Synthesis, Anti-Tumor Activity and Apoptosis-Inducing Effect of Novel Dimeric Keggin-Type Phosphotungstate

A dimeric Keggin-type phosphotungstate (ODA)10[(PW11FeO39)2O]·9H2O (abbreviated as ODA10[(PW11Fe)2], ODA = octadecyltrimethylammonium bromide) was synthesized and investigated comprehensively its antitumor activity on MCF-7 and A549 cells. The dimeric structure and amorphous morphology were characterized by FT-IR, UV-vis-DRS, SEM and XRD. The in vitro MTT assay of ODA10[(PW11Fe)2] showed anticancer activity on MCF-7 and A549 cells in a dose- and time-dependent manner, and the IC50 values for MCF-7 and A549 cells at 48 h were 5.83 μg/ml and 3.23 μg/ml, respectively. The images of the ODA10[(PW11Fe)2]-treated cells observed by inverted biological microscope exhibited the characteristic morphology of apoptosis. Flow cytometric analysis showed cell apoptosis and cycle arrested at S phase induced by ODA10[(PW11Fe)2]. The above results illuminated the main mechanism of the antitumor action of ODA10[(PW11Fe)2] on MCF-7 and A549 cells, indicating that this dimeric phosphotungstate is a promising anticancer drug.


INTRODUCTION
With the increasing morbidity and mortality, cancer has become a major killer that leads to healthand life-threatening for humans all over the world (Sun et al., 2019). At present, the main methods for the treatment of cancer are surgery, drug therapy, radiation therapy and cryotherapy. Among them, chemotherapy is also an effective method for cancer treatment (Xiao et al., 2019;Chen et al., 2020;Liu et al., 2020). Many chemotherapeutic agents, such as cisplatin (Weiss and Christian, 1993;Chen et al., 2018), fluorouracil (Arkenau et al., 2003) and capecitabine (Ssif et al., 2008), etc., have shown the potential for alleviating symptoms and curing the cancer (Cao et al., 2020;Geraldi, 2020;Zhao et al., 2020). However, most chemotherapeutic drugs possess inherent disadvantages, such as poor selectivity, severe side effect, low efficiency and drug resistance in cancer cells (Carr et al., 2008;Jonckheere et al., 2014;Hamis et al., 2018). Therefore, it is necessary to design drugs with high efficiency and low toxicity.
Polyoxometalates (abbreviated as POMs) are a series of transition metal oxygen anion clusters, which are mainly composed of molybdenum (Mo Ⅵ ), tungsten (W Ⅵ ), vanadium (V Ⅴ ), niobium (Nd Ⅴ ), and tantalum (Ta Ⅴ ) in their highest oxidation state bridged by oxygen atoms (Rhule et al., 1998;Wang et al., 2003). Intriguingly, many other elements can be incorporated into the framework of POMs, leading to the diversity in structures and properties (Dianat et al., 2015), such as redox potential, polarity, thermal stability and electronic properties, etc., making them attractive for application in the fields of catalysis (Mizuno et al., 2005;Hill, 2007), electrochemistry (Goura et al., 2020), material science (Du et al., 2010) and medicine (Muller et al., 1998). Jasmin et al. (1974) firstly reported the antivirus activity of (NH 4 ) 17 Na [NaSb 9 W 21 O 86 ] (HPA-23) against sarcoma virus. Since then, more POMs have been found to exhibit antitumor (Boulmier et al., 2017;Bijelic et al., 2019), antibacterial (Ma et al., 2020), antivirus (Qi et al., 2013), and antidiabetic activities (Liu W. J. et al., 2016). It is reported that POMs are significant antitumor drug candidates with high efficiency and low toxicity for curing most types of cancers, such as pancreatic cancer, breast cancer, leukemia, colon cancer, ovarian cancer and so on Hu et al., 2019).
The unique advantage of POMs over current drugs lies in the fact that the molecular structure and physiochemical properties of POMs are tunable and can be easily synthesized from readily available precursors in a few synthetic steps (Judd et al., 2001;Müller et al., 2006). POMs can be surface modified with synthetic organic compounds or natural molecules to effectively improve the biological activity in vitro and/or in vivo (Sun et al., 2016;Van Rompuy and Parac-Vogt, 2019). Electrostatic interaction, as a method of surface modification of POMs, combining organic countercations (such as quaternary ammonium salts) with POMs anions together, which makes the formed POMs take advantage of the synergistic effect and enhance the antitumor activity (Yu et al., 2014;Qu et al., 2017;Cheng et al., 2018). Quaternary ammonium salts are widely used as an antibacterial agent against a variety of bacteria, fungi and virus (Diz et al., 2001), which is based on the diversity in properties of low-molecular weight, outstanding cell membrane penetration, extended residence time, low toxicity, good biological activity and environmental stability (Dizman et al., 2006).
On the other hand, Keggin-type POMs were gaining increased interest as antitumor and antivirus agents due to the simple structure, small size and being easily synthesized (Shigeta et al., 2003;Zheng et al., 2009; (Dianat et al., 2013;Mathias et al., 2020) (Santos et al., 2012) were reported, the further studies on the antitumor efficacy and mechanism of dimeric Keggin-type phosphotungstate with quaternary ammonium cation are not very frequent.
The exact mechanism of cancer cells death induced by POMs is still unknown, but it is reported that the antitumor activity of POMs correlates with their biological activities, including immunomodulatory (Sun et al., 2010), apoptotic  and inhibition effects toward enzymes (Prudent et al., 2008). Due to the lack of a comprehensive research on the biological mechanism of POMs, compared to much more common organic drugs, POMs as inorganic drugs are still rarely applied in pharmacy field (Guo and Sadler, 1999). So, considerable attention has been paid to the cellular and molecular mechanisms between tumor cells and POMs.
In the present work, we have chosen the quaternary ammonium salt with relatively long alkyl chain of octadecyltrimethylammonium bromide (ODAB) as organic counteraction, which is expected to exhibit better biocompatibility and higher cell membrane penetration, because the biological activity of quaternary ammonium salts correlates with their molecular structure and the length of the carbon chain. The longer alkyl chain of the compounds contributes to higher antibacterial activity of theirs (Abel et al., 2002)

General Measurements
The content of P, W, Fe in the phosphotungstate was performed on an ICP-OES Plasma Spec (Thermo iCAP 6000), and the elemental analysis of C, H, N was tested on a CHN elemental analyzer (Perkin-Elmer 2400). Thermogravimetric analysis (TGA) was measured on a Shimadzu DTG-60 instrument using N 2 , with a heating rate of 6°C min −1 . Fourier Transform Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 632838 2 infrared (FT-IR) spectroscopy was tested by Nicolet-Impact 400 spectrometer using KBr disk. The UV-vis diffuse reflectance spectra (UV-vis DRS) were performed on a UV-Vis-NIR spectrometer (Agilent Technologies Cary Series) using BaSO 4 as reference. X-ray diffraction (XRD) data were tested by a SHIMADZU XRD-6000 X-ray diffractometer with Cu Kα radiation (λ 0.1548 nm). The morphology of the phosphotungstate was analyzed by scanning electron microscope (SEM) performed on a JSM-6360LV microscope. Particle size distribution was obtained by dynamic light scattering (DLS) using a Mastersizer 2000 laser particle size analyzer.

Cell Lines and Cell Culture Conditions
Human breast cancer cells MCF-7 and human non-small cell lung cancer cells A549 (ATCC) were incubated in RPMI 1640 medium with penicillin (100 U/ml), streptomycin (100 μg/ml) and FBS (10%) at 37°C with 5% CO 2 in an incubator.

Cell Viability Studies
The anti-proliferation effect of the phosphotungstate was detected by the MTT assay. The stock solution of ODA 10 [(PW 11 Fe) 2 ] with the concentration of 1 mg/ml was prepared in DMSO, and then sterilized by the methyl cellulose ester filter membrane with pore size 0.22 μm. At last, the stock solution was diluted by RPMI 1640 under sterile condition.
Cells were seeded into a 96-well plate at a density of 1 × 10 4 cells/well. 100 μl/well of RPMI 1640 medium was added to each well to incubate the cells for 24 h, and then the medium was replaced by various concentrations of ODA 10 [(PW 11 Fe) 2 ] (1, 3, 6, 12, 24 μg/ml). Each concentration has four duplicate samples. After 6, 12, 24 and 48 h, 20 μl of MTT (5 mg/ml) was added to each well, and then the plate continued to be incubated for 4 h in the incubator. Next, the formed formazan crystals were dissolved in 150 µl DMSO after removing the MTT medium. Finally, the absorbance was measured at a wavelength of 490 nm by an automatic microplate reader.

Morphological Observation
To observe whether the density and morphology of the tumor cells induced by the phosphotungstate changed or not, cells were seeded into a 6-well plate at a density of 2 × 10 5 cells/well for 24 h at 37°C, and then treated with different concentration of ODA 10 [(PW 11 Fe) 2 ] (1-24 μg/ml). After 24 h, the cellular density and morphology were observed by the inverted biological microscopy (XDS1C, Shanghai Wanheng Precision Instrument Co. Ltd., China).

Flow Cytometry Analysis of Cell Apoptosis
Cells were seeded into a six-well plate (2 × 10 5 cells/well) for 24 h, and then exposed to different concentration of ODA 10 [(PW 11 Fe) 2 ] (1-24 μg/ml). After 24 h, the cells were collected, washed thrice with cold PBS and then centrifugated. After discarding the supernatant, the cells were resuspended in Annexin-V-FITC/PI solution and remained in the dark for 15 min at room temperature. The cell apoptosis was determined on a FAC Scanto ™ flow cytometer (Becton Dickinson, United States).

Flow Cytometry Analysis of Cell Cycle Distribution
Cells were seeded into a six-well plate (10 6 cells/well) for 24 h at 37°C, and then exposed to different concentration of ODA 10 [(PW 11 Fe) 2 ] (1-24 μg/ml). After 24 h, the cells were collected using trypsin, centrifugated and then washed with PBS for two times. After being fixed by ice-cold 70% ethanol at 4°C overnight, the cells continued to be washed with cold PBS and resuspended in propidium iodide (PI) staining solution in the dark at 37°C for 30 min. Finally, the cell cycle was determined on a FAC Scanto ™ flow cytometer (Becton Dickinson, United States).

Synthesis and Characterization of the Dimeric Keggin-Type Phosphotungstate
The adjustment of pH is vital to the synthesis of the dimeric oxobridged [(PW 11 FeO 39 ) 2 O] 10anion, which usually exists at pH 3-5 . The dimeric phosphotungstate ODA 10 [(PW 11 Fe) 2 ] consists of two phosphotungstate units PW 11 Fe Ⅲ O 39 which are linked by Fe-O-Fe bond, the structure is shown in Figure 1A.
UV-vis DRS of the dimeric ODA 10 [(PW 11 Fe) 2 ] is shown in Figure 1C. The absorption at 260 nm was due to O→W charge transfer transition. The bands at 349 nm and 412 nm corresponded to O→Fe charge transfer transition of oxo-bridged di-iron complexes (Kurtz, 1990). Another evidence of Fe-O-Fe bond presented the characteristic absorptions at 466, 530 and 621 nm which were attributed to O→Fe charge transfer transitions (Kurtz, 1990). So, the IR and UV-vis DRS results all indicated that ODA 10 [(PW 11 Fe) 2 ] possessed dimeric structure.
The SEM image of the dimeric ODA 10 [(PW 11 Fe) 2 ] is shown in Figure 1D. The particles of ODA 10 [(PW 11 Fe) 2 ] were slightly irregular in shape and the particle size was about 510 nm measured by DLS ( Figure 1E), which was consistent with the result of SEM image. The obtained particles showed amorphous morphology, which might be caused by the relatively long carbon chain of organic countercations, making no crystalline feature observed in ODA 10 [(PW 11 Fe) 2 ], which was also proved by the results of XRD pattern of ODA 10 [(PW 11 Fe) 2 ] ( Figure 1F). The spectrum recorded for ODA 10 [(PW 11 Fe) 2 ] indicated an amorphous feature due to the lack of crystallinity caused by ODAB, which had strong diffraction peaks at the 2θ degree of 6.9°-10.3°and a weak broad peak at 14.3°-39.2°. The characteristic diffractions of Keggin-type polyoxometalate were detected at 8.28°, 8.9°, 9.1°, 27.9°and 28.9°, etc (Jalil et al., 2003), so it is concluded that ODA 10 [(PW 11 Fe) 2 ] was of Keggin-type structure. The broad peak at 14. 3°-39.2°m anifested again that the poor crystallinity of ODA 10 [(PW 11 Fe) 2 ] resulted from the longer carbon chain of quaternary ammonium cations. The above results of SEM and XRD proved that the dimeric ODA 10 [(PW 11 Fe) 2 ] had amorphous morphology and contained Keggin-type structure after quaternary ammonium cations combining with heteropoly anions.

Anticancer Activity Studies
The in vitro anti-proliferation activity of ODA 10 [(PW 11 Fe) 2 ] on MCF-7 and A549 cells was evaluated by the MTT assay. Cells were exposed to different concentrations of ODA 10 [(PW 11 Fe) 2 ] (1, 3, 6, 12, 24 μg/ml) for 6, 12, 24 and 48 h. As shown in Figures 2A,B,    O (abbreviated as TMA) containing relatively short alkyl chain at the same concentration, which was shown in Figures  2C,D. After treatment of the drugs for 24 h, ODA 10 [(PW 11 Fe) 2 ] showed the highest anticancer effect, while nearly no inhibition efficacy against MCF-7 and A549 cells was induced by TEA and TMA, that is because the longer alkyl chain in the quaternary ammonium cation of ODA 10 [(PW 11 Fe) 2 ] possessed better cell membrane penetration (Abel et al., 2002), which is beneficial for ODA 10 [(PW 11 Fe) 2 ] to interact with tumor cells, thus ODA 10 [(PW 11 Fe) 2 ] exhibited excellent antiproliferation effect on MCF-7 and A549 cells. The above results demonstrated that the dimeric Keggin-type POMs modified by quaternary ammonium cation with longer alkyl chain showed higher antitumor activity.
The anticancer activity of ODA 10 [(PW 11 Fe) 2 ] on MCF-7 and A549 cells was further compared with that of the clinical chemotherapeutic agents, such as cisplatin and carboplatin, under the same conditions, which was presented in Figure 3. After treatment of the drugs for 24 h, ODA 10 [(PW 11 Fe) 2 ] showed the strongest inhibitory effects against MCF-7 and A549 cells compared to cisplatin and carboplatin. From the above results, it can be demonstrated that ODA 10 [(PW 11 Fe) 2 ] exhibited an anti-proliferation effect on the tumor cells in a dose-and time-dependent manner, and can be utilized as an antitumor drug candidate for the treatment of cancer.

Cell Morphology
The changes of the cellular density and morphology of MCF-7 and A549 cells with the treatment of different doses of ODA 10 [(PW 11 Fe) 2 ] were directly detected using an inverted microscope ( Figure 4). After treatment of 24 h, cells of control group were nested distribution, flattened and showed normal cell architecture. The cell shape was regular polygon and few round cells existed. While the morphology of the ODA 10 [(PW 11 Fe) 2 ]-treated cells obviously changed. The cells were becoming round, shrunken, altered adherence, as well as the appearance of a large number of cell membrane blistering, which was the characteristic morphology of apoptosis. Moreover, the density of the cells was reduced with the drug concentration increasing, showing a dose-dependent effect. This phenomenon indicated that ODA 10 [(PW 11 Fe) 2 ] could induce cell apoptosis.

Flow Cytometry Analysis of Cell Cycle Distribution
In order to detect whether the antiproliferation effect against MCF-7 and A549 cells of ODA 10 [(PW 11 Fe) 2 ] is caused by cell cycle arrest, MCF-7 and A549 cells were treated with different  Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 632838 6 doses of ODA 10 [(PW 11 Fe) 2 ] (1, 3, 6, 12, 24 μg/ml) for 24 h with PI staining. The content of DNA was examined by flow cytometry. Generally, the process of cell replication is related to the doubling of DNA and other cellular contents. There are four distinct phases divided from cell cycle distribution: G 1 , S, G 2 and M phase, the entry to which is carefully regulated by different checkpoints. S phase is responsible for the synthesis of DNA. The cell prepares to divide during G 2 phase and division takes place during M phase. Then, the cells continue to divide after passing these checkpoints. Moreover, many external factors such as drugs, radiation and ROS (reactive oxygen species) can induce DNAdamage during S which causes the death of cells (Rodriguez-Vargas et al., 2012;Preya et al., 2017).
The results of cell cycle arrest of MCF-7 and A549 cells induced by different concentrations of ODA 10 [(PW 11 Fe) 2 ] from 1 to 24 μg/ml are shown in Figure 6. The cell population at S phase of MCF-7 cells in the drug-treatment group increased from 17.82 to 32.92%, in a dose-dependent effect, which was higher than that in control group (17.71%). The level of G 2 /M phase exhibited no obvious variations after treatment of ODA 10 [(PW 11 Fe) 2 ], accompanied by a significant reduction in G 1 phase ( Figure 6A). Similar to MCF-7 cells, as shown in Figure 6B, for A549 cells, the level of S phase increased from 13.8% in the control group to 16.89, 18.47, 36.63, 44.39, and 45.51%, respectively, with the proportions of G 1 and G 2 /M phase decreasing. The ratios of G 1 and G 2 /M phase decreased from 55.74 to 26.7% and 33.08 to 24.19%, respectively. Since DNA replicates during S phase, the above results manifested that DNA damaged at S phase and the antitumor mechanism on MCF-7 and A549 cells was S phase arrest.