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Alopecurone B reverses doxorubicin-resistant human osteosarcoma cell line by inhibiting P-glycoprotein and NF-kappa B signaling.

ABSTRACT

Doxorubicin (DOX) was first used in osteosarcoma in the early 1970s as a first-line antineoplastic drug. However, the occurrence of drug resistance in chemotherapeutic treatment has greatly restricted its use. When resistance to DOX treatment occurs, osteosarcoma may become not only resistant to the drug originally administered but also to a wide variety of structurally and mechanistically unrelated drugs. Thus, there is an urgent need to find ways of reversing DOX chemotherapy resistance in osteosarcoma. Plant-derived agents have great potential in preventing the onset of the carcinogenic process and enhancing the efficacy of conventional antitumor drugs. Alopecurone B (ALOB), a flavonoid, is isolated from Traditional Chinese Medicine Sophora alopecuroides L., and is reported to have potent inhibitory effect on multidrug resistance associated protein 1. In this study, a DOX-resistant osteosarcoma cell line (MG-63/DOX) was established by increasing the concentration gradient of DOX in a stepwise manner. MTT assay, flow cytometry analysis, dual-luciferase reporter gene assay, quantitative real-time polymerase chain reaction and Western blot analysis were applied to investigate the reversing effect of ALOB and its underlying mechanisms. The results indicated that ALOB mediated the resistance of MG-63/DOX cells to DOX by inhibiting P-glycoprotein function, transcription and expression. Besides, ALOB also enhanced the sensitivity of MG-63/DOX cells to other conventional chemotherapeutic drugs. Cell viability assay confirmed the reversing activity of ALOB. Furthermore, ALOB increased DOX-induced apoptosis at nontoxic concentration. In addition, ALOB showed inhibitory effect on NF-[kappa]B transcription in a DOX-independent manner. Furthermore, NF-[kappa]B signaling was suppressed by ALOB in an IKK-dependent manner. These studies not only demonstrate that ALOB is a potential agent for reversal of drug resistant cancers, but also testify that ALOB reverses multidrug resistance by inhibiting P-glycoprotein via NF-[kappa]B signaling.

Keywords:

Multidrug resistance

P-glycoprotein

Alopecurone B

Flavonoid

Doxorubicin

Nuclear factor-kappa B

Introduction

Multidrug resistance (MDR) remains a main hurdle of chemotherapy in the treatment of osteosarcoma which is a highly malignant tumor occurring frequently in children and adolescents (Wang et al. 2013b). The major cause of MDR is attributed to efflux pumps that reduce intracellular drug concentration. The efflux pumps are identified as ATP-binding cassette (ABC) transporters characterized with their homologous ATP-binding domains. P-glycoprotein (P-gp also known as MDR1 or ABCB1) is the most important ABC transporter. Numerous drugs in chemotherapy for osteosarcoma are the substrates of P-gp, including doxorubicin (DOX), daunorubicin, paclitaxel, vinblastine, vincristine and etoposide (Gottesman et al. 2002; Pluchino et al. 2012).

DOX was first used in osteosarcoma in the early 1970s. Currently data suggest that DOX acts through topoisomerase II by stabilizing the intermediary "cleavage DNA" product of topoisomerase II and inhibiting reconnection (Gill et al. 2013). When resistance to DOX treatment occurs, osteosarcoma may become not only resistant to the drug originally administered but also to a wide variety of structurally and mechanistically unrelated drugs. Mechanisms involve the MDR development including decreased drug uptake and increased drug efflux by P-gp, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis, mutated targets, etc. (Gillet and Gottesman 2012).

Among these mechanisms, nuclear factor kappa-B (NF-[kappa]B) signaling is thought to be highly related to P-gp expression. NF-[kappa]B is a mediator of inducible gene expression in response to inflammatory cytokines, pathogens and several stress signals, and is known for its crucial roles in the immune system, cell proliferation and transformation, apoptosis and tumor development (Assef et al. 2009). A constitutive NF-[kappa]B activity has been observed in several MDR malignancies. Phosphorylation of inhibitor [kappa]B-[alpha] (I[kappa]B-[alpha]) is required for NF-[kappa]B activation. A significant increase of p-I[kappa]B-[alpha] leads to the activation of NF-[kappa]B. Previous studies demonstrated that the activation of NF-[kappa]B leads to P-gp up-regulation (Wang et al. 2013a). Thus, inhibition of NF-[kappa]B signaling gives rise to down-regulation of P-gp and resensitization of MDR carcinomas to chemotherapeutic agents (Yang et al. 2012).

Although lots of MDR reversing agents have been found to overcome drug resistance in vitro, their results in vivo have been disappointing (Yang et al. 2011). Finding a cure for MDR osteosarcoma is extremely challenging. Flavonoids have been always attracted much attention about their MDR reversal activity. Previous studies reported that flavonoids increased the intracellular substrates accumulation and decreased the P-gp expression in several MDR cancer cells (Ni et al. 2014). A flavonoid was isolated from Traditional Chinese Medicine Sophora alopecuroides L. and its structure was identified in our laboratory as alopecurone B (ALOB) (Munekazu et al. 1995).

In this study, a DOX-resistant human osteosarcoma cell line was established, to evaluate the ability of ALOB to increase intracellular DOX accumulation and rhodamine 123 (Rh123) retention, and DOX-induced apoptosis. Furthermore, the effects of ALOB on the function and expression of P-gp were investigated. Finally, the suppression of ALOB on NF-[kappa]B activation was studied.

Materials and methods

Chemicals and drugs

ALOB was isolated by our laboratory and its structure was identified as reported previously (Munekazu et al. 1995; Ni et al. 2014) (Fig. 1). The compound was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO) as a stock solution of 50 mM and added to the extracellular solutions to obtain a desired concentration. The final concentration of DMSO was less than 0.1%, which did not affect the test. DOX, methotrexate, bleomycin and vincristine were from Santa Cruz Biotechnology (Santa Cruz, CA). Cisplatin, docetaxel, verapamil (Ver), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Rh123 and ammonium pyrrolidinedithiocarbamate (PDTC) were purchased from Sigma-Aldrich (St. Louis, MO). TNF-[alpha] was from Peprotech (Rocky Hill, NJ).

Cell culture and DOX resistance

The human osteosarcoma cell line MG-63 was purchased from Typical Culture Preservation Commission Cell Bank (Shanghai, China), and cultured in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 0.11 g/L sodium pyruvate (Life, Grand Island, NY) and 1.5 g/L NaHC[O.sub.3] at 37[degrees]C in a humidified atmosphere containing 5% C[O.sub.2].

[FIGURE 1 OMITTED]

A DOX resistant cell line (MG-63/DOX) was established from its parental cell line MG-63 by gradually increasing the concentration of DOX to which the cells were exposed in a stepwise manner over a period of 8 months. The concentration of DOX added to MG-63 cells was from 5 nM to 100 nM, after which the cells were maintained in culture medium containing 100 nM DOX and displayed 42.60-fold resistance to DOX compared with the corresponding parental sensitive cells. The cells were incubated in drug-free medium for at least 1 week before use.

Cytotoxicity assay

Chemosensitivity to conventional chemotherapeutic drugs was determined using MTT colorimetric assay as described previously (Gray et al. 2013; Wei et al. 2011 ; Zhao et al. 2012). Cells were treated with a full range of concentrations of conventional chemotherapeutic drugs and/or 10 [micro]M ALOB for 48 h. The absorbance in each well was read at 570 nm with background subtraction at 630 nm using a microplate reader (Molecular Devices). The resistance index (RI) was calculated using the following formula (Wei et al. 2011): resistance index (RI) = [IC.sub.50] (MG-63/DOX cells)/[IC.sub.50] (MG-63 cells). The reversal fold change, in terms of potency of reversal, was calculated using the following formula (Zhao et al. 2012): reversal fold change (RF) = [IC.sub.50] (MG-63/DOX cells)/[IC.sub.50] (MG-63/DOX cells treated with ALOB).

Cell viability assay

Cell viability assay was performed as described previously (Chiu et al. 2010).

Apoptosis assay

For the apoptotic measurements induced by ALOB or DOX, 1 x [10.sup.6] MG-63 or MG-63/DOX cells were suspended in 500 [micro]L binding buffer, and incubated with annexin V-FITC and propidium iodide (PI) (BD Biosciences PharMingen, San Diego, CA) at room temperature for 15 min in the dark and then analyzed on BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) within an hour.

Rh123 flow cytometry assay

MG-63 and MG-63/DOX cells were cultured with medium containing 5 [micro]M Rh123 at 37[degrees]C for 1.5 h, then washed three times with PBS, incubated with or without 10 [micro]M ALOB at 37[degrees]C for another 1.5 h (or with 10 [micro]M Ver as a positive control). After incubation, all cells were washed twice with ice-cold PBS, suspended and kept in the dark until flow cytometric analysis (FACS). Samples were analyzed on flow cytometer to detect the green fluorescence of Rh123. The data were analyzed by FlowJo 7.6.2 software (Tree Star Inc., Ashland, OR).

Intracellular DOX fluorescence assay

Intracellular DOX accumulation was measured by FACS. MG-63 and MG-63/DOX cells were treated with 10 [micro]M DOX for 3 h in the absence or presence of 10 [micro]M ALOB or Ver (positive control). Then cells were washed twice with PBS and suspended in PBS for FACS. Red fluorescence of DOX was observed with a 585 [+ or -] 40 nm band pass filter when the 488 nm argon laser was equipped. Statistical analysis was performed using FlowJo software.

[FIGURE 2 OMITTED]

P-gp flow cytometry assay

The expression of P-gp at the membrane surface was analyzed by flow cytometry. MG-63 and MG-63/DOX cells were incubated with 10 [micro]M ALOB for 0-48 h, and then harvested, washed twice with PBS, and labeled with FITC-conjugated mouse anti-human monoclonal antibody against P-gp or a control isotype according to the manufacturer's instructions (eBioscience, San Diego, CA).

Dual-luciferase reporter assay

Plasmid preparation was performed and as previously described (Wang et al. 2013a) with some modifications. MG-63 and MG-63/DOX cells were transfected using Opti-MEM (Life Technologies, Grand Island, NY) containing 10 [micro]g plasmids and Super Electroporator NEPA21 system (NEPAGENE, Japan). Thirty-six hours after transfection, cells were treated with 10 [micro]M ALOB for 8 h, or for 8 h with 1 [micro]M DOX and/or 10 [micro]M ALOB and for 4 h with 5 nM TNF-[alpha].

Quantitative real-time polymerase chain reaction analysis

The quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described (Kim et al. 2013). Data were normalized to the expression of GAPDH. The sequences of primers used were as follows: MDR1, sense 5'-AGAGTCAAGGAGCATGGCAC3' and antisense 5'-ACAGTCAGAGTTCACTGGCG-3'; and GAPDH, sense 5'-GAAAGCCTGCCGGTGACTAA-3' and antisense 5'-AGGAAAAGCATCACCCGGAG-3'.

Western blot analysis

Protein extracts were prepared from exponentially growing cells as described previously (Campbell et al. 2004). Protein of 20 or 38 [micro]g was used for SDS polyacrylamide gel electrophoresis. After transfer to PVDF membrane, the proteins were reacted with monoclonal anti-P-gp (Abeam Inc., Cambridge, MA) and anti-p-I[kappa]B-[alpha] (Ser32) (Cell Signaling Technology, Danvers, MA) followed by anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Laboratories, West Grove, PA). EasySee Western Blot Kit (TransGen Biotech, Beijing, China) was used to determine the levels of protein expression. Immunoreactive protein bands were detected with a ChemiDOC XRS+ (Bio-Rad Inc., Flercules, CA). Image Lab 4.0 (Bio-Rad, Inc., Hercules, CA) was used to quantify protein expression based on band intensity.

Statistical analysis

All quantitative results were reported as "mean [+ or -] SD" values from at least three experiments performed in a parallel manner. Statistical analysis was performed with the GraphPad Prism 5 software.

Results

The toxicity profiles of DOX stably resistant MG-63 cells

On MTT toxicity assay, the established MG-63 subline (Fig. 2 and Table 1) revealed its drug sensitivity in terms of [IC.sub.50] (inhibition concentration). The DOX-selected MG-63/DOX subline showed 42.6-fold higher resistance to DOX (in bold) and 5.58-8.91-fold higher resistance to other conventional chemotherapeutic drugs when compared with parental MG-63 cells. Which means MG-63/DOX subline was cross-resistant to the conventional chemotherapeutic drugs.

[FIGURE 3 OMITTED]

Reversal of drug resistance by ALOB

The reversing effect of ALOB was tested in MG-63/DOX cells. The [IC.sub.50] values of DOX in MG-63/DOX cells were 40.90 [+ or -] 1.96 and 2.85 [+ or -] 0.92 [micro]M for DOX plus vehicle and DOX plus 10 [micro]M ALOB, respectively. The reversal fold change of ALOB was 14.35. ALOB also enhanced the sensitivity of MG-63/DOX cells to other conventional chemotherapeutic drugs (Table 1).

Ver is a P-gp inhibitor and calcium channel blocker that has been used to characterize P-gp activity (Chiu et al. 2010). Therefore, Ver was used as a positive control. MG-63 and MG-63/DOX cells were pretreated with Ver or ALOB, and then challenged them with 100 nM DOX (maintained concentration of MG-63/DOX subline) to measure cell survival and analyze whether drug resistance can be reversed (Fig. 2A). When parental MG-63 cells were treated with up to 20 [micro]M Ver, the viability of cells was not altered. Interestingly, MG-63 cells treated with ALOB showed moderately increased viability. Addition of DOX to Ver-pretreated MG-63 cells did not enhance the sensitivity of the cells to DOX (cell viability at 20 [micro]M Ver > 70%). When DOX was added to ALOB-pretreated MG-63 cells, the viability was slightly reduced. In MG-63/DOX cells, Ver or ALOB had little effect on MG-63/DOX cells; upon addition of DOX, the Ver-pretreated MG-63/DOX cells re-sensitized to DOX toxicity with Ver concentration dependence. When MG-63/DOX subline was pretreated with ALOB, DOX toxicity was enough to significantly reduce the viability of MG-63/DOX cells. Apparently, Ver is capable of reversing DOX-induced resistance in MG-63/DOX subline, and the efficacy of re-sensitization by Ver was lower than that by ALOB under the same concentration (Fig. 2B).

Apoptosis induced by ALOB or DOX

To determine the cytotoxic effects of ALOB, MG-63 and MG63/DOX cells were treated with ALOB at concentrations ranging from 0 to 50 [micro]M for 48 h. At the concentration of 10 [micro]M, the viability of MG-63 and MG-63/DOX cells was more than 98%, respectively. ALOB at concentrations lower than 30 [micro]M did not significantly inhibit the number of viable cells (inhibition < 10%) as assayed by AV-FITC/P1 analysis. Even at the concentration of 40 [micro]M, the inhibitory effects of ALOB on cell viability were lower than 15% (Fig. 3A and C).

To investigate whether ALOB enhances DOX-induced apoptosis, MG-63 and MG-63/DOX cells were treated with DOX alone or DOX combined with Ver or ALOB. FACS indicated that a notable increase in early and late phase apoptosis was observed when MG-63 cells were treated with DOX alone (early and late apoptosis percentage, 33.42 [+ or -] 2.23%; normal cell percentage, 35.81 [+ or -] 4.76%) compared with untreated MG-63 cells (early and late apoptosis percentage, 0.83% [+ or -] 0.31%; normal cell percentage, 98.86 [+ or -] 1.62%). MG-63 cells were treated with DOX combined with Ver or ALOB showed moderate increase in early and late phase apoptosis (early and late apoptosis percentage: DOX combined with Ver, 34.92 [+ or -] 1.14%; DOX combined with ALOB, 37.75 [+ or -] 3.36%; normal cell percentage: DOX combined with Ver, 32.59 [+ or -] 3.91%; DOX combined with ALOB, 33.75 [+ or -] 2.45%) (Fig. 3B and D).

In contrast, a significant increase in early and late phase apoptosis was observed when MG-63/DOX ceils were treated with DOX combined with Ver or ALOB compared with DOX alone treatment (early and late apoptosis percentage: DOX, 16.95 [+ or -] 0.83%; DOX combined with Ver, 25.89 [+ or -] 2.50%; DOX combined with ALOB, 30.82 [+ or -] 1.76%; normal cell percentage: DOX, 71.93 [+ or -]3.53%; DOX combined with Ver, 52.61 [+ or -] 2.75%; DOX combined with ALOB, 43.16 [+ or -] 6.35%) (Fig. 3B and D).

Combined, these results indicate that under normal growth conditions, ALOB has little effect upon cell apoptosis in MG-63 and MG-63/DOX cells; cytotoxicity and cell apoptosis are induced only by DOX. Furthermore, ALOB shows more potent reversing effect than Ver.

[FIGURE 4 OMITTED]

Characterization of P-gp activity by DOX accumulation and Rh1233 efflux assay

In order to identify manner in which ALOB enhanced DOX-induced cell death, DOX and Rh123 were used as special substrates for P-gp to test P-gp activity (Yang et al. 2011) in MG-63 and MG-63/DOX cells. Low retention of fluorescent dye inside of the cells indicates high activity of the energy-dependent P-gp pump (Chiu et al. 2010). When MG-63 and MG-63/DOX cells were incubated with DOX (Fig. 4A and B) or Rh123 (Fig. 4C and D), the fluorescence level (PBS treatment, as a negative control) was lower in MG-63/DOX cells than that in MG-63 cells, which indicated high P-gp activity of MG-63/DOX subline. When MG-63 cells were treated with Ver or ALOB, the fluorescence level of DOX (Fig. 4A) or Rh123 (Fig. 4C) increased moderately compared with PBS treatment. The DOX (Fig. 4B) or Rh123 (Fig. 4D) fluorescence intensity increased significantly in MG-63/DOX cells after Ver or ALOB treatment compared with PBS treatment. Apparently, after treatment with ALOB, the fluorescence level of DOX or Rh123 was increased in MG-63/DOX cells. Therefore, it is logical to assume that pumping activity of P-gp transporter is inhibited effectively by ALOB.

ALOB sensitizes MG-63/DOX subline via inhibition of P-gp

To investigate high activity of P-gp associated with drug resistance, P-gp expression was analyzed. Firstly, the inhibitory effect of ALOB on MDR1 transcription in MG-63/DOX cells was determined. After incubation, ALOB suppressed MDR1 luciferase activity significantly (Fig. 5A). Next, after treatment with ALOB, MDR1 mRNA levels of MG-63 and MG-63/DOX cells at each time point were detected. Significantly, MG-63/DOX cells expressed very high levels of MDR1 mRNA. After treatment, ALOB showed slight effect on MDRI mRNA level in MG-63 cells; however, treatment of MG-63/DOX cells with ALOB indicated that the expression of MDR 1 mRNA was notably inhibited in a time-dependent manner (Fig. 5B). Thirdly, the P-gp levels at membrane surface detected by flow cytometry demonstrated similar results (Fig. 5C). At last, western blot experiment confirmed the inhibitory effect of ALOB on P-gp expression in MG-63/DOX cells treated under the same conditions (Fig. 5D). Combined, these data demonstrate that P-gp expression decreased by ALOB in MG-63/DOX cells correlates with low P-gp activity status.

[FIGURE 5 OMITTED]

Inhibitory effect of ALOB on NF-[kappa]B signaling

NF-[kappa]B activation regulates MDR1 gene expression (Yang et al. 2012), and DOX has the ability to inhibit NF-[kappa]B-dependent gene expression (Campbell et al. 2004). Thus, the effects of DOX with or without ALOB on NF-[kappa]B transcription were determined in MG-63 and MG63/DOX cells. Not surprisingly, dual-luciferase reporter experiments indicated that DOX significantly inhibited NF-[kappa]B transcriptional activity in MG-63 cells. ALOB showed a weaker inhibitory effect, but the combination of DOX and ALOB produced the most effective inhibition (Fig. 6A). In MG-63/DOX cells, the inhibitory effect of DOX on the NF-[kappa]B reporter plasmid was weaker than that observed in MG-63 cells; but ALOB significantly reduced NF-[kappa]B transcriptional activity. When combined with ALOB, DOX recovered its inhibitory effect (Fig. 6B).

To further investigate the inhibition of NF-[kappa]B transcriptional activity by DOX and ALOB, TNF-[alpha] was used to activate NF-[kappa]B. In MG-63 cells, treatment with TNF-[alpha] strongly enhanced the activity of transfected NF-[kappa]B luciferase reporter plasmid. DOX and ALOB significantly decreased subsequent NF-[kappa]B transcriptional activity induced by TNF-[alpha] (Fig. 6C). Similarly, in MG-63/DOX cells, ALOB enhanced the inhibitory effect of DOX (Fig. 6D).

It is interesting that DOX-induced MG-63 resistant subline shows high level of P-gp (Fig. 5B-D) whose expression is regulated by NF-[kappa]B. To verify the effect of DOX on MDRI expression, MG-63 cells were treated with DOX to detect MDR1 mRNA level at each time point. In response to DOX treatment, MDRI expression in MG-63 cells was inhibited at 12 h. After that time, DOX increased MDRI expression in a time-dependent manner (Fig. 6E).

To verify the inhibitory effect of ALOB on P-gp is regulated by NF-[kappa]B signaling, TNF-[alpha] was used to activate NF-[kappa]B signaling in MG-63 cells and PDTC was used to inhibit NF-[kappa]B signaling in MG-63/DOX cells (Yang et al. 2012). After treatment, TNF-[alpha] increased expression of P-gp in MG-63 cells, and ALOB decreased its stimulatory effect. In addition, PDTC significantly suppressed expression of P-gp in MG-63/DOX cells, and combination with ALOB demonstrated additive inhibition (Fig. 6F).

To further determine the effect of ALOB on NF-[kappa]B signaling, MG-63 and MG-63/DOX cells were treated with ALOB, and p-I[kappa]B-[alpha] protein level was detected. Western blot analysis indicated that I[kappa]B-[alpha] phosphorylation was significantly higher in MG-63/DOX cells than that in parental MG-63 cells. The phosphorylation of I[kappa]B-[alpha] was subsequently decreased following treatment with ALOB at 6 and 12 h (Fig. 6G). Furthermore, TNF-[alpha] significantly increased I[kappa]B-[alpha] phosphorylation in MG-63 cells, and this stimulatory effect was reduced by ALOB. Moreover, phosphorylation of I[kappa]B-[alpha] was decreased by PDTC, and treatment with PDTC combined with ALOB indicated extra suppression of p-I[kappa]B-[alpha] level in MG-63/DOX cells (Fig. 6H).

[FIGURE 6 OMITTED]

Discussion

P-gp has been most extensively examined. Overexpression of P-gp has been shown to correlate with overall poor chemotherapy response and prognosis (Leonard et al. 2003). Thus, we established a resistant MG-63 subline by using DOX, and found that the resistant subline showed higher P-gp level than that of parental cells (Fig. 5). In addition, we demonstrated that MG-63/DOX subline was also cross-resistant to other conventional chemotherapeutic drugs (Table 1). Apparently, the resistance is due, at least in part, to high activity of P-gp pump.

Numerous strategies to overcome P-gp-mediated MDR have been explored, including the design of novel drugs that evade recognition and efflux, inhibitors to block efflux and restore drug accumulation (Nobili et al., 2012). P-gp inhibitors have been used with limited clinical success, as the co-administration of a cytotoxic drug with an inhibitor often produces unpredictable or undesirable pharmacokinetics (Pluchino et al. 2012). In our manuscript, ALOB was tested for its reversing effect, cytotoxicity and inhibitory effect on MDR. We found that ALOB was nontoxic to MG-63 and MG-63/DOX cells (Fig. 3A), but showed significantly reversing effect on the resistance of MG63/DOX cells (Table 1 and Fig. 2). Inhibitory effect of ALOB on P-gp is the main reason for reversal (Figs. 4D and 5).

DOX is a topoisomerase II inhibitor, which accumulates in nucleus to inhibit reconnection of DNA (Gill et al. 2013). Due to the high level of P-gp, DOX was expelled and insufficient concentration was retained in MG-63/DOX cells (Fig. 4A and B). ALOB suppressed P-gp (Figs. 4 and 5) and increased apoptosis induced by DOX (Fig. 3B). We found that NF-[kappa]B signaling was activated in an IKK-dependent manner in MG-63/DOX cells (Fig. 6G). Following DOX stimulation, the transcriptional activity of NF-[kappa]B was suppressed in MG-63 cells (Fig. 6A and C), but DOX showed weak effect on MG-63/DOX cells (Fig. 6B and D). In Fig. 6, stimulation of cells with TNF-[alpha], which did provide the stimulus necessary to induce NF-[kappa]B transactivation (Vermeulen et al. 2002), and treatment with DOX and/or ALOB resulted in the inhibition of NF-[kappa]B-dependent, TNF-[alpha]-induced NF-[kappa]B transcriptional activity. We deduced that DOX and ALOB induced signals that convert NF-[kappa]B from being an activator of gene expression to being a repressor, possibly stimulating the recruitment of corepressor complexes (Campbell et al. 2004).

In Figs. 5 and 6E, alien effect of ALOB on P-gp expression compared with DOX revealed that as an inhibitor of P-gp, ALOB suppressed not only the transcriptional activity of NF-[kappa]B, but also NF-[kappa]B activation. Fig. 6F-H testified the conclusion. Obviously, ALOB showed to inhibit NF-[kappa]B in an IKK-dependent manner with phosphorylation of Ser32 of I[kappa]B-[alpha]. In order to verify the relationship between NF-[kappa]B and P-gp in MG-63 and MG-63/DOX cells, NF-[kappa]B signaling stimulator TNF-[alpha] and specific NF-[kappa]B signaling inhibitor PDTC were used (Yang et al. 2012). Our data indicated that the high level of P-gp induced by TNF-[alpha] was decreased by treating with ALOB in MG-63 cells. In PDTC treatment assay, additional inhibition of P-gp expression by ALOB was observed in MG-63/DOX cells (Fig. 6F). Besides, we confirmed that ALOB inhibited TNF-[alpha]-induced I[kappa]B-[alpha] phosphorylation in MG-63 cells, and showed further inhibitory effect on I[kappa]B-[alpha] phosphorylation in PDTC-treated MG-63/DOX cells (Fig. 6H). Based on the results, we make the consequence that ALOB reverses MDR by inhibiting P-gp via NF-[kappa]B signaling.

Furthermore, our previous study indicated that ALOB has potent inhibitory activity on multidrug resistance associated protein 1 (Ni et al. 2014).

In conclusion, we demonstrate for the first time that ALOB reversed DOX-induced resistant osteosarcoma cells by inhibiting P-gp and NF-[kappa]B signaling. ALOB enhanced DOX-induced apoptosis by promoting nuclear DOX accumulation in MG-63/DOX cells. Bearing its reversing effect on MDR and low toxicity, ALOB is a potential agent for reversal of drug resistant cancers.

ARTICLE INFO

Article history: Received 2 October 2014

Revised 12 December 2014

Accepted 21 December 2014

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Program No. 81202901); the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193).

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Zhao, X.Q., Xie, J.D., Chen, X.G., Sim, H.M., Zhang, X., Liang, Y.J., Singh, S., Talele, T.T., Sun, Y., Ambudkar, S.V., Chen, Z.S., Fu. L.W., 2012. Neratinib reverses ATP-binding cassette B1-mediated chemotherapeutic drug resistance in vitro, in vivo, and ex vivo. Mol. Pharmacol. 82, 47-58.

Yuan-Zheng Xia, Kai Ni, Chao Guo, Chao Zhang, Ya-Di Geng, Zhen-Dong, Wang Lei Yang *, Ling-Yi Kong *

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China

* Corresponding authors. Tel.: +86 25 8327 1405; fax: +86 25 8327 1405.

E-mail addresses: dorothyl9802003@gmail.com (L. Yang), cpu_lykong@126.com, lykong@cpu.edu.cn (L.-Y. Kong).

http://dx.doi.org/10.1016/j.phymed.2014.12.011
Table 1
The resistance indexes of MG-63/DOX cells and the reverse effects of
alopecurone B in MG-63/DOX cells for conventional chemotherapeutic
drugs.

Chemotherapeutic   [IC.sub.50]
drugs              ([micro]M) for
                   chemotherapeutic
                   drugs

                   MG-63

Bleomycin          5.16 [+ or -] 0.73
Cisplatin          1.64 [+ or -] 0.59
Docetaxel          0.62 [+ or -] 0.53
Doxorubicin        0.96 [+ or -] 0.37
Methotrexate       0.52 [+ or -] 0.17
Vincristine        0.75 [+ or -] 0.24

Chemotherapeutic   [IC.sub.50] ([micro]M) for
drugs              chemotherapeutic drugs

                   MG-63/DOX

                   +Vehicle              +Alopecurone B

Bleomycin          28.79 [+ or -] 1.97   12.62 [+ or -] 1.41
Cisplatin           9.86 [+ or -] 1.35    2.17 [+ or -] 0.87
Docetaxel           5.41 [+ or -] 1.96    1.81 [+ or -] 1.05
Doxorubicin        40.90 [+ or -] 1.96    2.85 [+ or -] 0.92
Methotrexate        3.97 [+ or -] 0.82    1.43 [+ or -] 0.76
Vincristine         6.68 [+ or -] 1.95    1.05 [+ or -] 1.29

Chemotherapeutic   Resistance index   Reversal fold
drugs

Bleomycin           5.58               2.28
Cisplatin           6.01               4.54
Docetaxel           8.72               2.99
Doxorubicin        42.60              14.35
Methotrexate        7.63               2.78
Vincristine         8.91               6.36

Cells were treated with a full range of concentrations of
conventional chemotherapeutic drugs with or without 10 [micro]M
alopecurone B. The values are "means [+ or -] S.D." of three independent
experiments.

Resistance index = [IC.sub.50] (MG-63/DOX cells)/[IC.sub.50] (MG-63
cells).

Reversal fold = [IC.sub.50] (MG-63/DOX cells)/[IC.sub.50] (MG-63/DOX
cells treated with alopecurone B).
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Title Annotation:nuclear factor kappa B
Author:Xia, Yuan-Zheng; Ni, Kai; Guo, Chao; Zhang, Chao; Geng, Ya-Di; Wang, Zhen-Dong; Yang, Lei; Kong, Lin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Mar 15, 2015
Words:5262
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