Bornyl caffeate induces apoptosis in human breast cancer cells in vitro via the ROS- and JNK-mediated pathways

Aim： To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms.
Methods： Human breast cancer cell line MCF-7 and other tumor cell lines （T47D, HepG2, HeLa, and PC12） were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential （MMP） was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species （ROS） were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis.

Results： Bornyl caffeate （10, 25, and 50 μmol/L） suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 （p38 inhibitor）, SP600125 （JNK inhibitor）, z-VAD （pan-caspase inhibitor） or the thiol antioxidant L-NAC.

Conclusion： Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways.     

Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo

Aim:

To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo.

Methods:

Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression.

Results:

Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC₅₀ value=40 μmol/L). Similarly, MMC inhibited the cell viability with an IC₅₀ value of 5 μmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 μmol/L), the IC₅₀ value of MMC was reduced to 5 μmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G₁ arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway.

Conclusion:

The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.     

RIP3 overexpression sensitizes human breast cancer cells to parthenolide in vitro via intracellular ROS accumulation

Aim： Receptor-interacting protein 3 （RIP3） is involved in tumor necrosis factor receptor signaling, and results in NF-KB-mediated prosurvival signaling and programmed cell death. The aim of this study was to determine whether overexpression of the RIP3 gene could sensitize human breast cancer cells to parthenolide in vitro. Methods： The expression of RIP3 mRNA in human breast cancer cell lines （MCF-7, MDA-MB-231, MDA-MB-435 and T47D） was detected using RT-PCR. Both MDA-MB-231 and MCF-7 cells were transfected with RIP3 expression or blank vectors via lentivirus. Cell viability was measured with MTT assay; intracellular ROS level and cell apoptosis were analyzed using flow cytometry. Results： RIP3 mRNA expression was not detected in the four human breast cancer cell lines tested. However, the transfection induced higher levels of RIP3 protein in MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of RIP3 decreased the IC50 values of parthenolide from 17.6 to 12.6 μmol/L in MCF-7 cells, and from 16.6 to 9.9 μmol/L in MDA-MB-231 cells. Moreover, overexpression of RIP3 significantly increased parthenolide-induced apoptosis and ROS accumulation in MCF-7 and MDA-MB-231 cells. Pretreatment with N-acetyl-cysteine abrogated the increased sensitivity of RIP3-transfected MCF-7 and MDA-MB-231 cells to parthenolide. Conclusion： Overexpression of RIP3 sensitizes MCF-7 and MDA-MB-231 breast cancer cells to parthenolide in vitro via intracellular ROS accumulation.     

Fluopsin C induces oncosis of human breast adenocarcinoma cells

Aim:

Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro.

Methods:

Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (Δψm). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining.

Results:

Fluopsin C (0.5-8 μmol/L) reduced the cell viability in dose- and time-dependent manners. Its IC₅₀ values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 μmol/L, respectively. Fluopsin C (2 μmol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and Δψm. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC₅₀ values at 24 h of 2.7 and 2.4 μmol/L, respectively.

Conclusion:

Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored.     

Modulation of the pentose phosphate pathway alters phase I metabolism of testosterone and dextromethorphan in HepG2 cells

Aim:

The pentose phosphate pathway (PPP) is involved in the activity of glucose-6-phosphate dehydrogenase (G6PD) and generation of NADPH, which plays a key role in drug metabolism. The aim of this study was to investigate the effects of modulation of the PPP on drug metabolism capacity in vitro.

Methods:

A pair of hepatic cell lines, ie, the cancerous HepG2 cells and normal L02 cells, was used. The expression of CYP450 enzymes, p53 and G6PD in the cells were analyzed. The metabolism of testosterone (TEST, 10 μmol/L) and dextromethorphan (DEM, 1 μmol/L), the two typical substrates for CYP3A4 and CYP2D6, in the cells was examined in the presence of different agents.

Results:

Both the expression and metabolic activities of CYP3A4 and CYP2D6 were considerably higher in HepG2 cells than in L02 cells. The metabolism of TEST and DEM in HepG2 cells was dose-dependently inhibited by the specific CYP3A4 inhibitor ketoconazole and CYP2D6 inhibitor quinidine. Addition of the p53 inhibitor cyclic PFT-α (5, 25 μmol/L) in HepG2 cells dose-dependently enhanced the metabolism of DEM and TEST, whereas addition of the p53 activator NSC 66811 (3, 10, 25 μmol/L) dose-dependently inhibited the metabolism. Furthermore, addition of the G6PD inhibitor 6-aminonicotinamide (5, 15 μmol/L) in HepG2 cells dose-dependently inhibited the metabolism of DEM and TEST, whereas addition of the PPP activity stimulator menadione (1, 5, 15 μmol/L) dose-dependently enhanced the metabolism.

Conclusion:

Modulation of p53 and the PPP alters the metabolism of DEM and TEST, suggesting that the metabolic flux pattern of PPP may be closely involved in drug metabolism and the individual variance.     

A novel sulfonamide agent,MPSP-001, exhibits potent activity against human cancer cells in vitro through disruption of microtubule

Aim:

To evaluate the anti-cancer effects of a new sulfonamide derivative, 2-(N-(3-chlorophenyl)-4-methoxyphenylsulfonamido)-N-hydroxypropanamide (MPSP-001).

Methods:

Human cancer cell lines (HepG2, THP-1, K562, HGC-27, SKOV3, PANC-1, SW480, Kba, HeLa, A549, MDA-MB-453, and MCF-7) were examined. The cytotoxicity of MPSP-001 was evaluated using the WST-8 assay. Cell cycle distribution was examined with flow cytometry. Mitotic spindle formation was detected using immunofluorescence microscopy. Apoptosis-related proteins were examined with Western blot using specific phosphorylated protein antibodies. Competitive tubulin-binding assay was performed to test whether the compound competitively bound to the colchicine site. Molecular docking was performed to explore the possible binding conformation.

Results:

MPSP-001 potently inhibited the growth of the 12 different types of human cancer cells with the IC₅₀ values ranging from 1.9 to 15.7 μmol/L. The compound exerted potent inhibition on the drug-resistant Kb/VCR and MCF-7/ADR cells, as on Kba and MCF-7 cells. In HeLa, HGC-27, A549, and other cells, the compound (5 μmol/L) caused cell cycle arrest at the G₂/M phase, and subsequently induced cell apoptosis. In Hela cells, it prevented the mitotic spindle formation. Furthermore, the compound dose-dependently inhibited polymerization of tubulin in vitro, and directly bound to the colchicine-site of β-tubulin. Molecular docking predicted that the compound may form two hydrogen bonds to the binding pocket. The compound showed synergistic effects with colchicine and taxol in blocking mitosis of HeLa cells.

Conclusion:

MPSP-001 shows a broad-spectrum of anti-tumor efficacy in vitro and represents a novel structure with anti-microtubule activity.     

Isothiafludine,a novel non-nucleoside compound,inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation

Aim： To investigate the action of isothiafludine （NZ-4）, a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus （HBV） in vitro and in vivo. Methods： HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA （pgRNA） and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 （25, 50 or 100 mg.k~l{1-1） for 15 d. Results： NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC~o value of 1.33 pmol/L, whereas the compound inhibited the cell viability with an IC5o value of 50.4 pmol/L. Furthermore, NZ-4 was active against the replication of various drug- resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 （5, 10, and 20 pmol/L） concentration- dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. Conclusion： NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat H BV infection.     

A novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN sustains high growth of breast cancer cells

Aim:

To investigate the endogenous signaling pathways associated with high proliferation potential of breast cancer cells.

Methods:

Breast cancer cell lines LM-MCF-7 and MCF-7 with high and low proliferation capability were used. The promoter activity of fatty acid synthase (FASN) was examined using luciferase reporter gene assay. The expression level of FASN mRNA was measured using RT-PCR and real time PCR, respectively. The level of leukotriene B4 (LTB4) was determined with ELISA. The expression levels of 5-lipoxygenase (5-LOX) was analyzed using RT-PCR and Western blot, respectively. 5-Bromo-20-deoxyuridine (BrdU) incorporation assay was used to study the proliferation of LM-MCF-7 and MCF-7 cells.

Results:

The promoter activity of FASN was significantly higher in LM-MCF-7 cells than MCF-7 cells. Treatment of LM-MCF-7 cells with ERK1/2 inhibitor PD98059 (30–50 μmol/L) or LOX inhibitor NDGA (25 μmol/L) abolished the activation of FASN. Moreover, treatment of LM-MCF-7 cells with the specific 5-LOX inhibitor MK-886 (20–40 μmol/L) or 5-LOX siRNA (50–100 nmol/L) decreased the promoter activity of FASN. The level of LTB4, the final metabolite produced by 5-LOX, was significantly higher in LM-MCF-7 cells than MCF-7 cells. Administration of exogenous LTB4 (1–10 nmol/L) was able to stimulate the promoter activity of FASN in MCF-7 cells. Treatment of LM-MCF-7 cells with the FASN inhibitor cerulenin (10 μmol/L) reduced all the levels of p-ERK1/2, 5-LOX, and LTB4. Treatment of LM-MCF-7 cells with cerulenin, PD98059, or MK-886 abolished the proliferation. Administration of exogenous LTB4 (10 nmol/L) significantly increased BrdU incorporation in MCF-7 cells.

Conclusion:

These results suggest a novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN contributes to the sustaining growth of breast cancer LM-MCF-7 cells.     

MG132, a proteasome inhibitor,enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression

Aim:

Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro.

Methods:

HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9.

Results:

Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03–3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone.

Conclusion:

Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.     

Bigelovin inhibits STAT3 signaling by inactivating JAK2 and induces apoptosis in human cancer cells

Aim:

To study the function and mechanism of bigelovin, a sesquiterpene lactone from the flower of Chinese herb Inula hupehensis, in regulating JAK2/STAT3 signaling and cancer cell growth.

Methods:

HepG2 cells stably transfected with the STAT3-responsive firefly luciferase reporter plasmid (HepG2/STAT3 cells), and a panel of human cancer cell lines were used to identify active compounds. Cell viability was measured using MTT assay. Western blotting was used to detect protein expression and phosphorylation. Kinase assays were performed and the reaction between bigelovin and thiol-containing compounds was analyzed with LC-MS.

Results:

Bigelovin (1–50 μmol/L) dose-dependently inhibited the IL-6-induced STAT3 activation in HepG2/STAT3 cells (IC₅₀=3.37 μmol/L) and the constitutive STAT3 activation in A549 and MDA-MB-468 cells. Furthermore, bigelovin dose-dependently inhibited JAK2 phosphorylation in HeLa and MDA-MB-468 cells, as well as the enzymatic activity of JAK2 in vitro (IC₅₀=44.24 μmol/L). Pretreatment of the cells with DTT (500 μmol/L) or GSH (500 μmol/L) eliminated the inhibitory effects of bigelovin on the IL-6-induced and the constitutive STAT3 activation. The results in LC-MS analysis suggested that bigelovin might react with cysteine residues of JAK2 leading to inactivation of JAK2. Bigelovin (5 and 20 μmol/L) had no effects on the signaling pathways of growth factors EGF, PDGF or insulin. Finally, bigelovin suppressed the cell viability and induced apoptosis in 10 different human cancer cell lines, particularly those with constitutively activated STAT3.

Conclusion:

Bigelovin potently inhibits STAT3 signaling by inactivating JAK2, and induces apoptosis of a variety of human cancer cells in vitro.     

Intratumoral estrogen sulfotransferase induction contributes to the anti-breast cancer effects of the dithiocarbamate derivative TM208

Aim:

Sulfotransferase-catalyzed sulfation is the most important pathway for inactivating estrogens. Thus, activation of estrogen sulfotransferase (EST) may be an alternative approach for the treatment of estrogen-dependent breast cancer. In this study we investigated the involvement of EST in anti-breast cancer effects of the dithiocarbamate derivative TM208 in vitro and in vivo.

Methods:

The viability of human breast cancer MCF-7 cells was determined using a SBB assay. Nude mice bearing MCF-7 cells were orally administered TM208 (50 and 150 mg·kg⁻¹·d⁻¹) for 18 days. The xenograft tumors and uteri were collected. The mRNA expression of EST was examined with real-time PCR. EST protein was detected with Western blot, ELISA or immunohistochemical staining assays. A radioactive assay was used to measure the EST activity. Uterotropic bioassay was used to examine the uterine estrogen responses.

Results:

Treatment with TM208 (10, 15 and 20 μmol/L) concentration-dependently increased EST expression in MCF-7 cells in vitro. Co-treatment with triclosan, an inhibitor of sulfonation, abolished TM208-induced cytotoxicity in MCF-7 cells. TM208 exhibited an apparent anti-estrogenic property: it exerted more potent cytotoxicity in E2-treated MCF-7 cells. In the nude mice bearing MCF-7 cells, TM208 administration time-dependently increased the expression and activity of EST, and blocked the gradual increase of E2 concentration in the xenograft tumors. Furthermore, TM208 administration blocked the estrogens-stimulated uterine enlargement. Tamoxifen, a positive control drug, produced similar effects on the expression and activity of EST in vitro and in vivo.

Conclusion:

The induction of EST and reduction of estrogen concentration contribute to the anti-breast cancer action of TM208 and tamoxifen. TM208 may be developed as anticancer drug for the treatment of estrogen receptor-positive breast cancer.     

Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208

Aim： To investigate the effects of a novel dithiocarbamate derivative TM208 on human breast cancer cells as well as the pharmacoki- netic characteristics of TM208 in human breast cancer xenograft mice. Methods： Human breast cancer MCF-7 and MDA-MB-231 cells were treated with TM208 or a positive control drug tamoxifen. Cell pro- liferation was examined using SRB and colony formation assays. Cell apoptosis was analyzed with Annexin V-FITC/PI staining assay. Protein expression was examined with Western blot, ELISA and immunohistochemical analyses. MCF-7 breast cancer xenograft nude mice were orally administered TM208 （50 or 150 mg.k$1〈1-1） or tamoxifen （50 mg.kgl〈t-~） for 18 d. On d 19, the tumors were collected for analyses. Blood samples were collected from the mice treated with the high dose of TM208, and plasma concentrations of TM208 were measured using LC-MS/MS. Results： Treatment of MCF-7 and MDA-MB-231 cells with TM208 dose-dependently inhibited the cell proliferation and colony formation in vitro （the IC~o values were 36.38＋3.77 and 18.13＋0.76 pmol/L, respectively）. TM208 （20-150pmol/L） dose-dependently induced apoptosis of both the breast cancer cells in vitro. In MCF-7 breast cancer xenograft nude mice, TM208 administration dose-depend- ently reduced the tumor growth, but did not result in the accumulation of TM208 or weight loss. TM208 dose-dependently inhibited the phosphorylation of EGFR and ERK1/2 in both the breast cancer cells in vitro as well as in the MCF-7 xenograft tumor. Conclusion： Inhibition of EGFR autophosphorylation plays an important role in the anticancer effect of TM208 against human breast cancer.     

Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway

Aim:

Telekin, isolated from the Chinese herb Carpesium divaricatum, has shown anti-proliferation effects against various cancer cells, including hepatocellular carcinoma cells. In this study, we investigated the anti-proliferation mechanisms of telekin in human hepatocellular carcinoma HepG2 cells in vitro.

Methods:

HepG2 cells were treated with telekin. Cell viability was evaluated using MTT assay. Flow cytometry was used to measure cell cycle profiles, ROS level and apoptosis. The protein expression levels were analyzed with Western blotting.

Results:

Telekin (3.75–30 μmol/L) dose-dependently inhibited the viability of HepG2 cells and induced l apoptosis. Furthermore, the treatment induced cell cycle arrest at G₂/M phase, accompanied by significantly increased the phosphorylation of Cdc25A and Cdc2, and decreased Cyclin B1 level. Moreover, the treatment significantly stimulated ROS production, and increased the phosphorylation of p38 and MAPKAPK-2 in the cells. Pretreatment with the antioxidant NAC (2.5, 5, and 10 mmol/L), or the p38 MAPK inhibitor SB203580 (2.5 and 5 μmol/L) dose-dependently attenuated these telekin-induced effects in the cells.

Conclusion:

Telekin suppresses hepatocellular carcinoma cells in vitro by inducing G₂/M phase arrest via activating the p38 MAPK pathway.     

Selective estrogen receptor modulator BC-1 activates antioxidant signaling pathway in vitro via formation of reactive metabolites

Aim:

Benzothiophene compounds are selective estrogen receptor modulators (SERMs), which are recently found to activate antioxidant signaling. In this study the molecular mechanisms of antioxidant signaling activation by benzothiophene compound BC-1 were investigated.

Methods:

HepG2 cells were stably transfected with antioxidant response element (ARE)-luciferase reporter (HepG2-ARE cells). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2-ARE cells was suppressed using siRNA. The metabolites of BC-1 in rat liver microsome incubation were analyzed using LC-UV and LC-MS.

Results:

Addition of BC-1 (5 μmol/L) in HepG2-ARE cells resulted in a 17-fold increase of ARE-luciferase activity. Pretreatment with the estrogen receptor agonist E₂ (5 μmol/L) or antagonist ICI 182,780 (5 μmol/L) did not affect BC-1-induced ARE-luciferase activity. However, transfection of the cells with anti-Nrf2 siRNA suppressed this effect by 79%. Addition of BC-1 in rat microsome incubation resulted in formation of di-quinone methides and o-quinones, followed by formation of GSH conjugates. BC-1 analogues with hydrogen (BC-2) or fluorine (BC-3) at the 4′ position did not form the di-quinone methides. Both BC-2 and BC-3 showed comparable estrogenic activity with BC-1, but did not induce ARE-luciferase activity in HepG2-ARE cells.

Conclusion:

Benzothiophene compound BC-1 activates ARE signaling via reactive metabolite formation that is independent of estrogen receptors.     

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

Aim:

To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions.

Methods:

A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination.

Results:

The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R²) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×10³(mol/L)^-1 and 59.5%, respectively.

Conclusion:

Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma.     

Bicyclol protects HepG2 cells against D-galacto-samine-induced apoptosis through inducing heat shock protein 27 and mitochondria associated pathway

Aim:

To study the inducing effect of bicyclol on heat shock protein 27 (HSP27) and its role on anti-apoptosis in HepG2 cells intoxicated with D-galactosamine (D-GaIN).

Methods:

HepG2 cells were treated with various concentrations of bicyclol and then subjected to D-GaIN intoxication. Apoptosis was assayed by hoechst 33258 staining and flow cytometry analysis. HSP27, cytochrome c, apoptosis inducing factor (AIF) and c-Jun N-terminal kinase (JNK) were assayed by Western blot. Heat shock factor 1 (HSF1) was determined by electrophoretic mobility shift assay and the interactions of HSP27 with cytochrome c and AIF were detected by co-immunoprecipitation.

Results:

The results showed that bicyclol induced HSP27 protein and mRNA expression in HepG2 cells in both time- and dose-dependent manners (the maximal response: 1.23 fold increase at 100 μmol/L). Bicyclol treatment stimulated HSF1 activation and increased the HSF1-HSE binding activity (the maximal response: 2.1 fold increase at 100 μmol/L). This inducing effect of bicyclol on HSP27 and HSF1 was markedly blocked by quercetin. Pretreatment of the cells with bicyclol markedly attenuated D-GaIN-induced apoptosis and the release of cytochrome c and AIF from mitochondria. The induced HSP27 by bicyclol suppressed the activity of caspase-3 and the phosphorylation of JNK caused by D-GaIN in HepG2 cells. All the above effect of bicyclol against D-GaIN-induced hepatocytes apoptosis were significantly reversed by quercetin.

Conclusion:

HSP27 is involved in the anti-hepatocytes apoptosis of bicyclol, and this effect of bicyclol-induced HSP27 is mainly through inhibition of mitochondria and JNK apoptotic pathways.     

Antitumor effects of concanavalin A and Sophora flavescens lectin in vitro and in vivo

Aim： Proteins with legume lectin domains are known to possess a wide range of biological functions. Here, the antitumor effects of two representative legume lectins, concanavalin A （ConA） and Sophora flavescens lectin （SFL）, on human breast carcinoma cells were investigated in vitro and in vivo. Methods： Human breast carcinoma MCF-7 cells and human normal mammary epithelial MCF-IOA cells were examined. Cell viability was detected using WST-1 and CCK-8 assays. Cell apoptosis was analyzed with Hoechst 33258 staining. Cell cycle was investigated using flow cytometry. The expression of relevant proteins was measured using Western blotting. Breast carcinoma MCF-7 bearing nude mice were used to study the antitumor effects in vivo. The mice were injected with ConA （40 mg/kg, ip） and SFL （55 mg/kg, ip） daily for 14 d. Results： ConA and SFL inhibited the growth of MCF-7 cells in dose- and time-dependent manners （ICso values were 15 and 20 pg/mL, respectively）. Both ConA and SFL induced apoptotic morphology in MCF-7 cells without affecting MCF-IOA cells. ConA and SFL dose- dependently increased the sub-G1 proportion in MCF-7 cells, while SFL also trip=~ered the G2/M phase cell cycle arrest. Both ConA and SFL dose-dependently increased the activities of caspase-3 and caspase-9 and release of cytochrome c from mitochondria into cytoplasm, up-regulated Bax and Bid, and down-regulated Bcl-2 and BcI-XL in MCF-7 cells. ConA reduced NF-KB, ERK, and JNK levels, and increased p53 and p21 levels, while SFL caused similar changes in NF-KB, ERK, p53, and p21 levels, but did not affect JNK expression. Administration of ConA and SFL significantly decreased the subcutaneous tumor mass volume and weight in MCF-7 bearing nude mice. Conclusion： ConA and SFL exert anti-tumor actions against human breast carcinoma MCF-7 cells both in vitro and in vivo.     

Ganoderma lucidum polysaccharides reduce methotrexate-induced small intestinal damage in mice via induction of epithelial cell proliferation and migration

Aim:

To study the effects of Ganoderma lucidum polysaccharides (Gl-PS) on methotrexate (MTX)-induced small intestinal damage in mice and the underlying mechanisms.

Methods:

BALB/c mice were used for in vivo study. The mice were administered with Gl-PS (50, 100, or 200 mg/kg, ig) for 10 d, and injected with MTX (50 mg/kg, ip) on d 7 and 8 to induce intestinal damage, and then sacrificed on d 11 for morphological study and tissue malondialdehyde (MDA) and superoxide dismutase (SOD) measurements. Before sacrificing, blood samples were collected to analyze immunoglobulin A (IgA). Rat intestinal IEC-6 cells were used for in vitro study. Cell proliferation and migration were assessed using MTT method and an in vitro wounding model, respectively. Transforming growth factor β (TGFβ) protein expression was determined using ELISA assay. Ornithine decarboxylase (ODC) and c-Myc mRNA expression profiles were determined using RT-PCR.

Results:

MTX treatment caused severe mucosal damage, significantly increased small intestine MDA levels, and decreased SOD and serum IgA levels in BALB/c mice. Gl-PS (100 and 200 mg/kg) markedly reversed the MTX effects. In IEC-6 cells, Gl-PS (0.1, 1, and 10 μg/mL) significantly stimulated the cell proliferation. Furthermore, Gl-PS (10 μg/mL) significantly stimulated the cell migration. In addition, Gl-PS (10 and 20 μg/mL) significantly increased the expression of ODC and c-Myc mRNAs. However, Gl-PS (up to 20 μg/mL) had no effect on the expression of TGFβ protein.

Conclusion:

The results suggest that Gl-PS protects small intestine against MTX-induced injury via induction of epithelial cell proliferation and migration.     

Autophagy counteracts apoptosis in human multiple myeloma cells exposed to oridonin in vitro via regulating intracellular ROS and SIRT1

Aim:

To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro.

Methods:

Human multiple myeloma RPMI8266 cells were used. The cell viability was assessed using MTT assay. Morphological changes of apoptosis and autophagy were observed under transmission electron microscope. TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs_605 nm-Anti-LC3 fluorescent probe. Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe. Protein levels of active caspase 3, Beclin 1 and SIRT1 were determined with Western blot analysis.

Results:

Exposure to oridonin (1-64 μmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC₅₀ value of 6.74 μmol/L. Exposure to oridonin (7 μmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells. Both the apoptosis and autophagy were time-dependent, and apoptosis was the main effector pathway of cell death. Exposure to oridonin (7 μmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner. The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis, autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 μmol/L). The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis, which was accompanied by increased intracellular ROS and decreased SIRT1.

Conclusion:

Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein. The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway, whereas the autophagy protects the cells from apoptosis.     

Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells

Aim:

To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata.

Methods:

Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus.

Results:

Treatment of SaOS2 cells with CEP (2.5–20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G₁ phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg⁻¹·d⁻¹, ip for 19 d) significantly reduced the volume and weight of the tumor.

Conclusion:

Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.