Ladder Formation Research Articles

Role of TRPM2 in H2O2-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H2O2)-induced cytosolic Ca2+ ([Ca2+]i) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca2+]i rise and whole-cell current change in response to H2O2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H2O2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H2O2-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca2+]i and whole-cell currents to H2O2. TRPM2 overexpression also aggravated the H2O2-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca2+ overload in response to H2O2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.

Carbon tetrachloride-induced nephrotoxicity and DNA damage in rats

In this study, the protective effects of vanillin were evaluated against carbon tetrachloride (CCl(4))-induced kidney damages in Wistar albino rats. CCl(4) (1 ml/kg, intraperitoneally [i.p.]) caused a significant induction of renal disorder, oxidative damage and DNA fragmentation as evidenced by increased plasma creatinine, urea and uric acid levels, increased lipid peroxidation (malondialdehyde [MDA]) and protein carbonyl. Furthermore, glutathione levels, catalase, superoxide dismutase, glutathione transferase and glutathione peroxidase activities were significantly decreased. A smear without ladder formation on agarose gel was also shown, indicating random DNA degradation. Pretreatment of rats with vanillin (150 mg/kg/day, i.p.), for 3 consecutive days before CCl(4) injection, protected kidney against the increase of MDA and degradation of membrane proteins compared to CCl(4)-treated rats and exhibited marked prevention against CCl(4)-induced nephropathology, oxidative stress and DNA damage. Kidney histological sections showed glomerular hypertrophy and tubular dilatation in CCl(4)-treated rats, however, in vanillin pretreated rats, these histopathological changes were less important and present a similar structure to that of control rats. These data indicated the protective role of vanillin against CCl(4)-induced nephrotoxicity and suggested its significant contribution of these beneficial effects.

Magnetic structure of quasi-one-dimensional antiferromagnetic TaFe1+yTe3

We report the magnetic structure of TaFe${}_{1+y}$Te${}_{3}$ single crystals by means of neutron diffraction measurements. TaFe${}_{1+y}$Te${}_{3}$ possesses a layered structure with a formation of two-leg zigzag ladders along the $b$ axis. We find that TaFe${}_{1+y}$Te${}_{3}$ undergoes an antiferromagnetic transition at 178 K with Fe1 spins of the intraladders ferromagnetically aligned and with Fe1 spins of the interladders antiferromagnetically coupled. Furthermore, spins of the neighboring interstitial Fe2 ($y$) ions align parallel to the Fe1 spins of each ladder. These findings are distinct from the magnetic structure of the recently discovered spin-ladder compound BaFe${}_{2}$Se${}_{3}$. TaFe${}_{1+y}$Te${}_{3}$ may serve as a model system for investigating the interesting physics of quasi-one-dimensional ferromagnetic systems.

A Dual Role for Poly(ADP-Ribose) Polymerase-1 During Caspase-Dependent Apoptosis

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway.

298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2012298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL Yuji Kusuda, Hideaki Miyake, Martin Gleave, and Masato Fujisawa Yuji KusudaYuji Kusuda Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.357AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clusterin is expressed in a variety of malignant tumors, including renal cell carcinoma (RCC), and associated with resistance to broad-spectrum treatment. OGX-011 is a second-generation antisense oligodeoxynucleotide (ODN) currently in late-stage clinical development that potently inhibits clusterin expression and enhances the efficacy of anticancer therapies in various human cancers. The objective of this study was to investigate whether inactivation of clusterin with OGX-011 potentiates the effect of sorafenib, a novel inhibitor of multiple tyrosine kinases, in a human RCC ACHN model. METHODS Effects of a combined treatment with OGX-011 and sorafenib on ACHN cells were examined by MTT assay, DNA fragmentation assay and Western bolt analyses of proteins involved in apoptosis and signal transduction. In vivo efficacy of this combined therapy was assessed in nude mice given subcutaneous injection of ACHN cells. RESULTS We initially revealed the dose-dependent inhibition of clusterin expression in ACHN cells by treatment with OGX-011. Although clusterin expression was increased in a dose-dependent manner by sorafenib, additional treatment of ACHN cells with OGX-011 significantly blocked the upregulation of clusterin expression induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN cells, OGX-011 treatment synergistically enhanced the sensitivity of ACHN cells to sorafenib in a dose-dependent manner, reducing the IC50 by more than 50%. Characteristic apoptotic DNA ladder formation was detected after combined treatment with OGX-011 and sublethal dose of sorafenib, but not either agent alone. Furthermore, combined treatment of ACHN cells with OGX-011 and sorafenib resulted in the marked downregulation of Mcl-1, phosphorylated Akt and p44/42 MAP kinase compared with treatment with either agent alone. In vivo systemic administration of OGX-011 and sorafenib synergistically decreased the subcutaneous ACHN tumor volume compared with that of control ODN plus sorafenib. CONCLUSIONS Collectively, these findings demonstrated that clusterin helps confer a resistant phenotype of human RCC cells to sorafenib through the inhibition of apoptosis and activation of major signal transduction pathways, and that combined use of OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib in patients with advanced RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e121 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuji Kusuda Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Martin Gleave Vancouver, Canada More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, •OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl4)-treated animals. It was observed that CCl4-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.

α-Tocopherol Sensitizes Human Leukemia HL-60 Cells to Apoptosis Induced by Benzyl Isothiocyanate

Pre-treatment with α-tocopherol (α-Toc) potentiated cytotoxicity induction by benzyl isothiocyanate (BITC). Biochemical events related to apoptosis, such as DNA ladder formation and caspase-3 activation, were also enhanced by α-Toc. These results suggest a significant role of the caspase-3 pathway in apoptosis induction regulated by α-Toc in combination.

Induction of apoptosis with mitochondrial membrane depolarization by a glycyrrhetinic acid derivative in human leukemia K562 cells

Glycyrrhetinic acid (GA) is the active compound in Glycyrrhizae radix, a famous traditional Chinese medicine. Recently the anticancer activity of GA became the focus of scientific interest and many GA derivatives were developed as anti-tumor lead compounds. We previously reported that AEGA, a GA derivative, has proliferation inhibition and apoptosis-inducing activity in various human tumor cells. The present study was undertaken to further investigate the molecular mechanisms involved in AEGA-induced apoptosis in human leukemia K562 cells. AEGA can inhibit the growth of K562 cells in dose- and time-dependent manners determined by the MTT assay. Induction of apoptosis was evidenced by morphological changes and biochemical markers such as cell shrinkage, chromatin condensation and DNA ladder formation. Further mechanistic analysis revealed that AEGA induced apoptosis through the collapse of mitochondrial membrane potential, the accumulation of the cytosolic cytochrome c and the activation of caspase-9 and caspase-3. The apoptosis induction by AEGA was associated with the alteration in the ratio of Bcl-2/Bax protein expression. These results suggest that AEGA may induce apoptosis through a mitochondria-mediated pathway, and might have the therapeutic value against hematological malignancies.

Involvement of Reactive Oxygen Species in Sonodynamically Induced Apoptosis Using a Novel Porphyrin Derivative

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

Involvement of Phosphatidylinositol 3-Kinase/Akt Pathway in Gemcitabine-Induced Apoptosis-Like Cell Death in Insulinoma Cell Line INS-1

This study demonstrated gemcitabine-induced cytotoxicity in the insulinoma cell line INS-1. Gemcitabine inhibited INS-1 cell proliferation and maintained consistent cell number for 24 h, and then caused apoptosis within 48 h of incubation. Since gemcitabine activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which is involved in the resistance of pancreatic exocrine cancer to gemcitabine, we investigated the participation of this pathway in gemcitabine-induced cytotoxicity in INS-1 cells. LY294002 and wortmannin, two PI3-K inhibitors, significantly prevented gemcitabine-induced cytotoxicity in INS-1 cells, indicating that the PI3-K/Akt pathway is involved in gemcitabine-induced cytotoxicity. Gemcitabine-induced Akt phosphorylation in INS-1 cells was prevented by LY294002. Although gemcitabine induced cell cycle arrest at the G1 and early S phases, LY294002 did not inhibit the cell cycle. These data suggest that PI3-K activation does not influence gemcitabine-induced cell cycle arrest. In gemcitabine-treated cells, nuclear fragmentation and DNA ladder formation were observed. These findings suggest that gemcitabine induced apoptotic cell death in INS-1 cells through the activation of the PI3-K/Akt pathway.

Induction of Apoptosis in Human Leukemia Cell Line (HL60) by Animal's Venom Derived Peptides (ICD-85).

Our previous studies revealed an inhibitory effect of ICD-85 (Venom derived peptides) on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope (TEM). DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC50-value of 0.04 μg/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio was observed. The characteristic DNA ladder formation of ICD-85-treated cells in agarose gel electrophoresis confirmed the results obtained through the electron microscopy. The results of the present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing cell apoptosis.

Synthesis of Oxovanadium Complexes and Their Apoptosis-Inducing Activity in Leukemia Cells

Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complexes-3b and 4-exhibited strong activities with IC(50) values of 6.02 and 3.90 μM respectively. Annexin V staining and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.

Procyanidin B2 cytotoxicity to MCF-7 human breast adenocarcinoma cells

Procyanidins have attracted some attention due to their demonstrated chemopreventive action, a relatively new and promising strategy to prevent cancer. Breast cancer is one of the leading causes of death in women worldwide and its treatment needs improvements. The aim of this work was to verify the procyanidin dimmer B2 cytotoxic effect to MCF-7 human breast cancer cells. MCF-7 cells were cultured in RPMI medium, containing 20% fetal bovine serum and antibiotics in a CO2 chamber. The cells were treated with different concentrations of B2 and its cytotoxic potential was assessed by the sulforhodamine B assay, morphologically through haematoxylin-eosin staining and by DNA fragmentation analysis. The significance of differences between experimental conditions was determined using the ANOVA test, followed by the Tukey test when P<0.05. Cell proliferation decreased in a concentration and time-dependent manner upon procyanidin dimmer B2 treatment, being 19.20 μM the IC50. Procyanidin dimmer B2 treatment displayed concentration and time-dependent decline in MCF-7 cells compared to control and also induced morphological alterations compatible with cell-death induction. Cell condensation and cell diameter decreased (3.5 folds compared to control cells), after 48 h cell-exposure to 50 μM procyanidin dimmer B2, but the DNA ladder formation was not observed. In conclusion, our results demonstrated that procyanidin dimmer B2 exhibits cytotoxic activity to MCF-7 cells and it could be a potential antineoplastic agent. Further studies are necessary to clarify the procyanidin dimmer B2 mechanism of action. The evaluation of biological efficacy of individual components is an important step towards drug discovery and development.

ChemInform Abstract: Supramolecular Coordination: Self‐Assembly of Finite Two‐ and Three‐Dimensional Ensembles

AbstractReview: 840 refs.

Induction of Mitochondrion-mediated Apoptosis by Semecarpus anacardium in the BCR–ABL+ 12B1 Leukemia Cell Line: Possible Mechanism of Therapeutic Action In Vivo

Background/Purpose Chronic myeloid leukemia (CML) is a clonal disorder leading to massive proliferation of hematopoietic cells. It is characterized by the BCR–ABL gene, resulting in production of a 210-kD chimeric tyrosine kinase that plays a major role in its etiopathogenesis. There is an increasing interest in identifying potent preventive and therapeutic agents for leukemia. Semecarpus anacardium Linn. nut milk extract (SA) has antileukemic activity in murine BCR–ABL+ leukemia in vivo. The present study aimed to elucidate the mechanism of action of SA by assessing its antiproliferative and apoptotic effects in the BCR–ABL+ 12B1 murine leukemic cell line. Methods Cell viability and apoptosis assay using MTT analysis, propidium iodide staining, DNA fragmentation, intracellular reactive oxygen species/calcium, mitochondrial membrane potential, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting were determined in 12B1 cells. Results Cell viability of SA-treated 12B1 cells was dose and time dependent. SA-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes seen on propidium iodide staining and DNA ladder formation on electrophoresis. A reduction in the BCR–ABL + mRNA levels, induced by SA, was observed by RT-PCR. This may have been the result of a reduction in the transcription of the BCR–ABL fusion gene, with concomitant activation of apoptosis. SA induced a rapid increase in intracellular Ca2+ ions, which led to increased levels of reactive oxygen species. Concomitantly, we observed a significant loss of mitochondrial membrane potential and release of cytochrome c into the cytosol in 12B1 cells. These effects of SA are accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of the caspase-3 and caspase-9 genes. Conclusion These results provide unprecedented evidence that SA triggers mitochondrial receptor pathways to induce apoptotic signals in 12B1 cells. This study highlights the beneficial effect of SA in inducing apoptosis in cancer cells and thereby revealing a possible mechanism of therapeutic action in vivo.

Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells, including colon, prostate, breast and leukemia. Among them, aspirin, a classical NSAID, shows promise in cancer therapy in certain types of cancers. We hypothesized that aspirin might affect the growth of liver cancer cells since liver is the principal site for aspirin metabolism. Therefore, we investigated the effects of aspirin on the HepG2 human hepatocellular carcinoma cell line in vitro and the HepG2 cell xenograft model in BALB/c nude mice. We found that treatment with aspirin inhibited cell growth and induced apoptosis involving both extrinsic and intrinsic pathways as measured by DNA ladder formation, alteration in the Bax/Bcl-2 ratio, activation of the caspase activities and related protein expressions. In vivo antitumor activity assay also showed that aspirin resulted in significant tumor growth inhibition compared to the control. Oral administration of aspirin (100 mg/kg/day) caused a significant reduction in the growth of HepG2 tumors in nude mice. These findings suggest that aspirin may be used as a promising anticancer agent against liver cancer.

Resveratrol induces apoptosis of KB human oral cancer cells

Resveratrol (trans-3,4′s,5,-trihydroxystilbene), a phytoalexin present in grape skin and red wine, suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms. However, the effects of resveratrol on oral cancer are not completely understood. Thus, effects of resveratrol on cell growth and apoptosis induction were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA fragmentation, immunoblotting, and determination of caspase activation in KB human oral cancer cells. Treatment with resveratrol induced inhibition of cell growth depending on the resveratrol treatment time and concentration in KB cells. Treatment with resveratrol induced DNA ladder formation in KB cells and promoted proteolytic cleavage of procaspase-3 and procaspase-7 with increases in the amount of cleaved caspases-3 and -7. Proteolytic processing of caspase-9 in KB cells was increased by resveratrol treatment. Activation of caspase-3/-7 was detected in living KB cells by fluorescence microscopy. These results suggest that the resveratrol can suppress cell growth and induce cell apoptosis in KB human oral cancer cells, and may have potential as an anti-cancer drug.

2-Anilinonicotinyl linked 2-aminobenzothiazoles and [1,2,4]triazolo[1,5-b] [1,2,4]benzothiadiazine conjugates as potential mitochondrial apoptotic inducers

A series of N-(2-anilino-pyridyl) linked 2-amino benzothiazoles (4a–n) and [1,2,4]triazolo [1,5-b]benzothiadiazine conjugates (5a–j) have been designed, synthesized and evaluated for their antiproliferative activity. Some of these compounds (4h–k, 4n, and 5e) have exhibited potent cytotoxicity specifically against human leukemia HL-60 cell lines with IC50 values in the range of 0.08–0.70μM. All these compounds were tested for their effects on the cell cycle perturbations and induction of apoptosis. Morphological evidences of apoptosis, including fragmentation of nuclei and inter nucleosomal DNA laddering formation were clearly observed after 24h exposure to compound 4i. Flow cytometry analysis revealed that compound 4i showed drastic cell cycle perturbations due to concentration dependant increase in the sub-G0 region which comprises of both the apoptotic and debris fraction, thus implying the extent of cell death. These compounds trigger the mitochondrial apoptotic pathway that results in the loss of mitochondrial membrane potential through activation of multiple caspases followed by activation of caspase-3, and finally cleavage of PARP. Further the mechanism of cell death was analysed by fluorescent microscopic analysis and also by scanning electron microscopy. The cytotoxicity of 4i correlated with induction of apoptosis, caspases activation and DNA damage and thus indicating the apoptotic pathway of anticancer effect of these compounds.

Ganoderma lucidum total triterpenes prevent radiation-induced DNA damage and apoptosis in splenic lymphocytes in vitro

The development of radioprotective agents has been the subject of intense research, especially in the field of radiotherapy. In this study, we examined the radioprotective activity of the total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst in mouse splenic lymphocytes in vitro. Using the MTT assay, Ganoderma triterpenes were found to have no effect on cell viability, indicating that they are non-toxic to splenic lymphocytes. The effect of the total triterpenes on DNA damage and apoptosis induced by radiation was analyzed using the comet assay, DNA ladder assay and flow cytometric analysis. Total triterpenes were found to be highly effective in preventing DNA laddering, even at low concentrations (25μg/ml). The comet assay demonstrated that the G. triterpenes effectively prevented DNA damage, and flow cytometry revealed a reduction in apoptotic cells. The effect of the total triterpenes on intracellular reactive oxygen species (ROS) level and endogenous antioxidant enzyme activity in splenic lymphocytes were determined to elucidate possible radioprotective mechanisms. Total triterpenes successfully reduced the formation of intracellular ROS and enhanced endogenous antioxidant enzyme activity in splenic lymphocytes following irradiation. Thus, these findings indicate that the total triterpenes isolated from G. lucidum have a remarkable ability to protect normal cells from radiation-induced damage, which suggests therapeutic potential.

Mitochondrial apoptosis contributes to the anti-cancer effect of Smilax glabra Roxb.

Smilax glabra Roxb. (SGR), a member of the Smilacaceae family and a rhizome of the Liliaceae plant, has shown anti-inflammation and detoxification properties, and a few studies reported its anti-cancer effect. In this study, we showed that SGR inhibited growth of human breast cancer cell line MCF7, colon carcinoma cell line HT-29, and gastric cancer cell line BGC-823 in a dose-dependent manner. Furthermore, SGR could inhibit tumor growth of HT-29 in Balb/c nude mice and murine hepatoma H22 cells in ICR mice. SGR elicited apoptotic cell death, as confirmed by DNA ladder formation, changes in nuclear morphology, and the increased FITC-Annexin-V/PI staining. Permeabilization of mitochondrial membrane (MMP), production of reactive oxygen species (ROS), elevation of intracellular [Ca2+], relocation of cytochrome c, and the activation of caspase-3 were found to be associated with the initiation of apoptosis by SGR treatment. Using microarray analysis, we found the changes in expression profiles of genes related to apoptosis, proliferation and cell cycle control in the cells treated with SGR. Our results demonstrated the mitochondrial regulation of apoptosis by which SGR exerts the anti-cancer effect.