Design, Synthesis, and Biological Activity of Sulfonamide Analogues of Antofine and Cryptopleurine as Potent and Orally Active Antitumor Agents

Due to their profound antiproliferative activity and unique mode of action, phenanthro­indolizidine and phenanthro­quinolizidine alkaloids, represented by antofine and cryptopleurine, have attracted attention recently as potential therapeutic agents. We have designed, synthesized, and evaluated the methanesulfonamide analogues of these natural alkaloids with the hope of improving their druglikeness. The analogues showed enhanced growth inhibition of human cancer cells compared with the parent natural products. In particular, a methanesulfonamide analogue of cryptopleurine (<b>5b</b>) exhibited improved bioavailability and significant antitumor activity, which suggests that <b>5b</b> is a promising new anticancer agent. Our studies suggest that the inhibition of cancer cell growth by <b>5b</b> is associated with the induction of G0/G1 cell cycle arrest via nicotinamide <i>N</i>-methyltransferase-dependent JNK activation in Caki-1 renal cancer cells. In addition, compound <b>5b</b> significantly inhibited the migration and invasion of Caki-1 cancer cells by modulating the p38 MAPK signaling pathway

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

<div><p>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 <i>Poncirus trifoliate</i>, 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 <i>in vitro</i> of 21α-MMD displaying growth-inhibiting potential with influence on MDR reversal in human lung cancer cells.</p></div

Subsequent downregulation of P-gp/MDR1 expression and inhibitory enhancement of 21α-MMD by stimulating mTOR and related signaling.

<p>(A) A549-PacR cells were incubated with 21α-MMD (25–100 μM) for 24 h. Whole-cell lysates were subjected to Western blot analysis using anti-ERK, anti-JNK, anti-p38, anti-Akt, anti-mTOR, anti-PI3K, anti-AMPK and antibodies related to their phosphorylated forms. The β-actin and phospho-protein relevant to the total protein bands confirmed the integrity and equal loading of total protein and phospho-proteins respectively. All protein levels were normalized to the β-actin levels. (B and C) Effect of mTOR knockdown was established first by transiently transfecting mTOR siRNA to A549-PacR cells for 24 h. Scramble siRNA was used as control separated from the mock control. mTOR and MDR1/P-gp protein and mRNA gene expressions were examined by Western blotting and PCR analysis respectively. (D and E) Cells were transiently transfected with mTOR siRNA or Scramble siRNA for 24 h followed by a 24 h exposure to 25 μM 21α-MMD. MDR1/P-gp protein and mRNA gene expression levels were confirmed by Western blotting and PCR analysis respectively. (F) After mTOR siRNA or Scramble siRNA transfections, cells were treated with 100 nM paclitaxel for various time courses and mTOR siRNA was subsequently incorporated with 25 μM 21α-MMD followed by cell viability assessment through MTT assay.</p

Regulation of the PI3K/AMPK/AKT, mTOR and MAPK signaling pathways by 21α-MMD in lung cancer cells.

<p>A549 or H1299 cells were incubated with various concentrations of 21α-MMD for 24 h. Cell lysates were subjected to Western blotting and probed using anti-ERK, anti-JNK, anti-p38, anti-Akt, anti-mTOR, anti-PI3K, anti-AMPK antibodies as well as antibodies for their phosphorylated forms. The β-actin and phospho-protein relevant to the total protein bands confirmed the integrity and equal loading of total and phospho-proteins respectively. All protein levels were normalized to the β-actin levels.</p

Localization of intracellular P-gp distribution after treatment with 21α-MMD in A549-PacR cells.

<p>Confocal analysis of the expression, distribution, and overlapping of P-gp (green) is shown for 12.5–100 μM 21α-MMD treatment in A549-PacR cells. The nuclei were stained with DAPI (blue). Images are representative of three independent experiments. Scale bars, 40 μm.</p

Intensification of paclitaxel and 5-FU cytotoxicity in A549 and A549-PacR cells by 21α-MMD.

<p>The cytotoxicity of 21α-MMD (6.25–100 μM), paclitaxel (12.5–200 μM), and 5-FU (2–60 μM) alone or in combination in A549 and A549-PacR cells was determined by MTT assay. Each point indicates the mean ± SD of three independent experiments, performed in triplicate. (A) Effects of 21α-MMD and paclitaxel (B) and 5-FU in A549 cells. (C) Effect of 21α-MMD (D) and paclitaxel (E) and 5-FU in A549-PacR cells. Cells were pretreated with or without 21α-MMD followed by various concentrations of paclitaxel or 5-FU for 24 h. MTT data were presented as the surviving cell viability after treatment regime. A negative control was used which identifies as the respective drug used in combination with 21α-MMD.</p

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.

<p>(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 H_<b>2</b>O_<b>2</b> 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. (*<i>p</i><0.05; **<i>p</i><0.01)</p

Induction of minimal G₀/G₁ cell cycle arrest by 21α-MMD.

<p>(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. (*<i>p</i><0.05; **<i>p</i><0.01)</p

Suppression of expression, function, and transcription of MDR1/P-glycoprotein (P-gp) by 21α-MMD in A549-PacR cells.

<p>(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. (*<i>p</i><0.05; **<i>p</i><0.01)</p

Anti-proliferative activity of compounds derived from <i>Poncirus trifoliate</i>.

<p>Anti-proliferative activity of compounds derived from <i>Poncirus trifoliate</i>.</p