naphthoquinones and Necrosis

Necrosis plays vital roles in living organisms which is related closely with various diseases. Non-invasively necrotic imaging can be of great values in clinical decision-making, evaluation of individualized treatment responses, and prediction of patient prognosis. This narrative review will demonstrate how the evolution of quinones for necrotic imaging has been promoted by searching for their active centers. In this review, we summarized the recent developments of various quinones with the continuous simplified π-conjugated cores in necrotic imaging and speculated their possible molecular mechanisms might be attributed to their intercalations with exposed DNA in necrotic tissues. We discussed their clinical challenges of necrotic imaging with quinones and their future translation studies deserved to be explored in personalized patient treatment.

Topics: Animals; Anthraquinones; Cells; DNA; Humans; Molecular Probes; Myocardial Infarction; Naphthoquinones; Necrosis; Quinones; Rats

Poly (ADP-ribose) polymerases (PARPs) are a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. PARPs play an important role in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, in view of the finding that certain tumors defective in homologous recombination mechanisms, may rely on PARP-mediated DNA repair for survival, and are sensitive to its inhibition. PARP inhibitors may also increase tumor sensitivity to DNA-damaging agents. Clinical trials of PARP inhibitors are investigating the utility of these approaches in cancer. The hyperactivation of PARP has also been shown to result in a specific programmed cell death pathway involving NAD+/ATP depletion, mu-calpain activation, loss of mitochondrial membrane potential, and the release of apoptosis inducing factor. Hyperactivation of the PARP pathway may be exploited to selectively kill cancer cells. Other PARP forms, including tankyrase 1 (PARP 5a), which plays an important role in enhancing telomere elongation by telomerase, have been found to be potential targets in cancer therapy. The PARP pathway and its inhibition thus offers a number of opportunities for therapeutic intervention in both cancer and other disease states.

Topics: Animals; Disease Models, Animal; DNA Repair; Enzyme Inhibitors; Epigenesis, Genetic; Humans; Molecular Targeted Therapy; Nanomedicine; Naphthoquinones; Necrosis; Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Transcription Factors

Shikonin, the main active ingredient of Lithospermum, and its derivatives have been proved to have antitumor effects, and the anti-tumor mechanisms involve multiple targets. Based on recent literatures, this review focuses on the antitumor effects and its mechanisms. More emphases are given on the aspects of induction of apoptosis, induction of necrosis, acting on matrix metalloproteinase, acting on the protein tyrosine kinase and antiangiogenesis. The current status and problems of shikonin derivatives in antitumor effects are simply summarized and lookout for the development of antitumor drugs with shikonin as leading compounds.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drugs, Chinese Herbal; Humans; Lithospermum; Matrix Metalloproteinase 9; Naphthoquinones; Necrosis; Neoplasms; Neovascularization, Pathologic; Plants, Medicinal; Protein-Tyrosine Kinases; Reactive Oxygen Species

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase expressed in multiple tumour types. ARQ 761 is a β-lapachone (β-lap) analogue that exploits the unique elevation of NQO1 found in solid tumours to cause tumour-specific cell death.. We performed a 3+3 dose escalation study of 3 schedules (weekly, every other week, 2/3 weeks) of ARQ 761 in patients with refractory advanced solid tumours. Tumour tissue was analysed for NQO1 expression. After 20 patients were analysed, enrolment was restricted to patients with NQO1-high tumours (H-score ≥ 200).. ARQ 761 has modest single-agent activity, which appears associated with tumour NQO1 expression. Principal toxicities include anaemia and possible methemoglobinaemia.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Cell Line, Tumor; DNA Damage; Female; Humans; Male; Middle Aged; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Necrosis; Neoplasms; Reactive Oxygen Species

Novel derivatives of aminophenyl-1,4-naphthoquinones, in which a pyrrolidine group was added to the naphthoquinone ring, were synthesized and investigated for the mechanisms of leukemic cell killing. The novel compounds, TW-85 and TW-96, differ in the functional (methyl or hydroxyl) group at the para-position of the aminophenyl moiety. TW-85 and TW-96 were found to induce concentration- and time-dependent apoptotic and/or necrotic cell death in human U937 promonocytic leukemia cells but only TW-96 could also kill K562 chronic myeloid leukemia cells and CCRF-CEM lymphoblastic leukemia cells. Normal peripheral blood mononuclear cells were noticeably less responsive to both compounds than leukemia cells. At low micromolar concentrations used, TW-85 killed U937 cells mainly by inducing apoptosis. TW-96 was a weaker apoptotic agent in U937 cells but proved to be cytotoxic and a stronger inducer of necrosis in all three leukemic cell lines tested. Both compounds induced mitochondrial permeability transition pore opening, cytochrome c release, and caspase activation in U937 cells. Cytotoxicity induced by TW-96, but not by TW-85, was associated with the elevation of the cytosolic levels of reactive oxygen species (ROS). The latter was attenuated by diphenyleneiodonium, indicating that NADPH oxidase was likely to be the source of ROS generation. Activation of p38 MAPK by the two agents appeared to prevent necrosis but differentially affected apoptotic cell death in U937 cells. These results further expand our understanding of the structure-activity relationship of aminophenyl-1,4-naphthoquinones as potential anti-leukemic agents with distinct modes of action.

Topics: Apoptosis; Cell Death; Humans; Leukemia; Leukemia, Myeloid; Leukocytes, Mononuclear; Naphthoquinones; Necrosis; Reactive Oxygen Species; U937 Cells

Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after.. We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro.. Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin.. Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP.. Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.

Topics: Apoptosis; Cell Line, Tumor; Cysteine; Humans; Melanoma; Naphthoquinones; Necrosis; Reactive Oxygen Species

The use of 3D models in various scientific applications is becoming more popular to replace traditional monolayers models. In this work, we used a three-dimensional in-house model of epidermis using HaCaT immortalized cells to evaluate the dermal toxicity induced by Basic Blue 99 and Basic Red 51, both present in commercial hair dye formulations. Our data show that cells cultured in the 3D model respond differently to those cultured in monolayer. Basic Red 51 dye induces apoptosis an DNA breaks in both models, however, these effects is more pronounced in cells cultured in monolayer. The toxic mode of action of Basic Blue 99 seems to be the induction of cell death, without genotoxic effects, but while the necrotic pathway is observed in HaCaT monolayer cell culture, was apoptosis seen in the Equivalent Human Epidermis (EHE) model. We could also confirm that cells in EHE model, an environment that could better mimic human effects, react differently to chemical stressors than the cells cultivated in 2D.

Topics: Apoptosis; Azo Compounds; Cell Culture Techniques; Cell Line; DNA Damage; Epidermis; Hair Dyes; Humans; Naphthoquinones; Necrosis; Quaternary Ammonium Compounds

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

Topics: Alarmins; Animals; Apoptosis; Autophagy; Cell Communication; Cell Line, Tumor; Chloroquine; Dendritic Cells; Female; Immunization; Immunologic Surveillance; Mice, Inbred BALB C; Models, Biological; Naphthoquinones; Necrosis; Neoplasm Metastasis; Up-Regulation

Shikonin, a natural small agent, has shown inhibitory effect in many kinds of cells, which increases intracellular reactive oxygen species (ROS) level and causes mitochondrial injury. In this study, shikonin showed good inhibitory effect on nasopharyngeal carcinoma CNE-2Z cells in vivo and vitro. The results presented here revealed that ROS levels increased markly after shikonin treated. The electron microscopy displays the change in ultrastructure of CNE-2Z cells after treatment for shikonin, which indicated that shikonin induced necroptosis. Shikonin-induced cell death was inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the activity was unaffected by the caspase inhibitor z-VAD-fmk. Furthermore, we have demonstrated that the activation of receptor-interacting kinase (RIP) led to necroptosis. Meanwhile, shikonin also significantly inhibited tumor growth in a CNE-2Z xenograft mouse model. Taken together, shikonin induced CNE-2Z cells death by producing ROS as a necroptosis inducer. It could serve as a new therapeutic agent for treating CNE-2Z cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Cell Line, Tumor; Heterografts; Humans; Mice; Mice, Nude; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Necrosis; Reactive Oxygen Species

Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.. Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.. Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.. Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Topics: A549 Cells; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 8; Cell Line, Tumor; Gene Silencing; Humans; Imidazoles; Indoles; Lithospermum; Lung Neoplasms; Macrolides; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation; RNA, Small Interfering; X-Ray Microtomography

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 μmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioma; Humans; Mitochondria; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Oxidative Stress; Protein Serine-Threonine Kinases; Rats; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Signal Transduction; Time Factors

Redox signaling plays a fundamental role in maintaining cell physiological activities. A deregulation of this balance through oxidative stress or nitrosative stress has been implicated in cancer. Here, we reported that 2-methoxy-6-acetyl-7-methyl juglone (MAM), a natural naphthoquinone isolated from Polygonum cuspidatum Sieb. et Zucc, caused hydrogen peroxide (H2O2) dependent activation of JNK and induced the expression of inducible nitric oxide synthase (iNOS), thereby leading to nitric oxide (NO) generation in multiple cancer cells. Nitrosative stress induced necroptosis in A549 lung cancer cells, but resulted in caspase-dependent intrinsic apoptosis in B16-F10 melanoma and MCF7 breast cancer cells. In addition, a decrease in GSH/GSSG levels accompanied with increased ROS production was observed. Reversal of ROS generation and cell death in GSH pretreated cells indicated the involvement of GSH depletion in MAM mediated cytotoxicity. In summary, a natural product MAM induced NO-dependent multiple forms of cell death in cancer cells mediated by H2O2-dependent JNK activation in cancer cells. GSH depletion might play an initial role in MAM-induced cytotoxicity.

Topics: Animals; Apoptosis; Cell Line, Tumor; Glutathione; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; MCF-7 Cells; Melanoma, Experimental; Mice; Naphthoquinones; Necrosis; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Reactive Oxygen Species

Natural products comprise valuable sources for new antiparasitic drugs. Here we tested the effects of a novel β-lapachone derivative on Trypanosoma cruzi parasite survival and proliferation and used microscopy and cytometry techniques to approach the mechanism(s) underlying parasite death. The selectivity index determination indicate that the compound trypanocidal activity was over ten-fold more cytotoxic to epimastigotes than to macrophages or splenocytes. Scanning electron microscopy analysis revealed that the R72 β-lapachone derivative affected the T. cruzi morphology and surface topography. General plasma membrane waving and blebbing particularly on the cytostome region were observed in the R72-treated parasites. Transmission electron microscopy observations confirmed the surface damage at the cytostome opening vicinity. We also observed ultrastructural evidence of the autophagic mechanism termed macroautophagy. Some of the autophagosomes involved large portions of the parasite cytoplasm and their fusion/confluence may lead to necrotic parasite death. The remarkably enhanced frequency of autophagy triggering was confirmed by quantitating monodansylcadaverine labeling. Some cells displayed evidence of chromatin pycnosis and nuclear fragmentation were detected. This latter phenomenon was also indicated by DAPI staining of R72-treated cells. The apoptotis induction was suggested to take place in circa one-third of the parasites assessed by annexin V labeling measured by flow cytometry. TUNEL staining corroborated the apoptosis induction. Propidium iodide labeling indicate that at least 10% of the R72-treated parasites suffered necrosis within 24 h. The present data indicate that the β-lapachone derivative R72 selectively triggers T. cruzi cell death, involving both apoptosis and autophagy-induced necrosis.

Topics: Apoptosis; Autophagy; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Naphthoquinones; Necrosis; Trypanocidal Agents; Trypanosoma cruzi

Many natural and synthetic quinones and naphthoquinones possess a variety of beneficial pharmacological properties. In plants, the cytotoxic properties of quinones serve in their defensive roles against invading bacteria, fungi and parasites. In this regard many quinones as well as polyphenols, exerting generally toxicity at high dosages, are able to induce favorable hormetic responses at a low dosage. The novel chloronaphthoquinone derivative of quercetin (CHNQ) showed a profound cytotoxicity followed by enhancement of intracellular generation of oxidants in human neonatal B-HNF-3 fibroblasts. Its synthetic precursors, quercetin and 2-chloro-3-hydroxy-[1,4]naphthoquinone, failed to induce these effects, and paradoxically, only CHNQ at a low concentration provided partial protection of the cells against oxidative challenge. Thus, the novel quinonoid-polyphenol CHNQ might have a merit in the search for new prospective agents in prevention and management of ageing and ageing-related pathologies.

Topics: Aging; Antioxidants; Apoptosis; Cells, Cultured; Flavonoids; Hormesis; Humans; Hydrogen Peroxide; Immunohistochemistry; Models, Molecular; Naphthoquinones; Necrosis; Neutral Red; Oxidants; Oxidative Stress; Polyphenols; Quinones; Tetrazolium Salts; Thiazoles

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Death; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Humans; Imidazoles; Indoles; Multiple Myeloma; Naphthoquinones; Necrosis; Proteasome Inhibitors; Purine Nucleosides; Pyrazines; Regulatory Factor X Transcription Factors; Transcription Factors; X-Box Binding Protein 1

Breast cancer, the most common cancer in the women, is the leading cause of death. Necrotic signaling pathways will enable targeted therapeutic agents to eliminate apoptosis-resistant cancer cells. In the present study, the effect of shikonin on the induction of cell necroptosis or apoptosis was evaluated using the T-47D breast cancer cell line. The cell death modes, caspase-3 and 8 activities and the levels of reactive oxygen species (ROS) were assessed. Cell death mainly occurred through necroptosis. In the presence of Nec-1, caspase-3 mediated apoptosis was apparent in the shikonin treated cells. Shikonin stimulates ROS generation in the mitochondria of T-47D cells, which causes necroptosis or apoptosis. Induction of necroptosis, as a backup-programmed cell death pathway via ROS stimulation, offers a new strategy for the treatment of breast cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Caspase 3; Cell Proliferation; Female; Humans; Mitochondria; Naphthoquinones; Necrosis; Reactive Oxygen Species; Tumor Cells, Cultured

The effects of shikonin on gastric cancer cells were investigated in this study. Exposure to shikonin reduced the viability of gastric cancer cells in a time- and dose-dependent manner. However, apoptosis was not observed in gastric cancer cell treatment with different concentrations of shikonin for 6h. By contrast, treatment with shikonin for 24h significantly induced apoptosis, as evidenced by the results of TUNEL assay and flow cytometry analysis in proportion to the concentration. Disruption of the mitochondrial membrane potential was observed in gastric cancer cells that were treated with shikonin for 6 and 24h. Pretreatment with necrostatin-1 recovered cell death and mitochondrial membrane potential in the 6h shikonin treatment, but not in the 24h shikonin treatment. Western blot results reveal enhanced p38 phosphorylation, downregulated AKT phosphorylation, and increased caspase3 and PARP cleavage in cells that were treated with shikonin for 24h, but not in cells treated for 6h. Shikonin also triggered reactive oxygen species (ROS) generation both in the 6 and 24h treatments. Pretreatment with N-acetylcysteine blocked shikonin-induced cell death. In summary, our findings suggest that shikonin, which may function as a promising agent in the treatment of gastric cancers, sequentially triggered necrosis or apoptosis through ROS generation in gastric cancer cells.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Flow Cytometry; Free Radical Scavengers; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Necrosis; Phosphorylation; Reactive Oxygen Species; Stomach Neoplasms; Time Factors

The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Proliferation; DNA, Neoplasm; Electrophoresis, Agar Gel; Fluorescence; HeLa Cells; Humans; Juglans; Naphthoquinones; Necrosis; Plant Extracts; Silver Staining; Staining and Labeling; Telomerase

β-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated β-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. β-Lapachone markedly induced cell death without caspase activation. β-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited β-lapachone-induced cell death and HMGB-1 release. In addition, β-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked β-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that β-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, β-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to β-lapachone. Taken together, our results demonstrate that β-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.

Topics: Antineoplastic Agents; Apoptosis Inducing Factor; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; HMGB1 Protein; Humans; Liver Neoplasms; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA Interference; RNA-Binding Proteins; Signal Transduction; Time Factors; Transfection

Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 and Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy.

Topics: Aminophenols; Aniline Compounds; Antineoplastic Agents; Ascorbic Acid; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Drug Synergism; Humans; Imidazoles; MAP Kinase Signaling System; Naphthoquinones; Necrosis; Oxidation-Reduction; Phenotype; Pyridines; Reactive Oxygen Species; Urinary Bladder Neoplasms

Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1μM after 6h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10μM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1μM SHK-induced apoptosis and converted 10μM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1μM and 85 genes by 10μM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1μM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10μM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Death; Drugs, Chinese Herbal; Gene Expression; Gene Expression Profiling; Glutathione; Humans; Naphthoquinones; Necrosis; Oxidative Stress; U937 Cells

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.. Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.. Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.. We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Glioma; Humans; Medicine, Chinese Traditional; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Rats; Reactive Oxygen Species; RNA-Binding Proteins

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of β-lapachone in 2 minutes. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. β-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs.

Topics: Breast Neoplasms; Catalase; DNA Breaks, Single-Stranded; DNA Damage; Female; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Necrosis; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species

Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.. MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.. The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).. Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Naphthoquinones; Necrosis; Neoplasm Transplantation; Nuclear Pore Complex Proteins; Osteosarcoma; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Calcium; Cell Line, Tumor; DNA Damage; Egtazic Acid; Humans; NAD; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Necrosis; Neoplasms; Oxidation-Reduction; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Quinones; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

(+)-Cordiaquinone J is a 1,4-naphthoquinone isolated from the roots of Cordia leucocephala that has antifungal and larvicidal effects. However, the cytotoxic effects of (+)-cordiaquinone J have never being explored. In the present study, the effect of (+)-cordiaquinone J on tumor cells viability was investigated, showing IC(50) values in the range of 2.7-6.6muM in HL-60 and SF-295 cells, respectively. Studies performed in HL-60 leukemia cells indicated that (+)-cordiaquinone J (1.5 and 3.0muM) reduces cell viability and 5-bromo-2-deoxyuridine incorporation after 24h of incubation. (+)-Cordiaquinone J showed rapid induction of apoptosis, as indicated by phosphatidylserine externalization, caspase activation, DNA fragmentation, morphologic changes, and rapid induction of necrosis, as indicated by the loss of membrane integrity and morphologic changes. (+)-Cordiaquinone J altered the redox potential of cells by inducing the depletion of reduced GSH intracellular content, the generation of reactive oxygen species and the loss of mitochondrial membrane potential. However, pre-treatment of cells with N-acetyl-l-cysteine abolished most of the observed effects related to (+)-cordiaquinone J treatment, including those involving apoptosis and necrosis induction.

Topics: Apoptosis; Caspase 3; Caspase 7; Cell Line, Tumor; Cordia; Glutathione; HL-60 Cells; Humans; Leukemia; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Necrosis; Oxidative Stress; Plant Roots; Reactive Oxygen Species

Mammalian thioredoxin reductase (TrxR), a ubiquitous selenocysteine containing oxidoreductase, catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx). TrxR has been suggested as a potential target for anticancer drugs development for its overexpression in human tumors and its diverse functions in intracellular redox control, cell growth and apoptosis. Mansonone F (MF) compounds have been shown to exhibit antiproliferative effects, but their complex mechanisms are unknown. In the present study, we have investigated the effects of some synthesized MF analogues on TrxR and HeLa cells. The studies of the mode of inhibition and the interactions of IG3, one of the most potent MF analogues, with TrxR showed MF compounds could be partly reduced by the C-terminal selenolthiol active site, and possibly by the N-terminal dithiol motif and/or FAD domain of TrxR simultaneously, accompanied by redox cycling with the generation of superoxide anion radicals. In addition, MF analogues exhibited the potential to inhibit the growth of HeLa cells and reduce TrxR activity in cell lysates. The cell cycle was arrested in G2/M phase and apoptosis was induced in a dose-dependent manner. Furthermore, our results showed that IG3-treated HeLa cells induced the change of intracellular ROS. Taken together, the reported results here suggest that inhibition of TrxR by MF analogues provides a possible complex mechanism for explaining the anticancer activity of MF compounds.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Flow Cytometry; HeLa Cells; Humans; Intracellular Space; Kinetics; NADPH Oxidases; Naphthoquinones; Necrosis; Reactive Oxygen Species; Sesquiterpenes; Superoxides; Thioredoxin-Disulfide Reductase

Quinones have diverse pharmacological properties including antibacterial, antifungal, antiviral, anti-inflammatory, antipyretic and anticancer activity. The cytotoxic potential of 1,4-naphthoquinone (NQ14) was studied against B16F1 melanoma cells grown in vitro. NQ14 treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay and lactate dehydrogenase leakage assay. Depletion in cellular glutathione levels after 1h incubation with NQ14 correlated with the corresponding increase in reactive oxygen species generation as determined by 2',7'-dicholorofluorescein diacetate assay suggests the role of oxidative stress in cell death. The frequency of micronucleated binucleate cells increased with increasing doses of NQ14 with a corresponding decrease in the cytokinesis block proliferation index indicating the drug induced genotoxicity and cell division delay. Further, a dose-dependent decrease in the clonogenic cell survival indicated the potential of NQ14 to inhibit cell proliferation contributing to cell death. The cell death after NQ14 treatment may be attributed to apoptosis as seen in DNA ladder pattern along with necrosis as indicated in flow cytometric analysis of Annexin V/PI stained cells. Results of the present study demonstrate the cytotoxic and genotoxic potential of NQ14 by the induction of oxidative stress mediated mechanisms leading to tumor cell kill.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Formazans; Glutathione; L-Lactate Dehydrogenase; Melanoma; Mice; Micronucleus Tests; Naphthoquinones; Necrosis; Oxidative Stress; Reactive Oxygen Species; Tetrazolium Salts

Degterev et al. previously demonstrated that death receptor mediated apoptosis could be diverted to necroptosis when apoptosis signaling was blocked, suggesting that necroptosis may function as a backup mechanism to insure the elimination of damaged cells under certain conditions when apoptosis was inhibited. Here, we show that shikonin-induced necroptosis can be reverted to apoptosis in the presence of necrostatin-1 (Nec-1), a specific necroptosis inhibitor and that the death mode switch is at least partially due to the conversion from mitochondrial inner membrane permeability to mitochondrial outer membrane permeability, which is associated with Bax translocation. The data combined with the previous reports support a notion that apoptosis and necroptosis may function as reciprocal backup mechanisms of cellular demise. To the best of our knowledge, this is the first study to document a conversion from necroptosis to apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Imidazoles; Indoles; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Naphthoquinones; Necrosis; Permeability

Utilizing primary rat hepatocytes we investigated the potential antimutagenic and anti-cytotoxic effects of carboxymethyl chitin-glucan (CM-CG) with respect to oxidative stress induced by the model free-radical-generating compounds hydrogen peroxide (H2O2) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Different kinds of CM-CG action were studied by two different treatment protocols: a. pre-incubation of freshly isolated hepatocytes with the potential anti-mutagen followed by exposure to the oxidant or b. simultaneous treatment of hepatocytes with the potential anti-mutagen and the oxidant.. As a measure of genotoxicity, the percentages of DNA in tails of comets by single cell gel electrophoresis were evaluated. The cytotoxicological endpoints analysed were the cell density (number of cells/cm2), and the percentages of apoptotic and necrotic cells.. H2O2 and DMNQ, causing DNA single-strand breaks via the formation of *OH radicals, have been demostrated to induce both genotoxic and cytotoxic effects in primary rat hepatocytes resulting in increased percentages of DNA in tails of comets, and increased frequencies of apoptotic and necrotic cells accompanied by a decreased cell density. Further investigations were therefore focussed on possible modifications of these parameters by CM-CG. The results obtained clearly demonstrate that CM-CG (applied before and during treatment) protects primary rat hepatocytes against the genotoxic and cytotoxic effects of oxidative stress (H2O2 or DMNQ), whereas CM-CG itself has no effect on the endpoints of genotoxicity and cytotoxicity studied.. Our results indicate that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that can inhibit the genotoxicity and cytotoxicity of experimentally induced oxidative stress in primary rat hepatocytes.

Topics: Animals; Antioxidants; Apoptosis; Cell Count; Cell Death; Cell Separation; Chitin; Comet Assay; DNA; DNA Damage; Female; Glucans; Hepatocytes; Hydrogen Peroxide; Mutagenicity Tests; Mutagens; Naphthoquinones; Necrosis; Oxidants; Protective Agents; Rats; Rats, Inbred F344

Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cell Death; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Mice; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While beta-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by beta-lapachone in a variety of cancer cells. We found that beta-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to beta-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked beta-lapachone-induced necrosis. Furthermore, necrotic cell death induced by beta-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy to eliminate cancer cells.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Damage; Female; HeLa Cells; Humans; Mice; Mice, Knockout; Naphthoquinones; Necrosis; Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Signal Transduction; Xenograft Model Antitumor Assays

Evidence is accumulating that capillary endothelial cells (cEC) and astrocytes play a pivotal role in neuroprotection, in particular with respect to counteract oxidative injury. Furthermore, differences among both cell types in response to oxidative stress have been shown and astrocytes seem to be more tolerant in terms of cytotoxicity, however, no reports exist on oxidative stress mediated genotoxicity in astrocytes. We investigated genotoxic and cytotoxic effects of oxidative stress in astrocytes and cECs induced by hypoxia/reoxygenation or by the redox cycling quinone DMNQ. Additionally, the dependence of these effects on glucose availabilty was also studied. On exposure to Hy/Re or 10 muM DMNQ for 24 hr, the frequency of micronucleated and apoptotic cells was significantly increasing, however, astrocytes proved to be more resistant to apoptosis induction, in particular on use of DMNQ. In astrocytes, the low background rates of necrotic cells were not affected and a significant necrosis induction was only detectable in cECs exposed to DMNQ for 24 hr. Short-term exposure to DMNQ (1 hr) had no effect in astrocytes but exerted significant geno- and cytotoxicity in cECs. Increasing the glucose concentration markedly reduced oxidative stress mediated geno- and cytotoxicity in astrocytes. Surprisingly, glucose deprivation (aglycemia) suppressed DMNQ induced micronucleus formation in astrocytes without affecting the frequency of apoptotic cells. Our results indicate that astrocytes are more resistant to oxidative stress than cECs, in particular regarding the potential to counteract genotoxicity as well as apoptosis induction mediated by a short term oxidative insult.

Topics: Analysis of Variance; Animals; Animals, Newborn; Apoptosis; Astrocytes; Cell Death; Cells, Cultured; Cerebral Cortex; Endothelial Cells; Glucose; Mice; Naphthoquinones; Necrosis; Oxidative Stress; Time Factors

Juglone (5-hydroxy-1,4-naphtoquinone) is a natural toxin produced by walnut trees. In this study we show that juglone differentially reduces viability of human cells in culture. Normal fibroblast were found to be especially sensitive to juglone and lost viability primarily through a rapid apoptotic and necrotic response. This response may have been triggered by DNA damage since juglone induced a rapid and strong phosphorylation of H2AX in all phases of the cell cycle. Furthermore, juglone inhibits mRNA synthesis in human fibroblasts in a dose-dependent manner. Surprisingly, juglone caused a drastic reduction of the basal level of p53 in human fibroblasts and this loss could not be fully rescued by proteasome and calpain I inhibitors. However, when cells were pretreated with UV light or ionizing radiation, juglone was not able to reduce the cellular levels of activated p53. Our results show that juglone has multiple effects on cells such as the induction of DNA damage, inhibition of transcription, reduction of p53 protein levels and the induction of cell death.

Topics: Apoptosis; Blotting, Western; Calpain; Cell Death; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytotoxins; Fibroblasts; Flow Cytometry; Histones; Humans; Naphthoquinones; Necrosis; Phosphorylation; Proteasome Endopeptidase Complex; RNA, Messenger; Tumor Suppressor Protein p53; Ultraviolet Rays

The mechanisms of the antitumor reactions of 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) to human lung adenocarcinoma A549 cells were investigated. A549 cells that received 1.25 microg/ml FNQ3 (IC(50) at 0.35 microg/ml) developed intensive mitochondrial H(2)O(2) production at 1 h. Selective structural mitochondrial swelling, alteration of mitochondrial membrane potential, and cytochrome c and caspase-9 release from the mitochondria occurred 18-24 h later. alpha-Tocopherol inhibited the alteration of both mitochondrial permeability and the leakage of procaspase-9. The caspase-9 was then activated in the cytosol. The expression of Bcl-2 oncoprotein was suppressed by FNQ3, and resulted in apoptosis. The higher dose of 5 microg/ml induced necrosis via severe mitochondrial breakage. These results showed that FNQ3 targets the mitochondria of A549 cells to produce a reactive oxygen species resulting in apoptosis and necrosis.

Topics: Adenocarcinoma; alpha-Tocopherol; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Blotting, Western; Caspase 9; Caspases; Cytochrome c Group; Cytoplasm; DNA Fragmentation; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Genes, bcl-2; Humans; Hydrogen Peroxide; Lung Neoplasms; Membrane Potentials; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Naphthoquinones; Necrosis; Permeability; Tumor Cells, Cultured

A wide spectrum of anti-cancer activity of genistein and beta-lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and beta-lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3-[4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment-induced growth inhibition and cytotoxicity and, annexin V-fluoresceine (FI) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-propidium iodide (PI) assays to determine potential treatment-induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and beta-lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment-induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with beta-lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or beta-lapachone alone. beta-lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2-M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether beta-lapachone-genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Apoptosis; Cell Division; Drug Synergism; Drug Therapy, Combination; Estrogens, Non-Steroidal; Humans; Isoflavones; Male; Naphthoquinones; Necrosis; Prostatic Neoplasms; Tumor Cells, Cultured

The enzyme DT-diaphorase mediates the two-electron reduction of quinones to hydroquinones. It has previously been shown that the toxicity of 2-methyl-1,4-naphthoquinone to rats is decreased by pre-treatment of the animals with compounds that increase tissue levels of this enzyme. In contrast, the severity of the haemolytic anaemia induced in rats by 2-hydroxy-1,4-naphthoquinone was increased in animals with high levels of DT-diaphorase. In the present experiments, the effect of alterations in tissue diaphorase activities on the toxicity of a third naphthoquinone derivative, 2,3-dimethyl-1,4-naphthoquinone, has been investigated. This compound induced severe haemolysis and slight renal tubular necrosis in control rats. Pre-treatment of the animals with BHA, a potent inducer of DT-diaphorase, diminished the severity of the haemolysis induced by this compound and abolished its nephrotoxicity. Pre-treatment with dicoumarol, an inhibitor of this enzyme, caused only a slight increase in the haemolysis induced by 2,3-dimethyl-1,4-naphthoquinone, but provoked a massive increase in its nephrotoxicity. Modulation of DT-diaphorase activity in animals may therefore not only alter the severity of naphthoquinone toxicity, but also cause pronounced changes in the site of toxic action of these substances. The factors that may control whether induction of DT-diaphorase in animals will decrease or increase naphthoquinone toxicity are discussed.

Topics: Administration, Oral; Animals; Butylated Hydroxyanisole; Dicumarol; Female; Hemolysis; Kidney Tubules; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Necrosis; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

2-Methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) has been reported to be more cytotoxic to human malignant tumor cell lines than are the corresponding normal epithelial cells. Therefore, we examined the dose response of FNQ3 against human cervical cancer HeLa cells in culture. When 1.25 mg/ml FNQ3 was applied, apoptosis was induced, as determined by an immunohistochemical staining of fragmented genome DNA and cell profiles. Significant inhibition of Bcl-2 oncogene protein expression by the same concentration of FNQ3 also was demonstrated by an immunohistochemical staining method to visualize the expressed cells and Western blot in polyacrylamide gel electrophoresis. Flow-cytometric spectra showed S-phase arrest in cell cycles and the appearance of sub-G1 phase consistent with apoptosis. On the other hand, concentrations of 5 microg/ml or more of FNQ3 induced necrosis. These results show that FNQ3 may act as an antitumor agent to induce apoptosis by affecting Bcl-2 expression and cell cycles, or necrosis as the result of primary mitochondrial injuries.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Cycle; Cervix Uteri; DNA; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Female; Flow Cytometry; HeLa Cells; Humans; Immunohistochemistry; Mitochondria; Naphthoquinones; Necrosis; Proto-Oncogene Proteins c-bcl-2; S Phase; Time Factors; Uterine Cervical Neoplasms

There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C.. We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2.. beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine.. beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma; Caspase 3; Caspases; Cell Death; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme Activation; Female; Humans; Male; Membrane Potentials; Mitochondria; Naphthoquinones; Necrosis; Tumor Cells, Cultured

Increasing concentrations (1-100 microM) of the redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), stimulated growth, triggered apoptosis, or caused necrosis of pancreatic RINm5F cells, depending on the dose and duration of the exposure. Following the exposure of RINm5F cells to 10 microM DMNQ, ornithine decarboxylase activity and polyamine biosynthesis increased. This was accompanied by enhanced cell proliferation. Conversely, exposure to 30 microM DMNQ for 3 h resulted in the inhibition of ornithine decarboxylase, intracellular polyamine depletion, and apoptotic cell killing. Pretreatment of the cultures with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, restored polyamine levels and prevented apoptosis. Exposure to the same DMNQ concentration for only 1 h, with subsequent re-incubation in growth medium, neither caused polyamine depletion nor resulted in apoptosis. Finally, exposure to an even higher DMNQ concentration (100 microM) for either 1 or 3 h caused rapid intracellular Ca2+ overload, ATP, NAD+, and glutathione depletion, and extensive DNA single strand breakage, which resulted in necrotic cell death. Our results show that a disturbance of polyamine biosynthesis occurred prior to cell growth or apoptosis elicited by oxidative stress. In addition, we show that effects as opposite as cell proliferation and deletion, by either apoptosis or necrosis, can be induced, in the same system, by varying the exposure to a prooxidant.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenosylmethionine Decarboxylase; Animals; Apoptosis; Cell Division; Cell Line; Cell Survival; Deoxyguanosine; Dose-Response Relationship, Drug; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Naphthoquinones; Necrosis; Oxidants; Polyamines; Tetradecanoylphorbol Acetate; Time Factors

HPLC analysis revealed that luteoskyrin administered orally to male mice accumulated selectively in the liver, with minor distribution to the serum and kidneys. Elevation of serum GOT and GPT values was maximal 3 days after administration. In mice administered this mycotoxin intravenously, selective accumulation was also observed in the liver, and the half-life of hepatic luteoskyrin in males was significantly longer than that in females. Increment of serum transaminases was also marked in males with maximum accumulation at 24 h after administration. Histopathologically, cellular membrane damage was an early effect of luteoskyrin on cell necrosis, and these morphological changes were also marked in males. Luteoskyrin also elevated hepatic lipid peroxides, the maximum elevation being 8 h after injection; this increase was suppressed by alpha-tocopherol and Bi(NO3)3. HPLC-ECD analysis indicated that the level of 8-hydroxy-deoxyguanosine, one of the markers of hydroxy-radical-mediated modification of DNA guanine residues, was increased in hepatic DNA. These findings indicate that luteoskyrin has a high affinity for the liver, resulting in induction of lipid peroxidation, hepatocellular membrane damage, and elevation of serum transaminase activities. It is suggested that the hydroxy radicals derived from this anthraquinone contribute to these toxicological changes.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alanine Transaminase; Animals; Aspartate Aminotransferases; Chromatography, High Pressure Liquid; Deoxyguanosine; Free Radical Scavengers; Hydroxylation; Lipid Peroxides; Liver; Male; Mice; Naphthoquinones; Necrosis; Time Factors

Topics: Animals; Body Weight; Carcinogens; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Chlorine; Diet; Dose-Response Relationship, Drug; Female; Lethal Dose 50; Liver; Liver Cirrhosis; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred Strains; Mycotoxins; Naphthoquinones; Necrosis; Oryza; Penicillium; Peptides, Cyclic; Sex Factors