Targeting mitochondria in cancer therapy could provide a basis for the selective anti-cancer activity

Abstract

To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells.

Fig 1

A, Structure of NSC130362. B, Dose response curve of NSC130362 in MIA PaCa-2 cells. Cells were pre-incubated with NSC130362 for 2 h followed by addition of ATO (5 μM) and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by a CellTiterGlo reagent. P<0.05.

Fig 2. Combined treatment of NSC130362 with pancreatic cancer drugs against PANC1 and YAPC cells.

Cells were pre-incubated with NSC130362 (10 μM) for 2 h followed by addition of drugs (10 μM) and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by a CellTiterGlo reagent. The first bar is NSC130362 alone. *, P<0.05.

Fig 3. Combined treatment of NSC130362 with ATO, docetaxel, and MDV3100 against prostate cancer cells.

Cells were pre-incubated with NSC130362 (10 μM) for 2 h followed by addition of drugs (10 μM) and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by a CellTiterGlo reagent. The first bar in the treatment of each cell line is NSC130362 alone. *, P<0.05.

Fig 4. Effect of DTT and hypoxic conditions on the activity of NSC130362/ATO combination in MIA PaCa-2 cells.

Cells were pre-incubated with NSC130362 (3 μM) in either normal (24% O₂) or hypoxic (0.5% O₂) conditions for 2 h in the presence or absence of DTT (3 mM) followed by addition of ATO (5 μM) and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by a CellTiterGlo reagent. P<0.05.

Fig 5. Structure of 1,4-NQ and quinoline-5,8-dione.

Fig 6. EC50 values and the selective cytotoxicity index of the NSC130362 analogs that contain 3-chloro substituent in MIA PaCa-2 cells and hepatocytes.

Doxorubicin was included as a reference compound. Standard deviation for all EC50 values does not exceed 10%. To calculate the selective cytotoxicity index for compounds that have the EC50 value above 90 μM, EC50 values were equated to 90 μM. P<0.05.

Fig 7. EC50 values and the selective cytotoxicity index of the NSC130362 analogs that do not contain 2-chloro substituent in MIA PaCa-2 cells and hepatocytes.

Standard deviation for all EC50 values does not exceed 10%. To calculate the selective cytotoxicity index for compounds that have the LC50 value above 90 μM, EC50 values were equated to 90 μM. P<0.05.

Fig 8. EC50 values and the selective cytotoxicity index of NSC130362 analogs in MIA PaCa-2 cells and hepatocytes.

Standard deviation for all EC50 values does not exceed 10%. P<0.05.

Fig 9. Activities of the nontoxic and toxic compounds.

Ratio of activity average values between the nontoxic and toxic compounds in cell based and in vitro assays described in Table 1. P value for difference in activities of the nontoxic and toxic compounds in each type of assay is indicated below the corresponding bar.

Fig 10. QSAR modeling of NSC130362 analogs.

A. 3-D alignment of NSC130362 analogs. Red, blue and green color denote oxygen, nitrogen, and chlorine atoms, respectively; B, the distribution of the non-toxic (selective cytotoxicity index above 20) and toxic (selective cytotoxicity index below 1) analogs in relation to AFP; C, an average of AFPs for the nontoxic and toxic compounds. P<0.05.