Glycometabolic adaptation mediates the insensitivity of drug-resistant K562/ADM leukaemia cells to adriamycin via the AKT-mTOR/c-Myc signalling pathway.

In human leukaemia, resistance to chemotherapy leads to treatment ineffectiveness or failure. Previous studies have indicated that cancers with increased levels of aerobic glycolysis are insensitive to numerous forms of chemotherapy and respond poorly to radiotherapy. Whether glycolysis serves a key role in drug resistance of leukaemia cells remains unclear. The present study systematically investigated aerobic glycolytic alterations and regulation in K562/adriamycin (ADM) multidrug­resistant (MDR) and ADM­sensitive K562 leukaemia cells in normoxia, and the association between drug resistance and improper glycometabolism. The cell proliferating activity was assessed with an MTT colorimetric assay, glycolysis, including glucose consumption, lactate export and key­enzyme activity was determined by corresponding commercial testing kits. The expression levels of hexokinase­II (HK­II), lactate dehydrogenase A (LDHA), glucose transporter­4 (GLUT­4), AKT, p­AKT473/308, mammalian target of rapamycin (mTOR), p­mTOR, c­Myc and hypoxia­inducible factor­1α (HIF­1α) were analyzed by western blot or reverse transcription­quantitative polymerase chain reaction (RT­qPCR). K562/ADM cells exhibited increased glucose consumption and lactate accumulation, increased lactate dehydrogenase, hexokinase and pyruvate kinase activities, and reduced phosphofructokinase activity. In addition, K562/ADM cells expressed significantly more HK­II and GLUT­4. Notably, inhibition of glycolysis effectively killed sensitive and resistant leukaemia cells and potently restored the sensitivity of MDR cells to the anticancer agent ADM. The AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (mTOR) signalling pathway, a crucial regulator of glycometabolic homeostasis, mediated over­activation and upregulation of c­Myc expression levels in K562/ADM cells, which directly stimulated glucose consumption and enhanced glycolysis. In conclusion, the present study demonstrated that MDR leukaemia cells exhibit increased aerobic glycolytic activity and that this may be responsible for resistance to chemotherapeutics in leukaemia MDR cells via activation of the AKT­mTOR­c­Myc signalling pathway. Therefore, inhibition of aerobic glycolysis may be a potential therapeutic strategy to efficiently treat multidrug resistance in relapsed or refractory leukaemia and cancers.

Differential expression and response to arsenic stress of MRPs and ASAN1 determine sensitivity of classical multidrug-resistant leukemia cells to arsenic trioxide.

There is no cross-resistance between arsenic trioxide and conventional chemotherapeutics. Classical multi-drug resistant (MDR) cells remain sensitive to arsenic trioxide, which may even reverse the drug resistance. Arsenic trioxide is also effective in leukemias/tumors that persist despite conventional cytotoxic or targeted drugs. We obtained a highly arsenic-resistant MDR leukemic cell line, HL-60/RS, by exposing leukemic HL-60 cells to adriamycin selection. We compared the arsenic sensitivity, and the expression and responses to arsenic of the arsenic-related transporters, MRP1, MRP2, and ASNA1, in paired parent/arsenic-resistant HL-60/RS/HL-60 and arsenic-sensitive/parental K562/ADM/K562 cells. Expression levels of MRP1, MRP2, and ASNA1 were negatively correlated with cell sensitivities to arsenic trioxide, and ASNA1 expression notably was highest in HL-60/RS cells and lowest in K562/ADM cells. Expression levels of MRP1, MRP2, and ASNA1 were significantly enhanced in HL-60/RS cells and inhibited in K562/ADM cells by arsenic trioxide treatment, compared with their parental sensitive cells, in accord with the high-resistance of HL-60/RS cells and high-sensitivity of K562/ADM cells. In conclusion, the cross-resistance of conventional chemotherapeutics-resistant leukemic cells to arsenic trioxide is determined by both levels of MRP1, MRP2, and ASNA1, and also by the responses of these transporters to arsenic stress.

Inhibition of thioredoxin reductase by auranofin induces apoptosis in adriamycin-resistant human K562 chronic myeloid leukemia cells.

Mammalian thioredoxin reductase (TrxR) catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx) and plays a central role in regulating cellular redox homeostasis, cell growth and apoptosis. Increasing evidence shows that TrxR is over-expressed or constitutively active in many tumor cells. Moreover, TrxR appears to contribute to increased tumor cell growth and a resistance to chemotherapy. In this study, we evaluated the activity of TrxR in adriamycin-resistant leukemic cells (K562/ADM) and adriamycin-sensitive parental lines (K562), and found that TrxR activity was higher in the drug resistant cell sublines K562/ADM than in K562 drug sensitive parental cells. Auranofin, a gold(I) compound clinically used as an antirheumatic agent, reduced TrxR activity and was more effective than adriamycin in decreasing cell viability in K562/ADM cells. In addition, auranofin induced apoptosis in dose-dependent manners, accompanied by caspase-3 activation in K562/ADM cells. Our results demonstrate that inhibition of TrxR and induction of apoptosis by auranofin provides its ability in overcoming adriamycin resistance in K562/ADM cells.

[siRNA silences mdr1 gene expression and reverses apoptosis resistance of K562/ADM cells line].

OBJECTIVE: To explore the effect of small interfering RNA(siRNA) on silence of mdr1 gene and reversal of apoptosis resistance in multidrug-resistant (MDR) human leukemia K562/ADM cell. METHODS: Human MDR leukemia cell line K562/ADM was used as the target cells. Two siRNAs (mdr1 siRNA-1 and mdr1 siRNA-2) targeted mdr1 gene were chemically synthesized and transfected into K562/ADM cells with liposome. Expression of mdr1 mRNA was determined by real-time PCR, P-glycoprotein (P-gp) expression and caspase-3 activity were measured with flow cytometry (FCM), and the cell apoptosis was observed by optical and electronic microscopy for morphology and Annexin V/PI staining. RESULTS: The mdr1 siRNA-1 and mdr1 siRNA-2 could markedly down-regulate the expression of mdr1 gene in K562/ADM cells, the expression of mdr1 mRNA decreased by 91.2% and 82.0% , and the P-gp by 74.1% and 84.4%, respectively. The caspase-3 activity was markedly enhanced, and the active caspase-3 in K562/ADM cells increased by about 40% compared to liposome alone and non-silencing controls. the sensitivity of K562/ADM cells to adriamycin-induced apoptosis was significantly augmented, the apoptotic rate of the cells treated with siRNA plus adriamycin increased by about 60% compared to adriamycin alone. CONCLUSION: siRNAs silence the expression of mdr1/P-gp to overcome the P-gp-mediated apoptosis resistance in drug-resistant K562/ADM cells.

Arsenic trioxide overcomes apoptosis inhibition in K562/ADM cells by regulating vital components in apoptotic pathway.

Extensive researches have revealed that arsenical can exert anti-tumor efficacy against several kinds of cancers including leukemia. Though, little is known about the effects of arsenical on leukemia resistant to chemotherapy, emerging as a serious clinical problem. In this study, we tested arsenic trioxide (As(2)O(3))-induced apoptosis in K562/ADM multidrug-resistant leukemic cells and investigated its possible mechanisms. Using microscopy, flow cytometry (FCM) and DNA electrophoresis, we found that As(2)O(3) could induce the cells to undergo G2/M phase arrest and apoptosis. Further, it was shown that the levels of FAS and P53 proteins increased and P-glycoprotein (P-gp) decreased upon drug action by employing FCM. Reverse transcription polymerase chain reaction (RT-PCR) detected increased mRNA product of FAS and caspase-3 genes and reduced MDR1 mRNA. CASPASE-3 activity was also enhanced after As(2)O(3) treatment. However, the expression of BCL-2 protein was not affected by the drug. Taken together, As(2)O(3) is able to reverse the apoptosis resistance in drug-resistant K562/ADM cells by modulating expression or activity of key factors associated with apoptosis induction.

Arsenic trioxide inhibits p-glycoprotein expression in multidrug-resistant human leukemia cells that overexpress the MDR1 gene.

OBJECTIVE: To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and p-glycoprotein (P-gp) expression of multidrug-resistant human leukemia cells. METHODS: Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry. RESULTS: Zero point five to 20 micromol/L As(2)O(3) inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than the parental K562 cells. As(2)O(3)-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As(2)O(3) significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin. CONCLUSIONS: As(2)O(3) induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells.

[Arsenic trioxide inhibits P-glycoprotein expression in multidrug-resistant human leukemia K562/ADM cell line that overexpresses mdr-1 gene and enhances their chemotherapeutic sensitivity].

OBJECTIVE: To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and P-glyco-protein (P-gp) expression of multidrug-resistant human leukemia K562/ADM cells, and the combined effects of As(2)O(3) with conventional chemotherapeutic agents. METHODS: Multidrug-resistant human leukemia cell line K562/ADM that overexpresses mdr-1 gene was used as the target cells. The cell proliferating activity was assessed with a MTT assay. Cell morphology was examined by light microscopy, confocal microscopy and electron-microscopy. P-gp expression, cell-cycle status were determined by flow cytometry. RESULTS: K562/ADM cells were highly resistant to adriamycin, and cross-resistant to daunorubicin and etoposide. As(2)O(3) at concentrations of 0.5 to 20 micromol/L inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than their parent K562 cells did. As(2)O(3) induced marked apoptosis of K562/ADM cells showed by typical apoptotic morphological changes and the appearance of high sub-G(1) cell population. As(2)O(3) significantly inhibited the P-gp expression in K562/ADM cells, and exerted a synergistic effect on the enhancement of the cell sensitivity to adriamycin, daunorubicin and etoposide. CONCLUSION: As(2)O(3) induces growth-inhibition and apoptosis of multidrug-resistant K562/ADM cells, and augments synergistically the sensitivity of the cells to conventional chemotherapeutic agents via down-regulation of P-gp expression.