Gynura procumbens Prevents Chemoresistance through Inhibition MDR1 Expression on MCF-7 Breast Cancer Cell Line and Sensitizes the Cells to Doxorubicin

The long-term exposure of doxorubicin (Dox) causes enhancement in MDR1 expression that leads to breast cancer cell resistance. This protein become a serious problem in cancer treatment and also well-known as negative prognostic factor in breast cancer malignancies. The new approach using natural chemopreventive substance was developed to inhibit this resistance progress. This study was aimed to investigate whether ethyl acetate fraction of Gynura procumnens (FEG) can prevent chemoresistance through suppressing the MDR1 protein expression. MCF-7 cell was used as chemoresistance cell model. The MCF-7 cells were maintained with 100 nM Dox-contained medium for ﬁ ve weeks. The chemoprevention effect of FEG was investigated by treated MCF-7/Dox with sub-toxic concentration of FEG. The cytotoxic properties of MCF-7 cells were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Immuno ﬂ uorescence and western blotting analysis was performed to detect the MDR1 expression. MCF-7/Dox cells need higher concentration for inhibiting cell growth, were compared with MCF-7, shown by IC 50 value. The MDR1 protein level elevated after Dox exposure in time dependent manner. The FEG treatment decreased MDR-1 protein level with dose dependent manner. FEG in combination with DOX potentiates the DOX effect on breast cancer cell growth inhibition. The FEG prevents the chemoresistance development in breast cancer cell line, MCF-7 induced by Dox through inhibiting MDR1 expression. The additional of FEG enhances Dox effect on cell death induction. Thus, FEG could be developed as co-chemotherapy agent for reverse multidrug resistance.


Introduction
Drug resistance becomes a major obstacle to the successful of cancer chemotherapy. There are several mechanisms mediate the drug resistance mechanism including drug inactivation, drug effl ux, drug target mutation, and the failure of apoptosis inisitian (Davis et al., 2003;Notarbartolo et al., 2005). Over expression of drug transporter such as P-gp plays a major role in causing multi drug resistance. P-gp is an adenosine triphosphatase (ATPase), a member of ATPbinding cassette (ABC) transporter encoded by MDR1 gene. In normal cells, P-gp licalized at ephitel cell and has a functioan to eliminate xenobiotic and metabolit endogen. However, in some human cancers P-gp overexpression has high corelation with the decreasing survival and become poor prognosis on cancer development (Leonessa and Clarke, 2003). There is positive corelation between overall MDR1 expression and grade of breast cancer tumor (Surowiak et al., 2005). This protein become a serious problem in cancer treatment and also well-known as negative prognostic factor in breast cancer malignancies.
P-gp substrates include substances in variable structure, chemical properties and drug mechanism. Several cancer chemotherapy drugs such as anthracyclines, taxanes, and epipodhophylotoxins already well-known as the substrate of this protein.
Long time exposure of these agents on cancer cells triggers the transcriptional activity of MDR1 lead the increasing of P-gp (Germann, 1996;Nielsen et al., 1996). The elevation of P-gp protein leads to chemotherapy failure due to cell resistance at several type of cancer. Doxorubicin which belongs to anthracyclin group could induce breast cancer multi drug resistance after long time exposure. DOX treatment increase P-gp expression level through Ras/Raf1 activation (Davis et al., 2003). P-gp-mediated drug effl ux decreases intracellular Dox uptake and decrease its effi cacy.
The development of P-gp inhibitors led to the third generation. Unfortunately, most of P-gp inhibitors that have been developed giving disappointing results. New approach using natural substances with moderate or low cytotoxic properties become a promising hope for reversing multi drug resistance due to MDR1 over expression. Natural fl avonoids have antitumor properties and others pharmacological activities related with its antitumor such as anti-infl ammatory and antioxidant. Flavonoid, especially flavones and flavonol show modulation effect on cancer cell growth through cell cycle arrest and apoptosis induction (Limtrakul et al., 2007). Quercetin and kaempferol down regulate multidrug resistance gene on human erythroleukemic K562/A cells. It causes decreasing of cells sensitivity to adriamycin (Yanqiu et al., 2011). Quercetin sensitizes cell resistance to daunorubicin through suppression on MDR1 expression and P-gp activity at human pancreatic carcinoma (Borska et al., 2010).
Gynura procumbens is commonly used as herbal medicines in South-East Asia, especially Indonesia, Malaysia, and Thailand. The extract and fraction of this plant have high anti-infl ammatory and antioxidant properties (Iskander et al., 2002;Rosidah et al., 2008). The ethyl acetate fraction of G. procumbens (FEG) may contain quercetin and kaempferol. FEG sensitizes MCF-7 breast cancer cells to Doxorubicin treatment led to cell death. FEG modulates the microtubule integrity followed by cell cycle arrest and cell growth inhibition . In this study, we subject FEG to doxorubicin-resistance cell. We use MCF-7 cells treated by 100 nM doxorubicin for fi ve week as a model of cell resistance. We investigate the modulation on their sensitivity to doxorubicin, P-gp level, and cell growth.

Plant material
The leaves of G. Procumbens were obtained from Balai Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (BP2TO2T) Indonesia. Dried powdered leaves were fi rst extracted with ethanol 96%, and concentrated by evaporation under reduced pressure and the temperature was kept below 40 o C. The extract was diluted in hot water and then fractionated with n-hexane. The aqueous fraction then was fractionated again with ethyl acetate. The ethyl acetate fraction was concentrated by evaporation under reduced pressure and the temperature was kept below 40 o C. The extract was stored at 4 o C until used.

Antibodies
Primary antibodies that used in western blotting and immunostaining were anti P-Glycoprotein (JSB-1) (GeneTex, Inc.), anti α-Tubulin (Santa Cruz), and secondary antibodies were diluted in PBS containing 5% skim milk, 0,05% Tween (blocking buffer). For P-gp detection, the anti-P-gp and its secondary antibody were diluted in Can Get Signal (CGS) solution (Cosmo Bio).

Cells growth assay
Cells (10 4 cells/well) were seeded in 96-well plate (100 μl/well). After 24 h of incubation, the medium was replaced with DOX, FEG, and its combination-containing medium. The medium was discarded and replaced with MTT-containing medium (0.5 mg/ml) after 24 h incubation and incubated again for further 4 h at 37 o C, 5% CO 2. The reaction was stopped using 10%SDS in 0.1 N HCl solution and was incubated for overnight in light protected chamber, to dissolve formasan salt. The absorbance of each well was measured by ELISA reader at 595 nm. The ratio between treated and control cells absorbance referred to percentage (%) of cells viability.

Gel electrophoresis and immunoblotting
Cells were recovered, washed in PBS, and lysed for 30 min on ice using lysis buffer (20 mM Tris-HCl, pH 8, 0.5 mM EDTA, 1% NP40, 25 mM NaCl, and complete inhibitors of protease. Cells extract were centrifuged at 15.000 rpm for 20 min at 4 o C to separate insoluble material. The protein concentration was determined using Bradford assay. Equal amount of each sample were mixed with SDS loading buffer, boiled for 3 min and subjected to SDS-PAGE at 120 V followed by electro blotting to Polyvinylidene fl uoride (PVDF) membranes for 2 h at 100 V. Membranes were blocked using 5% skim milk in PBS at room temperature for 1 hand subsequently probed with the primary antibody of interest. Blots were exposed by Chemilumi-oneSuper (Nacalay Tesque).

Long treatment of sub toxic concentration of DOX induced resistance cells development.
Breast cancer cells become less sensitive to chemotherapy agen during the cells resistance development. After five weeks induced by DOX, MCF-7 cells decreased its sensitivity to DOX treatment, as shown by IC 50 value (Table 1). DOX resulted cell growth inhibition more than 2-fold higher on MCF-7/DOX sub line than that of MCF-7 cell line ( Figure 1A). FEG treatment showed the opposite results. FEG caused cell growth inhibition stronger on MCF-7/DOX sub line compared with MCF-7 cell ( Figure 1B).

DOX induced the elevation of P-gp level
DOX-induced MCF-7 cells showed the elevation of P-gp expression level in time dependent manner (Figure 2A). expression level of this protein was correlated with less sensitivity of MCF-7/DOX cells to DOX. FEG treatment decreased P-gp level in dose dependent pattern ( Figure 2B). To make confirmation of this phenomenon, both immunobloting and immunofluorescence were conducted. FEG decreased the green fl uorescence intensity that refer P-gp protein decreasing (Figure 3). DOX induces breast cancer cell resistance due to increasing of P-gp expression level. The P-gp protein has 170 kDa molecule size. This protein is hyperglycosilated as such appears as a diffuse band on Western blot results (Patel et al., 2002). We also found the additional band below P-gp protein size (Figure 2). The lower molecules weight bands was detected using the JSB-1 antibody for P-gp represent degraded product of P-gp protein. JSB-1 antibody recognizes a cytoplasmic epitope of P-gp and does not cross-react with other protein such as MDR3 or muscle myosin, as does the C219 antibody (Leonessa and Clarke, 2003). . Cells (10 4 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX or FEG for 24 h incubation. Cells Viability was determined with MTT assay. This result represents from 3 independent experiments. The data was statistically analized by two tailed-T-test analysis with p < 0,05.

FEG reversed cell resistance and increased the sensitivity of MCF-7/DOX cells to DOX treatment
To investigate whether FEG could reverse the cell resistance development on MCF-7/DOX, MCF-7 cells were treated with DOX, FEG, or its combination and observed FEG reversal effect and its modulation on cell growth. Combination treatment of DOX+FEG suppressed cell growth o both of MCF-7 and MCF-7/DOX cells. The inhibition effect of combination treatment on MCF-7/ DOX cells more signifi cance compared to MCF-7 cells (Figure 4).

Discussion
P-glycoprotein (P-gp), a protein encoded by MDR-1 gene, has important function as ATP-dependent pump of drug effl ux. P-gp has specifi c substrate, such as vinca alkaloids, anthrasiclin, epipodhophylotoxins, and rhodamine. The exposure of These substrate to cancer cells could trigger transcriptional activity of MDR-1 (Germann, 1996;Lampidis et al., 1989;Nielsen et al., 1996).
Previous study have found the long time exposure of DOX induced cancer cells resistance through the increasing level of P-gp (Abolhoda et al., 1999). In this study MCF-7 cells were used as resistance model of breast cancer. This cells were developed by long term sub toxic concentration of DOX induction. MCF-7 cells are cell line that express MDR1 and Breast cancer resistance protein (BCRP) that will be triggered its expression after several chemotherapy agent induction (Faneyte et al., 2002). MDR-1 expression elevates at patient after long time chemotherapy agent administration and had strong correlation with cancer chemotherapy  failure (Leonessa and Clarke, 2003). This protein has identifi ed as negative prognostic in cancer diseases development. Sub toxic concentration of DOX for fi ve weeks decreased MCF-7 cells sensitivity to this agent, shown by IC 50 value become more than 2 fold higher compared with MCF-7 cells. DOX exposure to MCF-7 cells lead to increasing expression of P-gp in time dependent manner. MCF-7 cells after fi ve weeks DOX induction (MCF-7/DOX) shown the significance increasing of this protein level compared with MCF-7 cells. The P-gp level has strong correlation with the low sensitivity of MCF-7/DOX to DOX exposure.
The attempts to overcome drug resistance mediated by MDR-1 activation and elevation of P-gp level were developed the P-gp inhibitors. The agent that could suppress P-gp expression will increase uptake of chemotherapy agent and may influence its clinical efficacy. Flavonoids could modulate the expression and activation of P-gp. There are several mechanisms mediate their effect: changing membrane cells permeability, suppressing P-gp expression, inhibition ATPase activity, and occupation substrate binding site at P-gp, then changes its conformation (Arora et al., 2000;Boumendjel et al., 2002;Drori et al., 1995).
Natural compounds such as fl avonoids and polyphenol have signifi cant biological properties related to multidrug resistance development on cancer cells. Biochanin A and sylimarin inhibit drug effl ux from the cells by P-gp pump, and produce synergistic effect when combined with DOX at MCF-7 and MDA435/LCC6. Both compound decreased IC 50 value of daunomycin significantly at MCF-7/Adr cells (Zhang and Morris, 2003;Chung et al., 2005). (-)-Epigallocatechin (EGC), (-)-epicatechin gallate (ECG), dan (-)-epigallocatechin gallate (EGCG) increase daunomycin accumulation at P-gp overexpressing cells, KB-C2 (Kitagawa et al.,Figure 4. FEG increased the sensitivity of MCF-7/DOX cells to DOX. Cells (10 4 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX, FEG and its combination for 24 hours incubation. Cells Viability was determined with MTT assay. This result represents from 2 independent experiments with similar results. The data was statistically analized by two tailed-Ttest analysis. *p = 0,1 and **p = 0,028.  Figure 4. FEG increased the sensitivity of MCF-7/DOX cells to DOX. Cells (10 4 cells/well) were cultured at 96-well plate, then treated with indicated concentration of DOX, FEG and its combination for 24 h incubation. Cells Viability was determined with MTT assay. This result represents from 2 independent experiments with similar results. The data was statistically analized by two tailed-T-test analysis. *p = 0,1 and **p = 0,028. 2004). Flavonoid and polyphenol have a promising properties to be developed as P-gp inhibitor to prevent cell resistance.
FEG treatment enhances DOX cytotoxicity signifi cantly at MCF-7/DOX cells, higher than the effect on MCF-7 cells. The inhibition of cells growth exhibited synergistic effect of combination treatment DOX+FEG in dose dependent manner. FEG treatment decreases P-gp expression level at MCF-7/DOX cells linear to the given concentration. The ethyl acetate fraction of G. procumbens (FEG) was suggested have fl avon and or fl avonol with hydroxyl group modifi ed to be metoksi at 3 and 4', and asetoksi at 7 posisition (Sugiyanto et al., 2003). The ethyl acetate fraction of G. procumbens (FEG) may contain quercetin and kaempferol . FEG may modulate MDR-1 expression through the same mechanism with quercetin and kaempferol.
Quercetin inhibits the members of MDR1 family, such as : P-gp, MRP, and BCRP, competitively (Cooray et al., 2004;Scambia et al., 1994;van Zanden et al., 2005). Kaempferol and quercetin decrease P-gp expression and its activity at MDR KB-V1 cells (Limtrakul et al., 2005). Quercetin and kaempferol prevent cell resistance development through down regulate multidrug resistance gene on human erythroleukemic K562/A cells. It reverses cells sensitivity to adriamycin (Yanqiu et al., 2011). Quercetin suppresses MDR1 expression and P-gp activity at human pancreatic carcinoma. The net influx of daunorubicin increases when given together with quercetin (Borska et al., 2010). One study on structure activity relationship concludes fl avonoid with -OH group at 5 dan 7, with maximal have three total hydroxyl groups, and have strong inhibition on P-gp activity. Double bond at C2 and 3 as shown at fl avon generates its planarity, lead to intercalate with hydrophobic amino acids residue of P-gp (Sheu et al., 2010). Quercetin and kaempferol modulate both of MDR-1 gene and P-gp protein level and also its activity that reverse multidrug resistance on many type of cancer cell lines.
This study found that FEG decrease P-gp expression level at MCF-7/DOX cells. This results were suggested to the decreasing P-gp expression that may affect on the increasing of DOX accumulation at cancer cells. In the next study, monitoring the net uptake of DOX during co-treatment of FEG should be considered. This effect may precede through several possibility, such as suppression on transcriptional activity of MDR-1 gene, translation, or post translation.
As the conclusion, FEG prevents MCF-7 cells from developing resistance through down-regulating P-gp expression. FEG in combination with DOX potentiates the DOX effect on breast cancer cell growth inhibition. Thus, FEG could be developed as co-chemotherapy agent for reversing multidrug resistance.