A Rare Class of New Dimeric Naphthoquinones from Diospyros lotus have Multidrug Reversal and Antiproliferative Effects

Three new dimeric naphthoquinones, 5,4′-dihydroxy-1′-methoxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,5′,8′-tetraone (1), 5′,8′-dihydroxy-5-methoxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,1′,4′-tetraone (2) and 8,5′,8′-trihydroxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,1′,4′-tetraone (3), were isolated from the roots of Diospyros lotus. Their structures were elucidated by spectroscopic techniques, including 1D and 2D NMR, such as HSQC, HMBS, NOESY, and J-resolved. Compounds 1–3 were evaluated for their effects on the reversion of multidrug resistance (MDR) mediated by P-glycoprotein through use of the rhodamine-123 exclusion screening test on human ABCB1 gene transfected L5178Y mouse T-cell lymphoma. Compounds 1–3 were also assessed for their antiproliferative and cytotoxic effects on L5178 and L5178Y mouse T-cell lymphoma lines. Both 1 and 2 exhibited promising antiproliferative and MDR-reversing effects in a dose-dependent manner. The effects of the tested compounds on the activity of doxorubicin were observed to vary from slight antagonism to antagonism.


INTRODUCTION
The Diospyros genus in the Ebenaceae family consists of about 500 species. This genus is widely distributed in tropical and subtropical regions throughout the world and is native to the Himalayan region (Uddin et al., 2011). Diospyros lotus grows up to 9 m in height in semi-shaded areas . Diospyros species are known for their multiple uses in therapeutic and folk medicine. Different parts of this plant are used for different diseases: the leaves are used to treat lumbago, the fruits as a carminative to cure biliousness, the seeds as a sedative, and the bark as an astringent and febrifuge . A leaf extract of D. kaki (Japanese persimmon) in combination with jasmine is used in anti-tobacco smoking candies (Uddin et al., 2013). Various triterpenoids of the lupane, oleanane, and ursane series have been isolated and proved to exhibit antiinflammatory activity (Uddin et al., 2013). Diospyros species are used as traditional medicines, e.g., as an antifungal, to treat hiccough, for internal hemorrhage, for bedwetting in children, as a woman's drug for insomnia, as an antihypertensive, to treat dyspnea, as a vermicide and vermifuge, as a sedative, as an antifebrile, and as a bactericide (Tezuka et al., 1973;Ganapaty et al., 2006). A D. lotus extract and isolated compounds have been reported to display promising antiproliferative activity (Loizzo et al., 2009).
Multidrug resistance (MDR) is the main clinical challenge for the active treatment of cancer (chemotherapy; Szabo and Molnar, 1997). There are numerous mechanisms by which tumor cells develop resistance to cytotoxic secondary metabolites. One of them is produced by the overexpression of ATP-binding cassette (ABC) proteins or breast cancer resistance protein (BCRP). The ATP-binding cassette transporters represent the largest family of transmembrane proteins that bind ATP and use the energy to drive the transport of various molecules across cell membranes (Gottesman and Ambudkar, 2001;Leonard et al., 2003). ABC efflux transporters extrude a broad range of amphiphilic compounds against the concentration gradient in an energy-dependent fashion. Many of the ABC transporters have dedicated physiological functions, and afford normal tissue protection in the brain vessels, liver, and kidney (Gottesman et al., 2002;Sarkadi et al., 2006;Szakacs et al., 2006).
The firstly identified drug efflux protein was the P-glycoprotein (P-gp, MDR1, ABCB1), encoded by the ABCB1 gene. P-gp is composed of 1280 amino acids (170 kDa) organized in two transmembrane domains . This protein is overexpressed in several human tumors and can extrude a wide range of drugs (anticancer, antibiotics, antidepressants, antihistamines, antiarrhythmics, immunosuppressants, HIV protease inhibitors and steroids). Many drug molecules, such as tamoxifen, valspodar, dexniguldipine, and tariquidar, have been proposed to suppress the action of P-gp (Germann et al., 1993;Lopez and Martinez-Luis, 2014).
A second cellular pump is multidrug-resistant protein 1 (MRP1, ABCC1), described in Cole et al. (1992). MRP1 is an efflux pump originally discovered in doxorubicin-resistant lung carcinoma cells displaying a multidrug resistant phenotype without ABCB1 expression.
MRP1 is expressed ubiquitously in higher levels at the bloodbrain barrier, in the intestines and in the oral mucosa (He et al., 2011). MRP1 expression is higher in the lungs than in any other organ and it may have protective roles against air pollution and inhaled toxins (Sakamoto et al., 2013). The physiological substrates of MRP1 include bile acids, folic acid, leukotriene C4, and glutathione conjugates, and it confers resistance to vincristine, methotrexate, doxorubicin, and etoposide (Cole and Deeley, 2006;Li et al., 2008). A third cellular pump type is BCRP, first cloned in the drugresistant breast cancer cell line MCF-7 (Doyle et al., 1998). BCRP is a half-transporter member of the ABCG subfamily (ABCG2) with a size of 72 kDa. BCRP probably functions as a homodimer. The expression of BCRP overlaps largely with that of P-gp, because the protein can be found in tissues such as the placenta, prostate, small intestine, brain, colon, liver, and ovary (Doyle et al., 1998). Overexpression of BCRP is associated with resistance to a wide range of different anticancer agents: anthracyclines, mitoxantrone, flavopiridol, camptothecins, and antifolates (Assaraf, 2006;Bihorel et al., 2007;Robey et al., 2007). Several studies have demonstrated the frequent occurrence of drug efflux proteins in cancer tissue. Some authors have reported significant correlations between the overexpression of P-gp or MRP-1 and a poor treatment response in solid tumors and some leukemias (Brinkhuis et al., 2002;Diestra et al., 2003;Larkin et al., 2004;Damiani et al., 2006), and a prognostic significance for BCRP overexpression in specific forms of leukemia (Larkin et al., 2004).
The current study deals with the isolation of three new dimeric naphthoquinones (1-3) from the chloroform (CHCl 3 ) fraction of D. lotus. The isolated compounds (1-3) were evaluated for their effects on the reversion of MDR in mouse lymphoma and for their antiproliferative and cytotoxic effects on the L5178 and L5178Y mouse T-cell lymphoma cell lines. A combination assay was also applied to study the effects of the drug interactions between the dimeric naphthoquinone derivatives and the chemotherapeutic drug doxorubicin on the MDR mouse lymphoma cell line.

Cell Cultures
L5178 mouse T-cell lymphoma cells (ECACC cat. no. 87111908, U.S. FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described (Cornwell et al., 1987;Bak et al., 1989). The MDR1-expressing cell line L5178Y was selected by culturing the infected cells with colchicine. L5178 (parent) mouse T-cell lymphoma cells and the human MDR1-transfected subline were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated horse serum, 200 mM L-glutamine, and a penicillin-streptomycin mixture in 100 U/L and 10 mg/L concentrations, respectively. MDR was detected with a monoclonal antibody (Bak et al., 1989;Molnar et al., 1999). The cell lines were incubated in a humidified atmosphere (5% CO 2 , 95% air) at 37 • C. The L5178 mouse T-cell lymphoma cells (PAR; ECACC Cat. No. 87111908, obtained from FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described by Cornwell et al. (1987). The ABCB1-expressing cell line L5178Y (MDR) was selected by culturing the infected cells with colchicine (Cornwell et al., 1987). L5178 (parent) mouse T-cell lymphoma cells and the L5178Y human ABCB1-transfected subline were cultured in McCoy's 5A medium supplemented with 10% heatinactivated horse serum, 200 mM L-glutamine and a penicillinstreptomycin mixture in concentrations of 100 U/L and 10 mg/L, respectively.

Antiproliferative Assays
The antiproliferative effects of compounds 1-3 were determined in 96-well flat-bottomed microtiter plates (Molnar et al., 2004b;Gyemant et al., 2005). The antiproliferative potentials of the compounds were tested at a concentration of 1 μg/mL, using the L5178Y

Assays for Cytotoxic Effects
The effects of increasing concentrations of the drugs alone on cell growth were tested in 96-well flat-bottomed microtiter plates. The compounds were diluted in 100 μL of medium. 1 × 10 4 mouse T-cell lymphoma cells (PAR or MDR) in 50 μL of medium were then added to each well, with the exception of the medium control wells. The culture plates were further incubated at 37 • C for 24 h; at the end of the incubation period, 15 μL of MTT solution (from a 5 mg/mL stock) was added to each well. After

Assays for Reversal of MDR in Mouse Lymphoma Cells
The L5178Y MDR and L5178 parent cell lines were grown in McCoy's 5A medium containing 10% heat-inactivated horse serum, supplemented with L-glutamine and antibiotics. The cells were adjusted to a density of 2 × 10 6 mL, resuspended in serumfree McCoy's 5A medium and distributed in 0.5 mL aliquots into Eppendorf centrifuge tubes. The tested compounds were added at 0.1-1 μg/mL final concentrations, and the samples were incubated for 10 min at room temperature. Verapamil was applied as positive control (Cornwell et al., 1987) in 10 μg/mL concentration. Next, 10 μL ( (Gruber et al., 1988). The tested compounds were dissolved in DMSO, which was also used as solvent control. The percentage mean fluorescence intensity was calculated for the treated MDR and parental cell lines as compared with the untreated cells. The activity ratio was calculated via the following equation (Molnar et al., 2004a) on the basis of the measured fluorescence values: The results presented were obtained from a representative flow cytometric experiment in which 10 5 individual cells of the population were evaluated for the amount of rhodamine 123 retained. The data were analyzed with FlowJo software 3 .

Checkerboard Combination Assays
A checkerboard microplate method was applied to study the effects of the drug interactions between the dimeric naphthoquinone derivatives and the chemotherapeutic drug doxorubicin on the MDR mouse lymphoma cell line. The dilutions of doxorubicin were made in a horizontal direction in 100 μL, and the dilutions of the dimeric naphthoquinone compounds were made vertically in the microtiter plate in 50 μL. The cells were resuspended in culture medium and distributed into each well in 50 μL containing 6 × 10 3 PC3 MDR mouse T-cell lymphoma cells. The plates were incubated for 72 h at 37 • C in a CO 2 incubator. The cell growth rate was determined after MTT staining, as described above. The combination index (CI) values at 50% growth inhibition (ED 50 ) were determined by using CompuSyn software 4 (ComboSyn, Inc., Paramus, NJ. USA) to plot 4 or 5 data points for each ratio. CI values were calculated by means of the median-effect equation (Chou and Martin, 2005), where CI < 1, CI = 1, and CI > 1 represent synergism, an additive effect (or no interaction) and antagonism, respectively.

RESULTS AND DISCUSSION
The whole MeOH extract was suspended in water and successively partitioned with n-hexane, CHCl 3 , EtOAc, and n-BuOH. The CHCl 3 fraction was selected for phytochemical investigation due to the presence of a greater amount of compounds as indicated by the TLC profile. CC of the CHCl 3 fraction (30 g) on silica gel resulted in the isolation of three new dimeric naphthoquinones (1-3). The structures of these compounds were elucidated by spectroscopic techniques and comparisons with literature data.
Compound 1 was isolated as a yellow amorphous powder. It exhibited the molecular ion peak at m/z; 404.2100 a.m.u. (calcd. 404.2101) corresponding to the molecular formula C 23 H 16 O 7. The IR spectrum showed absorption bands at 3550 cm −1 for OH stretching, 2924 cm −1 for CH stretching, 1643 cm −1 for CO stretching and 1634, 160, and 1460 cm −1 for CH aromatic stretching. The UV spectrum exhibited absorptions at 253, 296, and 435 nm.
Dimeric naphthoquinones 1-3 were investigated for their potential properties as MDR efflux pump modulators. They were first screened for antiproliferative activity on human ABCB1 gene transfected mouse lymphoma cell line L5178Y, which specifically overexpresses a membrane-localized transporter (Pgp, ABCB1). In order to test the potential clinical application of compounds 1-3, they were evaluated as concerns the reversal of MDR (Csonka et al., 2013). The antiproliferative effects of compounds 1-3 were determined by the MTT method. Compounds 1 and 2 showed promising antiproliferative potential on the L5178Y mouse T-cell lymphoma cell line, with IC 50 values of 0.05 ± 0.004 μg/mL and 0.046 ± 0.005 μg/mL, respectively, whereas compound 3 had an IC 50 value of 0.26 ± 0.01 μg/mL ( Table 4).
The cytotoxic effects of the dimeric naphthoquinones on the PAR and MDR cell lines were studied. Evaluation of the cytotoxic activities of the compounds revealed that 1 and 3 were the most active against the parental mouse T-lymphoma cell line, with IC 50 values of <3 μg/mL. Both of these compounds were also active against the MDR cell line (Figure 5 and Table 5).
Compounds 1-3 were also evaluated for the reversion of the MDR of ABCB1 gene transfected mouse lymphoma cell line, followed by flow cytometry, which measures intracellular accumulation of rhodamine 123, a fluorescent substrate analog of epirubicin. The fluorescence activity ratio (FAR) value was used to evaluate the ABCB1 transporter modulating potential. When the tested compounds were investigated in concentrations of 0.1 to 1 μg/mL by flow cytometry (Table 6), the side scatter count and forward scatter count values increased, indicating that the compounds exerted membrane effects and the granulation of the cytoplasm was increased. The results revealed a special type of toxic effect on the reversal of MDR at toxic doses of 1 μg/mL. The FAR of compounds 1-3 differed: compound 1 proved to be a very effective MDR modulator, while compounds 2 and 3 did not exhibit significant effects in a short-time experiment. Verapamil, a calcium channel blocker and chemosensitizer, was used as a positive control. The results relating to MDR reversal activity in the current investigation are presented in Table 6. On MDR mouse lymphoma cells, compounds 1-3 were screened in two concentrations (1 and 0.1 μg/mL). Compound 1 was a fairly moderate modulator of the efflux pump activity (FAR = 1.72 at 1 μg/mL and 10.63 at 0.1 μg/mL), while compounds 2 (FAR = 1.1 at 1 μg/mL and 1.3 at 0.1 μg/ml) and 3 (FAR = 1.15 at 1 μg/mL and 0.84 at 0.1 μg/mL) were somewhat weaker ( Table 6).
The effects of the tested compounds on the activity of doxorubicin were observed to vary from slight antagonism to antagonism as shown in Table 7.
The tested dimeric naphthoquinones 1-3 display several structural differences, mainly relating to the positions of the aromatic linkages. Compound 3 differences from compounds 1 and 2 in containing an extra hydroxyl group rather than a methoxy substituent. It is clear that the joint presence of methoxy and hydroxy groups enhanced the activity ( Table 4). The positions of quinone moieties influenced the efflux pump activity, as demonstrated by the FAR values in Table 6.

CONCLUSION
Three new dimeric naphthoquinones (1-3) isolated from the CHCl 3 -soluble fraction of the roots of D. lotus, led to the reversal of MDR and exerted antiproliferative activity. They also exhibited promising antiproliferative effects in a dose-dependent manner on two cancer cell lines. This discovery strengthens our belief in the indigenous knowledge of traditional health cures against cancer diseases. The results suggest that research on naphthoquinone derivatives could possibly lead to the discovery of potent anticancer agents.

AUTHOR CONTRIBUTIONS
GU and AK were project supervisor. AR was performed isolation of compounds. BS and BA were gave the project idea. JM, AC, and DS were performed the activities of compounds. UF and AK involved in the useful discussion and participated in manuscript writing. All authors read and approved the final manuscript.