Ranolazine synthesis br Materials and methods br Results br

Materials and methods
Results
Discussion Despite the relative complexity of immunotherapy, such treatment may have an important role, in conjunction with other therapies in the treatment of cancer refractory to conventional treatments alone. Clinical studies have recently shown that adding immunotherapy to chemotherapy has survival benefits in comparison to chemotherapy alone [31]. Moreover, chemotherapeutic agents can sensitize tumors to immune cell-mediated killing [32], [33], [34]. For instance, some can increase sensitivity of tumor Ranolazine synthesis via up-regulation of death receptors DR5 and Fas, ligands to TRAIL and FasL respectively [6]. Similarly, Vγ2Vδ2 T cells could be selectively activated by naturally occurring phosphoantigens, which are accumulated in remarkably higher amount in stressed cells, or synthetic drugs offer new avenues for the development of effective T cell-based immunotherapies. Currently, several protocols based on the in vivo activation of Vγ2Vδ2 T cells with phosphoantigens or aminobisphosphonates, or the adoptive transfer of in vitro expanded Vγ2Vδ2 T cells are in development for the treatment of several tumors [6]. Several mechanisms were identified in tumor cells by which they defend against Vγ2Vδ2 T cells, including inhibiting recruitment of tumor infiltrating lymphocytes, secreting immunosuppressive molecules, promoting immunosuppressive activities such as blocking dendritic cell maturation and promote regulatory T cells [35]. All these studies do not address, the molecular signaling pathways activated in tumor cells upon Vγ2Vδ2 T cell induction. We anticipate that identifying Vγ2Vδ2 T cell-mediated signaling pathways, and establishing the molecular mechanism of their function would help develop a more effective Vγ2Vδ2 T cell-mediated therapy. The Vγ2Vδ2 T cells induce oxidative stress, which upregulates NKG2D ligand MICA [36], [37], [38]. Furthermore, MICA upregulation transmits its signal through the phosphoinositide 3-kinase (PI3K) related kinase pathway [39]. The PI3K/AKT/mTOR pathway can become abnormally activated in many human tumors and thereby contributes to cell growth, cell proliferation, and angiogenesis [40], [41], [42]. We previously shown that ovarian tumor cell lysis due to Vγ2Vδ2 T cell induction were mediated via AKT and ERK pathway [17]. Thus, our results showed an oxidative stress-mediated regulation of PI3K/AKT/mTOR pathway [43]. Importantly, it has been shown that ATM is activated by phosphorylation upon oxidative stress, and results in DNA damage response [21], [44], [45]. Oxidative stress results in DNA single- and double-strand breaks, DNA–DNA and DNA–protein cross links, and base modifications, resulting in activation of ATM kinase and its downstream signaling pathway. This damage response then activates DNA repair, cell cycle checkpoint, changes in gene expression profiles, and apoptosis [21], [46]. Even though a link between NKG2D ligand activation and ATM damage response pathway has been implicated [47], whether ATM signal transduction is directly modulated upon Vγ2Vδ2 T cell induction has not been established. Our results indicated that in sensitive ovarian tumor cells, ATM and ATR phosphorylation is enhanced (Fig. 1, left panel). Interestingly, in resistant cells ATM–Chk2 pathway is downregulated upon the recognition of Vγ2Vδ2 T cells (Fig. 1, right panel), which resulted downregulation of MICA and subsequent reduction of cytotoxicity and cell death. Our observation of downregulation of ATM and its substrates upon Vγ2Vδ2 T cell induction is striking and led us to do the experiments using ATM enhancer and inhibitors. We observed that ATM-inducing drugs were able to stimulate ATM level, which promotes cytotoxicity. On the other hand, when we inhibited ATM signaling pathway by using ATM-specific inhibitors, it reduced the cytotoxic lysis of resistant cells after Vγ2Vδ2 T cell induction. Thus, both ATM enhancers and inhibitors showed that ATM–Chk2 signaling pathway plays a critical role in enhancing the susceptibility of tumor cells against Vγ2Vδ2 T cells. We investigated the mechanism further, and revealed the cross-talk between the MICA and ATM pathway by examining the changes in the MICA expression level upon using ATM enhancers and inhibitors. We revealed that MICA expression is upregulated when ATM signaling pathway is upregulated and MICA expression is downregulated when ATM signaling is downregulated. These data support a direct cross-talk between ATM pathway and MICA regulation. At this point, we do not know how ATM influences MICA regulation, but it shows that ATM signal transductions are directly linked to MICA regulation and cytotoxicity to Vγ2Vδ2 T cells (Fig. 2, Fig. 3, Fig. 5). This direct link between ATM and MICA is further confirmed by using ATM knock down experiments (Fig. 5). It is highly likely that ATM upregulation might influence the apoptosis pathway, as it has been shown that ATM can promote apoptosis [48], [49], [50]. It will be highly interesting to elucidate further signaling mechanisms involved in the process of sensitization, which will be greatly helpful to develop improved combination therapies for ovarian cancer.