gastrointestinal tract. We have previously shown that deoxycholate induces apoptosis in colon epithelial cells and that these cells can be made resistant to deoxycholate-induced apoptosis. We now show that the nitric oxide synthase/nitric oxide/guanylate cyclase/cyclic guanosine monophosphate/cGMP-activated protein kinase (NOS/NO/GC/cGMP/PKG) signaling module contributes, in part, to the observed resistance of the cultured DOC-resistant colon epithelial cells (HCT-116R) using pharmacological inhibitors/antagonists (NS2028, Rp-8pCPT-cGMP, KT5823) of members of this signaling module. A novel finding from this study is the caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis of deoxycholate-sensitive HCT-116SA cells and the absence of guanylate cyclase alpha 1 cleavage in deoxycholate-treated HCT-116R resistant cells using Western blot analyses. This cleavage was specific to caspases as lysosomal, proteasomal, serine protease, cathepsin and calpain inhibitors failed to prevent the cleavage, whereas a general caspase inhibitor and a specific caspase-6 inhibitor did prevent guanylate cyclase alpha 1 cleavage.     

Protection of human colon epithelial cells against deoxycholate by rottlerin

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin. In elucidating its mechansim, rottlerin prevented the release of cytochrome c from mitochondria into cytosol, and also prevented the cleavage of caspase-3. Yet, rottlerin by itself markedly decreased mitochondrial membrane potential and increased mitochondrial superoxide production, but this did not result in cytochrome c release or in caspase-3 cleavage. At a higher test concentration, two other phenolic phytochemicals, namely, quercetin and resveratrol, were each able to largely prevent the occurrence of apoptosis in cells exposed to DOC. In contrast, epigallocatechin gallate, curcumin, and genistein were ineffective.     

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 microM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4',6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.     

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.     

Molecular ordering of ROS production,mitochondrial changes,and caspase activation during sodium salicylate-induced apoptosis

Salicylates and nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast, and leukemia. We examined the effects of sodium salicylate (NaSal) on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death. We demonstrate that NaSal mediates ROS production followed by a decrease in mitochondrial membrane potential (deltapsi(m)), release of cytochrome c, and activation of caspase-9 and caspase-3. However, expression of Bcl-2 or Bcl-x(L) prevents ROS production and subsequent loss of deltapsi(m), thereby inhibiting apoptotic cell death. The presence of ROS scavengers and an inhibitor of NADPH oxidase or expression of a dominant negative form of Rac1 blocks ROS production, deltapsi(m) collapse, and the subsequent activation of caspases. These observations indicate that NaSal mediates ROS production critical in the triggering of apoptotic tumor cell death through a Rac1-NADPH oxidase-dependent pathway. Our data collectively imply that NaSal-induced ROS are key mediators of deltapsi(m) collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in tumor apoptosis.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

Involvement of caspase-8 in chemotherapy-induced apoptosis of patient derived leukemia cell lines independent of the death receptor pathway and downstream from mitochondria

Resistance of leukemic cells to chemotherapy frequently occurs in patients with acute leukemia, which may be caused by alterations in common apoptotic pathways. Controversy exists whether cytostatic agents induce the mitochondrial or death receptor pathway of apoptosis. In the mitochondrial pathway cytochrome C release and caspase-9 activation play a central role in the induction of apoptosis, while formation of a Death Inducing Signaling Complex (DISC) and caspase-8 activation have been reported to be essential in death receptor-induced apoptosis. Here, we show in human derived myeloid and lymphoblastic leukemia cell lines that caspase-8 plays a more important role than previously expected in apoptosis mediated via the mitochondrial pathway. We demonstrated in these malignant cells chemotherapy-induced apoptosis independent of the death receptor pathway, since blocking this pathway using a retroviral construct encoding Flice inhibitory protein (FLIP) did not inhibit drug-induced apoptosis or caspase-8 activation, while overexpression of Bcl-2 completely inhibited both events. Furthermore, we showed that activation of caspase-8 by cytostatic agents occurred downstream from mitochondria. Since caspase-8 plays a central role in both death receptor- and chemotherapy-induced apoptosis of malignant cells from patients with acute leukemia, therapeutic strategies focusing at modulation and activation of caspase-8 may be successful in the treatment of drug-resistant malignancies. Supported by a grant of the Dutch Cancer Society/KWF Kankerbestrijding: 99-2122.     

Lipid alterations in human colon epithelial cells induced to differentiation and/or apoptosis by butyrate and polyunsaturated fatty acids

The present study highlights the important association between lipid alterations and differentiation/apoptotic responses in human colon differentiating (FHC) and nondifferentiating (HCT-116) cell lines after their treatment with short-chain fatty acid sodium butyrate (NaBt), polyunsaturated fatty acids (PUFAs), and/or their combination. Our data from GC/MS and LC/MS/MS showed an effective incorporation and metabolization of the supplemented arachidonic acid (AA) or docosahexaenoic acid (DHA), resulting in an enhanced content of the respective PUFA in individual phospholipid (PL) classes and an altered composition of the whole cellular fatty acid spectrum in both FHC and HCT-116 cells. We provide novel evidence that NaBt combined with PUFAs additionally modulated AA and DHA cellular levels and caused their shift from triacylglycerol to PL fractions. NaBt increased, while AA, DHA and their combination with NaBt decreased endogenous fatty acid synthesis in FHC but not in HCT-116 cells. Fatty acid treatment also altered membrane lipid structure, augmented cytoplasmic lipid droplet accumulation, reactive oxygen species (ROS) production and dissipation of the mitochondrial membrane potential. All these parameters were significantly enhanced by combined NaBt/PUFA treatment, but only in FHC cells was this accompanied by highly increased apoptosis and suppressed differentiation. Moreover, the most significant changes of ROS production, differentiation and apoptosis among the parameters studied, the highest effects of combined NaBt/PUFA treatment and a lower sensitivity of HCT-116 cells were confirmed using two-way ANOVA. Our results demonstrate an important role of fatty acid-induced lipid alterations in the different apoptotic/differentiation response of colon cells with various carcinogenic potential.     

Mitochondrial superoxide production contributes to vancomycin-induced renal tubular cell apoptosis

Vancomycin chloride (VCM), a glycopeptide antibiotic, is widely used for the therapy of infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is a major adverse effect in VCM therapy. In this study, we investigated the cellular mechanisms underlying VCM-induced renal tubular cell injury in cultured LLC-PK1 cells. VCM induced a concentration- and time-dependent cell injury in LLC-PK1 cells. VCM caused increases in the numbers of annexin V-positive/PI-negative cells and TUNEL-positive cells, indicating the involvement of apoptotic cell death in VCM-induced renal cell injury. The VCM-induced apoptosis was accompanied by the activation of caspase-9 and caspase-3/7 and reversed by inhibitors of these caspases. Moreover, VCM caused an increase in intracellular reactive oxygen species production and mitochondrial membrane depolarization, which were reversed by vitamin E. In addition, mitochondrial complex I activity was inhibited by VCM as well as by the complex I inhibitor rotenone, and rotenone mimicked the VCM-induced LLC-PK1 cell injury. These findings suggest that VCM causes apoptotic cell death in LLC-PK1 cells by enhancing mitochondrial superoxide production leading to mitochondrial membrane depolarization followed by the caspase activities. Moreover, mitochondrial complex I may play an important role in superoxide production and renal tubular cell apoptosis induced by VCM.     

Paradoxical effect of diphenyleneiodonium in inducing DNA damage and apoptosis

Diphenyleneiodonium (DPI) is often used as a molecular tool in unravelling redox-sensitive cellular events involving NADPH oxidase. However, to better understand unexpected actions of DPI, it was ascertained if DPI affects cellular DNA. DPI induced single-strand breaks in DNA of HCT-116 cells, although this only slightly increased GADD153 expression. Nevertheless, after sustaining DNA damage, the DPI-treated cells subsequently had features characteristic of apoptosis, such as translocated membrane phospholipid and nuclei containing condensed chromatin. Paradoxically, DPI attenuated the DNA damage and overall ROS production caused by sodium deoxycholate (DOC), although DPI did not inhibit DOC-induced generation of mitochondrial [image omitted] . Furthermore, DPI prevented the occurrence of apoptosis caused by DOC. However, other known chemical inhibitors of NADPH oxidase did not produce the same results as DPI in negating the effects of DOC. Collectively, these disparate findings suggest that DPI can act not in accord with conventional wisdom depending on the experimental conditions.     

Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos)

Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by macrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of copper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch.     

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.     

Dieckol,a natural polyphenolic drug,inhibits the proliferation and migration of colon cancer cells by inhibiting PI3K,AKT, and mTOR phosphorylation

This study investigated that dieckol (DKL), a natural drug, inhibits colon cancer cell proliferation and migration by inhibiting phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) phosphorylation in HCT-116 cells. The cells were treated with DKL in various concentrations (32 and 50 μM) for 24 h and then analyzed for various experiments. MTT (tetrazolium bromide) and crystal violet assay investigated DKL-mediated cytotoxicity. Dichlorodihydrofluorescein diacetate staining was used to assess the reactive oxygen species (ROS) measurement, and apoptotic changes were studied by dual acridine orange and ethidium bromide staining. Protein expression of cell survival, cell cycle, proliferation, and apoptosis protein was evaluated by western blot analysis. Results indicated that DKL produces significant cytotoxicity in HCT-116, and the half-maximal inhibitory concentration was found to be 32 μM for 24-h incubation. Moreover, effective production of ROS and enhanced apoptotic signs were observed upon DKL treatment in HCT-116. DKL induces the expression of phosphorylated PI3K, AKT, and mToR-associated enhanced expression of cyclin-D1, proliferating cell nuclear antigen, cyclin-dependent kinase (CDK)-4, CDK-6, and Bcl-2 in HCT-116. In addition, proapoptotic proteins such as Bax, caspase-9, and caspase-3 were significantly enhanced by DKL treatment in HCT-116. Hence, DKL has been considered a chemotherapeutic drug by impeding the expression of PI3K-, AKT-, and mTOR-mediated inhibition of proliferation and cell cycle-regulating proteins.     

Comparative proteomic analysis of indioside D-triggered cell death in HeLa cells

Medicinal plants represent a rich source of cancer drug leads. Indioside D, a furostanol glycoside isolated from Solanum mammosum, was found to possess antiproliferative activity toward a panel of human cancer cell lines. Proteomic analysis of indioside D-treated HeLa cells revealed profound protein changes related to energy production and oxidative stress, suggesting that mitochondria dysfunction plays a role in indioside D-induced apoptosis. Indioside D caused a rapid dissipation of mitochondrial transmembrane potential (DeltaPsim) and the generation of reactive oxygen species (ROS), leading to the activation of caspase-dependent apoptotic cell death. The Fas death receptor pathway was also activated following indioside D treatment, and triggered the activation of caspase-8 and cleavage of Bid, which also acted through the mitochondrial apoptosis pathway. These results suggest that indioside D induced apoptosis in HeLa cells via both intrinsic and extrinsic cell death pathways.