phenanthrenes and Liver-Neoplasms

Pancreatic cancer is the 10th leading cause of all new cancer cases for men and the fourth leading cause of death across genders, having very poor prognosis and survival rates. The current standard of care Gemcitabine fails to add any survival benefit for this disease (www.cancer.gov). Though the incidence of pancreatic cancer is found to be higher in developed countries, the aggressive biology of the cancer, its high rate of recurrence and chemo-resistance make it a formidable disease in all parts of the globe. Hepatocellular carcinoma (HCC) or liver cancer, on the other hand affects almost 750,000 people world wide with 84% of the cases coming from underdeveloped or developing countries. Our studies show that Minnelide, a water soluble pro-drug of triptolide (active compound from a chinese herb) is very effective against a number of malignant diseases.. The current study discusses the efficacy of this compound in pancreatic and liver cancer.

Topics: Animals; Antineoplastic Agents, Alkylating; Diterpenes; Epoxy Compounds; Humans; Liver Neoplasms; Organophosphates; Pancreatic Neoplasms; Phenanthrenes; Prodrugs

Topics: Animals; Antineoplastic Agents; Arginase; Benz(a)Anthracenes; Benzopyrenes; Carcinogens; Cell Division; Cell Transformation, Neoplastic; Deoxyribonucleases; DNA; Fluorouracil; Humans; Liver Neoplasms; Methylcholanthrene; Mice; Mutation; Neoplasms; Neoplasms, Experimental; Nucleosides; Oncogenic Viruses; Orotic Acid; Phenanthrenes; Protein Binding; Pyrimidinones; Rats; Skin Neoplasms; Thymine; Uracil

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of aflatoxin-contaminated foods, and are exposed to high levels of phenanthrene, a sentinel of hydrocarbon air toxics. Cruciferous vegetables, such as broccoli, contain anticarcinogens. Glucoraphanin, the principal glucosinolate in broccoli sprouts, can be hydrolyzed by gut microflora to sulforaphane, a potent inducer of carcinogen detoxication enzymes. In a randomized, placebo-controlled chemoprevention trial, we tested whether drinking hot water infusions of 3-day-old broccoli sprouts, containing defined concentrations of glucosinolates, could alter the disposition of aflatoxin and phenanthrene. Two hundred healthy adults drank infusions containing either 400 or < 3 micromol glucoraphanin nightly for 2 weeks. Adherence to the study protocol was outstanding; no problems with safety or tolerance were noted. Urinary levels of aflatoxin-N(7)-guanine were not different between the two intervention arms (P = 0.68). However, measurement of urinary levels of dithiocarbamates (sulforaphane metabolites) indicated striking interindividual differences in bioavailability. An inverse association was observed for excretion of dithiocarbamates and aflatoxin-DNA adducts (P = 0.002; R = 0.31) in individuals receiving broccoli sprout glucosinolates. Moreover, trans, anti-phenanthrene tetraol, a metabolite of the combustion product phenanthrene, was detected in urine of all participants and showed a robust inverse association with dithiocarbamate levels (P = 0.0001; R = 0.39), although again no overall difference between intervention arms was observed (P = 0.29). Understanding factors influencing glucosinolate hydrolysis and bioavailability will be required for optimal use of broccoli sprouts in human interventions.

Topics: Adult; Aflatoxins; Aged; Anticarcinogenic Agents; Beverages; Biological Availability; Brassica; Carcinoma, Hepatocellular; DNA Adducts; Female; Glucosinolates; Humans; Hydrolysis; Liver Neoplasms; Male; Middle Aged; Phenanthrenes; Placebos

Tripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown.. To identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS.. In vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal.. Flow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2K. Interferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 μg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury.. The TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects.

Topics: Animals; Carcinoma, Hepatocellular; Diterpenes; Histocompatibility Antigens Class I; Killer Cells, Natural; Lipopolysaccharides; Liver Neoplasms; Mice; Mice, Inbred C57BL; Phenanthrenes

Hepatocellular carcinoma (HCC) is an often-fatal malignant tumor with high lethality. Despite advances and significant efficacy in monotherapy, cancer therapy continues to pose several challenges. Novel combination regimens are an emerging strategy for anti-HCC and have demonstrated to be effective. Here, we propose a potential combination for HCC treatment named arsenic trioxide cooperate cryptotanshinone (ACCS). A remarkable synergistic therapeutic effect has been achieved compared with drugs alone in both in vivo and in vitro experiments. Mechanism study indicated that ACCS exerts its therapeutic actions by regulating macrophage-related immunity and glycolysis. ACCS potentiates the polarization of M1 macrophages and elevates the proportion of M1/M2 to remodel tumor immunity. Further molecular mechanism study revealed that ACCS intensifies the glucose utilization and glycolysis in the macrophage by increasing the phosphorylation of AMPK to activating the AMPK singling pathway. In conclusion, ACCS is a highly potential combination regimen for HCC treatment. The therapeutic potential of ACCS as a candidate option for anticancer drugs in restoring the balance of immunity and metabolism deserves further investigation.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Carcinoma, Hepatocellular; Cell Differentiation; Cytokines; Drug Combinations; Drug Synergism; Drugs, Chinese Herbal; Glycolysis; Humans; Immunity, Innate; Immunomodulation; Liver Neoplasms; Macrophage Activation; Macrophages; Mice; Mice, Inbred BALB C; Phenanthrenes; Th1 Cells

Hepatocellular carcinoma (HCC) is resistant to current immunotherapy. This poor outcome mainly results from the immunosuppressive characteristics of tumor microenvironment (TME). Accumulating evidence indicates that some chemotherapy agents trigger immunogenic cell death (ICD), providing a promising strategy to remodel the immunosuppressive TME. The role of Plumbagin (PLB, a naphthoquinone compound from Plumbago zeylanica L.) as the ICD inducer for HCC cells was confirmed in this study. Dihydrotanshinone I (DIH, a phenanthraquinone compound of Salvia miltiorrhiza) functioned as the ICD enhancer by generating the reactive oxygen species (ROS). A poly(D,L-lactic-co-glycolic acid) (PLGA)-based nanoparticle (NP) was used to co-encapsulate PLB, DIH and NH

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Furans; Lactic Acid; Liver Neoplasms; Mice; Nanoparticles; Naphthoquinones; Phenanthrenes; Quinones; Tumor Microenvironment

Triptolide (TPL) has been employed to treat hepatocellular carcinoma (HCC). However, the poor water solubility of TPL restricts its applications. Therefore, we prepared TPL-loaded cyclodextrin-based metal-organic framework (TPL@CD-MOF) to improve the solubility and bioavailability of TPL, thus enhancing the anti-tumor effect on HCC. The BET surface and the pore size of TPL@CD-MOF were 10.4 m

Topics: Carcinoma, Hepatocellular; Diterpenes; Drug Carriers; Epoxy Compounds; Excipients; gamma-Cyclodextrins; Humans; Liver Neoplasms; Metal-Organic Frameworks; Phenanthrenes

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemistry, Pharmaceutical; Diterpenes; Drug Carriers; Epoxy Compounds; Humans; Liposomes; Liver Neoplasms; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Particle Size; Phenanthrenes; Random Allocation; Signal Transduction; Surface Properties

In addition to early detection, early diagnosis, and early surgery, it is of great significance to use new strategies for the treatment of hepatocellular carcinoma (HCC). Studies showed that the combination of sorafenib (SFN) and triptolide (TPL) could reduce the clinical dose of SFN and maintain good anti-HCC effect. But the solubility of SFN and TPL in water is low and both drugs have certain toxicity. Therefore, we constructed a biomimetic nanosystem based on cancer cell-platelet (PLT) hybrid membrane camouflage to co-deliver SFN and TPL taking advantage of PLT membrane with long circulation functions and tumor cell membrane with homologous targeting. The biomimetic nanosystem, SFN and TPL loaded cancer cell-PLT hybrid membrane-camouflaged liquid crystalline lipid nanoparticles ((SFN + TPL)@CPLCNPs), could simultaneously load SFN and TPL at the molar ratio of SFN to TPL close to 10:1. (SFN + TPL)@CPLCNPs achieved long circulation function and tumor targeting at the same time, promoting tumor cell apoptosis, inhibiting tumor growth, and achieving a better "synergy and attenuation effect", which provided new ideas for the treatment of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomimetic Materials; Blood Platelets; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Diterpenes; Epoxy Compounds; Humans; Liposomes; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Phenanthrenes; RAW 264.7 Cells; Sorafenib

Cryptotanshinone (CPT) and dihydrotanshinone (DHT) are diterpenoid anthraquinone compounds extracted from traditional Chinese herbal medicine (TCM). Recent studies have shown that CPT regulates the signal transduction pathways via microRNA (miRNA) alterations. However, few studies have investigated the role of DHT in miRNA alterations affecting cell-signaling pathways. This study aimed to investigate the miRNA alterations and post-transcriptional regulation activities of DHT in comparison to CPT.. HepG2 and HT-29 cells were treated with DHT or CPT for 72 h. MiRNA, transcription factor encoding mRNA, and downstream gene expression were determined using real-time quantitative PCR. Protein expression was analyzed using western blotting.. The results revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via miR-15a-5p, miR-27a-5p, miR-100-5p, and miR-200a-5p alterations.In silico target predictions showed that downregulation of epidermal growth factor receptor (EGFR) mRNA expression by DHT might also suppress the expression of STAT family proteins and lead to anti-proliferation effects. We also found that, compared to CPT, DHT might possess higher potency in cell growth regulation via multi-miRNA and transcription factor alterations.. This study revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via alterations in miRNAs and transcription factors. In addition, the findings of this study suggest that DHT is more potent than CPT in cancer chemopreventive activities. Therefore, DHT at a low dose is a TCM compound with less toxic side effects and may contribute to the development of natural medicine as a potential cancer chemopreventive agent.

Topics: Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Proteins; Cell Proliferation; Colonic Neoplasms; Furans; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Hep G2 Cells; HT29 Cells; Humans; Liver Neoplasms; MicroRNAs; Phenanthrenes; Quinones; Signal Transduction; Transcription Factors; Transcriptome

To achieve improved drug delivery efficiency to hepatocellular carcinoma (HCC), biodegradable poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP), surface-modified with SP94 peptide, were designed for the efficient delivery of cryptotanshinone to the tumor for the treatment of HCC. Cryptotanshinone NP and SP94-NP were prepared by using nanoprecipitation. The physicochemical and pharmaceutical properties of the NP and SP94-NP were characterized, and the release kinetics suggested that both NP and SP94-NP provided continuous, slow release of cryptotanshinone for 48 h. The in vitro cellular experiment demonstrated that SP94-NP significantly enhanced the cellular uptake of cryptotanshinone and induced high cytotoxicity and cellular apoptosis of hepatocellular carcinoma (HepG2) cells. The in vivo detecting results of targeting effect using the Cy5.5 probe evidenced that SP94-NP showed an accumulation in tumor more efficiently than that of unconjugated ones. Meanwhile, SP94-NP exhibited the smallest tumor size than other groups and showed no toxicity to body. The results of this study provide a promising nanoplatform for the targeting of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Survival; Dose-Response Relationship, Drug; Drug Delivery Systems; Drugs, Chinese Herbal; Female; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Peptide Fragments; Phenanthrenes; Polyesters; Polyethylene Glycols

As a key glycolysis enzyme, the significance of pyruvate dehydrogenase kinase 1 (PDK1) in the development of colorectal cancer (CRC) remains unknown. This study revealed that the prognosis of CRC patients with high levels of PDK1 was poor, and PDK1 knockdown significantly reduced liver metastasis of CRC in both nude mice and immune competent BALB/C mice. When combined with cryptotanshinone (CPT), an inhibitor of STAT3-p-Y705, the liver metastasis was further inhibited. PDK1 knockdown obviously increased reactive oxygen species level in anoikis conditions and subsequently resulted in an elevated anoikis, but the combination of PDK1 knockdown and CPT showed a reduced effect on anoikis. Based on this discrepancy, the adherence ability of CRC cells to matrix protein fibronectin was further detected. It showed that PDK1 knockdown significantly decreased the adherence of CRC cells to fibronectin when combined with CPT. These results suggest that inhibition of PDK1 can decrease the surviving CRC cells in blood circulation via up-regulation of anoikis, and inhibition of STAT3-p-Y705 can prevent it to settle down on the liver premetastatic niche, which ultimately reduces liver metastasis.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phenanthrenes; Phosphorylation; Prognosis; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; STAT3 Transcription Factor; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Current therapies present significant limitations. Triptolide (TP) is highly effective against multiple cancers including HCC. However, high toxicity, low water solubility, and unknown therapeutic targets limit its clinical application. Herein, we designed galactosylated-chitosan-TP-nanoparticles (GC-TP-NPs) with high drug loading capacities for targeted delivery to HCC. In addition to a sustained release pattern, an efficient asialoglycoprotein receptor mediated cellular uptake in vitro, and high liver tumor accumulation in vivo, GC-TP-NPs showed lower systemic and male reproductive toxicities than free TP. Importantly, GC-TP-NPs retained the anti-cancer activities of the free TP, exerting the same pro-apoptotic and anti-proliferative effects on HCC cells in vitro, and displayed higher efficacies in reducing tumor sizes in vivo. Further investigation revealed that GC-TP-NPs induced cancer cell apoptosis via blocking TNF/NF-κB/BCL2 signaling. Collectively, GC-TP-NP represents a promising candidate in halting liver cancer progression while minimizing systemic toxicity.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Chitosan; Diterpenes; Epoxy Compounds; Galactose; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Nanoparticles; Phenanthrenes; Reproduction; Signal Transduction; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Two series of boehmeriasin A analogs have been synthesized in short and high yielding processes providing derivatives differing either in the alkaloid's pentacyclic scaffold or its peripheral substitution pattern. These series have enabled, for the first time, comparative studies into key biological properties revealing a new lead compound with exceptionally high activity against liver cancer cell lines in the picomolar range for both well (Huh7, Hep3B and HepG2) and poorly (Mahlavu, FOCUS and SNU475) differentiated cells. The cell death was characterized as apoptosis by cytochrome-C release, PARP protein cleavage and SubG1 cell cycle arrest. Subsequent testing associated apoptosis via oxidative stress with in situ formation of reactive oxygen species (ROS) and altered phospho-protein levels. Compound 19 decreased Akt protein phosphorylation which is crucially involved in liver cancer tumorigenesis. Given its simple synthetic accessibility and intriguing biological properties this new lead compound could address unmet challenges within liver cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Chemistry Techniques, Synthetic; Humans; Liver Neoplasms; Phenanthrenes; Quinolizidines

Cryptotanshinone (CT), a purified compound initially isolated from the dried roots of Salvia militorrhiza. Bunge, exhibits cytotoxic antitumor effects on many tumors. We have shown that CT possesses the dual capacities to concomitantly inhibit the proliferation of lung cancer cells and promote the generation of antitumor immunity. In this study, we investigated whether CT could be used to treat hepatocellular carcinoma (HCC) using a mouse Hepa1-6 model. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells in vitro by inducing Hepa1-6 cells apoptosis through the JAK2/STAT3 signaling pathway. In addition, CT activated macrophages and polarized mouse bone marrow-derived macrophages (BMM) toward an M1 phenotype in vitro, which depended on the TLR7/MyD88/NF-κB signaling pathway. Furthermore, CT significantly inhibited the growth of syngeneic Hepa1-6 hepatoma tumors, and, in combination with anti-PD-L1 cured Hepa1-6-bearing mice with the induction of long-term anti-Hepa1-6 specific immunity. Immunoprofiling of treated Hepa1-6-bearing mice revealed that CT-promoted activation of tumor-infiltrating macrophages and dendritic cells, induction of antitumor T cell response, and infiltration of effector/memory CD8 T cells in the tumor tissue. Importantly, the immunotherapeutic effects of CT and anti-PD-L1 depended on the presence of CD8 T cells. Thus, CT and anti-PD-L1 may provide an effective immunotherapeutic regimen for human HCC based on a combination of cytotoxic effects and induction of tumor-specific immunity.

Topics: Adaptive Immunity; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B7-H1 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Liver Neoplasms; Lymphocyte Activation; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Phenanthrenes; Salvia miltiorrhiza; Signal Transduction; Toll-Like Receptor 7; Treatment Outcome

Triptolide is a structurally unique diterpene triepoxide with potent antitumor activity. However,the effect and mechanism of triptolide on hepatocellular carcinoma (HCC) is not well studied.. Cells were treated with triptolide, and the anti-HCC activity of triptolide was evaluated using flow cytometry, western blot, and xenograft studies. MicroRNA microarray and quantitative reverse-transcription polymerase chain reaction was used to identify differential microRNAs induced by triptolide. Chromatin immunoprecipitation assay was employed to study the interaction between c-Myc and genomic regions of miR106b-25. MicroRNAs overexpression and knockdown experiments were performed to determine the role of these microRNAs in triptolide-induced apoptosis.. Triptolide inhibited cell proliferation and induced marked apoptosis in multiple HCC cell lines with different p53 status. Several signaling molecules involved in different pathways were altered after the treatment of triptolide. Xenograft tumor volume was significantly reduced in triptolide-treated group compared with vehicle control group. Two miRNA clusters, miR-17-92 and miR-106b-25, were significantly suppressed by triptolide, which resulted in the upregulation of their common target genes, including BIM, PTEN, and p21. In HCC samples, high levels of these miRNA clusters correlated with shorter recurrence free survival. Triptolide inhibited the expression of theses miRNAs in a c-Myc-dependent manner, which enhanced triptolide-induced cell death. We further showed that triptolide down-regulated the expression of c-Myc through targeting ERCC3, a newly identified triptolide-binding protein.. The triptolide-induced modulation of c-Myc/miRNA clusters/target genes axis enhances its potent antitumor activity, which indicates that triptolide serves as an attractive chemotherapeutic agent against HCC.

Topics: Animals; Antineoplastic Agents, Alkylating; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Diterpenes; DNA Helicases; DNA-Binding Proteins; Epoxy Compounds; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; MicroRNAs; Oncogenes; Phenanthrenes; Proto-Oncogene Proteins c-myc; RNA Interference; Xenograft Model Antitumor Assays

Juncunol is a phenanthrene isolated from the halophyte species Juncus acutus, with selective cytotoxic activity towards human hepatocarcinoma (HepG2) cells. However, its mechanism of action is unknown.. The in vitro cytotoxic mechanism of juncunol was evaluated on HepG2 cells through several methods to elucidate its potential to induce apoptotic features, decrease mitochondrial membrane potential, promote internal ROS production and influence cell cycle. We also report its haemolytic activity on human erythrocytes and in silico DNA-binding studies.. Juncunol reduced HepG2 cells proliferation through the induction of apoptotic cellular death, in a concentration-dependent manner. Apoptosis induction seems to be related with a decrease of the mitochondrial membrane potential but not with ROS production. Juncunol had no haemolytic activity and may act as a DNA intercalator. Our data suggests juncunol as a suitable candidate for more detailed studies, including in vivo experiments, in order to completely characterize its mode of action.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Computer Simulation; Dose-Response Relationship, Drug; Hemolysis; Humans; In Vitro Techniques; Liver Neoplasms; Membrane Potential, Mitochondrial; Molecular Docking Simulation; Phenanthrenes; Reactive Oxygen Species

Human hepatocellular carcinoma (HCC) is currently the second most common cancer and one of the leading causes of cancer-related mortality in Taiwan. Previous reports show that the expression of (E-type prostaglandin 2) EP2 and (E-type prostaglandin 4) EP4 are elevated in HCC and further demonstrate that Prostaglandin E2 (PGE2) induces HA22T cell proliferation and metastasis through EP2 and EP4 receptor. Danshen (root of Salvia miltiorrhiza Bunge) is a very important and popular traditional Chinese herbal medicine which is widely and successfully used against breast cancer, leukemia, pancreatic cancer, and head and neck squamous carcinoma cells. In this study, we used Cryptotansinone (Dsh-003) (MW 269.14) from Danshen to investigate their effect and corresponding mechanism of action in PGE2-treated HA22T cells. Dsh-003 inhibited HA22T cell viability and further induced cell apoptosis in PGE2-treated HA22T cells. Furthermore, Dsh-003 inhibited EP2, EP4, and their downstream effector such as p-PI3K and p-Akt expression in HA22T hepatocellular carcinoma cells. We also observed that Dsh-003 blocked PGE2-induced cell migration by down-regulating PGE2-induced β-catenin expression and by up-regulating E-cadherin and GSK3-β expression. All these findings suggest that Dsh-003 inhibit human HCC cell lines and could potentially be used as a novel drug for HCC treatment.

Topics: Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cell Survival; Dinoprostone; Humans; Liver Neoplasms; Phenanthrenes; Salvia miltiorrhiza; Signal Transduction; Tumor Cells, Cultured

The present study investigated the effect of triptolide on viability and apoptosis along with underlying mechanism in liver cancer cells. CCK-8 assay showed that triptolide treatment for 48 h significantly reduced the viability of HepG2 and QSG7701 cells at 50 µM concentration. Annexin V-FITC and propidium iodide staining showed that triptolide treatment of HepG2 cells at 50 µM concentrations induced apoptosis in 56.45% cells compared to only 2.36% cells in the control cultures. Western blot assay showed that treatment of HepG2 cells with 50 µM concentration of triptolide significantly induced phosphorylation of p53 in a 2 h-treatment. Phosphorylation of histone H2A.X indicator of DNA damage was induced by triptolide treatment after 12 h in HepG2 cells. The level of nuclear p53 in a 6 h-treatment with 0, 10, 20, 30, 40 and 50 µM concentration of triptolide was found to be 15.3, 19.6, 28.5, 43.7, 63.8 and 91.5%, respectively. Treatment of HepG2 cells with triptolide at 50 µM concentration caused a significant increase in the binding potential of p53 to DNA. Triptolide treatment of HepG2 cells caused a significant increase in the expression of p21, Bax and DR5 genes in HepG2 cells. It also increased the expression of miR-34b and miR-34c in HepG2 cells markedly. Treatment of HepG2 cells with p53 inhibitor, pifithrin-α prior to incubation with triptolide significantly prevented induction of cell apoptosis. Suppression of p53 expression by siRNA inhibited the expression of p53 as well as its target genes along with the prevention of apoptosis induction. In conclusion, triptolide inhibits viability and induces apoptosis in liver cancer cells through activation of the tumor suppressor gene p53. Thus, triptolide can be used for the treatment of liver cancer.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diterpenes; DNA Damage; DNA-Binding Proteins; Enzyme Activation; Epoxy Compounds; Hep G2 Cells; Humans; Liver Neoplasms; Phenanthrenes; Phosphorylation; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Arsenic trioxide (ATO) is approved for treating terminal-stage liver cancer in China. Cryptotanshinone (CT), a STAT3 inhibitor, has exhibited certain anti-tumor potency; however, the use of CT enhanced ATO for treating liver cancer has not been reported. Here we try to elucidate how CT could enhance the efficacy of ATO for treating liver cancer and its correlation to STAT3 in vitro and in vivo.. First we evaluated cell vitality, and our data indicated that the ATO combined with CT showed obvious growth inhibition of Bel-7404 cells compared to ATO or CT alone. Next we found that ATO combined with CT induced cell apoptosis in Bel-7404 cells and upregulated the activation of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-poly(ADP-ribose) polymerase in a time-dependent manner. Next, we found that ATO combined with CT not only inhibited the constitutive levels of phosphorylated-JAK2 and phosphorylated-STAT3. Our study provides strong evidence that CT could enhance the efficacy of ATO in treating liver cancer both in vitro and in vivo. Downregulation of phosphorylated-STAT3 expression may play an important role in inducing apoptosis of Bel-7404 cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Cell Survival; Down-Regulation; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Oxides; Phenanthrenes; Phosphorylation; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

BACKGROUND We investigated whether the plant-derived agent triptolide (TPL) could effectively inhibit the growth and invasion of human hepatocellular carcinoma (HCC) cells. MATERIAL AND METHODS MHCC-97H cells were treated with various concentration of TPL for various times. To detect the effect of NF-κB on TPL-induced signal pathways, MHCC-97H cells were transfected with p65 siRNA or p65 cDNA, then treated with TPL. We detected cell survival and apoptosis by MTT, soft-agar colony formation assay, flow cytometry, and TUNEL assay. Cell migration and invasion was determined by Matrigel invasion and a wound-healing assay. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); MMP-9 activity was detected by ELISA. Western blot and real-time PCR (RT-PCR) assays were used to detect p65 and MMP-9 protein and mRNA expression. A subcutaneously implanted tumor model of MHCC-97H cells in nude mice was used to assess the effects of TPL on tumorigenesis in vivo. RESULTS We showed that TPL treatment significantly suppressed growth and induced apoptosis of MHCC-97H cells in a dose- and time-dependent manner in vitro. Furthermore, TPL treatment inhibited invasion in vitro and inhibited the growth and lung metastasis of MHCC-97H cells in vivo. NF-κB and MMP-9 were inactivated with TPL treatment. Overexpression of p65 restored MMP-9 activity and inhibited the TPL anti-tumor effect on MHCC-97H cells. Knockdown of p65 blocked MMP-9 activation and enhanced TPL-induced cell apoptosis and survival inhibition, and TPL inhibition of migration and invasion in vitro. CONCLUSIONS TPL treatment inhibited MHCC-97H cell growth, invasion, and metastasis in vitro and vivo, suggesting that TPL could be developed as a potential therapeutic agent for the treatment of HCC.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Diterpenes; Epoxy Compounds; Female; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; NF-kappa B; Phenanthrenes; Random Allocation; Signal Transduction; Xenograft Model Antitumor Assays

Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3'-untranslated region (3' UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3' UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1.

Topics: 3' Untranslated Regions; Allosteric Regulation; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin D1; HSC70 Heat-Shock Proteins; Humans; Indolizines; Liver Neoplasms; Phenanthrenes; Protein Binding; Protein Processing, Post-Translational; Tumor Cells, Cultured

To determine the combined cytotoxic effect and the molecular basis of triptolide and sodium cantharidinate on hepatoma cell line 7721.
. After treating the hepatoma cell line 7721 with triptolide(9, 18, or 36 μg/mL) and/or sodium cantharidinate (2, 5, or 10 μg/mL), cell viability assay and apoptosis were examined by MTT and flocytometry, respectively. The protein levels of caspase 3 and nuclear factor κB were analyzed by Western blot.
. Viability of hepatoma cell line 7721 was inhibited by either the therapy of triptolide and/or sodium cantharidinate (P<0.05) in a time- and dose-dependent manner. The combined effects of both drugs were better than those of the single drug (P<0.05). The combined therapy down-regulated the expression of NF-κB p65 (P<0.05) while up-regulated the expression of caspase-3 (P<0.05).
. Triptolide and sodium cantharidinate exert a synergistic toxic effect on hepatoma cell line 7721, which is related to increasing capase-3 activity and suppression of NF- κB.. 目的：探讨斑蝥酸钠协同雷公藤甲素对人肝癌7721细胞生长的影响及机制。方法：采用系列浓度的斑蝥酸钠与雷公藤甲素单独或联合作用于人肝癌7721细胞，应用MTT法检测它们对人肝癌7721细胞生长抑制率的影响，荧光倒置显微镜法观察细胞形态的变化，流式细胞术检测细胞凋亡率，Western印迹法分别测定各组用药对人肝癌7721细胞中NF-κB p65和caspase-3表达的影响。结果：斑蝥酸钠和雷公藤甲素单独用药均可抑制人肝癌7721细胞的生长(P<0.05)，且呈剂量和时间依赖性。两药联合作用对人肝癌7721细胞生长抑制率和凋亡率均大于单独用药(P<0.05) ，并且两药联用可以显著下调NF-κB p65表达(P<0.05)，显著上调caspase-3的表达(P<0.05)。结论：斑蝥酸钠和雷公藤甲素均可抑制人肝癌7721细胞的生长，通过影响凋亡相关蛋白的表达诱导细胞凋亡；斑蝥酸钠可协同雷公藤甲素上调caspase-3的表达并下调NF-κB p65表达，联合用药效应明显。.

Topics: Apoptosis; Cantharidin; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Diterpenes; Down-Regulation; Drug Therapy, Combination; Epoxy Compounds; Humans; Liver Neoplasms; NF-kappa B; Phenanthrenes; Transcription Factor RelA

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6- tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine IC10, IC20 and IC50 levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC6) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the IC50 levels of 26.0±4.45 and 51.3±2.05 μM, respectively. For murine normal fibroblast NIH3T3 cells, the IC50 concentration was 81.3±10.1 μM. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

Topics: 3T3 Cells; Alkaloids; Animals; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Female; Hep G2 Cells; Humans; Lactams; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice; Mitochondria; Phenanthrenes; Plant Extracts; Reactive Oxygen Species

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors and histone deacetylase (HDAC) inhibitors have recently emerged as promising anticancer drugs. The aim of this study was to investigate the effect of combination treatment with the PARP inhibitor PJ34 and HDAC inhibitor SAHA on the proliferation of liver cancer cells. Cell proliferation and apoptosis were assessed in three human liver cancer cell lines (HepG2, Hep3B and HCC-LM3) treated with PJ34 (8 μmol/L) and SAHA (1 μmol/L), alone or combined, by Cell Counting Kit-8 assay and flow cytometry, respectively. The nude mice bearing subcutaneous HepG2 tumors were administered different groups of drugs (10 mg/kg PJ34, 25 mg/kg SAHA, 10 mg/kg PJ34+25 mg/kg SAHA), and the inhibition rates of tumor growth were compared between groups. The results showed that combined use of PJ34 and SAHA could synergistically inhibit the proliferation of liver cancer cell lines HepG2, Hep3B and HCC-LM3. The apoptosis rate of HepG2 cells treated with PJ34+SAHA was significantly higher than that of HepG2 cells treated with PJ34 or SAHA alone (P<0.05). In vivo, the tumor inhibition rates were 53.5%, 61.4% and 82.6% in PJ34, SAHA and PJ34+SAHA groups, respectively. The combined use of PJ34 and SAHA could significantly inhibit the xenograft tumor growth when compared with use of PJ34 or SAHA alone (P<0.05). It was led to conclude that PJ34 and SAHA can synergistically suppress the proliferation of liver cancer cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Mice; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Vorinostat; Xenograft Model Antitumor Assays

Miltirone (1), an abietane-type diterpene quinone isolated from Salvia miltiorrhiza, possesses anticancer activity in p-glycoprotein (P-gp)-overexpressing human cancer cells. Results of the current study suggest a dual effect of miltirone on P-gp inhibition and apoptotic induction in a human hepatoma HepG2 cell line and its P-gp-overexpressing doxorubicin-resistant counterpart (R-HepG2). Miltirone (1) elicited a concentration-dependent cytotoxicity, with a similar potency (EC50 ≈ 7-12 μM), in HepG2 and R-HepG2 cells. Miltirone (1) (1.56-6.25 μM) produced synergistic effects on doxorubicin (DOX)-induced growth inhibition of R-HepG2 (synergism: 0.3 < combination index < 0.5). Molecular docking studies illustrated that miltirone (1) interacted with the active site of P-gp with a higher binding affinity than DOX, suggesting that it was a P-gp inhibitor. Flow cytometric analysis confirmed miltirone (1) as a competitive inhibitor of P-gp. At non-necrotic concentrations (1.56-25 μM), miltirone (1) activated caspase-dependent apoptotic pathways and triggered the generation of reactive oxygen species (ROS) and ROS-mediated mitogen-activated protein kinase (MAPK) signaling pathways (e.g., p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase, and extracellular regulated kinase 1/2) in both HepG2 and R-HepG2 cells. Thus, we conclude that miltirone (1) is a dual inhibitor of P-gp and cell growth in human drug-resistant hepatoma cells.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Caspases; Cell Cycle; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Molecular Structure; Phenanthrenes; Salvia miltiorrhiza

Triptolide is a diterpenoid epoxide isolated from the herb Tripterygium wilfordii, which has long been used as a natural medicine in China for many diseases including cancer. However, its effect on liver cancer has not yet been evaluated. The aim of this study is to investigate the effect of triptolide on liver cancer cell lines.. The effect of triptolide with cancer chemotherapeutics such as cisplatin and fluorouracil (5-FU) in vitro and in vivo on liver cancer cells has been investigated. Cell viability and protein expression were determined by MTT assay and Western blotting respectively. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. The antitumor effects of triptolide alone or in combination were also measured in transplantation tumor models.. The combination of triptolide plus chemotherapeutics (Cisplatin, 5-FU) reduced cancer cell viability and enhanced apoptosis compared with single treatment in vitro. Furthermore, cells treated with triptolide plus chemotherapeutics exhibited marked production of intracellular ROS and caspase-3 activity compared with single treatment, which paralleled the incidence of apoptosis. Triptolide and chemotherapeutics treatment also induced Bax expression, and inhibited Bcl-2 expression. Triptolide also boosted the anticancerous effects of cisplatin and 5-FU in nude mice compared with cisplatin or 5-FU alone treated tumors.. Our study demonstrated that triptolide is associated with increased cellular sensitivity of liver cancer cells to cisplatin and 5-FU in vivo and in vitro, and may provide new liver cancer treatment strategies.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Diterpenes; Dose-Response Relationship, Drug; Drug Synergism; Drugs, Chinese Herbal; Epoxy Compounds; Female; Flow Cytometry; Fluorouracil; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Oxidative Stress; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tripterygium

Sorafenib is the only drug approved by the Food and Drug Administration for metastatic hepatocellular carcinoma (HCC). Triptolide, a diterpene triepoxide, exhibits antineoplastic properties in multiple tumor cell types. In this study, we examined the effects of these agents and their combination on HCC in vitro and in vivo models.. HuH-7 and PLC/PRF/5 cells were treated with triptolide (50 nM), sorafenib (1.25 or 2.5 μM), or a combination of both. Cell viability assay (CCK-8), caspase 3&7 activation, and nuclear factor κB assays were performed. For in vivo studies, 40 mice were implanted with subcutaneous HuH7 tumors and divided into four treatment groups (n = 10); saline control, sorafenib 10 mg/kg PO daily (S), Minnelide (a prodrug of triptolide) 0.21 mg/kg intraperitoneally7 daily (M), and combination of both (C). Tumor volumes were assessed weekly.. The combination of triptolide and sorafenib was superior to either drug alone in inducing apoptosis and decreasing viability, whereas triptolide alone was sufficient to decrease nuclear factor κB activity. After 2 weeks of treatment, tumor growth inhibition rates were S = 59%, M = 84%, and C = 93%, whereas tumor volumes in control animals increased by 9-fold. When crossed over to combination treatment, control mice tumor growth volumes plateaued over the following 4 weeks.. The combination of sorafenib and triptolide is superior to single drug treatment in increasing cell death and apoptosis in vitro. Combining sorafenib with Minnelide inhibited tumor growth with greater efficacy than single-agent treatments. Importantly, in vivo combination treatment allowed for using a lesser dose of sorafenib (10 mg/kg), which is less than 10% of currently prescribed dose for HCC patients. Therefore, combination treatment could have translational potential in the management of HCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diterpenes; Drug Synergism; Epoxy Compounds; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Nude; Models, Biological; NF-kappa B p50 Subunit; Niacinamide; Organophosphates; Phenanthrenes; Phenylurea Compounds; Prodrugs; Signal Transduction; Sincalide; Sorafenib; Translational Research, Biomedical; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) has one of the worst prognoses for survival as it is poorly responsive to both conventional chemotherapy and mechanism-directed therapy. This results from a lack of therapeutic concentration in the tumor tissue coupled with the highly toxic off-site effects exhibited by these compounds. Consequently, we believe the best packaging for holistic therapy for HCC will involve three components: a potent therapeutic, a rationally designed drug delivery vehicle to enrich the target site concentration of the drug, and a surface ligand that can enable a greater propensity to internalization by tumor cells compared to the parenchyma. We screened a library containing hundreds of compounds against a panel of HCC cells and found the natural product, triptolide, to be more effective than sorafenib, doxorubicin, and daunorubicin, which are the current standards of therapy. However, the potential clinical application of triptolide is limited due to its poor solubility and high toxicity. Consequently, we synthesized tumor pH-sensitive nanoformulated triptolide coated with folate for use in an HCC-subpopulation that overexpresses the folate receptor. Our results show triptolide itself can prevent disease progression, but at the cost of significant toxicity. Conversely, our pH-sensitive nanoformulated triptolide facilitates uptake into the tumor, and specifically tumor cells, leading to a further increase in efficacy while mitigating systemic toxicity.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Chemistry, Pharmaceutical; Diterpenes; Down-Regulation; Drug Liberation; Epoxy Compounds; Folic Acid; Humans; Hydrogen-Ion Concentration; Liver Neoplasms; Mice; Molecular Targeted Therapy; Nanostructures; Phenanthrenes; Survival Analysis; Xenograft Model Antitumor Assays

Stigmalactam, an aristolactam-type alkaloid extracted from Orophea enterocarpa, exerts cytotoxicity against several human and murine cancer cell lines, but the molecular mechanisms remain elusive. The aims of this study were to identify the mode and mechanisms of human cancer cell death induced by stigmalactam employing human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells as models, compared to normal murine fibroblasts. It was found that stigmalactam was toxic to HepG2 and MDA-MB-231 cells with IC50 levels of 23.0±2.67 μM and 33.2±4.54 μM, respectively, using MTT assays. At the same time the IC50 level towards murine normal fibroblast NIH3T3 cells was 24.4±6.75 μM. Reactive oxygen species (ROS) production was reduced in stigmalactam-treated cells dose dependently after 4 h of incubation, indicating antioxidant activity, measured by using 2',7',-dichlorohydrofluorescein diacetate and flow cytometry. Caspase-3 and caspase-9 activities were increased in a dose response manner, while stigmalactam decreased the mitochondrial transmembrane potential dose-dependently in HepG2 cells, using 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, indicating mitochondrial pathway-mediated apoptosis. In conclusion, stigmalactam from O. enterocarpa was toxic to both HepG2 and MDA-MB-231 cells and induced human cancer HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway.

Topics: Animals; Annonaceae; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Fibroblasts; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mitochondria; NIH 3T3 Cells; Phenanthrenes; Reactive Oxygen Species

Pancreatic cancer is one of the most lethal human malignancies, with an all-stage 5-year survival of <5%, mainly due to lack of effective available therapies. Cancer cell survival is dependent upon up-regulation of the pro-survival response, mediated by anti-apoptotic proteins such as Mcl-1.. Here we show that over-expression of Mcl-1 in pancreatic patient tumor samples is linked to advancement of the disease. We have previously shown that triptolide, a diterpene triepoxide, is effective both in vitro and in vivo, in killing pancreatic cancer cells. Decrease of Mcl-1 levels, either by siRNA or by treatment with triptolide results in cell death. Using pancreatic cancer cell lines, we have shown that miR-204, a putative regulator of Mcl-1, is repressed in cancer cell lines compared to normal cells. Over-expression of miR-204, either by a miR-204 mimic, or by triptolide treatment results in a decrease in Mcl-1 levels, and a subsequent decrease in cell viability. Using luciferase reporter assays, we confirmed the ability of miR-204 to down-regulate Mcl-1 by directly binding to the Mcl-1 3' UTR. Using human xenograft samples treated with Minnelide, a water soluble variant of triptolide, we have shown that miR-204 is up-regulated and Mcl-1 is down-regulated in treated vs. control tumors.. Triptolide mediated miR-204 increase causes pancreatic cancer cell death via loss of Mcl-1.

Topics: 3' Untranslated Regions; Animals; Antineoplastic Agents; Base Sequence; Binding Sites; Cell Death; Cell Line, Tumor; Diterpenes; Epithelium; Epoxy Compounds; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; Mice, SCID; MicroRNAs; Myeloid Cell Leukemia Sequence 1 Protein; Organophosphates; Pancreatic Ducts; Pancreatic Neoplasms; Phenanthrenes; RNA Interference; Up-Regulation; Xenograft Model Antitumor Assays

2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy.. We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it.. These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Topics: Acetylcysteine; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 9; Necrosis; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA Interference; Signal Transduction

Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc)-infected cells. In the current study, we have selected and characterized the gene encoding Cyclin A2 (CycA2). Deregulation of CycA2 has been implicated in many types of cancers, including hepatocellular carcinoma.. The effects of CycA2 on HCV propagation were investigated by siRNA-mediated knockdown assay, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay.. We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication. Moreover, we demonstrated that tylophorine, the natural plant product exerting a CycA2 inhibitory function, abrogated HCV replication.. HCV regulates CycA2 via NS5B protein for its own propagation. In addition, tylophorine may be a potential therapeutic agent for HCV.

Topics: Alkaloids; Antiviral Agents; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Cyclin A2; HEK293 Cells; Hepacivirus; Humans; In Vitro Techniques; Indolizines; Kidney; Liver Neoplasms; Phenanthrenes; RNA, Small Interfering; Viral Nonstructural Proteins; Virus Replication

Tanshinone IIA (Tan-IIA) is extracted from Danshen, Salviae miltiorrhizae Radix, which has been widely adopted in traditional herbal medicine to treat cardiovascular and hepatic diseases. Tan-IIA induces apoptosis and inhibits proliferation in human hepatocellular carcinoma (HCC) cells. However, the molecular mechanisms of Tan-IIA on human HCC cells are not understood clearly. In the present study, the cytotoxicity of Tan-IIA as well as its molecular mechanisms in human HCC J5 cells was investigated. The cytotoxicity was assayed by MTT. The protein expression of p53, p21, Bax, Bcl-2, Cdc25c, Cdc2, calreticulin, caspase 12, GADD153, caspase 3 and beta-actin in J5 cells were determined by Western blotting. The cell cycles were analyzed by FACS. The protein expression of caspase 12, GADD1533 and caspase 3 were detected by immunocytochemical staining. The results showed that Tan-IIA inhibited J5 cells in a dose- and time-dependent manner. The protein expression of p53, p21, Bax, calreticulin, caspase 12, caspase 3 and GADD153 were increased, but Bcl-2, Cdc25c and Cdc2 were decreased in J5 cells. In addition, the results also showed that Tan-IIA arrested J5 cells in the G2/M phase. Immunocytochemistry staining showed that J5 cells treated with Tan-IIA up-regulated the protein expression of caspase 12, 3 and GADD153. Taken together, the findings suggest that Tan-IIA inhibits and induces apoptosis in J5 cells through novel molecular targets, calreticulin, caspase 12 and GADD153.

Topics: Abietanes; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Calreticulin; Carcinoma, Hepatocellular; Caspase 12; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Drugs, Chinese Herbal; Humans; Liver Neoplasms; Male; Mitochondria; Phenanthrenes; Transcription Factor CHOP

To observe the effect of Tanshinone II A on the expression of epidermal growth facter (EGF) and epidermal growth facter recepter (EGFR) in human hepatocellular carcinoma cell line SMMC-7721.. The human hepatocellular carcinoma SMMC-7721 cells cultured in vitro was treated with different concentrations of Tanshinone II A. The proliferation of the cells was measured by MTT assay, and the apoptosis of the cells was investigated by flow cytometry and cytochemical staining with Hoechst 33342. The expression of EGF and EGFR was detected by immunocytochemistry method. The levels of EGF in medium were measured by radioimmunoassay.. Tanshinone II A inhibited the growth of SMMC-7721 cells remarkably in a dose-dependent manner. The inhibitory rate reached the peak (72.5%) after 0.5 microg/ml Tanshinone II A was used for 48 h, which was significantly higher than that in the controls (P<0.05). FCM analysis showed that when SMMC-7721 cells were treated with 0.5 microg/ml Tanshinone II A, the apoptosis rates for 24 h, 48 h and 72 h were (4.06+/-0.27)%, (7.58+/-0.56)% and (5.23+/-0.13)%, respectively which were markedly higher than those in the controls (all P<0.01). SMMC-7721 cell apoptosis with cell shrinkage, nuclear chromatin concentration and fragmentation as well as the formation of apoptotic bodies were observed by cytochemical staining when treated with Tanshinone II A. The immunocytochemistry showed that the expressions of EGF and EGFR were down regulated while the concentration of Tanshinone II A was increasing. The high expression rates for EGF and EGFR were 10%, 20%, respectively, and the gray scale was 181.52+/-1.63, 179.37+/-1.59, which were markedly higher than those in the controls (all P<0.05). The levels of EGF in medium measured by radioimmunoassay were decreased significantly after Tanshinone II A treatment.. Tanshinone II A can inhibit cell proliferation and induce apoptosis in hepatocellular carcinoma cell line SMMC-7721, which may be related to the down-regulation of EGF and EGFR protein expression.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Phenanthrenes

The role of reactive oxygen species (ROS) and p38 mitogen-activated protein kinases (MAPK) in tanshinones-induced apoptosis was investigated in HepG2 cells in this study. The major tanshinones (cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA), isolated from Salvia miltiorrhiza, inhibit cell growth and induce caspase-dependent apoptosis concentration-dependently, with dihydrotanshinone being the most potent. All four tanshinones were found to induce ROS generation, but only dihydrotanshinone can induce activation of p38 MAPK. The p38 MAPK activation by dihydrotanshinone was inhibited by N-acetyl cysteine pretreatment. It is thus concluded that ROS-mediated p38 MAPK activation plays a vital role in dihydrotanshinone-induced apoptosis in HepG2 cells.

Topics: Abietanes; Acetylcysteine; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Furans; Humans; Imidazoles; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Liver Neoplasms; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phenanthrenes; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Pyridines; Quinones; Reactive Oxygen Species; Time Factors

It has been suggested that poly(ADP-ribose) polymerase-l (PARP-l) plays an important role in DNA repair, cell death and proliferation, as well as in the stabilization of the genome. Pharmacological inhibition or genetic ablation of PARP-1 had a beneficial outcome in cancer chemotherapy since the cancer cells lacked PARP-1 and were sensitive to chemotherapeutic DNA damage. As a novel potent specific inhibitor of PARP-l, PJ34 has been reported to enhance chemotherapeutic effects in certain types of tumors. In a previous study, we found that PARP-1 expression was significantly increased in human hepatocellular carcinoma (HCC) compared to its surrounding liver tissue. This study investigated whether or not the inhibition of PARP-1 activity by PJ34 produces suppressive effects on human liver cancer cells and sensitizes the tumor cells to chemotherapeutic agents. We conclude that PJ34 significantly suppresses HepG2 cell growth in a dose-dependent manner, and inhibits HepG2 cell-derived tumor growth in nude mice. The suppressive effects of PJ34 are associated with increased cell apoptosis. Furthermore, PJ34 enhances suppressive effects of cisplatin in HepG2 cells. These results suggest that PJ34 may be developed into an effective agent for the treatment of human HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Proliferation; Cisplatin; Drug Synergism; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis.

Topics: Administration, Oral; Aflatoxin B1; Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; DNA Adducts; Heme Oxygenase-1; Humans; Imidazoles; Leukemia; Liver Neoplasms; Macrophages; Male; Mice; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); Nitric Oxide; Nitric Oxide Synthase Type II; Oleanolic Acid; Phenanthrenes; Rats; Rats, Inbred F344; Reactive Oxygen Species

Tanshinones are abietane type-diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicine in the treatment of cardiovascular diseases. Among the major diterpenes isolated, including cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone, tanshinone IIA had been shown to posses various pharmacological activities including antioxidant, protection/prevention from angina pectoris and myocardial infarction, and anticancer properties. Tanshinone IIA, usually the most abundant tanshinone present in the herb, has been the focus of studies in its clinical potential, among which its ability to inhibit the proliferation of cancer cell lines. The aim of this study was to study the cytotoxicity of the tanshinones on human HepG2 cells in vitro in relation to intracellular glutathione perturbation (reduced glutathione, GSH and oxidized glutathione, GSSG). Studies using MTT assay showed that all tanshinones decreased cell viability of HepG2 cells in a concentration-dependent manner, with the cell viability decreased to 60% and 35% after 24 h and 48 h treatment, respectively. Assessment of apoptotic cells with fragmented DNA by flow cytometry indicated that only tanshinone IIA (12.5 and 25 microM) induced apoptosis in the cancer cells. Tanshinone IIA and cryptotanshinone caused significant decreases in G(1) cells by 23% and 13%, respectively, after 24 h treatment. The declines in G(1) cells were compensated by increases in G(2)/M (15% for tanshinone IIA) and S cells (8% and 13% for tanshinone IIA and cryptotanshinone, respectively). All the tanshinones studied, except tanshinone IIA, elevated GSH/GSSG ratio at low concentrations (1.56 and 3.13 microM), but the ratio decreased, indicating oxidative stress at high concentrations (6.25-25 microM). Taken together, tanshinone IIA caused HepG2 cytotoxicity through apoptosis without influencing oxidative stress, while the other tanshinones showed lower efficacy in inducing apoptosis in the HepG2 cells.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Flow Cytometry; Glutathione; Humans; Liver Neoplasms; Mass Spectrometry; Oxidative Stress; Phenanthrenes; Salvia miltiorrhiza

To determine the effect of tanshinone IIA on the growth and apoptosis in human hepatoma cell line HepG2.. The human hepatoma cell line HepG2 was treated with tanshinone IIA at various concentrations for 72 h. The inhibition of proliferation was measured by MTT assay and apoptosis-related alterations in morphology measured by cytochemical staining (HT33258). DNA fragmentation was evaluated by agarose gel electrophoresis. Apoptotic rate and cell arrest were quantified by flow cytometry (FCM).. Tanshinone IIA inhibited the growth of HepG2 in a time- and dose- dependent manner. The semi-inhibitory concentration (IC50) value after the treatment with tanshinone IIA on HepG2 for 24, 48 and 72 h were 14.7, 7.4, and 3.9 microg/ mL, respectively. After the treatment with 0.5 - 10 microg/mL tanshinone IIA for 72 h, the formation of apoptotic bodies was observed. DNA ladder was shown in agarose gel electrophoresis, in addition to the cells treated by 1.0 microg/mL tanshinone IIA . The apoptotic rates at 0.5, 1.0, 2.0, 5.0, and 10.0 microg/mL for 72 h were 20.32%+/-2.16%, 28.0%+/-2.35%, 33.87%+/-3.43%, 46.73%+/-4.08% and 57.85%+/-3.74%, respectively, which were all significantly higher than those of the control group (P<0.05).. Tanshinone IIA can inhibit the proliferation of human hepatoma cell line HepG2 in a time- and dose- dependent manner, and the mechanism of growth inhibition of human hepatoma cells may be related to the induction of apoptosis.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Flow Cytometry; Humans; Liver Neoplasms; Microscopy, Fluorescence; Phenanthrenes; Time Factors

Tylophorine and its analogs are phenanthroindolizidine alkaloids, several of which have been isolated from the Tylophora genus of plants. Evaluation of (+)-S-tylophorine [DCB-3500 (NSC-717335)] and its analog DCB-3503 (NSC-716802) in the National Cancer Institute tumor screen showed a fairly uniform and potent inhibition of cell growth in all 60 cell lines (GI(50) approximately 10(-8) M). To further evaluate the antitumor potential of these compounds, we synthesized four tylophorine analogs, designated DCB-3500, DCB-3501, DCB-3502, and DCB-3503. All four tylophorine analogs exerted potent growth-inhibitory effects against HepG2, a human hepatocellular carcinoma cell line, and KB, a human nasopharyngeal carcinoma cell line. HepG2 cells were more sensitive than KB in terms of loss of clonogenicity. KB variants, which are resistant to etoposide, hydroxyurea, or camptothecin, have similar sensitivities to the tylophorine analogs, as do the parental KB cells. Treatment of nude mice bearing HepG2 tumor xenografts by i.p. injections of DCB-3503 at 6 mg/kg every 8 h on days 0 and 3 resulted in significant tumor growth suppression (P < 0.0001). Unlike conventional antitumor drugs, 3 micro M DCB-3503 did not cause DNA breaks or apoptosis in HepG2 cells. Tylophorine analogs induced albumin expression and decreased alpha-fetoprotein expression in HepG2 cells, which suggests that tylophorine analogs could induce HepG2 differentiation. Tylophorine analogs had an inhibitory effect on cyclic AMP response elements, activator protein-1 sites, or nuclear factor-kappaB binding site-mediated transcriptions. In summary, these tylophorine analogs are a unique class of antitumor compounds that have a mode of action different from known antitumor drugs.

Topics: Alkaloids; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Survival; Drug Design; Drug Resistance, Neoplasm; Genes, Reporter; Humans; Indolizines; KB Cells; Liver Neoplasms; Male; Mice; Mice, Nude; Microscopy, Confocal; Phenanthrenes; Structure-Activity Relationship; Transfection; Tumor Stem Cell Assay

To evaluate the effects of tanshinone II-A on inducing growth inhibition and apoptosis of human hepatocellular carcinoma (HCC) cells.. The human hepatocellular carcinoma cell line SMMC-7721 was used for the study. The cells were treated with tanshinone II-A at different doses and different times. Cell growth and proliferation were measured by MTT assay, cell count and colony-forming assay. Apoptosis induction was detected by microscopy, DNA ladder electrophoresis and flow cytometry.. In MTT assay, the inhibitory effect became gradually stronger with the passage of time, 24, 48, 72 and 96 h after treatment with tanshinone II-A, and the most significant effect was observed at 72 h. On the other hand, the increase of doses (0.125, 0.25, 0.5, 1.0 mg/L tanshinone II-A) resulted in enhanced inhibitory effect. The growth and proliferation of SMMC-7721 cells were obviously suppressed in a dose- and time-dependent manner. The results of cell count were similar to that of MTT assay. In colony-forming assay, the colony-forming rates were obviously inhibited by tanshinone II-A. In tanshinone II-A group, the morphology of cellular growth inhibition and characteristics of apoptosis such as chromatin condensation, crescent formation, margination and apoptotic body were observed under light and transmission electron microscopes. DNA ladder of cells was presented in electrophoresis. The apoptosis index (AI) was 16.9% (the control group was 4.6%) in flow cytometry. The cells were arrested in G(0)/G(1) phase, and the expressions of apoptosis-related genes bcl-2 and c-myc were down-regulated and fas, bax, p53 up-regulated.. Tanshinone II-A could inhibit the growth and proliferation of HCC cell effectively in vitro by apoptosis induction, which was associated with up-regulation of fas, p53, bax, expression and down-regulation of bcl-2 and c-myc.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Drugs, Chinese Herbal; Humans; Liver Neoplasms; Phenanthrenes

To investigate the effect of tanshinone IIA on the growth behavior of human hepatoma cell line BEL-7402 in vitro and explore the mechanism.. Human hepatoma cell line BEL-7402 was exposed to tanshinoneIIA at different concentrations for 72 h, and the suppression of the cell growth was observed under inverted-phase contrast microscope. Apoptosis-related alterations in the cell morphology and biochemistry were examined under fluorescence microscope and transmission electron microscope (TEM) and by DNA agarose gel electrophoresis, and the apoptotic rate was quantified by flow cytometry (FCM).. After treatment with 0-10 microg/ml tanshinone IIA for 72 h, the proliferation of BEL-7402 cells was significantly suppressed, and cell apoptosis occurred characterized by cell shrinkage, nuclear chromatin condensation and fragmentation, formation of membrane blebs and apoptotic bodies as observed under fluorescence microscope and TEM. DNA ladder was presented in DNA electrophoresis. FCM analysis yielded the cell apoptotic rates of (20.78+/-2.17) %, (24.64+/-2.07) %, (31.47+/-3.86) %, (43.65+/-4.04) % and (52.36+/-3.75) % at tanshinone IIA concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 microg/ml respectively, all significantly higher than those of the control group [(2.37+/-0.29)%].. Tanshinone IIA can inhibit the growth of human hepatoma BEL-7402 cells possibly through the mechanism of apoptosis induction.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Humans; Liver Neoplasms; Microscopy, Electron; Phenanthrenes

In order to study the effect of tanshinone II A on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone II A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6.28 micrograms/ml. After treatment with 1-10 micrograms/ml tanshinone II A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 micrograms/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32 +/- 0.16)%, (3.01 +/- 0.35)%, (3.87 +/- 0.43)%, (6.73 +/- 0.58)% and (20.85 +/- 1.74)% respectively, which were all significantly higher than those in the control group (1.07 +/- 0.13)%. It is concluded that Tanshinone II A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Drugs, Chinese Herbal; Flow Cytometry; Humans; Liver Neoplasms; Phenanthrenes

The environmental contaminant benzo[c]phenanthrene (B[c]Ph) has weak carcinogenic activity in rodent bioassays; however, the fjord region diol epoxides of B[c]Ph, B[c]Ph-3,4-diol 1,2-epoxides (B[c]PhDE), are potent carcinogens. To determine the role of cytochrome P450 isozymes in the activation of B[c]Ph in MCF-7 cells and the low activation of B[c]Ph in mouse skin, cells of the MCF-7 and the human hepatoma HepG2 cell lines were treated with the potent Ah receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to exposure to B[c]Ph for 24 h. Mice were treated topically with 1 microg of TCDD or vehicle (control) for 73 h and then with 2 micromol of B[c]Ph for 24 h. In MCF-7 cells, TCDD exposure increased B[c]PhDE-DNA adduct levels more than 3-fold with a 10-fold increase in the (-)-B[c]PhDE-2-dA(t) adduct. Treatment of HepG2 cells with TCDD prior to B[c]Ph application did not increase B[c]PhDE-DNA binding. Total B[c]PhDE-DNA adducts increased 3-fold in TCDD-treated mouse epidermis: the majority of the increase resulted from (+)-B[c]PhDE-1-dA adducts. Analysis of P450 enzymes by Western blotting detected a large increase of P4501B1 but almost no increase in P4501A1 in MCF-7 cells exposed to 10 microM B[c]Ph for 24 or 48 h. In HepG2 cells, there were no detectable levels of P4501A1 or P4501B1 after treatment with 10 microM B[c]Ph for 24 h. In contrast, topical application of 2 micromol of B[c]Ph to mouse skin for 48 or 72 h increased P4501A1, but no P4501B1 was detected. As a measure of P450 activity, the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) was analyzed in microsomes prepared from MCF-7 and HepG2 cells exposed to 0.1% DMSO, 10 microM B[c]Ph, or 10 nM TCDD for 24 or 48 h and from mouse epidermis treated with 1 microg of TCDD, or vehicle control for 72 h, or 2 micromol of B[c]Ph for 48 h. The levels of DMBA metabolites were low or undetectable in microsomes from B[c]Ph-treated MCF-7 and HepG2 cells, but a metabolite pattern consistent with P4501A1 metabolism of DMBA was present in B[c]Ph-exposed mouse epidermal microsomes. TCDD-treated MCF-7 cells, HepG2 cells, and mouse epidermis had DMBA metabolism patterns characteristic of P4501A1 activity. Microsomes from TCDD-treated human cells formed a higher proportion of the proximate carcinogenic metabolite DMBA-3,4-dihydrodiol (16% of total identified metabolites) than TCDD-treated mouse epidermis (2%). In mouse epidermis, the weak ability of B[c]Ph to increase hydrocarbon-metabolizing act

Topics: Animals; Biotransformation; Breast Neoplasms; Carcinogens; Carcinoma, Hepatocellular; Cytochrome P-450 Enzyme System; Enzyme Induction; Epidermal Cells; Epidermis; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred SENCAR; Phenanthrenes; Tumor Cells, Cultured

2-Acetylaminofluorene (AAF) is one of the most widely studied model carcinogens. It produces liver tumors in rats. Comparison with other arylamides shows that promutagenic DNA lesions are necessary but not sufficient to explain this tissue-specific effect. Mutagenicity of AAF was studied in AS52 cells and compared with that of 2-acetylaminophenanthrene and trans-4-acetylaminostilbene which are incomplete carcinogens in rat liver. The major mutations were G to T transversions in all cases. All three acetamides acted as initiators in an initiation-promotion experiment with phenobarbital as a promoter. Chronic toxic effects of AAF were attributed to specific effects of AAF metabolites on mitochondrial respiration. Electron drainage by 2-nitrosofluorene causes an uncoupling effect on oxidative phosphorylation in vitro. Corresponding compensatory effects were observed in vivo. Initiating as well as promoting properties of AAF are therefore considered responsible for the generation of rat liver tumors. The results support the hypothesis that genotoxic effects generate initiated cells which begin to proliferate only when microcirculation is disturbed due to cirrhotic alterations. These are triggered by non-genotoxic interference with mitochondrial respiration and oxidative phosphorylation.

Topics: 2-Acetylaminofluorene; Animals; Cell Survival; Liver Neoplasms; Mitochondria, Liver; Mutagens; NAD; Oxidation-Reduction; Phenanthrenes; Rats; Rats, Wistar; Stilbenes

The aim of this study was to find out the anti-cancer activity of Tanshinone and its mechanism of action. Human hepatic carcinoma cell line (SMMC-7721) and leukemia cell line (HL60) were treated with tanshinone the cancer cell proliferation indices were measured by Brdu Labeling and immuno-histochemical stain of PCNA. The Brdu labeling rates of human hepatic carcinoma and leukemia cells treated with tanshinone were 8.95% and 19.01%, which were lower than those of controls (28.0%, 25.57%) respectively (P < 0.01), PCNA positive rates were 57.0% and 30.32%, which were significantly lower than those of controls (74.3%, 47.05%) (P < 0.01). The results indicate that Brdu labeling and PCNA detection may have important utility in the studies of tumor cell proliferation and the relative factors affecting the cell proliferation. The inhibitory effect of tanshinone on cancer cell proliferation might be associated with inhibiting DNA synthesis, PCNA expression and activity of DNA polymerase delta of the tumor cells.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Bromodeoxyuridine; Carcinoma, Hepatocellular; Cell Division; DNA, Neoplasm; Drugs, Chinese Herbal; HL-60 Cells; Humans; Liver Neoplasms; Phenanthrenes; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured

Our earlier studies demonstrated that the beta-hydroxy-beta-methylglutaryl coenzyme A (HMG CoA) reductase gene is hypomethylated and overexpressed in hepatic nodules initiated by 1,2-dimethylhydrazine (1,2-DMH). The study presented here examined whether the pattern of DNA methylation of the HMG CoA reductase gene in hepatic nodules reflected carcinogen-DNA interaction during initiation. Accordingly, hepatic nodules were generated in male Fischer 344 rats with either 1,2-DMH or aristolochic acid (AA), which interact predominantly with the guanine and adenine bases in DNA, respectively. DNA from individual nodules was restricted with HpaII, MspI, and HhaI, and the fragments obtained were hybridized to a cDNA probe for HMG CoA reductase. The results indicated that the hypomethylation pattern in the reductase gene in the nodules initiated with these two carcinogens was similar, although they interacted with different bases in the DNA. The question still remained whether the DNA fragments obtained by digesting the two sets of nodules with the restriction endonucleases were from the same domains in the genome of HMG CoA reductase. To examine this, probes for the different domains of the HMG CoA reductase gene were generated from the cDNA using the restriction enzyme Accl. Three probes were obtained: (i) a 5'-end fragment corresponding to the membrane-spanning region, (ii) a second fragment corresponding to the 3'-end of the protein, and (iii) a third fragment spanning the region between (i) and (ii). Each of these probes was radiolabeled and hybridized to the HpaII- and HhaI-generated fragments from the DNA of hepatic nodules initiated with 1,2-DMH and AA. Irrespective of the carcinogen used for initiation, hypomethylation occurred in all three domains of the gene. More important, the pattern of hypomethylation was similar in the nodules initiated by the two carcinogens. Furthermore, an overall similarity was seen in the hypomethylation patterns in the c-myc and c-Ha-ras genes in the DNA of nodules initiated with either 1,2-DMH or AA. These results raise the possibility that the pattern of hypomethylation established in the hepatic nodules may not directly reflect the interaction between the initiating carcinogen and DNA but may represent a unique phenotype of hepatic nodules.

Topics: 1,2-Dimethylhydrazine; Animals; Aristolochic Acids; Carcinogens; Cell Cycle; Dimethylhydrazines; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes; Genes, myc; Genes, ras; Hydroxymethylglutaryl CoA Reductases; Liver Neoplasms; Male; Methylation; Phenanthrenes; Polymorphism, Restriction Fragment Length; Precancerous Conditions; Rats; Rats, Inbred F344

Aristolochic acid (AA), used as an anti-inflammatory agent in the past, is known to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis and urinary bladder. However, despite the induction of DNA adducts in the liver, no carcinogenic potential of AA has been reported in the latter organ. The present study was based on the rationale that the lack of carcinogenicity of AA to the liver could be because this chemical may not be necrogenic at the doses examined and liver cell proliferation has been established as an essential component for initiation of liver carcinogenesis in the rat. The results indicated that AA is non-necrogenic to the rat liver. However, a single non-necrogenic dose of AA (10 mg/kg b.w., i.p.) given 18 hours after 2/3 partial hepatectomy initiated liver cell carcinogenesis. The initiated cells are promotable with 1% dietary orotic acid, a liver tumor promoter, to form glutathione-S-transferase 7-7 positive hepatic foci and nodules.

Topics: Animals; Aristolochic Acids; Carcinogens; Cell Division; Cocarcinogenesis; Hepatectomy; Liver; Liver Neoplasms; Male; Necrosis; Orotic Acid; Phenanthrenes; Rats; Rats, Inbred F344

The mutagenic activities in Salmonella typhimurium and tumorigenic activities in newborn mice of 6-nitrochrysene (6-NC), 5-methyl-6-nitrochrysene (5-Me-6-NC), 11-methyl-6-nitrochrysene (11-Me-6-NC) and 5-methylchrysene (5-MeC) were compared. In S. typhimurium TA100 in the absence of rat liver 9000 g supernatant, 11-Me-6-NC was the most active compound followed by 6-NC; 5-Me-6-NC and 5-MeC were inactive. In the assays conducted in the presence of rat liver 9000 g supernatant, the order of activity was 11-Me-6-NC greater than 6-NC greater than 5-Me-6-NC approximately 5-MeC. In S. typhimurium TA98 a similar trend was observed. For the tumorigenicity studies, groups of mice were treated with the appropriate compounds in DMSO by i.p. injections on the 1st, 8th and 15th day of life. At a dose of 100 nmol/mouse 6-NC induced significantly more lung tumors than 5-MeC, which in turn was more active than 11-Me-6-NC and 5-Me-6-NC. All compounds induced significant numbers of liver tumors in treated males compared to controls; the order of activity was the same as that observed for lung tumor induction. The results of this study clearly indicate that bay region methyl substitution can either inhibit (5-position) or enhance (11-position) the mutagenic activity of 6-NC. In contrast, bay region methyl substitution (5- and 11-positions) inhibited the tumorigenic activity of 6-NC in newborn mice. Since ring oxidation and nitroreduction are involved in the metabolic activation of 6-NC in newborn mice, bay region methyl substitution may either inhibit the nitroreduction pathway or hinder the formation of the appropriate bay region diol epoxide. Steric factors may be important in determining the tumorigenicity of methylated nitrochrysenes.

Topics: Animals; Animals, Newborn; Biotransformation; Carcinogens; Chrysenes; Liver Neoplasms; Lung Neoplasms; Mice; Mutagenicity Tests; Mutagens; Phenanthrenes; Salmonella typhimurium; Structure-Activity Relationship

The tumorigenic activities of 6-nitrochrysene and its metabolites were evaluated in the newborn mouse model. Groups of mice were treated with the appropriate compounds in DMSO by i.p. injections on the 1st, 8th and 15th day of life. Seven hundred nmol/mouse of 6-nitrochrysene induced significant incidences and multiplicities of lung tumors in both sexes; only males were susceptible to liver tumor induction. At 100 nmol/mouse, 6-nitrochrysene had significant tumorigenicity in both lung and liver but was less active than at the higher dose. Administration of 100 nmol/mouse of 6-nitrochrysene, only on day 1, caused about the same tumor yield as was observed after treatment with 700 nmol/mouse given over 3 days. Among the metabolites of 6-nitrochrysene which were tested at 100 nmol/mouse, 6-nitrosochrysene and 6-aminochrysene were significantly less active in the lung and in the liver than 6-nitrochrysene. In the lung, trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene had activities comparable to those observed in mice treated with equimolar doses of 6-nitrochrysene. In the liver, trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene was more active than 6-nitrochrysene based on the number of tumors per mouse. These observations support our hypothesis that 6-nitrochrysene is metabolically activated by ring-oxidation to trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene, followed by nitro-reduction to trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene and, finally, oxidation to a diol-epoxide.

Topics: Animals; Animals, Newborn; Biotransformation; Chrysenes; Female; Liver Neoplasms; Lung Neoplasms; Male; Mice; Neoplasms, Experimental; Phenanthrenes

The cyclopenta(a)phenanthrene, 15,16-dihydro-11-methyl-cyclopenta(a)phenanthren-17-one, had potent mutagenic activity in cell-mediated mutation assays with V79 Chinese hamster cells as targets, and cells of the human hepatoma line HepG2 as mediators of activation. The compound was inactive when low-passage hamster embryo cells were used as activators. When the mutagenic activity of a series of cyclopenta(a)phenanthrenes was compared in mutation assays with HepG2 cells as activators, there was a good correlation between mutagenic activity in this system and carcinogenic activity in mouse skin in vivo. One exception was a noncarcinogenic compound, which is mutagenic in the Ames' test, and was also mutagenic in the mammalian cell assay.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Cricetulus; Humans; Liver Neoplasms; Lung; Mutagenicity Tests; Phenanthrenes

Comparison studies have been undertaken on the hepatocarcinogen 2-acetamidofluorene (AAF) and its nonhepatocarcinogenic analog 2-acetamidophenanthrene (AAP). Previous studies have shown that amount of compound acutely and persistently bound to rat liver DNA is comparable for the two compounds following single injections into adult Fischer rats, but that AAP fails to initiate tumors in weanling Sprague-Dawley rats. In this work we show that the amount of bound adduct from AAF and AAP is also comparable after three weeks of feeding compound to weanling Sprague-Dawley male rats. Three of the adducts found in RNA of AAP-treated rats cochromatographed on Sephadex LH-20 with two adenosine adducts and one guanosine adduct prepared by reaction of the nucleosides with N-acetoxy-N-trifluoroacetyl-2-aminophenanthrene at neutrality. Because of the lack of initiating ability of AAP in liver, we have also investigated early biochemical alterations in liver after various regimens. Feeding of either AAF or AAP to male weanling rats, followed by three weeks of DDT feeding produced no alteration in either histochemically detected gamma-glutamyltranspeptidase or in ganglioside complement in total liver homogenate. Partial hepatectomy after feeding of either AAF or AAP resulted in the appearance of new fucoganglioside and alteration in the distribution of the major gangliosides. DDT feeding after partial hepatectomy resulted in foci of elevated gamma-glutamyltranspeptidase in AAF-fed rats but not in AAP-fed rats. These results support a previous proposal that AAP may initiate tumorigenesis in rat liver, but that the promoting regimens now in use lack the ability to cause further progression of the initiated cells. The data also suggests that ganglioside synthesis may be a more sensitive marker for early stages in carcinogenesis than are the various histochemical stains now in use.

Topics: 2-Acetylaminofluorene; Animals; Diet; DNA; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Phenanthrenes; Rats; Rats, Inbred Strains; Structure-Activity Relationship

Topics: Adenoma; Animals; Animals, Newborn; Carcinogens; Chrysenes; Female; Liver Neoplasms; Lung Neoplasms; Lymphoma; Male; Mice; Neoplasms, Experimental; Phenanthrenes; Pregnancy; Structure-Activity Relationship

Benz(a)anthracene injected subcutaneously during the first 3 days of life caused a dose related increase in the incidence of liver and lung tumours in Swiss mice but over a similar dose range, the K region epoxide of benz(a)anthracene was less effective. Neonatally injected 7-methylbenz(a) was considerably more active than its K region epoxide in increasing the incidence of liver tumours in males. Both the parent compound and the epoxide slightly raised the incidence of lung tumours. Both chrysene and its K region epoxide increased liver tumour incidence but not lung tumour incidence. The K region epoxides of dibenz(a,h)-anthracene and 3-methylcholanthrene were without apparent effect on the incidence of liver, lung or other tumours despite indications from previously reported studies that the parent hydrocarbons are active at the same dose levels. The K region epoxide of phenanthrene had no effect on the incidence of any kind of neoplasm.

Topics: Animals; Animals, Newborn; Benz(a)Anthracenes; Chrysenes; Ethers, Cyclic; Female; Liver Neoplasms; Lung Neoplasms; Male; Methylcholanthrene; Mice; Neoplasms, Experimental; Phenanthrenes; Polycyclic Compounds; Structure-Activity Relationship

Topics: Administration, Oral; Administration, Topical; Animals; Female; Liver Neoplasms; Lung Neoplasms; Lymphoma; Male; Mice; Mice, Inbred Strains; Neoplasm Metastasis; Neoplasms, Experimental; Papilloma; Phenanthrenes; Sex Factors; Skin Neoplasms

Topics: Animals; Animals, Newborn; Carcinogens; Carcinoma, Hepatocellular; Cystadenoma; Female; Liver Neoplasms; Lung Neoplasms; Male; Mice; Neoplasms, Experimental; Phenanthrenes

Topics: Animals; Animals, Newborn; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Carcinoma, Hepatocellular; Liver Neoplasms; Lung Neoplasms; Lymphoma; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; Neoplasms; Neoplasms, Experimental; Phenanthrenes; Research; Toxicology