| The phenomenon of acquired multi-drug resistance (MDR) in leukemia is one of the major and pivotal impediments in leukemia therapy which has not yet been overcome. Over many years, a large number of extensive studies around leukemia drug-resistance, regulatory pathways and intervention measures have been launched but so far, the mechanisms remain incompletely understood and few effective or clinically practical countermeasures have been identified.Recently, the cardinal role of leukemia stem cells (LSC) in leukemia-MDR has attracted widespread attention and it has been speculated that they are the core factor underlying leukemia drug resistance and also the key cause of conventional chemotherapy failure and relapse in leukemia. Therefore, it is necessary to study the resistance characteristics of leukemia and its cellular and molecular mechanisms and to specifically explore ways to overcome these to provide new potential targets for clinical treatment of drug-resistant leukemia. In this study, human promyelocytic leukemia HL-60cells were selected for the adriamycin (ADM)-resistant phenotype, imitating the process of intra-individual chemotherapy, using long-term, intermittent and continuous stepwise increments of ADM concentration, to set up a stable MDR leukemia subline (HL-60/R). We then dynamically observed the effect on their drug sensitivity, expression of drug-resistance-related genes/proteins and their function, as well as their proteomic and LSC content. We found that after22months ADM-induction, the resistance of HL-60/R to ADM increased85.68-fold over that of their parental HL-60cells, which, meanwhile, cross-tolerated many other chemotherapeutics including cisplatin, daunorubicin, cytarabine, vincristine and etoposide. Additionally, drug-resistance was maintained even after culture in the absence of ADM for3months or6months’ refrigeration. Dynamic observations showed that growth speed, cell morphology and cell cycle distribution of HL-60/R cells were distinct from those of HL-60cells, and expression of the transporters ABCC1/MRP1, ABCB1/P-gp, ABCG2/BCRP, and especially mdr1/P-gp, gradually increased with increasing ADM concentration and induction time. Simultaneously, there was a sharp reduction in accumulation of intracellular ADM or rhodamine123(Rh123) and a marked efflux was observed. Protein expression spectrum (proteomics) preliminarily revealed a large number of differentially-expressed proteins in HL-60/R cells compared to parental HL-60cells, which, however, still need further identification by mass spectrometry. The proportion of CD34+CD38-CD123+LSC present, which remarkably enhanced colony-forming ability compared to parental cells, was synchronously increased with the process of ADM-induction in total HL-60cells, and then gradually dropped to almost10%after culture in the absence of ADM with increasing time in culture and as a result of cell multiplication, but increased again upon repeat exposure to ADM, indicating that LSC, due to their characteristics and natural resistance, survived to become the source of leukemia cell proliferation. Simultaneously, because of the repeated stimulus of the cytotoxic drugs, LSC became super-resistant and gained special regulation pathways that were transmitted to the daughter cells which, therefore, became the source of leukemia cells. Interestingly, different from other recognized chemo-resistant leukemia cell lines, the established HL-60/R sub-lines were also strongly cross-resistant to As2O3.As2O3is widely used in chemotherapy for most types of leukemia and solid tumors. It shows remarkable curative effects and has little cross-resistance with conventional chemotherapeutics. Studies show that most MDR leukemia cells do not manifest cross-resistance but have higher sensitivity to As2O3, and a possible explanation for this may be because As2O3is not a P-gp substrate and may even inhibit P-gp activity. Additionally, cellular arsenic resistance is closely related to arsenic transfer by MPR1, MPR2and ASNA1transporters. In this study, ADM induced MDR in HL-60/R cells. These cells also over-expressed mdrl/P-gp, and were quite resistant to As2O3, to which MDR K562/ADM cells, also induced by ADM and over-expressing P-gp, possess comparatively high sensitivity. To investigate the molecular mechanism underlying the resistance or sensitivity of MDR leukemia cells to As2O3, the drug-resistant K562/ADM cell line, HL-60/R cell line, their parental chemo-sensitive K562cells and HL-60cells were used as model cells to carry out paired-observation expression studies of arsenic transport-related proteins, as well as correlation with arsenic-sensitivity. The results revealed that expression levels of MRP1, MRP2and ASNA1were all lower in K562/ADM cells compared to their parental K562cells and to sensitive HL-60cells, but were highest in HL-60/R cells. As2O3could upregulate MRP1, MRP2and ASNA1in HL-60/R cells but inordinately suppress them in K562/ADM cells. Our study first confirmed whether or not cross-resistance to AS2O3in MDR leukemia cells existed, mainly related to intracellular contents of MPR1, MPR2and ASNA1arsenic transporters and the effect of AS2O3on them.Our studies showed an outstanding apoptosis-inducing effect and increased expression of GRP78in leukemia K562/ADM cells after arsenic treatment. To explore whether ERS (endoplasmic reticulum stress) is involved in As2O3-mediated apoptosis as well as the inhibitory effect on ABC transporters, we analyzed the effect of changes in expression of ERS-pathway-related genes and proteins including GRP78, CHOP/GADD153, XBP1, caspase-12and Bcl-2. We also investigated the influence of these changes on mdrl/P-gp and Ca2+signaling, comparing results in K562/ADM cells and their parental K562counterparts after As2O3treatment. The results proved that arsenic can induce ERS-related apoptosis in K562/ADM and K562cells by activating the ERS-response signaling pathway. Arsenic can also induce apoptosis in drug-resistant K562/ADM leukemia cells via down-regulation of mdrl/P-gp expression, thus strengthening the UPR(unfolded protein response)-triggered caspase-dependent cell-death pathway and down-regulating Bcl-2expression, consequently promoting Ca2+release and triggering calcium signaling in ERS-induced apoptosis.In conclusion, leukemia HL-60/R sub-lines acquired chemo-resistance as a result of intermittent and consecutive ADM-resistance-inducing methods and also developed characteristics of over-expression of resistance-related genes/proteins and changes in the protein expression spectrum as well as increases in the LSC population, which, distinct from classical leukemia MDR cells, are also a unique feature of high As2O3-resistance.The source of cellular resistance may be derived from surviving chemo-resistant LSC in the HL-60population subjected to long and consecutive stimulation with ADM. The LSC may adaptively mutate to gain characteristics of high chemo-reagent resistance or develop a special signal-regulated pathway that can be transmitted to their daughter cells to form a new chemo-resistant leukemia cell population. As2O3has been used in chemotherapy for most types of leukemia and solid tumors due to its remarkable curative effects and lack of cross-resistance with conventional chemotherapeutic drugs. However, whether drug-resistant leukemia cells possess cross-resistance to As2O3is closely related to their expression of the arsenic transporters MPR1, MPR2and ASNA1. As2O3induces expression of ERS. down-regulating mdr1/P-gp expression to strengthen the UPR-triggered caspase-dependent cell-death pathway and down-regulating Bcl-2expression. Consequently promoting Ca2+release and triggering calcium signaling in ERS-induced apoptosis are the major cellular and molecular mechanisms underlying the stronger apoptotic effect in some resistant leukemia cells, such as K562/ADM cells. |
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