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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

Induction of minimal G0/G1 cell cycle arrest by 21α-MMD.(A) Flow cytometry analysis was conducted in A549, H460, and H1299 cells after treatment with 21α-MMD for 24 h. (B) A549 and H1299 cells were treated with various concentrations of 21α-MMD for 24 h. DNA synthesis was measured by BrdU incorporation. (C) Cell cycle-related proteins cyclins A, D1, E, CDK2, CDK4, Rb, and phospho-Rb expressions were analyzed by Western blotting after exposure to various 21α-MMD concentrations for 24 h in A549 cells. (D) Cyclin E and CDK2 were selected as 21α-MMD targets to measure mRNA gene expression levels after treatment for 24 h based on the preliminary Western blot analysis results. mRNA levels of cyclin E and CDK2 were measured by RT-PCR and real-time PCR (E). Changes in the mRNA expression of the represented genes were determined by plotting the relative Ct ratio GAPDH, which was used as the internal control in real-time PCR. (*p<0.05; **p<0.01)
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pone.0127841.g002: Induction of minimal G0/G1 cell cycle arrest by 21α-MMD.(A) Flow cytometry analysis was conducted in A549, H460, and H1299 cells after treatment with 21α-MMD for 24 h. (B) A549 and H1299 cells were treated with various concentrations of 21α-MMD for 24 h. DNA synthesis was measured by BrdU incorporation. (C) Cell cycle-related proteins cyclins A, D1, E, CDK2, CDK4, Rb, and phospho-Rb expressions were analyzed by Western blotting after exposure to various 21α-MMD concentrations for 24 h in A549 cells. (D) Cyclin E and CDK2 were selected as 21α-MMD targets to measure mRNA gene expression levels after treatment for 24 h based on the preliminary Western blot analysis results. mRNA levels of cyclin E and CDK2 were measured by RT-PCR and real-time PCR (E). Changes in the mRNA expression of the represented genes were determined by plotting the relative Ct ratio GAPDH, which was used as the internal control in real-time PCR. (*p<0.05; **p<0.01)

Mentions: To further elucidate the underlying mechanism of 21α-MMD, we investigated whether it can induce cell cycle arrest and alter cell cycle regulatory molecules operative in the G0/G1 phase transition in various NSCLC cell lines. To determine whether 21α-MMD affects cells in a particular phase of the cell cycle we performed flow cytometry on A549, H460, and H1299 cells after treatment with 25 to 100 μM concentrations of 21α-MMD for 24 h (Fig 2A). In A549 and H460 cells, 21α-MMD caused a rapid but minimal accumulation in the number of cells in the G0/G1 phase in a concentration-dependent manner, while in H1299 treated cells, there was a slight inconsistent decrease of cells in the G0/G1 phase at 50 μM by 1.5%, nevertheless, the same phase was arrested with significant accumulation of cells at 100 μM. These findings suggest that 21α–MMD marginally induced G0/G1 phase cell cycle arrest by potentially and selectively modulating cell cycle regulators in human lung cancer cells. We also incorporated bromodeoxyuridine (BrdU) into A549 and H1299 cells to determine if there would be significant changes in the fraction of S phase cells after treatment with 21α-MMD (Fig 2B). As a result, 21α-MMD caused the inhibition of the S phase of both cells after 24 h treatment. To further examine the association of cell cycle regulatory proteins, Western blot analysis was conducted with the treatment of 21α-MMD (Fig 2C). A significant down-regulation of CDK2, CDK4, cyclin D1, Rb and pRb was found in A549 cells. It was also observed that CDK2, cyclin E, cyclin A, c-myc, and CDK4 were significantly suppressed with 100 μM 21α-MMD treatment for 24 h. Specific suppressive effects of 21α-MMD on cyclin E and CDK2 mRNA gene expressions were also evaluated through RT-PCR. Cyclin E and CDK2 mRNA levels were slightly affected showing detectable changes with 12% to 50% (CDK2), 18% to 34% (cyclin E) fold decrease in A549 cells, respectively. All mRNA levels were normalized to the internal control GAPDH (Fig 2D and 2E).


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)

Induction of minimal G0/G1 cell cycle arrest by 21α-MMD.(A) Flow cytometry analysis was conducted in A549, H460, and H1299 cells after treatment with 21α-MMD for 24 h. (B) A549 and H1299 cells were treated with various concentrations of 21α-MMD for 24 h. DNA synthesis was measured by BrdU incorporation. (C) Cell cycle-related proteins cyclins A, D1, E, CDK2, CDK4, Rb, and phospho-Rb expressions were analyzed by Western blotting after exposure to various 21α-MMD concentrations for 24 h in A549 cells. (D) Cyclin E and CDK2 were selected as 21α-MMD targets to measure mRNA gene expression levels after treatment for 24 h based on the preliminary Western blot analysis results. mRNA levels of cyclin E and CDK2 were measured by RT-PCR and real-time PCR (E). Changes in the mRNA expression of the represented genes were determined by plotting the relative Ct ratio GAPDH, which was used as the internal control in real-time PCR. (*p<0.05; **p<0.01)
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127841.g002: Induction of minimal G0/G1 cell cycle arrest by 21α-MMD.(A) Flow cytometry analysis was conducted in A549, H460, and H1299 cells after treatment with 21α-MMD for 24 h. (B) A549 and H1299 cells were treated with various concentrations of 21α-MMD for 24 h. DNA synthesis was measured by BrdU incorporation. (C) Cell cycle-related proteins cyclins A, D1, E, CDK2, CDK4, Rb, and phospho-Rb expressions were analyzed by Western blotting after exposure to various 21α-MMD concentrations for 24 h in A549 cells. (D) Cyclin E and CDK2 were selected as 21α-MMD targets to measure mRNA gene expression levels after treatment for 24 h based on the preliminary Western blot analysis results. mRNA levels of cyclin E and CDK2 were measured by RT-PCR and real-time PCR (E). Changes in the mRNA expression of the represented genes were determined by plotting the relative Ct ratio GAPDH, which was used as the internal control in real-time PCR. (*p<0.05; **p<0.01)
Mentions: To further elucidate the underlying mechanism of 21α-MMD, we investigated whether it can induce cell cycle arrest and alter cell cycle regulatory molecules operative in the G0/G1 phase transition in various NSCLC cell lines. To determine whether 21α-MMD affects cells in a particular phase of the cell cycle we performed flow cytometry on A549, H460, and H1299 cells after treatment with 25 to 100 μM concentrations of 21α-MMD for 24 h (Fig 2A). In A549 and H460 cells, 21α-MMD caused a rapid but minimal accumulation in the number of cells in the G0/G1 phase in a concentration-dependent manner, while in H1299 treated cells, there was a slight inconsistent decrease of cells in the G0/G1 phase at 50 μM by 1.5%, nevertheless, the same phase was arrested with significant accumulation of cells at 100 μM. These findings suggest that 21α–MMD marginally induced G0/G1 phase cell cycle arrest by potentially and selectively modulating cell cycle regulators in human lung cancer cells. We also incorporated bromodeoxyuridine (BrdU) into A549 and H1299 cells to determine if there would be significant changes in the fraction of S phase cells after treatment with 21α-MMD (Fig 2B). As a result, 21α-MMD caused the inhibition of the S phase of both cells after 24 h treatment. To further examine the association of cell cycle regulatory proteins, Western blot analysis was conducted with the treatment of 21α-MMD (Fig 2C). A significant down-regulation of CDK2, CDK4, cyclin D1, Rb and pRb was found in A549 cells. It was also observed that CDK2, cyclin E, cyclin A, c-myc, and CDK4 were significantly suppressed with 100 μM 21α-MMD treatment for 24 h. Specific suppressive effects of 21α-MMD on cyclin E and CDK2 mRNA gene expressions were also evaluated through RT-PCR. Cyclin E and CDK2 mRNA levels were slightly affected showing detectable changes with 12% to 50% (CDK2), 18% to 34% (cyclin E) fold decrease in A549 cells, respectively. All mRNA levels were normalized to the internal control GAPDH (Fig 2D and 2E).

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