Effect of 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride with antitumor activity on the cell cycle phases in the Jurkat model

Introduction

According to the World Health Organization (WHO), between 2022 and 2045, the number of diagnosed cancer cases worldwide will increase by 55% — from approximately 19.9 million cancer cases in 2022 to 30.9 million cases in 2045. As the incidence rises, cancer mortality could reach 16.6 million people by 2045. It is evident that a critical direction of modern pharmacology is to increase efficacy, reduce toxicity of known drugs, and develop new approaches for the treatment of recurrent cancers by searching for original compounds with antitumor activity.

One characteristic of the mechanism of action of antitumor agents is their ability to act on different phases of the cell cycle. The main phases are: G₁ phase lasting 4 to 24 hours (h); S phase (DNA synthesis) lasting 10–20 h; G₂ phase (pre-mitotic) lasting 2–10 h; followed by mitosis (M) lasting 0.5–1 h, and then the resting stage (G₀). The cell‑cycle phase specificity of DNA damage appears to be a predictor of the cell‑cycle arrest phase [1].

Several mechanisms of cytotoxic action have been identified for the anthracycline antibiotic doxorubicin, the main ones being DNA intercalation, inhibition of topoisomerase II leading to double‑strand DNA breaks [2, 3], free radical formation and oxidative stress, as well as membrane damage [4]. The cytotoxic effect of doxorubicin depends on concentration and duration of exposure; cell cycle blockade of tumor cells reaches a maximum during S phase and mitosis, while inhibition was also recorded at the G₂/M transition [5, 6].

At the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride (CHK-578) has been synthesized [7]. CHK-578 exhibits antitumor, anti-inflammatory, and antimetastatic activity in models of Lewis lung carcinoma (LLC) and B16 melanoma, with the greatest effect when co‑administered with doxorubicin [8, 9]. When co‑administered with gemcitabine, CHK-578 prevents hematopoietic suppression and increases survival in mice with Ca755 adenocarcinoma [10].

Objective

The aim of this work was to evaluate the effect of 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride (CHK-578) compared to doxorubicin on the cell cycle phases of Jurkat cells.

Materials and methods

Cell culture

Jurkat cells (human T‑lymphoblastic leukemia) from the cell bank of the Institute of General Pathology and Pathophysiology were thawed and cultured under sterile conditions in complete RPMI-1640 medium with glutamine (PanEco, Russia), 10% fetal calf serum (Biolot, Russia) inactivated for 30 min at 56 °C, supplemented with 500 µL gentamicin solution (PanEco, Russia) and amino acids for RPMI-1640 medium (PanEco, Russia) in a semi‑open system at 37 °C in a 5% CO₂ atmosphere until the required number of cells was obtained. Cultures containing 95% or more viable Jurkat lymphoblastic cells were used for the experiments. Cell viability was assessed by microscopy (Nikon Eclipse E200, Japan) using 0.4% trypan blue solution (Serva, USA).

Drugs and compounds

CHK-578 (substance, 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride), readily soluble in water, was synthesized at the Department of Medicinal Chemistry, Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies.

The antitumor drug doxorubicin (doxorubicin hydrochloride substance, DOX, Sigma-Aldrich, USA).

Study design

Cells were divided into groups: intact cell culture and cell cultures with the addition of the test substances. Cells in culture medium were seeded into 12‑well plates at 300,000 cells per well. Then DOX at a concentration of 10⁻⁵ M (in medium) or CHK-578 at concentrations of 10⁻⁴ M or 10⁻⁵ M (in medium) were added to the wells, and the plates were placed in a CO₂ incubator for 24 h and 48 h. The choice of concentrations of DOX and CHK-578 was based on literature data and previous studies [11–13]. Each group was duplicated, and the experiment was repeated twice. After incubation, cells were washed twice with 0.01 M sodium phosphate buffer (pH = 7.4) and fixed in 70% cold ethanol. The next day after fixation, cells were centrifuged for 5 minutes at 220 RCF, the supernatant was discarded, and 500 µL of propidium iodide solution with RNase A (BioInnIabs, Russia) was added, followed by incubation in the dark for 30 min for staining. After staining, 25,000 events per sample were recorded on a BD FACSCanto II flow cytometer. Data analysis was performed using FlowJo 10.5 software.

Statistical analysis of experimental data was performed using Statistica 13.5. The sum of data from both experiments was used for calculations: 8 observations per time point (2 experiments, each with 4 observations). All recorded characteristics are presented in tables as mean ± standard deviation (Mean±SD). Normality of distribution was tested using the Shapiro–Wilk test. Homogeneity of variances was assessed using Levene’s test. The significance of factor effects under homogeneous variance was determined by ANOVA followed by Tukey’s multiple comparisons test.

Results and discussion

To study the effect of doxorubicin and CHK-578 on different phases of the cell cycle, Jurkat cells were divided into 4 groups:

1. Intact cell culture;

2. Cell culture with DOX added at a concentration of 10⁻⁵ M;

3. Cell culture with CHK-578 added at a concentration of 10⁻⁴ M;

4. Cell culture with CHK-578 added at a concentration of 10⁻⁵ M.

The experimental results showed that culturing Jurkat cells with doxorubicin and CHK-578 for 24 h (Table 1) did not lead to changes in the cell cycle compared to the intact control.

Table 1. Effect of doxorubicin and CHK-578 on the cell cycle after 24‑h incubation

When cells were cultured for 48 h under the action of doxorubicin at 10⁻⁵ M and CHK-578 at 10⁻⁴ M, the proportion of cells in the G₁ phase of the cell cycle increased significantly, and the proportion of cells in the S phase decreased significantly (Table 2).

Table 2. Effect of doxorubicin and CHK-578 on the cell cycle after 48‑h incubation

Notes: n — number of observations; * — p < 0.05 by Tukey’s test compared to the control; ᵃ — p < 0.05 by Tukey’s test compared to the CHK-578 10⁻⁴ M group.

Acute T‑cell lymphoblastic leukemia, a disease of the hematopoietic system accounting for approximately 15% of acute lymphoblastic leukemia cases in children and 25% in adults, is aggressive and resistant to chemotherapy [11, 12]. The human Jurkat lymphoblastoid cell line, derived from peripheral blood T‑lymphocytes, is used as a model system to study apoptotic cell death in acute T‑cell leukemia in response to antitumor therapy, as well as to study differential sensitivity to cytostatic effects [13, 14]. According to the literature, upon treatment of Jurkat cells with doxorubicin at 10⁻⁵ M, the proportion of cells in the S phase of the cell cycle was 26±2.9% [15, 16].

In our studies, doxorubicin‑induced cell cycle blockade reached a maximum after 48 h incubation in the S phase, which is consistent with the work of Mendivil‑Perez M et al. (2015), in which doxorubicin caused death of acute lymphoblastic leukemia cells through oxidative stress‑induced apoptosis and direct DNA damage [16]. However, inhibitory effects of doxorubicin on DNA synthesis were also noted in other phases of the cell cycle, but at significantly lower concentrations [5].

Culturing Jurkat cells for 48 h with CHK-578 only at a concentration of 10⁻⁴ M resulted in an increased proportion of cells in the G₁ phase and a reduced proportion in the S phase, indicating a unidirectional effect of doxorubicin and CHK-578 on DNA synthesis in the S phase of the cell cycle.

In previous studies of the antitumor properties of CHK-578 in the B16 melanoma model in C57BL/6 mice, it was shown that CHK-578 co‑administered with doxorubicin significantly suppressed tumor growth on days 11, 15, and 21 of tumor development, without causing a significant reduction in tumor volume when administered separately. Co‑administration of CHK-578 (10 mg/kg) and doxorubicin (4 mg/kg) produced a maximal antimetastatic effect, with a metastasis inhibition index of 98.9% [8]. The data obtained on the Jurkat cell line confirm the possibility of enhancing the antitumor effect with the combined use of 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride (CHK-578) and doxorubicin.

Conclusion

Thus, 2-isobutyl-4,6-dimethyl-5-oxypyrimidine hydrochloride (CHK-578) and doxorubicin, when cultured with Jurkat cells, act on DNA synthesis in the S phase of the cell cycle. The obtained data confirm the possibility of enhancing the antitumor effect with the combined use of CHK-578 and DOX, which was previously recorded in in vivo model experiments.