Journal: Molecular cancer research : MCR

Article Title: α v β 3 INTEGRIN MEDIATES RADIORESISTANCE OF PROSTATE CANCER CELLS THROUGH REGULATION OF SURVIVIN

doi: 10.1158/1541-7786.MCR-18-0544

Figure Lengend Snippet: (A) cRGD peptide blocks αvβ3 integrin mediated adhesion to VN, but not to FN (P<0.01). Adhesion of αvβ3-LNCaP cells to FN or VN was performed in the presence or absence of cRGD peptide (1.74 μM) or control peptide cRAD (1.74 μM). Triplicate experiments were performed. (B) SRB assay also showed that cRGD alone caused reduction of survival of αvβ3-LNCaP cells. Surviving fraction due to different treatments was calculated as OD of treated cells / OD of cells treated with control cRAD. cRGD (2 μM) alone caused significant reduction of cell survival (P<0.01). For comparison, the absolute surviving fraction of cells treated with IR (4 Gy) or combination of cRGD and IR (4 Gy) were shown. (C) cRGD produced synergistic inhibition of αvβ3-LNCaP cell survival in combination with IR at dose-dependent manner. αvβ3-LNCaP cells were collected, counted and incubated with either cRGD or control cRAD for 1 hour prior to IR. Immediately after IR, cells were plated in 96 well plates and incubated at 37 °C in 5% CO2 in the presence of the peptide. A fresh peptide contained medium was exchanged every 3 days during the incubation. To determine the synergistic effect of peptide in combination with IR, survival curves were corrected for the drug effect of the peptide itself: surviving fraction = OD of irradiated cells with the peptide / OD of non-irradiated cells with the peptide. Results were achieved in two experiments using two clones of αvβ3-LNCaP. (D) cRGD in combination with IR completely inhibits anchorage-independent growth of αvβ3-LNCaP cells. αvβ3-LNCaP or mock-LNCaP cells were incubated in the presence or absence of 2 μM cRGD 1 hour prior to irradiation. After either 0 or 5 Gy, 50,000 cells were seeded in a 6-cm soft agar dishes with duplicates. The experiment was repeated twice with 2 different clones of αvβ3-LNCaP with similar results. (E) Representative images of anchorage-independent growth of LNCaP transfectants (0 Gy vs 5 Gy) are presented. The scale bar (equivalent to 400 μ) was added using a software Freehand. (F) Soft-agar assay was used to determine anchorage-independent cell growth. PC-3 cells were incubated in the presence or absence of 2 μM cRGD 1 hour prior to irradiation up to 8 Gy. The experiment was repeated twice. The differences between cRGD and cRAD (control) treatments are significant at high dose of IR (P<0.01). (G) Representative images of anchorage-independent growth of PC-3 cells (0 Gy vs 8 Gy) are presented. The scale bar equals to 400 μ as measured using a microscope.

Article Snippet: Cyclo (-Arg-Gly-Asp-D-Phe-Val) (cRGD) and the control Cyclo (-Arg-Ala-Asp-D-Phe-Val) (cRAD) peptides were from Bachem (Beidendorf, Switzerland).

Techniques: Control, Sulforhodamine B Assay, Comparison, Produced, Inhibition, Incubation, Irradiation, Clone Assay, Software, Soft Agar Assay, Microscopy