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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

Increased generation of intracellular ROS, loss of mitochondrial transmembrane potential (∆ѱm), and regulation of H2O2-mediated oxidative stress by 21α-MMD in lung cancer cells.(A and B) After treatment with various concentrations of 21α-MMD for 24 h, the cells were exposed to the oxidative fluorescent dye DCFDA. ROS levels were interpreted as percentage of fluorescent intensity measured by FACS. DCFDA stained cells were observed under fluorescence microscopy to observe intracellular ROS distribution (C) A549 cells were treated with 10 μM NAC and 6 μM 21α-MMD alone or in combination for 24 h. Treated and non-treated cells were subjected to Western blotting for the detection of Akt and phospho-Akt protein expression. (D) A549 cells were treated with 0.6 mM H2O2 and 12.5 μM 21α-MMD alone or in combination for 24 h. Treated or non-treated cells were subjected for Western blotting for the detection of ERK and p-ERK protein expression. (E) A549 cells were treated as in (C). Treated or non-treated cells were subjected to MTT assay to examine changes in cell growth rate. (F) A549 cells were treated with 25 μM 21α-MMD for 24 h followed by ∆ѱm determination by flow cytometric analysis. (*p<0.05; **p<0.01)
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pone.0127841.g003: Increased generation of intracellular ROS, loss of mitochondrial transmembrane potential (∆ѱm), and regulation of H2O2-mediated oxidative stress by 21α-MMD in lung cancer cells.(A and B) After treatment with various concentrations of 21α-MMD for 24 h, the cells were exposed to the oxidative fluorescent dye DCFDA. ROS levels were interpreted as percentage of fluorescent intensity measured by FACS. DCFDA stained cells were observed under fluorescence microscopy to observe intracellular ROS distribution (C) A549 cells were treated with 10 μM NAC and 6 μM 21α-MMD alone or in combination for 24 h. Treated and non-treated cells were subjected to Western blotting for the detection of Akt and phospho-Akt protein expression. (D) A549 cells were treated with 0.6 mM H2O2 and 12.5 μM 21α-MMD alone or in combination for 24 h. Treated or non-treated cells were subjected for Western blotting for the detection of ERK and p-ERK protein expression. (E) A549 cells were treated as in (C). Treated or non-treated cells were subjected to MTT assay to examine changes in cell growth rate. (F) A549 cells were treated with 25 μM 21α-MMD for 24 h followed by ∆ѱm determination by flow cytometric analysis. (*p<0.05; **p<0.01)

Mentions: Regulating changes in the levels of ROS proved to be relatively significant in various cellular functions including cell growth and survival [30]. To further understand the mechanism of the redox regulatory activity of 21α–MMD on A549 and H1299 cells, the levels of ROS after treatment with 21α–MMD (25–100 μM) were determined (Fig 3A). Flow cytometric analysis showed that the proportion of cells with high fluorescence intensity was increased in cells after treatment with 21α–MMD in a concentration-dependent manner for 24 h, indicating that the levels of intracellular ROS in A549 and H1299 were significantly increased with observed 15% to 34% increase in generation levels in A549 cells while 4.5% to 30.8% increase in H1299 cells respective of the increasing concentrations of 21α-MMD from 25 to 100 μM. To further evaluate the involvement of ROS in the activity of 21α-MMD, we incorporated the ROS scavenger NAC with or without 21α-MMD treatment in A549 and H1299 cells for 24 h. Increase of the percentage of ROS production was significantly blocked by NAC at 10 mM concentration with 9.8% and 6.8% generation levels in A549 and H1299 cells, respectively, while 10 mM NAC together with 25 μM 21α-MMD significantly decreased the production with detected 3.1% and 2.4% production in both cells, respectively. Previous studies have revealed that the phosphorylation of Akt, which frequently is hyperactivated in cancer, found to be crucial in ROS-dependent autophagy and contributes to tumor cell resistance to cytotoxic chemotherapies [31]. Akt is also involved in the induction of the accumulation of oxygen radicals, which when exploited can selectively kill cancer cells with high-level Akt [32]. Thus, it is of interest whether 21α-MMD targets Akt phosphorylation to regulate changes in ROS generation. 21α-MMD caused slight inhibition of total Akt expression in A549 cells at 6 μM. However when combined with the ROS inhibitor NAC at 10 mM, total Akt and its phosphorylation were significantly inhibited. This further supports evidences on the involvement of Akt in redox regulation (Fig 3C). Therefore, the activity of 21α-MMD on mitochondrial depolarization might be mediated by ROS and the regulation of its intracellular levels might act as a dependent key factor in affecting the mechanistic activity of 21α-MMD in lung cancer cells. To functionally characterize the ability of 21α-MMD to regulate intracellular ROS generation, we further examined whether 21α-MMD modulates H2O2-mediated oxidative stress in A549 cells. It has been previously reported that inhibition of ERK significantly increased cell death after H2O2 treatment in various cell models including epithelial and neuronal cells [33]. We then next identified the possible mechanism for this effect through ROS generation and ERK phosphorylation. 21α-MMD (12.5 μM) significantly down-regulated phospho-ERK expression when combined with H2O2 (0.6 mM) compared to H2O2 treatment alone in A549 cells (Fig 3D). Furthermore, it was found that treatment of 0.6 mM H2O2 with 12.5 μM 21α-MMD increased the oxidative-stress induced cytotoxicity significantly with observed 8% viable cells compared to 54% viable cells when treated 0.6 mM H2O2 alone, in A549 cells (Fig 3E). These findings suggest that 21α-MMD enhances the H2O2-mediated cytotoxicity by regulation of ERK phosphorylation. Previous studies showed that differences in the intrinsic mitochondrial membrane potential (∆ѱm) are linked to the sensitivity of tumor cells to various cytotoxic chemopreventive agents [34]. We therefore performed an assay to measure mitochondrial transmembrane potential using the lipophilic cationic dye TMRE to evaluate the effect of 21α-MMD on the mitochondrial membrane integrity of A549 cells. The treatment of 21α-MMD (25 µM) for 24 h was found to induce significant blocking of hyperpolarization of the mitochondrial membrane reflected by decrease in fluorescence intensity compared to untreated cells, suggesting that treatment with 21α-MMD resulted in dissipation of ∆ѱm. After 24 h treatment, the mitochondrial damage resulted in 44.8% reduction in the accumulation of TMRE in the organelle in A549 cells, thus implying a decreased permeability threshold. All mitochondrial integrity values were subtracted and relative to control (Fig 3F). These results suggest that 21α-MMD might act by disrupting the mitochondrial membrane.


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)

Increased generation of intracellular ROS, loss of mitochondrial transmembrane potential (∆ѱm), and regulation of H2O2-mediated oxidative stress by 21α-MMD in lung cancer cells.(A and B) After treatment with various concentrations of 21α-MMD for 24 h, the cells were exposed to the oxidative fluorescent dye DCFDA. ROS levels were interpreted as percentage of fluorescent intensity measured by FACS. DCFDA stained cells were observed under fluorescence microscopy to observe intracellular ROS distribution (C) A549 cells were treated with 10 μM NAC and 6 μM 21α-MMD alone or in combination for 24 h. Treated and non-treated cells were subjected to Western blotting for the detection of Akt and phospho-Akt protein expression. (D) A549 cells were treated with 0.6 mM H2O2 and 12.5 μM 21α-MMD alone or in combination for 24 h. Treated or non-treated cells were subjected for Western blotting for the detection of ERK and p-ERK protein expression. (E) A549 cells were treated as in (C). Treated or non-treated cells were subjected to MTT assay to examine changes in cell growth rate. (F) A549 cells were treated with 25 μM 21α-MMD for 24 h followed by ∆ѱm determination by flow cytometric analysis. (*p<0.05; **p<0.01)
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476707&req=5

pone.0127841.g003: Increased generation of intracellular ROS, loss of mitochondrial transmembrane potential (∆ѱm), and regulation of H2O2-mediated oxidative stress by 21α-MMD in lung cancer cells.(A and B) After treatment with various concentrations of 21α-MMD for 24 h, the cells were exposed to the oxidative fluorescent dye DCFDA. ROS levels were interpreted as percentage of fluorescent intensity measured by FACS. DCFDA stained cells were observed under fluorescence microscopy to observe intracellular ROS distribution (C) A549 cells were treated with 10 μM NAC and 6 μM 21α-MMD alone or in combination for 24 h. Treated and non-treated cells were subjected to Western blotting for the detection of Akt and phospho-Akt protein expression. (D) A549 cells were treated with 0.6 mM H2O2 and 12.5 μM 21α-MMD alone or in combination for 24 h. Treated or non-treated cells were subjected for Western blotting for the detection of ERK and p-ERK protein expression. (E) A549 cells were treated as in (C). Treated or non-treated cells were subjected to MTT assay to examine changes in cell growth rate. (F) A549 cells were treated with 25 μM 21α-MMD for 24 h followed by ∆ѱm determination by flow cytometric analysis. (*p<0.05; **p<0.01)
Mentions: Regulating changes in the levels of ROS proved to be relatively significant in various cellular functions including cell growth and survival [30]. To further understand the mechanism of the redox regulatory activity of 21α–MMD on A549 and H1299 cells, the levels of ROS after treatment with 21α–MMD (25–100 μM) were determined (Fig 3A). Flow cytometric analysis showed that the proportion of cells with high fluorescence intensity was increased in cells after treatment with 21α–MMD in a concentration-dependent manner for 24 h, indicating that the levels of intracellular ROS in A549 and H1299 were significantly increased with observed 15% to 34% increase in generation levels in A549 cells while 4.5% to 30.8% increase in H1299 cells respective of the increasing concentrations of 21α-MMD from 25 to 100 μM. To further evaluate the involvement of ROS in the activity of 21α-MMD, we incorporated the ROS scavenger NAC with or without 21α-MMD treatment in A549 and H1299 cells for 24 h. Increase of the percentage of ROS production was significantly blocked by NAC at 10 mM concentration with 9.8% and 6.8% generation levels in A549 and H1299 cells, respectively, while 10 mM NAC together with 25 μM 21α-MMD significantly decreased the production with detected 3.1% and 2.4% production in both cells, respectively. Previous studies have revealed that the phosphorylation of Akt, which frequently is hyperactivated in cancer, found to be crucial in ROS-dependent autophagy and contributes to tumor cell resistance to cytotoxic chemotherapies [31]. Akt is also involved in the induction of the accumulation of oxygen radicals, which when exploited can selectively kill cancer cells with high-level Akt [32]. Thus, it is of interest whether 21α-MMD targets Akt phosphorylation to regulate changes in ROS generation. 21α-MMD caused slight inhibition of total Akt expression in A549 cells at 6 μM. However when combined with the ROS inhibitor NAC at 10 mM, total Akt and its phosphorylation were significantly inhibited. This further supports evidences on the involvement of Akt in redox regulation (Fig 3C). Therefore, the activity of 21α-MMD on mitochondrial depolarization might be mediated by ROS and the regulation of its intracellular levels might act as a dependent key factor in affecting the mechanistic activity of 21α-MMD in lung cancer cells. To functionally characterize the ability of 21α-MMD to regulate intracellular ROS generation, we further examined whether 21α-MMD modulates H2O2-mediated oxidative stress in A549 cells. It has been previously reported that inhibition of ERK significantly increased cell death after H2O2 treatment in various cell models including epithelial and neuronal cells [33]. We then next identified the possible mechanism for this effect through ROS generation and ERK phosphorylation. 21α-MMD (12.5 μM) significantly down-regulated phospho-ERK expression when combined with H2O2 (0.6 mM) compared to H2O2 treatment alone in A549 cells (Fig 3D). Furthermore, it was found that treatment of 0.6 mM H2O2 with 12.5 μM 21α-MMD increased the oxidative-stress induced cytotoxicity significantly with observed 8% viable cells compared to 54% viable cells when treated 0.6 mM H2O2 alone, in A549 cells (Fig 3E). These findings suggest that 21α-MMD enhances the H2O2-mediated cytotoxicity by regulation of ERK phosphorylation. Previous studies showed that differences in the intrinsic mitochondrial membrane potential (∆ѱm) are linked to the sensitivity of tumor cells to various cytotoxic chemopreventive agents [34]. We therefore performed an assay to measure mitochondrial transmembrane potential using the lipophilic cationic dye TMRE to evaluate the effect of 21α-MMD on the mitochondrial membrane integrity of A549 cells. The treatment of 21α-MMD (25 µM) for 24 h was found to induce significant blocking of hyperpolarization of the mitochondrial membrane reflected by decrease in fluorescence intensity compared to untreated cells, suggesting that treatment with 21α-MMD resulted in dissipation of ∆ѱm. After 24 h treatment, the mitochondrial damage resulted in 44.8% reduction in the accumulation of TMRE in the organelle in A549 cells, thus implying a decreased permeability threshold. All mitochondrial integrity values were subtracted and relative to control (Fig 3F). These results suggest that 21α-MMD might act by disrupting the mitochondrial membrane.

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