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  • Over the past decade targeting DNA repair proteins has emerg

    2020-07-28

    Over the past decade, targeting DNA repair proteins has emerged as an effective tool to enhance chemosensitivity of malignant cells [9]. However, identification and selection of the proper target of DNA repair pathways is critical to increase cytotoxic activity of chemotherapeutic agents in cancer cells. It has been previously reported that DNA-PK expression and NHEJ activity are quite elevated in primary leukemic cells from ALL patients [8]. This prompted us to evaluate the effect of DNA-PK inhibition on sensitization of BCP-ALL cells to DNA-damaging chemotherapeutic agents. We found that DNA-PK inhibitor NU7441 increased susceptibility to doxorubicin-induced apoptosis in BCP-ALL cell lines NALM-6 and SUP-B15. There is a lot of evidence showing the radio- and chemosensitizing potential of NU7441 in cancer cells. Yang et al. demonstrated that NU7441 enhances radiosensitivity of liver cancer cells through inhibition of cell growth and interfering with cholecystokinin receptor checkpoint [17]. In another study, it was shown that NU7441sensitizes lung tumor cells to carbon ion irradiation [18]. In the present study, NU7441 could enhance the chemosensitization of BCP-ALL cells to doxorubicin by inhibiting cell growth, blocking cell cycle progression and inducing apoptosis. However, we detected no such effects in terms of cytotoxicity and apoptosis in normal human PBMCs (Figs. 1 c and 4 e). NU7441 alone suppressed the growth of BCP-ALL cell lines in a time-and dose-dependent manner while its combination with doxorubicin resulted in a massive inhibition of cell growth (Fig. 1, Fig. 2). The potent suppression of cell growth found in NU7441-treated cells may be due to disruption of cell cycle distribution. Our results showed that cells treated with NU7441 alone were mainly arrested in the G0/G1 phase (Fig. 3), consistent with previous studies showing that NU7441 blocks breast and prostate cancer cells before S phase entry [12], [13]. Furthermore, combination of NU7441 with doxorubicin caused a significant decrease of S phase population in both cell lines compared to control. CDKs have a highly complex role in both the activation of DNA damage checkpoint signaling and the initiation of DNA repair [19]. Multiple lines of studies suggest that CDK inhibition predisposes cancer cells to DNA damage via a variety of mechanisms [19], [20]. Cyclin D3 in conjunction with CDK4/6 has unique role in lymphocyte development and oncogenic transformation of lymphoid cells [21]. In addition, CDK inhibitor p21 can suppress cell cycle progression through interacting with cyclin D3/CDK6 complex [22]. NU7441 significantly down-regulated protein levels of cyclin D3 and CDK6, while caused a remarkable increase in mRNA and protein levels of p21 (Fig. 6). These findings support this idea that checkpoint signaling molecules could be potential targets to enhance sensitivity of cancer cells to DNA-damaging agents. In keeping with this idea, Crescenzi et al. revealed that CDK inhibitor, roscovitine inhibits DNA repair pathways and sensitizes cancer cells to doxorubicin [23]. DNA damage rapidly induces GADD45 expression resulting in cell cycle arrest, apoptosis or DNA repair [24]. Interestingly, Ramachandran K et al. demonstrated that overexpression of GADD45 enhanced sensitivity of prostate cancer cell lines to genotoxic drugs [25]. The expression of GADD45 is negatively regulated by c-Myc, which is a transcriptional activator implicated in the control of cell proliferation [26], [27]. Recent studies revealed that the down-regulation of c-myc expression can enhance sensitivity to chemotherapeutic drugs [28], [29]. Our data demonstrated that potentiation of growth inhibition by NU7441 was associated with up-regulation of GADD45 and down-regulation of c-Myc protein. PCNA is also a c-Myc-regulated gene that plays a critical role in DNA replication and cell cycle regulation [30]. The decrease of PCNA expression could induce cell cycle arrest and cell proliferation inhibition of malignant cells [31].