This article is mentioned in:

Introduction

Chronic myeloid leukemia (CML) is a well-described hematopoietic malignancy as a result of the generation of the BCR-ABL fusion oncogene (1). Systemic chemotherapy is the main treatment method for patients with CML. Although the majority of the CML patients respond to therapy, >62.5% of the patients will experience multidrug resistance (MDR) (2). MDR is the main reason for tumor chemotherapeutic failure, and finding selective MDR reversal agents has become a research focus. MDR is often associated with overexpression of drug efflux transporters belonging to adenosine triphosphate (ATP)-dependent binding cassette (ABC) protein family (3). A large number of studies revealed that multidrug resistance 1 (MDR1), also known as P-glycoprotein or ABCB1, and multi-drug resistance-associated protein 1 (MRP1), also known as ABCC1, which are ABC transporters and work as drug efflux pumps, play crucial roles in MDR of human CML (2–7).

The phosphoinositide 3-kinase/serine-threonine kinase (PI3K/Akt) signaling pathway is a well-known fundamental intracellular signaling transduction pathway involved in multiple biological processes both in normal and cancer cells, including gene transcription and translation, cell growth, proliferation and survival, cell metabolism, cell cycle progression, apoptosis and autophagy (8–10). Besides, there is an increasing amount of preclinical data supporting that the PI3K/Akt signaling pathway is also involved in the drug resistance of different types of human malignant cells, including CML (2,4,5,12–15). A previous study demonstrated that the resistant CML cell line K562/ADM presented higher PI3K/Akt activity than the sensitive one and was in accordance with the MDR phenotype (2). Moreover, there is a positive relationship between the levels of P-gp and MRP1 and the activity of PI3K/Akt signaling pathway (4). The increasing level of p-Akt showed high activation of PI3K/Akt signaling pathway in drug-resistant CML cell lines. Blocking the PI3K/Akt signaling pathway with LY294002 (a PI3K-specific inhibitor) or Akt siRNA could downregulate the expression of P-gp and MRP1 and restore drug sensitivity (2,4). Although the exact mechanisms that underlie the role of PI3K/Akt signaling pathway activation remain unclear, these data clearly support that PI3K/Akt pathway plays an important role in the pathogenesis of MDR in CML.

Rhizoma Anemarrhenae (Zhimu in Chinese), a well-known traditional Chinese medicinal herb, which is officially listed in the Chinese Pharmacopoeia, has been effectively used for febrile diseases in oriental clinical practices. Timosaponin A-III (TAIII), a steroidal saponin isolated from the rhizomes of Anemarrhena asphodeloides (AA), has been credited with a wide spectrum of bioactivities, including improving learning and memory (16), inhibiting inflammation (17), suppressing allergic reaction (18), controlling hyperglycemic (19,20), activating autophagy (21) and inducing cancer cells apoptosis (22–24). However, the ability of TAIII to reverse MDR has not been reported. In this study, for the first time, we explored the effects of TAIII on the reversal of multidrug resistant and demonstrated its molecular mechanism.

Materials and methods

Chemicals and reagents

TAIII (purity >98%, Yuanye Bio-Technology Co. Ltd., Shanghai, China), wortmannin (a specific PI3K inhibitor, Beyotime Institute of Biotechnology, Shanghai, China), rhodamine-123 (Rho-123, Sigma Chemical Co., St. Louis, MO, USA) and 5(6)-carboxyfluorescein diacetate (CFDA, Sigma Chemical Co.) were dissolved in dimethyl sulfoxide (DMSO, Amresco, USA) at the concentrations 200 μM, 10 mM, 1 mg/ml and 1 mM respectively aliquoted and stored at −20°C. The stock solutions of TAIII contained a final DMSO concentration of <0.1%. Adriamycin (ADM) purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China) was dissolved in a concentration of 2 g/l with ddH2O and stored at −20°C. Rabbit polyclonal antibodies including anti-MDR1, MRP1, total-Akt and phosphor-Akt (Ser473) were purchased from Beijing Bioss Bio-Technology Co., Ltd. (Beijing, China). Rabbit polyclonal antibodies against GADPH was obtained from Goodhere Biotechnology Co., Ltd. (Hangzhou, China).

Cell lines and cell culture

The human CML K562 cells were obtained from Key Laboratory of Tumour Molecular Biology of Binzhou Medical University (Binzhou, China) and its MDR subline K562/ADM was purchased from the Department of Pharmacology, the Institute of Hematology of Chinese Academy of Medical Sciences (Tianjin, China). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, both from Hyclone, USA), 100 U/ml of penicillin and 0.1 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. K562/ADM cells were cultured in the same medium with an additional 4 mg/l ADM. Before the experiment, K562/ADM cells were cultured in drug-free medium for 72 h.

Multidrug resistance determination in K562/ADM cells

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Shanghai, China) was used to determine the survival rate of cells incubated with ADM. The cells were seeded in a 96-well plate at a density of 5×103 cells/well in RPMI-1640 containing 10% FBS. Then, various concentrations of ADM (0.2–1.6 mg/l for K562 cells and 16–128 mg/l for K562/ADM cells) were added. After the cells were incubated at 37°C in 5% CO2 for 24 h, 10 μl of CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance was measured at 570 nm with a fluorescence spectrofluorometer (F-7000; Hitachi HighTechnologies Corp., Tokyo, Japan). A blank well containing only medium and drugs was used as a control. The 50% inhibition of cell growth (IC50) produced by ADM was calculated.

TAIII intrinsic cytotoxic activity determination in K562/ADM cells

CCK-8 assay was also used to determine the direct cytotoxic activity of TAIII as described above. TAIII of different concentrations at 1–16 μM were added to each well for 24 h. Relative survival rate (%) of each group = absorbance of the experimental group/the absorbance of the control group x 100%. The calculated TAIII concentration at 90% survival rate was IC10. The concentrations below IC10 were selected as the experimental concentration for TAIII to reverse drug-resistance.

Reversal efficacy of TAIII determination

Briefly, the K562/ADM cells were seeded into a 96-well plate, then 1 or 2 μM TAIII with or without various concentrations of ADM (4–32 μg/ml) was added to each well accordingly. Then, the quantity of viable cells were determined by CCK-8 assay according to the manufacturer's instructions. ADM IC50 was calculated using the untreated cells as the 100% viable control. The reversal fold (RF) values, as potency of reversal, were obtained from the following formula: RF=IC50 of ADM only/IC50 of ADM with TAIII.

Cellular uptake of ADM

K562/ADM cells were plated in 6-well plates at a concentration of 1×106 cells in 1 ml growth medium. After incubation alone or with TAIII (1 and 2 μM) at 37°C for 24 h, 3 mg/l ADM was added to designated K562/ADM cells for another 1 h at 37°C. Then, the cells were harvested by centrifugation and washed twice with ice-cold phosphate-buffered saline (PBS). The cell-associated mean fluorescence intensity (MFI) of ADM was detected by flow cytometer using a FACSCalibur (Beckman Coulter, Brea, CA, USA) with excitation/emission wavelengths of 485/580 nm.

Rho-123 and CFDA accumulation assay

Rhodamine-123 (Rho-123) and 5(6)-carboxyfluorescein diacetate (CFDA) were, respectively, used to evaluate the transport function of P-gp and MRP1 in K562/ADM cells by flow cytometric analysis. Rho-123 was a special substrate for P-gp which contains yellow-green fluorophores. CFDA was used as a model MRP1 substrate to evaluate the function of MRP1 (25). A total of 6×105 cells were seeded into 6-well plates which were pretreated with TAIII (1 and 2 μM) for 24 h followed by combined-treatment with Rho-123 (2 μg/ml) or CFDA (1 μM) for another 30 min at 37°C in 5% CO2. Cells with the equivalent amount of DMSO, without Rho-123, CFDA and TAIII were used to evaluate cell auto-fluorescence. Then, the cells were harvested and washed twice with cold PBS and subsequently analyzed by flow cytometry. The values were expressed by the mean fluorescence intensity of Rho-123 and CFDA.

RT-PCR analysis

After treatment with 1 or 2 μM TAIII for 24 h, ~3×106 cells were harvested for RT-PCR analysis. Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Total RNA was reverse transcribed to cDNA and stored at −20°C. Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Primers used in RT-PCR were as follows: MDR1 forward, 5′-GGAGCCTACTTGGTGGCACATAA-3′; reverse, 5′-TGGCATAGTCAGGAGCAAATGAAC-3′. MRP1 forward, 5′-CTGGGAACATGATTAGGAAGC-3′; reverse, 5′-GAGGATTTCCCAGAGCCGAC-3′. GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse, 5′-GAAGATGGTGATGGGATTTC-3′. RT-PCR was performed on an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) by using SYBR Green reaction kit (Takara, Bio, Otsu, Shiga, Japan). The reaction system of PCR was: SYBR green reagent, forward primer, reverse primer, template cDNA and nuclease-free distilled water. PCR programs were carried out as follows: 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec, 60°C for 30 sec. GAPDH served as an internal control. The PCR products were separated by 1% agarose gels. The gels were scanned and analyzed by the Gel Imaging System. RT-PCR for each gene of each cDNA sample was assayed in triplicate.

Western blot analysis

After different treatments, the cells were harvested and washed with PBS. Lysis buffer (100 μl) (Beyotime Biotechnology) was added and the protein concentration of the lysate was determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Biotechnology). The lysed samples containing 50 μg were separated by 6–10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime Biotechnology) with a constant voltage of 80 V for 0. 5 h and 120 V for another 1.5 h. The resolved proteins were electrophoretically transferred to polyvinylidine difluoride membranes (EMD Millipore, Bedford, MA, USA) and blocked with 5% skimmed milk for 2 h. Subsequently, the membranes were incubated overnight at 4°C with specific antibodies. The primary antibodies were rabbit polyclonal antibodies against P-gp (1:500), MRP-1 (1:500), total-Akt (1:500), p-Akt (1:500), GADPH (1:1,000). The following day, the membranes were incubated in horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (1:5,000) for 2 h at room temperature. Finally, images were captured by a FluorChem FC2 gel imaging system (Alpha Innotech, San Leandro, CA, USA). The intensity of each band was normalized by GADPH for their respective lanes.

Data analysis

Statistical analyses were performed using SPSS17.0 software (IBM SPSS, Armonk, NY, USA). Data were expressed as the means ± SD. Statistical comparisons were evaluated by one-way ANOVA. Values of P<0.05 was considered statistically significant.

Results

Multi-drug resistance of K562/ADM cells

Compared with its parental cells, K562/ADM cells showed clear drug-resistant property. As shown in Table I, the IC50 of ADM on K562 and K562/ADM cells were 1.013 and 32.176 mg/l, respectively. The drug-resistance was 31.78-fold (P<0.05).

Table I Determination of multidrug resistance according to the sensitivity of K562/ADM and K562 cells toward ADM (means ± SD of triplicate experiments).

Table I

Determination of multidrug resistance according to the sensitivity of K562/ADM and K562 cells toward ADM (means ± SD of triplicate experiments).

Treament	K562/ADM IC50 (μg/ml)	K562 IC50 (μg/ml)	Resistant fold
ADM	32.176±3.1718a	1.013±0.1008	31.7784

a P<0.05 vs. K562 cells.

Direct cytotoxic activity of TAIII

TAIII inhibited viability of K562/ADM cells in a dose-dependent manner (Fig. 1). TAIII of 1 μM had no significant cytotoxicity (cell growth inhibition <5%), and 2 μM TAIII had a very weak cytotoxicity (cell growth inhibition <10%). To minimize TAIII itself on K562/ADM cells growth, 1 and 2 μM was selected.

Figure 1 K562/ADM cells were treated with various concentrations of TAIII for 24 h. Proliferation of K562/ADM cells was determined using CCK-8 assay, and the inhibition rate (%) was calculated. The data are shown as mean ± SD for three independent experiments. *P<0.05.

Reversal of drug resistance by TAIII

As shown in Fig. 2, the reversal effect of TAIII on K562/ADM cells was dose-dependent. The IC50 values of ADM in K562/ADM cells were 30.74±2.77, 20.05±1.18 and 12.19±2.17 mg/l for ADM plus vehicle, ADM plus 1 μM TAIII and ADM plus 2 μM TAIII, respectively. The reversal fold-change of TAIII of 1 and 2 μM was 1.50 and 2.52, respectively (Table II, P<0.05).

Figure 2 TAIII treatment altered ADM-induced cytotoxicity of K562/ADM cells. Growth curves are based on data from CCK-8 assays in K562/ADM cells after 24-h culture in the absence or presence of 1 and 2 μM TAIII. The results showed increasing drug sensitivity in a dose-dependent manner in K562/ADM cells. Results are expressed as mean ± SD for three independent experiments. *P<0.05.

Table II Effect of TAIII on the sensitivity of K562/ADM cells toward ADM by CCK-8 assay (means ± SD of triplicate experiments).

Table II

Effect of TAIII on the sensitivity of K562/ADM cells toward ADM by CCK-8 assay (means ± SD of triplicate experiments).

Treament	IC50 ( μg/ml)	Reversal fold (RF)
ADM alone	30.743±2.7661
ADM+1 μM TAIII	20.524±1.1798a	1.50
ADM+2 μM TAIII	12.192±2.1667a	2.52

a P<0.05 vs. ADM alone.

Effect of TAIII on the intracellular accumulation of ADM

The intracellular accumulation of ADM decreased significantly in K562/ADM cells compared to the parental K562 cells (26,27). We determined that TAIII increased the intracellular accumulation of ADM in K562/ADM cells. Our results indicated that TAIII elevated the sensitivity of K562/ADM cells toward ADM through increasing intracellular ADM accumulation (Fig. 3A; P<0.05). As show in Fig. 3D, the fluorescence intensity of ADM in TAIII-treated K562/ADM cells increased in a dose-dependent manner compared to untreated K562/ADM cells.

Figure 3 The effect of TAIII on the intracellular accumulation of adriamycin (ADM), rhodamine-123 (Rho-123) and 5(6)-carboxyfluorescein diacetate (CFDA) of K562/ADM cells. (A) TAIII significantly increased the ADM-associated MFI in K562/ADM cells in a dose-dependent manner. (B) Rho-123 was used as a special substrate for P-gp to test P-gp activity in K562/ADM cells. The FCM results showed that there was an increasing intracellular accumulation of Rho-123 in K562/ADM cells in a dose-dependent manner. (C) CFDA was used as a model MRP1 substrate to evaluate the function of MRP1. The FCM results showed that there was an increased intracellular accumulation of CFDA in K562/ADM cells. (D) Enhanced uptake of ADM, Rho-123 and CFDA presented in terms of mean fluorescence intensity value. Bars means ± SD. n=4. P>0.05, compared to the control group; *P<0.05, compared to the control group.

TAIII inhibits P-gp and MRP1-mediated transport

To assess the impact of TAIII treatment on the function of P-gp and MRP1 as efflux pump in K562/ADM cells, we examined the P-gp-mediated Rho-123 and MRP1-mediated CFDA transports in the cells treated with TAIII. As shown in Fig. 3B, Fig. 3C and D, TAIII treatment significantly increased the intracellular accumulation of Rho-123 and CFDA in a dose-dependent manner (P<0.05).

TAIII decreases expression of P-gp and MRP1 in K562/ADM cells via the PI3K/Akt signaling pathway

P-gp and MRP1 are ABC transporters, which are overexpressed in many drug-resistant cells (8,12,13). K562/ADM cells express higher levels of P-gp and MRP1 than K562 cells (26,28). In this study, K562/ADM cells expressed P-gp and MRP1 protein at high level. After incubation alone or with TAIII (1 and 2 μM) for 24 h, the expression levels of P-gp and MRP1 were determined by RT-PCR and western blotting (Figs. 4 and 5). The results of RT-PCR are shown in the Fig. 4, the MDR1 and MRP1 mRNA expression in K562/ADM cells significantly decreased in a dose-dependent manner (P<0.05). The results of western blotting are shown in Fig. 5, compared to the negative control group, TAIII was able to induce a significant downregulation of P-gp and MRP1 protein expression in a dose-dependent manner. These results indicated that TAIII could modulate P-gp and MRP1 gene expression, thus increasing the intracellular ADM accumulation.

Figure 4 TAIII treatment decreases the expression of P-gp and MRP1 mRNA in K562/ADM cells in a dose-dependent manner. P-gp and MRP1 mRNA levels were evaluated after 24 h of 1 and 2 μM TAIII treatment. Total RNA was isolated and used in RT-PCR to determine changes in P-gp and MRP1 mRNA levels after normalization to GADPH expression. The results are expressed as the mean ± SD for three independent experiments. Bars means ± SD. n=3. P>0.05, compared to the control group; *P<0.05, **P<0.01, ***P<0.001 versus control group.

Figure 5 The effect of TAIII on expression of drug resistance related proteins in K562/ADM cells. After TAIII (0, 1 and 2 μM) treatment for 24 h, western blotting results showed decreased protein expression of P-gp, MRP1 and p-Akt in human CML K562/ADM cells. GADPH was the internal control. The results are expressed as the mean ± SD for three independent experiments. Bars means ± SD. n=3. P>0.05, compared to the control group; *P<0.05, **P<0.01 compared to the control group.

PI3K/Akt signaling pathway played a vital role in the development of CML MDR, and was closely related to high expressions of P-gp and MRP1 (2,4). In order to further analyze the inhibitory effects of TAIII on the expression of P-gp and MRP1, we analyzed the alteration of total expression and activity of Akt (Akt Ser473) in K562/ADM cells. Akt is one of the most important modulators in PI3K/Akt pathway. As shown in Fig. 5, after treatment with TAIII for 24 h, the expression of total-Akt did not change apparently, whereas the expression of Akt phosphorylation (Akt Ser473) changed significantly (P<0.05, Fig. 5). These data suggested that the PI3K/Akt pathway is involved in the TAIII mediated reversal of multidrug resistance of K562/ADM cells.

Blocking PI3K/Akt pathway modulates the chemosensitivity of K562/ADM cells

To further determine the causal relationship between chemosensitivity and TAIII-inactivated PI3K/Akt signaling pathway on cell MDR, specific inhibitor of PI3K/Akt wortmannin was selected to treat K562/ADM cells which overexpressed P-gp and MRP1. The protein levels of total-Akt, Akt Ser473, P-gp and MRP1 were measured. As shown in Fig. 6, K562/ADM cells with the inhibitor wortmannin treatment showed significantly decreasing protein levels of Akt Ser473, P-gp and MRP1, whereas total Akt was not affected by inhibition (P<0.05).

Figure 6 Effect of PI3K/Akt signaling pathway inhibitor on drug resistance related protein expression in K562/ADM cells. Cells were pretreated with or without PI3K/Akt inhibitor wortmannin (1 μM) for 24 h, and then the relative expression level of p-Akt, P-gp and MRP1 was measured by western blotting. The downregulated expression of p-Akt, P-gp and MRP1 was calculated after normalization to GADPH expression. Bars means ± SD. n=3. P>0.05, compared to the control group; *P<0.05, **P<0.01, ***P<0.001 compared to the control group.

The inhibition of PI3K/Akt pathway plays a crucial role in the multidrug-resistance reversal of TAIII

In order to confirm the role of PI3K/Akt signaling pathway in TAIII-mediated reversal effect of multidrug resistance, we analyzed the proteins level of total-Akt, Akt Ser473, P-gp and MRP1 in K562/ADM cells by western blotting. The cells were treated with wortmannin (1 μM) alone and combined-treatment with TAIII (1 and 2 μM) for 24 h. The results of western blotting are shown in Fig. 7, compared with wortmannin (1 μM) alone group, wortmannin (1 μM) combined with TAIII (1 μM) group and wortmannin (1 μM) combined with TAIII (2 μM) group did not attenuate the expression of p-Akt, P-gp and MRP1. Since TAIII did not strengthen the inhibitor function, we may conclude that PI3K/Akt signaling pathway was mainly responsible for the drug resistance reversal effect of TAIII.

Figure 7 The effect of wortmannin (1 μM) combined-treatment with TAIII (1 and 2 μM) on expression of drug resistance related proteins in K562/ADM cells. Cells were treated with wortmannin (1 μM) alone and combined-treatment with TAIII (1 and 2 μM) for 24 h and then the relative expression level of p-Akt, P-gp and MRP1 were measured by western blotting. The effects of wortmannin in inhibiting of the level of p-Akt, P-gp and MRP1 did not change significantly after treatment with TAIII in K562/ADM cells. Bars means ± SD. n=3. P>0.05, compared to the control group.

Discussion

CML is one of the most genetically homogeneous malignancies characterized by clonal myeloid cells with an abnormal fusion protein, BCR-ABL, which has tyrosine kinase activity. Since biological sample collection is a non-invasive process, CML is one of the extensively studied diseases for gene expression profiling, which needs elucidation of the BCR-ABL downstream mechanisms involved in CML progression and the pathways involved in therapy resistance. Although the BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) has shown significant progress toward treatment against CML, the drug did not successfully cure patients of the disease (9,29). MDR has become a main obstacle for chemotherapy of CML. Mechanism of MDR is associated with altered expression of ATP-binding cassette (ABC) family of transporters on cell membrane, the most common cause of multidrug resistance (MDR) (1). While as many as 18 ABC transporters have been observed to export chemotherapy drugs using in vitro experimental systems, only 3 transporters have been implicated as major contributors to MDR in cancer: P-glycoprotein (Pgp; ABCB1; MDR1), multidrug resistance-associated protein (MRP1; ABCC1) and breast cancer resistance protein (BCRP; ABCG) (30). Moreover, among the three transporters, the expression of P-gp and MRP1 was extensively measured, studied in CML and its expression levels were correlated with multidrug resistance (31).

The PI3K/Akt signaling pathway has become an important player in the pathogenesis of MDR CML and a promising target for systemic therapy (1,2,4,5,9–11). Many cancer cells have mutations in the PI3K/Akt pathway that leads to hyper-activation of this pathway. In previous studies, it was reported that the PI3K/Akt signaling pathway was always prominently activated by many regulators and was strongly linked to pro-survival in cancer cells (9). Moreover, several lines of evidence implicated that the activating of PI3K/Akt signaling pathway had enhanced drug efflux by ATP-binding cassette (ABC) transporters (2). The maintaining of MDR in tumor cells by PI3K/Akt signaling pathway was most correlated with P-gp and MRP1 (4). Blocking the PI3K/Akt signaling pathway results in the downregulation of the expression of P-gp and MRP1, the sensitivity to chemotherapy drugs was regained (4).

TAIII, one of the main active constituents of Chinese medicinal herb Anemarrhena asphodeloides Bunge, was reported to possess a variety of pharmacological functions. There is accumulating evidence that TAIII is a pronounced activator of autophagy (21,32,33). Preclinical study reported that TAIII was efficacious in inducing autophagy in various cancer cells correlating with a potent inhibitory effect on the activity of mammalian target of rapamycin (mTOR) (31–33). Moreover, it has been reported that the PI3K/Akt/mTOR signaling pathway played a suppressive role in autophagy (10). Furthermore, TAIII can also induce tumor cells apoptosis by inhibition of mTORC1 (34). Nuclear factor-kappa B (NF-κB) is a transcription factor universally present in eukaryocytes, and it can enter into the nucleus and bind to some specific κB sequences thus inducing or upregulating expression of certain genes (27). Previous studies demonstrated that NF-κB could bind to the specific κB sequences on the first exon of the MDR1 promoter region and then upregulated the expression of P-gp to induce drug resistance (27,35). Studies have shown that TAIII has strong anti-inflammatory properties by inhibiting the expression of inflammatory cytokines via inhibition of NF-κB activation (16,17). However, NF-κB was also reported as a downstream signal molecule of PI3K/Akt signaling pathway (36,37). Based on these studies, it is possible to conclude that TAIII may have an inhibition effect on PI3K/Akt signaling pathway.

In this study, we investigated the effects of MDR reversal in a CML multidrug-resistant cell line K562/ADM using TAIII. Firstly, we determined the non-toxic concentration of TAIII to reverse drug-resistance. We found that TAIII of 1 and 2 μM showed very weak cytotoxcity (cell growth inhibition <10%). Then, our data demonstrated that TAIII and ADM combined treatment sensitized K562/ADM cells and induced apoptosis in a dose-dependent manner, decreasing P-gp and MRP1 gene expression. Additionally, the combined treatment reduced the expression of phosphorylated Akt (p-Akt) without affecting the expression of total-Akt, that means the activity of PI3K/Akt signaling pathway was downregulated. Moreover, we found that the levels of p-Akt, P-gp and MRP1 in K562/ADM cells decreased after exposure to the specific inhibitor of PI3K/Akt wortmannin. However, TAIII treatment combined with wortmannin did not exhibit strengthen effect on downregulation of P-gp and MRP1 expression. Therefore, it is reasonable to believe that TAIII plays a suppressive role in the expression of P-gp and MRP1 via inhibition of the PI3K/Akt signaling pathway.

The above studies show that TAIII could increase the intracellular accumulation of ADM in K562/ADM cells at non-toxic concentrations by downregulating P-gp and MRP1 expressions, function and transcription via a mechanism involving the inhibition of the PI3K/Akt signaling pathway. The studies provide evidence in support of further investigation into the clinical application of TAIII as new, potent, and clinically relevant MDR reversal agent in cancer chemotherapy.

Acknowledgements

This study was supported by the Shandong Science and Technology Committee (no. 2010GSF10264), the Foundation of Shandong Educational Committee (nos. J10LC60 and J11LC01), Natural Science Foundation of Shandong Province (no. ZR2014HL032) and Projects of Medical and Health Technology Development Program in Shandong province (no. 2014WS0183).

References