Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells.

Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.

Improved tympanic membrane regeneration after myringoplastic surgery using an artificial biograft.

Tympanic membrane perforations are due to common otologic problems. The current treatments to heal tympanic membrane perforation, such as myringoplasty, have some disadvantages, including the need for autologous grafting, which is rapidly absorbed by the organism before perforation recovery is complete. To improve the structural and functional tympanic membrane healing after surgery, we propose a new branch of artificial grafts. In this study, we report the development of artificial grafts using electrospun bioabsorbable polymers. Polymers such as poly (l-lactic acid) and poly (lactic-co-glycolic acid) acted as the scaffold for cell growth in a co-culture of fibroblasts and keratinocytes. This co-culture promoted the growth of an epithelial-equivalent tissue over the electrospun scaffold, which was used as an alternative graft in myringoplasty. The in vivo study was performed in Sprague Dawley rats. Ear endoscopy was performed 30days after surgery and showed that tympanic membrane perforations treated with artificial grafts healed naturally, completely and with the possibility of maintaining their actual functionality. In conclusion, our study described a new artificial graft created specifically to fulfill the requirements of perforated tympanic membrane healing processes, which are compatibility, proper durability and less intense side effects following myringoplasty.

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFß/SMAD (transforming growth factor-ß/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.