In Vitro Antitumor Activity of a Keggin Vanadium-Substituted Polyoxomolybdate and Its ctDNA Binding Properties

A Keggin vanadium-substituted polyoxomolybdate, K 5 PMo 10 V 2 O 40 (PMo 10 V 2 ), has been synthesized and it’s antitumor effect against Hela cells was investigated. The calf thymus DNA (ctDNA) binding ability of PMo 10 V 2 was also evaluated by UV-Vis absorption spectra and fluorescence spectra. The identity and high purity of PMo 10 V 2 was confirmed by elemental analysis and IR analysis. And the antitumor activity test of PMo 10 V 2 was carried out on Hela cancer cells line by MTT assay. The results of MTT assay show that PMo 10 V 2 significantly reduced the viability of Hela cells in a dose-dependent manner and exhibited stronger inhibitory activity against Hela cells at an IC 50 of 800 μg/mL, which is more effective than the positive control, 5-Fu (P < 0.05). The results of the UV-Vis absorption spectra and fluorescence spectra indicated the groove or outside stacking binding between PMo 10 V 2 and ctDNA.These results show that the antitumor activity of PMo 10 V 2 may be caused by the interactions between DNA and PMo 10 V 2 .


Introduction
The current interest in polyoxometalates (POMs) keeps on increasing worldwide, not only because of their enormous structural variety, but also their potential applications in multitudinal fields such as catalysis, medicine, and functional materials [1,2].Especially, the medicinal properties of POMs have been a subject of interest in drug discovery.In the literature, POMs serve as a few kinds of inorganic medicinal candidates that have been documented to exhibit antiviral activities and antitumor activities [3][4][5][6].For instance, the PM-8 polyoxometalate synthesized by Yamase and coworkers and that has been evaluated for in vivo and in vitro antitumor activities found to induce apoptotic cell death in human pancreatic cancer cells, AsPC-1, and possesses potent antitumor activity against human Co-4 colon, MX-1 breast, and lung cancer [7][8][9][10].In addition, the group also proposed mechanism that a single electron reduction\oxidation cycle of PM-8 in tumor cells could inhibit the ATP generation [11].On the other hand, these compounds are less expensive than those of organic medicines, attracting great attention on the medicinal exploration.During the last 20 years, it has been established that the size, shape, and charge density of many polyoxoanions are strongly relative to their biological activities and toxicity [12,13].
Cancer is difficult to treat and developing efficient antitumor medicines remain one of the most important goals of modern medicinal chemistry research [14].Cancer starts due to prototype causative factors in a group of cells, leading to DNA damage, thus causing genetic mutations or chromosome structure, the number of different changes, which in turn leads to irreversible changes.DNA is very important genetic substance in any organism and a primary intracellular target for antitumor drugs.The interactions between DNA and small molecules can cause damage in cancer cells and lead to cell death [15].In recent year the interaction of drugs with DNA and investigation of new effective DNA probes have been focused on the study of small molecular interacting with DNA [16].POMs are seldom researched in the agents that directly interact with DNA, [17][18][19][20], which have shown the ability on inhibiting tumor growth by affecting the mitotic index, the synthesis of DNA, the activity of succinct 2 Journal of Chemistry hydrogenase, cytochrome oxidase, and acid phosphatase of tumor cells [21].
As a part of our ongoing antitumor drug discovery, a number of POMs have been synthesized and evaluated for their potential antitumor activities [22][23][24][25].Herein, a Keggin vanadium-substituted polyoxomolybdate, K 5 PMo 10 V 2 O 40 (PMo 10 V 2 ), was prepared.The antitumor activity against Hela cells of PMo 10 V 2 were investigated in vitro.The results indicate that PMo 10 V 2 exhibits strong antitumor activity against the Hela cells, indicating that it is a potential antitumor candidate.The calf thymus DNA (ctDNA) binding ability of PMo 10 V 2 was also investigated, using UV-Vis absorption spectra and fluorescence quenching.

Experiment
2.1.General Procedures.All solvents and chemicals were used as obtained from commercial supplies.The ctDNA and ethidium bromide (EB) were purchased from Sigma Company.The solution of ctDNA was prepared by dissolving ctDNA in phosphate buffer solution at 4 ∘ C under intense stirring for more than 24 h to get a homogeneous solution.Solutions of ctDNA in 10 mM phosphate buffer solution (PBS, 10 mM) of pH 7.4 gave a ratio of UV absorbance at 260 nm and 280 nm;  260 / 280 was 1.82, indicating that the ctDNA was sufficiently free of protein and need no more further purification.The ctDNA concentration per nucleotide was determined by UV absorbance at 260 nm ( 260 = 6600 L mol −1 cm −1 ).And Hela cancer cell lines, DMEM (Hyclone), Fetal bovine serum (sijiqing), penicillin-streptomycin (Sigma), 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma), DMSO (Sigma), and phosphate buffer saline (Sigma) were used for determination of POM cytotoxicity.The elemental analysis of PMo 10 V 2 was estimated by a Leaman inductively coupled plasma (ICP) spectrometer.Infrared spectrum was recorded in the range 400-4000 cm −1 on a Nicolet Magna 560 FT/IR Spectrophotometer using KBr pellets.UV-Vis spectra of PMo 10 V 2 in different concentrations of ctDNA were recorded on a Shimadzu U-3010 Spectrophotometer with a 1 cm path length cell.The fluorescence spectra were recorded on a shimadzu RF-5301PC spectrofluorophotometer.The optical absorbance was read on a plate reader (BIO-RAD Co., USA) at a wavelength of 570 nm for MTT.

Synthesis of K
was synthesized according to the literature [26] and identified by IR spectrum and elemental analysis.

Cytotoxicity and the Antitumor Activity In Vitro Assay.
The antitumor activity of the PMo 10 V 2 on the human cancer cells were tested by the MTT method as described in literature [27].MTT is a dye that can accept a hydrogen atom.The surviving tumor cells can reduce the yellow MTT to a blue formazan, but the dead tumor cells do not possess this capability.Thus the number of survival cells could be calculated by the change of optical density.Hela cells line was used for in vitro MTT assay.DMEM medium supplemented with 10% fetal bovine serum and 50 g/mL penicillin-streptomycin. K 5 PMo 10 V 2 O 40 and 5-fluorouracil (5-Fu) were sterilized by filtration prior to use.Each agent was dissolved in DMEM to generate the appropriate doses for experimentation.Nontreated cells (DMEM alone) were used as negative controls.The Hela cells were plated on the 96-well cell culture plates at a density of 5 × 10 3 cells well −1 for 24 h at 37 ∘ C in a 5% CO 2 incubator.After that the dilutions of PMo 10 V 2 and 5-Fu at different concentrations were added and allowed to incubate for 24 h.After that the MTT solution (0.02 mL, 5 mg/mL in 0.01 M PBS) was added to each well.The cells were incubated for another 4 h at 37 ∘ C and then added DMSO 100 L per well and shaked for 10 min at room temperature.The samples containing PMo 10 V 2 was dissolved in DMEM medium at 800, 400, 200, 100, 50, 25, and 12.5 g/mL concentrations.The 5-Fu was used as the positive control group with the concentration of 400 and 200 g/mL in DMEM.The inhibition concentration was determined for each experimental group by comparison with MTT results from nontreated (DMEM alone) cells and calculated using the following formula: Mean of the samples Mean of control group ) × 100%. (1)

ctDNA Binding Experiments
2.4.1.UV-Vis Absorption Spectra.The absorption titration experiments were performed in absence and presence of ctDNA by sequential addition of specified volume of ctDNA stock solution into a 1 cm path length cuvette containing PMo 10 V 2 solution (100 M/L) on a U-3010 UV-Vis spectrophotometer.After each addition of ctDNA solution, the absorption spectra were recorded from 200 to 700 nm.The intrinsic binding constant of PMo 10 V 2 with ctDNA was calculated by using the following equation: where,   ,   , and   correspond to  obs /[PMo 10 V 2 ] and the extinction coefficient of free and fully bound PMo 10 V 2 , respectively [19].A plot of [ctDNA]  /(  −   ) versus [ctDNA]  , where [ctDNA]  is the total concentration of ctDNA in the base pairs unit, gives   as the ratio of the slope to intercept [18,28].

Fluorescence Spectra.
The interaction pattern of the PMo 10 V 2 with ctDNA was determined by fluorescence titration method.Fluorescence measurements were carried out by keeping the concentration of PMo 10 V 2 constant and varied concentrations of ctDNA.The fluorescence quenching experiment was performed with EB and the experimental data were plotted according to the Stern-Volmer equation (3): ( 0 and  are the fluorescence intensities in the absence and presence of the PMo 10 V 2 , respectively, [] is the concentration of PMo 10 V 2 to ctDNA, and  SV is the Stern-Volmer quenching constant which is dependent on the ratio of the bound concentration of EB to the concentration of ctDNA.)Excitation and emission slit widths were set as 5 and 10 nm, respectively.In competition binding experiments, the concentrations of EB and ctDNA were 100 M and 450 M, respectively, while PMo 10 V 2 concentration varied from 0 to 80 M.The 522 nm was chosen as the excitation wavelength and the emission spectra were recorded from 200 to 700 nm.The binding mode of PMo 10 V 2 with ctDNA was obtained on the basis of previous published methods [19].).The results of the IR spectra analysis to the compound PMo 10 V 2 within the range from 400 cm −1 to 4000 cm −1 using KBr pellets are shown in Figure 1.As shown in Figure 1, the IR spectrum of PMo 10 V 2 showed the characteristic asymmetric stretching vibrational peaks at 776, 588, 505 (Mo-O  -Mo), 869 (Mo-O  -Mo), 960 (Mo-O  ), 1066 and 1214 cm −1 (splitting of the triply degenerate PO4).These peaks suggest that the PMo 10 V 2 are successfully synthesized.

Cytotoxicity and the Antitumor Activity In Vitro Assays.
In order to evaluate the antitumor activity of PMo 10 V 2 , the MTT method was used to detect the activity for Hela cancer cell lines in vitro.A wide range of concentrations (12.5-800 g/mL) of PMo 10 V 2 and 5-Fu (200, 400 g/mL) were tested for its toxicity on the Hela cancer cell line where a dose-dependent cytotoxicity of PMo 10 V 2 was observed.
The DMEM without drugs was used as control group.It shows that higher concentration of PMo 10 V 2 possesses high toxicity on the Hela cancer cells; at a lower concentration as 12.5 g/mL there was no obvious sign of cell death.There is a significant difference between control group and experimental group.The inhibitory effect of PMo 10 V 2 and 5-Fu is given in Table 1 and the IC 50 value of PMo 10 V 2 is 800 g/mL, which is more effective than the positive control, 5-Fu ( < 0.05).It can be concluded that the PMo 10 V 2 possesses the antitumor activity with a dose-dependent decrease in the Hale cells viability, as shown in Table 1 and Figure 2.

Absorption Titration.
The interaction of PMo 10 V 2 with DNA was analyzed by UV-Vis absorption and fluorescence spectroscopic titration.Absorption titrations experiments were carried out to determine the binding mode of PMo 10 V 2 with ctDNA by monitoring the absorption spectra of PMo 10 V 2 in the absence and presence of ctDNA [29].
Figure 3 shows the absorption spectra of PMo 10 V 2 in the presence of different concentrations of ctDNA.In the UV region, the PMo 10 V 2 performed an intense absorption band around 254 nm.Upon the addition of increasing concentration of ctDNA to PMo 10 V 2 solution, a continuing increase in the absorption peak of PMo 10 V without any red or blue shift was observed, shown in the Figure 3.The spectroscopic change suggests that there are interactions between PMo 10 V 2 and ctDNA.
The titration absorption data were analyzed to evaluate the intrinsic binding constant   , which can be calculated based on (2), which was given by the ratio of slope to the intercept in plots from the equation.As shown in the inset ) is much smaller than those reported characteristics of classical intercalators (4 × 10 4 M −1 ) [30].The phenomenon is indicating a weaker binding of ctDNA with PMo 10 V 2 than with the classical intercalation interaction.According to the above results, the groove or outside stacking coordinate covalent binding is more probable than intercalative binding between PMo 10 V 2 and ctDNA.

Fluorescence Spectroscopic Experiments.
To further explore the PMo 10 V 2 interaction with ctDNA, a competition experiment with EB, known as the typical intercalator with ctDNA, was investigated.Fluorescent probe technology is a high sensitivity, selectivity detection methods.EB and NR are common used fluorescent probes.EB is a typical indicator of intercalation since it can form soluble complexes with nucleic acids emitting intense fluorescence in the presence of ctDNA due to its strong intercalation between the adjacent ctDNA base pairs.The fluorescence will decrease when the EB dissociate from ctDNA.It was previously reported that the enhanced fluorescence can be quenched by the addition of a second molecule which could replace the bound EB or break the secondary structure of the ctDNA [12,31].The Electron transfer or replacement of the molecular fluorophores mechanism has been proposed to account for the quenching effect.For further exploration of ctDNA-PMo 10 V 2 interaction, the fluorescence emission of bound EB to ctDNA has been investigated in the presence of PMo 10 V 2 .The results in Figure 4 show the quenching effect of PMo 10 V 2 .Upon the addition of increasing concentration of PMo 10 V 2 , the fluorescence intensity can be quenched.And the reduction of fluorescence intensity depended on the addition of the PMo 10 V 2 .In the inset of Figure 4, the plots of   / versus [PMo 10 V 2 ] was a straight line.According to the Stern-Volmer equation, the value of quenching constants was 4.89 × 10 3 M −1 which is much lower than the corresponding value of EB.PMo 10 V 2 does not show any fluorescence whether in ctDNA absence or presence.The maximum binding constant of PMo 10 V 2 to ctDNA is much lower than EB, according to the UV-Vis analyses.Hence PMo 10 V 2 is not expected to replace the strong bound EB.Therefore, the binding mode of PMo 10 V 2 with ctDNA is not intercalation interaction.The result is consistent with the findings obtained from UV-Vis spectral studies.

Conclusion
In this study, the synthesis, characterization, antitumor activities, and ctDNA interaction of PMo 10 V 2 were investigated.
The results of combined spectroscopic methods reveal that PMo 10 V 2 bounds to ctDNA by groove or outside stacking binding mode and proved the presence of any direct coordinate covalent bond formation.This binding mode may be explained by considering the high capacity of this PMo 10 V 2 for accepting electrons.These results show that PMo 10 V 2 as antitumor agents possess high efficacy on Hela cells.The researches of pharmacodynamics, toxicology, and the interaction with DNA on PMo 10 V 2 are being further tested.

Table 1 :
The inhibitory effect of PMo 10 V 2 .