Research of EL-3 on anti-lung cancer mechanism
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Objective To investigate the mechanism of extract of limax(EL-3) on anti-lung cancer.Methods(1) BrdU infiltration method was used to observe the inhibiting rates of EL-3 on the human lung cancer A549 cells.(2) The morphology of A549 cells apoptosis induced by EL-3 was determined and counted with the acridine orange/ethidium bromide(AO/EB) staining,and the cell cycle and apoptosis were analyzed with FCM.(3) The ladder-like DNA bands of apoptosis cells was observed by the DNA garose gelatin electrophoresis.(4) The protein expression levels of PCNA,VEGF and CD3 affected by EL-3 were detected with Immunochistochemical technique and DAB staining.Results(1) EL-3 using only in concentration 100 μg/mL to induce A549 cells,the inhibiting rate was 63.59%.,which was significant compared with black group(0) and solvent group(7.54%)(P0.01).(2) EL-3 was more effective combined with chemotherapeutics and enhanced the effect of CDDP on nucleic acid synthesis inhibition rate of A549.EL-3 in low,middle and high dose groups,the IR of DDP used only were increased to14.01% 44,5%,48.96% and 67.05%(P0.01);when IC50 in 20.62 μg/mL,EL-3 used with paclitaxel significantly improved the inhibition of A549 cell nucleic acidsynthesis.EL-3 in low,middle and high dose groups enhanced the IR of TAX used only on A549 cell were respectively increased from 21.79% to 58.35%,66.13% and 73.67%(P0.05),and the IC50 was 7.41 μg/mL.(3) EL-3 was effective to induce apoptosis in A549 cells;(4) EL-3 was effective to inhibit the expression of PCNA,VEGF and CD31 protein expression in rats.Conclusion anti-cancer mechanism of EL-3 is the multi-target,such as(1) inhibiting lung cancer cell nucleic acid synthesis;(2) inducing lung cancer cells apoptosis;and(3) inhibiting foci of angiogenesis and cancer cells to vascular wall adhesion.Keywords:
Acridine orange
Ethidium bromide
IC50
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To study the effects of RNA interference (RNAi)-mediated insulin-like growth factor I receptor (IGF-IR) gene silencing on human lung cancer cells.Plasmids expressing IGF-IR shRNA1 and IGF-IR shRNA2 were constructed. Human non-small cell lung cancer cells of the line A549 were cultured and transfected with sequence-specific shRNA. RT-PCR was used to monitor the IGF-IR mRNA expression. Western blotting was used to detect the expression of IGF-IR, bcl-2 and caspase-3, associated with apoptosis, and IGF-IR signaling pathways-associated proteins, total and phospho-ERK1/2 and Akt. MTT assay and flow cytometry were used to examine the cell activity and cell cycle. Twelve nude mice were injected subcutaneously with A549 cells, 20 days later the mice were randomly divided into 3 groups to be injected into the tumor with IGF-IR, PBS, or blank plasmid respectively 4 times with the interval of 5 days. Five days after the 4th injection the mice were killed and the tumors were taken out. TUNNEL assay was used to detect the apoptotic cell in the tumor.RT-PCR showed that the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA1 was only 24% +/- 4% that of the A549 cells transfected with blank plasmid (P < 0.05); however, the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA2 was 78% +/- 5% that of the A549 cells transfected with blank plasmid (P > 0.05). The IGF-IR protein expression level of the A549 cells of the IGF-IR shRNA1 group was only 10.2% +/- 2.8% that of the A549 cells of the blank plasmid group (P < 0.05). Western blotting showed that the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA1 group were 46% +/- 6% and 156% +/- 8% those of the negative controls (both P < 0.05); however, the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA2 group were not different from those of the negative control cells. The Akt kinase and ERK phosphorylation levels of the A549 cells of the IGF-IR shRNA1 group were 10% and 36% +/- 3% those of the negative control cells respectively (both P < 0.05). Since 48h after the transfection the active cell number of the IGF-IR shRNA1 group was 64% +/- 7% that of the negative group (P < 0.05), and this decrease effect lasted to 72 h after (67% +/- 6% that of the negative cells, P < 0.05). 48 h after the transfection the percentage of cells at G(0)/G(1) phase of the IGF-IR shRNA1 group was 77.5%, significantly higher than that of the negative control group, and the percentages of the cells at S and G(2)/M phases of the IGF-IR shRNA1 group were 15.7% and 7.3% respectively, both significantly lower than those of the negative control group (23.0% and 29.9% respectively). Since the second injection the tumor size of the mice of IGF-IR shRNA group was 40% - 50% that of the PBS group (P < 0.05), and the tumor size of the mice of the PBS group was 90% that of the control group. TUNNEL assay showed that the number of apoptotic cells in the tumors of the IGF-IR shRNA1 group mice was 118 +/- 8/high power, significantly higher than that of the control group (70 +/- 9, P < 0.05).RNAi technique effectively inhibits the expression of IGF-IR, thus decreasing the NSCLC cell proliferation inducing apoptosis and inhibiting the tumor growth.
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AIM:To explore the proliferation inhibition and apoptosis inducing effects of matrine (Mat) on U937 cells and its mechanism. METHODS:The proliferation inhibition rate of U937 cells was assayed by MTT analysis; cell cycle was evaluated by flow cytometry (FCM); cell apoptosis rate was calculated by Annexin/PI double staining method. The Bcl-2 mRNA expression of U937 cells treated with Mat was assayed by RT-PCR. RESULTS:After U937 cells were treated with Mat (0.2-0.5 g/L) for 24,48 and 72 h,the cell proliferation was markedly inhibited in a dose-dependent and time-dependent manner (P0.05 or P0.01),and the 5% inhibiting concentration (IC50) was 0.4 g/L. Cell cycle analysis showed that it was arrested in the S phase after 72 h. When the U937 cells were treated by Mat (0.2-0.5 g/L) for 72 h,the corresponding apoptosis rate (3.25%,5.32%,8.17%,11.20%) indicated by AnnexinV-FITC double staining,had statistical significance compared with the control group (1.84%) and the 0.1 g/L Mat-treated group (1.95%,P0.05 or P0.01). The Bcl-2 mRNA expression in U937 cells was decreased 72 h after treated by Mat (0.2-0.5 g/L) with a concentration-effect relationship. CONCLUSION:Mat can suppress the U937 cell proliferation in a concentration-dependent and time-dependent manner. The effect has a relation with the U937 cell cycle arrested in the S phase. Mat can down-regulate the expression of Bcl-2 and induce the apoptosis.
U937 cell
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Objective To investigate the effect of anti-cancer bioactive peptide (ACBP) on human breast cancer cell line nm231 and its mechanisms. Methods After being treated with different concentrations of ACBP (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, and 0.25 μg/ml respectively) for 72 hours, the proliferative activity of the nm231 cells were detected by MTT. And the nm231 cells that treated with 0.25 μg/ml ACBP was observed under a light microscope at 4, 6, 24, 48, and 72 hours respectively, and examined by transmission electron microscopy at 48 hours. Gel electrophoresis of DNA and flow cytometry using AV/PI double staining were used to detect the effect of ACBP (0.25 μg/ml) on the cell apoptosis at different time points, and its effect on the cell cycle at 48 hours. In all the experiments, the nm231 cells in the control group were cultured in RPMI 1640 medium instead of ACBP. Results The proliferation of nm231 cells was markedly inhibited by 0.1 μg/ml ACBP with an inhibitory rate of 10.3%. The rate reached 35.1% in the cells treated with 0.25 μg/ml ACBP. The inhibitory effect showed a concentration-dependent manner. After being treated with 0.25 μg/ml ACBP for 24 hours, apoptotic features could be detected in the nm231 cells under a light microscope. Then, at 48 hours, a large number of typical apoptotic cells could be observed by both light microscopy and transmission electron microscopy. DNA gel electrophoresis showed DNA fragmentation in the cells treated with 0.25 μg/ml ACBP for 24 hours, and typical DNA ladder in those treated for 48 hours. Flow cytometry found that the proportions of AV+/PI- and AV+/PI+ cells increased with culture time. In the nm231 cells treated with ACBP for 48 hours, the proportion of the cells in G0/1 phase was significantly higher than that in the control, while the proliferation index was significantly lower than in the control. (both P 0.01). Conclusion ACBP can inhibit the proliferation of nm231 cells by inhibiting the DNA synthesis and inducing cell apoptosis.
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Objective To observe the effects of anti-proliferation and inducing apoptosis on A-549 cell line induced by eicosapentaenoic acid(EPA) and its synergistic effect with cisplatin(DDP) in vitro.Methods A-549 cell line was cultured in vitro.MTT assay was used to determine the growth inhibitory effect of EPA on A-549 cells.Morphological changes of the apoptosis of A-549 cells were observed by using inverted microscope.TUNEL was used to detect cell cycle distribution and apoptotic rate of A-549 cells.Results The results of experiments showed that when A-549 cells were exposed to EPA for 24h,48h,72h and 96h respectively at final concentration of(60-120) μg/mL,the growth of cells was inhibited and showed in dose and time dependent manner.EPA combined with DDP showed significant synergistic anti-tumor effect on A-549 cells.When the concentration of EPA altered from 60μg/mL to 120μg/mL,the apoptotic index(AI) of A-549 cells elevated accordingly by the method of terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL),the apoptotic indexes were(81.52±1.96)%,(83.74±2.21)%,(85.30±2.38)%,(86.26±2.44)% respectively and showed statistical significance compared between different concentration(P0.05).Conclusion EPA inhibits growth of A-549 cell line in vitro in dose and time dependent manner;EPA combined with DDP shows significant synergistic anti-tumor effect on A-549 cell line.
Terminal deoxynucleotidyl transferase
MTT assay
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AIM:To study the effect of vitamin K3(VK3) on the induction of apoptosis in androgen-independent prostate cancer cell PC-3M in vitro.METHODS:Cell viability was estimated by MTT assay.AO/EB staining was performed to detect apoptotic cells.Apoptosis and the changes of cell cycle were detected by flow cytometry.NAC was used to observe the effect of growth inhibition by VK3.RT-PCR was used to confirm the changes in gene expression.Levels of intracellular peroxides were estimated by using an oxidation-sensitive fluorescent probe DCFH-DA.RESULTS:PC-3M cells growth was significantly inhibited by VK3(≥60 μmol/L,P0.05).The inhibitory effect was time and dosage dependent.The result of AO/EB staining showed that apoptosis of PC-3M cells were induced by VK3.A typical subdiploid peak before G0/G1 phase was observed after treated for 12 h with VK3(60 μmol/L) by flow cytometry.The effect of growth inhibition treated with VK3 was antagonized by antioxygen NAC(5,10,20,40,80 μmol/L).An increase in the level of DCF fluorescence after PC-3M cells were treated for 1-2 h with VK3 was observed.Antioxidase GSH-Px and CAT were run-down after treated with VK3.CONCLUSION:The results indicate that apoptosis in PC-3M cells is induced through oxidative stress by VK3.
Viability assay
Growth inhibition
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Objective To study the effect of exogenous human interleukin-8 on human ovarian cancer SKOV-3 cell migration and the intervention role of cisplatin on it; to preliminary explore the mechanisms of DDP inhibiting cell migration.Methods SKOV-3 cells were treated with DDP.Optimal concentration and effect time of DDP was determined with MTT assay.Transwell assay were used to study the intervention effect of DDP.Western blot assay was applied to detect NF-κB protein expression levels.Results MTT assay: compared with the control group, the best concentration of DDP which had a significant inhibitory effect on cell proliferation was 100 μg / ml( P 0.05).The concentration of DDP in the range of 200 μg / ml to 400 μg / ml also had the intervention effect,but cell growth inhibition rate between these groups showed no significant difference( P 0.05).DDP( 100 μg / ml) were treated on SKOV-3 cells 24 h,48 h,72 h respectively,the inhibition rate of different times showed no significant difference( P 0.05).The migratory ability of cells SKOV-3 was increased when IL-8( 100 ng / L) was added in.DDP( 100 μg/ml) inhibited IL-8 induced SKOV-3 cell migration,and as the concentration increased the number of cell migration was reduced from 241.67 to 155.99( P 0.05).Cells dealed with DDP,which NF-κB protein expression level were reduced significantly( 64.04 ± 4.6).Conclusion IL-8 can promote the migration of human ovarian cancer cells SKOV-3.DDP can inhibit the migration,and this process probably depends on the activating of the NFκB pathway.
MTT assay
Gentamicin protection assay
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OBJECTIVE:To investigate the proliferation and cell apoptosis effects of combination treatment with L-carnitine and EPI on lung adenocarcinoma GLC-82 cell.METHODS: The cell proliferation was examined by MTT assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.The expression of Bcl-2,Bcl-xl and Bax proteins were measured by Western blot.RESULTS: After 24 h and 48 h,the proliferation rate of L-carnitine(20 μg/mL) were 37.56%(F=46.287,P=0.001) and 33.33%(F=41.638,P=0.001),respectively.Cells were treated with L-carnitine and EPI,which reduced the inhibitory effects on GLC-82 cell.After 24 h and 48 h,the antagonized rate of combination treatment were 12.14%(F=20.527,P=0.002) and 18.52%(F=27.269,P=0.001),respectively.The difference was significant.The expression of Bcl-2 and Bcl-xl proteins was up-regulated by L-carnitine.The expression of Bax protein was no difference.The expression of Bcl-2 and Bcl-xl proteins were significantly increased by combination treatment with L-carnitine and EPI.CONCLUSIONS:Combination treatment with L-carnitine and EPI can weaken the cytotoxicity of EPI on GLC-82 cells.After treated with L-carnitine,the proportion of G_0/G_1 phase is significantly increased,S-phase cells is reduced,the phenomenon of induced early apoptosis is disappeared and cell apoptosis is significantly decreased by upregulating the expression of Bcl-2 and Bcl-xl.
MTT assay
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Objective To observe the effect of pure puerarin and pueraria crude extract on H446 cell proliferation, and explore its mechanism. Methods H446 cells grew in different concentrations of SP and CP, cell growth inhibition was detected by MTT assay.Cell cycle and apoptosis were detected by flow cytometry.Bcl-2 and Bax protein were detected by immunohistochemistry. Results The CP for 72 h 50% inhibitory concentration (IC50) was 435 μg/ml, for SP was 1 403 μg/ml、 435 μg/ml on H446 for 72 h,the apoptosis rate induced by CP and SP were respectively 14.71% and 2.61%, the percentages of G0/G1 phase cell numbers were respectively 79.20% and 72.20%, those of G2/M phase were respectively 8.94% and 10.50%, there was significant difference compared with control group(P0.05). Compared with the control group the Bcl-2 protein expression was lower, Bax expression is increased, P0.05. The expression proportion of Bax/Bcl-2 was higher than control group. Conclusions SP and CP inhibit H446 tumor cell proliferation. SP and CP can induce apoptosis, which may be related to block the cell cycle in the G0/G1 phase, up Bax Expression, lower Bcl-2 expression.
Puerarin
MTT assay
IC50
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Objective To observe the effects of anti-proliferation and inducing apoptosis on A-549 cell line induced by eicosapentaenoic acid(EPA)and its synergistic effect with cisplatin(DDP).Methods A-549 cell line was cultured in vitro.A-549 cells were divided into group A and group B,the group B was divided into EPA 15(B1 group),30(B2 group),60(B3 group),DDP 30 μg/ml(B4 group),DDP30 μg/ml +EPA 15 μg/ml(B5 group),DDP 30 μg/ml +EPA 30 μg/ml(B6 group) and DDP 30 μg/ml+EPA 60 μg/ml(B7 group).The group A added control fluid 500 μl to replace the drugs,the experiment steps were alike.MTT assay was used to determine the effect of EPA on A-549 cells growth inhibitory.Morphological changes of the apoptosis of A-549 cells were observed by using inverted microscope.TUNEL was used to detect cell apoptotic index of A-549.Results ①in the group B1～B7,the growth of A-549 cell were inhibited and in dose and time dependent manner.EPA combined with DDP shows significant synergistic anti-tumor effect on A-549 cells.②The A-549 cells in group B showed morphological changes such as cellular volume decreased and become round,nuclear chromatin condensed,plasma membrance bleb formed,also the apoptotic body were observed by microscope nuclear chromatin condensation,loose between the cells and adherent ability decrease under invert microscope.③The apoptotic index(AI) of A-549 cells in group B1～B3 was 5.57%±0.82%,11.68%±1.26%,24.53%±1.68%,respectively,there was statistic significance compared with different concentration(P0.01).Conclusions EPA inhibited growth of A-549 cell line in vitro in dose and time dependent manner,EPA combined with DDP shows significant synergistic anti-tumor effect on A-549 cell line.
MTT assay
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Objective: To study the growth inhibitory effect and mechanism of Astragalus Injection to apoptosis of colon cancer SW480 cell lines.Methods: The control group,the oxaliplatin control group and the experimental group were included in this study.The influence of Astragalus Injection on the growth proliferation and apoptosis of SW480 cell in vitro was detected with MTT assay.The apoptosis was detected with flow cytometry(FCM) and PI staining.Results: The inhibition of the proliferation of SW480 cell lines in experimental group was more obviously compared with the control group,and which was directly proportional to the action concentration(P=0.0000.01).FCM showed that the apoptosis rates in groups of 125 μg/ml,250 μg/ml,500 μg/ml,1 000 μg/ml were 24.2%,42.6%,64.1%,84.0% respectively after Astragalus Injection on SW480 cell after 48 hours.And which were significantly higher than the control group,the differences were significant(P=0.0000.01).Conclusion: Astragalus Injection can inhibit the growth of SW480 cell and induce apoptosis of colon cancer cell SW480.
Astragalus
Growth inhibition
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