Functional and mutational analysis after radiation and cetuximab treatment on prostate carcinoma cell line DU145

Background Epidermal Growth Factor Receptor is often overexpressed in advanced prostate carcinoma. In-vitro-studies in prostate carcinoma cell line DU145 have demonstrated increased sensibility to radiation after cetuximab treatment, but clinical data are not sufficient to date. Methods We analyzed effects of radiation and cetuximab in DU145 and A431 using proliferation, colony-forming-unit- and annexin-V-apoptosis-assays. Changes in protein expression of pEGFR and pERK1/2 after radiation and cetuximab treatment were analyzed. Using NGS we also investigated the impact of cetuximab long-term treatment. Results Cell counts in DU145 were reduced by 44% after 4 Gy (p = 0.006) and 55% after 4 Gy and cetuximab (p < 0.001). The surviving fraction (SF) was 0.69 after 2 Gy, 0.41 after 4 Gy and 0.15 after 6 Gy (each p < 0.001). Cetuximab treatment did not alter significantly growth reduction in 4 Gy radiated DU145 cells, p > 0.05 or SF, p > 0.05, but minor effects on apoptotic cell fraction in DU145 were detected. Using western blot, there were no detectable pEGFR and pERK1/2 protein signals after cetuximab treatment. No RAS mutation or HER2 amplification was detected, however a TP53 gen-mutation c.820G > T was found. Conclusions Radiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking MAP-Kinase-pathway using cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab treatment did not induce resistance-mutations. Further research must clarify which combination of anti-EGFR treatment strategies can increase radiation-sensitizing-effects.

TPX2 Enhanced the Activation of the HGF/ETS-1 Pathway and Increased the Invasion of Endocrine-Independent Prostate Carcinoma Cells

The prognosis for endocrine-independent prostate carcinoma is still poor due to its highly metastatic feature. In the present work, TPX2 (the targeting protein for Xklp2), which is known as a micro-tubulin interacted protein, was identified as a novel coactivator of ETS-1, a transcription factor that plays a central role in mediating the metastasis of human malignancies. TPX2 enhanced the transcription factor activation of ETS-1 and increased the expression of ETS-1’s downstream metastasis-related genes, such as mmp3 or mmp9, induced by HGF (hepatocyte growth factor), a typical agonist of the HGF/c-MET/ETS-1 pathway. The protein-interaction between TPX2 and ETS-1 was examined using immunoprecipitation (IP). TPX2 enhanced the accumulation of ETS-1 in the nuclear and the recruitment of its binding element (EST binding site, EBS) located in the promoter region of its downstream gene, mmp9. Moreover, TPX2 enhanced the in vitro or in vivo invasion of a typical endocrine-independent prostate carcinoma cell line, PC-3. Therefore, TPX2 enhanced the activation of the HGF/ETS-1 pathway to enhance the invasion of endocrine-independent prostate carcinoma cells and thus it would be a promising target for prostate carcinoma treatment.

Synergistic In-Vitro Effects through Radiation and Blocking the Epidermal Growth Factor Receptor (EGFR) with the Monoclonal Antibody Cetuximab in Prostate Carcinoma Cell Line DU145

Background: The EGF-receptor is often overexpressed in advanced prostate carcinoma. In-vitro studies in prostate carcinoma cell-line DU145 demonstrated increased sensibilities to radiation with Cetuximab; in-vivo effects were not detected. Methods: In vitro, we analyzed the effect of radiation and Cetuximab in cell-lines DU145 and A431 (reference), using a proliferation assay, colony-forming unit assay, and Annexin-V apoptosis assay. We analyzed changes in the protein expression of pEGFR and pERK1/2 post-radiation and Cetuximab. Additionally, we investigated the impact of Cetuximab long-term treatment on the development of secondary-resistance-mutations. Results: DU145 cell counts were reduced by 44% after 4Gy (p=0.006) and by 55% after 4Gy and Cetuximab (p<0.001). The surviving fraction was 0.69 after 2Gy; 0.41, 4Gy; and 0.15, 6Gy (p<0,001). The additional Cetuximab-treatment did not significantly alter the impact on growth reduction or on the surviving fraction. After radiation and Cetuximab-treatment minor effects on the apoptotic cell-fraction in DU145 were detected. Using western blot, there were no pEGFR and pERK1/2 protein signals after Cetuximab-treatment. While no mutations of RAS, BRAF, PI3KCA and no amplifications of HER2 were detected, there were a TP53 mutation before and after long-term treatment with Cetuximab. Conclusion: Radiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking EGFR-MAP-Kinase pathway with Cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab-treatment did not cause typical resistance mutations in DU145. Further research must clarify whether a combination of anti-EGFR therapeutics and immune-oncological approaches can increase the radiation-sensitizing-effect.

Sclerin, a New Cytotoxic Cyclononapeptide from Annona scleroderma

A new cytotoxic cyclononapeptide, sclerin, cyclo(–Dab1–Ser2–Tyr3–Gly4–Thr5–Val6–Ala7– Ile8–Pro9–) (1), was isolated from the methanol extract of the seeds of Annona scleroderma, together with the known metabolite, cyclosenegalin A, cyclo(–Pro1–Gly2–Leu3–Ser4–Ala5–Val6–Thr7–) (2). The planar structures for the two compounds were established by comprehensive analysis of NMR and ESI-HRMS data, and the absolute stereochemistry was stablished by Marfey’s method. Compound 1 showed moderate cytotoxic activity against the human prostate carcinoma cell line DU-145 at µM concentration.

Photoactivated inhibition of cathepsin K in a 3D tumor model

Collagenolytic activity of cathepsin K is important for many physiological and pathological processes including osteoclast-mediated bone degradation, macrophage function and fibroblast-mediated matrix remodeling. Here, we report application of a light-activated inhibitor for controlling activity of cathepsin K in a 3D functional imaging assay. Using prostate carcinoma cell line engineered to overexpress cathepsin K, we demonstrate the utility of the proteolytic assay in living tumor spheroids for the evaluation and quantification of the inhibitor effects on cathepsin K-mediated collagen I degradation. Importantly, we also show that utilizing the ruthenium-caged version of a potent nitrile cathepsin K inhibitor (4), cis-[Ru(bpy)2(4)2](BF4)2 (5), offers significant advantage in terms of effective concentration of the inhibitor and especially its light-activated control in the 3D assay. Our results suggest that light activation provides a suitable, attractive approach for spatial and temporal control of proteolytic activity, which remains a critical, unmet need in treatment of human diseases, especially cancer.