Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia

Abstract

Background Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. Methods Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2–5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia–telangiectasia mutated (ATM). Results Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. Conclusions The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production. Electronic supplementary material The online version of this article (10.1186/s12885-019-5377-y) contains supplementary material, which is available to authorized users.

Hexokinase II inhibition by 3-bromopyruvate sensitizes myeloid leukemic cells K-562 to anti-leukemic drug, daunorubicin

“…Interestingly, our results also indicate that HK-II inhibition using 3-BP results in substantial decrease in the K-562 cell proliferation through the induction of S-phase (4 h) and G 2 -M phase cell cycle arrest (24 h) augmented by the DNR in the CT (Figure 4). DNR induced cell cycle arrest in G 2 /M phase has been reported in cancer cells including acute myeloid leukemic cells [48]. However, data presented here suggest that the combined DNR and 3-BP treatment leads to profound accumulation of cells in both S phase and G 2 /M phase resulting in significantly higher growth delay.…”

Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells

“…DNR is an anthracycline chemotherapeutic drug that inhibits DNA and DNA-dependent RNA synthesis ( 22 ), and has been extensively used in the treatment of leukemia. Moreover, DNR is a non-specific cell cycle agent, and thus exerts cytotoxic effects at every phase of the cell cycle, including G 0 , G 1 , S, G 2 and M ( 23 ). The results of the present study suggested that low CREBBP expression potentially resulted in DNR chemoresistance via regulation of cell cycle progression.…”

Antibiotics for cancer treatment: A double-edged sword

“…Both daunorubicin and doxorubicin have an anthracyclic plane, which can be inserted between DNA base pairs and tightly bound to DNA, becoming an obstacle of DNA spatial structure, thus inhibiting DNA and DNA-dependent RNA synthesis, and can selectively act on purine nucleosides [13]. At the same time, with the effects of cytotoxicity, daunorubicin and doxorubicin can also inhibit the activity of nuclear topoisomerase, interfere with the DNA fracture-reconnection reaction caused by topoisomerase, leading to DNA double strand break, as well as DNA single strand break [14]. Mitomycin has anticancer effect on a variety of cancers, also works as a non-specific drug in cell cycle [15].…”