DNA Ladder Formation Research Articles

An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

Singlet oxygen causes the cytotoxic process of tumour cells in photodynamic therapy. The mechanism by which singlet oxygen damages cells is, however, not fully understood. To address this issue, we synthesized and used two types of endoperoxides, MNPE (1-methylnaphthalene-4-propionate endoperoxide) and NDPE (naphthalene-1,4-dipropionate endoperoxide), that generate defined amounts of singlet oxygen at 37 degrees C with similar half lives. MNPE, which is more hydrophobic than NDPE, induced the release of cytochrome c from mitochondria into the cytosol and exhibited cytotoxicity, but NDPE did not. RBL cells, a rat basophil leukaemia-derived line, that overexpress phospholipid hydroperoxide glutathione peroxidase in mitochondria were found to be highly resistant to the cytotoxic effect of MNPE. MNPE treatment induced much less DNA ladder formation and nuclear fragmentation in cells than etoposide treatment, even though these treatments induced a similar extent of cellular damage. Singlet oxygen inhibited caspase 9 and 3 activities directly and also suppressed the activation of the caspase cascade. Collectively, these data suggest that singlet oxygen triggers an apoptotic pathway by releasing cytochrome c from mitochondria via the peroxidation of mitochondrial components and results in cell death that is different from typical apoptosis, because of the abortive apoptotic pathway caused by impaired caspase activation.

Aloe Emodin‐Induced Apoptosis in t‐HSC/Cl‐6 Cells Involves a Mitochondria‐Mediated Pathway

Abstract: The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t‐HSC/Cl‐6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t‐HSC/Cl‐6 cells with 12.5, 25, or 50 μM aloe emodin inhibited t‐HSC/Cl‐6 cell viability in a dose‐ and time‐dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v‐propidium iodide flow‐cytometric analysis. Aloe emodin treatment of t‐HSC/Cl‐6 cells caused activation of caspase‐3 and caspase‐9, detected with a caspase activity assay, although no change was observed in caspase‐8 activity. Western blotting showed caspase‐3 and caspase‐9 active forms and the subsequent proteolytic cleavage of poly(ADP‐ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time‐dependent manner. Increased Bax and unchanged Bcl‐2 levels resulted in an increased Bax/Bcl‐2 ratio. Thus, our research provides evidence that aloe emodin‐induced apoptosis involves a mitochondria‐associated apoptosis pathway.

Aloe Emodin-Induced Apoptosis in t-HSC/Cl-6 Cells Involves a Mitochondria-Mediated Pathway

The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t-HSC/Cl-6 cells with 12.5, 25, or 50 microM aloe emodin inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. Aloe emodin treatment of t-HSC/Cl-6 cells caused activation of caspase-3 and caspase-9, detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time-dependent manner. Increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. Thus, our research provides evidence that aloe emodin-induced apoptosis involves a mitochondria-associated apoptosis pathway.

A novel anti-tumor agent, polyoxomolybdate induces apoptotic cell death in AsPC-1 human pancreatic cancer cells

Anti-tumoral polyoxomolybdates have been investigated in the course of study of the medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH 3Pr i] 6[Mo 7O 24]·3H 2O (PM-8) has been recognized as one of significantly anti-tumoral polyoxomolybdates. PM-8 inhibited the cell growth of human pancreatic cells (AsPC-1) depending on the dose. DNA ladder formation and DNA fragmentation were observed by Hoechst and TUNEL staining and flowcytometry analysis. The ratio of apoptotic cells were 29%, 35%, and 57% with treatment of PM-8 after 24, 48, and 72 h, respectively, which suggested that the anti-tumor activity of PM-8 results from the activation of the apoptotic pathway. Polyoxomolybdates provide promising, novel anti-tumor agent, especially for the treatment of cancers that are difficult to treat.

Petrotetrayndiol A induces cell cycle arrest and apoptosis in SK-MEL-2 human melanoma cells through cytochrome c-mediated activation of caspases

We investigated the possible mechanisms by which petrotetrayndiol A, a polyacetylene from the sponge Petrosia sp., exerts its anti-proliferative activity in cultured SK-MEL-2 human melanoma cells. Petrotetrayndiol A-treated SK-MEL-2 cells showed growth inhibition and induction of apoptosis in a dose-dependent manner as measured by MTT assay, fluorescent microscopy and flow cytometric analysis. Flow cytometric analysis revealed that petrotetrayndiol A resulted in G2/M arrest in the cell cycle progression which was associated with a marked decrease in the protein expression of cyclin B1 and its activating partner Cdc2 with concomitant inductions of p21 WAF1/ CIP1 . The increase in apoptosis was associated with a dose-dependent up-regulation of cytosolic factor, such as Bax and release of cytochrome c, and down-regulation of Bcl-2. We also observed activation of caspase-9 and caspase-3, DNA ladder formation, proteolytic degradation of poly(ADP-ribose)-polymerase (PARP), and selective down-regulation of cIAP-1. The apoptotic manifestations, such as PARP cleavage and DNA fragmentation, were abolished in the presence of the tripeptide caspase inhibitor z-VAD-fmk and a caspase-3-specific inhibitor Ac-DEVD-cho. Our data thus demonstrate that petrotetrayndiol A-induced apoptosis and growth inhibition of SK-MEL-2 cells is dependent on caspase activation.

Glycerol enhances radiosensitivity in a human oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 gene via Bax-mediated induction of apoptosis

Radiotherapy for oral squamous cell carcinomas is limited in its efficacy and in its ability to improve the survival rate in patients at an advanced stage. A protocol is described here which may elevate the therapeutic efficacy of radiation for these cells. The addition of glycerol to the culture medium prior to irradiation of an oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 (mp53) gene was found to increase the radiosensitivity of these cells. A colony formation assay was used to evaluate the effect of glycerol on the radiation sensitivity of Ca9-22 cells. Apoptosis was analyzed using Hoechst 33342 staining, Western blotting, and a DNA ladder formation assay. Glycerol, when present in the culture medium, enhanced the radiation sensitivity and extent of apoptosis following X-irradiation in the Ca9-22 cells, although neither X-rays or glycerol alone increased the extent of apoptosis. Bax protein was accumulated after treatment with X-rays plus glycerol, but not after exposure to X-rays or glycerol alone. A gel mobility-shift assay showed that glycerol restored the DNA-binding activity of mp53 for a p53-consensus sequence to levels similar to that of wild-type p53. These findings suggest that pre-treatment with glycerol may enhance the effectiveness of radiotherapy against oral squamous cell carcinomas bearing an mp53 gene mutation.

Effects of vitamin restriction and supplementation on rat intestinal epithelial cell apoptosis

Diet influences intestinal growth and function and vitamins modulate intestinal cell turnover. We have assessed the effects of chronic, moderate (50% of control) vitamin restriction and supplementation on intestinal epithelial cell (IEC) apoptosis and the relevance of this to alterations in tissue oxidative stress and antioxidant status. Feeding a vitamin-restricted diet to male, weanling WNIN rats for 20 weeks significantly increased IEC apoptosis, but only in the villi region, as evident from increased annexin V staining, M30 positivity, histological observations, DNA ladder formation, and reduced expression of Bcl-2. This was associated with elevated levels of lipid peroxides and protein carbonyls in the intestinal mucosa despite the increased activities of superoxide dismutase, catalase, and glutathione peroxidase. Consistent with the increased oxidative stress and apoptosis, structural and functional integrity of the villi were compromised as evident from the lowered ratio of villus height:crypt depth and the decreased activities of the membrane marker enzymes alkaline phosphatase and Lys-Ala dipeptidyl aminopeptidase. These changes were reversed by supplementation with a vitamin mixture or vitamin E alone, whereas riboflavin or folic acid supplementation reduced the apoptotic rates, but only partially. Further, oxidative stress was the least in vitamin E- or vitamin mixture-supplemented rats and correlated well with their IEC apoptotic rates. Increased tissue oxidative stress seems to mediate the vitamin-restriction-induced apoptosis of the IECs in rats.

Activation of p38 Mitogen-Activated Protein Kinase duringent-11α-Hydroxy-16-kauren-15-one-Induced Apoptosis in Human Leukemia HL-60 Cells

Kaurene-type diterpenes possess various biological activities including antitumor and anti-inflammatory effects. Indeed, we have found that an ent-kaurene diterpene, ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis via caspase-8 activation in human promyelocytic leukemia HL-60 cells. However, the mechanism of caspase-8 activation by KD is not clear. In this study, we investigated the involvement of p38 mitogen-activated protein kinase (p38 (MAPK)) in KD-induced apoptosis. p38 (MAPK) was activated by treatment with KD parallel to DNA ladder formation. Pretreatment with SB203580, a specific inhibitor of p38 (MAPK), attenuated induction of apoptosis by KD and inhibited activation of caspase-8. Cleavage of Bid, a typical substrate of caspase-8, was also inhibited by treatment with SB203580, suggesting that activation of p38 (MAPK) occurs upstream of caspase-8 during KD-induced apoptosis.

Cisplatin-Induced Apoptosis Is Enhanced by Hypoxia and by Inhibition of Mitochondria in Renal Collecting Duct Cells

Cisplatin is a widely used chemotherapeutic agent. Here we show that cisplatin induces apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells) in a dose-dependent manner. Additionally, we studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1F(o)-ATP synthase or by uncoupling. The role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, cisplatin-induced apoptosis was markedly enhanced. Mitochondrial blockade led to enhanced production of lactic acid. Also, anoxia potentiated the cisplatin-induced caspase-3 activation. Neither intra- nor extracellular pH had an influence on caspase-3 activation at low cisplatin concentrations. Acidic conditions (pH 6.8) potentiated the caspase-3 activation when high (100 microM) cisplatin concentrations were used. We demonstrate that intact mitochondria are important to prevent cisplatin-induced apoptosis in MDCK-C7 cells and that acidic conditions can aggravate the toxic effects of cisplatin.

Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide

The main anticancer action of doxorubicin (DOX) is believed to be due to topoisomerase II inhibition and free radical generation. Our previous study has demonstrated that TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H 2O 2 generation mediated by NAD(P)H oxidase activation [H. Mizutani et al. J. Biol. Chem. 277 (2002) 30684-30689]. Therefore, to clarify whether DOX functions as an anticancer drug through the same mechanism or not, we investigated the mechanism of apoptosis induced by DOX in the human leukemia cell line HL-60 and the H 2O 2-resistant sub-clone, HP100. DOX-induced DNA ladder formation could be detected in HL-60 cells after a 7 h incubation, whereas it could not be detected under the same condition in HP100 cells, suggesting the involvement of H 2O 2-mediated pathways in apoptosis. Flow cytometry revealed that H 2O 2 formation preceded the increase in ΔΨm and caspase-3 activation. Poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase inhibitors prevented DOX-induced DNA ladder formation in HL-60 cells. Moreover, DOX significantly induced formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an indicator of oxidative DNA damage, in HL-60 cells at 1 h, but not in HP100 cells. DOX-induced apoptosis was mainly initiated by oxidative DNA damage in comparison with the ability of other topoisomerase inhibitors (TAS-103, amrubicin and amrubicinol) to cause DNA cleavage and apoptosis. These results suggest that the critical apoptotic trigger of DOX is considered to be oxidative DNA damage by the DOX-induced direct H 2O 2 generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H 2O 2 generation through PARP and NAD(P)H oxidase activation, leading to the ΔΨm increase and subsequent caspase-3 activation in DOX-induced apoptosis.

Angiogenesis Inhibitor Z24 Induces Endothelial Cell Apoptosis and Suppresses Tumor Growth and Metastasis

Z24, a small molecular compound with similar chemical structure to SU5416 designed and synthesized by our lab, has been proved to be an angiogenesis inhibitor. In this study, Z24 was shown to induce human umbilical venous endothelial cell (HUVEC) apoptosis confirmed by morphologic changes including the presence of apoptotic bodies, significant apoptotic sub-G1 peak upon flow-cytometric analysis, formation of DNA ladders upon agarose gel electrophoresis, and TUNEL (TdT mediated X-dUTP nick-end labeling) results. Systemic administration of Z24 at non-toxic dose in nude mice resulted in inhibition of subcutaneous tumor growth of human colon cancer HCT-8, while it did not inhibit this cell line in vitro, with 100-fold more potent growth-inhibition against endothelial cells. The immunohistochemical results showed that the microvessel density of tumor tissue of the Z24 group was significantly lower than that of the control groups (P<0.05), which supported its anti-angiogenic property. We further found that Z24 inhibited the pulmonary metastasis of mouse lung adenocarcinoma LA795, with fewer surface lung metastases (89.6%, P<0.0001) and decreased lung weights (38.5%, P<0.01) compared to the vehicle group. All these findings support that Z24 is a promising angiogenesis inhibitor for limiting tumor growth and metastasis.

.BETA.-D-Xylopyranosyl-(1.RAR.3)-.BETA.-D-glucuronopyranosyl Echinocystic Acid Isolated from the Roots of Codonopsis lanceolata Induces Caspase-Dependent Apoptosis in Human Acute Promyelocytic Leukemia HL-60 Cells

We previously demonstrated that beta-D-xylopyranosyl-(1-->3)-beta-D-glucuronopyranosyl echinocystic acid (codonoposide 1c), a biologically active compound isolated from the roots of Codonopsis lanceolata, is cytotoxic to cancer cells. In the present study, we investigated the effects of codonoposide 1c on the induction of apoptosis, and its putative action pathway in HL-60 human promyelocytic leukemia cells. Codonoposide 1c-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. We observed that codonoposide 1c caused activation of caspase-8, caspase-9, and caspase-3. A broad caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-3 inhibitor (z-DEVD-fmk) almost completely suppressed codonoposide 1c-induced DNA fragmentation. We further found that codonoposide 1c induces mitochondrial translocation of Bid from cytosol, reduction of cytosolic Bax, and cytochrome c release from mitochondria. Interestingly, codonoposide 1c also triggered the mitochondrial release of Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) into cytosol, and a reduction in X-linked inhibitor of apoptosis protein (XIAP). Taken together, our data indicate that codonoposide 1c is a potent inducer of apoptosis and facilates its activity via Bid cleavage and translocation to mitochondria, Bax reduction in cytosol, release of cytochrome c and Smac/DIABLO into the cytosol, and subsequently caspase activation, providing a potential mechanism for the cytotoxic activity of codonoposide 1c.

Membrane Toxicity Accounts for Apoptosis Induced by Realgar Nanoparticles in Promyelocytic Leukemia HL-60 Cells

Previous studies have demonstrated that realgar nanoparticles might provide a less toxic agent for antineoplasia by suppressing angiogenesis. Here, we addressed the question of whether the size effects on apoptosis induction mainly contribute to the comparably higher concentration of easily soluble As2O3 present in realgar nanoparticles. Results revealed that treatment with realgar nanoparticles resulted in considerably low cell viability and produced characteristic apoptotic events in HL-60 cells, including morphological changes, DNA ladder formation, and increased number of cells with sub-G1-phase, whereas raw realgar particles with the same As2O3 concentration failed to induce apoptosis. On the other hand, the effects of realgar nanoparticles and raw realgar particles on cell membrane were examined. Realgar nanoparticles had acute toxicity to cell membrane, potentiating lipid peroxidation, increasing lactate dehydrogenase (LDH) release, and reducing membrane fluidity, whereas raw realgar particles had little effect on cell membrane besides a similar reduction of membrane fluidity. These results suggest that the promotion of lipid peroxidation and membrane permeability might play an important role in the process of apoptotic induction by realgar nanoparticles. However, raw realgar particles are not sufficient to elicit apoptosis, although they can reduce membrane fluidity in HL-60 cells.

Lidocaine-induced apoptosis and necrosis in U937 cells depending on its dosage

Local anesthetics are known to affect a variety of cellular responses other than the action of anesthetics through the Na(+) channel blockade. In this study, we examined the effect of a common local anesthetic lidocaine on the cellular activity and viability of human histiocytic lymphoma U937 cells. The cellular activity and viability were assessed by WST-1 reduction activity and trypan blue exclusion test, respectively. Induction of apoptosis was monitored by DNA ladder formation, reduction of mitochondrial transmembrane potential (DeltaPsim), caspase-3 activity and nuclear morphology. Lidocaine at concentrations below 12 mM induced apoptosis characterized by DNA fragmentation and chromatin condensation dose- and time-dependently. A pan-caspase inhibitor and a caspase-3 inhibitor blocked DNA ladder formation followed by the reduction of cell death. However, the caspase inhibitors did not affect the DeltaPsim, but cyclosporin A inhibited the collapse of DeltaPsim followed by a reduction of cell death. Lidocaine-induced apoptosis was mitochondria- and caspase-dependent, but the collapse of DeltaPsim was independent of caspase activation. At concentrations above 15 mM, lidocaine induced necrosis with early disruption of membrane integrity. These results indicate that lidocaine induced apoptosis and necrosis in U937 cells depending on its dosage.

Induction of Apoptosis in Human Oral Cancer Cell Lines, OC2 and TSCCa, by Chingwaysan

Chingwaysan, a Chinese herbal formula, contains Cimicfugae Rhizoma, Rehmanniae Radixet Rhizoma, Moutan Radicis Cortex, Coptidis Rhizoma and Angelicae Sinensis Radix. This medicine is well-known for its curing power for ulcerated gums, toothaches, cheek boils and bleeding gingiva. However, no reports can be found on its application in the treatment of oral cancers. We are therefore interested in whether Chingwaysan is capable of causing abnormal apoptosis processes, and whether this condition can be rectified through Chingwaysan herb treatment. We used aqueous extract to treat OC2 and TSCCa cells (both are human oral cancer cell lines) with different Chingwaysan concentrations (0, 10, 25, 50, 75 and 100 microl/ml). The MTT (3, (4, 5-dimethyl-thiazol) 2, 5-diphenyl-tetraxolium bromide) reduction assay was employed to quantify the differences in cell activity and viability. DNA ladder formation on agarose electrophoresis was also performed. The bax expression level was monitored using immunoblotting techniques. The patterns of the changes in expression were scanned and analyzed by NIH image 1.56 software. Taken together, drastic morphological changes, reduced cell viability and the presence of inter-nucleosomal DNA fragmentation all indicated that Chingwaysan is capable of inducing apoptosis in OC2 and TSCCa cell lines. Furthermore, the accumulation of wild type bax protein significantly increased in a dose-dependent manner upon treatment with Chingwaysan. In conclusion, Chingwaysan can induce apoptosis via a bax-dependent pathway in cells from these two particular oral cancer cell lines.

Role of stress-activated MAP kinase P38 in cisplatin- and DTT-induced apoptosis of the esophageal carcinoma cell line Eca109

To study the role of P38 kinase in esophageal cancer cell apoptosis induced by genotoxin, cisplatin and the unfolded protein response (UPR) inducer, dithiothreitol (DTT). Esophageal carcinoma cell line Eca109 was cultured in RPMI 1640 medium to 70% confluency and treated with either cisplatin, DTT, or cisplatin plus DTT in the presence or absence of P38 inhibitor, SB203580. The untreated cells served as the control. The esophageal carcinoma cell apoptosis was detected by agarose gel DNA ladder analysis and quantified by flow cytometry. The P38 phosphorylation was detected by immunohistochemistry using antibodies specific to phosphorylated P38 protein. (1) Both cisplatin and DTT induced apoptosis in the esophageal cancer cell line Eca109 as shown by DNA ladder formation; (2) As detected by antibodies specific for the phosphorylated P38 protein (p-P38), both cisplatin and DTT treatments activated the stress-activated enzyme, MAP kinase P38. The number of positive cells was about 50% for the treatment groups, comparing to that of 10% for untreated group. DTT treatment, but not cisplatin treatment, induces nuclear localization of p-P38; (3) As measured by flow cytometry, inhibition of P38 activity by SB203580 blocks DTT- and cisplatin-induced apoptosis. The rates for DTT, cisplatin, and DTT plus cisplatin-induced apoptosis were 16.8%, 17.1%, and 21.4%, respectively. Addition of the SB compound during the incubation reduced the apoptotic rate to about 7.6% for all the treatment groups, suggesting that P38 activation is essential for cisplatin- and DTT-induced apoptosis in Eca109 cells. (1) Both DTT and cisplatin were able to induce apoptosis in esophageal cancer cell line Eca109; (2) P38 MAP kinase is essential for DTT- and cisplatin-induced apoptosis in Eca109 cells; (3) P38 activation may be the common signaling component relaying the multiple upstream signaling events to the downstream cell death program.

Induction of cell-specific apoptosis and protection from Dalton′s lymphoma challenge in mice by an active fraction from Emilia sonchifolia

Objective: To isolate an active anticancer fraction from Emilia sonchifolia and to determine the mechanism of its anticancer activity. Materials and methods: The anticancer principle was separated using thin layer chromatography (TLC) from the most active n-hexane extract and chemically analysed. The anticancer efficacy of n-hexane extract was determined in mice using Dalton's lymphoma ascitic (DLA) cells. Cytotoxicity of the extracts and isolates to macrophages, thymocytes and DLA cells was measured using Trypan blue exclusion method, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, DNA ladder assay and DNA synthesis in culture. Short-term toxicity evaluation of the active fraction was also carried out in mice. Results: The hexane extract was found to be most active and it showed in vitro cytotoxicity to DLA and thymocytes, but not to macrophages. In a concentration and time-dependent manner, it induced membrane blebbing, nuclear condensation, DNA ladder formation, and formation of apoptotic bodies which are characteristic to apoptotic cell death. The n-hexane fraction protected 50% of mice challenged intraperitoneally with 106 DLA cells. This fraction did not exhibit conspicuous adverse toxic symptoms in mice. An active terpene fraction was separated from the n-hexane extract by TLC. This isolate induced apoptotic cell death in DLA cells at 0.8 µg per mL level. Conclusion: An anticancer terpene fraction was isolated by TLC from Emilia sonchifolia that induced cell-specific apoptosis and appears to be a promising anticancer agent.

A Potent Apoptosis-Inducing Activity of a Sesquiterpene Lactone, Arucanolide, in HL60 Cells: a Crucial Role of Apoptosis-Inducing Factor

Six main sesquiterpene lactones (germacranolides) from Calea urticifolia were evaluated for in vitro cytotoxicity against human tumor cell lines HL60 and SW480 cells. Among them, arucanolide and parthenolide displayed marked cytotoxicity against both cell lines. Arucanolide exhibited a low IC50 in HL60 cells. The cytotoxic activity of arucanolide was observed at lower concentrations compared to that of parthenolide, which has been reported to be a typical and simple germacranolide. The activity was found to be mainly due to apoptosis that was assessed by morphological findings, DNA ladder formation (24 – 36 h), and flow cytometric analysis in HL60 cells. Western blotting and an apoptosis inhibition assay using caspase inhibitors did not demonstrate the activation of any caspases tested. However, the mitochondrial membrane potential of HL60 cells was lost after 24-h treatment with arucanolide, and concurrently apoptosis-inducing factor (AIF) released from mitochondria was detected by Western blot analysis. The inactivation of nuclear factor-κB, which has been commonly shown in parthenolide-induced apoptosis, did not occur in arucanolide-induced apoptosis. Taken together, the findings presented here indicate that arucanolide induced marked apoptosis in HL60 cells mainly by dissipating mitochondrial membrane potential, which would trigger AIF-induced apoptosis.

Correlation of Apoptosis with Comet Formation Induced by Tea Polyphenols in Human Leukemia Cells

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.

Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells

Asiatic acid is a pentacyclic triterpene contained in medicinal plants. The cytotoxic effect of this compound and its augmentative effect on the anticancer drug irinotecan hydrochloride (CPT-11) were investigated in the human colon adenocarcinoma cell line HT-29. Asiatic acid dose-dependently showed cytotoxicity in HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were observed in a dose-dependent manner. A caspase-3 inhibitor suppressed the DNA ladder formation in a concentration-dependent manner. Bcl-2 and Bcl-XL proteins were decreased by asiatic acid treatment. These results indicate that asiatic acid induced apoptosis in HT-29 cells via caspase-3 activation. Cytotoxic effects of combined treatment with CPT-11 and asiatic acid on HT-29 cells were further examined. Simultaneous treatment or sequential exposure first to asiatic acid and then to CPT-11 showed an additive effect. Synergism was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid can be used as an agent for increasing sensitivity of colon cancer cells to treatment with CPT-11 or as an agent for reducing adverse effects of CPT-11.