RESEARCH ARTICLE Reversal of Multidrug Resistance and Computational Studies of Pistagremic Acid Isolated from Pistacia integerrima

Pistagremic acid (PA) is a bioactive triterpenoid isolated from various parts of Pistacia integerrima plants. The aim of this research was to investigate PA for reversion of multidrug resistant (MDR) mediated by P-glycoprotein using rhodamine-123 exclusion study on a multidrug resistant human ABCB1 (ATP-binding cassette, sub-family B, member 1) gene-transfected mouse T-lymphoma cell line in vitro . Results were similar to those with verapamil as a positive control. Docking studies of PA and standard Rhodamine123 were carried out against a P-gp crystal structure which showed satisfactory results. Actually, PA cannot bind exactly where co-crystallized ligand of P-gp is already present. However, the docking study predicted that if a compound gives a lesser score then it may have some potency. The docking scores of PA and Rhodamine were similar. Therefore, we can conclude that there are certain important chemical features of PA which are responsible for the inhibiting potency of P-gp.


Introduction
Cancer is one of the leading causes of morbidity and mortality throughout the world. But the treatment of cancer is limited due to the development of multidrug-resistance (MDR) phenotype (Ferlay et al., 2015;Ferreira et al., 2015). One of the MDR-related mechanisms consequences from efflux pumps related to the family of ATP-binding cassette (ABC) proteins (Gottesman, 2002;Jin et al., 2012).
Among them, the P-glycoprotein (P-gp) belongs to the ABC superfamily of transporters. Members of this family are broadly divided into three groups. These proteins play a vital function in the in the MDR of pathogen yeasts and bacteria against drugs (Horio et al., 1988). The P-gp is mostly targeted for the treatment of cancer and other human diseases, P-gp is probably the best known of the ABC proteins and thus, it can be considered as a paradigmatic model for this family of transporters (Jones and George, 2004). The P-gp is long poly peptide protein has 1280 amino acids residues which are arranged as a single chain, with two homologous halves having 43% amino acid identity. A linker region of ~60 amino acids connects the two halves of the protein. Each half has six transmembrane domains (TM) and a hydrophilic domain containing an ATP-binding site, known as nucleotide binding domain (NBD) (Jara et al., 2013).
The primary binding region for Rhodamine123 in mouse P-gp was found to be a hydrophobic pocket involving binding site residues such as Ser218, Phe299, Val334, Leu335, Phe339, which have already been proposed to play a crucial role for substrate binding on experimental bases (Li et al., 2010).
Pistacia integerrima belongs to family Anacardiaceae. It is commonly known as kakar singhi. It is distributed in the eastern Himalayan range from Indus to Kumaon (Anonymous, (1998)) at a height of 12000 to 8000 feet. It is a medium sized deciduous tree that can achieve a height of forty feet. P. integrrima is a significant medicinal plant and is used as anti-inflammatory, antidiabetic agent, a blood cleanser, a tonic for gastrointestinal disorders, as cough expectorant (Upadhye, 2010;Pant., 2010). In Pakistan, galls of P. integrrima are used for the treatment of hepatitis and other liver disorders (Ahmad., 2010). It has also been reported to possess CNS depressant activity (Ansari., 1994a). The aim of current study was to isolated (PA) from Pistacia integerrima (Rauf et al., 2014a;Rauf et al., 2014b) and screen for multidrug resistance-reversal effects.

Materials and Methods
Experimental Plant material P. integrrima plant material was collected from Toormang, Razagram area of district Dir, Khyber Pukhtun Khawa province of Pakistan in the month of February 2010. The plant material was identified by Prof. Dr. Abdur Rashid of the department of Botany, University of Peshawar, Pakistan. A voucher specimen no (Bot.20037 (PUP) was deposited in the herbarium of the mentioned department.

Assay for reversal of MDR in mouse lymphoma cells
The L5178 MDR and L5178Y parent cell lines were grown in McCoy`s 5A medium containing 10% heatinactivated horse serum, was completed with L-glutamine and antibiotics. The cells were adjusted to a density of 2×10 6 mL resuspended in serum-free McCoy`s 5A medium and distributed in 0.5 mL aliquots into Eppendorf centrifuge tubes. The tested compound was added at 4 μg/ ml final concentrations, and the samples were incubated for 10 minutes at room temperature. Verapamil was applied as a positive control [Cornwell MM, Pastan I and Gottesman MM: Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein in 10 µg/ml concentration.
Next, 10 µL (5.2 µM final concentration) of the indicator rhodamine 123 (Sigma, St Louis, MO, USA) was added to the samples and the cells were incubated for a further 20 minutes at 37ºC, washed twice and resuspended in 0.5 mL PBS for analysis. The fluorescence of the cell population was measured with a Partec CyFlow flow cytometer (Münster, Germany). The tested compound was dissolved in DMSO, which was also used as a solvent control. The percentage of mean fluorescence intensity was calculated for the treated MDR and parental cell lines as compared with the untreated cells.

Molecular docking studies
The crystal structure of mice P-glycoprotein (P-gp) (PDB code= 4Q9L resolution 3.80Å) was obtained from protein data bank (PDB) (Berman et al., 2000). The structure was subjected to the energy refinement by swiss PDB viewer v4.1.0 program (Guex and Peitsch, 1997). The ligands structure ligands were through Chem sketch (Li et al., 2004) and Avogadro,s software (Hanwell et al., 2012).
The docking of Pistagremic acid and standard Rhodamine123 were carried out through Autodock Vina (Trott and Olson, 2010) and i-GEMDOCKv 2.1 software's (Hsu et al., 2011). The docking method was optimized by an already co-crystallized ligand of the P-gp receptor.

Results and Discussion
Shade dried and crushed plant materials of P. integerrima were repeatedly extracted with MeOH (X5) at room temperature. The extracts were suspended in water and successively partitioned with the various organic solvent to obtained CHCl3, EtOAc fractions. The EtOAc fraction (30g) was subjected to repeated chromatography using silica gel and further purification by recrystallization process. To obtain pure and larger crystals, these crystals were re-grown from a mixture of n-hexane-acetone and chloroform (70:30) and thus obtained a compound named Pistagremic acid (50 mg).

Reversal of Multidrug Resistance Mouse Lymphoma cells
On the basis of their previous anti-cancer potencies (Rauf et al., 2013), PA was selected for flow-cytometric cell cycle screening at the concentration (20 μg/ml). The effect in multidrug resistant mouse lymphoma cells is displayed in Table 1.
The fluorescence activity ratio (FAR) value was used to evaluate the ABCB1 transporter modulating potential. The values of SSC (side scatter count) and FSC (forward scatter count) were increased in the flow cytometry which Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 2313 DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.2311 Reversal of Multidrug Resistance and Computational Studies of Pistagremic Acid Isolated from Pistacia integerrima showed that the compounds (PA) had membrane effect and the granulation of cytoplasm was increased. The FAR values obtained indicated that PA is very effective MDR modulator in a short time experiment. Verapamil, which is a calcium channel blocker and chemo sensitizer, was used as a positive control. On MDR mouse lymphoma cells the PA was screen in one concentration (20 μg/ml). The PA was strong modulators of the efflux-pump activity (FAR 12.27, 20 µg/ml) (Table 1).
Molecular docking studies have important applications in the field of drug discovery. It initially recognized the inhibiting potency of new compounds against the targeted receptors. Our molecular docking studies can co-relate with the in-vitro results of Pistagremic acid. The docking studies of Pistagremic acid and standard Rhodamine123 was carried out against the crystal structure of P-gp. The docking of Pistagremic acid gives very satisfactory results as it is confirmed from the docking Table 1 and from Figure 1. Actually, Pistagremic acid cannot bind exactly where already co-crystallized ligand of P-gp is present. Generally, docking study predicts that, if a compound gives lesser interaction energy then that compound has higher activity. Hence the docking scores of Pistagremic acid are near to the Rhodamine (Table 2). Therefore, we can conclude that there are certain important structural features of Pistagremic acid which are responsible for the inhibiting potency of P-gp from mice.
The detailed interactions (Figure 1) of the Pistagremic acid against the P-gp shows us that there is a total of eight hydrophobic contacts observed from the residues i.e Ile864, Gly868, Glu871, Met872, Leu875, Phe934, Phe938 and Met945. But no hydrogen bond interaction has been observed from any residues in the binding pocket of P-gp. So this may be the reason for such a satisfactory result of Pistagremic acid as comparatively with the standard Rhodamine.
In conclusion, it is concluded that there are certain important structural features of PA which are responsible for the inhibiting potency of P-gp from mice. The docking scores of PA and Rhodamine are similar. This study directed the researcher to isolate PA and synthesize their derivatives and to enhance their anticancer potential.  Figure 1. The Predicted Docked Poses of Pistagremic Acid (PA). In the above figure "A" the encircle line represent sdocked PA shown by green colors ball and sticks while the standard Rhodamine123 is shown by the red color ball and sticks. The co-crystallized ligand in the binding pocket of P-gp is observed from the cyan color sticks. The "B" shows the 3-D schematic representation of the docked Pistagremic acid while the "C" shows the 2-D interactions of important residues in the binding pocket of Pistagremic acid