Printer Friendly

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza.

Objective: Multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine.

Methods: The inhibitory effects of tanshinones on P-gp function were compared using digoxin bidirectional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones.

Results: Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity.

Conclusions: Taken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.





Multidrug resistance

Salvia miltiorrhiza


Multidrug resistance (MDR), the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic agents following exposure to a single cytotoxic compound, is an obstacle to successful cancer chemotherapy (Persidis, 1999). One of the important mechanisms conferring multidrug resistance is the overexpression of membrane transporters (Gottesman et al., 2002), a group of membrane-associated proteins that govern the transport of drugs and other xenobiotics into and out of the cells (Giacominietal., 2010).

P-glycoprotein (P-gp), a 1701 kDa protein encoded by human ABCB1 gene, belongs to the ATP-binding cassette (ABC) superfamily of membrane transporters (Bosch and Croop, 1998). P-gp is expressed in a variety of normal tissues such as the apical membrane of blood-brain barrier (BBB), small intestine and kidney proximal tubule epithelial cells, protecting the body from xenobiotics and regulating drug absorption and disposition (Fenner et al., 2009; Giacomini et al., 2010). However, overexpression of P-gp on cancer cell membrane can mediate the efflux of cytotoxic drugs and decrease their intracellular accumulation, leading to multidrug resistance and chemotherapy failure (Szakacs et al., 2006). A variety of chemically unrelated anticancer drugs including paclitaxel, doxorubicin and vinblastine are P-gp substrates (Szakacs et al., 2006). Inhibition of P-gp function represents a logical approach to overcome MDR in cancer chemotherapy.

Since the discovery of the inhibition of P-gp activity by verapamil in 1981, at least three generations of P-gp inhibitors have been identified (Darby et al., 2011). Some representative P-gp inhibitors include quinidine, verapamil, cyclosporine A, dexverapamil, dexniguldipine, tariquidar, zosuquidar and laniquidar (Palmeira et al., 2012). However, so far none of the P-gp inhibitors have been used in clinical practice due to their relatively low affinities, unpredictable drug-drug interactions and severe side effects (Darby et al., 2011). Thus, there has been substantial research effort to investigate the use of natural products, with good safety profile, to inhibit P-gp as a means to reverse MDR (Zhang et al., 2009). Bioactive components isolated from various herbs including ginseng, hawthorn, garlic, grapefruit juice, green tea, and St. John's Wort have been showed to exhibit different inhibitory effects on P-gp function both in vitro and in vivo (Zhou et al., 2004), suggesting a new strategy for the development of novel P-gp inhibitors.

Tanshinones are a series of abietane diterpenes isolated form Salvia miltiorrhiza (Danshen), a traditional Chinese medicine that has been used in Asian countries for the prevention and treatment of cardiovascular diseases (Wu and Wang, 2012). Nowadays, the potential applications of tanshinones in cancer treatment especially when used as an adjuvant therapy have been described (Zhou et al., 2005). However, the role of tanshinones in circumvention of multidrug resistance has not been fully studied yet. The clarification of this potential therapeutic action could further extend the clinical applications of tanshinones in cancer chemotherapy. Therefore, in the present study, the inhibitory effects of five tanshinones, namely tanshinone I, tanshinone 1IA, cryptotanshinone, dihydrotanshinone and miltirone on P-gp function were evaluated in colon cancer cells, in which the MDR usually develops during the course of treatment. Furthermore, the reversal of P-gp mediated MDR by effective tanshinones and the possible underlying mechanisms of action were investigated, with an aim to develop potential P-gp inhibitors and further investigate the potential applications of tanshinones in cancer therapy.

Materials and methods


Tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone were purchased from Chengdu Cogon Bio-tech Co., Ltd. (Sichuan, China); miltirone was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China). The chemical structures of tanshinones were shown in Fig. 1, their purities were greater than 98% as determined by HPLC-UV. Digoxin, verapamil, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma-Aldrich Corporation (St. Louis, MO, USA). Doxorubicin was purchased from LC laboratories (Woburn, MA, USA). The primary monoclonal P-gp antibody was from Calbiochem (Billerica, MA, USA). Anti-mouse IgG horseradish peroxidase conjugate was obtained from Invitrogen (Carlsbad, CA, USA). The P-gp ATPase assay system was purchased from Promega (Madison, Wl, USA). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), Hanks balanced salt solution (HBSS), non-essential amino acids (NEAA), phosphate buffered saline (PBS) and Penicillin-Streptomycin were obtained from Gibco (Carlsbad, CA, USA).

Cell culture

Human colon cancer cell line Caco-2 was obtained from the American Type Culture Collection (Manassas, VA, USA). SW620 and its drug selected P-gp overexpressing SW620 Ad300 subline were kindly provided by Dr. Susan Bates from the National Cancer Institute (N1H, Bethesda, MD, USA). The resistant subline was developed from its parental cancer cell line by stepwise selection in increasing concentrations of doxorubicin. The resistance phenotype was stable for at least three months in drug-free medium (To et al., 2013). The cells were maintained in DMEM (Caco-2) or RPMI 1640 (SW620 and SW620 Ad300) medium, supplemented with 10% FBS, lOOU/ml penicillin and 100p,g/ml streptomycin at 37[degrees]C in a humidified atmosphere with 5% C02.

Cell viability assay

Cytotoxicity of tanshinones on all the cell lines, as well as the growth inhibitory effects of doxorubicin and irinotecan, with or without the concomitant treatment of effective tanshinones, on SW620 and its resistant subline were evaluated by MTT assay. In brief, cells were seeded into 96-well plates at a density of 5000 cells/well and allowed to incubate overnight. After drug treatment as designed, MTT at the final concentration of 0.5mg/ml was added into each well, followed by incubation at 37[degrees]C for another 4h. Finally, purple formazan product was dissolved in 100 pi DMSO, the absorbance was determined at 570 nm using a Benchmark Plus microplate reader (Bio-Rad, Hercules, USA).

Bi-directional transport assay in Caco-2 cells

Caco-2 cells were seeded onto six-well Transwell permeable supports (0.4 [micro]m pore size, 4.67 [cm.sup.2] growth area, Corning, NY, USA) at a density of 2.5 x [10.sup.5] cells/ml and cultured for 21 days to differentiate into functionally polarized monolayers. The transepithelial electrical resistance (TEER) values of the monolayers were measured to evaluate their integrities, only the monolayers of which the TEER values were greater than 200 [ohm] [cm.sup.2] were employed for the transport assay. Before the transport experiments, the Caco-2 cell monolayers were pre-incubated with tanshinones, verapamil (positive control) or 1% DMSO (vehicle control) diluted with HBSS for 1 h. To start the assay, digoxin (50 [micro]M) was added to the donor chamber in each well, then 500 [micro]l samples were removed from the receiver chambers at 15,30,45,60,90, and 120 min, respectively. Withdrawn sample was replaced with 500 p.1 pre-warmed HBSS to maintain the liquid volume in each chamber. After the transport assay, the TEER values were measured again to monitor the integrities of the monolayers.

The concentration of digoxin in the samples was determined by high-performance liquid chromatography (HPLC) analysis as previously described, with minor modifications (Hughes and Crowe, 2010). Briefly, the Hewlett-Packard (HP) 1050 Series pumping system with a multiple wavelength detector was used for the determination. Digoxin and phenacetin (internal standard) were separated using a reversed-phase Agilent ZORBAX XDB-C8 column (150 mm x 4.6 mm, 5 pm), equipped with an Eclipse XDB-C8 guard column. The mobile phase consisted of water and acetonitrile (72:28, v/v) at a flow rate of 1 ml/min. The wavelength of UV detection was 220 nm.

Drug efflux assay by flow cytometry

The inhibition of P-gp mediated doxorubicin (DOX) efflux in SW620 Ad300 cells by effective tanshinones was studied using flow cytometry as previously described (To et al., 2013). Briefly, the cells were trypsinized and incubated with DOX in the presence or absence of effective tanshinones in complete culture medium (Phenol red-free) at 37[degrees]C for 30 min. Subsequently, the cells were washed twice with ice-cold PBS, followed by incubation with effective tanshinones in DOX-free medium at 37[degrees]C for another 1 h. Finally, the cells were washed with cold PBS and placed on ice in the dark until analysis by flow cytometry.

Samples were analyzed using a BDLSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA). DOX fluorescence was detected with a 488 nm argon laser and a 610 nm bandpass filter. For each sample, 10,000 events were collected and cell debris was eliminated by gating on forward versus side scatter. All the data were analyzed using Flow Jo 7.6.1 software (Tree Star, Inc., Ashland, OR).

P-gp protein expression by western blot analysis

Regulation of P-gp protein expression in SW620 Ad300 cells by effective tanshinones was studied by western blot analysis. The cells were seeded into 60 mm dishes at a density of 2.5 x [10.sup.5] cells/ml and allowed to incubate overnight. The cells were then harvested for western blot analysis following exposure to effective tanshinones. Protein concentration was determined using BCA protein assay kit (Pierce biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. Equal amounts of protein were resolved by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, CA, USA). The membranes were incubated at 4[degrees]C overnight with a mouse monoclonal anti-P-gp antibody diluted at 1:500 in 5% BSA in washing buffer. Afterwards, the membranes were incubated with HRP-conjugated anti-mouse secondary antibody at room temperature for 2 h. Chemiluminescent signals were then developed with LumiGLO reagent and Peroxide (Cell Signaling Technology, #7003) and detected by the ChemiDoc XRS gel documentation system (Bio-Rad). GAPDH was used as the loading control.

Immunofluorescence for P-gp by confocal microscopy

Cells grown on coverslips were fixed with 4% (v/v) paraformaldehyde for 15 min at room temperature, and permeabilized with methanol at -20[degrees]C for 10 min. The cells were then covered with 10% (v/v) goat serum for 60 min at room temperature followed by incubation with diluted primary antibody against P-gp at 4[degrees]C overnight. Cells were then probed with Alexa Fluor 568 Conjugate secondary antibody at room temperature for 2h in dark. Subsequently, cells were washed with PBS and incubated with DAPI for another 15 min. Fluorescent signals were detected using a confocal fluorescence microscope (Nikon EZ-C1, Nikon, Tokyo, Japan).

Reverse transcription and quantitative real-time PCR

Quantitative real-time PCR was performed to determine the relative mRNA expression level of ABCB1 in SW620 Ad300 cells following treatment with effective tanshinones at a range of concentrations for 24 h. The total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, and the concentration was measured by a Thermo NanoDrop 2000c Spectrophotometer (Wilmington, DE, USA) at 260 nm. The same amount of total RNA (1 [micro]g) was used to synthesize cDNA by reverse transcription using the PrimeScript RT Master Mix Kit (TaKaRa-Bio, Dalian, China). The human GAPDH mRNA was amplified in parallel as the internal control. The specific primers used were as follows: ABCB1 forward: 5'-GGGAGCTTAACACCCGACTTA3' and reverse: 5'-GCCAAAATCACAAGGGTTAGCTT-3'; GAPDH forward: 5'-AGCCACATCCCTCAGACAC-3' and reverse: 5' GTTCAAACTTCTGCTCCTGA-3'. Real-time PCR was performed at 95[degrees]C for 30 s, followed by 40 cycles of 95[degrees]C for 5 s and 60cC for 30 s. Data analysis was carried out using the [2.sup.-[DELTA][DELTA]Ct] method for relative quantification. All samples were normalized to GAPDH.

P-gp ATPase assay

A Pgp-Glo[TM] Assay System was used to test the inhibitory effects of effective tanshinones on the activity of P-gp ATPase stimulated by verapamil (a known P-gp substrate). Briefly, the test compounds were pre-incubated with recombinant human P-gp membranes in assay buffer at 37[degrees]C for about 5 min. The ATP hydrolysis reaction was then initiated by adding 5 mM MgATP, followed by incubation at 37[degrees]C for 40 min on a plate shaker. After the incubation, the reaction was terminated by adding ATP detection reagent, and then incubated at room temperature for 20 min to allow the luminescent signals to develop. Subsequently, the luminescence was determined using a GloMax 20/20

Luminometer (Promega Corporation, Madison, WI, USA).

Data analysis

The apparent permeability ([]) of digoxin transport across Caco-2 cell monolayer was calculated using the following equation:

[] = (Vr/[C.sub.0]) (1/S) (dC/dt)

where Vr is the volume of medium in the receiver chamber, [C.sub.o] is the initial concentration of digoxin in the donor chamber, S is the surface area of the monolayer, dC/dt is the linear slope of the digoxin concentration in the receiver chamber versus time after modification for dilution (Hubatsch et al., 2007).

The efflux ratio (PE) was defined by the following equation:

[R.sub.E] = [P.sub.BA]/[P.sub.AB]

where [P.sub.BA] and [P.sub.AB] represent the [] of digoxin transport from the basolateral to apical and apical to basolateral side of the monolayer, respectively (Hubatsch et al., 2007).

Statistical analysis of the data was carried out using Prism 5.0 (GraphPad Software, CA, USA). All the data were expressed as Mean [+ or -] standard error of mean (SEM). The significance of difference between groups was estimated by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. All the assays were performed in three independent experiments.


Inhibition of P-gp-mediated digoxin bi-directional transport across Caco-2 cell monolayer by tanshinones

The inhibitory effects of five tanshinones on digoxin (P-gp substrate) bi-directional transport across Caco-2 cell monolayer were evaluated, with an aim to screen for potential MDR-reversing tanshinones. All the compounds showed no cytotoxicity during the transport experiments as determined by MTT assay (data not shown). Due to the efflux function of P-gp, which is located on the apical side of the Caco-2 cell monolayer, the transport of digoxin from basolateral to apical side was much easier than its transport from apical to basolateral side. As shown in Table 1, the efflux ratio of digoxin bi-directional transport across Caco-2 cell monolayer was 12.59, which was consistent with the values (4-14) indicated in the U.S. FDA guideline for drug interactions studies (Huang et al., 2007). At 25[micro]M, both cryptotanshinone and dihydrotanshinone decreased the efflux ratio of digoxin bi-directional transport significantly, indicating their inhibitory effects on P-gp efflux function. Furthermore, the effects of cryptotanshinone and dihydrotanshinone were shown to be concentration-dependent (Table 1). Verapamil, a known P-gp inhibitor, also showed significant inhibitory effect. However, tanshinone 1, tanshinone HA and miltirone showed no inhibitory effect.

Potentiation of doxorubicin and irinotecan cytotoxicities in P-gp overexpressing SW620 Ad300 cells by cryptotanshinone and dihydrotanshinone

Reversal of doxorubicin (DOX) and irinotecan resistance in SW620 Ad300 cells by cryptotanshinone and dihydrotanshinone was evaluated by MTT assay. The cytotoxicity of cryptotanshinone and dihydrotanshinone was first examined in SW620 Ad300 cells. When tested at the concentration used in the combination treatment, these two tanshinones alone did not exhibit any appreciable cytotoxicity (Fig. 2A and B). As indicated in Fig. 2C and D, P-gp overexpressing cells were remarkably resistant to DOX, as compared with its respective SW620 parental cells. However, both cryptotanshinone and dihydrotanshinone could significantly potentiate the cytotoxicity of DOX in the resistant cells. Besides, the cytotoxicity of irinotecan, another P-gp substrate anticancer drug widely used in combination chemotherapy for colon cancer (Bansal et al., 2009), was compared in both SW620 parental and resistant cells. As shown in Fig. 2E and F, P-gp overexpressing cells also showed resistance to irinotecan, which could be reversed by the co-administration of cryptotanshinone and dihydrotanshinone.

Increased intracellular accumulation of DOX in P-gp overexpressing SW620 Ad300 cells by cryptotanshinone and dihydrotanshinone

To investigate whether the reversal of P-gp mediated MDR by cryptotanshinone and dihydrotanshinone was due to the increased intracellular concentration of P-gp substrate, the effects of two effective tanshinones on intracellular accumulation of DOX in SW620 parental and P-gp overexpressing cells were evaluated using flow cytometry analysis. As shown in Fig. 3A, the mean fluorescence of DOX was much lower in P-gp overexpressing SW620 Ad300 cells, as compared with the parental cells, indicating less intracellular accumulation of DOX in the resistant cells due to the efflux function of P-gp. Besides, both cryptotanshinone and dihydrotanshinone showed no effects on the mean fluorescence of DOX in SW620 parental cells. However, as shown in Fig. 3B and C, the mean fluorescence of DOX in P-gp overexpressing SW620 Ad300 cells was significantly increased by cryptotanshinone and dihydrotanshinone in a concentration-dependent manner, thereby indicating their inhibitory effects on P-gp mediated efflux of DOX. Verapamil was used as positive control.

Regulation of P-gp protein and mRNA levels in SW620 AdSOO cells by cryptotanshinone and dihydrotanshinone

Since the reversal of P-gp mediated MDR might be associated with the alteration of protein expression, the P-gp protein level in cells was examined. As shown in Fig. 4A, there was much more P-gp expression in SW620 Ad300 cells, when compared with the SW620 parental cells. P-gp expression was not affected following the treatment with either tanshinone for 6 and 12 h, respectively (Fig. 4A). However, P-gp protein level was significantly down-regulated by cryptotanshinone and dihydrotanshinone at 25 p,M after 24 h (Fig. 4B). Further studies also showed that down-regulation of Pgp expression by two tanshinones was concentration-dependent (Fig. 4C and D). Besides, as shown in Fig. 5, the immunofluorescence staining data for P-gp was also consistent with the total P-gp protein expression obtained from the western blot analysis.

The relative ABCB1 mRNA expression was further analyzed in SW620 Ad300 cells. As shown in Fig. 6, the ABCB1 mRNA level was down-regulated by cryptotanshinone and dihydrotanshinone in a concentration-dependent manner following 24 h of exposure.

Inhibition of verapamil stimulated P-gp ATPase activity by cryptotanshinone and dihydrotanshinone

To further understand the mechanisms of P-gp inhibition by cryptotanshinone and dihydrotanshinone, the P-gp ATPase activity was determined. In the assay, the residual ATP after the hydrolysis reaction was measured as luminescence in relative light units (RLU). Since P-gp depends on ATP hydrolysis to provide energy for its efflux function, a greater amount of unreacted ATP after the assay would indicate lower P-gp ATPase activity. As shown in Fig. 7A, upon exposure to cryptotanshinone at a range of concentrations, the luminescence signal was increased in a concentration-dependent manner, suggesting the inhibition of Pgp ATPase activity by cryptotanshinone. Similarly, the other P-gp inhibiting tanshinone, dihydrotanshinone, was also found to inhibit P-gp ATPase activity, albeit with a weaker inhibitory effect than cryptotanshinone (Fig. 7B).


It has been reported that more than 7 million people died from cancer per year around the world (Jemal et al., 2011). Virtually, many of the deaths are due to chemotherapy failure caused by MDR derived in cancer cells. The underlying mechanisms of MDR mainly include overexpression of membrane transporters, cancer stem cells, altered expression of target proteins, alteration in the apoptotic signaling pathway and physical barriers to drug delivery (Szakacs et al., 2006). The most commonly observed mechanism contributing to MDR is the overexpression of ABC transporters mainly including P-gp, breast cancer resistant protein (BCRP) and multidrug resistance-associated protein 1 (MRP1) in cancer cells (Fenner et al., 2009). In fact, more than 70% of the P-gp inhibitors reported in the last decade were natural products and their synthetic derivatives, suggesting an important and effective strategy for the development of novel P-gp inhibitors (Zhou et al., 2005). Hence, the potential circumvention of MDR by tanshinones, the lipophilic components isolated from Danshen, was evaluated in this study.

The effects of tanshinones on P-gp efflux function were first investigated using digoxin bi-directional transport assay, a method recommended by the U.S. FDA for P-gp inhibition studies (Hubatsch et al., 2007). According to the results, among the five tanshinones tested, only cryptotanshinone and dihydrotanshinone decreased the efflux ratio of digoxin bi-directional transport across Caco-2 cell monolayer, indicating their inhibition on P-gp function. The reversal of P-gp mediated MDR by cryptotanshinone and dihydrotanshinone was further studied using a pair of parental colon cancer cell line SW620 and its drug resistant subline SW620 Ad300. The resistant SW620 Ad300 cells have been quite extensively characterized. Among most important drug transporters, only P-gp was found to be overexpressed in this resistant cell line, thus enabling the study of P-gp-inhibitory effect by the tanshinones. Both cryptotanshinone and dihydrotanshinone were found to potentiate the cytotoxicity of doxorubicin, a P-gp substrate anticancer drug, in the P-gp overexpressing cells (SW620 Ad300). Moreover, the cytotoxicity of irinotecan was also enhanced by two effective tanshinones. Importantly, cryptotanshinone and dihydrotanshinone were found to be more potent in inhibiting P-gp than verapamil, a commonly used P-gp competitive inhibitor as control in in vitro systems. Moreover, both cryptotanshinone and dihydrotanshinone showed equal cytotoxicity potency in SW620 and P-gp overexpressing SW620 Ad300 cells. Thus, unlike doxorubicin and irinotecan, these two tanshinones might not be transported by P-gp, or they could kill cancer cells through different cell death mechanisms in SW620 parental and resistant cells. This finding indicates the great potential of the two tanshinones in cancer therapy for the circumvention of MDR.

The intracellular accumulation of a P-gp substrate (DOX) was examined in SW620 parental and resistant cells so as to provide an explanation for the potentiation of cytotoxicity. Consistent with the drug sensitization assay, both cryptotanshinone and dihydrotanshinone increased the intracellular accumulation of DOX in SW620 Ad300 cells, but not in the parental cells. Since DOX is known to interact with P-gp via more than one drug binding sites, it remains not clear as to whether the tested tanshinones can affect DOX binding at one or more of these binding sites, which would require further investigation.

To fully understand the underlying mechanisms of P-gp inhibition by cryptotanshinone and dihydrotanshinone, P-gp protein and mRNA expression as well as P-gp ATPase activity were further studied. Down-regulation of P-gp protein expression may contribute to the inhibition of P-gp function. The current findings showed that P-gp protein level was significantly down-regulated in SW620 Ad300 cells by cryptotanshinone and dihydrotanshinone at 25 p,M after 24 h of incubation. Further study by RT-PCR analysis revealed that relative ABCB1 mRNA expression was also down-regulated by these tanshinones. As mentioned earlier, one of the physiological roles of P-gp is to protect the body from xenobiotics, via limiting the absorption of toxins through the intestinal mucosa and facilitating the excretion of drugs and their metabolites into the bile and urine. Thus, down-regulation of P-gp expression in normal tissues may lead to impaired defensive mechanism in the body. To this end, it remains to be determined whether cryptotanshinone and dihydrotanshinone could differentially down-regulate P-gp expression in resistant cancer cells versus normal cells.

Since the efflux function of ABC transporters is ATP-dependent, measuring ATPase activity is another widely used approach to study the inhibition of ABC transporters. However, at 25 p,M, only cryptotanshinone showed weak but significant inhibitory effect on P-gp ATPase activity. Higher concentration is needed to achieve marked and significant inhibitory effect for both drugs, suggesting that the inhibition of P-gp function by cryptotanshinone and dihydrotanshinone may not totally depend on the inhibition of ATPase activity.

In conclusion, this study evaluated the inhibition of P-gp function in colon cancer cells by five tanshinones. Cryptotanshinone and dihydrotanshinone showed potent P-gp inhibitory effects and they were also found to apparently reverse MDR in P-gp overexpressing colon cancer cells. The two tanshinones were shown to circumvent P-gp-mediated resistance by down-regulating P-gp mRNA/protein expression and inhibiting P-gp ATPase activity, thereby increasing intracellular accumulation of the transporter substrate anticancer drugs. These two tanshinones may therefore be the promising candidates as P-gp inhibitors. In this regard, they could be used as an adjuvant therapy together with the current chemotherapies in cancer treatment, in particular to those cancers with P-gp-mediated MDR.

Conflict of interest

No conflict to disclose.


Article history:

Received 21 February 2014

Received in revised form 2 May 2014

Accepted 19 June 2014


This work was partially supported by ITF and GRF grants by the Hong Kong Government. Tao Hu receives the postgraduate student scholarship from The Chinese University of Hong Kong.


Bansal, T., Mishra, G., Jaggi, M., Khar, R.K., Talegaonkar, S., 2009. Effect of Pglycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats. Eur. J. Pharm. Sci. 36, 580-590.

Bosch, I., Croop, J.M., 1998. P-glycoprotein structure and evolutionary homologies. Cytotechnology 27, 1-30.

Darby, R.A., Callaghan, R., McMahon, R.M., 2011. P-glycoprotein inhibition: the past, the present and the future. Curr. Drug Metab. 12, 722-731.

Fenner, K.S., Troutman, M.D., Kempshall, S., Cook, J.A., Ware, J.A., Smith, D.A., Lee, C.A., 2009. Drug-drug interactions mediated through P-glycoprotein: clinical relevance and in vitro-in vivo correlation using digoxin as a probe drug. Clin. Pharmacol. Ther. 85, 173-181.

Giacomini, K.M., Huang, S.M., Tweedie, D.J., Benet, L.Z., Brouwer, K.L., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., Hoffmaster, K.A., ishikawa, T., Keppler, D., Kim, R.B., Lee, C.A., Niemi, M., Polli, J.W., Sugiyama, Y., Swaan, P.W., Ware, J.A., Wright, S.H., Yee, S.W., Zamek-Gliszczynski, M.J., Zhang, L, 2010. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215-236.

Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48-58.

Huang, S.M., Temple, R., Throckmorton, D.C., Lesko, L.J., 2007. Drug interaction studies: study design, data analysis, and implications for dosing and labeling. Clin. Pharmacol. Ther. 81, 298-304.

Hubatsch, I., Ragnarsson, E.G., Artursson, P., 2007. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2, 2111-2119.

Hughes, J., Crowe, A., 2010. Inhibition of P-glycoprotein-mediated efflux of digoxin and its metabolites by macrolide antibiotics. J. Pharmacol. Sci. 113, 315-324.

Jemal, A.. Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. CA Cancer. J. Clin. 61, 69-90.

Palmeira, A., Sousa, E., Vasconcelos, M.H., Pinto, M.M., 2012. Three decades of Pgp inhibitors: skimming through several generations and scaffolds. Curr. Med. Chem. 19, 1946-2025.

Persidis, A., 1999. Cancer multidrug resistance. Nat. Biotechnol. 17, 94-95.

Szakacs, G., Paterson, J.K., Ludwig, J.A., Booth-Genthe, C., Gottesman, M.M., 2006.

Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5, 219-234.

To, K.K., Ren, S.X., Wong, C.C., Cho, C.H., 2013. Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells. Peptides 40, 13-21.

Wu. W.Y., Wang, Y.P., 2012. Pharmacological actions and therapeutic applications of Salvia miltiorrhiza depside salt and its active components. Acta Pharmacol. Sin. 33, 1119-1130.

Zhang, W., Han, Y., Lim, S.L, Lim, L.Y., 2009. Dietary regulation of P-gp function and expression. Expert Opin. Drug Metab. Toxicol. 5, 789-801.

Zhou, L., Zuo, Z., Chow, M.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 45, 1345-1359.

Zhou, S., Lim, L.Y., Chowbay, B., 2004. Herbal modulation of P-glycoprotein. Drug Metab. Rev. 36, 57-104.

Tao Hu (a), Kenneth K.W. To (b), Lin Wang (b), Lin Zhang (a), Lan Lu (a), Jing Shen (a), Ruby L.Y. Chan (a), Mingxing Li (a), John H.K. Yeung (a), Chi Hin Cho (a) *

(a) School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

(b) School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

* Corresponding author at: Lo Kwee Seong Integrated Biomedical Sciences Building, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong. China. Tel.: +852 3943 6886; fax: +852 2603 5139.

E-mail addresses:, (C.H. Cho).

Table 1
Effects of tanshinones on apparent permeability ([])
and efflux ratio of digoxin bi-directional transport across
Caco-2 cell monolayer.
                                    [] (x [10.
                                    sup.-6] cm/s)
Compounds           (pM)            [P.sub.AB]

Vehicle control     --              0.89 [+ or -] 0.10
Tanshinone I        25              0.95 [+ or -] 0.20
Tanshinone IIA      25              0.98 [+ or -] 0.22
Cryptotanshinone    25              1.59 [+ or -] 0.26 ***
                    12.5            1.70 [+ or -] 0.16 ***
                    6.25            1.40 [+ or -] 0.13 **
Dihydrotanshinone   25              1.49 [+ or -] 0.04 **
                    12.5            1.39 [+ or -] 0.12 **
                    6.25            1.33 [+ or -] 0.28 *
Miltirone           25              1.02 [+ or -] 0.18
Verapamil           100             2.29 [+ or -] 0.19 ***

                    (x [10.sup.-6]

Compounds           [P.sub.BA]               Efflux ratio

Vehicle control     11.21 [+ or -] 0.95      12.59 [+ or -] 0.48
Tanshinone I        11.27 [+ or -] 1.19      11.86 [+ or -] 1.26
Tanshinone IIA      11.81 [+ or -] 1.21      12.07 [+ or -] 1.40
Cryptotanshinone    8.36 [+ or -] 1.90 *     5.26 [+ or -] 1.18 ***
                    9.31 [+ or -] 0.44       5.48 [+ or -] 0.22 ***
                    11.01 [+ or -] 0.50      7.23 [+ or -] 0.57 **
Dihydrotanshinone   8.28 [+ or -] 0.51 *     5.56 [+ or -] 0.47 ***
                    10.48 [+ or -] 0.83      7.54 [+ or -] 0.94 ***
                    10.89 [+ or -] 1.42      8.19 [+ or -] 1.49 *
Miltirone           11.30 [+ or -] 1.79      11.08 [+ or -] 1.52
Verapamil           3.16 [+ or -] 0.29 ***   1.38 [+ or -] 0.10 ***

AB, apical to basolateral; BA, basolateral to apical.

* p < 0.05, ** p < 0.01 and *** p < 0.001, compared
with the control group.
COPYRIGHT 2014 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Hu, Tao; To, Kenneth K.W.; Wang, Lin; Zhang, Lin; Lu, Lan; Shen, Jing; Chan, Ruby L.Y.; Li, Mingxing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
Previous Article:Pro-angiogenic effects of Carthami Flos whole extract in human microvascular endothelial cells in vitro and in zebrafish in vivo.
Next Article:Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters