Aim To study the effects of tetrodotoxin （TTX） combined with acetylsalicylic acid （ASA） on nociceptive stimulus in mice. Methods To assess the antinociceptive effects of TTX, ASA or TTX plus ASA, the acetic acid-i...Aim To study the effects of tetrodotoxin （TTX） combined with acetylsalicylic acid （ASA） on nociceptive stimulus in mice. Methods To assess the antinociceptive effects of TTX, ASA or TTX plus ASA, the acetic acid-induced abdominal constriction test and formalin pain test were used. Results TTX （0.5 - 4.0 μg· kg^-1 ） or ASA （25 - 200 mg· kg^-1 ） im produced a significant inhibition of acetic acid-induced abdominal constriction. The median inhibitory doses （ID508） were 2.1 μg· kg^-1 for TTX（ and 64 mg· kg^-1 for ASA. TTX and ASA also showed a dose-dependent inhibition of the second phase response in the formalin pain model, the ID508, being 2.3μg·kg^-1 and 74.2 mg· kg^-1, respectively. The ihteraction between TTX and ASA was synergistic, as evidenced by the fact that （1） when ASA alone compared with the combination of TTX （0.79 μg · kg^-1 or 0.39μg· kg^-1 ） and ASA, the ID508, of ASA reduced from 64.0 mg· kg^-1 to 5.8 mg· kg^-1 or 12.6 mg· kg^-1, and from 74.2 mg· kg^-1 to 7.4 mg· kg^-1 or 13.0 mg· kg^-1 on tile two models of nociceptive tests, respectively; and that （2） synergism in the analgesic effects was shown by isobiolographic analysis. Conclusion TTX, ASA and the combination of the two drags produce analgesic effects in acetic acid-induced abdominal constriction test and formalin-induced pain test. The interactions between TTX and ASA may be useful in developing novel analgesic agents.展开更多
AIM:To investigate the gene knock-down effect by the phosphoinositide-3-kinase,catalytic,alpha polypeptide(PIK3CA)-targeted double-stranded RNA(dsRNA) and its effect on cell proliferation and cycle distribution in SW9...AIM:To investigate the gene knock-down effect by the phosphoinositide-3-kinase,catalytic,alpha polypeptide(PIK3CA)-targeted double-stranded RNA(dsRNA) and its effect on cell proliferation and cycle distribution in SW948.METHODS:Two PIK3CA-targeted dsRNAs were constructed and transfected into SW948 cells.Transfections were performed using lipofectamine TM 2000.The transfection effectiveness was calculated basing on the rate of fluorescence cell of SW948 at 6 h after transfection.Total messenger RNA was extracted from these cells using the RNeasy kit,and semiquantitative reverse transcription polymerase chain reaction was performed to detect the down-regulation of PIK3CA,AKT1,MYC,and CCND1 gene expression.Cells were harvested,proteins were resolved,and western blot was employed to detect the expression levels of PIK3CA,AKT1,MYC,and CCND1 gene.Cell proliferation was assessed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay and the inhibition rate was calculated.Soft agar colony formation assay was performed basing on colonies greater than 60 μm in diameter at ×100 magnification.The effect on cell cycle distribution and apoptosis was assessed by flow cytometry.All experiments were performed in triplicate.RESULTS:Green fluorescence was observed in SW948 cell transfected with plasmid Pgenesil-1,and the transfection effectiveness was about 65%.Forty-eight hours post-transfection,mRNA expression of PIK3CA in SW948 cells was 0.51 ± 0.04 vs 0.49 ± 0.03 vs 0.92 ± 0.01 vs 0.93 ± 0.03(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of AKT1 was 0.50 ± 0.03 vs 0.48 ± 0.01 vs 0.93 ± 0.04 vs 0.92 ± 0.02(P = 0.000) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of MYC was 0.49 ± 0.01 vs 0.50 ± 0.04 vs 0.90 ± 0.02 vs 0.91 ± 0.03(P = 0.001) in the four groups respectively.mRNA expression of CCND1 was 0.45 ± 0.02 vs 0.51 ± 0.01 vs 0.96 ± 0.03 vs 0.98 ± 0.01(P = 0.001) in the four groups respectively.The protein level of PIK3CA was 0.53 ± 0.01 vs 0.54 ± 0.02 vs 0.92 ± 0.03 vs 0.91 ± 0.02(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.The protein level of AKT1 in the four groups was 0.49 ± 0.02 vs 0.55 ± 0.03 vs 0.94 ± 0.03 vs 0.95 ± 0.04,P = 0.000).The protein level of MYC in the four groups was 0.51 ± 0.03 vs 0.52 ± 0.04 vs 0.92 ± 0.02 vs 0.95 ± 0.01(P = 0.000).The protein level of CCND1 in the four groups was 0.54 ± 0.04 vs 0.56 ± 0.03 vs 0.93 ± 0.01 vs 0.93 ± 0.03(P = 0.000).Both Pgenesil-CA1 and Pgenesil-CA2 plasmids significantly suppressed the growth of SW948 cells when compared with the negative or blank group at 48 h after transfec-tion(29% vs 25% vs 17% vs 14%,P = 0.001),60 h after transfection(38% vs 34% vs 19% vs 16%,P = 0.001),and 72 h after transfection(53% vs 48% vs 20% vs 17%,P = 0.000).Numbers of colonies in negative,blank,CA1,and CA2 groups were 42 ± 4,45 ± 5,8 ± 2,and 10 ± 3,respectively(P = 0.000).There were more than 4.5 times colonies in the blank and negative control groups as there were in the CA1 and CA2 groups.In addition,the colonies in blank and negative control groups were also larger than those in the CA1 and CA2 groups.The percentage of cells in the CA1 and CA2 groups was significantly higher in G 0 /G 1 phase,but lower in S and G 2 /M phase when compared with the negative and control groups.Moreover,cell apoptosis rates in the CA1 and CA2 groups were 5.11 ± 0.32 and 4.73 ± 0.32,which were significantly higher than those in negative(0.95 ± 0.11,P = 0.000) and blank groups(0.86 ± 0.13,P = 0.001).No significant difference was found between CA1 and CA2 groups in cell cycle distribution and apoptosis.CONCLUSION:PIK3CA-targeted short hairpin RNAs can block the phosphoinositide 3-kinase-Akt signaling pathway and inhibit cell growth,increase apoptosis,and induce cell cycle arrest in the PIK3CA-mutant colon cancer SW948 cells.展开更多
AIM： To investigate the ability of hexahydrocurcumin （HHC） to enhance 5-fluorouracil （5-FU） in inhibiting the growth of HT-29 cells by focusing on cyclooxygenase （COX）-2 expression.METHODS： Antiproliferative e...AIM： To investigate the ability of hexahydrocurcumin （HHC） to enhance 5-fluorouracil （5-FU） in inhibiting the growth of HT-29 cells by focusing on cyclooxygenase （COX）-2 expression.METHODS： Antiproliferative effects of HHC and 5-FU, alone and in combination, on growth of HT-29 human colon cancer cells were assessed using 5-diphenyltetrazolium bromide （MTT） reduction assay. In combinationtreatment, low doses of 5-FU were used combined with various concentrations of HHC to minimize the toxic- ity and side effects of 5-FU. The therapeutic effects of these drugs on down-regulation of COX-2 mRNA and protein expression were examined using semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analysis.RESULTS： Ml-I- reduction assay indicated that HHC alone markedly decreased the viability of HT-29 human colon cancer cells compared to control. Semi-quantitative RT-PCR analysis indicated that HHC is a selective COX-2 inhibitor. This finding was supported by the ob- servation that HHC significantly down-regulates COX-2 mRNA expression compared to the control （control： 100.05% ± 0.03% vs HHC： 61.01% ± 0.35%, P 〈 0.05） but does not alter COX-1 mRNA. In combined treatment, addition of HHC to a low dose of 5-FU exerts a synergistic effect against the growth of HT-29 cells by markedly reducing cell viability to a greater degree than monotherapy. Semi-quantitative RT-PCR indicated that 5-FU at the concentration of 5 pmol/L in combina- tion with HHC at the concentration of 25 pmol/L signifi- cantly down-regulates COX-2 mRNA expression when compared with values in cells treated with 5-FU or HHC alone （HHC ＋ 5-FU： 31.93% ± 5.69%, 5-FU： 100.66% ± 4.52% vs HHC： 61.01% ±0.35%, P 〈 0.05）.CONCLUSION： HHC together with 5-FU exerts a synergistic effect and may prove chemotherapeutically useful in treating human colon cancer.展开更多
It aims to analyze the impact heme oxygenase -1 （heme oxygenase 1, HO-1） on regulating factors of human hepatoma cell HepG2＇s cell cycle, through constructing recombinant vector of pcDNA3.1 containing wild-type and...It aims to analyze the impact heme oxygenase -1 （heme oxygenase 1, HO-1） on regulating factors of human hepatoma cell HepG2＇s cell cycle, through constructing recombinant vector of pcDNA3.1 containing wild-type and mutant HO-1 gene （＋）-wtHO-1 and pcDNA3.1 （＋）-mHO-1G143H. By using the method of liposome-mediated, the recombinant vector was transfected hepatoma cell line HepG2. And the transfected one with empty vector was treated as a control group. By the selection of G418, stable expression of wild-type and mutant HO-1 in HepG2 liver cancer cell lines were established. Use the blot of semi-quantitative RT-PCR and Western to test transfected cell lines expressing levels of riO-1 mRNA and protein. As HO-1 expression in stably transfected cell lines altered, we use Western blot to test transfected cell lines P21, P27 protein expression levels. As result shows, we got 1 HO-over-expression of wild-type and mutant in HepG2 cells; wild- type and mutant＇s over expression of HO-1 can induce the expression of tumor suppressor genes p21 and p27.we got the conclusion that HO-l＇s over-expression of tumor suppressor genes p21 and p27 is unrelated to the expression of heme decomposition products. HO-1 may regulate the expression of p21 and p27 through other mechanisms.展开更多
文摘Aim To study the effects of tetrodotoxin （TTX） combined with acetylsalicylic acid （ASA） on nociceptive stimulus in mice. Methods To assess the antinociceptive effects of TTX, ASA or TTX plus ASA, the acetic acid-induced abdominal constriction test and formalin pain test were used. Results TTX （0.5 - 4.0 μg· kg^-1 ） or ASA （25 - 200 mg· kg^-1 ） im produced a significant inhibition of acetic acid-induced abdominal constriction. The median inhibitory doses （ID508） were 2.1 μg· kg^-1 for TTX（ and 64 mg· kg^-1 for ASA. TTX and ASA also showed a dose-dependent inhibition of the second phase response in the formalin pain model, the ID508, being 2.3μg·kg^-1 and 74.2 mg· kg^-1, respectively. The ihteraction between TTX and ASA was synergistic, as evidenced by the fact that （1） when ASA alone compared with the combination of TTX （0.79 μg · kg^-1 or 0.39μg· kg^-1 ） and ASA, the ID508, of ASA reduced from 64.0 mg· kg^-1 to 5.8 mg· kg^-1 or 12.6 mg· kg^-1, and from 74.2 mg· kg^-1 to 7.4 mg· kg^-1 or 13.0 mg· kg^-1 on tile two models of nociceptive tests, respectively; and that （2） synergism in the analgesic effects was shown by isobiolographic analysis. Conclusion TTX, ASA and the combination of the two drags produce analgesic effects in acetic acid-induced abdominal constriction test and formalin-induced pain test. The interactions between TTX and ASA may be useful in developing novel analgesic agents.
基金Supported by Grants from Science and Technology of Guangdong Province Funds,No. 2010B080701038
文摘AIM:To investigate the gene knock-down effect by the phosphoinositide-3-kinase,catalytic,alpha polypeptide(PIK3CA)-targeted double-stranded RNA(dsRNA) and its effect on cell proliferation and cycle distribution in SW948.METHODS:Two PIK3CA-targeted dsRNAs were constructed and transfected into SW948 cells.Transfections were performed using lipofectamine TM 2000.The transfection effectiveness was calculated basing on the rate of fluorescence cell of SW948 at 6 h after transfection.Total messenger RNA was extracted from these cells using the RNeasy kit,and semiquantitative reverse transcription polymerase chain reaction was performed to detect the down-regulation of PIK3CA,AKT1,MYC,and CCND1 gene expression.Cells were harvested,proteins were resolved,and western blot was employed to detect the expression levels of PIK3CA,AKT1,MYC,and CCND1 gene.Cell proliferation was assessed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay and the inhibition rate was calculated.Soft agar colony formation assay was performed basing on colonies greater than 60 μm in diameter at ×100 magnification.The effect on cell cycle distribution and apoptosis was assessed by flow cytometry.All experiments were performed in triplicate.RESULTS:Green fluorescence was observed in SW948 cell transfected with plasmid Pgenesil-1,and the transfection effectiveness was about 65%.Forty-eight hours post-transfection,mRNA expression of PIK3CA in SW948 cells was 0.51 ± 0.04 vs 0.49 ± 0.03 vs 0.92 ± 0.01 vs 0.93 ± 0.03(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of AKT1 was 0.50 ± 0.03 vs 0.48 ± 0.01 vs 0.93 ± 0.04 vs 0.92 ± 0.02(P = 0.000) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.mRNA expression of MYC was 0.49 ± 0.01 vs 0.50 ± 0.04 vs 0.90 ± 0.02 vs 0.91 ± 0.03(P = 0.001) in the four groups respectively.mRNA expression of CCND1 was 0.45 ± 0.02 vs 0.51 ± 0.01 vs 0.96 ± 0.03 vs 0.98 ± 0.01(P = 0.001) in the four groups respectively.The protein level of PIK3CA was 0.53 ± 0.01 vs 0.54 ± 0.02 vs 0.92 ± 0.03 vs 0.91 ± 0.02(P = 0.001) in Pgenesil-CA1,Pgenesil-CA2,negative and blank group respectively.The protein level of AKT1 in the four groups was 0.49 ± 0.02 vs 0.55 ± 0.03 vs 0.94 ± 0.03 vs 0.95 ± 0.04,P = 0.000).The protein level of MYC in the four groups was 0.51 ± 0.03 vs 0.52 ± 0.04 vs 0.92 ± 0.02 vs 0.95 ± 0.01(P = 0.000).The protein level of CCND1 in the four groups was 0.54 ± 0.04 vs 0.56 ± 0.03 vs 0.93 ± 0.01 vs 0.93 ± 0.03(P = 0.000).Both Pgenesil-CA1 and Pgenesil-CA2 plasmids significantly suppressed the growth of SW948 cells when compared with the negative or blank group at 48 h after transfec-tion(29% vs 25% vs 17% vs 14%,P = 0.001),60 h after transfection(38% vs 34% vs 19% vs 16%,P = 0.001),and 72 h after transfection(53% vs 48% vs 20% vs 17%,P = 0.000).Numbers of colonies in negative,blank,CA1,and CA2 groups were 42 ± 4,45 ± 5,8 ± 2,and 10 ± 3,respectively(P = 0.000).There were more than 4.5 times colonies in the blank and negative control groups as there were in the CA1 and CA2 groups.In addition,the colonies in blank and negative control groups were also larger than those in the CA1 and CA2 groups.The percentage of cells in the CA1 and CA2 groups was significantly higher in G 0 /G 1 phase,but lower in S and G 2 /M phase when compared with the negative and control groups.Moreover,cell apoptosis rates in the CA1 and CA2 groups were 5.11 ± 0.32 and 4.73 ± 0.32,which were significantly higher than those in negative(0.95 ± 0.11,P = 0.000) and blank groups(0.86 ± 0.13,P = 0.001).No significant difference was found between CA1 and CA2 groups in cell cycle distribution and apoptosis.CONCLUSION:PIK3CA-targeted short hairpin RNAs can block the phosphoinositide 3-kinase-Akt signaling pathway and inhibit cell growth,increase apoptosis,and induce cell cycle arrest in the PIK3CA-mutant colon cancer SW948 cells.
基金Supported by National Research Council of ThailandPartial support from The Strategic Basic Research Grant of The Thailand Research Fund
文摘AIM： To investigate the ability of hexahydrocurcumin （HHC） to enhance 5-fluorouracil （5-FU） in inhibiting the growth of HT-29 cells by focusing on cyclooxygenase （COX）-2 expression.METHODS： Antiproliferative effects of HHC and 5-FU, alone and in combination, on growth of HT-29 human colon cancer cells were assessed using 5-diphenyltetrazolium bromide （MTT） reduction assay. In combinationtreatment, low doses of 5-FU were used combined with various concentrations of HHC to minimize the toxic- ity and side effects of 5-FU. The therapeutic effects of these drugs on down-regulation of COX-2 mRNA and protein expression were examined using semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analysis.RESULTS： Ml-I- reduction assay indicated that HHC alone markedly decreased the viability of HT-29 human colon cancer cells compared to control. Semi-quantitative RT-PCR analysis indicated that HHC is a selective COX-2 inhibitor. This finding was supported by the ob- servation that HHC significantly down-regulates COX-2 mRNA expression compared to the control （control： 100.05% ± 0.03% vs HHC： 61.01% ± 0.35%, P 〈 0.05） but does not alter COX-1 mRNA. In combined treatment, addition of HHC to a low dose of 5-FU exerts a synergistic effect against the growth of HT-29 cells by markedly reducing cell viability to a greater degree than monotherapy. Semi-quantitative RT-PCR indicated that 5-FU at the concentration of 5 pmol/L in combina- tion with HHC at the concentration of 25 pmol/L signifi- cantly down-regulates COX-2 mRNA expression when compared with values in cells treated with 5-FU or HHC alone （HHC ＋ 5-FU： 31.93% ± 5.69%, 5-FU： 100.66% ± 4.52% vs HHC： 61.01% ±0.35%, P 〈 0.05）.CONCLUSION： HHC together with 5-FU exerts a synergistic effect and may prove chemotherapeutically useful in treating human colon cancer.
文摘It aims to analyze the impact heme oxygenase -1 （heme oxygenase 1, HO-1） on regulating factors of human hepatoma cell HepG2＇s cell cycle, through constructing recombinant vector of pcDNA3.1 containing wild-type and mutant HO-1 gene （＋）-wtHO-1 and pcDNA3.1 （＋）-mHO-1G143H. By using the method of liposome-mediated, the recombinant vector was transfected hepatoma cell line HepG2. And the transfected one with empty vector was treated as a control group. By the selection of G418, stable expression of wild-type and mutant HO-1 in HepG2 liver cancer cell lines were established. Use the blot of semi-quantitative RT-PCR and Western to test transfected cell lines expressing levels of riO-1 mRNA and protein. As HO-1 expression in stably transfected cell lines altered, we use Western blot to test transfected cell lines P21, P27 protein expression levels. As result shows, we got 1 HO-over-expression of wild-type and mutant in HepG2 cells; wild- type and mutant＇s over expression of HO-1 can induce the expression of tumor suppressor genes p21 and p27.we got the conclusion that HO-l＇s over-expression of tumor suppressor genes p21 and p27 is unrelated to the expression of heme decomposition products. HO-1 may regulate the expression of p21 and p27 through other mechanisms.
