LNO3 AND L3 Are Associated With Antiproliferative And Pro-Apoptotic Action In Hepatoma Cells

Abstract

The identification of antitumoral substances is the focus of intense biomedical research. Two structural analogues of thalidomide, LNO3 and L3, are two synthetic compounds that might possess such antitumor properties. We evaluated the toxicological effects of these substances, including cytotoxicity, genotoxicity and induction of apoptosis in HTC cells. Additionally, the production of free radicals (nitric oxide and superoxide) was investigated, and the expression of caspases genes 3, 8, and 9 were determined by RT-qPCR. The compounds exhibited cytotoxic effects that resulted in inhibited cell proliferation. LNO3 showed to be more effective and toxic than L3 in all assays. LNO3 stimulated the release of NO and superoxide, which was accompanied by the formation of peroxynitrite. Apoptosis was induced in a dose-dependent manner by both compounds; however, the expression of caspases 3, 8 and 9 was unchanged. These results suggested that L3 and LNO3 possess antiproliferative and pro-apoptotic effects in HTC cells. Additionally, although they exhibited cytotoxicity, L3 and LNO3 might be useful coadjuvants in tumor treatment studies.

Figure 1. Chemical structures of Thalidomide, L3 and LNO3.

Figure 2. Nitric oxide concentrations in DMEM. Average concentration of nitric oxide (NO) in the culture medium of HTC cells exposed to 500 μg/mL of L3 or LNO3 for 60 min. NO concentration (μM) measured by chemiluminescence. The bars represent the mean ± SD of three independent experiments. Significant differences: *p ≤ 0.05 compared with control cells.

Figure 3. Superoxide production. Quantification of average concentration of superoxide anions (O₂ ^-) in the culture medium of HTC cells exposed to 500 μg/mL of L3 or LNO3 for 15 min (1 reading per second). The O₂ ^- concentration (μM) was measured by chemiluminescence. PMA (phorbol myristate acetate; 2 μg/mL) was used as a positive control. The results were expressed as the integral area. The bars represent the mean ± SD of three independent experiments. Significant differences: *p ≤ 0.05 compared with control cells.

Figure 4. Cell growth curve determined by counting HTC cells after 24, 48, 72 and 96 h of exposure to L3 or LNO3. Doxorubicin (10 μg/mL) was used a control. The bars represent the mean ± SD of two independent experiments. CTRL: HTC cell control, DXR: doxorubicin.

Figure 5. Assay of induction of apoptosis in situ in HTC cells after 24 h of exposure to L3 or LNO3. Camptothecin (10 μg/mL) was used as a positive control. The bars represent the mean ± SD of three independent experiments. Significant differences: *p ≤ 0.05 compared with control cells.

Figure 6. Average of number of micronuclei (MN) in HTC cells exposed to L3 or LNO3 for 26 h. The treatments were as follows: control HTC cells, HTC cells with Benzo[a]pyrene (20 μg/mL) (DNA-damage inducer), HTC cells with L3 or LNO3. The bars represent the mean ± SD of three independent experiments. Significant differences: *p ≤ 0.05 compared with control HTC cells.

Figure 7. Relative expression of the Casp3 (caspase), Casp8 and Casp9 genes using RT-qPCR after 12 h of exposure to 250 μg/mL L3O or LNO3. The bars represent the mean ± SD of three independent experiments.