OBJECTIVETo explore the effect and mechanism of DNA polymerase β expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone.

METHODSPolβ wild-type cells (polβ+/+), polβ overexpressed cells (polβ oe) and polβ null cells (polβ-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone.

RESULTSWith the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with polβ+/+ cells, the rate of apoptosis in polβ-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with polβ+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in polβ-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with polβ+/+ cells, fluorescent intensity of polβ-/- cells treated with different dosages of hydroquinone increased, while which of polβ oe cells decreased (P < 0.05). Compared with polβ+/+ cells, ·OH level of polβ-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of polβ oe cells decreased sharply (P < 0.05). Under the same concentrations of hydroquinone, the activity of SOD and GSH-Px were decreased most rapidly in polβ-/- cells. The activity of SOD and GSH-Px in polβ oe cells decreased slower than in the polβ-/- cells.

CONCLUSIONHydroquinone could induced apoptosis by the generation of ROS and decrease of ΔΨm; polβ could protect cells from apoptosis induced by hydroquinone through decrease of ROS level and depolarization of mitochondria.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA Polymerase beta ; metabolism ; Hydroquinones ; toxicity ; Membrane Potential, Mitochondrial ; drug effects ; Mice

This study was aimed to investigate the changes of mitochondrial membrane potential (DeltaPsim) of HL-60 cells induced by cytarabine and the correlation between mitochondrial membrane potential and apoptosis of HL-60 cells. HL-60 cells were stained with Rhodamine 123; change of mitochondrial membrane potential of HL-60 cells was detected by flow cytometry. AO/EB staining and flow cytometry were used to examine the apoptosis of HL-60 cells. The results showed that the levels of HL-60 cell DeltaPsim in experimental groups decreased after cultured for 6 hours. In Ara-C 0.05 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 117.9+/-7.6, 100.9+/-7.7, 87.6+/-10.7, respectively, there was significant difference between the different culture groups (p<0.05). In Ara-C 0.1 mg/ml group, rhodamine 123 fluorescence intensity in mitochondria of HL-60 cells at 6, 12, 24 hours were 111.9+/-10.1, 86.6+/-9.2, 68.4+/-12.2, respectively, there was significant difference between the different culture groups (p<0.05); rhodamine 123 fluorescence intensity was significantly different between the two groups at 12, 24 hours (p<0.05). In Ara-C 0.05 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (41.2+/-3.0)%, (53.7+/-5.1)%, (65.8+/-2.6)% respectively, there was significant difference between the different culture groups (p<0.01); In Ara-C 0.1 mg/ml group, the apoptosis rate of HL-60 cells at 6, 12, 24 hours were (45.7+/-4.1)%, (58.2+/-4.3)%, (70.1+/-2.3)% respectively, there was significant difference between the different culture groups (p<0.01); the apoptosis rates showed no significantly difference between the two groups at same time. The changes of mitochondrial membrane potential and apoptosis rate of HL-60 cells were significantly negatively correlated. In Ara-C 0.05 mg/ml group, r was -0.89, p<0.01, while in Ara-C 0.1 mg/ml group, r was -0.76, p<0.01. It is concluded that the mitochondrial membrane potential on HL-60 cells decrease at HL-60 cells apoptosis induced by Ara-C, therefore the reduction of mitochondrial membrane potential may be one of the important mechanisms at the induced apoptosis.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; drug effects

OBJECTIVETo study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism.

METHODSMTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay.

RESULTSMTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells.

CONCLUSION3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Female ; Glycolysis ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Pyruvates ; pharmacology ; Reactive Oxygen Species ; metabolism

The aim of the present study was to investigate the anti-proliferation and pro-apoptosis effect of Coix lachrymajobi L varma-yuan on acute T lymphoblast leukemia cell line Jurkat cells and its mechanism. Jurkat cells were treated with Coix lachrymajobi L varma-yuan of various concentrations (0, 0.4, 0.8, 1.6 mg/ml) for 24h. The inhibitory ratio was measured by Cell Counting Kit-8. The effects of Coix lachrymajobi L varma-yuan on apoptosis of Jurkat cells were determined by Hoechst 33258, PI and Annexin V-FITC/PI double staining. The mitochondrial membrane potential was analyzed by JC-1 staining. The results demonstrated that Coix lachrymajobi L varma-yuan inhibited the proliferation of Jurkat cells, and induced chromatin condensation and fragmentation (characteristic of apoptosis) and loss of mitochondrial membrane potential. In conclusion, Coix lachrymajobi L varma-yuan can inhibit the cell proliferation and induce the apoptosis of Jurkat cells. These effects relate to loss of mitochondrial membrane potential. These results suggest that Coix lachrymajobi L varma-yuan may be of value in treating lymphoma.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Coix ; chemistry ; Humans ; Jurkat Cells ; Membrane Potential, Mitochondrial ; Plant Oils ; pharmacology

OBJECTIVETo study the apoptotic effect and mechanisms of methylmercury (MeHg) on HL-7702 cell line in vitro.

METHODSIn this study, the cell apoptosis was observed by AO/EB method and FCM method; the mitochondrial membrane potential was detected by FCM; and the expression of proteins related to apoptosis was measured by immunocytochemical method.

RESULTSAfter exposure to MeHg for 24 h in different doses, apoptotic rate ascended with the increasing of MeHg concentration. By AO/EB method, cell apoptotic ratio of negative control group was (2.62 +/- 0.19)%, cell apoptotic ratio of 10-50 micromol/L exposure groups were (7.97 +/- 0.64)%, (12.66 +/- 0.76)%, (19.16 +/- 0.87)%, (18.42 +/- 0.88)%, and (11.52 +/- 0.63)%, there were significant differences between the exposure and negative control groups (q values were 17.057, 32.009, 52.732, 50.373, 28.375; P<0.05). Mitochondrial membrane potential descended with the increase of MeHg, mitochondrial membrane potential of negative control group was (10.23 +/- 3.43) mV, mitochondrial membrane potential of 10-50 micromol/L exposure groups were (3.25 +/- 0.66), (3.03 +/- 0.35), (1.68 +/- 1.26), (1.69 +/- 1.13) and (1.77 +/- 0.88) mV, and there was significant differences between exposure and negative control groups (q values were 9.569, 9.871, 11.722, 11.708, 11.598; P<0.05). The expression of Bax, Bcl-2, CytC, Caspase-3 and AIF enhanced with the increase of MeHg, Bax/Bcl-2 ratio also appeared a trend of increase. Bax expression integral optical density (IOD) of negative control group was (21295.86 +/- 1969.81), Bax expression IOD of 10, 20, 30 micromol/L groups were 42807.87 +/- 4416.64, 55651.65 +/- 4662.72, and 72708.56 +/- 910.10, there were significant differences in Bax expression between 10, 20, 30 micromol/L groups and negative control group (q values were 14.191, 14.320, 33.917; P<0.05); Bcl-2 expression IOD of negative control group was (12588.33 +/- 4091.02), Bcl-2 expression IOD of 10, 20, 30 micromol/L groups were 20539.16 +/- 4906.09, 23689.97 +/- 2281.42, and 28692.80 +/- 4655.86, there were significant differences in Bcl-2 expression between 10, 20, 30 micromol/L groups and negative control group (q values were 4.322, 6.035, 8.754; P<0.05); and AIF expression IOD of negative control group was (12942.72 +/- 457.94), AIF expression IOD of 10, 20, 30, 40 micromol/L groups were 16973.57 +/- 1922.87, 29998.91 +/- 6803.58, 52467.16 +/- 1916.25 and 106342.53 +/- 1273.19, there were significant differences in AIF expression between 20, 30 and 40 micromol/L groups and negative control group (q values were 11.449, 26.530, 62.692; P<0.05).

CONCLUSIONMeHg could induce apoptosis on HL-7702 cell line in vitro. The mechanisms could be related to mitochondrial pathway in apoptosis.

Apoptosis ; drug effects ; Cell Line ; Flow Cytometry ; Hepatocytes ; cytology ; drug effects ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Methylmercury Compounds ; pharmacology ; Mitochondrial Proteins ; metabolism

OBJECTIVETo study effects of three pentacyclic triterpenoids, oleanolic acid (OA), ursolic acid (UA) and asiatic acid (AA) on Ca(2+)-induced liver mitochondrial permeability transition (MPT).

METHODEffects of three compounds on liver MPT induced by Ca2+ were assessed by measuring the change in mitochondrial swelling, mitochondrial membrane potential and release of matrix Ca2+ in vitro.

RESULTObvious mitochondrial swelling, loss of mitochondrial membrane potential and release of matrix Ca2+ occurred after the addition of 50 micromol x L(-1) Ca2+. However, preincubation with 50 mg x L(-1) OA, UA or AA significantly blocked the above changes. In addition, it was also found that there are differences in the inhibitions of three compounds on liver MPT induced by Ca2+.

CONCLUSIONThree pentacyclic triterpenoids, OA, UA and AA, have significant mitochondrial protection through blocking on liver MPT and the inhibition on liver MPT of AA is stronger than that of UA and OA.

Animals ; Calcium ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Swelling ; drug effects ; Pentacyclic Triterpenes ; pharmacology

OBJECTIVEThis study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes.

METHODSCell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis.

RESULTSGerminal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes.

CONCLUSIONN-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms.

Animals ; Apoptosis ; drug effects ; Environmental Pollutants ; toxicity ; Female ; Fertilization ; drug effects ; Hexanes ; toxicity ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Oocytes ; drug effects ; growth & development

OBJECTIVETo study in vitro sperm damage caused by trichloroethylene in male rats.

METHODSSperms of Sprague-Dawley (SD) rats were collected 4 hours after being contaminated by trichloroethylene of 0, 2, 4, 6, 8, and 10 mmol/L in vitro. Giemsa staining was performed to observe the morphological changes of sperms, and flow cytometer was used to detect the changes in mitochondrial membrane potential.

RESULTSThe sperm motilities in 6, 8, and 10 mmol/L trichloroethylene groups decreased significantly compared with that in control group (P <0.01); the sperm aberration rates in 8 and 10 mmol/L trichloroethylene groups were significantly higher than that in control group (P<0.01). With the increase in exposure dose, the proportion of sperms with reduced mitochondrial membrane potential increased, and there were significant differences in sperm apoptosis rate between the 4, 6, 8, and 10 mmol/L trichloroethylene groups and control group (P<0.01).

CONCLUSIONIn vitro exposure to trichloroethylene can reduce sperm motility and increase the aberration rate and apoptosis rate of sperms in male SD rats.

Animals ; Apoptosis ; drug effects ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sperm Motility ; drug effects ; Spermatozoa ; cytology ; drug effects ; Trichloroethylene ; toxicity

OBJECTIVE: To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids. METHODS: MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting. RESULTS: At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells. CONCLUSIONS In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.
3T3 Cells ; Adenosine Triphosphate ; Animals ; Apoptosis ; Cell Death ; drug effects ; Dexamethasone ; Membrane Potential, Mitochondrial ; Mice ; Microscopy, Electron ; Mitochondria ; ultrastructure ; Necrosis

OBJECTIVETo detect the effect of andrographolide on apoptosis of Candida albicans biofilm dispersion cells.

METHODThe morphological changes of apoptotic C. albicans biofilm cells were observed by using Hoechst 33258 staining Fluorescence microscope; changes of mitochondrial membrane potential (MMP) of C. albicans biofilm cells were detected by rhodamine 123 staining flow cytometry; and reactive oxygen species (ROS) was detected by DHR staining flow cytometry.

RESULT1 000, 100 micromol x L(-1) of andrographolide could cause pyknosis and dense staining of C. albicans biofilm cells, 1 000, 100, 10 micromol x L(-1) of andrographolide could decrease MMP and increase ROS of C. albicans biofilm cells.

CONCLUSIONAndrographolide of appropriate concentrations could induce apoptosis of dispersion cells of C. albicans biofilms.

Antifungal Agents ; pharmacology ; Apoptosis ; drug effects ; Biofilms ; Candida albicans ; drug effects ; physiology ; Diterpenes ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism