Fig 1.
RT53 induces rapid cancer cells membranolysis.
(A) Non-malignant (HaCat and MRC-5) and cancerous (U2OS, Lu1205, MCA205, Jurkat, HUT78 and HL-60) cells were treated with 20 μM of RT53. Plasma membrane permeability was evaluated at the designed time points by measuring extracellular LDH into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. (n = 3) (B) U2OS cells were treated with 20 μM of RT53 or the control peptide RT53M for 3 h. Plasma membrane permeability was evaluated as in (A). (C) Mice red blood cells were incubated with 20 μM of RT53. Released hemoglobin was detected by densitometry at 540 nm. Hemoglobin release by cells treated with 1% Triton X-100 was used as 100% lysis control.
Fig 2.
RT53 induces unregulated necrotic cell death.
(A) Ultrastructural analysis of RT53-mediated cell death. U2OS cells were left untreated (Control) or exposed to 15 μM of RT53 for 30 min. Cells were then analyzed by transmission electron microscopy following osmium tetroxide staining. (B) U2OS cells were exposed to 20 μM of RT53 in the presence or absence of 50 μM zVAD-fmk, 50 μM Necrostatin-1 (Nec-1) or 100 mM cyclosporin A (CsA) for 1 h. Necrotic cell death was monitored by lactate dehydrogenase (LDH) release from cells into the culture medium. The obtained values were normalized to those of the maximum LDH released (completely lysed) control. Data are means±s.e.m. (n = 3).
Fig 3.
RT53 triggers calreticulin exposure as well as the release of HMGB1 and ATP.
(A) U2OS cells stably expressing CRT-GFP were treated with 10 μM of RT53 or 1 μM mitoxantrone for 6 h, in the presence or absence of zVAD-fmk. Cells were then, fixed, stained for DNA, and examined by fluorescence microscopy. (B) U2OS cells were left untreated or treated with 10 μM of RT53 or 200 nM thapsigargin (TG) as a positive control for 6 h. Cell lysates were analyzed by Western blot for phosphorylated and total protein eIF2α. (C) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular HMGB1 was then measured in the culture supernatant. Data are means±s.e.m. (n = 3). (D) U2OS cells were left untreated or exposed to increasing concentrations of RT53 for 3h. Extracellular ATP was then measured in the culture supernatant. Data are means±s.e.m. (n = 3).
Fig 4.
RT53 treatment prevents tumor growth.
(A) Closed circle: RT53-treated cancerous cells induce anti-tumor immunogenicity in a prophylactic tumor vaccination assay. Syngeneic C57BL/6 mice were injected s.c. on the left flank with 2x106 MCA205 cells treated in vitro with RT53. Eight days later, the mice were challenged subcutaneously on the right flank with 0.5x106 live MCA205 cells and tumor growth was monitored. (n = 6 per group). Open circles: effect of intratumoral injection of RT53 on the growth of established tumors. Palpable MCA205 tumor on C57BL/6 mice were injected intratumorally with 300 μg RT53 once a day for 3 days. Open triangles represent the growth in saline-treated controls. Tumor growth was monitored on day 1 of injections (left panel). (n = 6 per group). (B) Percentage of tumor-free mice at day 21.
Fig 5.
Intratumoral administration of RT53 induces tumor necrosis, immune cells infiltration and inflammation.
(A) Plasma was harvested 4, 24, 48 or 96 h following single intratumoral injection of normal saline (control) or 300 μg RT53 in normal saline and IL-1β (left panel) as well as IL-6 (right panel) levels were estimated using ELISA. Data from 3 animals are presented for each time point as mean±SEM. (B) Established MCA205 fibrosarcomas were surgically excised 24 h or 96 h post intratumoral injection with normal saline (control) or 300 μg RT53 in normal saline and sections subjected to H&E staining (top) or stained for CD3 or rabbit IgG isotype control (middle and bottom, respectively). (C) Number of infiltrating CD3-positive cells per view field following CD3 staining. Data from 3 animals are presented as mean±SEM. (D) Relative transcription of CCL2 and CXCL10 (normalized to GAPDH) as determined by real-time RT-PCR on samples of tumors 24 h after RT53 or normal saline injection. Values of CCL2 and CXCL10 are represented as fold change relative to untreated tumors, set to 1 (mean±SEM; n = 3).