Shikonin attenuates H2O2-induced oxidative injury in HT29 cells via antioxidant activities and the inhibition of mitochondrial pathway-mediated apoptosis.

Shikonin, a natural naphthoquinone extracted from the roots of Lithospermumery throrhizon, possesses multiple pharmacological properties, including antioxidant, anti-inflammatory and antitumor effects. It has been hypothesized that the properties of shikonin are associated with its oxygen free radical scavenging abilities. However, the mechanism underlying the antioxidant activity of shikonin is not completely understood. The aim of the present study was to investigate the effect of shikonin against H2O2-induced oxidative injury in HT29 cells and to explore the underlying molecular mechanism. The concentration and duration of H2O2 treatment to cause maximal damage, and the effects of shikonin (2.5, 5 or 10 µg/ml) on the activity of H2O2-induced HT29 cells were determined by MTT assay. The apoptotic rate in HT29 cells was determined by annexin V/propidium iodide staining. HT29 cell cycle alteration was also analyzed by propidium iodide staining. Reactive oxygen species (ROS) production was assessed by monitoring 2',7'-dichlorofluorescin in diacetate fluorescence. Mitochondrial membrane potentials were determined by JC-1 staining. The activities of malondialdehyde, superoxide dismutase, caspase-9 and caspase-3 were measured using spectrophotometric assays. The expression levels of Bcl-2, Bax and cytochrome c were determined by western blotting. The results suggested that shikonin increased cell viability, reduced cell apoptosis and increased the proliferation index in H2O2-treated HT29 cells. Shikonin also significantly inhibited increases in intracellular reactive oxygen species (ROS), restored the mitochondrial membrane potential, prevented the release of lactic dehydrogenase and decreased the levels of superoxide dismutase and malondialdehyde in H2O2-induced HT29 cells. Furthermore, shikonin significantly decreased caspase-9 and caspase-3 activities, increased Bcl-2 expression and decreased Bax and cytochrome c expression levels in H2O2-induced HT29 cells. The results indicated that shikonin protected against H2O2-induced oxidative injury by removing ROS, ameliorating mitochondrial dysfunction, attenuating DNA oxidative damage and inhibiting mitochondrial pathway-mediated apoptosis.

[Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway].

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G0/G1 or G2/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

The induction of autophagy against mitochondria-mediated apoptosis in lung cancer cells by a ruthenium (II) imidazole complex.

In the present study, it was found that the ruthenium (II) imidazole complex [Ru(Im)4(dppz)]2+ (Ru1) could induce significant growth inhibition and apoptosis in A549 and NCI-H460 cells. Apart from the induction of apoptosis, it was reported for the first time that Ru1 induced an autophagic response in A549 and NCI-H460 cells as evidenced by the formation of autophagosomes, acidic vesicular organelles (AVOs), and the up-regulation of LC3-II. Furthermore, scavenging of reactive oxygen species (ROS) by antioxidant NAC or Tiron inhibited the release of cytochrome c, caspase-3 activity, and eventually rescued cancer cells from Ru1-mediated apoptosis, suggesting that Ru1 inducing apoptosis was partially caspase 3-dependent by triggering ROS-mediated mitochondrial dysfunction in A549 and NCI-H460 cells. Further study indicated that the extracellular signal-regulated kinase (ERK) signaling pathway was involved in Ru1-induced autophagy in A549 and NCI-H460 cells. Moreover, blocking autophagy using pharmacological inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhanced Ru1-induced apoptosis, indicating the cytoprotective role of autophagy in Ru1-treated A549 and NCI-H460 cells. Finally, the in vivo mice bearing A549 xenografts, Ru1 dosed at 10 or 20 mg/kg significantly inhibited tumor growth.

The studies on the cytotoxicity in vitro, cellular uptake, cell cycle arrest and apoptosis-inducing properties of ruthenium methylimidazole complex [Ru(MeIm)4(p-cpip)](2.).

A new ruthenium methylimidazole complex [Ru(MeIm)4(p-cpip)](2+) (Ru1, p-cpip=2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, MeIm=1-methylimidazole) has been synthesized and characterized. The cellular uptake, in vitro cytotoxicities, cell cycle arrest and apoptosis-inducing mechanism of this Ru(II) complex have been extensively explored by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, Comet assay, inverted fluorescence microscope as well as Western blotting experimental techniques. Notably, Ru1 displayed relatively high cytotoxic activity against lung cancer A549 cells and had high selectivity between tumor and normal cells in comparison with cisplatin. Further studies showed that Ru1 caused cell cycle arrest at G0/G1 phase and induced apoptosis via the mitochondrial pathway, which involved reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and Bcl-2 and caspase correlative family member activation. For providing more information about the possible antitumor mechanism, the in vitro DNA binding studies have been also investigated by different spectrophotometric methods, thermal denaturation and viscosity measurements.

EIF4E over-expresses and enhances cell proliferation and cell cycle progression in nasopharyngeal carcinoma.

Eukaryotic translation initiation factor 4E (eIF4E) is involved in integration and amplification of many carcinogenesis signals in tumors. However, it remains unclear whether eIF4E over-expresses in NPC and whether it is associated with the development of NPC. Here, we analyzed the expression state of eIF4E, c-Myc, and MMP9 in 24 nasopharyngitises and 64 nasopharyngeal carcinomas (NPC) tissues and studied the influences of eIF4E on the proliferation and cell cycle in NPC cell lines. The results indicate that eIF4E might over-express in NPC and the over-expression of eIF4E promotes NPC growth and cell cycle progression through enhancing the translational expression of c-Myc and MMP9. The finding certainly adds new knowledge in the understanding of the carcinogenesis of NPC and provides a potential molecular target for the NPC therapy and prevention.

[Level of SDF-1/CXCR4 in children with acute leukemia and its significance].

The objective of this study was to detect the level of plasma stromal cell-derived factor-1 (SDF-1) and the expression of CXCR4 (SDF-1 receptor in bone marrow cells) in children with Acute Leukemia (AL) and to investigate the relationship between the expression of CXCR4 and extramedullary infiltration. 48 children with acute leukemia and 20 with non-hematologic malignancies were selected into the AL group and the control group respectively. The peripheral plasma and bone marrow cells were collected. The level of SDF-1 in peripheral plasma was detected by ELISA and the expression of CXCR4 in bone marrow cells was determined by flow cytometry. The results showed that the levels of SDF-1 in peripheral plasma and the expression of CXCR4 in bone marrow cells of AL group was significantly higher than that of control group, among which the level of SDF-1 of the acute lymphoblastic leukemia (ALL) group was also higher than that of the acute myeloid leukemia (AML) group, the expression level of CXCR4 in the bone marrow cells of the extramedullary infiltration (EI) group was higher than that of the non-extramedullary Infiltration (NI) group, and all the differences between the both groups were significant. It is concluded that SDF-1 and CXCR4 express a high level in children with AL, which closely relates with the type of leukemia and the migration and infiltration of leukemia cells in bone marrow.

[Changes of Foxp3 and IL-10 and TGF-beta in aging of mice].

AIM: To compare the expression levels of Foxp3 mRNA in the spleen PBMC (peripheral blood mononuclear cell) in the progress of aging mice and analyse the relevance with serum IL-10 and TGF-beta. METHODS: Healthy male BALB/c mice were divided into 3 groups at random (8 mice each group): 2m group with 2 months mice (young), 6m group with 6 months mice (middle-aged) and 15m group with 15 months mice (aged). The phenotype of CD4(+);CD25(+);Foxp3(+); T cells derived from normal murine SPL were analyzed by FCM. Real-time fluorescence quantitative RT-PCR was used to determine the expression levels of Foxp3 mRNA in the spleen PBMC. ELISA was used to detect the concentration of IL-10 and TGF-beta. RESULTS: The subsets of CD4(+);CD25(+);Foxp3(+); T lymphocytes from murine splenocytes were significantly higher in 15m group than that in 2m group(P<0.01). The expression levels of Foxp3 mRNA in the spleen PBMC were significantly higher in 15m group than that in 2m group(P<0.01).The serum concentration of IL-10 and TGF-beta were significantly higher in 15m group than that in 2m group(P<0.01).There was no correlation between serum concentration of IL-10 and TGF-beta and Foxp3 mRNA expression levels in the spleen PBMC in 15m group. CONCLUSION: The level of the CD4(+);CD25(+);Foxp3(+); T cells and Foxp3 mRNA expression levels were up-regulated and IL-10 and TGF-beta contents were increased in the progress of aging mice. Foxp3 and IL-10 and TGF-beta may be play a part in the process of immunosenescence. Quantitative detection of Foxp3 mRNA by the method of SYBR Green I real-time fluorescence quantitative RT-PCR is stable and reliable.

Effects of a JAK inhibitor, AG490, on proliferation and apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z.

BACKGROUND AND OBJECTIVE: Abnormal activation of Janus kinas/signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway is closely related to malignant transformation of cells. This study was to investigate the effects of a JAK inhibitor, AG490, on the proliferation, apoptosis, and expressions of apoptosis-related survivin and Mcl-1 genes, thus to explore the role of JAK-STAT3 signaling pathway in the regulation of cell apoptosis in nasopharyngeal carcinoma (NPC) cell line CNE-2Z. METHODS: CNE-2Z cells were treated with different doses of AG490. The cell proliferation was measured using MTT array. Cell apoptosis was assessed by flow cytometry (FCM) and Hoechst33342 fluorescence staining. The mRNA levels of STAT3, survivin and Mcl-1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein contents of STAT3, phosphoralated STAT3 (p-STAT3), survivin and Mcl-1 were measured by western blot. RESULTS: The inhibition rates of CNE-2Z cell proliferation by 100 micromol/L AG490 were 37.95% and 52.99% at 24 and 48 h after treatment. After incubation with 50 micromol/L and 100 micromol/L AG490 for 24 h, the apoptotic rates of CNE-2Z cells were increased from 1.37% to 9.30% and 9.00%, respectively (p < 0.01); typical changes in apoptotic morphology were observed under fluorescence microscopy; moreover, activation of STAT3 was inhibited, mRNA and protein levels of Mcl-1 and survivin were down-regulated. CONCLUSION: Blocking of JAK-STAT3 signaling pathway in CNE-2Z cells could remarkably inhibit cell proliferation and inactivate STAT3, as well as promote cell apoptosis by down-regulating Mcl-1 and survivin.

Effect of protein kinase C on proliferation and apoptosis of T lymphocytes in idiopathic thrombocytopenic purpura children.

It is well-documented that T lymphocyte proliferation and apoptosis are abnormal in idiopathic thrombocytopenic purpura (ITP) children. However, the underlying regulation mechanisms especially in terms of signal transduction remain unknown. In this paper, we reported the changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes and the effect of PKC on T lymphocyte proliferation and apoptosis. We demonstrated that in ITP children, the activator (PMA) and inhibitor (H-7) of PKC affected on T lymphocyte proliferation and apoptosis dramatically, but they altered little in healthy children. PKC activity was significantly enhanced in ITP children together with an increased expression of FasL on CD3+T, CD4+T and CD8+T cells, resulting in a positive correlation between PKC activity and the expression of FasL on T cells. While the PKC activity and the platelet count were negatively correlated. Taken together, our findings suggest that the PKC activation may enhance T lymphocytes activity, suppress T cell apoptosis and be involve in thrombocytes damage as a mechanism related to immune pathogenesis of ITP.

[Knockdown of bcl-xL expression with RNA interference induces nasopharyngeal carcinoma cells apoptosis].

OBJECTIVE: To study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction. METHODS: Small interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Amnion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue (MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry. RESULTS: Green fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10% - 70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4. CONCLUSIONS: The synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti-nasopharyngeal carcinoma gene therapy.

[Effect of IL-15 on the proliferation, differentiation and anti-apoptosis of CD34+ cells in patients with MDS].

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.

[Influence of cytochrome C on apoptosis induced by daunorubicine in acute myeloid leukemia (AML) cells].

The purpose was to study the responses of AML cell treated with cytochrome C and to explore the influence of cytochrome C on apoptosis of AML cell induced by daunorudicine (DNR). The differentiation of AML cell was detected by Wright-Giemsa staining and NBT test, the apoptosis of AML cell was assayed by flow cytometry and fluorescence microscopy. The results showed as follows: (1) different concentrations of cytochrome C could induce different effects on AML cells. Concentration of cytochrome C for differentiation was 10 microl/ml, for apoptosis was 20 microl/ml, and for necrosis was 40 microl/ml. (2) the apoptosis of AML cells decreased with the administration of cytochrome C in 10.0 microg/ml before treating AML cells with DNR (P < 0.01), but no change was shown with the administration of cytochrome C in 20.0 microg/ml (P > 0.05). (3) in reverse sequence, administrating of cytochrome C in 10 microl/ml and 20 microl/ml after treating AML cells with DNR, two different concentrations of cytochrome C could increase the apoptosis of AML cells (P < 0.01). It is suggested that cytochrome C may probably affect the apoptosis of AML cells induced by DNR.

[Effects of adenosine preconditioning on cardiac myocyte apoptosis and expression of nuclear factor-kappaB in ischemia/reperfusion rats].

OBJECTIVE: To investigate the effect of adenosine preconditioning on cardiac myocyte apoptosis and the expression of nuclear factor-kappaB (NF-kappaB)during myocardial ischemia/reperfusion (I/R) in rats. METHODS: A model of I/R injury (I/R group) and pretreatment with adenosine (AP group) was reproduced in rats. The morphologic features of apoptosis were identified histochemically by TdT-mediated dUTP nick end (TUNEL) staining. Expression of NF-kappaB in apoptosis myocardiac cells was observed by immunohistochemical stain and image analysis. RESULTS: The percentage of cardiac myocyte apptosis and the expression of NF-kappaB in AP group were (2635.0+/-316.0)% and (33.21+/-16.91)%, they were significantly lower than those in I/R group (5013.0+/-503.7) % and (59.30+/-10.36) % respectively (P<0.05 or P<0.01), but higher than those in control group (68.7+/-50.3) % and (10.98+/-4.65)% respectively (both P<0.01). CONCLUSION: Adenosine preconditioning can pretect from myocardial I/R injury and relieve cardiac myocyte apoptosis and expression of NF-kappaB.

[Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: It was reported that chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1(SDF-1) were involved in the proliferation, differentiation, and metastasis of tumor. This study was designed to observe the expression of CXCR4 in NPC cells with different differentiation grade and proliferative ability to primitively clarify the relationship between CXCR4 and the malignity of NPC cells. METHODS: After treated with all-trans-retinoic acid(RA) and telomerase antisense oligodeoxynucleotide (ASODN) respectively, the expression of CXCR4 mRNA and CXCR4 protein in NPC CNE1 and CNE2Z cells were determined by in situ hybridization and immunohistochemistry, respectively; the distribution of cell cycle was examined with flow cytometry and the proliferation of cells was identified by MTT method. RESULTS: CXCR4 mRNA and CXCR4 protein were strongly expressed in both CNE1 and CNE2Z cells, and their expression in CNE2Z cells was stronger than that in CNE1 cells. After treated with 1x10(-5) mol/L and 1x10(-4) mol/L RA, CNE1 cells were arrested in G1 phase and CNE2Z cells in S phase, while the CXCR4 mRNA expression was significantly decreased in both CNE1 and CNE2Z cells compared with control group cells (P< 0.01). The effect of 1x10(-4) mol/L RA was more powerful than that of 1x10(-5) mol/L RA. After treated with ASODN, the proliferation of CNE1 and CNE2Z cells was inhibited, and the expression of CXCR4 protein was decreased compared with the control (P< 0.01). CONCLUSION: CXCR4 is highly expressed in NPC cells,and its expression was associated with differentiation grade and proliferation ability of NPC cells.

[Apoptotic effect of bcl-XL antisense oligodeoxynucleotide mediated by lipofectin on cell strain CNE-2Z].

BACKGROUND & OBJECTIVE: Recent studies have shown that overexpression of bcl-XL was detected in human nasopharyngeal carcinoma (NPC) cell strain CNE-2Z, suggesting it may play a pivotal role in tumorigenesis of NPC. The current study was designed to explore the effect of bcl-XL antisense oligodeoxynucleotide (ASODN) on CNE-2Z. METHODS: A 20-mer gapmer ASODN with a full phosphorothioate backbone targeting a sequence unique of the bcl-XL coding region was artificially synthesized. Bcl-XL ASODN was transfected into CNE-2Z cells through lipofectin. The survival rate was assessed by MTT assay and internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. Apoptotic changes after treatment with ASODN were observed by fluorescence microscopy and flow cytometry. RESULTS: MTT assay showed that the proliferation of CNE-2Z cells decreased significantly after treatment with ASODN/Lip as compared with control (P < 0.01). ASODN/Lip reduced the proliferation of CNE-2Z in a dose-dependent manner. After treatment with ASODN/Lip for 36 hours, most cells stained with Hoechst 33258/Pl exhibited apoptotic cell morphology such as cell shrinkage, nuclear condensation, and nuclear fragmentation under fluorescence microscope; a apoptotic peak appeared on flow cytometry; a ladder-like pattern of DNA fragmentation appeared on agarose gel electrophoresis. CONCLUSION: ASODN can inhibit proliferation of CNE-2Z cells and induce apoptosis of CNE-2Z cells. The results suggest that bcl-XL is a promising target for gene therapy of NPC.

[Effects of telomerase sense and antisense oligodeoxynucleotides on growth and differentiation of nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: It was reported that telomere and telomerase were associated with the development of cancers. Telomerase antisense oligodeoxynucleotides(ASODN) directly act on telomerase, or induce tumor cells to differentiate, result in telomerase activity decreases, and cells growth inhibited. However, whether the inhibition of telomerase activity by ASODN could induce tumor cells to differentiate is still unknown. Telomerase RNA acts as a template for synthesis of telomere, so telomerase sense oligodeoxynucleotides(SODN) can be hybridized to the telomere DNA. Whether this reaction could affect the growth of tumor cells is worth being studied. This research was just to investigate the effects of telomerase SODN and ASODN on the growth and differentiation of nasopharyngeal carcinoma(NPC) cells. METHODS: After transfecting telomerase SODN and ASODN into NPC CNE1 and CNE2Z cells by lipofectin, the proliferation of the cells was identified by MTT method, and the expression of keratin was detected by immunohistochemistry. Meanwhile, the morphological changes of the cells were observed. RESULTS: SODN inhibited the growth of CNE1 and CNE2Z cells as well as ASODN in dose- and time-dependent manner. After being treated with 1.5 mumol/L, 3.0 mumol/L, 4.5 mumol/L, and 6.0 mumol/L SODN, the inhibition rates in CNE1 cells for 6 h were 16.28%, 19.38%, 22.48%, and 23.26%, respectively; for 48 h were 26.26%, 38.89%, 39.90%, and 38.89%, respectively; the inhibition rates in CNE2Z cells for 6 h were 7.69%, 8.24%, 18.13%, and 20.32%, respectively, and for 48 h were 28.84%, 28.88%, 32.89%, and 31.54%, respectively. The keratin expressions in cells of SODN and ASODN groups were significantly increased and the cells tended to be mature. CONCLUSION: The telomerase SODN and ASODN could both inhibit the growth of NPC cells and induce cells to differentiate.