BV6, an IAP Antagonist, Activates Apoptosis and Enhances Radiosensitization of Non- small Cell Lung Carcinoma In Vitro  Wenyan Li, MD, PhD, Bo Li, MD, Nicholas J. Giacalone, BS, Artour Torossian, BS, Yunguang Sun, PhD, Kathy Niu, BS, Opal Lin-Tsai, BA, Bo Lu, MD, PhD  Journal of Thoracic Oncology  Volume 6, Issue 11, Pages 1801-1809 (November 2011) DOI: 10.1097/JTO.0b013e318226b4a6 Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1. HCC193 is more sensitive than H460 to BV6. To determine relative sensitivity to BV6 treatment for the two non-small cell lung cancer cell lines, we measured cell survival with MTS assays after DMSO (control) or indicated concentrations of BV6 for 24 hours. Cell survival percentage, measured relative to the control, was significantly more decreased for HCC193 compared with H460. Experiments were performed in triplicate, and the histogram shows the mean ± SD. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2. BV6 reduced levels of cIAP1 and XIAP in both H460 and HCC193. To measure the effect of BV6 on its target proteins, western blots for cIAP1 and XIAP were performed with Actin as the loading control. A, Cells were treated with DMSO (control) or BV6 (1 μM for HCC193 and 5 μM for H460) and harvested at indicated time points. BV6 reduced cIAP1 within 1 hour and gradually decreased XIAP with increased incubation time for both cell lines. B, Cells were treated with DMSO (control) or indicated concentrations of BV6 for 24 hours for HCC193 and 48 hours for H460. BV6 reduced cIAP1 with 0.25 μM BV6 and gradually decreased XIAP with increasing concentration for both cell lines. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3. BV6-induced apoptosis in HCC193 and H460 cell lines. To measure the amount of apoptosis, a Western blot detected caspase-3 over indicated time points after incubation with DMSO (control) or 1 μM BV6 for HCC193 and 5 μM BV6 for H460 cells. Actin was used as the loading control. A higher concentration and incubation time with BV6 was needed for H460 compared with HCC193 to produce cleaved caspase-3. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4. BV6 sensitized HCC193 and H460 non-small cell lung cancer cell lines to radiation treatment. To measure the radiosensitization effects of BV6, clonogenic assays were performed. Both cell lines were treated with DMSO (control), 1 μM BV6 for 24 hours, and, for an additional group of H460 cells, 5 μM BV6 over 48 hours. Within 1 hour of vehicle or BV6 administration, 0, 2, 4, or 6 Gy radiation was applied. A significant dose enhancement ratio (DER) was evident for HCC193 treated with 1 μM BV6 (DER = 1.38) and for H460 treated with 5 μM BV6 (DER = 1.4) (p < 0.05). Each experiment was performed in triplicate, and error bars represent ± SD. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5. BV6 and radiation combination treatment results in increased apoptosis in HCC193 and H460 non-small cell lung cancer cell lines. A, To investigate mechanism of cell death involved in radiosensitization, Western blots detected pro- and cleaved caspase-3. Each group was treated with DMSO (control), BV6 (1 μM for 24hours for HCC193 and 5 μM for 48 hours for H460), 6 Gy radiation, or a combination of the BV6 and radiation treatments. Actin was used as the loading control. Both cell lines produced more cleaved caspase-3 when given the combination treatment than when given BV6 or radiation alone. B, To confirm the presence of apoptosis, Hoechst staining revealed apoptotic characteristics of condensed or fragmented nuclei. The same procedures were used, but an additional set of H460 cells treated with 1 μM BV6 for 24 hours is also shown. There are significantly more apoptotic cells when given combination treatment compared with drug alone or radiation alone treatments. Error bars represent the SE (n = 3). *p < 0.05. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 6. HCC193 predominantly used the extrinsic pathway of apoptosis and H460 predominantly used the intrinsic pathway during radiosensitization. A, To investigate the pathway of BV6- and radiation-induced apoptosis, western blots were performed to detect pro- and cleaved caspase-8 as well as pro- and cleaved caspase-9. Cells were treated in the same manner as in Figure 5A. HCC193 cells increased cleaved caspase-8 while H460 increased cleaved caspase-9 after BV6 or combination treatment. B, To measure amount of TNF-α, an activator of the extrinsic pathway, culture media were analyzed by ELISA following a 12-hour treatment with DMSO (control), 1 or 5 μM BV6 for HCC193, and H460 cells. Tests were performed in triplicate with error bars representing the SD. Significance was defined as p < 0.05, as compared with control. C, To investigate whether the response to BV6 would be diminished in the presence of a TNF-α neutralizing antibody, we measured cell survival by MTS assays in the presence of infliximab. These were performed with DMSO, 1 μM BV6, or 5 μM BV6 with or without 10 μg/ml infliximab and a 24-hour incubation period. In the absence of infliximab, HCC193 cells demonstrated 80% and 61% survival with 1 and 5 μM BV6, respectively. In the presence of HCC193 cells, the survival rates were 84% and 70%, respectively. In H460 cells, the presence of infliximab resulted in a generalized decrease in cell survival for all groups; this drop in survival was also seen in the HCC193 DMSO group. *p < 0.05 and **p < 0.01. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 7. Schematic overview showing the effects of radiation and BV6 on the apoptotic signaling cascade in HCC193 and H460 cell lines. A, In HCC193 cells, apoptosis is primarily activated through the extrinsic pathway. Cell surface death receptors are activated by ligands such as TNF-α and TRAIL, leading to the activation of the extrinsic apoptotic signaling cascade through initiator caspase-8. IAPs exert their antiapoptotic activity by inhibiting both this cascade as well as TNF-α-mediated NF-κB signaling. BV6 blocks these effects by antagonizing IAPs, inducing their degradation, and enhancing TNFα secretion. B, In H460 cells, apoptosis is primarily mediated through the intrinsic pathway. Here, cell stress triggers mitochondria to release the Apaf-1/cytochrome c complex, leading to the activation of the intrinsic pathway through caspase-9. BV6 prevents IAPs from inhibiting this cascade and also induces their proteasomal degradation. Journal of Thoracic Oncology 2011 6, 1801-1809DOI: (10.1097/JTO.0b013e318226b4a6) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions