Nodosin induces apoptosis of HepG2 cells by increasing Apaf-1 expression and activating caspase-3

AIM:To investigate the effects of nodosin on apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS:HepG2 cells were treated with nodosin at different concentrations (1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L) for 24 h. The morphological changes of the HepG2 cells were observed by Hoechst 33258 staining and electron microscopy. The apoptotic rates were analyzed by flow cytometry. The mRNA expression of apoptotic protease-activating factor-1 (Apaf-1) was detected by RT-qPCR. The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:HepG2 cells showed obvious cell shrinkage and nucleus drift when treated with nodosin as the concentration was increased. Many apoptotic bodies were observed in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups. The mRNA expression of Apaf-1 was increased in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups as compared with control group (P<0.05). The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were increased with the increasing dose of nodosin (P<0.05). CONCLUSION:Nodosin induces the apoptosis of HepG2 cells. This effect was related to increasing Apaf-1 mRNA expression and subsequently promoting the activation of caspase-3.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

Protective effect of senegenin on retinal ganglion cells in oxidative stress induced injury

AIM: To evaluate the effect of senegenin (Sen) on H₂O₂-treated retinal ganglion cells (RGCs) and to explore its underlying mechanisms. METHODS: RGCs were retrograde labeled by injection of fluorogold into the superior colliculi of SD rats on the postnatal day 3. On the postnatal days 6 to 8, the retinas were dissociated with papain and cultured. Primary RGCs cultured in vitro were treated with H₂O₂ and/or various doses of Sen. The viability of RGCs was evaluated by counting the fluorescence-labeled neurons under microscope. The morphological changes of the nuclei in the retinal neurons were observed by Hoechst 33258 staining. Western blotting was applied to determine the expression of cleaved caspase-3, cytochrome C and Bcl-2 in cultured retinal neurons. RESULTS: Compared with the control cells, Sen at doses of 10, 20 or 40 μmol/L had no toxicity to RGCs (P>0.05). However, Sen at doses of 80 and 160 μmol/L had significant toxicity to RGCs (P<0.01). Compared with H₂O₂-injured group, Sen at doses of 10, 20 and 40 μmol/L effectively protected against H₂O₂-induced injury in RGCs (P<0.05) with the best efficiency at 40 μmol/L. Hoechst 33258 staining showed that the neuronal apoptosis caused by H₂O₂ was reduced by Sen. The results of Western blotting showed an up-regulation of Bcl-2, and decreased cytochrome C and cleaved caspase-3 levels by Sen in H₂O₂-treated retinal neurons. CONCLUSION: Sen is able to protect RGCs from H₂O₂-induced injury by enhancing Bcl-2 expression and inhibiting cell apoptosis.     

Role of juglone in regulating oxidative stress and apoptosis in ovarian cancer SKOV3 cells

AIM: To study the cytotoxicity of juglone on ovarian cancer SKOV3 cells. METHODS: The activity of SKOV3 cells was detected by MTT assay. The cells apoptosis was examined by flow cytometry. The level of reactive oxygen species (ROS) was measured by DCF-DA staining. The protein levels of cytochrome C (Cyt C) and activated caspase-3 were evaluated by Western blot. RESULTS: MTT assay showed that juglone significantly inhibited the growth of SKOV3 cells in dose- and time-dependent manners (P<0.05). The early apoptotic rate and late apoptotic rate of SKOV3 cells in 50 μmol/L juglone group at 24 and 48 h were higher than those in control group (P<0.01).Moreover, juglone induced ROS accumulation, and increased the protein levels of Cyt C and activated caspase-3.CONCLUSION: Juglone inhibits the cell growth and induces the apoptosis of SKOV3 cells by ROS accumulation.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Hydrogen sulfide protects H9c2 cardiomyocytes against high glucose-induced injury by inhibiting necroptosis

AIM: To study whether hydrogen sulfide(H₂S) protects H9c2 cardiomyocytes against high glucose(HG)-induced injury by inhibiting necroptosis. METHODS: The protein levels of RIP3(an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot. The cell viability was measured by CCK-8 assay. The intracellular le-vels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP) was examined by rhodamine 123 staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cells were treated with HG(35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h. Pretreatment of the cells with 400μmol/L NaHS(a donor of H₂S) or co-treatment of the cells with necrostatin-1(Nec-1; a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG. Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the generation of ROS and MMP loss. On the other hand, pretreatment with NaHS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes. CONCLUSION: H₂S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

T0901317 induces apoptosis of human breast cancer MDA-MB-231 cells by up-regulating LXRα expression

AIM: To investigate the pro-apoptotic effect of T0901317, an artificial agonist of liver X receptor α (LXRα), on human breast cancer MDA-MB-231 cells and its mechanism. METHODS: MDA-MB-231 cells were treated with different concentrations (0, 10, 20 and 40 μmol/L) of T0901317 for different time (0, 12, 24 and 48 h). The cell apoptosis was determined by Annexin V/propidium iodide staining and Hoechst 33342 staining. The expression of apoptosis-related proteins, such as Bcl-2, caspase 3 and cleaved caspase-3, and LXRα was determined by Western blot. The mRNA expression of Bcl-2 and LXRα was analyzed by RT-qPCR. RESULTS: T0901317 induced the cell apoptosis in a dose-and time- dependent manner. The expression of cleaved caspase-3 and LXRα was up-regulated, but Bcl-2 was down-regulated by T0901317. The mRNA expression of Bcl-2 was down-regulated, while LXRα was up-regulated by T0901317.CONCLUSION: T0901317 up-regulates LXRα expression and induces the apoptosis of MDA-MB-231 cells.     

Effects of propofol on biological characteristics of colorectal cancer cells

AIM: To investigate the effect of propofol on the viability, invasion ability and apoptosis of colorectal cancer cells.METHODS: Propofol at 10, 25, 50 and 100 μmol/L was used to treat LoVo cells for 72 h, and propofol at 100 μmol/L was used to treat the LoVo cells for 12, 24, 48 and 72 h. The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay. The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, Notch1 and hairy and enhancer of split 1 (Hes1) were determined by Western blot.RESULTS: Propofol inhibited LoVo cell viability. The cell invasion ability, S stage cells, and the protein levels of MMP-2, MMP-9, Notch1 and Hes1 in propofol group were significantly lower than those in control group, and the apoptotic rate, G₀/G₁ cells and the protein level of cleaved caspase-3 were significantly higher than those in control group (P<0.01).CONCLUSION: Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells, blocks cell cycle and induces apoptosis. The mechanism is related to down-regulation of Notch1 signaling pathway.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Induction of apoptosis in mouse fibroblast cell line L929 by arachidonic acid

AIM: To observe whether arachidonic acid (AA) could induce apoptosis in mouse fibroblast cell line L929 and the potential mechanism involved. METHODS: The viability and damaged degree of L929 was monitored by MTT and the release of lactate dehydrogenase (LDH). Lipid peroxidation in L929 was measured as malondialdehyde (MDA) content by colorimetric assay. Hoechst 33258 staining was used to observe AA-induced morphological changes. Agarose gel electrophoresis was used to detect DNA fragmentation. RESULTS: Treatment of L929 cell with AA for 24 h, in the range of 40-160 μmol/L, caused a great decrease in cell survival and increased MDA contents and the release of LDH simultaneously (P＜0.01). Cells treated by 160 μmol/L AA showed typical morphological changes of apoptosis. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length fragments was observed 24 h after AA treatment. CONCLUSION: Higher concentration of arachidonic acid (80-160 μmol/L) induced apoptosis in mouse fibroblast cell line L929. The mechanism of its action might be related to lipid peroxidation.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

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.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

N-acetylcysteine protects H9c2 cells against injuries induced by methyl-glyoxal

AIM: To investigate the protective effect of N-acetylcysteine(NAC) on H9c2 cells from injuries induced by methylglyoxal(MG) and the potential mechanism. METHODS: H9c2 cells were divided into control group, MG treatment group, NAC + MG treatment group, SP600125 pretreatment + MG group, NAC group and SP600125 group. The viability of the H9c2 cells was measured by CCK-8 assay. The protein levels of p-JNK and t-JNK were tested by Western blot. The changes of intracellular reactive oxygen species(ROS) were evaluated by 2', 7'-dichlorofluorescein diacetate(DCFH-DA) staining. Mitochondrial membrane potential(MMP) was measured by rhodamine 123(Rh123) staining. The morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining. RESULTS: Du-ring 100~800μmol/L concentration range, MG caused significantly reduced viability of the H9c2 cells in a dose-dependent manner. NAC had a protective effect on H9c2 cells against the injuries induced by MG during 500~1500μmol/L concentration range through raising cell viability, inhibiting cellular oxidative stress and improving MMP(P<0.01). SP600125, an inhibitor of JNK, showed the protective effect similar to NAC on H9c2 cells against MG-induced injuries, including attenuating oxidative stress, improving MMP and suppressing apoptosis.CONCLUSION: N-acetylcysteine offers obvious protective effect on H9c2 cells against the injuries induced by methylglyoxal. The underlying mechanisms may be associated with decreasing the production of ROS, ameliorating MMP, inhibiting the activation of JNK and suppressing apoptosis.     

Artesunate induces apoptosis of human hepatocellular carcinoma HepG2 cells by increasing generation of reactive oxygen species

AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47^phox and p22^phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47^phox and p22^phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.     

Tumor necrosis factor-related apoptosis-inducing ligand participates in injury of vascular endothelial cells induced by rapamycin in vitro

AIM: To investigate the effects of rapamycin on apoptosis, proliferation, migration ability and tumor related apoptosis inducing ligand(TRAIL) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: Cultured HUVECs were treated with rapamycin at the concentrations of 0, 1, 10 and 100 μg/L for 24 h. The cell proliferation was measured by CCK-8 method. The cell migration ability was detected by Transwell chambers and wound healing test. The apoptotic index of HUVECs was quantitatively determined by measuring the activation of caspase-3. The morphological changes of the apoptotic cells were observed by DAPI staining. The expression of TRAIL was detected by Western blotting. RESULTS: A 24 h-incubation with rapamycin(1-100 μg/L) caused significant cell loss associated with the increase in apoptosis, as quantified by the determination of caspase-3 activity(P<0.01) in HUVECs. Obvious apoptotic morphology was observed by DAPI staining in HUVECs incubated with rapamycin. Rapamycin at the concentrations of 1-100 μg/L also impaired the migration ability of HUVECs(P<0.01). In addition, rapamycin(10-100 μg/L) inhibited the proliferation of HUVECs, whereas rapamycin at 1 μg/L had no such effect(P<0.01). Rapamycin(10-100 μg/L) also induced TRAIL expression in a dose-dependent manner(P<0.01). CONCLUSION: Rapamycin induces apoptosis, and inhibits the proliferation and migration of HUVECs. The up-regulation of TRAIL might be related to the injury of vascular endothelial cells caused by rapamycin.     

Effects of curcumin analogue T83 on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM：To compare the effect of T83 (a 4-arylidene curcumin analogue) on the apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance. METHODS：The effects of T83 on the viability, apoptosis, mitochondrial membrane potential (MMP), expression of procaspase-3/procaspase-9/Cyt-C proteins and relative PTEN/Akt/p27 mRNA expression in CNE-2R cells and CNE-2 cells were detected and compared by the methods of MTT assay, Hoechst staining, flow cytometry, Western blotting and qRT-PCR. RESULTS：T83 inhibited the viability of CNE-2R cells with the IC50 of 0.9,0.4 and 0.2 μmol/L for 24 h, 48 h and 72 h,respectively, which was more effective than that inhibiting the viability of CNE-2 cells with the IC50 of 1.8,0.5 and 0.4 μmol/L, respectively. After treated with T83 for 48 h, chromatin condensation, margination and splitting into a massive structure were observed in CNE-2R cells and CNE-2 cells,and the late apoptotic rate of CNE-2R cells was increased from 1.57% to 27.26%, which was higher than that of CNE-2 cells (1.74% to 8.15%). After treated with T83 for 36 h, the MMP in CNE-2R cells decreased by 87.71% and that decreased by 50.47% in CNE-2 cells. After treated with T83 for 48 h, the protein levels of procaspase-3 and procaspase-9 were decreased, and the protein level of Cyt-C was increased, which were more susceptible in CNE-2R cells than those in CNE-2 cells. After treated with T83 for 24 h, the relative mRNA expression of PTEN and p27 was significantly up-regulated, and the mRNA expression of Akt was down-regulated, which were more susceptible in CNE-2R cells than those in CNE-2 cells. CONCLUSION：Compared with CNE-2 cells, the inhibitory effect of T83 on the viability of CNE-2R cells is more specific by starting the mitochondrial apoptotic pathway, which is due to the inhibition of PTEN/Akt/p27 signaling pathway.