Rabbit Anti-Human S6 monoclonal antibody, clone 0I9M3 [Alexa Fluor 647]

Renal cell carcinoma (RCC) is a urologic malignant cancer and often diagnosed at an advanced stage, which results in high mortality. Targeted therapy may improve the quality of life and survival of patients who are not suitable for nephrectomy. Everolimus, an mTOR inhibitor, is currently used as sequential or second-line therapy for RCC refractory to Sunitinib or sorafenib. However, its efficiency is palliative. In this study, we evaluated whether the antitumor activity of everolimus against RCC is enhanced by Selumetinib, a selective MEK1 inhibitor. We discovered that everolimus in combination with Selumetinib synergistically inhibited the proliferation of Caki-1, 786-O and 769-P cells in vitro. Mechanistically, this combination decreased p-RPS6 and p-4E-BP1 dramatically, which causes G1 cell cycle arrest and prevents reactivation of AKT and ERK. In vivo, the antitumor efficacy and pharmacodynamic biomarkers of the combination therapy were recapitulated in Caki-1 xenograft model. In addition, this combination treatment potently inhibited angiogenesis in xenograft models by impairing VEGF secretion from tumor cells. Our findings provide a sound evidence that combination of everolimus and Selumetinib is a potential dual-targeted strategy for renal cell carcinoma.

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen-or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.