Celecoxib suppresses autophagy and enhances cytotoxicity of imatinib in imatinib-resistant chronic myeloid leukemia cells

Chronic myelogenous leukemia (CML) is a hematological stem cell disorder responsible for 15–20 % of newly diagnosed leukemia in adults. More than 90 % of CML patients are characterized by the Philadelphia chromosome carrying the Bcr-Abl fusion gene [1, 2]. Current therapeutic regimens include treatment by tyrosine kinase inhibitors (TKIs) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) [3, 4]. Imatinib, recommended as a frontline therapy for chronic phase CML, gives a good clinical response. Unfortunately, some patients, especially in the progressive phase of the disease, show no response or relapse due to resistance. The major mechanism of imatinib resistance is a Bcr-Abl kinase domain mutation [5] and the existence of leukemia stem cells [6]. Allo-HSCT is recommended as an initial treatment for younger patients with HLA-matched donors and is the only curative treatment as of today. However, not all patients are eligible for allo-HSCT. Therefore, it is critical to continue research for novel therapeutic approaches.

Celecoxib is a non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase-2 (COX-2) inhibitor that is used to treat osteoarthritis and rheumatoid arthritis. Recent studies show that celecoxib can regress colon cancer xenografts [7] and enhance the efficacy of chemotherapy [8] and/or radiation treatment [9]. Celecoxib has been approved by the FDA for the treatment of familial adenomatous polyposis (FPA) to reduce the incidence of colon and rectal cancer [10]. Furthermore, its effects on esophagus, liver, breast, lung and prostate cancers have also been confirmed [11‐15]. The antitumor effect of celecoxib is associated with cell cycle arrest, apoptosis and autophagy induction [16, 17]. However, there are only few studies on the effects of celecoxib on hematological malignancies. These studies revealed that celecoxib could inhibit the proliferation of lymphoma, acute leukemia or CML cells [18‐21]. Celecoxib was reported to have anti-tumor effects on K562 and HL-60 leukemia cells demonstrated by cell-cycle arrest and cell apoptosis and the effects were synergistic with other chemotherapy medicines [19, 21]. However, the molecular mechanism of these anti-tumor effects has not been well elucidated.

Autophagy is a conserved catabolic process where cell constituents are incorporated into autophagosomes that fuse with lysosomes after which the contents are degraded and recycled [22]. The adaptor protein p62 and LC3 are autophagy associated proteins [23, 24]. In tumorgenesis the function of autophagy is complex, it is not always pro-death but can also be pro-survival under conditions of cellular stress [25, 26]. Celecoxib could induce autophagy in solid tumors such as urothelial carcinoma and colorectal cancer and studies revealed that autophagy inhibition enhances cancer cell apoptosis [16, 27]. However, the effect on autophagy of celecoxib in hematopoietic tumors has not been well established.

In this study, we examined the effect of celecoxib on cell proliferation, necrosis, apoptosis and autophagy in CML cell lines KBM5 and KBM5-T315I. The results showed that celecoxib had cytotoxic effect on KBM5 and imatinib-resistant KBM5-T315I cells. Apoptosis and necrosis was induced as reported, however contrary to other reports [16, 17], autophagy was inhibited by celecoxib in the two cell lines. Furthermore, this study found that celecoxib could suppress the autophagic flux by preventing the lysosome function. At last, celecoxib was demonstrated to strengthen the cytotoxicity of imatinib in imatinib-resistant CML cells. So, celecoxib might serve as a new tool to enhance the antitumor activity of conventional therapeutic agents in imatinib-resistant CML cells.

Celecoxib is a selective COX-2 inhibitor and its anti-tumor effect has been reported in various cancers [7‐9]. In this paper, we demonstrated that the anti-tumor activities of celecoxib included cell cycle arrest, necrosis, apoptosis and autophagy suppression in KBM5 and KBM5-T315I cells. KBM5-T315I cell is a mutation line of KBM5 with a threonine to isoleucine mutation at position 315 in the Abl fragment of the Bcr-Abl kinase domain. This leads to an alteration of the enzymes active site and makes these cells resistant to the first and second generation of TKI [35]. Results showed that celecoxib caused cytotoxic effect in the two CML cell lines which was dose and time-dependent. When extending the celecoxib incubation time, the inhibition effect was stronger in KBM5-T315I cells than in KBM5 cells (Fig. 1), indicating that celecoxib might be used as a new therapeutic agents in imatinib-resistant CML. In accordance with other reports [19, 21], our findings also confirmed that the anti-tumor effect was mediated by cell-cycle arrest at G1 phase (Fig. 3), necrosis and apoptosis induction (Fig. 4) in CML cell lines.

It was interesting to find that celecoxib had an opposite effect on autophagy in CML to that in solid tumors as previously reported. Huang et al. [16] reported that celecoxib induced autophagy in human colon cancer cells and that autophagy inhibitors augmented drug-induced apoptosis. Huang et al. [27] also showed the same results in human urothelial carcinoma cells. However, in these studies, autophagy formation was demonstrated by looking at the LC3-II or Atg12–Atg5 protein levels but not by looking at the levels of the p62 protein. Autophagy is a degradative process that leads to the decomposition of intracellular material in lysosomes [36] and the adaptor proteins p62 and LC3 are the most important markers of this process [23, 24]. The autophagic process can be divided into three steps, the formation of the autophagosomes, the fuse of autophagosomes and lysosomes and the degradation of the autophagic cargo in autolysosomes. Protein p62 can bind to LC3 and ubiquitinated proteins to facilitate autophagic clearance and was degraded in autolysosome [37]. The level of p62 can be regulated by autophagy and accumulates in autophagy deficient cells [38]. Thus, p62 accumulates when autophagy is inhibited and decreased levels can be observed when autophagy takes place. In our study, a variety of approaches were used to determinate the effect of celecoxib on autophagy. Western blot displayed that the ratio of LC3-II to LC3-I and the level of p62 protein were all increased when cells were treated with celecoxib (Fig. 5a, b). The same results were obtained in CML patient samples (Fig. 5c). Given that detection the expression of LC3-II and the fluorescence intensity of LysoTracker probes are measuring different biological events in the autophagic process, they were surprisingly both up-regulated during autophagic process [32, 39]. In our study, celecoxib or CQ treatment both enhanced the LysoTracker fluorescence intensity, indicating an increased autophagosome amount (Fig. 6a). Multiple signal transduction mechanisms are known to regulate autophagy and the mTOR pathway is the most important one for autophagy formation [30]. However, the phosphorylated mTOR and 4EBP proteins, which indicate an activated mTOR pathway, were not changed in the system (Fig. 5a), demonstrating that autophagy was not induced by mTOR pathway. Therefore, these results imply that celecoxib inhibits the autophagic flux at its late stage.

CQ, an inhibitor of autophagy, prevents the fusion between autophagosomes and dilated lysosomes by altering the lysosomal pH [31]. In our study, the effect on autophagy by CQ was the same as the effect by celecoxib indicating that celecoxib worked as an autophagy inhibitor (Fig. 5). Furthermore, the result that celecoxib did not promote the LC3-II level in CML cells which were pretreated with CQ sustained celecoxib to be an autophagy inhibitor (Fig. 5d). To move forward, the weakened fluorescence intensity of the LysoSensor in CML cells treated with celecoxib demonstrated that celecoxib could prevent lysosomal function by altering the lysosomal pH just like CQ to suppress autophagy (Fig. 6b). Furthermore, LysoTraker and LysoSensor dyes were stained simultaneously to CML cells treated with celecoxib or CQ. The result showed that LysoTraker fluorescence increased with LysoSensor fluorescence no significant change which was not consistent with the result of Fig. 6b (Additional file 1: Figure S1). We think the two dyes maybe have interaction effect when stained the CML cells simultaneously, or other unknown mechanisms need further investigation.

Autophagy has been suggested to play a paradoxical role in various cancers. In some types of tumor, autophagy shows a protective role against anticancer treatment, however, others go through autophagic cell death [40, 41]. In hematology, it has become clear that autophagy is one of the survival mechanisms for CML stem cells which are insensitive to all three generations of TKIs [42, 43]. Studies have shown that TKI induced autophagy in CML [44‐46]. Sheng et al. [45] showed that Bcr-Abl up-regulated activating transcription factor 5 (ATF5) to stimulate mTORC1 transcription and that imatinib could promote autophagy by inhibition of the PI3 K/Akt/mTORC1 pathway. Meanwhile, Yu et al. [46] showed that imatinib induced autophagy through inhibition of the expression of microRNA-30a and by up-regulation of BECLIN1 and ATG5 expression, independent of the mTORC1 pathway. So, it is suggested that imatinib or other chemotherapeutic agents could benefit from the addition of autophagy inhibitors in the treatment of CML. Helgason et al. [44] showed that autophagy inhibitors, such as CQ and 3-MA, could increase CML cell death with anti-leukemia drugs including crotoxin, SAHA, bafetinib, imatinib and dasatinib. In this study, the result showed that celecoxib enhanced the cytotoxicity of imatinib in KBM5-T315I cells but not in KBM5 cells (Fig. 7a). It may because that imatinib itself has good cytotoxicity effect on KBM5 cells, but has only slightly inhibition effect on KBM5-T315I cells. So, when combination of imatinib with celecoxib, the addictive effect was just showed in KBM5-T315I cells. Nevertheless, apoptosis in cells treated with imatinib was enhanced by celecoxib (Fig. 7b). In common with other studies [47‐49], imatinib could induce autophagy which was alleviated by celecoxib (Fig. 7b).

In summary, the present study has demonstrated several features of celecoxib-induced cytotoxicity in both imatinib-sensitive and imatinib-resistant CML cells, namely cell cycle arrest, apoptosis and necrosis induction and autophagy inhibition. It is the first time to reveal that celecoxib acts as an autophagy inhibitor through its effects on the function of lysosomal in CML cell lines. Besides its role in cell cycle arrest and apoptosis induction, celecoxib appears to enhance the cytotoxicity of imatinib in imatinib-resistant CML cells. Thus, the use of celecoxib in combination with imatinib appears to be a promising approach for imatinib-resistant CML therapy, and deserves further investigation.