DNA Fragmentation In HT-29 Cells Research Articles

Induction of HT-29 Colon Cancer Cells Apoptosis by Pyrogallol with Growth Inhibiting Efficacy Against Drug-Resistant Helicobacter pylori.

Colon cancer is the most aggressive form of cancers, that causes 0.5 million deaths per year around the globe. Targeting colon cancer by conventional therapeutic options elicits toxicity. Traditional medicines take a lead to alleviate the existing clinical challenges. To investigate antibacterial activity against Helicobacter Pylori and in vitro anti-colon cancer activity by Acacia nilotica extract (ACE) and its active constituent pyrogallol. Pyrogallol isolated from A. nilotica by column chromatography and HPLC and structure was elucidated by spectral analysis. Antibacterial activity was done by flow cytometry. Cytotoxicity was measured by MTT assay. Apoptotic morphology and nuclear fragmentation were assessed with AO/ethidium bromide and DAPI staining. DNA fragmentation was done by electrophoresis. Western blot used to analyze the molecular mechanism of apoptosis. Cell cycle arrest was determined using flow cytometry of propidium iodide stained cells. Cell migration was determined by wound healing assay. ACE (20 µg/ml) and pyrogallol (10 µg/ml) treatment reduced the survival of H.pylori at 61% and 62%, respectively. MTT results show that HT-29 cells are more sensitive to pyrogallol with an IC50 value of 35μg/ml compared to ACE. Pyrogallol treated HT-29 cells reached dead state i.e. late apoptotic state with severe nuclear fragmentation. Pyrogallol elicits dose dependent DNA fragmentation in HT-29 cells. Pyrogallol induced apoptosis by simultaneous down-regulation of Bcl-2 and up-regulation of BAX and cytochrome c. Pyrogallol arrested HT-29 cells in S and G2/M phase of cell cycle. Further pyrogallol exhibited marked antimetastatic potential by inhibiting the migration of HT-29 cells dose dependently. Both ACE and pyrogallol repressed the growth of H.pylori and as significant anti-colon cancer agent.

Chemopreventive and Anticancer Activities of Allium victorialis var. platyphyllum Extracts.

Background:Allium victorialis var. platyphyllum is an edible perennial herb and has been used as a vegetable or as a Korean traditional medicine. Allium species have received much attention owing to their diverse pharmacological properties, including antioxidative, anti-inflammatory, and anticancer activities. However, A. victorialis var. platyphyllum needs more study.Methods:The chemopreventive potential of A. victorialis var. platyphyllum methanol extracts was examined by measuring 12-O-tetra-decanoylphorbol 13-acetate (TPA)-induced superoxide anion production in the differentiated HL-60 cells, TPA-induced mouse ear edema, and Ames/Salmonella mutagenicity. The apoptosis-inducing capabilities of the extracts were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, 4’,6-diamidino-2-phenylindole staining, and the DNA fragmentation assay in human colon cancer HT-29 cells. Antimetastatic activities of the extracts were also investigated in an experimental mouse lung metastasis model.Results:The methanol extracts of A. victorialis var. platyphyllum rhizome (AVP-R) and A. victorialis var. platyphyllum stem (AVP-S) dose-dependently inhibited the TPA-induced generation of superoxide anion in HL-60 cells and TPA-induced ear edema in mice, as well as 7,12-dimethylbenz[a]anthracene (DMBA) and tert-butyl hydroperoxide (t-BOOH) -induced bacterial mutagenesis. AVP-R and AVP-S reduced cell viability in a dose-related manner and induced apoptotic morphological changes and internucleosomal DNA fragmentation in HT-29 cells. In the experimental mouse lung metastasis model, the formation of tumor nodules in lung tissue was significantly inhibited by the treatment of the extracts.Conclusions:AVP-R and AVP-S possess antioxidative, anti-inflammatory, antimutagenic, proapoptotic, and antimetastatic activities. Therefore, these extracts can serve as a beneficial supplement for the prevention and treatment of cancer.

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells.

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

P-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis

Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells.

Aglycon of Rhizochalin from the Rhizochalina incrustata Induces Apoptosis via Activation of AMP-Activated Protein Kinase in HT-29 Colon Cancer Cells

Rhizochalin is a two-headed sphingolipid-like compound isolated from the sponge Rhizochalina incrustata. It has been reported that rhizocalin and its derivates have a chemopreventive and chemotherapeutic effect. However, the molecular mechanism of these effects is not understood. Here, we demonstrate that aglycon of rhizochalin (AglRhz) from the Rhizochalina incrustata induces AMP-activated protein kinase (AMPK) phosphorylation, and thereby inhibits mammalian target of rapamycin (mTOR)-p70S6 kinase-extracellular signal-regulated kinase (ERK) signaling and activator protein 1 (AP-1) activity via phosphorylation of Raptor in HT-29 cells. In addition, AglRhz induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP), and DNA fragmentation in HT-29 cells, leads to induction of apoptosis as well as suppression of tumorigenicity of HT-29 cells. Notably, AglRhz inhibits insulin-like growth factor (IGF)-1-induced AP-1 activity and cell transformation in JB6 Cl41 cells. Overall, our findings identify AMPK as an important target protein for mediating the anti-tumor properties of AglRhz in HT-29 colon cancer cells and have important implication for sponges, the most important marine source, in colon cancer.

24-Hydroxyursolic Acid from the Leaves of theDiospyros kaki(Persimmon) Induces Apoptosis by Activation of AMP-activated Protein Kinase

There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

Sulindac activates nuclear translocation of AIF, DFF40 and endonuclease G but not induces oligonucleosomal DNA fragmentation in HT-29 cells

Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase- 3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100–300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.