Acquired multidrug resistance in human K562/ADM cells is associated with enhanced autophagy.

Autophagy, as a necessary process for survival in mammalian cells deprived of nutrients or growth factors, will be activated in many tumor cells while treated with chemotherapeutic drugs, but the role of autophagy in acquired multidrug resistance of human acute myelogenous leukemia to adriamycin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental acute myelogenous leukemia cell line K562 and resistant sub cell line, K562/ADM, which were obtained by treating adriamycin with increasing concentrations. K562/ADM and K562 cells were exposed to PBS culture medium for 3 hours, then the stress-induced autophagy was measured. Real-time quantitative RT-PCR revealed the expression of LC3 mRNA was higher in K562/ADM than in K562 cells. LC3-II, as an autophagosomal marker, was more abundant in K562/ADM than in K562 cells measured by Western blotting. To determine the effect of 3-MA, a known specific inhibitor of autophagy, on overcoming acquired multidrug resistance induced by adriamycin, the MTT assay and flow cytometry were performed. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of adriamycin in acquired resistant cells (K562/ADM). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in multidrug resistance of K562/ADM cells induced by adriamycin.

[Apoptosis effects of drug sensitivity leukemia cells induced by nano-realgar].

OBJECTIVE: To explore apoptosis-inducing effects of realgar nanoparticle (nano-realgar) on drug-sensitive leukemia cells. METHOD: Preparation of nano-realgar was mechanical milled using a high-energy planetary ball mill. Using drug-sensitive leukemia cells (K562) as target cells, MTT assay was used to detect the proliferating activity of K562 cells, and the cellular apoptosis was investigated with double staining of FITC-Annexin V and propidium iodide (PI) by flow cytometry. Flow cytometry (FCM) was employed to detect expression of intracellular Bax, Bcl-2, P-53 protein and the activity of Caspase-3. RESULT: The raw realgar was made to ultra-fine powder by ball milling, and the average diameter of the nanoparticle was (72.72 +/- 22.18) nm measured with electron microscopes. Nano-realgar significantly inhibited the proliferation of K562 cells, Treated for 24, 48 and 72 hours, the 50% inhibitory concentration (IC50) was 43.48, 20.52, 16.07 mg x L(-1). After exposure to 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 hours, the apoptosis of K562 cells detected by Annexin V/PI staining was increased, the apoptotic rate of K562 cells was 10. 52% and 73.25%. After the target cells were treated with 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 h, the expression of P-53, Bax, Bcl-2 markedly increased in a time and dose-dependent manner. After administration of 20 mg x L(-1) and 50 mg x L(-1) nano-realgar for 48 h, the percentage of BCRP+, P-gp+ and co-expressing P-gp and BCRP cell population in K562 cells incrased dramatically. CONCLUSION: Nano-Realgar significantly induced apoptosis of drug-sensitive leukemia cells.

Antitumor effect of arsenic trioxide in human K562 and K562/ADM cells by autophagy.

Arsenic trioxide (As(2)O(3)) has been reported to have potent antitumor effects in vitro and in vivo by inducing cell death via cell cycle arrest and apoptosis in leukemia cells, but the mechanisms of As(2)O(3)-mediated cell death are not fully understood. In this study, we provided in vitro evidence that As(2)O(3) was a potent inducer of autophagy in leukemia K562 and its drug-resistant line K562/ADM cells. As(2)O(3) significantly activated autophagic cell death (programmed cell death type II) in leukemia cell lines. Numerous large cytoplasmic inclusions, abundant autophagic vacuoles, phagocytizing cytoplasm and organelles were observed in As(2)O(3)-treated cells using electron microscope. MDC-labeled autophagic vacuoles were observed by fluorescent inverted phase contrast microscopy and the enhanced MDC fluorescent staining was detected by flow cytometry in As(2)O(3)-treated cells. Furthermore, real-time quantitative RT-PCR revealed that the expression levels of Beclin-1 and LC3 genes, which play key roles in autophagy, increased in As(2)O(3) treated samples than in controls, indicating that autophagy can potentially be involved in the antitumor properties of As(2)O(3). The expression level of Bcl-2 gene, an anti-apoptotic molecule, decreased in As(2)O(3) treated samples than in controls, suggesting that Bcl-2 may be involved in accumulating Beclin-1 and triggering autophagic cell death in As(2)O(3)-treated leukemia cells. Western blotting also showed that As(2)O(3) up-regulated Beclin-1. Altogether, our data provide direct evidence that autophagic cell death is critical for the effects of As(2)O(3) on acute myelogenous leukemia cells.

Parthenolide and arsenic trioxide co-trigger autophagy-accompanied apoptosis in hepatocellular carcinoma cells.

Although arsenic trioxide (ATO) shows a strong anti-tumor effect in the treatment of acute promyelocytic leukemia, it does not benefit patients with hepatocellular carcinoma (HCC). Thus, combination therapy is proposed to enhance the efficacy of ATO. Parthenolide (PTL), a natural compound, selectively eradicates cancer cells and cancer stem cells with no toxicity to normal cells. In this study, we chose PTL and ATO in combination and found that nontoxic dosage of PTL and ATO co-treatment can synergistically inhibit the in vitro and in vivo proliferation activity of HCC cells through suppressing stemness and self-renewal ability and inducing mitochondria-dependent apoptosis. More importantly, USP7-HUWE1-p53 pathway is involved in PTL enhancing ATO-induced apoptosis of HCC cell lines. Meanwhile, accompanied by induction of apoptosis, PTL and ATO evoke autophagic activity via inhibiting PI3K/Akt/mTOR pathway, and consciously controlling autophagy can improve the anti-HCC efficacy of a combination of PTL and ATO. In short, our conclusion represents a novel promising approach to the treatment of HCC.

Vacuolating Cytotoxin A Triggers Mitophagy in Helicobacter pylori-Infected Human Gastric Epithelium Cells.

Helicobacter pylori (H. pylori)-derived vacuolating cytotoxin A (VacA) causes damage to various organelles, including mitochondria, and induces autophagy and cell death. However, it is unknown whether VacA-induced mitochondrial damage can develop into mitophagy. In this study, we found that H. pylori, H. pylori culture filtrate (HPCF), and VacA could activate autophagy in a gastric epithelial cell line (GES-1). VacA-caused mitochondrial depolarization retards the import of PINK1 into the damaged mitochondria and evokes mitophagy. And, among mass spectrometry (LC-MS/MS) identified 25 mitochondrial proteins bound with VacA, Tom20, Tom40, and Tom70, TOM complexes responsible for PINK1 import, were further identified as having the ability to bind VacA in vitro using pull-down assay, co-immunoprecipitation, and protein-protein docking. Additionally, we found that the cell membrane protein STOM and the mitochondrial inner membrane protein PGAM5 also interacted with VacA. These findings suggest that VacA captured by STOM forms endosomes to enter cells and target mitochondria. Then, VacA is transported into the mitochondrial membrane space through the TOM complexes, and PGAM5 aids in inserting VacA into the inner mitochondrial membrane to destroy the membrane potential, which promotes PINK1 accumulation and Parkin recruitment to induce mitophagy. This study helps us understand VacA entering mitochondria to induce the mitophagy process.

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.

Suppression Of Aberrant Activation Of NF-κB Pathway In Drug-resistant Leukemia Stem Cells Contributes To Parthenolide-potentiated Reversal Of Drug Resistance In Leukemia.

Although many drugs that targeted the specific features of leukemia stem cells (LSCs) have substantial application in the clinical treatment of leukemia, the LSCs relapsed and caused drug-resistant leukemia. Therefore, it is necessary to identify the unique features of LSCs in relapsing and drug-resistant leukemia and also to explore the drugs that directed at these features. Our clinical data have indicated that relapsed patients with acute myeloid leukemia have more abundant proportion of LSCs with enhanced breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) expression when compared to the untreated patients. The results showed that compared with LSCs derived from sensitive K562 cells, LSCs from drug-resistant K562/ADM cells have much higher chemotherapeutic resistance, and so we termed these cells as "drug-resistant LSCs". Subsequently, aberrant activation of NF-κB pathway in drug-resistant LSCs was further using gene chip analysis. Also, parthenolide (PTL), which is a specific NF-κB inhibitor, effectively eliminated drug-resistant LSCs and enhanced the sensitivity of K562/ADM cells to doxorubicin-induced apoptosis by down-regulating NF-κB pathway-mediated P-gp expression. These findings make the research area of LSCs more abundant and provide a potential therapeutic strategy for the treatment of refractory and relapsed leukemia.

[The relationship between multi-drug resistance and proportion of leukemia stem cells and expression of drug transporters in drug-resistant leukemia K562/ADM cells].

OBJECTIVE: To investigate the drug resistance, proportion of leukemia stem cells (LSC) and expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-sensitive and multidrug-resistant leukemia cell population. METHODS: The multidrug-resistant leukemia K562/ADM cell and its parental K562 cell were used as the model cells. The drug sensitivity was tested with a MTT assay. Flow cytometry was employed to detect the immunophenotype of stem cells and the expression of P-gp and BCRP. The self-renewal and proliferating potential were examined with methylcellulose colony-forming unit assay. RESULTS: K562/ADM cells were highly resistant to adriamycin, daunorubicin and etoposide. The amount of CD34+, CD123+ and CD34+ CD38- cells in K562/ADM cells was much higher than that in K562 cells, and the proportion of CD34+ CD38- CD123+ cells (LSC) in K562/ADM cells was (5.23 +/- 0.21)% versus (1.27 + 0.17)% in K562 cells, which was 4.12-fold higher than that in K562 cells. Both P-gp and BCRP were overexpressed in K562/ADM cells relative to K562 cells, and the K562/ADM cells coexpressing P-gp and BCRP were 11.25-fold higher over K562 cells. The proportion of CD34+ CD38- CD123+ BCRP+ and CD34+ CD38- P-gp+ BCRP+ cells in K562/ADM cells were (4.13 +/- 0.40)% and (5.80 +/- 1.19)% respectively, which were 3.66- and 11.37-fold higher than the same cells in K562 cells [(1.13 +/- 0.15)% and (0.51 +/- 0.01)%]. Furthermore, drug-resistant K562/ADM cells displayed 4.17-time greater colony-forming ability over the parent K562 cells, corresponding to the proportion of LSCs in K562/ADM cells. CONCLUSIONS: The ABC transporters-overexpressing LSC population exists in drug-resistant leukemic K562/ADM cells relative to drug-sensitive K562 cells, and the drug-resistant LSCs may be the source of chemotherapeutic resistance of leukemia.

[The analysis of HLA-A, B and DRB1 allelic polymorphism in Han race population in Lanzhou region of China].

OBJECTIVE: To investigate the HLA-A, B and DRB1 allele polymorphism of the Han race population in Lanzhou area. METHODS: Polymerase chain reaction-sequence specific primer was used to detect HLA-A, B and DRB1 alleles in 200 unrelated healthy Han individuals from Lanzhou region, Northwest China, and the results were compared with those of Han populations in North, South and Northwest China, and Hui, Uigur and Tibetan population in China. RESULTS: Fourteen of alleles were detected and identified for HLA-A; 32 for HLA-B; and 13 for HLA-DRB1. HLA- A*01, A*02,A*11,A*24, A*30, A*31, A*33; HLA- B*13, B*15, B*40, B*44, B*46, B*51, B*58, B*60; HLA- DRB1*04,. DRB1*07, DRB1*08, DRB1*09, DRB1*11, DRB1*12, DRB1*14 and DRB1*15 were the most common alleles. The frequencies of HLA-A, B and DRB1 genes of Lanzhou Han race were close to that of North China Hans and Hui population in Northwest China, and a little different to that of South China Hans. The HLA-DRB1 alleles were significantly different to those of Uigur and Tibetan race population of China. CONCLUSION: The allelic polymorphism of HLA-A,B and DRB1 loci of Han race population in Lanzhou area was between North and South Han race of China, close to Northwest China Hui, and markedly different to Northwest China Uigur and Tibetan race populations.

Ebb-and-flow of macroautophagy and chaperone-mediated autophagy in Raji cells induced by starvation and arsenic trioxide.

Autophagy is crucial in the maintenance of homeostasis and regenerated energy of mammalian cells. Macroautophagy and chaperone-mediated autophagy(CMA) are the two best-identified pathways. Recent research has found that in normal cells, decline of macroautophagy is appropriately parallel with activation of CMA. However, whether it is also true in cancer cells has been poorly studied. Here we focused on cross-talk and conversion between macroautophagy and CMA in cultured Burkitt lymphoma Raji cells when facing serum deprivation and exposure to a toxic compound, arsenic trioxide. The results showed that both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of nutrition deprivation, and then gradually decreased to near baseline. With nutrient deprivation persisted, CMA progressively increased along with the decline of macroautophagy. On the other hand, in arsenic trioxide-treated Raji cells, macroautophagy activity was also significantly increased, but CMA activity was not rapidly enhanced until macroautophagy was inhibited by 3-methyladenine, an inhibitor. Together, we conclude that cancer cells exhibit differential responses to diverse stressor-induced damage by autophagy. The sequential switch of the first-aider macroautophagy to the homeostasis-stabilizer CMA, whether active or passive, might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular stressors. These findings must be helpful to understand the characteristics, compensatory mechanisms and answer modes of different autophagic pathways in cancer cells, which might be very important and promising to the development of potential targeting interventions for cancer therapies via regulation of autophagic pathways.

Arsenic trioxide induces autophagy and antitumor effects in Burkitt's lymphoma Raji cells.

Although it is generally acknowledged that auto-phagy plays an important role in tumorigenesis and therapy, studies of autophagy in different cell types and under different conditions have led to conflicting theories regarding the influence of autophagy on cell death. In the present study, we explored the role of autophagy and its underlying mechanism in the inhibitory effects of arsenic trioxide (As2O3) on Burkitt's lymphoma Raji cells. The results showed that As2O3 significantly inhibited the proliferation of Raji cells in a dose- and time-dependent manner, induced G2/M phase cell cycle arrest and apoptosis. Moreover, As2O3 also promoted the formation of autophagic vacuoles, as well as increased the degradation of autophagy substrate P62 protein, which was accompanied by an upregulation of Beclin-1 gene and a downregulation of Bcl-2 gene expression. 3-Methyladenine, an autophagy inhibitor, not only increased cell viability through inhibiting autophagic cell death and apoptosis, but also reversed the upregulation of Beclin-1 gene and the downregulation of Bcl-2 gene in the Raji cells induced by As2O3. These results may lead to a better understanding of the action of As2O3 and may provide evidence that autophagy plays an important role in the regulation of cell death. Therefore, regulation of autophagic activity may be a promising therapy for patients with Burkitt's lymphoma.

Insulin resistance reduces sensitivity to Cis-platinum and promotes adhesion, migration and invasion in HepG2 cells.

The liver is normally the major site of glucose metabolism in intact organisms and the most important target organ for the action of insulin. It has been widely accepted that insulin resistance (IR) is closely associated with postoperative recurrence of hepatocellular carcinoma (HCC). However, the relationship between IR and drug resistance in liver cancer cells is unclear. In the present study, IR was induced in HepG2 cells via incubation with a high concentration of insulin. Once the insulin-resistant cell line was established, the instability of HepG2/ IR cells was further tested via incubation in insulin-free medium for another 72h. Afterwards, the biological effects of insulin resistance on adhesion, migration, invasion and sensitivity to cis-platinum (DDP) of cells were determined. The results indicated that glucose consumption was reduced in insulin-resistant cells. In addition, the expression of the insulin receptor and glucose transportor-2 was downregulated. Furthermore, HepG2/IR cells displayed markedly enhanced adhesion, migration, and invasion. Most importantly, these cells exhibited a lower sensitivity to DDP. By contrast, HepG2/IR cells exhibited decreased adhesion and invasion after treatment with the insulin sensitizer pioglitazone hydrochloride. The results suggest that IR is closely related to drug resistance as well as adhesion, migration, and invasion in HepG2 cells. These findings may help explain the clinical observation of limited efficacy for chemotherapy on a background of IR, which promotes the invasion and migration of cancer cells.

[Genetic characterization of human parainfluenza virus 3 circulating in Gansu and Shaanxi Provinces from 2009 to 2011].

To investigate the genetic characterization of Human parainfluenza virus-3 (HPIV-3) circulating in Gansu and Shaanxi Provinces of China, 719 throat swabs were collected from pediatric patients with acute respiratory infections from 2009-2011. Multiplex RT-PCR was used to screen common respiratory viral pathogens. For HPIV-3-positive specimens, nested RT-PCR was used to amplify the HN gene of HPIV-3. The nucleotides of Hemagglutinin-neuraminidase(HN)gene of 13 HPIV-3 positive strains identified in Gansu and Shaanxi Provinces were successfully sequenced and compared with those downloaded from GenBank. The phylogenetic analysis based on the nucleotides sequence of HN gene showed that 13 HPIV-3 strains belonged to sub-cluster C3 with little sequence variation (overall nucleotide divergence of 0.2%-2.3% and amino acid divergence at 0-1.1%). Compared with the complete gene of HPIV-3 strains from U.S.A., Canada, and Australia, the biggest divergence of the nucleotide and amino acid lovels was 6.0% and 3.4%, respectively. The nucleotide divergence between shaanxi09-2 and shaanxi10-H0091 was 0.9%, while the nucleotide divergence between shaanxi10-H005 and gansull-62110372 was 0.5%, between shaanxi09-2 and BJ/291/09 was 0.6%. However, there was no amino acid divergence among them. It is likely that HPIV-3 virus had been transmitting in Gansu and Shaanxi Provinces for several years. Human parainfluenza virus-3 (HPIV-3) circulated in Gansu and Shaanxi Provinces from 2009 to 2011 belonged to sub-cluster C3.

[Effects of 5-bromotetrandrine and daunorubicin on apoptosis and expression of survivin in K562/A02 cells].

The aim of this study was to investigate the potential benefit of combination therapy with 5-bromotetrandrine (5-BrTet) and daunorubicin (DNR) on chronic leukemia. The apoptosis of K562/A02 cells treated by DNA, BrTet and BrTet combined with DNR for 48 hours was detected by flow cytometry; the expressions levels of survivin mRNA and protein K562/A02 cells treated by DNR, BrTet and BrTet combined with DNR and in untreated K562 cells for 48 hours were measured by RT-PCR and Western blot respectively. The results showed that the combination of BrTet with DNR increased apoptotic rate of K562/A02, down-regulated the expression levels of survivin mRNA and protein in K562/A02 cells as compared with blank control and cells treated by BrTet or DNR alone, the survivin expression in K562/A02 cells was higher than that in K562 cells. It is concluded that the combination of BrTet with DNR can effectively reverse the multidrug resistance of K562/A02 cells, promote the apoptosis of K562/A02 cells, the mechanism of which may be related with down-regulation of survivin expression. Survivin may be a target for the treatment of MDR in hematopoietic malignancies.

The crystal structures of copper(II), manganese(II), and nickel(II) complexes of a (Z)-2-hydroxy-N'-(2-oxoindolin-3-ylidene) benzohydrazide--potential antitumor agents.

Mononuclear complexes of Cu(II), Ni(II), and Mn(II) with a new Schiff base ligand derived from indoline-2,3-dione and 2-hydroxybenzohydrazide, [Cu(II)(L)(2)], [Ni(II)(L)(2)], and [Mn(II)L.(AcO).2C(2)H(5)OH] [HL=(Z)-2-hydroxy-N'-(2-oxoindolin-3-ylidene)benzohydrazide], have been prepared. The complexes have been structurally characterized by X-ray crystallography. Among the three complexes, the Cu(II) complex had the novel highest antitumor activity.

[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.

Sensitization and apoptosis augmentation of K562/ADM cells by anti-multidrug resistance gene peptide nucleic acid and antisense oligodeoxyribonucleotide.

AIM: To investigate the reversal effect and apoptosis enhancement of peptide nucleic acid (PNA) and antisense oligodeoxyribonucleotide (ASODN) targeted to multidrug resistance gene (mdr1) on human multidrug resistant leukemia K562/ADM cells. METHODS: A 15-mer PNA and the same sequence of ASODN, complementary to the 5' end of the AUG initiator codon-containing region of mdr1 messenger RNA (MDR1-PNA, MDR1-ASODN), were designed and synthesized. Proliferation and sensitivity to adriamycin of K562/ADM cells treated with MDR1-PNA- and MDR1-ASODN were analyzed with a MTT colorimetric assay. Apoptotic morphologies, P-glycoprotein (P-gp) expression, intracellular adriamycin accumulation, and cell cycle were measured. RESULTS: MDR1-PNA 1 to 10 micromol/L and MDR1-ASODN 2 to 20 micromol/L alone had no inhibitory effects on the proliferation of K562/ADM cells, but significantly inhibited the growth of K562/ADM cells cultured in adriamycin-containing medium. After treatment with MDR1-PNA and MDR1-ASODN, intracellular adriamycin accumulation in K562/ADM cells increased greatly and P-gp synthesis was strikingly reduced. The resistance to adriamycin of the drug-resistant cells was partly reversed and the cells were induced to apoptosis by adriamycin. The reversal efficacy of MDR1-PNA was 3.1-fold higher than that of the same sequence of MDR-ASODN, but neither MDR1-PNA nor MDR1-ASODN could completely block the mdr1/P-gp expression. CONCLUSION: Sequence-special PNA targeted to mdr1 gene more effectively than the same sequence of MDR1-ASODN inhibited the expression of P-glycoprotein to overcome the drug-resistance.

[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.