Lycopene protects primary mouse cerebrocortical neurons against t-BHP-induced damage in vitro

AIM: To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons exposed to tert-butyl hydroperoxide (t-BHP) and its mechanisms of in vitro.METHODS: Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group, t-BHP group, lycopene+t-BHP group and lycopene group. The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay. ROS content was measured by flow cytometry, and the protein levels of Bax, Bcl-2, caspase-3, cleaved caspase-3 and cytochrome C were examined by Western blot.RESULTS: The primary mouse cortical neurons expressed MAP-2 protein. Lycopene at concentration of 4 μmol/L reversed the decrease in cell viability. Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure. In addition, the protein level of Bcl-2 was increased, and the expression of Bax, cleaved caspase-3 and cytochrome-C was suppressed in lycopene+t-BHP group.CONCLUSION: The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuronal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway.     

Tert-butyl hydroperoxide damages mitochrondria and induces apoptosis in cortical neurons

AIM: To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in rat cortical neurons. METHODS: Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis and mitochondrial transmembrane potential (ΔΨm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 and Bax protein, cytosolic cytochrome c, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were detected by Western blotting. RESULTS: After exposure of cortical neurons to tBHP (25-400 μmol/L), the cell viability was reduced. ΔΨm and cellular GSH content were also decreased significantly. The level of Bcl-2 protein was reduced and Bax was elevated. Meanwhile, tBHP exposure resulted in cytochrome c release, caspase-3 and PARP proteolysis, DNA fragmentation and eventually neuron apoptosis. CONCLUSION: Mitochondrial damage may mediate tBHP- induced apoptosis in cortical neurons.     

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63

AIM: To investigate the effect of Ginsenoside Rh2(Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS: The cell viability was determined by MTT assay. MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining. The expression of Bcl-2, Bax, cytochrome C(Cyt C) and cleaved caspase-3 were measured by Western blot.RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner. Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased, while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group(P<0.05). The protein level of cleaved caspase-3 was also increased(P<0.05).CONCLUSION: Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway, suggesting that Rh2 is a novel approach for the treatment of osteosarcoma.     

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.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.     

Growth-inhibitory and apoptosis-inducing effects of andrographolide on acute lymphoblastic leukemia cells

AIM To explore the effect of andrographolide (AND) on the growth and apoptosis of acute lymphoblastic leukemia （ALL） cells. METHODS CCK-8 assay was used to assess the viability of human acute lymphoblastic leukemia CEM-C1 cells treated with AND for 12 h, 24 h and 48 h. The cell morphological changes were observed by Wright-Giemsa staining. The cell apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide (PI) staining, and the cell cycle was examined by flow cytometry with PI staining. The expression levels of apoptosis-related proteins were examined by Western blot. The mitochondrial membrane potential (MMP) of the cells was determined by JC-1 assay. RESULTS The results of CCK-8 assay indicated that AND inhibited the viability of CEM-C1 cells in a dose- and time-dependent manner. After administration of AND for 24 h, CEM-C1 cells shrank, the cytoplasm turned red and the cell numbers were significantly reduced. Incubation of AND for 24 h resulted in G₂-phase arrest and apoptosis. Treatment with AND for 24 h increased the protein levels of cleaved caspase-3, cleaved caspase-7 and Bax, and down-regulated Bcl-2 in the CEM-C1 cells (P<0.05). The ratios of cleaved caspase-3/caspase-3, cleaved caspase-7/caspase-7 and Bax/Bcl-2 were elevated with the increase in the concentration of AND. Collapsed MMP in CEM-C1 cells was observed after AND administration for 24 h. Treatment with AND in vivo suppressed the growth of the xenograft tumor and increased the protein level of cleaved caspase-3. CONCLUSION Andrographolide exerts growth-inhibitory and apoptosis-inducing effects on ALL cells both in vitro and in vivo.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.     

Nisin induces apoptosis of human osteosarcoma MG63 cells via oxidative stress

AIM To investigate the effect of nisin on apoptosis of human osteosarcoma MG63 cells and its related oxidative stress mechanism. METHODS The MG63 cells were cultured in the medium containing different concentrations of nisin with or without antioxidant N-acetyl-L-cysteine （NAC）. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry with annexin-V/PI staining. The production of intracellular reactive oxygen species （ROS） was detected by redox-sensitive dye 2',7'-dichlorofluorescein diacetate （DCFH-DA）. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide （JC-1） was used to detect the changes of mitochondrial membrane potential （MMP）. The protein levels of apoptosis-associated molecules Bax, Bcl-2 and cleaved caspase-3 were determined by Western blot. RESULTS Nisin decreased the viability of MG63 cells and promoted the apoptosis in a dose-dependent manner. It also up-regulated the protein level of cleaved caspase-3, increased the protein expression ratio of Bax/Bcl-2, triggered a large amount of intracellular ROS generation and reduced the MMP （P<0.05）. Moreover, antioxidant NAC significantly inhibited nisin-induced apoptosis of MG63 cells, down-regulated the protein level of cleaved caspase-3, decreased Bax/Bcl-2 ratio, reduced intracellular ROS level, and restored the MMP （P<0.05）. CONCLUSION Nisin may promote oxidative stress in human osteosarcoma cells, activate mitochondrial apoptosis pathway, and eventually induce apoptosis.     

Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.     

Influence of hydrogen sulfide on ileal epithelial cell apoptosis in rats with intestinal ischemia-reperfusion injury

AIM: To investigate the influence of hydrogen sulfide (H₂S) on intestinal epithelial cell mitochondrial morphology and function and the expression of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax in rats with intestinal ischemia-reperfusion (I/R) injury. METHODS: Wistar rats (n=24) were randomly divided into 3 groups (8 in each group): sham group, I/R group and I/R+sodium hydrosulfide (NaHS) group. The animal model of intestinal I/R injury was established. The rats in I/R+NaHS group received NaHS (100 μmol/kg bolus +1 mg·kg^-1·h^-1 infusion) 10 min prior to the onset of reperfusion, whereas the rats in I/R group and sham group received equal volume of normal sodium. Ileum epithelial mitochondrial morphology and function were measured. Plasma H₂S was detected by sensitive sulfide electrode. The expression of Bcl-2 and Bax mRNA was studied by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax were tested by Western blot.RESULTS: The area, volume density, maximum diameter, minimum diameter and equivalent diameter of mitochondria, and the expression of cleaved caspase-3, Cyt C and Bax in I/R group were significantly higher than those in I/R+NaHS and sham groups (P<0.01). The mitochondrial count, circumference, specific surface area, area density and population density, plasma H₂S, respiratory control rate (RCR), the ratio of P/O, R₃ , R₄, and the expression of Bcl-2 in I/R group were sharply lower than those in I/R+NaHS and sham groups (P<0.01). H₂S was negatively correlated with caspase-3, cleaved caspase-3, Cyt C and Bax (P<0.01), and was positively correlated with Bcl-2 (P<0.01). CONCLUSION: H₂S has a protective effect on mitochondrial morphology and function in rats with intestinal I/R injury by down-regulating cleaved caspase-3, Cyt C and Bax and up-regulating Bcl-2.     

Effect of rosuvastatin on cerebral microvascular endothelial cell injury induced by oxygen-glucose deprivation/reoxygenation

AIM: To explore the effect of rosuvastatin on the oxygen-glucose deprivation (OGD)/reoxygenation induced injury of cerebral microvascular endothelial cells (BMECs). METHODS: BMECs derived from BALB/c mice were isolated and cultured. BMECs were pretreated with rosuvastatin, followed by OGD for 3 h or 6 h and reoxygenation for 24 h. The morphological changes of BMECs were observed under light microscope. MTT assay was used to measured the cell viability, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to assess the proliferation of BMECs. The protein levels of cleaved caspase-3 was observed by immunofluorescence staining. The protein levels of Bcl-2, Bax, matrix metalloproteinase (MMP) 2, MMP9, phosphorylated nuclear factor kappa B (p-NF-κB), phosphorylated P38 mitogen-activated protein kinase (p-P38) and phosphorylated c-Jun N-terminal kinase (p-JNK) were determined by Western blot. RESULTS: Rosuvastatin at 10 μmol/L improved the viability of the BMECs with OGD/reoxygenation-induced damage, and maintained the structure of BMECs. Moreover, rosuvastatin significantly prohibited the protein levels of cleaved caspase-3, MMP2, MMP9, p-NF-κB, p-P38 and p-JNK, and up-regulated the ratio of Bcl-2/Bax (P<0.05). CONCLUSION: Rosuvastatin reduces OGD/reoxygenation-induced injury of BMECs by inhibiting the expression of apoptosis-related proteins and MMPs, suggesting that rosuvastatin has potential value for the maintenance of blood-brain barrier.     

Tanshinone ⅡA induces apoptosis via JNK signaling pathway in human osteosarcoma HOS cells

AIM: To explore the effect of tanshinone ⅡA on human osteosarcoma HOS cells and the underlying mechanism.METHODS: The cell viability and the appropriate dose of tanshinone ⅡA were determined by CCK-8 assay. Colony formation assay and Transwell assay were used to investigate the proliferation and migration abilities of the HOS cells treated with tanshinone ⅡA. The apoptosis of the HOS cells was monitored by Hoechst 33258 staining, transmission electron microscopy and flow cytometry. The protein levels of apoptosis-related molecules and JNK signaling-associated proteins were determined by Western blot. Meanwhile, a JNK inhibitor was added for confirming the relationship between the pathway and apoptosis mentioned above.RESULTS: Tanshinone ⅡA inhibited both HOS cell proliferation and migration in a dose-and time-dependent manner. Exposure of the HOS cells to tanshinone ⅡA resulted in the activation of apoptosis. Tanshinone ⅡA treatment increased the protein levels of cleaved caspase-3, Bax and JNK signaling-associated proteins, and decreased the protein level of Bcl-2, which were reversed by JNK inhibitor SP600125. Moreover, the result of CCK-8 assay revealed that tanshinone ⅡA-induced cell death was alleviated by JNK inhibitor.CONCLUSION: Tanshinone ⅡA induces cell growth inhibition and the activation of apoptosis via JNK signaling pathway in human osteosarcoma HOS cells.     

Relationship between histone H3K36 trimethylation and acute renal injury induced by ischemia/reperfusion

AIM: To observe the expression change of H3K36 trimethylation (H3K36me3) in acute kidney injury (AKI) induced by ischemia/reperfusion (IR) and analyze its correlation with SMYD2 and renal injury, and to provide a new target for clinical treatment of IR-AKI. METHODS: The ICR mice (n=30) were randomly divided into IR group (n=15) and sham operation group (sham group, n=15). The AKI model was established by clamping bilateral renal pedicles for 45 min. The serum and renal tissues of the mice were collected after the model was established for 24 h. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical method. The pathological changes of the kidney were observed by HE staining. The changes of NGAL and cleaved caspase-3 were observed by immunohistochemical (IHC) staining. The protein levels of NGAL, SMYD2, H3K36me3, p-P53, P53, cleaved caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JNK and p-JNK1/2/3 in the renal tissues were determined by Western blot. The relationship between H3K36me3 and SMYD2/NGAL was analyzed by Pearson correlation method. RESULTS: Compared with sham group, BUN and SCr levels of the mice in IR group were significantly increased (P<0.05). HE staining showed significant edema, abscission and necrosis in renal tubular epithelial cells of the micc in IR group, and abscission of brush border and large amount of cell debris in the lumen were also observed. The IHC staining results showed that the protein levels of NGAL and cleaved caspase-3 in IR group were significantly increased. The results of Western blot showed that the protein levels of NGAL, SMYD2, H3K36me3, p-P53, cleaved caspase-3, Bax, STAT3, p-STAT3, JNK and p-JNK1/2/3 in IR group were significantly up-regulated (P<0.05), while the Bcl-2 levels were significantly down-regulated (P<0.05). The correlation analysis showed that H3K36me3 was positively correlated with the levels of SMYD2 and NGAL. CONCLUSION: H3K36me3 is closely related to SMYD2 and renal injury in IR-AKI mice, and the up-regulated expression of H3K36me3 may be involved in the regulation of IR-AKI occurrence and development together with the activation of STAT3 and JNK signaling pathways.     

Effects of aspirin on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To investigate the effects of aspirin on the apoptosis of nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance and its potential mechanism. METHODS: The effects of aspirin on the cell viability, apoptosis, and protein levels of procaspase-3, cleaved caspase-3, procaspase-9, procaspase-12, PARP and cleaved PARP, PI3K p110α, Akt, Bcl-2, Bax and p27 in the CNE2R cells and CNE2 cells were detected by the methods of MTT assay, flow cytometry and Western blot. RESULTS: Aspirin inhibited the viability of homologous nasopharyngeal carcinoma cell lines CNE2 and CNE2R (with the IC₅₀ to CNE2 cells of 6.18, 3.92 and 3.06 mmol/L for 24 h, 48 h and 72 h, respectively; and with the IC₅₀ to CNE2R cells of 7.05, 3.90 and 2.20 mmol/L for 24 h, 48 h and 72 h, respectively). After treated with aspirin for 48 h, the apoptotic rate of CNE2R cells was higher than that of CNE2 cells (P<0.05). After treated with aspirin for 48 h, the protein levels of procaspase-3, procaspase-9, procaspase-12 and PARP were decreased, the protein levels of cleaved caspase-3, cleaved PARP and p27 were increased, and the protein levels of PI3K p110α, Akt and Bcl-2/Bax were decreased. CONCLUSION: Aspirin inhibits the viability of homologous nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance. Aspirin also induces the apoptosis of CNE2 and CNE2R cells, which is more effective in CNE2R cells. The underlying mechanisms may be involved in affecting PI3K/Akt signaling pathway, Bcl-2/Bax and p27 expression.     

Establishment of gastric cancerous multi-drug resistance cell stain BGC823/5-FU and its resistance mechanism

AIM: To establish the gastric cancerous multidrug resistance cell stain BGC823/5-FU and investigate the relationship between the resistance and the expression of apoptosis related protein Survivin, Bcl-2, Bax and caspase-3. METHODS: Human gastric cancer cell line BGC823 was induced into MDR cell line by intermittent administration of high dose of 5-FU. MTT assay was used to detect the sensitivity of these MDR cells to some chemotherapeutic agents. Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells. Western blotting was used to detect the expression of Survivin, Bcl-2, Bax and caspase-3. RESULTS: The resistance cell stain BGC823/5-FU was established, which possessed the ability of 10.82 fold resistance to 5-FU and cross-resistance to adriamycin, mitomycin C and cisplatin. The expression of P-glucoprotein was higher in BGC823/5-FU cells than that in BGC823 cells, while the accumulative value of intracellular DNR was decreased in BGC823/5-FU cells. Compared with its parent cells, expressions of Bax and caspase-3 in BGC823/5-FU cells were significantly down-regulated, surviving and Bcl-2 were upregulated in BGC823/5-FU cells. CONCLUSION: Gastric cancer cell line BGC823 has been induced into MDR cell line BGC823/5-FU. P-glucoprotein, Survivin, Bcl-2/Bax ratio and caspase-3 may play an important role in MDR of BGC823/5-FU cells.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effect of component II of broccoli polypeptide on glioma cell apoptosis

AIM：To explore the effect of component II of broccoli polypeptide on the apoptosis in glioma cells. METHODS：Human glioma SHG-44 cells were cultured and divided into control group and 3, 10, 30 and 100 mg/L component II of broccoli polypeptide groups. Cell viability was detected by MTT assay. The apoptotic rates were examined by Annexin V/PI staining. The morphological changes of the cells were observed under inverted microscope. The protein expression of Bax and Bcl-2 was detected by immunocytochemistry and Western blotting. The protein level of caspase-3 was also examined by Western blotting. RESULTS：Treatment with component II of broccoli polypeptide for 24 h, 48 h or 72 h induced significant inhibition of viability of SHG-44 cells in a time- and dose-dependent manner. The results of Annexin V/PI staining showed that the apoptotic rates were increased in treatment groups in a dose-dependent manner. The density of glioma cells was decreased after treated with increasing concentrations of the drug, and the apoptotic bodies were observed under inverted microscope at 72 h. The results of immunocytochemistry and Western blotting showed that the expression of Bax protein was increased but Bcl-2 protein expression was decreased, and the ratio of Bax/Bcl-2 was increased significantly compared with control group (P<0.05 or P<0.01). The level of caspase-3 protein was increased in 30 and 100 mg/L component II of broccoli polypeptide groups compared with control group (P<0.01). CONCLUSION：The component II of broccoli polypeptide increases the ratio of Bax/Bcl-2 and activates caspase-3 protein, thus inducing the apoptosis of glioma cells.     

Reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells

AIM:To explore the reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells and its potential mechanism. METHODS:The proper conditions of treatment with shikonin and cisplatin were determined by CCK-8 assay. The cell cycle and apoptotic rate were analyzed by flow cytometry. The protein levels of cell cycle-and apoptotic-related molecules, such as cyclin D1, cyclin-dependent kinases 2 (CDK2), P18, p-Rb, Bcl-2, Bax and cleaved caspase-3, were determined by Western blot. RESULTS:The results of CCK-8 assay showed that compared with cisplatin group, combined treatment with shikonin and cisplatin had a better inhibitory effect on the growth of cisplatin-resistant SKOV3/DDP cells. The cell cycle G₁/S transition was inhibited, while early apoptotic rate was increased after combined use of shikonin and cisplatin. The results of Western blot showed that compared with cisplatin group, the cells in combination group had lower protein levels of cyclin D1, CDK2, p-Rb and Bcl-2, accompanied with higher protein levels of P18, Bax and cleaved caspase-3. CONCLUSION:Shikonin reverses the cisplatin resistance of ovarian cancer SKOV3/DDP cells. The mechanism may be related to the regulation of cell cycle-and apoptotic-related molecules, and further inhibition of cell viability and promotion of cell apoptosis.