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Suppression of MAPK Signaling and Reversal of mTOR-Dependent MDR1-Associated Multidrug Resistance by 21α-Methylmelianodiol in Lung Cancer Cells.

Aldonza MB, Hong JY, Bae SY, Song J, Kim WK, Oh J, Shin Y, Lee SH, Lee SK - PLoS ONE (2015)

Bottom Line: Interplay between PI3K/AMPK/AKT and MAPK pathways is a crucial effector in lung cancer growth and progression.Here, we described whether 21α-Methylmelianodiol (21α-MMD), an active triterpenoid derivative of Poncirus trifoliate, can display anticancer properties by regulating these signals and modulate the occurrence of multidrug resistance in NSCLC cells.Employing the established paclitaxel-resistant A549 cells (A549-PacR), we further found that 21α-MMD induced a MDR reversal activity through the inhibition of P-gp/MDR1 expressions, function, and transcription with regained paclitaxel sensitivity which might dependently correlate to the regulation of PI3K/mTOR signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul, Korea.

ABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide and remains the most prevalent. Interplay between PI3K/AMPK/AKT and MAPK pathways is a crucial effector in lung cancer growth and progression. These signals transduction protein kinases serve as good therapeutic targets for non-small cell lung cancer (NSCLC) which comprises up to 90% of lung cancers. Here, we described whether 21α-Methylmelianodiol (21α-MMD), an active triterpenoid derivative of Poncirus trifoliate, can display anticancer properties by regulating these signals and modulate the occurrence of multidrug resistance in NSCLC cells. We found that 21α-MMD inhibited the growth and colony formation of lung cancer cells without affecting the normal lung cell phenotype. 21α-MMD also abrogated the metastatic activity of lung cancer cells through the inhibition of cell migration and invasion, and induced G0/G1 cell cycle arrest with increased intracellular ROS generation and loss of mitochondrial membrane integrity. 21α-MMD regulated the expressions of PI3K/AKT/AMPK and MAPK signaling which drove us to further evaluate its activity on multidrug resistance (MDR) in lung cancer cells by specifying on P-glycoprotein (P-gp)/MDR1-association. Employing the established paclitaxel-resistant A549 cells (A549-PacR), we further found that 21α-MMD induced a MDR reversal activity through the inhibition of P-gp/MDR1 expressions, function, and transcription with regained paclitaxel sensitivity which might dependently correlate to the regulation of PI3K/mTOR signaling pathway. Taken together, these findings demonstrate, for the first time, the mechanistic evaluation in vitro of 21α-MMD displaying growth-inhibiting potential with influence on MDR reversal in human lung cancer cells.

No MeSH data available.


Related in: MedlinePlus

Suppression of expression, function, and transcription of MDR1/P-glycoprotein (P-gp) by 21α-MMD in A549-PacR cells.(A) P-gp protein expression was determined by Western blotting after 24 and 48 h treatment with 21α-MMD at various indicated concentrations (B) The effect of 21α-MMD interaction with MDR1 mRNA gene expression levels in A549-PacR cells was determined by RT-PCR analysis. Cells were treated with 21α-MMD for 24 and 48 h. (C) Effect of 21α-MMD on intracellular Rhodamine-123 (Rho-123) accumulation in A549-PacR was quantitatively measured by flow cytometry. Cells were treated with various concentrations of 21α-MMD for 24 h and 48 h followed by the exposure to 1 μg/ml of Rho-123 dye for 90 min. Parental A549 cells was used as positive control. Each column shows the mean ± SD of three independent experiments, performed in triplicate. (*p<0.05; **p<0.01)
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pone.0127841.g006: Suppression of expression, function, and transcription of MDR1/P-glycoprotein (P-gp) by 21α-MMD in A549-PacR cells.(A) P-gp protein expression was determined by Western blotting after 24 and 48 h treatment with 21α-MMD at various indicated concentrations (B) The effect of 21α-MMD interaction with MDR1 mRNA gene expression levels in A549-PacR cells was determined by RT-PCR analysis. Cells were treated with 21α-MMD for 24 and 48 h. (C) Effect of 21α-MMD on intracellular Rhodamine-123 (Rho-123) accumulation in A549-PacR was quantitatively measured by flow cytometry. Cells were treated with various concentrations of 21α-MMD for 24 h and 48 h followed by the exposure to 1 μg/ml of Rho-123 dye for 90 min. Parental A549 cells was used as positive control. Each column shows the mean ± SD of three independent experiments, performed in triplicate. (*p<0.05; **p<0.01)

Mentions: Overexpression of MDR1 mRNA and P-glycoprotein (P-gp) levels is associated with phenotype multi-drug resistance (MDR). P-gp, encoded by the ABCB1/MDR1 gene, functions as an ATP-driven efflux pump transporter [40,41]. The effect on the expression and efflux pump activity of MDR1/P-gp in A549-PacR cells was examined accordingly after treatment with various concentrations of 21α-MMD. To elucidate potential P-gp suppression activity, P-gp protein expression was examined by Western blotting. P-gp expression was suppressed in A549-PacR in a time-dependent manner at higher 21α-MMD concentrations while revealing less or no expression of P-gp in parental A549 cells (Fig 6A). However, at 25 μM, 21α-MMD caused slight induction of P-gp protein expression which can be attributed to the specific characteristic of the P-gp efflux pump being stimulated by inhibitors at lower cytotoxic doses but significantly inhibited at higher concentrations. One might consider 21α-MMD as in the same category of andrographolide, berberin, glycyrrhizin, etc. by which their characterization as both P-gp (MDR1) inducers and inhibitors are given by their “biphasic protein modulation” [33]. Consistent with this, 21α-MMD significantly suppressed MDR1 mRNA expression and levels in A549-PacR cells in both same manners with observed 17.2% to 69.0% (24 h) and 38.7% to 85.5% fold decrease in mRNA levels (Fig 6B and 6C). To further examine the suppressive effects of 21α–MMD on P-gp expression and whether it is functionally associated with the recovery of drug accumulation in A549-PacR cells, Rho-123 accumulation assay was conducted. Rho-123 dye was used as a substrate to determine the efflux function of P-gp in MDR1/P-gp overexpressing A549-PacR cells since the P-gp-dependent efflux of fluorescent Rho-123 was extensively used in determining efflux from drug-resistant cell lines expressing P-gp. The intracellular accumulation of Rh-123 was detected using flow cytometer. The parental A549 cells, which do not express P-gp, occurred with high accumulation of intracellular Rho-123 (the peaks were shifted to the right side of histogram). In A549-PacR cells, which overexpress P-gp, however, accumulated relatively lower levels of Rho-123 due to the efflux pump action of P-gp. In line with P-gp inhibition, 21α–MMD increased the intracellular accumulation of Rho-123 at higher concentrations in a time-dependent manner indicating that 21α–MMD supports its suppressive effects on P-gp/MDR1 expression and function, revealing elevated P-gp efflux function by 58.24% to 162.8% (24 h) and 52.8% to 169.4% (48 h). Moreover, to examine enhanced Rho-123 efflux, the degree of enhancement by fluorescence intensity was also determined. We found an enhanced uptake of Rho-123 in 21α-MMD-treated A549-PacR cells in a dose-dependent manner for 24 h compared to the non-treated A549-PacR cells with almost no fluorescent visibility while competent when compared to parental A549 cells (Fig 6D). These findings suggest that 21α-MMD inhibits P-gp efflux function in A549-PacR cells. To demonstrate the localization of P-gp in A549-PacR cells, confocal microscopy was also conducted. Immunofluorescence analysis demonstrated that P-gp, which is labeled with green fluorescence (FITC), is mainly overexpressed in the cellular plasma membrane and cytoplasmic regions of A549-PacR cells while less expressed in parental A549 cells (Fig 7). Nuclei of the cells were labeled with blue fluorescence (DAPI). After treatment with 21αα-MMD at various concentrations for 24 h, the amount of P-gp fluorescence was significantly decreased dose-dependently.


Suppression of MAPK Signaling and Reversal of mTOR-Dependent MDR1-Associated Multidrug Resistance by 21α-Methylmelianodiol in Lung Cancer Cells.

Aldonza MB, Hong JY, Bae SY, Song J, Kim WK, Oh J, Shin Y, Lee SH, Lee SK - PLoS ONE (2015)

Suppression of expression, function, and transcription of MDR1/P-glycoprotein (P-gp) by 21α-MMD in A549-PacR cells.(A) P-gp protein expression was determined by Western blotting after 24 and 48 h treatment with 21α-MMD at various indicated concentrations (B) The effect of 21α-MMD interaction with MDR1 mRNA gene expression levels in A549-PacR cells was determined by RT-PCR analysis. Cells were treated with 21α-MMD for 24 and 48 h. (C) Effect of 21α-MMD on intracellular Rhodamine-123 (Rho-123) accumulation in A549-PacR was quantitatively measured by flow cytometry. Cells were treated with various concentrations of 21α-MMD for 24 h and 48 h followed by the exposure to 1 μg/ml of Rho-123 dye for 90 min. Parental A549 cells was used as positive control. Each column shows the mean ± SD of three independent experiments, performed in triplicate. (*p<0.05; **p<0.01)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476707&req=5

pone.0127841.g006: Suppression of expression, function, and transcription of MDR1/P-glycoprotein (P-gp) by 21α-MMD in A549-PacR cells.(A) P-gp protein expression was determined by Western blotting after 24 and 48 h treatment with 21α-MMD at various indicated concentrations (B) The effect of 21α-MMD interaction with MDR1 mRNA gene expression levels in A549-PacR cells was determined by RT-PCR analysis. Cells were treated with 21α-MMD for 24 and 48 h. (C) Effect of 21α-MMD on intracellular Rhodamine-123 (Rho-123) accumulation in A549-PacR was quantitatively measured by flow cytometry. Cells were treated with various concentrations of 21α-MMD for 24 h and 48 h followed by the exposure to 1 μg/ml of Rho-123 dye for 90 min. Parental A549 cells was used as positive control. Each column shows the mean ± SD of three independent experiments, performed in triplicate. (*p<0.05; **p<0.01)
Mentions: Overexpression of MDR1 mRNA and P-glycoprotein (P-gp) levels is associated with phenotype multi-drug resistance (MDR). P-gp, encoded by the ABCB1/MDR1 gene, functions as an ATP-driven efflux pump transporter [40,41]. The effect on the expression and efflux pump activity of MDR1/P-gp in A549-PacR cells was examined accordingly after treatment with various concentrations of 21α-MMD. To elucidate potential P-gp suppression activity, P-gp protein expression was examined by Western blotting. P-gp expression was suppressed in A549-PacR in a time-dependent manner at higher 21α-MMD concentrations while revealing less or no expression of P-gp in parental A549 cells (Fig 6A). However, at 25 μM, 21α-MMD caused slight induction of P-gp protein expression which can be attributed to the specific characteristic of the P-gp efflux pump being stimulated by inhibitors at lower cytotoxic doses but significantly inhibited at higher concentrations. One might consider 21α-MMD as in the same category of andrographolide, berberin, glycyrrhizin, etc. by which their characterization as both P-gp (MDR1) inducers and inhibitors are given by their “biphasic protein modulation” [33]. Consistent with this, 21α-MMD significantly suppressed MDR1 mRNA expression and levels in A549-PacR cells in both same manners with observed 17.2% to 69.0% (24 h) and 38.7% to 85.5% fold decrease in mRNA levels (Fig 6B and 6C). To further examine the suppressive effects of 21α–MMD on P-gp expression and whether it is functionally associated with the recovery of drug accumulation in A549-PacR cells, Rho-123 accumulation assay was conducted. Rho-123 dye was used as a substrate to determine the efflux function of P-gp in MDR1/P-gp overexpressing A549-PacR cells since the P-gp-dependent efflux of fluorescent Rho-123 was extensively used in determining efflux from drug-resistant cell lines expressing P-gp. The intracellular accumulation of Rh-123 was detected using flow cytometer. The parental A549 cells, which do not express P-gp, occurred with high accumulation of intracellular Rho-123 (the peaks were shifted to the right side of histogram). In A549-PacR cells, which overexpress P-gp, however, accumulated relatively lower levels of Rho-123 due to the efflux pump action of P-gp. In line with P-gp inhibition, 21α–MMD increased the intracellular accumulation of Rho-123 at higher concentrations in a time-dependent manner indicating that 21α–MMD supports its suppressive effects on P-gp/MDR1 expression and function, revealing elevated P-gp efflux function by 58.24% to 162.8% (24 h) and 52.8% to 169.4% (48 h). Moreover, to examine enhanced Rho-123 efflux, the degree of enhancement by fluorescence intensity was also determined. We found an enhanced uptake of Rho-123 in 21α-MMD-treated A549-PacR cells in a dose-dependent manner for 24 h compared to the non-treated A549-PacR cells with almost no fluorescent visibility while competent when compared to parental A549 cells (Fig 6D). These findings suggest that 21α-MMD inhibits P-gp efflux function in A549-PacR cells. To demonstrate the localization of P-gp in A549-PacR cells, confocal microscopy was also conducted. Immunofluorescence analysis demonstrated that P-gp, which is labeled with green fluorescence (FITC), is mainly overexpressed in the cellular plasma membrane and cytoplasmic regions of A549-PacR cells while less expressed in parental A549 cells (Fig 7). Nuclei of the cells were labeled with blue fluorescence (DAPI). After treatment with 21αα-MMD at various concentrations for 24 h, the amount of P-gp fluorescence was significantly decreased dose-dependently.

Bottom Line: Interplay between PI3K/AMPK/AKT and MAPK pathways is a crucial effector in lung cancer growth and progression.Here, we described whether 21α-Methylmelianodiol (21α-MMD), an active triterpenoid derivative of Poncirus trifoliate, can display anticancer properties by regulating these signals and modulate the occurrence of multidrug resistance in NSCLC cells.Employing the established paclitaxel-resistant A549 cells (A549-PacR), we further found that 21α-MMD induced a MDR reversal activity through the inhibition of P-gp/MDR1 expressions, function, and transcription with regained paclitaxel sensitivity which might dependently correlate to the regulation of PI3K/mTOR signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul, Korea.

ABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide and remains the most prevalent. Interplay between PI3K/AMPK/AKT and MAPK pathways is a crucial effector in lung cancer growth and progression. These signals transduction protein kinases serve as good therapeutic targets for non-small cell lung cancer (NSCLC) which comprises up to 90% of lung cancers. Here, we described whether 21α-Methylmelianodiol (21α-MMD), an active triterpenoid derivative of Poncirus trifoliate, can display anticancer properties by regulating these signals and modulate the occurrence of multidrug resistance in NSCLC cells. We found that 21α-MMD inhibited the growth and colony formation of lung cancer cells without affecting the normal lung cell phenotype. 21α-MMD also abrogated the metastatic activity of lung cancer cells through the inhibition of cell migration and invasion, and induced G0/G1 cell cycle arrest with increased intracellular ROS generation and loss of mitochondrial membrane integrity. 21α-MMD regulated the expressions of PI3K/AKT/AMPK and MAPK signaling which drove us to further evaluate its activity on multidrug resistance (MDR) in lung cancer cells by specifying on P-glycoprotein (P-gp)/MDR1-association. Employing the established paclitaxel-resistant A549 cells (A549-PacR), we further found that 21α-MMD induced a MDR reversal activity through the inhibition of P-gp/MDR1 expressions, function, and transcription with regained paclitaxel sensitivity which might dependently correlate to the regulation of PI3K/mTOR signaling pathway. Taken together, these findings demonstrate, for the first time, the mechanistic evaluation in vitro of 21α-MMD displaying growth-inhibiting potential with influence on MDR reversal in human lung cancer cells.

No MeSH data available.


Related in: MedlinePlus