In vitro cytotoxic effect of 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’- trinitroethoxy)-1,3,5-triazine on human fibroblasts, peripheral blood mononuclear cells and breast cancer cells

In vitro cytotoxic effect of 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’trinitroethoxy)-1,3,5-triazine on human fibroblasts, peripheral blood mononuclear cells and breast cancer cells LV Limareva, PV Iliasov, AA Gidaspov, VA Zalomlenkov, AS Sustretov, AI Sizova, VV Rossinskaya. 2-(Morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine having QSAR-predicted anti-tumor activity was tested for the cytotoxicity using MTT and LDH cell viability tests. The experiments were conducted using human fibroblasts, peripheral blood mononuclear cells and breast cancer cells and allowed to identify effective cytotoxic concentration ant therapeutic range of this compound. The data obtained suggest the feasibility of the further studies of the test compound as a potential anti-cancer agent.

Phase II but was ultimately refused due to its high toxicity, poor stability and solubility, despite good efficacy for a number of malignancies.
Therefore, there is a need in such compounds having good solubility and stability as well as low toxicity for normal cells. At the same time, partial replacement of amines for groups comprising polynitromethyl moieties could improve NO donor activity.
This compound was tested for physicochemical properties, potential biological effects and therapeutic targets using PASS Online (http://www.way2drug.com/passonline/index.php, [14]) web resource. A significant effect was assumed to meet activity level (Ра) ≥ 0.7 for separate enzymes and metabolic pathways and ≥ 0.6 for cytotoxicity towards tumor cell lines.
The compound was dissolved in 50 µL of dimethyl sulfoxide (Panreac Quimica SAU, Spain) and used ex tempora in appropriate concentrations. Doxorubicin (LENS Farm LLC, Russia) commonly used in standard cancer chemotherapy regimen was a reference drug. All experiments were performed at least in triplicate.
Human primary dermal fibroblast culture, PHA activated peripheral blood mononuclear cells (PBMC) and BT474 HEP2-positive breast cancer cell line were used to assess the compound cytotoxic activity. Such an approach would ensure comparison of the test compound effect on the malignant target cells, connective tissue cells presenting in most organs and body parts, and blood cells inherently contacting any substance being administered by parenteral mode.
Fibroblasts were grown using a primary explant technique [15] with complete cell culture medium (199 medium supplemented with 10% fetal calf serum and 40 µg/mL gentamycine, PanEco Biolot, Russia) in 96-well plates inoculated with 2×10 4 cells/cm 2 in МСО-17АI СО 2 incubator (Sanyo, Japan) at 37°С, 5% СО 2 and constant humidity. Before test, the culture was identified and characterized using morphological and genetical methods. This examination confirmed that the cells were unipotential fibroblastic lineage cells. PCR showed no culture contamination, including by mycoplasms and cytomegalovirus. Upon obtaining a confluent monolayer, the appropriate wells were added with the test compound in various concentrations and incubated for 5 days at 37°С, 5% СО 2 and constant humidity.
Mononuclear blood cells (lymphocytes) were isolated from the heparinized venous blood of adult healthy volunteers by gradient centrifugation with 1.077 g/cm 3 Ficoll solution (PanEco, Russia). Cell were counted and assessed for viability in a counting chamber using 0.1% Tripan Blue, considering >90% viability satisfactory.
Lymphocytes were incubated with 5 µg/mL phytohemagglutinin P (PHA, Sigma-Aldrich, USA) and the test compound for 5 days in the complete RPMI-1640 medium supplemented with L-glutamine, streptomycin, 20 mg/mL HEPES, 10% fetal calf serum (PanEco, Russia) at 37°С, 5% СО 2 . Lymphocyte count was 800000 cells/400 µL. After 5 days, the plates were removed from an incubator and used for cytotoxicity testing. The wells with cells and no test compounds were used as control samples, and the wells without both cells and test compound were blanks.
Cytotoxicity tests used in our study included MTT and LDH tests [16,17].
MTT test is based on a tetrazolium dye readily penetrating cell membranes and being converted to a colored formazan by mitochondrial enzymes in live cell. These enzymes are inactive in dead cells and cannot conduct such a conversion, therefore the color intensity is directly proportional to the number of live (i.e., survived after the exposure to an agent) cells in the well.
The test was performed as follows.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Merck, USA) was dissolved in the growth media for a concentration of 3 mg/mL. From each well, 100 µL of growth media was drawn and replaced for 100 µL of tetrazolium dye solution. The plate was covered, carefully stirred by circular motion, placed to a thermostat at 37°С and incubated for 3 hours.
After incubation, the medium was carefully removed from each well while holding the plate at an angle of 45 degrees. Then each well was added with 250 µL of dimethyl sulfoxide and pipetted until complete dissolution of the formazan.
Color intensity was measured at 540 nm on Tecan Infinite М200 plate reader (Tecan Instruments, Austria). Cell viability was calculated using a formula = − × 100%, wherein A exp, A c and A b are absorbance values in test, control and blank wells, respectively.
LDH activity test is based on the measurement of activity of lactate dehydrogenase (LDH), an enzyme which is usually located within cells and only enters the medium upon the cell destruction. Thus, LDH activity in the medium is directly proportional to the number of destroyed (dead) cells.
This test was performed as follows.
At the first step, the medium from each well was transferred to a separate 1. This step allowed to estimate LDH activity in a solution due to dead cell lysis.
Then the survived cells remaining in the well were lysed by adding 250 µL of distilled water followed by 3 freeze-thaw cycles, and the LDH activity of these cells (A2) was measured in the resulting solution by a similar manner.
This allowed to estimate the number of survived cells. After this, a percentage of survived cells from the total number of both survived and dead cells was calculated using a formula: The data were statistically processed in Microsoft Office Excel 2016. The results considered significant at p < 0.05.

Results and Discussion
Estimated solubility of the test compound in aqueous media is ~10 -4 М (ALOGPS 2.1, http://www.vcclab.org/lab/alogps/). The predicted bioactivitiy and cytotoxicity values are listed in Table 1. Table 1 Predicted   The test results were essentially the same as for MTT test (Figure 3)  concentrations of about 100 nM caused almost 100% cell death [18]. This discrepancy seems to be associated with shorter incubation time (5 days) in our experiments.
Note that other malignant cell lines are characterized by other effective doxorubicin concentrations vs. the above ones, e.g., LNCaP and HepG2 cells (prostate and liver cancer, respectively) are only susceptible to doxorubicin in micromolar range [19,20], and doxorubicin therapeutic index (a ratio of 75% inhibitory to 10% lethal dose) in mice is about 0.3 [21].