Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh₂

Abstract

Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh₂ was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh₂ on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC₅₀ values of ginsenoside Rh₂ and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh₂ treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh₂. The results showed that ginsenoside Rh₂, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh₂ in cancer therapy, especially for multidrug resistance reversal in clinic.

Introduction

The exposure of cancer cells to a single anti-cancer drug, including vinka alkaloids, epipodophylotoxins, and anthracyclines, may lead to resistance to a wide range of structural dissimilar drugs [1]. This phenomenon is known as multidrug resistance (MDR) and has been attributed to the over-expression of the MDR-1 gene which encodes permeability glycoprotein (P-gp). P-gp is a 170-kDa protein, function of which is to export kinds of structural and functional unrelated drugs from cells [2]. Overexpression of P-gp will decrease the accumulation of anticancer drugs within tumor cells [3-7]. Therefore, inhibition of P-gp transporter by pharmacological agents would improve the treatment effect of existing chemotherapy against tumors [8].

As one of the major obstacles for cancer chemotherapy, there are many mechanisms responsible for MDR, including increased drug efflux, DNA repair, and apoptotic signaling pathways [9]. Among all the mechanisms, apoptosis, which plays a key role in controlling cell quantity, is a very important factor of cell death to response to toxicity. Previous study showed that [10] resistance to anticancer drugs was always closely related with a propensity of apoptosis. Generally, apoptosis is characterized with mitochondrial membrane depolarization, cytochrome C release, and caspase activation [11]. In malignancy, the upstream apoptotic signals have been disabled. Although, numerous agents restored the apoptotic process has been described [12], clinical applications are always limited by the deleterious toxicities or the low efficacy. Some more powerful and harmless therapies are imperative.

To solve this problem, researchers and clinician have turned to natural products for help [13-15]. Among kinds of natural products, one of the most widely used natural tonics in Oriental countries Panax ginseng, had gained popularity in West countries during the last decades without any significant toxicity [16,17]. The major active components of Panax ginseng are ginsenosides [18,19]. Previous studies [20,21] have shown that crude fractions of ginsenosides extracted from roots of P. ginseng could induce a phenotypic reverse transformation in cultured Morris hepatoma cells.

Ginsenoside Rh₂ (G-Rh₂), found only in red ginseng, is a plant glycoside with a dammarane skeleton and has exhibited potent cytotoxicity against several types of tumors [22]. Hasegawa [23] had hypothesized that G-Rh₂ might interact with P-gp because Rh₂ had low membrane permeability and potential efflux mediated by ATP-binding cassette transporters, making G-Rh₂ mainly distributed in the intestine [24] where P-gp is highly expressed. Moreover, G-Rh₂ has been reported to involve in the activation of cyclin A/Cdk2 which induces the apoptosis [25,26] and reserve the MDR process as well [27]. While more and more attention has been paid to the anti-tumor effect of G-Rh₂, the underlying mechanisms of G-Rh₂ in regulating tumor cells, especially for the MDR process, still remains unclear.

In the present study, we established adriamycin-resistant human mammary carcinoma cell line MCF-7/ADM in vitro [28,29] and assessed the effects of G-Rh₂ on the apoptotic process and Caspase-3 activity in MCF-7 and MCF-7/ADM cell lines. The cytotoxicity of G-Rh₂ on MCF-7 and MCF-7/ADM cultures was estimated by methyl thiazolyl tetrazolium (MTT) test; the retention of P-gp in the two cell lines after treatment with G-Rh₂ was illustrated by Rhodamin 123 assay; flow cytometry and real time polymerase chain reaction (RT-PCR) were conducted to determine the expression levels of P-gp and multidrug resistance gene 1 (MDR1) in the two cell lines after exposure to G-Rh₂. We hoped that our results would reveal the underlying mechanism of G-Rh₂ in regulating the tumor cells, especially for the process of reverse of MDR.

Materials and methods

Preparation of chemicals and MCF-7/ADM cell line

G-Rh₂ (purity > 98%) was obtained from Department of Chemistry, School of Basic Medical Science, Jilin University; adriamycin (ADM), MTT, rhodamin 123, and verapamil were purchased from Sigma.

Human mammary carcinoma cell line MCF-7 provided by Tianjin Medical University Cancer Institute & Hospital was used to induce the resistance against adriamycin. All the procedures were approved by the Ethics Committee of Tianjin Medical University Cancer Institute & Hospital. The cell line was exposed in stepwise increased concentration of ADM to develop the resistant cell line MCF-7/ADM.

Inhibition of the growth, induction of apoptosis, and improving of caspase-3 activity in MCF-7 and MCF-7/ADM cell lines by G-Rh₂

MTT assay was performed according to the method of Lau et al [30]. Briefly, 50 μL exponentially growing cells (2 × 10⁵ cells/ml) were seeded into a 96-well plate in triplicate. Then the cells were treated with increasing concentrations of G-Rh₂ [0 μmol/L (control), 0.625 μmol/L, 1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, 40 μmol/L, 80 μmol/L], and each concentration was repeated in triplicate. The experiment was conducted for three times with different G-Rh₂ treatment time, including 24 h, 48 h, and 72 h. After G-Rh₂ treatment, 5 mg/ml MTT was added to each well and incubated for 4 h at 37°C. The survival rates of different cell lines were measured at 570 nm with a Microplate Reader.

Apoptosis of MCF-7 and MCF7/ADM induced by G-Rh₂ was determined using flow cytometry by annexin-V-FITC/PI dual staining. For this experiment, MCF-7 and MCF7/ADM cell lines were prepared as described above, but the working concentrations of G-Rh₂ was 0 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L.

Influence of G-Rh₂ on caspase-3 activities in the two cell lines was investigated by measuring the levels of p-nitroaniline (pNA), which was the production of Caspase-3 reacting with its substrate. The cells were treating with 0 μmol/L and 40 μmol/L G-Rh₂ for 16 h. The production levels of pNA in different treatments were detected with Microplate Reader at 562 nm. The caspase-3 activity was determined as (production of pNA/incubation time in hours) × (100 µL sample volume/µg protein).

Effect of Rh₂ on the sensitivity of MCF-7/ADM cell line against ADM

The sensitivity of MCF-7/ADM cell line against ADM after treated with G-Rh₂ was determined with MTT assay as described above. The working concentrations of Rh₂ used were 5 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L and MCF-7/ADM cell line was treated for 72 h. Resistance index was calculated as IC₅₀ of MCF-7/ADM of each treatment/IC₅₀ of MCF-7. The IC₅₀ of MCF-7 cell line against ADM was 1.12 ± 0.14 μM, which was measured previously in our lab.

Detections of expression level of MDR1 and P-gp in MCF-7/ADM influenced by Rh₂

The effect of G-Rh₂ on the expression of MDR1 and P-gp was determined by RT-PCR and flow cytometry. The concentrations of G-Rh₂ used here were 0 μmol/L (control), 20 μmol/L, 40 μmol/L, and 80 μmol/L and treatment time was 2 h. And MCF-7 cells were selected as negative control.

Total RNA was extracted by Trizol reagent (Invitrogen life technologies USA) according to operation manual and reverse translated into cDNA template in PE-9600 system. The expression level of MDR1 was determined with β-actin gene as reference gene (Table S1). Final reverse transcribed reaction mixture of 20 µL includes 10 µL of 2 × Premix Ex Taq^TM, 0.4 µL of each primer, 0.4 µL of 50 × ROX Reference Dye, 1 µL RNA template, and 7.8 µL ddH₂O. The RT-PCR program consisted of 95°C for 15 s, 40 cycles of 95°C for 30 s and 59°C for 30 s, and 72°C for 45 s. The program was conducted in Bio-Rad CFX96TM Real-time system.

The expression levels of P-gp in different cell lines were detected with flow cytometry. Approximately 1 × 10⁶ targeted cells were incubated in RPMI 1640 with 10% FBS at 37°C for 24 h. Then the cells were washed twice with PBS, added with 0.25% pancreatin, and incubated at 37°C for 10 min. After washed by PBS for another two times, the cells were labeled with 20 µlanti-mouse IgG₁ and incubated in dark for 30 min before examination with flow cytometer (Beckman coulter. EPICS. XL). Isotype control were set up for MCF-7 and MCF-7/ADM cell lines, respectively. All the experiments were conducted in triplicate.

Effect of Rh₂ on the level of cell retention of rhodamine 123 in MCF-7/ADM cell line

The retention studies were performed with confluent epithelial monolayers of cell lines grown on 24-well plates. The working concentrations of G-Rh₂ used for the treatment of MCF-7/ADM cells were 20 μmol/L, 40 μmol/L, 80 μmol/L, 160 μmol/L, and 320 μmol/L; verapamil was selected as positive control for assessing the effect of G-Rh₂ and the working concentrations of verapamil were 10 μmol/L, 100 μmol/L, 200 μmol/L, and 400 μmol/L; MCF-7/ADM cells without treatment were used as negative control. In brief, cultured cells were washed and then pre-incubated in RPMI 1640 containing 0.25% pancreatin at 37°C for 1-3 min. The RPMI 1640 with 10% FBS was added to inhibit the activity of pancreatin. The cultured cells were incubated with 5 μL Rhodamine 123 at 37°C for 1 h. The concentration of Rhodamine 123 in different cell lines were determined using flow cytometry as described above. All the experiments were conducted in triplicate.

Statistical analysis

All the data were expressed in the form of mean ± SD. ANOVA and LSD tests were performed with significant level of 0.05. All the statistical analysis were conducted using SPSS version 16.0 (IBM, Armonk, NY, USA).

Results

Effect of G-Rh₂ on the growth, apoptotic process, and caspase-3 activities inMCF-7 and MCF-7/ADM cell lines

The results of MTT assay showed that G-Rh₂ had a significant inhibiting effect on the proliferation of MCF-7 and MCF-7/ADM cell lines. The inhibiting effect was time-dependent and significant dose-dependent in the treatment of 48 h and 72 h. For the treatment time of 24 h, we inferred that dose-dependent would be detected with G-Rh₂ higher than 80 μmol/L, and more work would be conducted in future studies. Compared with MCF-7/ADM cell lines, the inhibiting effect of G-Rh₂ on MCF-7 was more powerful (Tables 1 and 2).

Table 1.

Inhibiting effect of G-Rh₂ on the growth of MCF-7 cell lines (mean ± SD)

Concentration (μmol/L)	24 h		48 h		72 h
	OD	Inhibiting Rate (%)	OD	Inhibiting Rate (%)	OD	Inhibiting Rate (%)
0	0.399 ± 0.010	N/A	0.660 ± 0.041	N/A	0.768 ± 0.050	N/A
5	0.360 ± 0.035	9.77	0.556 ± 0.055	15.71*	0.670 ± 0.007	12.72
10	0.363 ± 0.030	9.11	0.557 ± 0.047	15.61*	0.448 ± 0.034	41.60*
20	0.363 ± 0.038	9.11	0.519 ± 0.054	21.31*	0.255 ± 0.018	66.83*
40	0.206 ± 0.003	48.37*	0.170 ± 0.012	74.24*	0.193 ± 0.016	74.82*
80	0.160 ± 0.013	59.98*	0.122 ± 0.024	81.46*	0.188 ± 0.008	75.51*

^*Significantly different from G-Rh₂ concentration of 0 μmol/L.

Table 2.

Inhibiting effect of G-Rh₂ on the growth of MCF-7/ADMcell lines (mean ± SD)

Concentration (μmol/L)	24 h		48 h		72 h
	OD	Inhibiting Rate (%)	OD	Inhibiting Rate (%)	OD	Inhibiting Rate (%)
0	0.260 ± 0.010	——	0.295 ± 0.020	——	0.458 ± 0.043	——
5	0.236 ± 0.007	9.11	0.237 ± 0.016	19.86*	0.379 ± 0.048	17.26*
10	0.236 ± 0.023	9.11	0.233 ± 0.007	21.22*	0.345 ± 0.042	24.69*
20	0.235 ± 0.011	9.37	0.233 ± 0.026	21.11*	0.289 ± 0.040	36.78*
40	0.223 ± 0.012	14.12	0.192 ± 0.017	34.99*	0.246 ± 0.079	46.17*
80	0.138 ± 0.007	46.85*	0.123 ± 0.016	58.24*	0.202 ± 0.062	55.79*

^*Significantly different from G-Rh₂ concentration of 0 μmol/L.

The concentrations of G-Rh₂ which had influence on the apoptotic process were 20 μmol/L and 40 μmol/L. The effect on inducing apoptosis also differed between MCF-7 and MCF-7/ADM cell lines. For MCF-7 cell line, G-Rh₂ resulted in necrosis in most cells while for MCF-7/ADM cell line, G-Rh₂ leaded to apoptosis in most cells (Table 3; Figures 1 and 2).

Table 3.

Effect of G-Rh₂ on the apoptotic process of MCF-7 and MCF-7/ADM cell lines (mean ± SD)

Concentration (μmol/L)	Apoptosis (%)		Necrosis (%)
	MCF-7	MCF-7/ADM	MCF-7	MCF-7/ADM
0	1.43 ± 0.22	1.75 ± 0.23	1.59 ± 0.19	1.96 ± 0.2
10	1.95 ± 0.47	4.36 ± 1.25	1.52 ± 0.2	1.94 ± 0.28
20	7.31 ± 1.86*	6.21 ± 0.91*	3.85 ± 0.49	1.83 ± 0.26
40	11.33 ± 0.66*	26.49 ± 2.42*	31.9 ± 2.55*	7.28 ± 0.20*

^*Significantly different from G-Rh₂ concentration of 0 μmol/L.

Figure 1.

Inducing effect of G-Rh₂ on apoptotic process of MCF-7 cell lines: A: MCF-7/ADM + 0 μmol/L G-Rh₂; B: MCF-7/ADM + 10 μmol/L G-Rh₂; C: MCF-7/ADM + 20 μmol/L G-Rh₂; D: MCF-7/ADM + 40 μmol/L G-Rh₂.

Figure 2.

Inducing effect of G-Rh₂ on apoptotic process of MCF-7/ADM cell lines: A: MCF-7/ADM + 0 μmol/L G-Rh₂; B: MCF-7/ADM + 10 μmol/L G-Rh₂; C: MCF-7/ADM + 20 μmol/L G-Rh₂; D: MCF-7/ADM + 40 μmol/L G-Rh₂.

Significant activation of caspase-3 was detected in MCF/ADM cell line after treatment with 40 μmol/L G-Rh₂ but not in MCF-7 cell line (Table 4).

Table 4.

Effect of G-Rh₂ on the production of caspase-3

	Concentration of Caspase-3 (pmol/μL)
	MCF-7	MCF-7/ADM
0 μmol/L	13.02 ± 1.79	15.50 ± 0.15
40 μmol/L	8.07 ± 1.07	26.28 ± 0.39*

^*Significantly different from G-Rh₂ concentration of 0 μmol/L.

Effect of G-Rh₂ on sensitivity of MCF-7/ADM cell line against ADM

The data showed that the exposure to of G-Rh₂ could significantly decrease the ADM IC₅₀ at the concentration of 20 μmol/L and 40 μmol/L. The effect existed even at the concentration of 10 μmol/L and 5 μmol/L but statistically insignificant (Table 5). The resistance index was reduced to 1.91 under the concentration of 40 μmol/L.

Table 5.

Effect of G-Rh₂ on the IC₅₀ of MCF-7/ADM cell line against ADM

	Concentration of Rh2 (μmol/L)
	0	5	10	20	40
ADM IC50 (μM)	65.43 ± 3.94	60.35 ± 1.88	58.05 ± 2.95	36.02 ± 2.28*	2.14 ± 2626*
Resistance Index	58.42	53.88	51.83	32.16	1.91

^*Significantly different from G-Rh₂ concentration of 0 μmol.

Effect of Rh₂ on expression level of MDR1 gene and P-gp in MCF-7/ADM

MDR1 was highly expressed in MCF-7/ADM cells but no expression of MDR1 was detected in MCF-7 cells (Figure 3). After treatment with G-Rh₂, the expression of MDR1 in MCF-7/ADM didn’t change significantly (Figure 3). The results indicated the existence of MDR process in MCF-7/ADM cell line but not in MCF-7 cell line.

Figure 3.

Expression of MDR1 in MCF-7 and MCF-7/ADM cell lines after exposure to different concentration of G-Rh₂.

With the flow cytometry detection, it was revealed that the expression level of P-gp was significantly different between MCF-7 and MCF-7/ADM cell lines (Figure 4). However, the treatment with G-Rh₂ had no influence on the P-gp expression level in the MCF-7/ADM cells (Figure 5).

Figure 4.

Expression of P-gp in MCF-7 and MCF-7/ADM cell lines: A: Isotype control of MCF-7 cell line; B: MCF-7 cell line; C: Isotype control of MCF-7/ADM cell line; D: MCF-7/ADM cell line.

Figure 5.

Effect of Rh₂ on the expression of P-gp in MCF-7/ADM cell line: A: Isotype control of MCF-7/ADM cell line + 20 μmol/L G-Rh₂; B: MCF-7/ADM cell line + 20 μmol/L G-Rh₂; C: Isotype control of MCF-7/ADM cell line + 40 μmol/L G-Rh₂; D: MCF-7/ADM cell line + 40 μmol/L G-Rh₂; E: Isotype control of MCF-7/ADM cell line + 80 μmol/L G-Rh₂; F: MCF-7/ADM cell line + 80 μmol/L G-Rh₂.

Effect of G-Rh₂ on the level of cell retention of rhodamine 123 in MCF-7/ADM cell line

The fluorescence intensity of rhodamine 123 within MCF-7/ADM cell line was significantly strengthened after the treatment of G-Rh₂ and the effects were dose-dependent at the concentrations < 80 μmol/L and the activity was decreased when the concentrations higher than 80 μmol/L (Figure 6). In addition, when comparison to verapamil, the improvement of G-Rh₂ on cell retention level of rhodamine 123 was more powerful at the concentrations of 40 μmol/L and 80 μmol/L (Figure 6).

Figure 6.

Effect of Rh₂ on the cell retention level of rhodamine 123 in MCF-7/ADM cell line: A: MCF-7/ADM + 0 μmol/L G-Rh₂; B: MCF-7/ADM + 20 μmol/L G-Rh₂; C: MCF-7/ADM + 40 μmol/L G-Rh₂; D: MCF-7/ADM + 80 μmol/L G-Rh₂; E: MCF-7/ADM + 160 μmol/L G-Rh₂; F: MCF-7/ADM + 320 μmol/L G-Rh₂; G: MCF-7/ADM + 10 μmol/L verapamil; H: MCF-7/ADM + 100 μmol/L verapamil; I: MCF-7/ADM + 290 μmol/L verapamil; J: MCF-7/ADM + 400 μmol/L verapamil.

Discussion

MDR is one of the major factors resulting in the failure of chemotherapy in treating the tumors. Lots of clinical commonly used medicines, including adriamycin, vincristine, vinblastine, actinomycin, and mitomycin can induce MDR. The underlying mechanism of the occurrence of MDR is complicated [9]. However, increasing studies have confirmed the major characteristic of MDR is the overexpression of MDR1 which encodes the P-gp. Therefore, inhibition of P-gp transporter by pharmacological agents has been taken as a potential improvement of the existing chemotherapy against tumors.

In the present study, it was found that G-Rh₂ was highly effective in inducing the apoptosis and reversing the MDR of adriamycin-resistant human mammary carcinoma cell line MCF-7/ADM in vitro. The results of MTT assay showed that the IC₅₀ of MCF-7 and MCF-7/ADM after being treated with Rh₂ were significantly decreased. The effect also had the characteristics of time and dose-dependence, which showed strong inhibition on the growth of MCF-7 and MCF-7/ADM cells. Moreover, in our experiment, G-Rh₂ could active the apoptotic process of different cells with the lowest concentration of 20 μmol/L (Table 3; Figures 1 and 2). These results seemed to be identical with previous studies conducted in MCF-7 and SK-HEP-1 cells [25,31,32], which concluded that G-Rh₂ induced the apoptosis of human cells by a mechanism that involves the activation of cyclin A-Cdk2 by caspase-3-mediated cleavage of p21^WAF1/CIP1. However, the patterns of apoptosis between MCF-7 and MCF-7/ADM cells in the present study were quite different (Figures 1 and 2). Moreover, the effect of G-Rh₂ on the activity of caspase-3 was also significantly different between the two cell lines, and it was shown that the treatment with G-Rh₂ had no influence on the activity of caspase-3 in MCF-7 cell line (Table 4). Although we couldn’t give a more comprehensive explanation to this difference, it was inferred that for human mammary carcinoma, the apoptotic induction effect of G-Rh₂ might not directly act on caspase-3. More detailed work would be conducted in the future to highlight the underlying mechanism.

The ability of G-Rh₂ to reverse MDR has been tested with quartz crystal microbalance (QCM) [27]. However, the detail mechanism was not discussed. In the study of Rg₃ [33], it was found that the reversal of MDR was attributed to the binding of Rg₃ with P-gp. This competitive binding resulted in a decline of the excretion level of other substrates that bind to P-gp. The concentration of Rg₃ that significantly inhibited the excretion of Rhodamine 123 (more powerful than the effect of verapamil) in the study of Kim et al was higher than 200 μmol/L. In the present study, our data revealed that Rh₂ could increase the sensitivity of MCF-7/ADM against ADM with the lowest concentration of 5 μmol/L (Table 4), which was a sign of the reversal of MDR. Compared with Rg₃, the concentration G-Rh₂ that could significantly inhibit the excretion of Rhodamine 123 was as low as 20 μmol/L, showing a more efficient and safer ability in binding to P-gp (Figure 6). However, our results also illustrated a decline trend in the Rhodamine 123 concentration within MCF-7/ADM cells when the concentration of G-Rh₂ was higher than 160 μmol/L. This might result from the cytotoxicity of the high concentration G-Rh₂ (Figure 6). Therefore, we recommended the applicable concentration of G-Rh₂ in clinical practice should be lower than 160 μmol/L. In addition, we also confirmed that the treatment with G-Rh₂ did not change the expression level of MDR1 as well as the production of P-gp (Figures 3 and 5). The mechanism was similar with that of Rg₃. However, except for competitive binding, other studies also infer some other mechanisms for the effect of ginsenosides like Rh₂ and Rg₃, such as blocking the calcium channel, disabling the chloride channel, and inhibiting the pathway activated by PKC [34-38]. Therefore, further studies need to be carried out in the future to elucidate the underlying mechanism of Rh₂ reversing the MDR.

In conclusion, our study demonstrated that as a Chinese medicine monomer, Rh₂ had effective ability to induce the apoptosis process in breast tumor cells and reverse the MDR by specifically binding to P-gp. Considering other features including lipid solubility and small molecular weight of G-Rh₂, more work should be carried out on G-Rh₂ to facilitate the application of this natural product as a promising anti-cancer medicine in the future.

Disclosure of conflict of interest

None.