Reversal Effect and Mechanisms of Recombinant Human Tumor Necrosis Factor-NC Against the Doxorubicin Resistance in Leukemia K562/Doxorubicin Cells 
ZHOU Jing-hong1, CHEN Bo-hua2
1.Principle Scientist, Hutchison Medi Pharma Ltd., Shanghai, 201203, China
2 Senior Scientist, The Naval Medical Research Institute, Shanghai, 200433, China
 
ABSTRACT
Objective: To explore the reversal effect and mechanisms of recombinant human tumor necrosis factor-NC (rhTNF-NC) against the doxorubicin (Dox) resistance in chronic myelogenous leukemia (CML) K562/Dox cells. Methods: The chemo-sensitivity of tumor cells dealt with different concentrations of rhTNF-NC to Dox was detected by tetrazolium dye assay (MTT). The intra-cellular Dox accumulation represented by fluorescence intensity was determined by flow cytometry (FCM) at the excitation wave length of 488 nm and emission wave length of 550 nm. The expression of multidrug resistance (MDR)-related genes and proteins was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Results: After being exposed to gradually increasing concentrations of Dox for 10 consecutive months, K562/Dox cells were more resistant to Dox (nearly 132 times) than Dox-sensitive K562 cells. The IC50 of Dox for K562 and K562/Dox cells were (0.04±0.01) and (5.55±0.08) μmol/L, respectively. When K562/Dox cells were treated with rhTNF-NC at 500, 2 500 or 5 000U/mL, the IC50 of Dox was decreased to (2.22±0.34), (1.41±0.13) and (1.04±0.09) μmol/L, respectively. The concentration-response curves were moved upward by the treatment of rhTNF-NC (P<0.01). FCM analysis displayed that intra-cellular accumulation of Dox was significantly increased when combing Dox with rhTNF-NC. After treatment with rhTNF-NC, the expression of MDR gene (MDR1), MDR-associated protein (MRP), glutathione S transferase π (GSTπ) mRNA, P glycoprotein (P-gp) and protein kinase Cα (PKCα) protein was down-regulated, while topoisomerase IIa (TopoIIa) mRNA expression was up-regulated. Conclusion: rhTNF-NC can effectively augment the drug accumulation in tumor cells. This is due to the up-regulation of TopoIIα and down-regulation of MDR1, MRP and GSTπ at mRNA expression as well as reduction of P-gp and PKCα expression.
Keywords: Recombinant human tumor necrosis factor; Multidrug resistance; Doxorubicin; K562 cells
 
Introduction
Discovered by Carswell et al.[1], tumor necrosis factor (TNF) exists in the serum of mice injected by Bacille Calmette Guerin (BCG) or infected by bacterialTNF can inhibit the activity of tumor proliferation, promote tumor apoptosis, block tumor angiogenesis so as to result in tumor shrinkage and regression[2]. However, in terms of its reversal of multidrug resistance (MDR), further mechanisms are needed to be explored. endotoxin and can cause the tumor necrosis in tumor-bearing mice. At present, it has been confirmed that by modulating various signaling pathways,
 
MDR is one of the most serious problems responsible for the failure of cancer chemotherapy. The typical MDR phenotype includes cross-resistance to anthracyclines, vinca alkaloids, podophyllotoxin, taxans and various cytotoxic compounds with increased expression of the membrane protein, P-glycoprotein (P-gp). P-gp functions as an efflux pump on the surface membranes of resistant cells and transports chemotherapeutic agents out of the cancer cell[3-4]. Besides, MDR-related protein (MRP), glutathione S-transferase π (GSTp), and topoisomerase IIa (TopoIIa), protein kinase Cα (PKCα) and other factors are also very import in mediating multidrug resistance[5-6].
 
To enhance the response of tumors to chemotherapy, much attention has been paid on a wide variety of pharmacological agents, including calcium channel blockers, cyclosporins, cardiovascular drugs, steroid analogs, antibiotics, calmodulin inhibitors and antimalarials. Although there are hundreds of reversal drugs found in vitro, but their clinical application has been limited because of their toxicity. Several reports indicate that cytokines like tumor necrosis factor-alpha (TNF-α) applied alone or in combination with other chemotherapeutic agents might be capable of decreasing this MDR process[7-8]. The new recombinant human tumor necrosis factor-NC (rhTNF-NC) with higher efficacy and less toxicity than its original counterpart-TNF-α has been approved to be a new drug in clinic. In this report, the reversal effect of rhTNF-NC on the doxorubicin (Dox) resistance in chronic myelogenous leukemia (CML) K562/Dox cells and its underlying mechanisms were studied.
 
Materials and Methods
Reagents
The human chronic myelogenous leukemia (CML) cell line K562 was purchased from American Type Culture Collection (ATCC). Dox resistant K562/Dox cells were induced in our laboratory. Fetal bovine serum (FBS) and RPMI1640 culture solution were provided by Gibco Company. rhTNF-NC was from Shanghai Weike Biopharmaceutical Co., Ltd. Dox and ethidium bromide were obtained commercially from Sigma Chemical Co. (St. Louis, MO, USA) and reverse transcription-polymerase chain reaction (RT-PCR) kits were purchased from Takara. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2- H-tetrazolium bromide (MTT) was from Fluka. PKCα monoclonal antibody and horseradish peroxidase-conjugated anti-rabbit IgG were from Santa Cruz. P-gp monoclonal antibody was purchased from Chemicon, Temecula, CA. ECL Western blotting kit was bought form Amersham Corp.
 
This experiment was performed in accordance with the Declaration of Helsinki and approved by Ethics Committee of Hutchison Medi Pharma Ltd Institutional Animal Care and Use Committee (IACUC)。
 
Methods
Cell culture: The human CML cell line K562 was preserved in our laboratory. Cells were cultured in 90% RPMI1640 and 10% FBS containing 100 U/mL penicillin and 100 μg/mL streptomycin and incubated in a humidified incubator with 5% CO2 at 37℃. Gradually increasing concentrations of Dox were added to cell culture for 10 consecutive months to force K562 cells to turn into K562/Dox cells, which exhibited prominent phenotype of resistance to Dox cytotoxicity.   K562/Dox cells could grow stably in cell culture with 2 μmol/L Dox. Dox was removed from the culture media 2 weeks before the experiment.
 
Cytotoxicity assay by MTT: The K562/Dox cells were plated in 96-well plates at a density of 4×103 cells/well in 180 μL drug-free RPMI1640 with 10% FBS and incubated at 37℃ for 24 h. After 24-h incubation, Dox alone or in combination with rhTNF-NC was added to the wells at the indicated concentration and the final volume was 200 μL per well. The cells in the positive control well were cultured in culture medium without drugs and the blank control without cells would provide background optical density (OD). The cells were incubated for an additional 72 h. 20 μL MTT reagent (5 mg/mL solution in PBS) was added to all wells and the cells were further incubated at 37℃ for 4 h. When the incubation was ended, the supernatant fluid was removed. The reaction was terminated by adding 150 μL/DMSO per well, followed by reading OD at 570 nm using an enzyme-linked immune-sorbent assay (ELISA) plate reader. The inhibition rate was calculated by the following equation: Inhibition rate (%) = 1 - (the mean OD of drug-treated cells - the mean OD of blank)/(the mean OD of untreated cells - the mean OD of blank)×100%. The IC50 for Dox was defined as a Dox concentration in which the growth inhibition rate was 50%.
 
Dox accumulation detected by flow cytometry (FCM): Accumulation of the fluorescent anthracycline Dox was measured as a functional index of P-gp activity. K562/Dox cells (1×109/L) were cultured for 1 h at 37℃ in the presence of Dox (1.25 or 5 μmol/L) alone or in combination with rhTNF-NC. Cells were then harvested and washed twice with cold (0℃) PBS, then placed in ice-water to block the reaction until analysis. Fluorescence intensity of 2×104 cells was then determined by FCM at the excitation wave length of 488 nm and emission wave length of 550 nm.
 
Gene expression analyzed by RT-PCR:Cells were plated at a density of 105 cells/mL and treated with rhTNF-NC at 2 500 U/L for 24, 48 and 72 h. Total RNA was extracted from the treated cells or control samples with the TRIzol system (Gibco BRL). The synthesis of cDNA by reverse transcription was performed utilizing 1 mg of total cellular RNA, 5 U AMV reverse transcriptase, 0.125 mM oligodT-adaptor primer, 1 U/mL RNAse inhibitor, 1 mM dNTP, 5 mM MgCl2, 2 mL 1×RNA PCR buffer in a total volume of 20 mL. The tubes were incubated at 42℃ for 60 min, then at 99℃ for 5 min, before being quickly chilled on ice. The cDNA was stored at -20℃ until required for analysis.
 
PCR amplification was performed using 3.5 mL cDNA in a 50 mL reaction volume containing 0.012 U/mL Taq DNA polymease (Promega) and a couple of primers. The PCR conditions for amplification were: an initial denaturation at 96℃ for 5 min, then 7 cycles at 96℃ for 1 min, 57℃ for 90 s and 72℃ for 1 min, then 32 cycles at 96℃ for 30 s, 57℃ for 60 s, and 72℃ for 30 s, with a final extension at 72℃ for 5 min. The amplified fragments were detected by 2% (w/v) agarose gel electrophoresis and staining with 0.3 g/mL ethidium bromide. Each band was analyzed on image analysis system IS1000 (Alpha Innotech, San Leandro, CA, USA). The levels of specific gene expression were determined semiquantitatively by calculating the ratio of density metric value from specific genes by comparison to the control b-actin expression. The primer sequences and fragment lengths were shown in Table 1.
Table 1 Primer Sequences and Fragment Lengths
Genes
Primer sequence (base pairs)
Fragment length (base pairs)
MDR1
5?-GTT GCC ATT GAC TGA AAG AAC-3?
5?-ACA GGA GAT AGG CTG GTT TGA-3?
157
GSTπ?
5?-ATG CTG CTG GCA GAT CAG-3?
5?-GTA GAT GAG GGA GAT GTA TTT GCA-3?
270
MRP
5?-GTA CAT TAA CAT GAT CTG GTC-3?
5?-CGT TCA TCA GCT TGA TCC GAT-3?
256
TopoIIα?
5’AAC TTT GGC TGT TTC AGG 3’
5’ATC ATT ATC TTC CCA TAA CGA AGC G3’
216
β-Actin
5’CAC GTC ACA CTT CAT GAT GG 3’
5’ATG TTT GAG ACC TTC AAC AC 3’
494
 
P-gp and PKCα expression analyzed by Western blot: Cells were pretreated at 500, 2 500 and 5000 U/mL of rhTNF-NC for 48 h, respectively, and then were lysed for 30 min at 4℃ with lysis buffer [Tris-HCl (PH7.5) 50 mM, NaCl 25 mM, DTT 100 mM, EDTA 2 mM, 2% SDS, 1% Triton X-100, PMSF 1 mM, pH7.4). The cell lysates were centrifuged at 10 000 g for 5 min at 4℃. After measurement of protein concentrations, equivalent amounts of protein (100 mg) in extracts of K562/Dox cells were then mixed with SDS sample buffer (0.375 M Tris-HCI, 4% SDS, 20% glycerol, 3.1% dithiothreitol, pH 6.8) and heated in boiling water for 5 min. Samples were subjected to 8% SDS-PAGE, and transferred electrophoretically to a polyvinylidene difluoride membrane for 1 h at room temperature. After blocking for 1 h at room temperature with 10% non-fat dry milk (w/v), the membrane was incubated with anti-human p-gp and PKCa monoclonal antibodies at 4℃ overnight, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h at room temperature. Immunodetection was done by using ECL Western blotting kit. The relative density of the protein bands was quantified using Scoin Image software (Scoin Corp.).
 
Statistical analysis
All the data were calculated and analyzed by SAS. Statistical significance was determined by Student’s t-test, and the data were expressed as the mean ± standard deviation (x±s). All the data from cell culture experiments were on the basis of 3 individual cell preparations at least. The association between the concentration-response curves was examined using the linear regression analysis. P<0.05 was considered to be statistically significant.
 
Results
Cytotoxicity in K562 and K562/Dox cells
Cytotoxicity was expressed as the percentage of growth inhibition compared with the untreated control cells. MTT assay showed that K562/Dox cells were more resistant to Dox (nearly 132 times) than Dox-sensitive K562 cells. The IC50 for rhTNF-NC in K562 cells was similar to that for tamoxifen in K562/Dox cells (P>0.05) (Table 2).
Table 2 Cytotoxicity of Several Agents in K562 and K562/Dox Cells (x±s)
Agents
Unit
IC50 in K562
IC50 in K562/Dox
Dox
μmol/L
0.04±0.01
5.55±0.08
rhTNF-NC
103U/mL
73.59±13.24
84.14±15.01
Tamoxifen
μmol/L
11.27±0.98
13.09±4.03
 
Enhancement of chemo-sensitivity via rhTNF-NC
The concentration-dependent reversal effect of rhTNF-NC was determined by incubating the cells with increasing concentrations of Dox in the presence of a fixed concentration of rhTNF-NC. When rhTNF-NC was fixed at 0, 500, 2 500 or 5 000 U/mL, the IC50 of Dox was (2.22±0.34), (1.41±0.13) and (1.04±0.09) U/mL in K562/Dox cells, respectively, and there was statistical significance by comparison to Dox alone (P<0.05). The reversal fold of Dox resistance was 14.2 using tamoxifen. Above experimental results showed that 500, 2 500 and 5 000 U/mL rhTNF-NC significantly increased the sensitivity of K562/Dox cells to Dox in a concentration-dependent manner, the reversal fold was 2.51-5.33 in K562/Dox cells (Table 3). However, in K562 cells, rhTNF-NC did not increase the chemo-sensitivity to Dox.
 
Concentration-response curves revealed that rhTNF-NC made the response curve of Dox move upward in a parallel manner. The regression equations of the concentration-response curves calculated for rhTNF-NC 5 000, 500 and 0 U/mL in K562/Dox cells were: Y= - 0.0156+1.0713lg(x), Y= - 0.3632+1.0611lg(x) and Y= - 0.9221+1.2398lg(x) (Figure 1). By comparison to the slopes of dose-response curves, it was shown no significant difference (P>0.05), but the differences between the IC50 of dose-response curves were statistically significant (P<0.01).
Table 3 Reversal Effect of Agents in K562/Dox Cells (x±s)
Agents
Concentration (μmol/L or U/mL)
In K562/Dox(reversal fold)
Dox
5.55±0.08
rhTNF-NC
500
2.22±0.34 (2.51)
2 500
1.41±0.13 (3.94)
5 000
1.04±0.09 (5.33)
Tamoxifen
5
0.39±0.08 (14.2)
 
 
 
 
 
 
 
 
 
 
 
 

Figure 1 Reversal Effect of rhTNF-NC at 0, 500, 5 000 U/mL in K562/Dox Cells (n=3)
 
Dox accumulation in K562/Dox cells
To assess MDR1 expression at the functional level, accumulation of the fluorescent Dox was measured and quantified in K562/Dox cells treated by 1.25 or 5 μmol/L Dox alone or in combination with 500, 2 500 or 5 000 U/mL rhTNF-NC for 1 h using FCM. As shown in Table 5, the mean fluorescence intensity (representing the accumulation of Dox) in K562/Dox cells incubated with Dox for 1 h, increased significantly in rhTNF-NC-treated cells in a dose-dependent manner compared with Dox alone. The fluorescence intensity was 7.76±1.02, 10.38±0.69, 12.06±3.21 and 14.31±2.96 for 0, 500, 2 500 and 5 000 U/mL rhTNF-NC combined with 1.25 μmol/L Dox as well as 21.92±1.23, 24.54±3.96, 25.89±2.91 and 27.09±3.09 for rhTNF-NC combined with 5 μmol/L Dox.
Table 4 Dox Accumulation (Fluorescence Intensity) in K562/Dox Cells
Concentration of rhTNF
       Dox (μmol/L)
U/mL
1.25
5
0
7.76±1.02
21.92±1.23
500
10.38±0.69*
24.54±3.96
2 500
12.06±3.21*
25.89±2.91*
5 000
14.31±2.96**
27.09±3.09**
Compared with the corresponding control (rh-TNF-NC 0 U/mL), *P<0.05, **P<0.01.
 
Alteration of MDR-related gene expression at mRNA level
The experimental results showed that MDR1 mRNA was overexpressed in K562/Dox cells, and MRP was moderately expressed. It could be seen that after treatment with 2 500 U/mL rhTNF-NC, MDR1 and MRP mRNA expression was reduced at 48 h, and the amplitude of reduction were 59.52% and 65.52% respectively, then recovered mildly at 72 h. GSTπ mRNA expression was decreased by 57.93% at 72 h. TopoIIα mRNA expression was increased from 0.37±0.11 to 0.77±0.07 (Figure 2). These results indicated the significant alteration of MDR-related gene expression.
 
   
  Marker 0 h 24 h 48 h 72 h                Marker 0 h 24 h  48 h 72 h
       
  Marker 0 h 24 h 48 h 72 h                Marker 0 h  24 h 48 h 72 h
 
 
 
Figure 2 Effect of Each Exposure Time to 2 500 U/mL rhTNF-NC on MDR1, MRP, GST π, TopoⅡa mRNA expression in 562/Dox cells (n=3)
Note: *P<0.05: compared mRNA expression at 24, 48 and 72 h with that at 0 h.
β-actin
Alteration of MDR-related protein expression
P-gp is a member of a superfamily of adenosine triphosphate-binding cassette transporter proteins and acts as a transporter to excrete chemotherapeutic drugs. It could be activated by PKCα. As expected, rhTNF-NC could significantly inhibit p-gp and PKCα expression, leading to drug accumulation (Figure 3 and 4)         
Figure 3 Effect of Different Concentrations of rhTNF-NC on P-gp Expression in K562/Dox Cells
Figure 4 Effect of rhTNF-NC on PKCα Expression in K562/Dox Cells
Note: A: K562, B: K562/Dox, C: K562/Dox cells treated by 5 000 U/mL rhTNF-NC, D: K562/Dox cells treated by 2 500 U/mL rhTNF-NC.
 
Discussion
Cytokines, such as TNF-α and interleukin-2 (IL-2), have been demonstrated to alter the MDR phenotypes in vitro and in vivo experiments. They are potential chemosensitizers of tumor cells in the context of a combined application of MDR-associated chemotherapeutic drugs, such as Dox and vincristine[8]. rhTNF–NC has been approved by China Food and Drug Administration. The results of clinical studies have displayed that the patients can tolerate rhTNF-NC much better than its wild-type TNF-α, and rhTNF-NC is similar to TNF-α in the cytotoxic effect on tumor cells, but with less toxicity to the host in vivo.
Many studies on the reversal of MDR by TNF-α have been performed, but it is still far from the profound understanding of the mechanisms. Liu et al.[9] made an experiment about the reversal of TNF-α in MDR hepatocellular carcinoma cells and found that TNF-α could down-regulate the expression of MDR1, lung resistant protein (LRP) and GSTπ genes, up-regulate the expression of TopoIIα genes and there existed a time-effect relationship. The changes of gene expression were significant 24-48 h after TNF-α action, and the protein expression had the greatest change at 48 h.
Chen et al.[10] found that after transfecting TNF-α cDNA into MDR breast cancer cell line MCF/ADR, both MDR1 mRNA and P-gp expression in the cells and drug resistance to Dox decreased dramatically, but the sensitivity increased.Brouckaert et al.[11] reported that TNF-α augmented the tumor response in B16-BL6 melanoma-bearing mice treated with stealth liposomal Dox. PKC almost participates in the regulation of all MDR mechanisms. For instance, PKC makes P-gp phosphorylation, and P-gp is activated to promote drug efflux. Additionally, PKC can also make MRP, LRP, GST and GSTπ phosphorylation to enhance their activity[6]. In order to verify whether MDR was controlled by PKC, the PKC isoforms were evaluated. It was indicated that PKCα played a key role in HCT15/DOX cells. Inhibition of PKCα with Go6976, a specific inhibitor of classical PKC, or knockdown of PKCα by siRNA led to reduced MDR expression and increased Dox-induced apoptosis[12]. PKCα is also involved in P-gp phosphorylation and vincristine accumulation in human MDR glioma cells[13]. Besides, it may contribute to MDR mechanism in KBV200 cells[14], gastric MGC803 cells[15] and several other tumor cell lines. In fact, not only PKCα activate p-gp, other protein kinases, such as PKCα could also regulate P-gp. Many PKC inhibitors are in preclinical stage. Phase I/II clinical trials have revealed that a part of PKC inhibitors exert a certain therapeutic effect on the drug resistance of lung cancer, and further investigations into its action mechanisms are conductive to great breakthroughs of drug resistance of lung cancer[16].
 
The experimental results were nearly in accordance with those reported in previous studies. It displayed that rhTNF-NC could strongly augment the drug accumulation in tumor cells. This is due to the up-regulation of TopoIIα and down-regulation of MDR1, MRP and GSTπ at mRNA expression as well as reduction of P-gp and PKCα expression at protein level. At present, a very encouraging tumor response has been reported in the patients with lung cancer. The domestic rhTNF can improve the quality of life, mean survival time and 1-year survival of patients, even though it can’t improve 2 and 3-year survivals[17]. How to combine rhTNF-NC with chemotherapy scientifically and efficiently is worth studying further.
 
Declaration
The authors of this manuscript declare that they have no conflict of interest.
 
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