lignans and Necrosis

In this study, five neolignans were isolated from Saururus cernuus-threo-dihydroguaiaretic acid (1), threo-austrobailignan-6 (2), threo-austrobailignan-5 (3), verrucosin (4), and saucernetin (5)-and have their cytotoxic effects evaluated in prostate cancer cell lines (PC3 and DU145). Initially, using an in silico approach, tested compounds were predicted to be absorbed by the gastrointestinal tract, be able to permeate the blood-brain barrier and did not show any alert in PAINS (pan-assay structures interference). In vitro assays showed that compounds 2, 4, and 5 reduced cell viability of DU145 cell line at 100 μmol/L after 48 h while compounds 1 and 3 showed to be inactive at the same conditions. Furthermore, compounds 4 and 5 reduced cell number as early as in 24 h at 50 μmol/L and compound 2 showed effects at 100 μmol/L in 24 h against both cancer cell lines PC3 and DU145. Studies using flow cytometry were conducted and indicated that compound 4 induced strong necrosis and apoptosis whereas compound 5 induced strong necrosis. Otherwise, less active compound 2 did not show evidence of induction of apoptosis or necrosis, suggesting that its mechanism of action involves inhibition of cell proliferation. In conclusion, compounds 4 and 5 have been shown to be promising cytotoxic agents against prostate cancer cell lines and can be used as a starting point for the development of new drugs for the treatment of prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Lignans; Male; Necrosis; Prostatic Neoplasms; Saururaceae

Toll-like receptor 4 (TLR4) and C5aR1 (CD88) have been recognized as potential therapeutic targets for the reduction of inflammation and secondary damage and improvement of outcome after ischemia and reperfusion (I/R). The inflammatory responses which induce cell apoptosis and necrosis after I/R brain injury lead to a limited process of neural repair. To further comprehend how these targets function in I/R state, we investigated the pathological changes and TLR4 and C5aR1 signaling pathways in vitro and in vivo models of I/R brain injury in this study. Meanwhile, we explored the roles of schisantherin A on I/R brain injury, and whether it exerted neuroprotective effects by regulating the TLR4 and C5aR1 signaling pathways or not. The results showed that schisantherin A significantly reduced the neuronal apoptosis induced by oxygen and glucose deprivation and reperfusion (OGD/R) injury in primary culture of rat cortical neurons. Also, schisantherin A alleviated neurological deficits, reduced infarct volume, attenuated oxidation stress, inflammation and apoptosis in ischemic parietal cortex of rats after middle cerebral artery occlusion and reperfusion (MCAO/R) injury. Moreover, the activated TLR4 and C5aR1 signaling pathways were inhibited by schisantherin A treatment. In conclusion, TLR4 and C5aR1 played a vital role during I/R brain injury in rats, and schisantherin A exhibited neuroprotective effects by TLR4 and C5aR1 signaling pathways. These findings also provided new insights that would aid in elucidating the effect of schisantherin A against cerebral I/R and support the development of schisantherin A as a potential treatment for ischemic stroke.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Survival; Cerebral Cortex; Cyclooctanes; Dioxoles; Inflammation; Lignans; Necrosis; Neurons; Neuroprotective Agents; Oxidative Stress; Parietal Lobe; Primary Cell Culture; Rats, Sprague-Dawley; Receptor, Anaphylatoxin C5a; Reperfusion Injury; Signal Transduction; Toll-Like Receptor 4

Arctigenin has previously been identified as a potential anti-tumor treatment for advanced pancreatic cancer. However, the mechanism of how arctigenin kills cancer cells is not fully understood. In the present work we studied the mechanism of toxicity by arctigenin in the human pancreatic cell line, Panc-1, with special emphasis on the mitochondria. A comparison of Panc-1 cells cultured in glucose versus galactose medium was applied, allowing assessments of effects in glycolytic versus oxidative phosphorylation (OXPHOS)-dependent Panc-1 cells. For control purposes, the mitochondrial toxic response to treatment with arctigenin was compared to the anti-cancer drug, sorafenib, which is a tyrosine kinase inhibitor known for mitochondrial toxic off-target effects (Will et al., 2008). In both Panc-1 OXPHOS-dependent and glycolytic cells, arctigenin dissipated the mitochondrial membrane potential, which was demonstrated to be due to inhibition of the mitochondrial complexes II and IV. However, arctigenin selectively killed only the OXPHOS-dependent Panc-1 cells. This selective killing of OXPHOS-dependent Panc-1 cells was accompanied by generation of ER stress, mitochondrial membrane permeabilization and caspase activation leading to apoptosis and aponecrosis.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Endoplasmic Reticulum Stress; Furans; Glycolysis; Humans; Lignans; Membrane Potential, Mitochondrial; Necrosis; Oxidative Phosphorylation; Oxygen Consumption

Honokiol is a pharmacologically active small molecule with multifunctional antitumor effects. Although plenty of literature is available on honokiol-triggered apoptosis and programmed necrosis, few studies have investigated the potential existence of death mode transition from apoptosis to programmed necrosis. In the current study, we demonstrated that the necrotic cell population (PI-positive) gradually increased and the early-stage apoptotic cell population (PI-negative and AV-positive) decreased in a dose- and time-dependent manner following honokiol treatment. Furthermore, we demonstrated that these PI-positive cells were under necrotic cell death, since no late-apoptosis characteristics including conspicuous chromatin condensation or DNA ladder patterns were detected. These results demonstrated that cells suffered death mode transition from early-stage apoptosis to programmed necrosis with the increase of honokiol dose or treatment time. The protein expression of RIP3 markedly increased in parallel with HNK-triggered death mode transition, while the expression of RIP1 decreased. Cyclophilin D expression increased during cell death mode transition, and inhibition of cyclophilin D by cyclosporin A clearly blocked HNK-triggered programmed necrosis. These data indicated that honokiol-induced programmed necrosis and death mode transition are potentially RIP3‑dependent, cyclophilin D-regulated. Further results showed that blocked cyclophilin D by cyclosporin A inhibited HNK-induced necrosis, but did not affect HNK-induced RIP3 overexpression. This indicated that cyclophilin D was a potential modulator at downstream of RIP3. In conclusion, honokiol triggers a potential RIP3-dependent cell death mode transition from early-stage apoptosis to programmed necrosis, which is highly regulated by cyclophilin D.

Topics: Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cyclophilins; Humans; Lignans; Necrosis; Peptidyl-Prolyl Isomerase F; Receptor-Interacting Protein Serine-Threonine Kinases

The medicinal herb formulation Da-Cheng-Qi decoction (DCQD) has been shown to ameliorate the severity of acute pancreatitis by regulating an apoptosis-necrosis switch in cells. The active components responsible for this effect and their detailed mechanism of action remain unclear. Here we determine the pharmacokinetic characteristics of the four most abundant compounds in DCQD using a rat model of severe acute pancreatitis. Acute pancreatitis-like symptoms were first induced in rats and then they were given DCQD orally. Rhein was found in rat serum at much higher levels than magnolol, hesperidin, or naringin, even though it was the least abundant of the four compounds in the DCQD. We also examined pharmacodynamics in AR42J cells stimulated with 10(-8) M cerulein as a cellular model of acute pancreatitis. After pretreating AR42J cells with individual compounds and then exposing them to cerulein, we determined cell viability, levels of apoptosis and necrosis, and numbers of cells positive for reactive oxygen species (ROS). Pretreatment with any of the four DCQD compounds increased cell viability and the apoptosis index, while also reducing necrosis and ROS generation. The compounds showed maximal effect in AR42J cells around the same time that they showed maximum serum concentration in rats. Although all four components appear to play a role in an apoptosis-necrosis cellular switch in vitro, rhein may be the most bioactive DCQD ingredient.

Topics: Animals; Anthraquinones; Apoptosis; Biphenyl Compounds; Cells, Cultured; Disease Models, Animal; Drugs, Chinese Herbal; Flavanones; Hesperidin; Lignans; Male; Necrosis; Pancreas; Pancreatitis, Acute Necrotizing; Phytotherapy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Our previous report has shown that honokiol (HNK), a constituent of Magnolia officinalis, induces a novel form of non-apoptotic programmed cell death in human leukemia NB4 and K562 cells. In this study, we further explored the relationship between the cell death pathway and cytoplasmic vacuolization and studied the underlying mechanism of leukemia cell death mediated by honokiol. The results showed that low concentrations of honokiol activated an novel alternative cell death fitted the criteria of paraptosis, such as cytoplasmic vacuolization derived from endoplasmic reticulum swelling, lack of caspase activation, and lack of apoptotic morphology. Results further indicated that the cell death was time- and concentration-dependent. In addition, honokiol-induced paraptosis did not involve membrane blebbing, chromatin condensation and phosphatidylserine exposure at the outer of the plasma membrane. The mechanism of the cell death may be associated, at least in part, with the increased generation of reactive oxygen species. Further analysis showed that honokiol induces cell death predominantly via paraptosis and to a certain extent via apoptosis in NB4 cells, and predominantly via apoptosis and to a certain extent via paraptosis in K562 cells. These observations suggest that cell death occurs via more than one pathway in leukemia cells and targeting paraptosis may be an alternative and promising avenue for honokiol in leukemia therapy.

Topics: Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cell Membrane; Cytoplasm; Endoplasmic Reticulum; Humans; Leukemia; Lignans; Metabolic Networks and Pathways; Necrosis; Phosphatidylserines; Reactive Oxygen Species; Vacuoles

Selectively eradicating cancer cells with minimum adverse effects on normal cells is a major challenge in the development of anticancer therapy. We hypothesize that nutrient-limiting conditions frequently encountered by cancer cells in poorly vascularized solid tumors might provide an opportunity for developing selective therapy. In this study, we investigated the function and molecular mechanisms of a natural compound, arctigenin, in regulating tumor cell growth. We demonstrated that arctigenin selectively promoted glucose-starved A549 tumor cells to undergo necrosis by inhibiting mitochondrial respiration. In doing so, arctigenin elevated cellular level of reactive oxygen species (ROS) and blocked cellular energy metabolism in the glucose-starved tumor cells. We also demonstrated that cellular ROS generation was caused by intracellular ATP depletion and played an essential role in the arctigenin-induced tumor cell death under the glucose-limiting condition. Furthermore, we combined arctigenin with the glucose analogue 2-deoxyglucose (2DG) and examined their effects on tumor cell growth. Interestingly, this combination displayed preferential cell-death inducing activity against tumor cells compared to normal cells. Hence, we propose that the combination of arctigenin and 2DG may represent a promising new cancer therapy with minimal normal tissue toxicity.

Topics: Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Arctium; Cell Death; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Energy Metabolism; Furans; Glucose; Humans; Lignans; Mitochondria; Necrosis; Reactive Oxygen Species; Tumor Microenvironment

Antioxidants have been used as adjuvant with anticancer therapy to synergize the potential of the anti-neoplastic therapeutics. Based on the fact, we have studied the effect of three natural antioxidants curcumin, silymarin and acteoside on AP9-cd (standardized lignan composition from Cedrus deodara) induced cytotoxicity in human leukemia HL-60 cells. The antioxidant potential of individual test compounds was first evaluated with ferric reducing antioxidant power (FRAP) test, which revealed that all four molecules behave as antioxidants. The apoptotic potential of AP9-cd was significantly enhanced in HL-60 cells in the presence of curcumin, silymarin and acteoside. It was confirmed by using various models like MTT assay, DNA fragmentation, nuclei condensation, sub-Go DNA population, Annexin-V-FITC binding, ROS depletion and immunoblotting in HL-60 cells. AP9-cd and individual antioxidants alone at low doses (10μg and 10μM, respectively) have meager or no cytotoxicity in HL-60 cells, whereas in mutual combinations, there were 2-3 times enhancement in Annexin-V-FITC and sub-Go DNA population. Moreover, prominent DNA ladders were observed at low doses of AP9-cd in combinations with various antioxidants. The Hoechst staining of the nucleus also revealed the same results for the HL-60 cells treated with AP9-cd and different antioxidants. The molecular diagnostics revealed that the combinations induced a strong antioxidant effect which was correlated with the downregulation of NF-κB expression in the nucleus. Out of the three antioxidants, curcumin was found to be more potent than acteoside and silymarin in terms of enhancing the apoptotic potential of AP9-cd. These results propose an important role of natural antioxidant as adjuvant to enhance the anticancer potential of AP9-cd and more likely other anti-neoplastic therapeutics.

Topics: Annexin A5; Antineoplastic Agents; Antioxidants; Apoptosis; Biological Products; Cedrus; Cell Cycle; Cell Proliferation; DNA Fragmentation; Down-Regulation; Drug Synergism; Ferric Compounds; Fluorescein-5-isothiocyanate; Fluoresceins; HL-60 Cells; Humans; Leukemia; Lignans; Membrane Potential, Mitochondrial; Necrosis; NF-kappa B; Nitric Oxide; Oxidation-Reduction; Reactive Oxygen Species

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W-2 thyroid carcinoma cells. After 24 h treatment with 80 microM magnolol in serum-containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome-c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase-3 and -7) and of cleaved poly (ADP-ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time-dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol-induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol-induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome-c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation.

Topics: Apoptosis; Apoptosis Inducing Factor; Biphenyl Compounds; Blotting, Western; Caspase 3; Caspase 7; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Humans; Immunohistochemistry; Lignans; Necrosis; Poly(ADP-ribose) Polymerases; Signal Transduction; Thyroid Neoplasms

Previous reports have shown that honokiol induces apoptosis in numerous cancer cell lines and showed preclinical efficacies against apoptosis-resistant B-cell chronic lymphocytic leukemia and multiple myeloma cells from relapse-refractory patients. Here, we show that honokiol can induce a cell death distinct from apoptosis in HL60, MCF-7, and HEK293 cell lines. The death was characterized by a rapid loss of integrity of plasma membrane without externalization of phosphatidyl serine. The broad caspase inhibitor z-VAD-fmk failed to prevent this cell death. Consistently, caspase activation and DNA laddering were not observed. The death was paralleled by a rapid loss of mitochondrial membrane potential, which was mechanistically associated with the mitochondrial permeability transition pore regulated by cyclophilin D (CypD) based on the following evidence: (a) cyclosporin A, an inhibitor of CypD (an essential component of the mitochondrial permeability transition pore), effectively prevented honokiol-induced cell death and loss of mitochondrial membrane potential; (b) inhibition of CypD by RNA interference blocked honokiol-induced cell death; (c) CypD up-regulated by honokiol was correlated with the death rates in HL60, but not in K562 cells, which underwent apoptosis after being exposed to honokiol. We further showed that honokiol induced a CypD-regulated death in primary human acute myelogenous leukemia cells, overcame Bcl-2 and Bcl-X(L)-mediated apoptotic resistance, and was effective against HL60 cells in a pilot in vivo study. To the best of our knowledge, this is the first report to document an induction of mitochondrial permeability transition pore-associated cell death by honokiol.

Topics: Adult; Aged; Antineoplastic Agents, Phytogenic; Apoptosis Inducing Factor; Biphenyl Compounds; Cell Death; Cell Line, Tumor; Cell Nucleus; Cyclophilins; Female; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Lignans; Male; Membrane Potential, Mitochondrial; Middle Aged; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Neoplasms; Reactive Oxygen Species

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.

Topics: Animals; Apoptosis; Cedrus; Cell Division; Cell Line, Tumor; Cells, Cultured; Flow Cytometry; Hepatocytes; Humans; Leukemia; Lignans; Membrane Potentials; Necrosis; Nitric Oxide; Peroxides; Rats; Reactive Oxygen Species

Carbon tetrachloride (CCl(4)) -induced hepatotoxicity is a commonly used model for investigating lipid peroxidation-related tissue injury. In the present study, the effect of flaxseed extract was observed on histological sections, glutathione-content and DNA strand breaks. Lignan-containing flaxseed extract (1.6 g/kg body weight/day) was daily administered with intragastric injection to rats for three days, on the fourth day, CCl(4) (2 g/kg) was intraperitoneally injected. Liver tissue was sampled at 24 hr after administering CCl(4). Liver-necrosis was observed in CCl(4)-injected rats without pretreatment of flaxseed extract. Pretreatment of flaxseed extract reduced extent of the necrosis found 24 hr after the intraperitoneal administration of CCl(4). Pretreatment of flaxseed extract protect against CCl(4)-induced decrease of reduced glutathione-content measured from reactions with 5,5'-dithiobis-(2-nitrobenzoic acid) and also protect against the elevation of DNA strand breaks in the liver cells measured by comet assay. Flaxseed-extract appears to protect liver cells against CCl(4)-induced necrosis.

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; DNA Damage; Drug Administration Schedule; Flax; Glutathione; Lignans; Liver; Liver Diseases; Necrosis; Plant Extracts; Rats; Rats, Inbred Strains

Four lignans: podophyllotoxin (A), 4'-demethyl-podophyllotoxin (B), alpha-peltatin (C), and beta-peltatin (D) are the podophyllin ingredients producing lesional necrosis after application on condylomata acuminata. This study evaluates comparatively their destructive potency on rabbit skin after 10-day repeated applications. Any visible changes or increase on skinfold thickness induced by each of the drugs tested as 0.01, 0.05, 0.1, and 0.5% ethanolic preparations were followed. These variables demonstrated a close covariation; the analyses revealed a significant difference with respect to influence on measurements according to the following ranking order: A greater than C greater than D = B.

Topics: Administration, Topical; Animals; Antineoplastic Agents, Phytogenic; Condylomata Acuminata; Humans; Kinetics; Lignans; Necrosis; Plant Extracts; Podophyllotoxin; Rabbits; Skin; Skinfold Thickness