The severe acute respiratory syndrome‐ 2 (SARS‐CoV‐2) caused the coronavirus disease COVID‐19 responsible for the current pandemic presenting a high mortality rate. The development of vaccines provided a transient relief to the social, economic, and health care systems. However, the virus is constantly mutating to survive, generating new pathogenic variants that jeopardize the ongoing efforts and up‐to‐date accomplishments against it. The TMPRSS2 is a crucial player for SARS‐CoV‐2 cell entry. This transmembrane serine protease intervenes with the viral Spike/Angiotensin‐Converting Enzyme 2 (ACE2) host receptor axis, enabling to initiate the SARS‐CoV‐2 entry to target cells. The subsequent viral replication causes pathophysiological changes including a major complication of the COVID‐19 known as the inflammatory storm (IS). This is characterized by an enhanced release of inflammatory modulators leading to a hyper‐inflammation. TMPRSS2 is expressed in the epithelial cells of several tissues, including the epithelium of the respiratory tract, that is the focus of our study. However, this catalytic enzyme shows poor anchoring in the cell membrane and is consequently released into the extracellular space with the potential to activate or cleave neighboring cells dependent on serine proteases. An example of this cellular scenario is represented by the Protease Activated Receptor‐2 (PAR‐2), associated with activation of inflammatory pathways upon cleavage of the N‐terminus by a serine protease. We hypothesized: 1) that TMPRSS2 co‐localizes with PAR‐2 at the cell membrane of target epithelial cells; 2) TMPRSS2 activates PAR‐2 and associated proinflammatory signaling pathways; 3) the SARS‐CoV‐2 chaotic inflammation together with PAR‐2 activation will worsen the resulting IS. Based on our lab prior work on PAR‐2 and its antagonist, we anticipate that by inhibiting PAR‐2 activation, we will observe a decrease in the COVID‐19 associated IS. In this study, we used the MCF‐7 cell line to standardize the protocols, since this cell line expresses both proteins. They are more resilient to adverse conditions in the lab (due to the pandemic consequences at research labs) and will allow us to establish a proof of concept to be compare with the human primary airway epithelial cells that we will subsequently use. Using MCF‐7 as a model, we established the presence of PAR‐2 and TMPRSS2 via western blot. In addition, by using immuno‐cyto‐fluorescence we confirmed that these two proteins colocalize at the cell membrane. Activation of intracellular calcium‐dependent pathways, detected by intracellular calcium fluorogenic assay, showed that after treating cells with trypsin (Gold Standard for PAR‐2 activation), TMPRSS2, PAR‐2 agonist, and PAR‐2 antagonist, trypsin caused a significant (p < 0.05) increase compared to TMPRSS2 either alone, with the PAR‐2 agonist or the PAR‐2 antagonist with a decreasing of 50 % trypsin activity. Importantly, BrdU assay results showed that PAR‐2 antagonist decreased the proliferation of MCF‐7 cells when compa...

Endogenous proteases are associated with the ovulation process and inflammation, among other human physiological processes. Still their role in the transition from normal tissue to cancer development remains uncertain. For example, the Protease activated receptor 2 (PAR‐2) is a G Protein Couple receptor expressed in mostly in epithelial cells, including the female reproductive system. Previous work from our lab showed that the PAR‐2 antagonist‐FSLLRY‐NH2 inhibited the cell‐mediated inflammation in the airways. The aim of this study is to evaluate the effect of the PAR‐2 antagonist when a normal cell progresses to a cancerous one, using trypsin as a gold standard enzyme for Par‐2 activation. In this case, the transition from normal endometrium, to endometriosis, ovarian. We hypothesized that PAR‐2 antagonist will decrease the activation of its receptor reducing the transition to endometriosis and ovarian cancer. To test this, we performed western blot to determine PAR‐2 expression in endometriosis (12Z) cells, human endometrial stromal cells (HESC) cells, ovarian cancer (A2780) cells, and breast cancer (MCF‐7) cells. In addition, we quantified the activation of PAR‐2 receptor using a Fluorogenic intracellular calcium assay in human normal endometrial (uterine) epithelial cells (HEUEC), ovarian cancer (Caov‐3), and MCF‐7 cells as internal positive control. We also performed the BrdU proliferation assay on these cells. Our results showed a higher expression (p<0.05) of PAR‐2 in A2780 cells (ovarian aggressive cancer) when compared with the other cells lines. The intracellular calcium assay resulted in a significant reduction (p<0.05) once exposed to the PAR‐2 antagonist in the HEUEC cells and Caov‐3 when compared to cells treated with trypsin (p<0.05). In contrast, there was not significant difference in the MCF‐7 cell line upon exposure to treatments. We observed a significant decrease in MCF‐7 proliferation when treated with the PAR‐2 antagonist in comparison to trypsin treatment. Our results suggested that PAR‐2 antagonist can be used as treatment or adjunct therapy for specific female reproductive cancers. Next steps of this study will include the active full‐length PAR‐2 recombinant and test other concentrations of the antagonist to determine the LD50 in the selected cells. Proliferating markers will be measured for all the previous cell lines as well for endometrial cancer.