Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis

Abstract In this study, we investigated the effects and molecular mechanisms of 2‐phenylbenzimidazole‐5‐sulphonic acid (PBSA), an ultraviolet B protecting agent used in sunscreen lotions and moisturizers, on ovarian cancer cell responses and tumour angiogenesis. PBSA treatment markedly blocked mitogen‐induced invasion through down‐regulation of matrix metalloproteinase (MMP) expression and activity in ovarian cancer SKOV‐3 cells. In addition, PBSA inhibited mitogen‐induced cell proliferation by suppression of cyclin‐dependent kinases (Cdks), but not cyclins, leading to pRb hypophosphorylation and G1 phase cell cycle arrest. These anti‐cancer activities of PBSA in ovarian cancer cell invasion and proliferation were mediated by the inhibition of mitogen‐activated protein kinase kinase 3/6‐p38 mitogen‐activated protein kinase (MKK3/6‐p38MAPK) activity and subsequent down‐regulation of MMP‐2, MMP‐9, Cdk4, Cdk2 and integrin β1, as evidenced by treatment with p38MAPK inhibitor SB203580. Furthermore, PBSA suppressed the expression and secretion of vascular endothelial growth factor in SKOV‐3 cells, leading to inhibition of capillary‐like tubular structures in vitro and angiogenic sprouting ex vivo. Taken together, our results demonstrate the pharmacological effects and molecular targets of PBSA on modulating ovarian cancer cell responses and tumour angiogenesis, and suggest further evaluation and development of PBSA as a promising chemotherapeutic agent for the treatment of ovarian cancer.

and integrins mediates aberrant activation of their downstream signalling pathways that triggers uncontrolled and imbalanced responses, suggesting the rational strategy and pharmacological efficacy of RTK/integrin-targeted therapeutics for the treatment of ovarian cancer. [3][4][5] In addition, matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) and cellular components in the tissue microenvironment, are closely associated with cancer cell growth and progression as well as normal tissue remodelling. 6,7 High expression and activity of MMPs have been reported to contribute to aggressive phenotypes and poor survival of ovarian cancer. 8,9 However, inhibition of MMP activity without sufficient knowledge about substrate specificity or physiological roles in cancer biology has been disappointing in various cancer clinical trials. 10 Therefore, intensive investigations of cellular and molecular networks underlying the progression of ovarian cancer may provide insights into effective therapeutic targets and strategies for the prevention and treatment of cancer.
2-Phenylbenzimidazole-5-sulphonic acid (PBSA) has widely been used as a sunscreen agent to protect skin from ultraviolet (UV) radiation, resulting in reduction of sunburn, early skin ageing and skin cancer. In parallel to the protective effect against UV-induced cyclobutane pyrimidine dimers, PBSA has also been reported to generate a variety of free radicals and exhibit photosensitizing activity, raising the possibility of phototoxic damage to cellular components including DNA, proteins and lipids. [11][12][13][14] Although the potential side effects and risks of PBSA to ecosystem as well as human health have been proposed in several safety and toxicity studies, chronic exposure to PBSA did not significantly induce pathological changes in the liver or gonads of rainbow trout, a common toxicity model organism. 15 Furthermore, the phototoxic potential of PBSA could be reduced by complexation with hydroxypropyl-β-cyclodextrin. 16 However, the effects and molecular mechanisms of PBSA on cell responses including invasion, adhesion and proliferation have not yet been elucidated in detail. In this study, we report the regulatory roles and molecular mechanisms of PBSA in ovarian cancer cell fates including invasion and proliferation, and tumour-derived angiogenic responses.
F I G U R E 1 PBSA inhibits cell invasion and adhesion. A, The chemical structure of PBSA. B, Cell invasion, (C) adhesion and (D) viability assays were performed as described in Materials and methods. Quiescent SKOV-3 cells were pre-treated with PBSA for 30 min, followed by 10% FBS stimulation for 16 h (invasion), 2 h (adhesion) or 24 h (viability). Results from at least three independent experiments (mean ± SD) are presented as (B) the numbers of invasive cells, (C) the numbers of adherent cells or (D) the percentage of viable cells of total cell counts. Statistical significance is indicated (**P < .01, compared with 10% FBS-treated cells)
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and used between passages 4 and 6 for all experiments.

| RNA purification and reverse transcriptasepolymerase chain reaction (RT-PCR)
Subconfluent SKOV-3 cells in 100 mm dishes (1 × 10 6 cells/well, SPL Life Sciences Co.) were serum-starved for 24 hours in basal DMEM to synchronize cells in G 1 /G 0 phase of the cell cycle and pre-treated with PBSA (10, 50 μmol/L) for 30   subjected to Western blot analysis as described previously. 17 All

| Western blot analysis
Western blots are representative of at least three independent experiments. Bands of interest were integrated and quantified by the use of NIH ImageJ version 1.51j8 software. were rinsed with PBS to remove any residual PBSA and further incubated with 10% FBS for another 12 hours. Cell viability was determined by a Muse ™ cell analyser using cell count and viability assay kit (Merck Millipore), and the cell proliferation was quantified as previously described. 18 The results from triplicate determinations (mean ± standard deviation) are presented as the percentage of viable cells of total cell counts or the fold increase of the untreated controls.  The results (mean ± standard deviation) are presented as the numbers of adherent cells. 19

| Cell invasion assay
The upper side of the transwell insert (Costar Ⓡ , 6.5 mm diame-

| Zymogram analysis
Activities of MMPs were measured by zymography. 21

| Tube formation assay
Tube formation assay was performed to examine the ability of con-

| Statistical analysis
Statistical analysis was performed using Student's t test and was based on at least three different experiments. The results were considered to be statistically significant when P < .05.

| PBSA inhibits cell invasion through the regulation of MMP expression and activity
Cell invasion, migration and adhesion are closely associated with cancer progression and aggressiveness. 6 We first examined the ability of   reversible ( Figure 3D). Collectively, these findings indicate that PBSA possesses anti-invasive, anti-adhesive and anti-proliferative activity against ovarian cancer SKOV-3 cells. F I G U R E 6 PBSA inhibits endothelial tube formation through the downregulation of VEGF. A, Quiescent SKOV-3 cells were pre-treated with PBSA for 30 min, followed by 10% FBS stimulation for (left panel) 24 h or (right panel) 48 h. RT-PCR and ELISA analyses were performed as described in Materials and methods. B, HUVEC tube formation assay was performed using conditioned media from SKOV-3 cell culture treated as described above (A, left panel). Values represent the mean ± SD of at least three independent experiments. Statistical significance is indicated (*P < .05, **P < .01, compared with 10% FBS-treated cells). C, Quiescent HUVECs were incubated for 24 h in complete media containing growth factors with or without PBSA. Results from at least three independent experiments are presented as the fold increase of the untreated controls (left panel) or the percentage of viable cells of total cell counts (right panel)

| PBSA inhibits endothelial cell tube formation in vitro and angiogenic sprouting ex vivo through p38 MAPK -dependent regulation of VEGF expression and secretion in ovarian cancer cells
In the tumour microenvironment, cancer cells produce and secrete numerous biologically active molecules such as growth factors and cytokines, which are closely associated with angiogenic and metastatic responses. [28][29][30] We analysed the changes in the expression and secretion of a key angiogenic factor VEGF in PBSA-treated SKOV-3 cells. As shown in Figure 6A,

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S' CO NTR I B UTI O N S
MSK, JHK, KBK and DWS designed and performed research and wrote the manuscript; EKA, YRC, SH and CHL performed research; GUB and JSO analysed data; KBK and DWS were responsible for supervising the entire project and writing the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.