Optimization of the Lactam Side Chain of 7‑Azaindenoisoquinoline Topoisomerase I Inhibitors and Mechanism of Action Studies in Cancer Cells

Optimization of the lactam ω-aminoalkyl substituents in a series of 7-azaindenoisoquinolines resulted in new anticancer agents with improved Top1 inhibitory potencies and cancer cell cytotoxicities. The new compounds <b>14–17</b> and <b>19</b> exhibited mean graph midpoint cytotoxicity (GI₅₀) values of 21–71 nM in the NCI panel of 60 human cancer cell cultures. Ternary 7-azaindenoisoquinoline–DNA–Top1 cleavage complexes that persist for up to 6 h were detected in HCT116 colon cancer cells. Ternary complexes containing 7-azaindenoisoquinolines were significantly more stable than those in which camptothecin was incorporated. DNA content distribution histograms showed S-phase block 3 h after drug removal. Drug-induced DNA damage in HCT116 cells was revealed by induction of the histone γ-H2AX marker. The 7-azaindenoisoquinolines were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. Molecular modeling studies indicate that the 7-azaindenoisoquinolines intercalate at the DNA cleavage site in DNA–Top1 covalent complexes with the lactam side chain projecting into the major groove. Overall, the results indicate that the 7-azaindenoisoquinolines are promising anticancer agents that merit further development

P2Y₁₄ expression in MDCK-C11 cells.

<p>(a) RT-PCR analysis of IC markers including the V-ATPase a4 subunit (V0A4), the V-ATPase B1 subunit (V1B1) and AE1, and the principal cell marker aquaporin 2 (AQP2), as well as P2Y₁₄ in MDCK-C11 cells. (b) Representative immunoblots following plasma membrane biotinylation showing cell surface versus total protein expression of the V-ATPase B1 and A subunits, and actin. (c) RT-PCR detection of P2 receptors in MDCK-C11 cells. (d) Immunoblot profile of P2Y₁₄ expression in MDCK-C11. Plasma membrane (left) and total cell expression (right) are represented under control conditions (C) and after treatment with endoglycosydase H (H) and PNGase F (F). (e) X-Z confocal microscopy representation of MDCK-C11 cells grown on filter, showing P2Y₁₄ expression (red). Plasma membrane is labeled with biotin-streptavidin FITC (green). The merge panel shows partial co-localization of P2Y₁₄ with biotin in the apical membrane (orange/yellow) as well as sub-apical localization (red). Scale bars = 4 μm. (f) Concentration-dependent inhibition of [³H]UDP-glucose binding to MDCK-C11 membranes by unlabeled ligands. Membranes (15 μg protein) were incubated for 3 hours at 22C with [³H]UDP-glucose (3 nM) and increasing concentrations of UDP-glucose or ATP. Each point represents the average of 4 independent experiments performed in triplicate. The data are expressed as values relative to the total binding observed in the absence of unlabeled ligand and are corrected for non specific binding determined in the presence of a saturating concentration of UDP-glucose (10 μM).</p

RT-PCR detection of P2 receptors.

<p>Lower panel shows RT-PCR in whole kidney and upper panel shows RT-PCR in EGFP(+) cells isolated by FACS from B1-EGFP mouse kidneys. GAPDH was used as a positive control. NC = no template control.</p

Quantitative PCR analysis of EGFP(+) cells isolated from the kidney cortex and medulla.

<p>(A) Detection of IC markers (B1, AE1, pendrin) in EGFP(+) vs EGFP(−) cells isolated from renal cortex and medulla. Values are normalized to GAPDH and represented as fold changes relative to the values obtained in cortical EGFP(+) cells. (B) Relative P2 receptor mRNA levels, analyzed by quantitative PCR in medullary and cortical EGFP(+) cells. Data are normalized for GAPDH and are expressed as mean ± SEM (n = 3), *P<0.05, **P< 0.001.</p

Flow cytometry analysis of immune cell infiltration in the kidneys of mice 48 h after injection with saline (sham) or 100 μM UDP-glucose (UDP-glu).

<p>Changes in kidney medulla (A) or cortex (B) infiltrated immune cell counts are represented as % changes relative to control. Values are means of percent of each cell population ± SEM from 4–6 animals. *P<0.05.</p

Immunofluorescence localization of P2Y₁₄ in mouse kidney.

<p>Cortical (A) and medullary (B) sections double-labeled for P2Y₁₄ (green) and the V-ATPase B1 subunit (red). P2Y₁₄ was detected in ICs identified by their positive labeling for the V-ATPase (yellow in the merge panels shown in A and B). No P2Y₁₄ was detected in distal tubule cells, which also express the V-ATPase (red in the merge panel shown in A). The P2Y₁₄ staining was abolished after pre-incubation of the P2Y₁₄ antibody with its immunizing peptide in the cortex (C) and medulla (D). Scale bars = 25 μm.</p

Quantitative PCR detection of pro-inflammatory mediators in EGFP(+) cells.

<p>EGFP(+) cells were isolated by FACS from B1-EGFP mice 4h after an i.v. injection with saline (sham) or with saline containing 100 μM UDP-glucose (UDP-glu). All values are normalized to GAPDH. Data are represented as % changes relative to control. Values are mean ± SEM (n = 4), *P<0.05, ** P<0.001.</p

P2Y₁₄ activation by UDP-glucose increases ERK1/2-phosphorylation in MDCK-C11 cells.

<p>Representative immunoblots showing triplicates of ERK1/2 phosphorylation (upper lane) versus total ERK1/2 (lower lane) in cells pretreated with vehicle or the P2Y₁₄ antagonist PPTN (10 μM), in the absence (CTRL) or presence of 100 μM UDP-glucose (UDP-glu). Quantification of the ratio of p-ERK/total ERK showed that UDP-glucose induced a significant increase in ERK1/2 phosphorylation (lower left panel, n = 7) and that PPTN prevented the increase in ERK1/2 phosphorylation induced by UDP-glucose (lower right panel, n = 5). Values are represented, relative to either control or PPTN alone, as means ± SEM, * p < 0.005.</p

Expression of P2Y₁₄ in EGFP(+) cells.

<p>(A) Representative immunoblot profile of P2Y₁₄ in two EGFP(+) cell samples isolated by FACS. (B) Binding of [³H]UDP-glucose to total membranes prepared from FACS isolated EGFP(+) and EGFP(−) cells in the presence or absence of a saturating concentration (10^–5 M) of unlabeled UDP-glucose or ATP. Data are represented as fold changes compared to the binding measured in the presence of unlabeled UDP-glucose. Each bar represent the average of 3 independent experiments each performed in triplicate. Values are expressed as mean ± SEM, * P<0.05.</p

Sequence of the primers used for RT-PCR detection.

<p>Sequence of the primers used for RT-PCR detection.</p