OBJECTIVE： This study investigated the effects of allylpyrocatechol （APC）, the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphyloc...OBJECTIVE： This study investigated the effects of allylpyrocatechol （APC）, the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host. METHODS： Effects of APC on expressions of genes encoding catalase （katA）, superoxide dismutases （SODs）, including soda and sodM, and alkyl hydroperoxide reductase （ahpC） in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species （ROS） levels were determined using the nitroblue tetrazolium （NBT） reduction assay. RESULTS： APC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL （P〈0.05）. Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells （P〈0.05）. Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 （A310nm） compared to untreated cells which was 0.394 （A310nm）. In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10^-4 （U/L or μmol/（min.L）） compared to untreated cells, which was 4.8 × 10^-4 U/L （P〈0.05）. Absorbance readings （A575nm） for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively （P〈0.05）. CONCLUSION： APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O2, presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternatepathway involving ahpC to reduce stress invoked by O2- and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.展开更多
Objective： The combination effect of Piperbetle （PB） and 5-fluorouracil （5-FU） in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods： HT29 and HCT1...Objective： The combination effect of Piperbetle （PB） and 5-fluorouracil （5-FU） in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods： HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography （HPLC） was performed to exclude any possible chemical interaction between the compounds. Results： In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose （IC^0 12.5 pmol/L） and a shorter time （24 h） in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. Conclusions： In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway.展开更多
基金the Faculty of Health Sciences,MARA University of Technology(UiTM) SelangorPuncak Alam and UiTM LESTARI grant(600-IRMI/ MYRA 5/3/LESTARI(16/2016) for the funds provided to complete this study
文摘OBJECTIVE： This study investigated the effects of allylpyrocatechol （APC）, the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host. METHODS： Effects of APC on expressions of genes encoding catalase （katA）, superoxide dismutases （SODs）, including soda and sodM, and alkyl hydroperoxide reductase （ahpC） in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species （ROS） levels were determined using the nitroblue tetrazolium （NBT） reduction assay. RESULTS： APC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL （P〈0.05）. Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells （P〈0.05）. Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 （A310nm） compared to untreated cells which was 0.394 （A310nm）. In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10^-4 （U/L or μmol/（min.L）） compared to untreated cells, which was 4.8 × 10^-4 U/L （P〈0.05）. Absorbance readings （A575nm） for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively （P〈0.05）. CONCLUSION： APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O2, presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternatepathway involving ahpC to reduce stress invoked by O2- and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.
基金supported by the Ministry of Higher Education,Malaysia(No.UKM-JJ-03-FRGS0044-2010)Universiti Kebangsaan Malaysia(No.UKM-DPP-2014-131)
文摘Objective： The combination effect of Piperbetle （PB） and 5-fluorouracil （5-FU） in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods： HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography （HPLC） was performed to exclude any possible chemical interaction between the compounds. Results： In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose （IC^0 12.5 pmol/L） and a shorter time （24 h） in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. Conclusions： In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway.
