A small library of synthetic di-substituted 1, 4-naphthoquinones induces ROS-mediated cell death in murine fibroblasts

Abstract

Synthesis of compound libraries and their concurrent assessment as selective reagents for probing and modulating biological function continues to be an active area of chemical biology. Microwave-assisted solid-phase Dötz benzannulation reactions have been used to inexpensively synthesize 2, 3-disubstituted-1, 4-naphthoquinone derivatives. Herein, we report the biological testing of a small library of such compounds using a murine fibroblast cell line (L929). Assessment of cellular viability identified three categories of cytotoxic compounds: no toxicity, low/intermediate toxicity and high toxicity. Increased levels of Annexin-V-positive staining and of caspase 3 activity confirmed that low, intermediate, and highly toxic compounds promote cell death. The compounds varied in their ability to induce mitochondrial depolarization and formation of reactive oxygen species (ROS). Both cytotoxic and non-cytotoxic compounds triggered mitochondrial depolarization, while one highly cytotoxic compound did not. In addition, all cytotoxic compounds promoted increased intracellular ROS but the cells were only partially protected from compound-induced apoptosis when in the presence of superoxide dismutase, catalase, or ascorbic acid suggesting utilization of additional pro-death mechanisms. In summary, nine of twelve (75%) 1, 4-naphthoquinone synthetic compounds were cytotoxic. Although the mitochondria did not appear to be a central target for induction of cell death, all of the cytotoxic compounds induced ROS formation. Thus, the data demonstrate that the synthesis regime effectively created cytotoxic compounds highlighting the potential use of the regime and its products for the identification of biologically relevant reagents.

Figure 1. Structures of the synthetic 2, 3-disubstituted-1, 4-naphthoquinone library.

All compound structures are based on the core structure (upper left), except for 1, 8, and 10. Compound 9 is vitamin K1.

Figure 2. The synthetic naphthoquinone compounds vary in their ability to inhibit cellular viability.

L929 cells were cultured for 48 hrs in the absence or presence of decreasing concentrations of vehicle control (DMSO) or decreasing concentrations of the indicated compound. The cells were then assessed for viability by a MTT assay. The compounds presented with no toxicity (Group I), low to intermediate toxicity (Group II), and high toxicity (Group III) (as described in the text). Compounds 2, 5, 8, and 11 are representative examples of each group. The data is presented as the mean ± SEM of triplicate wells and is one of three representative experiments.

Figure 3. The cytotoxic compounds promote abnormal cellular morphology.

The cellular morphology of L929 cells, grown in chamber well slides for 24 hrs in the presence of media alone (A), vehicle control (DMSO) (B), compound 2 (C), 5 (D), 9 (E), or H₂O₂ (F), was assessed by light microscopy (40 X). The cells were treated with the IC₅₀ of each compound, an equivalent volume of DMSO (vehicle control), or 600 µM H₂O₂ (positive control).

Figure 4. The cytotoxic compounds promote PS externalization and activate caspase 3.

L929 cells were cultured for 48 hrs in the presence of media, vehicle control (DMSO), or the indicated compound (concentration  =  IC₅₀) (compound 5, plumbagin, served as an internal positive control). The cells were harvested, washed, stained with Annexin V-FITC and were assessed by flow cytometry within 30 minutes of staining (A). Percentage of apoptotic non-treated cells (background) was approximately 10–20%. The data is plotted as the fold increase in apoptosis above that induced by the vehicle control. In parallel experiments, cells grown in the presence of media, vehicle control (DMSO) or the indicated compound were assessed for the presence of active caspase 3 using a colorimetric assay kit (BioSource) (B). Although all compounds were tested, compounds 2, 6, and 9 are presented as representative compounds for intermediate, high, or no toxicity, respectively. Compound 5 (plumbagin) served as an internal positive control. Data is presented as the mean ± SEM of duplicate wells and is one of three representative experiments. (A) ** p≤0.001, *** p≤0.0004, **** p≤0.0001. (B) * p≤0.049, ** p≤0.0076.

Figure 5. The synthetic naphthoquinone compounds vary in their ability to induce mitochondrial depolarization.

L929 cells were in the presence of media (-), DMSO vehicle control (V), a positive control (+) or the indicated compound (concentration  =  IC₅₀). Compound 7 (jugalone) functioned as an internal control. The cells were harvested, washed, stained with DePsipher to assess mitochondrial depolarization and were assessed by flow cytometry within 30 minutes of staining. The upper and lower right hand quadrants of the dot plots (represented in panel B) together constitute the percentage of cells that underwent mitochondrial depolarization. Data is plotted as the mean ± SEM percentage of depolarized cells from duplicate samples (A) or as representative dot plots (B). The data is taken from one of two representative experiments. * p≤0.036, ** p≤0.0066, *** p≤0.0002.

Figure 6. Compound-induced cell death associates with formation of ROS.

L929 cells were cultured in the presence of media, vehicle control (DMSO), or the indicated compound (concentration  =  IC₅₀). At 24 hrs, the cultures were assessed for the formation of intracellular reactive oxygen species (ROS) utilizing a fluorometric assay (Molecular Probes) over a 350 minute time period (A). The assay was conducted for all twelve naphthoquionones, however the only representative compounds are shown. The data is presented as the mean ± SEM of triplicate wells and is taken from one of three representative experiments. In separate experiments, the cells incubated with the compounds were also treated with catalase (1 mg/ml), superoxide dismutase (SOD, 0.5 mg/ml), or a combination of the two. A separate group of cells were pre-treated for 4 hours with 10 µM ascorbic acid, were washed and then grown in the presence of media, vehicle control (DMSO), or the indicated compound (concentration  =  IC₅₀). At 48 hrs, all groups of cells were assessed for viability (B). The data is presented as the mean ± SEM of five replicate wells and is taken from one of three representative experiments. (A) * p< .001. (B). * p≤0.043, *** p≤0.0004, **** p≤0.0002, (each compound treatment, solid black, is compared to the DMSO vehicle control).