OBJECTIVE: To investigate the antitumor activity of decoction and study its liver and kidney toxicity and its effect on the immune system in a tumor-bearing mouse model. METHODS: Hepatoma H22 tumor-bearing mouse models were randomized into model group, cyclophosphamide (CTX) group, and low-, moderate-, and high-dose decoction groups (JW-L, JW-M, and JW-H groups, respectively). The antitumor activity of decoction was assessed by calculating the tumor inhibition rate and pathological observation of the tumor tissues. Immunohistochemistry was used to detect the expressions of Bax, Bcl-2, Bax/Bcl-2 and caspase-3 in the tumors. The liver and kidney toxicity of decoction was analyzed by evaluating the biochemical indicators of liver and kidney functions. The immune function of the tumor-bearing mice were assessed by calculating the immune organ index, testing peripheral blood routines, and detection of serum IL-2 and TNF-α levels using enzyme-linked immunosorbent assay. RESULTS: Compared with that in the model group, the tumor mass in CTX, JW-M and JW-H groups were all significantly reduced ( < 0.05) with cell rupture and necrosis in the tumors. Immunohistochemistry revealed obviously up-regulated expressions of Bax and caspase-3 and down- regulated expression of Bcl-2 protein with an increased Bax/Bcl-2 ratio in CTX, JW-M and JW-H groups. Treatment with decoction significantly reduced Cr, BUN, AST and ALT levels, improved the immune organ index, increased peripheral blood leukocytes, erythrocytes and hemoglobin levels, and up-regulated the levels of TNF-α and IL-2 in the tumor-bearing mice. These changes were especially significant in JW-H group when compared with the parameters in the model group ( < 0.01). CONCLUSIONS decoction has a strong anti-tumor activity and can improve the liver and kidney functions of tumor-bearing mice. Its anti-tumor effect may be attributed to the up-regulation of Bax, caspase-3, TNF-α and IL-2 levels and the down-regulation of Bcl-2 expression as well as the enhancement of the non-specific immune function.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; immunology ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; drug effects ; Liver ; drug effects ; pathology ; Liver Neoplasms ; drug therapy ; immunology ; metabolism ; pathology ; Mice ; Necrosis ; Neoplasm Proteins ; metabolism ; Random Allocation ; Up-Regulation

OBJECTIVE: To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms. METHODS: The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively. RESULTS: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells. CONCLUSIONS COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.
Biomarkers, Tumor ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Celastrus ; chemistry ; Cell Hypoxia ; drug effects ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cobalt ; Down-Regulation ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; Signal Transduction ; drug effects

Considering that high levels of nitric oxide (NO) exert anti-cancer effect and the derivatives of oleanolic acid (OA) have shown potent anti-cancer activity, new O-vinyl diazeniumdiolate-based NO releasing derivatives (5a-l, 11a-l) of OA were designed, synthesized, and biologically evaluated in the present study. These derivatives could release different amounts of NO in liver cells. Among them, 5d, 5i, 5j, 11g, 11h, and 11j released more NO in SMMC-7721 cells and displayed stronger proliferative inhibition against SMMC-7721 and HepG2 cells than OA and other tested compounds. The most active compound 5j showed almost 20-fold better solubility than OA in aqueous solution, released larger amounts of NO in liver cancer cells than that in normal ones, and exhibited potent anti-hepatocellular carcinoma activity but little effect on the normal liver cells. The inhibitory activity against the cancer cells was significantly diminished upon addition of an NO scavenger, suggesting that NO may contribute, at least in part, to the activity of 5j.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Azo Compounds ; chemistry ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drug Screening Assays, Antitumor ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; pathology ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Nitric Oxide ; chemistry ; Nitric Oxide Donors ; chemical synthesis ; chemistry ; pharmacology ; Oleanolic Acid ; analogs & derivatives ; chemistry ; pharmacology

OBJECTIVETo investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo.

METHODSTransonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.

RESULTSHPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.

CONCLUSIONSToosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; ultrastructure ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Female ; Immunohistochemistry ; Liver Neoplasms ; drug therapy ; pathology ; ultrastructure ; Male ; Melia ; chemistry ; Mice, Inbred BALB C ; Mitochondria ; drug effects ; metabolism ; Neoplasm Transplantation ; Plant Extracts ; therapeutic use ; Reference Standards ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVE: To investigate the effects of LCL161, a Smac mimetic, on the proliferation and apoptosis in hepatocellular carcinoma cells and the underlying mechanisms. 
 METHODS: The effect of LCL161 on the cell viability of HepG2 and SMMC7721 cells was measured by MTT assay. The effect of LCL161 at lower concentrations on the proliferation in hepatocellular carcinoma (HCC) cells was detected by colony formation assay. Apoptosis was assessed by flow cytometry with PI staining. The mitochondrial membrane potential was measured by JC-1 staining. The expression of PARP, p-Akt, cIAP1 and XIAP protein was analyzed by Western blot.
 RESULTS: LCL161 displayed notable antiproliferative activity on HCC cells at the concentrations of 1-16 μmol/L (P<0.01), with IC50 values of 4.3 and 4.9 μmol/L for HepG2 and SMMC7721 cells, respectively, after treatment for 48 h. LCL161 at lower concentrations obviously inhibited the colony formation of HCC cells. LCL161 induced significant apoptosis in HCC cells (P<0.01), and resulted in the apoptotic rate at (1.5±0.8)% or (1.8±0.6)% , (15.2±2.8)% or (12.2±2.4)%, (28.7±3.0)% or (22.4±2.7)%, (34.6±2.3)% or (30.2±2.4)% for HepG2 cells or SMMC7721 cells at the concentration of 0, 2, 4 or 8 μmol/L, respectively. The result of JC-1 staining indicated that the mitochondrial membrane potential of HCC cells was reduced by LCL161. In addition, LCL161 promoted the cleavage of PARP, down-regulated the protein expression of p-Akt, and degraded cIAP1.
 CONCLUSION LCL161 possesses significant anti-proliferative activity and pro-apoptotic action in HepG2 and SMMC7721 cells, which might be correlated with reduction in mitochondrial membrane potential, down-regulation of p-Akt and degradation of cIAP1.
Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; Down-Regulation ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Liver Neoplasms ; Membrane Potential, Mitochondrial ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; Thiazoles ; pharmacology ; Ubiquitin-Protein Ligases ; metabolism ; X-Linked Inhibitor of Apoptosis Protein

OBJECTIVETo investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells.

METHODSMDR HCC Huh7.5.1/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. CCK-8 assay was used to determine the cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the expression level of cancer stem cell marker CD133. Clone formation assay and mouse subcutaneous xenograft tumors were used to investigate the tumorigenicity in vitro and in vivo. Western blotting (WB) was used to analyze the changes of expressions of CD133, Smad3, Bcl-2, Bax and p-Smad3 in different conditions.

RESULTSADM treatment of HCC cells in vitro resulted in a development of subline, Huh7.5.1/ADM cells, with CSC phenotypes: stable MDR phenotype (besides ADMc Huh7.5.1/ADM cells were also more resistant to some other anticancer drugs including VCR, MMC and CTX ) (IC50: 0.215 ± 0.018 vs. 0.123 ± 0.004, 0.145 ± 0.009 vs. 0.014 ± 0.002, 1.021 ± 0.119 vs. 0.071 ± 0.006, 27.007 ± 1.606 vs. 1.919 ± 0.032) (unit: µg/ml) (P<0.05). Huh7.5.1/ADM cells enriched the cancer stem-like cell fraction (CD133-positive subpopulation) (76.06 ± 2.948% vs. 25.38 ± 4.349%) (P<0.05), had stronger tumorigenicity in vivo and colony formation ability, and activated the Smad3 activity. Inhibition of Smad3 activity by SIS3 decreased stemness of the Huh7.5.1/ADM cells: CD133-positive subpopulation (48.49 ± 2.304% vs. 76.06 ± 2.948%) (P<0.05); ADM IC50: (0.112 ± 0.019 vs. 0.215 ± 0.018), VCR IC50 (0.065 ± 0.013 vs. 0.145±0.009), MMC IC₅₀ (0.749 ± 0.121 vs. 1.021 ± 0.119), CTX IC50 (10.576 ± 1.248 vs. 27.007 ± 1.606) (unit: µg/ml) (P<0.05), and decreased tumorigenicity and colony formation ability.

CONCLUSIONSIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.

AC133 Antigen ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; pathology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Glycoproteins ; metabolism ; Heterografts ; Humans ; Isoquinolines ; pharmacology ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; drug effects ; Peptides ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyridines ; pharmacology ; Pyrroles ; pharmacology ; Smad3 Protein ; antagonists & inhibitors ; metabolism ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma.

METHODSThe inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis.

RESULTSThe inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs.

CONCLUSIONTAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.

Acetogenins ; chemistry ; pharmacology ; therapeutic use ; Animals ; Annona ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Organ Specificity ; drug effects ; Spleen ; drug effects ; Thymus Gland ; drug effects ; Xenograft Model Antitumor Assays

OBJECTIVETo evaluate the effect of Bear Bile Powder(, BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity.

METHODSHepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay.

RESULTSThe treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells (P<0.01). In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells (including early and late apoptotic cells) were 18.0%±1.3%, 34.9%±2.2%, 33.9%±2.8%, 37.4%±2.8% and 46.0%±2.5%, respectively (P<0.05); and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%, 8.5%±0.8%, 13.5%±1.6%, 17.6%±2.3% and 46.7%±3.6%, respectively (P<0.01). Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05).

CONCLUSIONSBBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.

Animals ; Apoptosis ; drug effects ; Bile ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Caspases ; metabolism ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Ursidae

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in Korea. Curative treatment is only possible when the disease is diagnosed at the early stage. The prognosis of patients with HCC is even dismal in advanced stages. No systemic cytotoxic chemotherapy has proven to be beneficial in overall survival. Recently, the understanding of the molecular pathogenesis led to the development of new therapies. With the evidence of dysregulation of critical genes associated with cellular proliferation, growth factor signaling, cell cycling, apoptosis, and angiogenesis in HCC, a number of molecular target agents are under clinical trials. Sorafenib is the first systemic anticancer drug which has proven to gain survival benefit in the global as well as Asia-Pacific trials. However, the survival gain is still modest, and further efforts to improve outcomes in patients with HCC are necessary by developing novel drugs or combining other forms of therapies. This article will review signaling pathways in HCC and introduce molecular target agents under investigation currently.
Antineoplastic Agents/therapeutic use ; Carcinoma, Hepatocellular/*drug therapy/metabolism/pathology ; Humans ; Liver Neoplasms/*drug therapy/metabolism/pathology ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Niacinamide/analogs & derivatives/therapeutic use ; Phenylurea Compounds/therapeutic use ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Wnt Proteins/antagonists & inhibitors/metabolism

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in Korea. Curative treatment is only possible when the disease is diagnosed at the early stage. The prognosis of patients with HCC is even dismal in advanced stages. No systemic cytotoxic chemotherapy has proven to be beneficial in overall survival. Recently, the understanding of the molecular pathogenesis led to the development of new therapies. With the evidence of dysregulation of critical genes associated with cellular proliferation, growth factor signaling, cell cycling, apoptosis, and angiogenesis in HCC, a number of molecular target agents are under clinical trials. Sorafenib is the first systemic anticancer drug which has proven to gain survival benefit in the global as well as Asia-Pacific trials. However, the survival gain is still modest, and further efforts to improve outcomes in patients with HCC are necessary by developing novel drugs or combining other forms of therapies. This article will review signaling pathways in HCC and introduce molecular target agents under investigation currently.
Antineoplastic Agents/therapeutic use ; Carcinoma, Hepatocellular/*drug therapy/metabolism/pathology ; Humans ; Liver Neoplasms/*drug therapy/metabolism/pathology ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Niacinamide/analogs & derivatives/therapeutic use ; Phenylurea Compounds/therapeutic use ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Wnt Proteins/antagonists & inhibitors/metabolism