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Guggulsterone of Commiphora mukul resin reverses drug resistance in imatinib-resistant leukemic cells by inhibiting cyclooxygenase-2 and P-glycoprotein.


The purpose of this study was to investigate the effects of guggulsterone on cyclooxygenase-2 and P-glycoprotein mediated drug resistance in imatinib-resistant K562 cells (K562/IMA). MTT cytotoxicity assay, flow cytometry, western blot analysis, and ELISA were performed to investigate the antiproliferative effect, the reversal action of drug resistance, and the inhibitory effect on cyclooxygenase-2, P-glycoprotein, BCR/ABL kinase, and PGE2 release in K562/IMA cells by guggulsterone. The results showed that co-administration of guggulsterone resulted in a significant increase in chemo-sensitivity of K562/IMA cells to imatinib, compared with imatinib treatment alone. Rhodamine123 accumulation in K562/IMA cells was significantly enhanced after incubation with guggulsterone (60,120 [micro]M), compared with untreated K562/IMA cells (p < 0.05). When imatinib (1 [micro]M) was combined with guggulsterone (60, 120 [micro]M), the mean apoptotic population of K562/IMA cells was 15.47% and 24.91%. It was increased by 3.82 and 6.79 times, compared with imatinib (1 [micro]M) treatment alone. Furthermore, guggulsterone had significantly inhibitory effects on the levels of cyclooxygenase-2, P-glycoprotein and prostaglandin [E.sub.2]. However, guggulsterone had little inhibitory effect on the activity of BCR/ABL kinase. The present study indicates guggulsterone induces apoptosis by inhibiting cyclooxygenase-2 and down-regulating P-glycoprotein expression in K562/IMA cells.



Commiphora mukul

Imatinib resistant

K562/IMA cells




The presence of multidrug resistance (MDR) is the major factor responsible for chemotherapy failure in cancer patients who are undergoing chemotherapy. The MDR phenotype is characterized by the over-expression of P-glycoprotein in plasma membrane that works as a pump to extrude anticancer drugs from cells. However, in addition to the over-expression of P-glycoprotein, the MDR phenotype is associated with other modifications of cell biology that make cancer cells more resistant to many other mechanisms of cell damage (Breier et al. 2005). Cyclooxygenase-2 (COX-2), an inducible isoform of enzyme, responsible for generation of prostaglandins from arachidonic acid, is constitutively expressed in a number of cancer cells. Recently, a close association between MDR1 and COX-2 has been reported in human cancer cells in several studies (Bai et al. 2010; Kalle et al. 2010). These studies revealed that COX-2 and MDR-1 over-expression were responsible for the development of resistance to chemotherapy in cancer cells and COX-2 inhibitor induced apoptosis of these cells by down-regulating the expression of COX-2 and MDR-1. It suggests that COX-2 modulates MDR1 expression, and is involved in the development of the MDR phenotype.

Guggulsterone [4,17(20)-pregnadiene-3,16-dione] (Fig. 1), the active component of gugulipid, is derived from the gum resin of the tree Commiphora mukul, and has cis- and trans- isomers. This gum resin has been used for centuries in Ayurvedic medicine to treat obesity, arthritis, and hyperlipidemia (Sinai and Gonzalez 2002). Experimental studies showed that guggulsterone had inhibitory effects on COX-2 expression in cancer cells (Macha et al. 2011; Sarfaraz et al. 2008). Furthermore, our previous research showed that guggulsterone could inhibit P-glycoprotein-mediated MDR in P-glycoprotein over-expressed human cancer cell lines (Xu et al. 2009,2011,2012). The mechanism underlying the antitumor activity of guggulsterone is thought to involve inhibition of COX-2 enzyme activity, but it is unclear whether COX-2 inhibition is required to reverse MDR.

In the present study, imatinib-resistant K562 cells (K562/IMA) were developed by continuous exposure of cells to imatinib. In an effort to elucidate the possible mechanism of resistance, we examined the expression of P-glycoprotein and COX-2 and the results demonstrated that P-glycoprotein and COX-2 were over-expressed in K562/IMA cells compared to K562 cells. We analyzed the reversal effect of guggulsterone on K562/IMA cells, and the possible involvement of COX-2 in the development of resistance to imatinib. These results revealed synergistic effect of guggulsterone and imatinib in inducing apoptosis in K562/IMA cells, by a mechanism involving the inhibition of COX-2 and down-regulation of P-glycoprotein expression.

Materials and methods

Chemicals and drugs

Z-guggulsterone was purchased from Steraloids (Newport, R.I., USA) and its structure was identified as reported previously (Agrawal et al. 2004) (Fig. 1). The compound was dissolved in dimethylsulphoxide (DMSO) as a stock solution of 100 mM and added to the extracellular solutions to obtain a desired concentration. The final concentration of DMSO was <0.1%, which did not affect the test. Imatinib, Rhodamine 123 (Rh123), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other reagent grade chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). FITC-Annexin V and propidium iodide (PI) were obtained from BD Bioscienses (Franklin Lakes, NJ, USA). Anti-COX-2 polyclonal antibody, anti-P-glycoprotein monoclonal antibody, and anti-Tyr were from Abeam (Cambridge, UK). Cell culture media and supplements were products of Gibco-BRL (Rockville, MD, USA).

Cell lines and culture conditions

K562/IMA cells were developed by serial prolonged exposures of K562 cells to increasing concentrations of imatinib starting from 1 nM to 1 [micro]M. Cells were grown in RPM1-1640 supplemented with 10% heat inactivated fetal bovine serum, 100IU/ml penicillin, 100mg/ml streptomycin and 2mM L-glutamine. K562/IMA cells were grown in medium containing 1 [micro]M imatinib. Cultures were maintained in a humidified atmosphere with 5% C[O.sub.2] at 37[degrees]C.

Cytotoxicity and multidrug resistance reversal assay

To determine the reversal effect of guggulsterone on resistant tumor cells, the cytotoxicity of guggulsterone toward K562/IMA cells was first measured by MTT method. Briefly, K562/IMA cells (5 x [10.sup.4] per well) were seeded in 96-well plates. After 24 h incubation, the cells were treated with various concentrations of guggulsterone for 24 h. Cell viability was assessed by MTT assay as reported (Flansen et al. 1989).

The reversal effect of guggulsterone was further investigated with the same method. Cells seeded into 96-well plates were treated with varying concentrations of imatinib in the absence or presence of guggulsterone for 24 h, respectively. [IC.sub.50] values for imatinib (concentration resulting in 50% inhibition of cell growth) were calculated from plotted results using untreated cells as 100%. Control medium included equivalent amount of DMSO (as solvent control), but the applied dose did not exhibit modulation effects on the cell growth or drug sensitivity in these studies.

Measurement of Rh123 accumulation

Rh123 is a known substrate for MDR1, and can be used as a function assay for P-glycoprotein. Cells were seeded into 6-well plates at a density of 1 x [10.sup.6] per well, and pretreated with guggulsterone (30, 60 and 120 [micro]M) for 24 h. After the pretreatment, cells were incubated with Rh123 (5 [micro]M) in the dark at 37[degrees]C in 5% C[O.sub.2] for 2 h. After Rh123 accumulation, the intracellular mean fluorescence intensity (MFI) associated with Rh123 was measured with FACScan flow cytometry (Beckton Dickinson). Excitation was performed by an argon ion laser operating at 488 nm and the emitted fluorescence was collected through a 530 nm pass filter. Data analysis was performed using Cell Quest software.

Apoptosis assay

Double staining for Annexin V-FITC/PI was performed according to Vermes method (Gong et al. 1994). Cells (5 x 105 per well) were seeded into 6-well plates and then treated with 1 [micro]M imatinib in the absence or presence of guggulsterone (30, 60 and 120 [micro]M). After 24 h, the cells were harvested and washed twice with icecold PBS (0.01 M, pH 7.2). After 5 min of centrifuging at 200 g, Annexin V/FITC and PI double-staining were performed according to manufacturer's instruction. Cell apoptosis was analyzed on a FACScan flow cytometry. Annexin V-positive, PI-negative cells were scored as apoptotic. Double-stained cells were considered either as necrotic or as late apoptotic.

Western blot analysis

K562/IMA cells treated with guggulsterone for 24 h were lysed in a lysis buffer containing 20 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40,0.5% deoxycholic acid, 1 mM [beta]-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 10 [micro]g/ml leupeptin, and 20 [micro]g/ml aprotinin. After 30 min of shaking at 37[micro]C, the mixtures were centrifuged (10,000 x g) for 10 min, and the supernatants were collected as the whole cell extracts. The protein content was determined according to the Bradford method (Bradford 1976). An equal amount of total cell lysate (100 [micro]g) was resolved on 8-12% SDS-PAGE gels along with protein molecular weight standards, and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat dry milk and then incubated with the relevant antibodies in antibody-diluted buffer (1 x Trisbuffered saline and 0.05% Tween 20 with 5% milk) with gentle shaking at 4[micro]C for 8-12 h and then incubated with peroxidase conjugated secondary antibodies. Equal protein loading was detected by probing the membrane with [beta]-action antibodies.

[PGE.sub.2] estimation

The [PGE.sub.2] release from K562/IMA cells treated with imatinib (1 [micro]M) in the absence or presence of guggulsterone (30, 60 and 120 [micro]M) was estimated as per manufacturer's instructions (Cayman, USA).

Statistical analyses

Data were expressed in the form of mean [+ or -] S.D., and the significance of the difference between groups was determined by ANOVA followed by the Bonferroni post hoc test. p < 0.05 was considered significant.


Drug resistance modulation

We used MTT assay to determine the cytotoxicity of a combination of imatinib with different concentrations of guggulsterone. As shown in Table 1, guggulsterone could significantly increase imatinib toxicity in K562/IMA cells in a concentration-dependent manner. The [IC.sub.50] of imatinib for K562/IMA cells was decreased from 14.58 [micro]M to 5.37 [micro]M in the presence of 120 [micro]M guggulsterone. However, no such activity was found in K562 cells. These finding indicated that guggulsterone enhanced the potency of imatinib against K562/IMA cells, whereas, had little effects on K562 cells, supporting the notion that guggulsterone might reverse drug resistance of K562/IMA cells.

To examine whether the enhanced cytotoxicity with guggulsterone was due to reversal effect and not due to cytotoxicity of itself, the effect of guggulsterone on the proliferation of sensitive and resistant K562 cell lines was determined. After cells were treated with guggulsterone for 24 h, the cellular proliferation was hardly influenced (data not shown). These data indicated that guggulsterone was nontoxic at the concentration needed to reverse drug resistance in vitro.

Effect on imatinib-induced apoptosis

We next examined the mechanism involved in guggulsterone-induced cytotoxicity in K562/IMA cells (Fig. 2). Apoptosis was quantified by using Annexin V-FITC staining assay. Treatment of K562/IMA cells with 120 [micro]M guggulsterone resulted in 2.70% cells undergoing apoptosis (Fig. 2b), meanwhile with imatinib at 1 [micro]M alone showed 4.05% of K562/IMA cells undergoing apoptosis (Fig. 2c). However, when cells were treated with both imatinib (1 [micro]M)and guggulsterone (30,60 and 120 [micro]M), there was a significant increase in the percent apoptosis of K562/IMA cells (Fig. 2d-f). The finding suggested that imatinib resistance of K562/IMA cells might be reversed to a considerable extent if guggulsterone treatment was involved.

Rh123 accumulation

Rh123 acts as a good substrate for MDR1, and agents that block P-glycoprotein have been found to increase the retention of Rh123 in MDR cells (Yoshimura et al. 1990). Therefore, we further investigated the effect of guggulsterone on the accumulation of Rh123 in K562/IMA cells. As shown in Fig. 3, K562/IMA cells in the absence of guggulsterone exhibited a significant decrease of Rh123 compared to K562 cells, while a notable increase was seen in K562/IMA cells in the presence of guggulsterone. The short time (2 h) treatment with guggulsterone induced the increase of Rh123 in K562/IMA cells, suggesting that guggulsterone had the ability to inhibit the drug-transport activity of P-glycoprotein.

Effects of guggulsterone on COX-2 and P-glycoprotein expression in K562/IMA cells

Recently, a causal link between COX-2 and MDR1 gene expression, implicated in cancer chemo-resistance, has been demonstrated. Thus, the expression of COX-2 and the down stream enzyme involved in PGE2 biosynthesis was correlated with Pglycoprotein, the product of MDR1. Our previous studies revealed that guggulsterone-induced apoptosis in MDR cells was through the down-regulation of P-glycoprotein (Xu et al. 2009, 2011). In the light of this, the expression of COX-2 and P-glycoprotein in the absence or presence of guggulsterone was monitored by western blot analysis. K562/IMA cells showed over-expression of both COX-2 and P-glycoprotein (Fig. 4), suggesting a possible role for COX-2 and P-glycoprotein in the development of resistance in K562 cells against imatinib. Meanwhile, the results indicated the down-regulation in the protein levels of COX-2 and P-glycoprotein by guggulsterone in K562/IMA cells (Fig. 5).

Effect of guggulsterone on PGE2 release in K562/IMA cells

To examine more closely the involvement of COX-2, the PGE2 (a product formed from arachidonic acid by the action of COX-2) release from K562/IMA cells was determined by ELISA method. The results clearly showed a significant increase in the PGE2 levels in K562/IMA cells compared to K562 cells and a significant decline in the levels of [PGE.sub.2] in cells treated with guggulsterone (Fig. 6).

Effect of guggulsterone on BCR/ABL kinase in K562/IMA cells

We next examined whether guggulsterone inhibited the kinase activity of BCR/ABL. As shown in Fig. 7, guggulsterone showed no effect on tyrosine phosphorylation of BCR/ABL kinase in K562/IMA cells. Taken together, these results indicate that guggulsterone reversed MDR in K562/IMA cells is through a mechanism not involving direct inhibition of BCR/ABL kinase.


Previous studies have shown that the development of the MDR phenotype is associated with the constitutive expression of COX-2 in carcinoma cell lines. The relationship between COX-2 and P-glycoprotein suggests that inhibiting COX-2 activity may improve results of chemotherapy by increasing the sensitivity of tumor cells to anticancer drugs. Previous studies revealed that guggulsterone down-regulated nuclear factor kappaB (NF-kB) and COX-2 expression in cancer cells. Furthermore, our previous studies revealed that guggulsterone inhibited P-glycoprotein overexpression and function in doxorubicin-resistant human leukemia K562/DOX cells and breast cancer MCF-7/DOX cells (Xu et al. 2009, 2011, 2012). When combined with doxorubicin, guggulsterone significantly promoted the sensitivity of K562/DOX cells and MCF-7/DOX cells toward doxorubicin through increasing intracellular accumulation of doxorubicin, as compared with doxorubicin treatment alone. Further study demonstrated that the inhibitory effect of guggulsterone on P-glycoprotein activity was the major cause of increased stagnation of doxorubicin inside K562/DOX cells and MCF-7/DOX cells, indicating that guggulsterone might effectively reverse multidrug resistance in drug-resistant cancer cells via inhibiting expression and drug-transport function of Pglycoprotein. It is unclear that guggulsterone reversing multidrug resistance is associated with COX-2. Thereby, in the present study, we investigated the effects of guggulsterone on COX-2 and Pglycoprotein expression in a drug-resistant cancer cell lines.

Our present study demonstrated that COX-2 and P-glycoprotein over-expression played a role in the development of resistance to imatinib in K562 cells. Guggulsterone increased imatinib-induced apoptosis of K562/IMA cells by inhibiting COX-2 and P-glycoprotein expression. The results from flow cytometry analysis of K562/IMA cells treated with imatinib and guggulsterone in combination correlated with the synergistic induction of apoptosis. Guggulsterone at different concentration also significantly enhanced the cytotoxic effects of imatinib on K562/IMA cells. This synergy is in sharp contrast to earlier report that many antileukemic agents could not synergize with imatinib in inhibiting the growth of imatinib-resistant cells (Hoover et al. 2002; La Rosee et al. 2004). P-glycoprotein couples ATP hydrolysis at two ATP-binding sites to transport of a wide variety of neutral or cationic lipophilic compounds. Rh123 is a special substrate for P-glycoprotein. The uptake of Rh123 is resulted from passive inward diffusion (Lehnert et al. 1996; Shapiro and Ling 1998; Krishna and Mayer 2000), while the efflux is known to be P-glycoprotein-dependent. Rh123 has been used extensively as an indicator of P-glycoprotein activity in drug-resistant cell lines with P-glycoprotein over-expression (Green et al. 2001; Galski et al. 2006). Herein, Rh123 was also used to assess the modulating ability of guggulsterone in drug transport function of P-glycoprotein. The results showed that the fluorescent intensity from Rh123 in K562/IMA cells was remarkably increased after guggulsterone treatment, indicating that guggulsterone might elevate the cytotoxic effects of imatinib on K562/IMA cells through suppressing the drug-transport activity of P-glycoprotein. The accumulation level of Rh123 was hardly varied in K562 cells no matter with or without guggulsterone treatment, supporting the notion that guggulsterone enhanced imatinib-induced cytotoxicity toward K562/IMA was attributed to its inhibition activity to P-glycoprotein.

From a mechanistic perspective, expression of the BCR/ABL oncogene up-regulates multiple downstream signaling pathways (Steelman et al. 2004). Of these pathways, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade plays a pivotal role in Abl oncogene-mediated proliferation, survival, and transformation (Kharas et al. 2004; Neshat et al. 2000; Skorski et al. 1997). Recent evidence indicates that PI3K inhibitors have been shown to synergize with imatinib in inhibiting leukemia cell growth (Klejman et al. 2002). The phosphatidylinositol 3'-kinase/PDK-1/Akt signaling cascade represents a convergence point for a plethora of receptor tyrosine kinase and cytokine-mediated pathways that regulate cell proliferation and offers a framework to account for the ability of many extracellular trophic factors to maintain cell survival (Cantley 2002; Vivanco and Sawyers 2002). However, in the present study, we failed to observe any inhibitory effect of guggulsterone on BCR/ABL kinase activity. These results indicate that guggulsterone-induced apoptosis is not mediated though the inhibition of BCR/ABL kinase directly, perhaps indirectly mediated though the inhibition of its downstream effector pathway.

COX-2 inhibitors have been investigated for cancer chemoprevention and chemotherapy (Taketo 1998; Ziemann et al. 2002). There is also evidence that COX-2 inhibitors can be effective in cells with minimal COX-2 expression and that many inhibitory responses on cell growth induced by these compounds are COX2 independent (Taketo 1998). Moreover, COX-2 over-expression induces the expression of MDR-1, which causes multidrug resistance (Bai et al. 2010; Kalle et al. 2010), suggesting that COX-2 inhibition might reduce the chemoresistance phenotype. Previous data showed that bone marrow COX-2 levels were elevated in chronic-phase chronic myeloid leukemia and were associated with reduced survival (Yamazaki et al. 2002). The data presented here also indicate an over-expression of COX-2 and P-glycoprotein in imatinib-resistant cells, but not in the sensitive cells, and thereby increasing the survival of these cells despite imatinib treatment at high concentrations. Guggulsterone in the present study inhibited the expression of both COX-2 and P-glycoprotein, which may be responsible for the development of resistance, thus sensitizing K562/IMA cells to the cytotoxic effects of imatinib. The fact that COX-2 specific inhibitor such as celecoxib blocks the COX-2-mediated increase in MDR-1 expression and activity supports such a possibility.

Ziemann et al. (2006) demonstrated induction of MDR1 over-expression in primary rat hepatocytes by PGE2 treatment. Arunasree et al. showed co-expression of MDR1 and COX-2 in human leukemia cells and suggested regulation of MDR1 by prostaglandins. In the present study, intend to check for the possible mechanism of regulation of MDR1 mediated by [PGE.sub.2], western blot analysis in the presence of guggulsterone in K562/IMA cells demonstrated down-regulation of [PGE.sub.2] release which making the cells sensitive to imatinib. Results are also in agreement with previous studies on leukemia cells, where [PGE.sub.2] mediated induction of MDR1 expression was down-regulated by COX-2 inhibitor (Arunasree et al. 2008).

Antileukemic effects of guggulsterone have been observed previously in doxorubicin resistant leukemia cells (Xu et al. 2009). For the first time we observed more potent effects of guggulsterone in imatinib-resistant cells than in imatinib-sensitive K562 cells. This enhanced potency of guggulsterone in K562/IMA cells may be mediated by the COX-2-dependent mechanism, because our findings provide evidence that COX-2 and P-glycoprotein overexpression are responsible for the development of resistance to imatinib in K562/IMA cells and guggulsterone induces apoptosis of K562/IMA cells by down-regulating the expression of COX-2 and P-glycoprotein expression. This study suggests the possible use of guggulsterone along with imatinib in overcoming the drug-resistance in imatinib-resistant chronic myelogenous leukemia.


Article history:

Received 29 October 2013

Received in revised form 9 January 2014

Accepted 24 February 2014


This work was partly supported by National Natural Science Foundation of China (No. 81073104).


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Hong-Bin Xu *, Lu-Zhong Xu, Xia-Ping Mao, Jun Fu

Department of Clinical Pharmacy, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China

* Corresponding author. Tel.: +86 2166302761: fax: +86 2166307668.

E-mail addresses:, (H.-B. Xu).


Table 1
Effect of guggulsterone on imatinib cytotoxicity in K562 and K562/IMA

Compound                   K562 cells           K562/IMA cells
                           ([IC.sub.50]         ([IC.sub.50]
                           [micro]M)            [micro]M)

Imatinib                   0.19 [+ or -] 0.16   14.58 [+ or -] 0.16
Imatinib + guggulsterone   0.22 [+ or -] 0.09   11.93 [+ or -] 0.16
  (30 [micro]M)
Imatinib + guggulsterone   0.20 [+ or -] 0.21    8.16 [+ or -] 0.16
  (60 [micro]M)
Imatinib + guggulsterone   0.18 [+ or -] 0.19    5.37 [+ or -] 0.16
  (120 [micro]M)

Effects of guggulsterone on the sensitivity of K562 and K562/1MA
cells toward imatinib were examined by MTT method. The cells were
treated with imatinib in the presence or absence of guggulsterone
(30,60 and 120 [micro]M). IC50 values for imatinib were calculated.
The results are presented as meaniS.D. from four independent
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Author:Xu, Hong-Bin; Xu, Lu-Zhong; Mao, Xia-Ping; Fu, Jun
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jun 15, 2014
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