Introduction
In recent years, the incidence of breast cancer has become one of the most rapidly increaing among malignant tumours. With progress in understanding of the nature of breast cancer, diagnosis and treatments have been improved. Chemotherapy has become more and more important and is considered to be a major treatment to avoid the recurrence of cancer after surgery [
1]. Although the remission rate is higher in previously untreated patients, relapse occurs soon. It has been reported that most initially responsive patients acquire a multidrug resistance (MDR) phenotype. Some other patients show MDR even in their first treatment. In metastatic breast cancer, the development of a MDR phenotype is primarily responsible for insensitivity to a new drug [
2].
Attention has been focused recently on the study of the agents for reversing MDR. Although hundreds of compounds have been found
in vitro to be able to modulate the MDR phenotype, their clinical application was limited owing to high toxicity
in vivo. The key to the clinical use of reversal agents therefore lies in searching for agents with low toxicity and high reversal activity [
3]. In the past, many progestogen compounds, such as MG and medroxyprogesterone, have been shown to have reversal effects
in vitro [
4,
5,
6,
7]. Unfortunately, the effective concentration to reverse drug resistance
in vivo is very difficult to achieve [
8]. Nomegestrol (NOM), a derivative of megestrol (MG), has very low toxicity. There is almost no incidence of liver lesions, pituitory-inhibiting activity, cortisol-like activity or oestrogen-like activity, which are common in other progestogen compounds [
9]. NOM might therefore be suitable for clinical use.
In many previous studies on MDR reversal by compounds related to NOM, attention has been paid to the modulation of P-glycoprotein (P-gp) function. The reversal mechanism consists of, for example, binding directly to P-gp, inhibiting the transport function of drug efflux, increasing the intracellular accumulation of the drug and changing lipid mobility in the plasma membrane [
4,
10,
11,
12]. However, different compounds have different mechanisms. No results on the reversal effect of NOM or on its mechanism were available. The present study was directed to the reversal activity of NOM on MDR in adriamycin (ADR)-resistant (MCF7/ADR) cells, and to compare NOM with classic reversal agents [verapamil (VRP), tamoxifen (TAM), droloxifene (DRO) and MG]. A possible reversal mechanism was explored by studying the cell cycle, intracellular drug accumulation and the expression of mRNA and proteins of MDR1, glutathione S-transferase Pi (GSTπ), MDR-related protein (MRP) and topoisomerase IIα (TopoIIα).
Discussion
The overexpression of P-gp in tumour cell membrane is considered to be the major mechanism of MDR. P-gp is able to pump various anticancer drugs out of cells, thus resulting in a low intracellular drug concentration that is insufficient to kill tumour cells [
18]. To use compounds with low toxicity, or with none at all, to bind P-gp and block its transport function is the most common method of reversing MDR [
19]. Progesterone and MG belong to the progestogen group. Although progesterone is not a substrate for P-gp, it can directly bind to P-gp and block the transport of drug efflux [
20]. MG is a strong reversal agent: its capacity to increase intracellular accumulation of vincristine is 2-3-fold that of progesterone [
6]. Previous results have shown that a daily dose of 800 mg MG resulted in a plasma concentration of 2 μM [
21]; however, to reverse MDR, 5 μM is necessary in theory. Because high doses of MG induce vomiting, oedema, dizziness and androgen-like side-effects, the ideal effective concentration is difficult to obtain
in vivo. Therefore, the search for new drugs with low toxicity is in progress to meet an urgent need for clinical applications.
NOM is a progestogen compound used in family planning. In recent years, biological activities other than contraception have been found [
22]. Animal experiments have demonstrated that NOM has almost no toxicity. Its full chemical name is 3,20-diketo-6-methyl-17-α-hydroxy-19-norpregna-4,6-diene [
9].
The present study has demonstrated that NOM significantly sensitizes the MCF7/ADR cell line to ADR in a concentration-dependent manner, but has no similar effect on MCF7/WT cells. The reversal effects of 20, 10 and 5 μM NOM were 21-fold, 12-fold and 8-fold, respectively. The enhancement of chemosensitivity to ADR by NOM was stronger than that by MG, a precursor of NOM, at corresponding concentrations (P <0.05). The reversal effect of NOM was similar to that of VRP, which is a classical reversal agent with a high reversal activity; NOM therefore also has a strong reversal activity.
Both TAM and DRO are anti-oestrogen agents. TAM is a first-line drug used in endocrinotherapy for breast cancer. Anti-oestrogens can inhibit P-gp function by binding it [
23]. In our study, the effect of NOM was significantly greater than that of DRO.
The development of MCF7/ADR cells was induced by treating MCF7/WT with ADR. Its MDR phenotype is not altered after 3 months of serial passaging in drug-free medium [
24,
25]. MCF7/ADR cells have been demonstrated to overexpress MDR1/P-gp, as well as the GSTπ and MRP genes. They are therefore good cell models for studying the effect of NOM on breast cancer MDR.
Semiquantitative RT-PCR analysis is a sensitive and specific method [
26,
27]. A variety of methods using either quantitative or semequantitative RT-PCR have been used to determine relative initial target mRNA levels in samples. However, in all of these methods undefined variations in amplification complicated the interpretation of results. Most investigators use internal amplification standards in an attempt to correct for variations between tubes. In the present study, as in many other studies, we chose to use endogenous standards. The reference mRNA and target mRNA are usually processed together throughout the experiments, from RNA extraction until PCR amplification. This tends to minimize differences in RNA yields between samples.
It has been considered that the overexpression of MDR1/P-gp is a major mechanism of MDR in tumour cells [
28]. A few studies have reported that some reversal agents can inhibit MDR1/P-gp expression. Stein
et al [
29] have found that cytokines such as interleukin-2, interferon-γ and tumour necrosis factor-α were capable of decreasing the expression of MDR1 mRNA in the colon carcinoma cell lines HCT15 and HCT16. Liu
et al [
30] treated MCF7/ADR cells with the Chinese herb
Fructus psoraleae for 48 h and found that P-gp expression became undetectable. However, no results on whether or not progestogen can modulate the expression of MDR1/P-gp have been reported. The present study has demonstrated that NOM significantly inhibited MDR1/P-gp expression on the third day after treatment. The MDR mRNA expression level was very low, and that of P-gp almost ceased.
It is worthy to note that on the fifth day after NOM treatment, the expression of MDR1/P-gp began to increase again. This phenomenon was also observed when treating colon carcinoma cell line with cytokines [
29]. Some studies have also found that P-gp antagonists such as VRP, cyclosporin A and reserpine could induce MDR1/P-gp expression in the colon carcinoma cell line LS180-Ad50 [
31,
32]. A few papers have reported MDR1 expression after treatment with progesterone. Lee
et al [
33] showed that progesterone interacted with P-gp, inducing its expression in the granulose cell in preovulatory follicles to modulate steroid efflux. Our results show that NOM modulated the expression of MDR1 in a time-dependent manner. Over 5 days of treatment with NOM, MDR1 expression became elevated. From these findings it seems that NOM and other progestogen compounds show an effect of upregulated MDR1 expression in long-term treatment. It is unclear how NOM effects this time-dependent modulation of the expression of MDR1/P-gp. If a similar modulation of the MDR phenotype occurs
in vivo, then the duration of treatment with NOM becomes important. It will be of prime importance to verify the time course for modulation by reversal agents and to design appropriate clinical trials.
In investigating the P-gp-induced MDR phenotype, the overexpression of some non-P-gp MDR-related proteins have been demonstrated. These proteins are important in drug resistance in some tumour cell lines [
34,
35]. We also observed the expression of the non-P-gp MDR-related genes GSTπ, TopoIIα and MRP. We found that NOM markedly decreased the level of expression of GSTπ mRNA and protein with a time course similar to that of MDR1/P-gp. The results suggest that NOM possibly acts as a reversal agent of GSTπ. MRP belongs to the superfamily of ATP-binding cassette protein transporters. Like P-gp, MRP is located in the plasma membrane of resistant tumour cells; however, the mechanism of MRP might be fundamentally different from that of P-gp. The extrusion of several drugs by MRP requires glutathione. MRP might be identical to the GS-X pump [
36]. MRP-related MDR was not reversed by classic reversal agents of P-gp such as VRP and cyclosporin A [
37,
38,
39]. Our results suggest that NOM cannot modulate MRP gene expression either.
TopoII, as a kind of nuclease, is important in the processes of DNA metabolism such as transcription, replication and chromosome partitioning during cell division. TopoIIα is present mainly in the S phase [
40]. TopoIIα in tumour cells is a target enzyme of some anticancer agents. If TopoIIα activity decreases or a TopoIIα gene mutation develops, TopoIIα-related MDR results [
41,
42].
Our results showed that TopoIIα expression in MCF7/ADR cells was weaker than in MCF7/WT cells. Because TopoIIα is a target enzyme of ADR, MDR to ADR in MCF7/ADR cells might to some extent be related to TopoIIα. However, no change in TopoIIα gene expression level was detected in NOM-treated MCF7/ADR cells, indicating that the reversal effect of NOM on MDR in MCF7/ADR cells cannot result from modulating the gene expression of TopoIIα.
P-gp belongs to the ATP-dependent transporters. It acts as a pump or hydrophobic vacuum cleaner, effectively increasing drug efflux and decreasing drug influx [
43]. Many studies have demonstrated that progestogen compounds directly bind P-gp and block the function of the pump efflux [
10,
44]. We studied the effect of NOM on intracellular ADR accumulation in MCF7/ADR cells by using FCM assays. ADR can emit fluorescence; its intensity represents its accumulation [
25]. After 2 h of treatment with NOM, the intracellular fluorescence intensity of ADR was markedly enhanced, suggesting that NOM directly inhibits the P-gp pump efflux function in the same way as other progestogen compounds do.
It has been reported that ADR induces dose-dependent G
2M arrest and that cells in this phase are particularly sensitive to chemical drugs [
45]. Cell-cycle DNA content assays by FCM suggest that NOM enhances the blocking activity of ADR on the cell cycle. This enhancement of blocking activity might be partly responsible for the reversal effect of NOM.
In brief, our results indicate that in MCF7/ADR cells NOM can significantly sensitize their chemosensitivity to ADR, down-regulate MDR1/P-gp and GSTπ expression levels in time-dependent manner, markedly increase intracellular ADR accumulation and enhance the cell cycle blocking activity of ADR. At present, although a variety of agents inhibit the function of P-gp in vitro, their clinical use is also limited by the toxicity associated with the doses required to reverse MDR. Because NOM can be safely administered at high doses, it might be a good candidate for an MDR reversal agent in clinics.