N. M. Kunda1, J. Qin1, G. Qiao1, B. Prabhakar2, A. V. Maker1,2 1University Of Illinois At Chicago,Division Of Surgical Oncology, Department Of Surgery, College Of Medicine,Chicago, IL, USA 2University Of Illinois At Chicago,Department Of Microbiology & Immunology, College Of Medicine,Chicago, IL, USA
We have previously demonstrated that human and murine colon cancer cells undergo near complete cell death in vitro and in vivo upon direct exposure to PV-10 (10% rose bengal disodium), a synthetic dye currently in clinical trails for intralesional therapy of in-transit melanoma. Occasional bystander responses have raised the possibility that PV-10 induced cell death can generate an anti-tumor immune response. The mechanism of PV-10 cell death in not known, and it is critical to determine, if rose bengal disodium is to be used as an immunotherapeutic strategy for solid tumors.
Murine colon cancer CT-26 cells were treated with PV-10 concentrations of 0 – 300 μM for 24 hours. For cell cycle studies, cells were fixed with 80% ethanol and DNA staining was performed using DAPI. DNA content was measured with FACS and cell cycle distribution was analyzed. GRP78 and LC3 were analyzed using Western blot. Protein levels of GRP78 were measured to evaluate ER (Endoplasmic Reticulum) integrity, and the conversion of LC3-I to LC3-II was determined as an indicator of stress-induced autophagy.
At clinical concentrations of PV-10, the fraction of cells in the G2-M phase increased from 21%-43% (p=0.05), and the fraction of cells in the G1 phase decreased correspondingly from 58% to 30% (p=0.06), consistent with cell cycle growth arrest. The fraction of cells in the sub-G1 phase increased over 4-fold from 3% to 14% (p=0.04), consistent with DNA degradation and apoptosis. GRP78 expression increased almost 2-fold from 1 to 1.83 upon PV-10 exposure, indicative of ER stress. Similarly, LC3-II expression increased almost 5-fold from 1 to 4.53 with PV-10 exposure, consistent with autophagy-induced cell death (Figure 1).
Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis. In order for cytotoxic agents to act as potential immunotherapeutic strategies in the treatment of solid tumors, immunogenic cell death targeting the endoplasmic reticulum (ER), leading to ER stress may be critical. Therefore, based on these results, further evaluation of PV-10 as a potential agent to stimulate immunologic cell death in solid tumors is warranted.