A novel thiophene-3-carboxamide analog of annonaceous acetogenin exhibits antitumor activity via inhibition of mitochondrial complex I

Abstract

Previously we synthesized JCI-20679, a novel thiophene-3-carboxamide analog of annonaceous acetogenins which have shown potent antitumor activity, with no serious side effects, in mouse xenograft models. In this study, we investigated the antitumor mechanism of JCI-20679. The growth inhibition profile (termed "fingerprint") of this agent across a panel of 39 human cancer cell lines (termed "JFCR39") was measured; this fingerprint was analyzed by the COMPARE algorithm utilizing the entire drug sensitivity database for the JFCR39 panel. The JCI-20679-specific fingerprint exhibited a high similarity to those of two antidiabetic biguanides and a natural rotenoid deguelin which were already known to be mitochondrial complex I inhibitors. In addition, the fingerprint exhibited by JCI-20679 was not similar to that displayed by any typical anticancer drugs within the database, suggesting that it has a unique mode of action. In vitro experiments using bovine heart-derived mitochondria showed direct inhibition of mitochondrial complex I by JCI-20679 and associated derivatives. This inhibition of enzymatic activity positively correlated with tumor cell growth inhibition. Furthermore, a fluorescently labeled derivative of JCI-20679 localized to the mitochondria of live cancer cells in vitro. These results suggest that JCI-20679 can inhibit cancer cell growth by inhibiting mitochondrial complex I. Our results show that JCI-20679 is a novel anticancer drug lead with a unique mode of action.

Keywords: Antitumor compound; COMPARE analysis; complex I; mitochondria; natural polyketide.

Figure 1

Growth inhibition profile against a panel of 39 human cancer cell lines. The mean graph was produced by computer processing of the 50% growth inhibition (GI ₅₀) values as described under “Materials and Methods”. Logarithmic GI ₅₀ values for each cell line are indicated. In the plot, columns to the right of zero indicate sensitivity of the cell line to the compound, and columns to the left indicate resistance to the compound. The x‐axis represents the logarithm of the difference between the mean GI ₅₀ values for 39 cell lines and the GI ₅₀ value for each cell line in the JFCR39 panel. The mean graph of JCI‐20679 is very similar to that of solamin (Pearson correlation coefficients; r = 0.616), phenformin (Pearson correlation coefficients; r = 0.704), and deguelin (Pearson correlation coefficients; r = 0.695). MG‐MID, the mean of log GI ₅₀ values for 39 cell lines; Delta, the logarithm of the difference between the MG‐MID and the log GI ₅₀ of the most sensitive cell line; Range, the logarithm of the difference between the log GI ₅₀ of the most resistant cell line and the log GI ₅₀ of the most sensitive cell line. One scale represents one logarithm difference. Quantification of the GI ₅₀ value was represented as the mean of at least two different experiments. Br, breast; CNS, central nervous system; Co, colon; Lu, lung; Me, melanoma; Ov, ovarian; Re, renal; St, stomach; xPg, prostate. Chemical structure of the compounds are shown in below.

Figure 2

Dose–response curves with IC ₅₀ values for JCI‐20679 (A), solamin (B), phenformin (C), deguelin (D), and rotenone (E) in respect to antibody‐captured bovine mitochondrial complex I activity. The results are expressed as percentage of complex I activity with no inhibitors. The calculated regression curve is indicated as a line. Data are mean ± SEM (n = 3).

Figure 3

NCI‐H23 cells were treated with the indicated concentration of either JCI‐20679 (A) or rotenone (B) for 1 h and cellular oxygen consumption rate measured as described under “Materials and Methods”. The values are expressed as percentages of the nontreated control. Data are mean ± SEM (n = 3). NCI‐H23 cells were treated with the indicated concentration of JCI‐20679 for 6 h and cellular ATP content (C) and viable cell number (D) were measured as described under “Materials and Methods”. Each values are expressed as percentages of the nontreated control. Data are mean ± SEM (n = 3). (E) a scatter plot of concentrations of the annonaceous acetogenin derivatives (JCI‐20425, JCI‐20679, JCI‐13160 and JCI‐13163) and the related compounds (solamin, phenformin, buformin, deguelin, and rotenone) that inhibited 50% of mitochondrial complex I activity (IC ₅₀) versus the logarithm of 50% growth inhibition (GI ₅₀) of NCI‐H23 cells were shown. A high and statistically significant positive correlation (r = 0.857, Pearson correlation coefficient, and two‐tailed P = 0.0032) was observed between the two activities (n = 9). (F) a scatter plot of concentrations of the annonaceous acetogenin derivatives and the related compounds that inhibited 75% of intracellular ATP inhibition activity (IC ₇₅) versus the logarithm of (GI ₅₀) of NCI‐H23 cells were shown. A high and statistically significant positive correlation (r = 0.915, Pearson correlation coefficient, and two‐tailed P = 0.0005) was observed between the two activities (n = 9).

Figure 4

Chemical structure (A) and visualization of intracellular localization (B–D) of JCI‐13291. NCI‐H23 cells were pretreated with 2 μmol/L JCI‐13291 for 5 h before the cells were stained with organelle markers. Subsequently, NCI‐H23 cells were stained with MitoTracker Red (B), ER‐Tracker Red (C) or LysoTracker Red (D) and observed under a fluorescence microscope. The signal derived from JCI‐13291 (left panels) and organelle dyes (middle panels), along with merged images (right panels) are shown. The white bar indicates 50 μm. Results are representative of those obtained in at least two independent experiments.

Figure 5

Characterization and JCI‐20679 sensitivity of ρ0 cells. (A) mitochondrial DNA‐encoded transcripts (MTND6 for ND6, MTCO2 for COX2) in the parental (ρ+) and mitochondrial DNA‐deficient ρ0) NCI‐H23 cells were examined by real‐time RT‐PCR analysis. Relative expressions of MTND6 or MTCO2 mRNA in the ρ+ and ρ0 cells are shown. Data were normalized by 18S rRNA expression. Baseline MTND6 or MTCO2 mRNA expression in parental NCI‐H23 cells were set as controls, respectively. Results shown are the means of three independent experiments; bars, ±SEM; N.D., Not detected. (B) Western blot analysis of mitochondrial DNA‐encoded proteins (ND6 and COX2) in ρ+ and ρ0 NCI‐H23 cells. The levels of β‐actin were used to control for loading. (C) visualization of mitochondrial membrane potential by fluorescence microscopy. ρ+ and ρ0 NCI‐H23 cells were stained with JC‐1. Red fluorescence indicates an area of high membrane potential on the functional mitochondria. DIC, differential interference contrast. White bar indicates 50 μm. (D) ρ+ and ρ0 NCI‐H23 cells were treated with indicated concentrations of JCI‐20679 for 48 h and the cellular growth was determined via a sulforhodamine B assay. Percentage of control values is indicated. Data are mean ± SEM (n = 3).