Manumycin inhibits activity of pancreatic duct cancer cell line Panc-1 via inducing apoptosis

AIM: In this study, we investigated the anticancer effect and mechanisms of manumycin on pancreatic cancer cell line-Panc-1 and the role of p38MAPK pathway in apoptosis. METHODS: The test of anticancer effect was performed by MTT assay. Apoptosis was induced in the cells by manumycin and then treated with SB203580, a specific p38MAPK inhibitor. A quantitative caspase-3 activity assay kit was used in this experiment. RESULTS: Manumycin (6 μmol/L, 18 μmol/L, 54 μmol/L) significantly inhibited cell growth of pancreatic cancer cell line Panc-1. The inhibition rates 24 h after treatment with 6 μmol/L, 18 μmol/L and 54 μmol/L manumycin were 8.9%, 21.9% and 67.0%, respectively. Compared with the control group, the survival levels of the last two groups were of significant statistical difference (P<0.01). The anticancer effects also showed dosage-effect relationship, the value of IC50 24 h after treatment was 34.7 μmol/L. In addition, this reagent simultaneously activated caspase-3 protein, which was partly blocked by p38MAPK specific inhibitor, SB203580. CONCLUSION: Manumycin exerted anticancer effect on Panc-1 cell line via inducing cell apoptosis, which was partly regulated by p38MAPK.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨ_m), reactive oxygen species (ROS) and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured by the method of colorimetry. The levels of cytochrome C, Bcl-2, Bax, β-catenin and phosphorylated low-density lipoprotein receptor-related protein 6 (p-LRP6) were determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨ_m, and increased the levels of ROS, cytochrome C and[Ca²⁺]_i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.     

Effect of curcumin on the proliferation and apoptosis in LNCap cells

AIM:To observe the effect of curcumin on the proliferation and apoptosis of prostate cancer cell line LNCap. METHODS:LNCap cells were treated with different doses (10 μmol/L, 20 μmol/L, 30 μmol/L, 40 μmol/L) of curcumin and its effects were analyzed in cell growth and apoptosis by microscope, MTT colorimetric assay and flow cytometry. The expression of prostate specific antigen (PSA) was measured by AXSYMTM system-chemical luciferase methods and expression of androgen receptor (AR) was detected by Western blotting. RESULTS:The results showed that curcumin inhibited the proliferation of LNCap cells. The cell growth was inhibited by curcumin in a dose dependent manner and the optimal dose and time was 40 μmol/L, 24 h. Curcumin induced apoptosis in LNCap cells, the most dramatic dose was 40 μmol/L curcumin, at this dose the apoptosis rate was 9.23%. Curcumin inhibited the expression of PSA in LNCap cells and the most dramatic dose and time was 40 μmol/L, 24 h. The PSA in this group was 20% of the control group. Curcumin inhibited the expression of AR on prostate cancer cells. CONCLUSION:Curcumin decreases proliferation and induces apoptosis in LNCap cells in a dose-dependent manner. Curcumin also inhibites the expression of PSA and AR on LNCap cells.     

Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Effects of oleanolic acid on apoptosis and cell cycle of HL-60 cells in vitro

AIM: To study the mechanism of oleanolic acid induced apoptosis and its influence on cell cycle in HL-60 cells in vitro. METHODS: The HL-60 cells were treated with different concentrations of oleanolic acid and then cultured for 12 h, 24 h, 48 h and 72 h, respectively. MTT assay was used to evaluate the inhibitory effect of oleanolic acid on HL-60 cells in vitro. The argarose gel electrophoresis was used to detect the chromatin DNA fragmentation. FACS was used to analyze the cell cycle of HL-60 cells. Western blotting was used to detect the activation of caspase-3 which has been confirmed the last execution of apoptosis pathway. RESULTS: MTT assay showed that oleanolic acid dramatically inhibited the growth of HL-60 cells in vitro, more than 50% HL-60 cells were inhibited when the cells were treated with 80 μmol/L oleanolic acid for 48 h; the apparent DNA ladder was detected after exposure of HL-60 cells to oleanolic acid for 48 h. FACS analysis showed that cell cycle of HL-60 cells was arrested in G₁ phase, the inhibition ratio of HL-60 cells achieved 63.24% and 67.90% after treated with oleanolic acid for 48 h and 72 h correspondingly. Western blotting detected the activation of caspase-3 after exposure of HL-60 cells to 80 μmol/L oleanolic acid for 48 h. CONCLUSION: These results suggest that oleanolic acid induces apoptosis and the cell cycle of HL-60 cells is arrested in G₁ phase.     

Effects of marine fungal metabolites 1386A from the South China Sea on proliferation,apoptosis and mitochondrial membrane potential in gastric cancer cell line MCG-803

AIM: To investigate the effects of marine fungal metabolites 1386A from the South China Sea(1386A) on gastric cancer cell line MCG-803.METHODS: The effects of 1386A on cell viability and cell growth were detected by MTT method and colony-forming assay, respectively. The changes of cell cycle, cell apoptosis and mitochondrial membrane potential were determined by flow cytometry.RESULTS: The IC₅₀ of cell viability on MCG-803 cells after treated with 1386A for 24 h, 48 h and 72 h was 29.70 μmol/L, 14.07 μmol/L or 13.47 μmol/L, respectively. The IC₅₀ of the cell growth on MCG-803 cells after treated with 1386A for 48 h was 3.27 μmol/L. After treated with 1386A for 48 h, the MCG-803 cells in S stage was increased from 25.6% to 56.8%. After treated with 1386A for 24 h, the early apoptosis of MCG-803 cells was increased from 3.34% to 8.39%, and the mitochondrial membrane potential of MCG-803 cells was decreased by 56.26%.CONCLUSION: 1386A has an inhibitory effect on the cell growth in MCG-803 cells through the mitochondrial pathway.     

Epigallocatechin gallate induces apoptosis of ovarian cancer cell line SKOV-3 via regulation of SIRT1-P53 pathways

AIM: To study the effect of epigallocatechin gallate (EGCG) on the growth and apoptosis of ova-rian cancer cell line SKOV-3 and its molecular mechanism. METHODS: SKOV-3 cells were treated with different concentrations of EGCG (0~50 μmol/L), SRT1720 (1 μmol/L) or EX527 (1 μmol/L) for 24 h. The cell activity was evaluated by CCK-8 assay. The apoptosis of SKOV-3 cells was analyzed by flow cytometry. The mRNA expression of Bcl-2 and Bax was detected by real-time PCR. SIRT1 deacetylase fluorometric assay kit was used to detect the activity of SIRT1. The protein levels of SIRT1 and acetylated P53 (Ac-P53) were determined by Western blot. RESULTS: EGCG or EX527 decreased the deacetylase activity and protein expression of SIRT1, and increased the level of Ac-P53 in a dose-dependent manner. Moreover, SRT1720 abrogated the effects of EGCG on the activity, apoptosis and SIRT1-P53 pathways in ovarian cancer cell line SKOV-3. CONCLUSION: EGCG inhibits the activity and induces apoptosis of ovarian cancer cell line SKOV-3 by regulating SIRT1-P53 pathways.     

Manumycin inhibits the activity of breast cancer cell line SK-BR-3 via inducing apoptosis

AIM: To investigate the anticancer effect of manumycin on abdominal metastatic breast cancer cell line - SK-BR-3 and its relationship with p38 MAPK. METHODS: The test of anticancer effect was performed by the method of MTT, apoptosis induced by manumycin and affected by SB203580, a specific p38 MAPK inhibitor, were examined by caspase-3 activity assay kit, and the protein expression was detected by immunoblotting assay. RESULTS: The inhibition rates at 24 h after treatment with manumycin of 6 μmol/L, 18 μmol/L, 54 μmol/L were (7.4±3.9)%, (21.0±4.4)% and (64.7±4.1)%, respectively and showed dosage-effect relationship. Compared with the control group, the survival rates of the last two treatment groups were decreased significantly (P<0.01). The value of IC₅₀ 24 h after treatment with manumycin was 42.5 μmol/L. Manumycin simultaneously activated caspase-3 protein, which was partly blocked by p38 MAPK inhibitor, SB203580. The results of immunoblotting showed that manumycin increased p38 MAPK protein phosphorylation. CONCLUSION: Manumycin exerts anticancer effect on SK-BR-3 cell line via inducing cell apoptosis, which is partly regulated by p38 MAPK.     

Effects of andrographolide on invasion and apoptosis of ovarian cancer SKOV-3 cells

AIM:To investigate the effects of andrographolide on the invasion and apoptosis of ovarian cancer cell line SKOV-3,and to explore the possible mechanisms.METHODS:SKOV-3 cells were treated with different concentrations (0,5,10,20 or 40 μmol/L) of andrographolide for different time (12,24,36 or 48 h),and then the cell viability was determined by CCK-8 assay.The cell invasion ability was analyzed by Transwell assay and cell apoptosis was detected by TUNEL staining.The protein levels of p-PI3K,p-Akt and p-mTOR were examined by Western blot.RESULTS:The results of CCK-8 assay revealed that andrographolide inhibited the growth of SKOV-3 cells in a dose-and time-dependent manner.Treatment with andrographolide at 20 μmol/L for 36 h significantly decreased the invasion ability of SKOV-3 cells,while increased cell apoptosis.In addition,the protein levels of p-PI3K,p-Akt and p-mTOR were reduced after andrographolide treatment.CONCLUSION:Andrographolide inhibits the growth and invasion of ovarian cancer SKOV-3 cells by suppression of PI3K/Akt/mTOR signaling pathway.     

Effect of proteasome inhibitor MG132 on proliferation and cell cycle distribution of NK/T cell lymphoma cells

AIM: To study the influence of MG132 on the proliferation and cell cycle distribution of NK/T cell lymphoma cells, and to investigate the potential role of proteasome inhibitor on the treatment of NK/T cell lymphoma. METHODS: NK/T cell lymphoma cells HANK1 were treated with proteasome inhibitor MG132, and the proliferation was evaluated by MTT assay. The morphological changes were observed under inverse microscope. The cell cycle distribution and apoptosis were detected using flow cytometry. RESULTS: The growth inhibitory rate of HANK1 cells was(57.72±7.44)% after cultured for 24 h with 1 μmol/L MG132 and was just(3.98±0.07)% after cultured for 24 h with 0.1 μmol/L MG132. The positive relationship between the concentration of MG132 and growth inhibitory rate was observed. On the other hand, after cultured for 24 h with 1μmol/L MG132, the cells in G1 and G2 phases were(72.33±3.44)% and(12.86±1.29)%, respectively, much higher than those in control group(63.63%±2.29% and 7.94%±1.91%, respectively). The early and late apoptosis rates in MG132 group were 33.57%±2.10% and 16.66%±0.47%, respectively, much higher than that in control group (7.18%±0.82% and 3.81%±1.06%, respectively). CONCLUSION: MG132 inhibits cell proliferation and induces cell cycle arrested at G1 and G2 phases, and cell apoptosis in NK/T cell lymphoma cells in a concentration dependent manner. Proteasome inhibitor may be a good drug to treat patients with advanced NK/T cell lymphomas.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Selenium dioxide induces leukemia cell apoptosis and changes of intracellular reactive oxygen species (ROS) and Ca2+ levels

AIM: The effects of selenium dioxide (SeO2) on proliferation, apoptosis, intracellular reactive oxygen species (ROS) and Ca2+ levels in three leukemia cell lines NB4, K562 and HL-60 were investigated. METHODS: Three leukemia cell lines were treated with 3-30 μmol/L SeO2. Flow cytometry was used to detect apoptosis rate, and analyze the changes of ROS and Ca2+ level within cells. RESULTS: SeO2 at 10 and 30 μmol/L inhibited proliferation in three leukemia cell lines. Treatment with 30 μmol/L SeO2 for 48 h induced 54.0%, 46.5%, 49.6% apoptosis in NB4, K562, and HL-60 cells, respectively, and also markedly decreased ROS and Ca2+ levels among three cell lines. The rate of ROS positive cells in NB4 and HL-60 decreased with the increase in SeO2 concentrations. ROS was clearly reduced with 30 μmol/L SeO2 in K562. Ca2+ levels were tardily declined with 10, 30 μmol/L SeO2 in NB4 and HL-60 cells. Ca2+ levels were clearly reduced with 30 μmol/L SeO2 in K562. CONCLUSION: SeO2 induces apoptosis in three leukemia cells. The declines of intracellular ROS and Ca2+ levels are involved in apoptosis induced by SeO2.     

Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages

AIM: To investigate the effects and possible mechanisms of quercetin (Que) on endoplasmic reti-culum stress (ERS)-related apoptosis induced by thapsigargin (TG) in RAW264.7 cells. METHODS: ER stress of RAW264.7 cells were induced by TG at concentration of 1 μmol/L for 24 h. After treated with different concentrations of Que (80, 120 and 160 μmol/L), the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) were determined by flow cytometry, and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by Western blotting. The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3-kinase (PI3K) inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting. RESULTS: Que suppressed ER stress-related injury induced by TG in RAW264.7 cells. Compared with TG group, the cell viability increased (P<0.05), apoptotic rate and [Ca²⁺]_i decreased (P<0.05) and the changes of apoptotic morphology were alleviated. The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que (P<0.05). The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002 (P<0.05), but they failed to exhibit additive suppression. CONCLUSION: Que suppresses the ER stress induced by TG in RAW264.7 cells. The protective effect may be related to its suppression on PI3K signaling pathway.     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Effects of ouercetin on lipopolysaccharide induced hepatocyte injury in vitro

AIM: To study the effects of quercetin on lipopolysaccharide (LPS) induced hepatocyte injury and the expression of TNF-α in vitro. METHODS: Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. LPS at concentration of 40 mg/L was used to induce injury to the cultured cells, and 0.5-10 μmol/L quercetin was added at the same time. After 24 h of incubation, the cell apoptosis rates were detected by MTT and PI-AnnexinV. LDH and TNF-α were measured by kits. RESULTS: 40 mg/L LPS caused a 27% growth inhibition. The apoptosis rate was 30.2%. LDH leakage was 20 folds higher than normal. TNF-α expression significantly increased. Treated with quercetin at doses of 0.5-10 μmol/L, the apoptosis rate, LDH leakage and TNF-α expression in hepatocytes were attenuated in a dose dependent manner. CONCLUSION: 0.5-10 μmol/L of quercetin protects hepatocytes from injury induced by LPS, which is associated with suppression of the inflammatory cytokine TNF-α.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Effect of vitamin K3 on apoptosis induced by androgen-independent prostate cancer cell PC-3M

AIM: To study the effect of vitamin K₃ (VK₃) on the induction of apoptosis in androgen-independent prostate cancer cell PC-3M in vitro.METHODS: Cell viability was estimated by MTT assay. AO/EB staining was performed to detect apoptotic cells. Apoptosis and the changes of cell cycle were detected by flow cytometry. NAC was used to observe the effect of growth inhibition by VK₃. RT-PCR was used to confirm the changes in gene expression. Levels of intracellular peroxides were estimated by using an oxidation-sensitive fluorescent probe DCFH-DA. RESULTS: PC-3M cells growth was significantly inhibited by VK₃ (≥60 μmol/L, P<0.05). The inhibitory effect was time and dosage dependent. The result of AO/EB staining showed that apoptosis of PC-3M cells were induced by VK₃. A typical subdiploid peak before G₀/G₁ phase was observed after treated for 12 h with VK₃ (60 μmol/L) by flow cytometry. The effect of growth inhibition treated with VK₃ was antagonized by antioxygen NAC (5, 10, 20, 40, 80 μmol/L). An increase in the level of DCF fluorescence after PC-3M cells were treated for 1-2 h with VK₃ was observed. Antioxidase GSH-Px and CAT were run-down after treated with VK₃. CONCLUSION: The results indicate that apoptosis in PC-3M cells is induced through oxidative stress by VK₃.