Silvia Caggia

We have previously shown that heterotrimeric G-protein subunit alphai2 (Gα_i2) is essential for cell migration and invasion in prostate, ovarian and breast cancer cells, and novel small molecule inhibitors targeting Gα_i2 block its effects on migratory and invasive behavior. In this study, we have identified potent, metabolically stable, second generation Gα_i2 inhibitors which inhibit cell migration in prostate cancer cells. Recent studies have shown that chemotherapy can induce the cancer cells to migrate to distant sites to form metastases. In the present study, we determined the effects of taxanes (docetaxel), anti-androgens (enzalutamide and bicalutamide) and histone deacetylase (HDAC) inhibitors (SAHA and SBI-I-19) on cell migration in prostate cancer cells. All treatments induced cell migration, and simultaneous treatments with new Gα_i2 inhibitors blocked their effects on cell migration. We concluded that a combination treatment of Gα_i2 inhibitors and chemotherapy could blunt the capability of cancer cells to migrate and form metastases.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gα_i2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gα_i2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 μΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 μΜ blocked the activation of Gα_i2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gα_i2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gα_i2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gα_i2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Background: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells.Methods: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin‐insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes.Results: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF‐induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR.Conclusion: We suggest that EGF‐induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G‐protein coupled receptor (GPCR), Giα2, PI3‐kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF‐1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF‐stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1‐ and oxytocin‐induced migratory behavior and PI3‐kinase activation without affecting EGF‐induced PI3‐kinase activation and cell migration. Basal‐ and EGF‐induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3‐kinase–AKT–Rac1 axis.

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on melanoma cancer, the present study was undertaken to investigate the biological activity of the resinous exudate of aerial parts from Psoralea glandulosa, and its active components (bakuchiol (1), 3-hydroxy-bakuchiol (2) and 12-hydroxy-iso-bakuchiol (3)) against melanoma cells (A2058). In addition, the effect in cancer cells of bakuchiol acetate (4), a semi-synthetic derivative of bakuchiol, was examined. The results obtained show that the resinous exudate inhibited the growth of cancer cells with IC₅₀ value of 10.5 μg/mL after 48 h of treatment, while, for pure compounds, the most active was the semi-synthetic compound 4. Our data also demonstrate that resin is able to induce apoptotic cell death, which could be related to an overall action of the meroterpenes present. In addition, our data seem to indicate that the apoptosis correlated to the tested products appears, at least in part, to be associated with an increase of reactive oxygen species (ROS) production. In summary, our study provides the first evidence that P. glandulosa may be considered a source of useful molecules in the development of analogues with more potent efficacy against melanoma cells.