Synthesis, characterization and antiproliferative effects of naphtho [2,3-b] thiophen-4,9-quinone on bladder tumor cells

Abstract Naphthoquinones are natural plants products or synthesized compounds. They have α, β-cyclic aromatic dienones structure with a naphthalene skeleton. Little is known about naphthoquinone and nothing about naphtho [2,3-b] thiophen-4,9-quinone effects on bladder cancer. In this study, a naphthoquinone containing a hetero sulfur atom was synthesized using classical synthetic method. The molecular structure was elucidated by NMR techniques and the antitumor effects were evaluated on bladder tumor cell lines with different TP53 status using tripan blue and MTT cytotoxic method, quantification of reactive oxygen species (ROS), wound healing, cell morphology and cell cycle progression assays. The results showed selective cytotoxicity, colonies reduction, morphological change, inhibition of the cell migration process, induction of ROS production and cell cycle arrest. Naphtho [2,3-b] thiophen-4,9-quinone presents antiproliferative activity regardless TP53 status and may be a promising agent in the treatment of bladder cancer, as they have an oxidizing effect and interfere with cell cycle. Graphical Abstract


Introduction
Bladder cancer is the 10th most common cancer and the 13th most deadly in the world (Saginala et al. 2020). Mutations in the TP53 gene are frequently found in bladder cancer cells and the therapeutic response depends on the presence and type of TP53 mutation and the treatment used. Currently, different protocols for treatment are available (Saginala et al. 2020), however the high failure rates and toxicity observed encourage the search for potential natural products or synthetic molecules inspired by natural product as a therapeutic alternative (Demain and Vaishnav 2011).
In the scenario of research on new molecules with potential antitumor activity, naphthoquinones are of great interest. Naphthoquinones are natural products biosynthesized primarily in monocotyledonous angiosperm plant species (Thomson 1991). Structurally, they are naphthalene-derived molecules with two carbonyl groups. They have several structural forms which 1,4-naphthoquinone is the most stable (Qiu et al. 2018).
Although naphthoquinones have a history of activity against several types of cancer cells, little is reported about their activity on bladder tumor cells. In this context, the aims of this study were to elucidate the molecular structure of the naphthoquinone naphtho [2,3-b] thiophen-4,9-quinone, a synthetic naphthoquinone, and investigate the antineoplastic potential in bladder carcinoma cell lines with different TP53 status by exploring its effect on cell proliferation, clonogenic survival, production of reactive oxygen species (ROS), migration, cell morphology and cell cycle progression.
The synthesis of naphtho [2,3-b] thiophen-4,9-quinone was performed employing the commercial reagents phthalic anhydride (Sigma-AldrichV R ) and thiophene (Sigma-Aldrich V R ). The synthesis was done according to Zani et al. (1997). In a round bottom flask, phthalic anhydride (3.0 g, 20.3 mmol) was dissolved in 30 mL of dichloromethane and then 5 ml of aluminum chloride suspension (22.5 mmol) was added drop by drop. After stirring the mixture for 30 min at room temperature, 1.0 mL (12.5 mmol) of thiophene was slowly added under vigorous addition. After 3 hours, the reaction was stopped by the addition of 100 mL of HCl (4.0 M). The reaction mixture was transferred to a separatory funnel and was extracted three times with 50 ml of dichloromethane. The combined organic phases were extracted with a NaOH solution (1.0 M). Thenoylbenzoic acid was precipitated by acidification with 2.0 M HCl. Thenoylbenzoic acid and was subjected to cyclization and oxidation by heating in concentrated sulfuric acid (100 C, 2 h). The reaction mixture was incubated at room temperature. Then it was incubated in ice leading to the precipitation of thiophen-4,9-naphthoquinone. The precipitate was recrystallized in a methanol:acetic acid solution (0.1%) with a final yield of 48% (Figure 1). The chemical structure was characterized by NMR. The 1 H NMR and 13 C NMR spectra were obtained from the Departamento de Farm acia, Universidade Federal de Ouro Preto, in Bruker V R Avance DRX400 equipment (400 MHz), at 25 C, using deuterated chloroform (CDCl 3 ) to solubilize the samples. From the data obtained by the NMR 1 H, NMR 13 C, DEPT-135, HSQC and HMBC spectra it was possible to characterize the naphtho [2,3-b] thiophen-4,9-quinone chemical structure. The data are presented in the Table 1 and Figures S1 to S8.

Cytotoxicity, clonogenic survival assay and quantification of ROS
In relation to cytotoxicity, the IC 50 values (0.62 mg/mL for RT4 cells, 0.43 mg/mL for T24 cells and 4.54 mg/mL for MRC5 cells) and the selectivity indexes (7.32 for RT4 cells and 10.55 for T24 cells) were calculated based on results of MTT assay and shown in the Table S1. The results suggest selective cytotoxicity of NQ for bladder tumor cells. In addition, the results of the trypan blue exclusion assay reinforce the cytotoxic capacity of NQ in these cell lines. After treatment with 0.7, 0.9, 1.25 and 2.50 mg/mL of NQ, the percentage of viable cells in relation to the untreated control was 38.8, 42.7, 33.3 and 14% for RT4 respectively and 24.4, 24.0, 13.1 and 6.5% for T24 ( Figure S9).
A cancer cell that loss the ability to produce a large clone, after exposition to a chemotherapy agent, is also considered a dead cell. Thus, the most suitable assay to measure tumor cell death is the clonogenic assay which permits to observe the relationship between the insult-producing agent and the proportion of cell survival curve (Munshi et al. 2005). Survival curves are generated for many established cell lines growing in culture, neoplastic or normal, from various origins, including humans and rodents. In this assay, in relation to RT4 cells, there was a significant decrease in the number of colonies in the four NQ concentrations tested (0.7, 0.9, 1.25 and 2.5 mg/mL). In the T24 cells, there was a significant decrease in the number of colonies only at the 2.5 mg/mL concentration compared to the untreated control ( Figure S10). Thus, the results observed could indicate that compound cause a reduction in cellular reproductive capacity, suggesting that it induce DNA lesions that interfere with clonogenic potential, prevent the cell to proliferate indefinitely and decrease tumor aggressiveness. Some studies using this assay show the ability of some naphthoquinones to reduce cell proliferation in tumor cells. For example, juglone, [2-(4-methoxyanilino)-1,4naphthoquinone] and [2-(4-hydroxyanilino)-1,4-naphthoquinone], both associated with ascorbate, induced a decrease in cell colonies of the T24 bladder tumor cells (Kviecinski et al. 2012;Felipe et al. 2013). The reactive oxygen species (ROS) quantification assay showed that NQ significantly induced the production of ROS at the four concentrations tested in the two bladder tumor lines. At the highest concentration (2.5 mg/ml), in RT4 and T24 cells, there was an increase of approximately five times in ROS production compared to the untreated control ( Figure S11). ROS are by-products of mitochondrial metabolism in eukaryotic cells. They are reactive molecules that have unpaired electrons and that, in low concentrations, perform essential functions in cells, such as controlling the action of caspases during the apoptosis process. When there is an imbalance in ROS concentrations in the cell, it generates oxidative stress causing DNA damage, which is associated with the carcinogenesis process. An unbalanced increase in ROS can lead to cancer cell death. Therefore, a greater increase in ROS caused by naphthoquinones may have a desirable cytotoxic action and be a potential antitumor candidate (Veskoukis et al. 2012;Wellington 2015;Pereyra et al. 2019).
Naphthoquinones can be reduced to semiquinones (free radicals) and hydroxyquinone. Under aerobic conditions, an electron is reduced, generating free radicals as an intermediate, such as superoxide (O2 -). Two electron reduction can also occur in an anaerobic situation through the enzyme NADPH quinone oxireductase 1 (Pereyra et al. 2019). The b-lapachone naphthoquinone, for example, presents a selective cytotoxicity that may be linked to the overexpression of NADPH quinone oxireductase 1 in tumor cells (Pink et al. 2000). A study conducted by Kumar et al. (2009) demonstrated the potential cytotoxic and genotoxic effect of 1,4-naphthoquinone through the induction of oxidative stress in B16F1 melanoma tumor cells.
It is possible to suggest, based on experimental data and those reported in the scientific literature, that the cytotoxic effects caused by NQ to RT4 and T24 cells may be associated with the ability of this naphthoquinone to produce a significant amount of ROS.

Wound healing, cell morphology and cell cycle progression assays
The wound healing assay revealed that NQ inhibited migration in T24 high tumor grade cells (mutated TP53, Figure S12-B). In RT4 low grade cells (TP53 wide type), there was no change in cell migration compared with the untreated control cells ( Figure  S12-A). The detailed mechanism by which the naphthoquinones exert effects on cell migration remains unclear. However, previous study has suggested that the anti-metastatic properties of this family of compounds could be mediated by the reduction in the activities of matrix metalloproteinase (MMP) enzyme (Liew et al. 2014), commonly secreted by tumor cells and related to the process of metastasis in cancers (Zhang et al. 2015). Zeng et al. (2016) observed that the MMP-9 levels were significantly increased in patients with higher grade tumors, which justifies the results observed.
After the treatment with NQ both cell lines showed change in cell morphology (elongated cells) ( Figure S13). According to Talman et al. (2011), the changes at morphology might be correlated with reduced cell-to-cell contact and cell death. Furthermore, similar changes in HeLa cell morphology have been reported as a consequence of the inhibition of the PI3K/AKT/ERK signaling pathway (Mishra et al. 2010).
The values of the nuclear division index (NDI) showed that NQ decreases cell cycle progression in the two tested cells, confirming the antiproliferative effect of this molecule. In RT4 cells there was a significant decrease in NDI at concentrations of 1.25 and 2.5 mg/mL, while in cells T24 the significant decrease was found to the concentrations of 0.7, 1.25 and 2.5 mg/mL ( Figure S14). There are studies in the literature showing naphthoquinones limiting cell cycle progression in tumor cells such as HepG2 (Castro et al. 2021) and B16F1 Kumar et al. 2009).

Experimental section
See supplementary material.

Conclusion
In conclusion, naphthoquinones, especially the naphtho [2,3-b] thiophen-4,9-quinone, have antiproliferative activity regardless TP53 status and may be a promising agent in the treatment of bladder cancer, as they have an oxidizing effect and interfere with cell cycle.