Jung Mo Lee1,2, Hey Soo Kim1, Arum Kim1, Yoon Soo Chang1, Jin Gu Lee3, Jaeho Cho4, Eun Young Kim5. 1. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. 2. Department of Internal Medicine, National Health Insurance Service Ilsan Hospital, Goyang, Korea. 3. Department of Thoracic and Cardiovascular Surgery, Yonsei University College of Medicine, Seoul, Korea. 4. Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Korea. 5. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. narae97@yuhs.ac.
Lung cancer is a leading cause of cancer mortality worldwide. The American Cancer Society estimated that, in the year 2021, 235760 new cases of lung cancer will occur and 131880 patients will die of this devastating disease in the United States of America.1 In recent years, there has been increasing awareness of the molecular pathways that drive malignancy, particularly in lung adenocarcinoma, and the development of agents that target these pathways. Although the availability of new systemic therapeutic agents represents substantial progression, these agents are not sufficiently efficacious.2 Moreover, the median and 5-year survival rates for patients with all stages of non-small cell lung cancer (NSCLC) and small cell lung cancer are poor; and compared to the progress made for other types of cancers, improvements in survival in recent years have been marginal at best.Radiotherapy is one of the main treatment modalities in lung cancer, and it has an important role in all stages of lung cancer, as either definitive or palliative therapy. However, tumor radioresistance and dose-limiting toxicity restrict the curative potential of radiotherapy.34 Therefore, novel approaches are needed to overcome these limitations and to augment the effects of radiation. Ionizing radiation provokes the activation of signal transducer and activator of transcription 3 (STAT3), which contributes to the development of radiation resistance through various mechanisms, including the upregulation of antiapoptotic genes, the alteration of cell cycle, the promotion of proliferation, and the induction of epithelial to mesenchymal transition.5 Aberrations in the apoptotic pathway after irradiation can attenuate the therapeutic effects of radiation. Therefore, manipulation of radiation-induced apoptosis is a strategy to overcome radioresistance in NSCLC.Apoptosis involves a cascade of caspase proteases released from the mitochondria and regulated by Bcl-2 proteins. The Bcl-2 family is comprised of antiapoptotic members, such as Bcl-2, Mcl-1, and Bcl-xL, and proapoptotic members, such as Bax, Bak, and Bid.6 The balance between cell survival and cell death is modulated by the ratios and interactions of antiapoptotic and proapoptotic Bcl-2 family proteins.7 Defects in the apoptotic pathway correlate with cellular resistance to therapy and are frequently observed in NSCLC.89 Moreover, the overexpression of Bcl-2 or Bcl-xL has been observed in several cancers, and elevated expression of Bcl-2, Mcl-1, and Bcl-xL is related to resistance to radiation-induced apoptosis.1011BH3 mimetics show a biochemical affinity for specific antiapoptotic BCL-2 proteins, which is linked to their ability to kill specific cells.1213 ABT-737, a “first-in-class” BH3 mimetic, is a small molecule inhibitor that binds with high affinity to Bcl-xL, Bcl-2, and Bcl-w, inducing the mitochondrial pathway of apoptosis that begins with the permeabilization of the mitochondrial outer membrane. Some studies have shown that ABT-737 can enhance the radiosensitivity of various solid cancers. However, to the best of our knowledge, the radiosensitizing effect of ABT-737 has yet to be investigated in NSCLC.In the current study, we hypothesized that the inhibition of Bcl-2 might enhance the tumoricidal effects of radiation in lung cancer. Therefore, we explored an approach to overcome radiation resistance by targeting antiapoptotic Bcl-2 family proteins using a combination of ABT-737 with radiation in K-ras mutant NSCLC cell lines and a lung cancer-induced LSL K-ras G12D and p53fl/fl mouse model.
MATERIALS AND METHODS
Cell lines, chemicals, and antibodies
A549 and H460 cells were purchased from ATCC (Manassas, VA, USA) in 2012. ABT-737 was obtained from AdooQ Bioscience (Irvine, CA, USA), and its chemical and crystal structures are described elsewhere.1415 Antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), unless stated otherwise.
Cell Irradiation Immunoblotting
A549 and H460 lung cancer cells were irradiated at doses specified in each experiment of up to 6 Gy using an X-rad 320 irradiator (Precision X-Ray, North Branford, CT, USA). The cells were harvested on ice using 2× Laemmli sample buffer containing protease and phosphatase inhibitors (Sigma–Aldrich, St. Louis, MO, USA). After sonication, 30–50 mg of lysate was separated by gel electrophoresis on 7.5%–12% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories Inc., Richmond, CA, USA). The expression level of each protein was measured using ImageJ (NIH, Bethesda, MD, USA) and quantified relative to that of β-actin.
Immunofluorescence
A549 and H460 lung cancer cells were irradiated with a dose of 4 Gy. After irradiation, the cells were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air for 24 h. After incubation, the cells were fixed and permeabilized with cold 100% MeOH and subsequently incubated overnight with primary antibodies against Bcl-xL, LC3A/B, and p62 in antibody diluent at a 1:100 dilution. After this step, the cells were subsequently incubated for 90 min with secondary antibodies against Alexa Fluor 488 and Alexa Fluor 555 in antibody diluent at a 1:1000 dilution, followed by incubation with DAPI in phosphate-buffered saline (PBS) for DNA staining for 1 min. Images were taken using a confocal microscope (Zeiss; LSM780). All experiments were replicated three times. The corrected total cell fluorescence (CTCF) was measured using ImageJ.
Clonogenic assay
A549 and H460 lung cancer cells were treated with 0.1% dimethylsulfoxide (DMSO) or 1 µM ABT-737 and then irradiated with a dose of 2 Gy. After irradiation, the cells were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air for 10–14 days. After incubation, the cells were rinsed with PBS and stained for 30 min with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet.16 After staining, colonies were counted using a cutoff of 50 viable cells. Experiments were conducted in triplicate, and means, standard deviations, and p values were calculated using Student’s t-tests.
Reverse transcription polymerase chain reaction
A549 and H460 cells were irradiated with a dose of 2 Gy. After irradiation, total RNA was extracted using TRI reagent (Ambion, Austin, TX, USA). Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using TaqMan gene expression assay reagents (Thermo Fisher Scientific, Waltham, MA, USA) and a StepOnePlus RT-PCR system (Applied Biosystems, Carlsbad, CA, USA) using an inventoried primer-probe set. Three independent experiments were conducted.
Mouse irradiation and imaging
LSL K-ras G12D and p53fl/fl mice were obtained from the NCI mouse repository (http://mouse.ncifcrf.gov/), bred, and genotyped according to the supplier’s guidelines. AdCre virus was obtained from the Gene Transfer Vector Core of the University of Iowa (Iowa City, IA, USA). LSL K-ras G12D and p53fl/fl mice inhaled 5×107 PFU AdCre virus at 8 weeks after birth. Then, 12±2 weeks after AdCre particle inhalation, the mice were randomized and treated with either vehicle or ABT-737 (50 mg/kg, intraperitoneal injection, daily) for 3 days. The mice underwent microcomputed tomography (micro-CT) using a small animal eXplore Locus micro-CT (GE Healthcare, Little Chalfont, UK) under isoflurane anesthesia (45 µm resolution, 80 kV, 450 µA current), and were irradiated in the left lung with 10 Gy using an X-rad 320 irradiator (Precision X-Ray, North Branford, CT, USA). After 2 weeks, the second round of micro-CT was performed to determine the therapeutic effects, and lungs were harvested for histological analysis.Treatment response was evaluated by CT image analysis. We detected all measurable tumors on CT images. To measure the changes in tumor size, the longest diameter of the tumor on each side of the lung was quantified using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Changes in tumor size (%) were calculated as follows: (diameterbefore–diameterafter)/diameterbefore×100 for each side of the lung.
Immunohistochemistry
Mice were sacrificed after the second micro-CT, and the expression of activated caspase-3 was analyzed by immunohistochemistry (IHC) using the LABS2 System (Dako, Carpinteria, CA, USA) according to the manufacturer’s instructions. In brief, sections were deparaffinized, rehydrated, immersed in 3% H2O2 in methanol solution. Then, they were incubated overnight with primary antibodies against activated caspase-3 in antibody diluent (Dako) at a 1:100 dilution. Sections were incubated for 10 min with biotinylated linker and processed using avidin/biotin IHC techniques. 3,3'-Diaminobenzidine (DAB) was used as a chromogen in conjunction with the Liquid DAB Substrate kit (Novacastra, Newcastle, UK). The number of apoptotic bodies per high-power field (400×) was counted in five fields, and the mean numbers were compared using Student’s t-tests. Protein expression was measured and quantified using ImageJ.
Statistical analysis
Independent sample t-tests were used for univariate analysis of continuous variables. Differences in tumor area changes among groups were analyzed by Mann-Whitney U tests. Predictive factors for overall survival were calculated using the Kaplan-Meier estimate. SPSS software (v. 23; IBM Corp., Armonk, NY, USA) was used for statistical analysis. All statistical analyses were two-tailed, and p values of less than 0.05 were considered statistically significant.
Ethical approval and consent to participate
This animal study was approved by the Institutional Animal Care and Use Committee of the Department of Laboratory Animal Resources, Yonsei Biomedical Research Institute, Yonsei University College of Medicine (2015-0307), and followed the guidelines of the American Association for the Assessment and Accreditation of Laboratory Animal Care.
RESULTS
Irradiation treatment increased the expression of antiapoptotic proteins in A549 and H460 cells
Overexpression of Bcl-2 family antiapoptotic proteins is associated with resistance to radiotherapy. To identify antiapoptotic molecules influenced by irradiation, A549 and H460 cells were irradiated, and the expression of antiapoptotic proteins of the Bcl-2 family was evaluated by immunoblotting at 24 h and 48 h after irradiation. At 24 h after irradiation, the expression levels of Bcl-xL, Bcl-w, Mcl-1, and γ-H2AX increased as the dose of radiation increased. Similar results were obtained at 48 h after irradiation, but this change was more pronounced after 24 h (Fig. 1A and B). To determine the effects of irradiation in vivo, LSL K-ras G12D and p53fl/fl mice were irradiated in the left lung with 10 Gy (Fig. 1C). To evaluate the effects of radiation therapy on the same subject, only the left lung of the mouse was treated with radiation, and the irradiated lung (Lt) and nonirradiated lung (Rt) were compared. After the first mouse was sacrificed, the lungs were harvested 4 h after irradiation, and the second mouse was sacrificed 24 h after radiation treatment. The expression of antiapoptotic proteins was evaluated by immunoblotting using lung tissue lysates at 4 h and 24 h after irradiation. Similar to the results in A549 and H460 cells, the expression levels of Bcl-xL, Bcl-2, Bcl-w, and Mcl-1 were increased in the irradiated lungs at 4 and 24 h after irradiation (Fig. 1D). Confocal microscopy revealed an increase in Bcl-xL expression as a response of A549 and H460 cells to 4 Gy of radiation. The expression of Bcl-xL (CTCF) was increased after 4 Gy of irradiation in both cell lines (p<0.05) (Fig. 1E). Additionally, we performed IHC analysis of Bcl-xL. The percentage of Bcl-xL-positive cells was calculated by randomly selecting five different sites. A total of 28.14% of cells in the control lung (right lung) and 57.45% of cells in the irradiated lung (left lung) were positive for Bcl-xL (p<0.001), indicating that radiation augmented a subset of antiapoptotic molecules (Fig. 1F).
Fig. 1
Irradiation treatment increased the expression of antiapoptotic proteins in A549 and H460 cells. (A and B) A549 and H460 cells were irradiated, and the expression of antiapoptotic members of the Bcl-2 protein family was evaluated by immunoblotting 24 h and 48 h after irradiation. (C) Schematic depiction of the radiation protocol. (D) LSL K-ras G12D and p53fl/fl mice were irradiated only in the left lung, and the irradiated lung (Lt) and nonirradiated lung (Rt) were compared. The expression of antiapoptotic members of the Bcl-2 protein family was evaluated by immunoblotting using lysates from left and right lung tissues. (E) Confocal microscopy images of A549 and H460 cells after immunofluorescence for Bcl-xL (green) protein at 24 h after irradiation (magnification: 800×). (F) H&E staining and Bcl-xL immunohistochemical analysis of both lungs of the mice. For immunohistochemistry, DAB was used as a chromogen (magnification: 400×). Group averages are graphed, and error bars represent the S.E.M.
Upregulation of Bcl-2 family proteins was caused by irradiation-mediated STAT 3 activation and induction of the autophagic pathway
To evaluate changes in the expression of antiapoptotic molecules at the transcriptional level after irradiation, A549 and H460 cells were irradiated, and RT-PCR was used to quantify the mRNA expression of antiapoptotic molecules. The mRNA expression levels of Bcl-w and Bcl-xL were increased in A549 cells; the increase in Bcl-w mRNA was more prominent than those of other molecules (Fig. 2A). Bcl-2 mRNA was not detected in A549 cells, consistent with the known reference data (http://www.proteinatlas.org/ENSG00000171791-BCL2/cell/CAB000003). In H460 cells, only the Bcl-2 mRNA level rose, and there was no clear increase in the mRNA expression of other molecules at 6 h and 24 h after irradiation (Fig. 2A). Taken together, these findings did not adequately explain why irradiation induced overexpression of antiapoptotic members of the Bcl-2 protein family at the transcriptional level; another mechanism or mixed mechanism was expected to modulate protein expression.
Fig. 2
Upregulation of Bcl-2 family proteins was caused by irradiation-mediated STAT3 activation and induction of the autophagic pathway. (A) A549 and H460 cells were irradiated with a dose of 2 Gy, and mRNA expression of antiapoptotic molecules was evaluated by real-time PCR at 6 h and 24 h after irradiation. *p<0.05. (B) A549 and H460 cells were irradiated with a dose of 1 Gy, and the phosphorylation of Src, JAK2, and STAT3 was evaluated by immunoblotting at the indicated times after irradiation. (C) A549 and H460 cells were exposed to 1 Gy of radiation and/or 50 nM dasatinib for 2 h. The levels of phospho (p)-Src, p-JAK2, p-STAT3, and p-H2AX (Ser139) were evaluated by immunoblotting 2 h after treatment. Actin was used as a loading control. (D) A549 and H460 cells were irradiated, and the expressions of LC3A/B, Atg 7, and p62 were evaluated by immunoblotting 24 h after irradiation. (E) Confocal microscopy images of A549 and H460 cells after immunofluorescence for LC3A/B (green) and p62 (red) proteins at 24 h after 4 Gy of radiation (magnification: 800×). *p<0.05. (F) A549 and H460 cells were exposed to 2 Gy of radiation and/or 2.5 µM wortmannin for 24 h. The levels of LC3A/B, Atg 7, p62, p-H2AX (Ser139), and antiapoptotic proteins were evaluated by immunoblotting 24 h after treatment. Actin was used as a loading control.
Therefore, we next tested whether the augmented expression of the antiapoptotic protein was mediated by STAT3 activation. To this end, we evaluated the levels of phosphorylated Src, Janus kinase (JAK) 2, and STAT3 by immunoblotting after 1 Gy irradiation in A549 and H460 cells. The expression of p-Src, p-JAK2 and p-STAT3 was increased after irradiation in H460 cells. In A549 cells, the levels of phosphorylated JAK2 were increased after irradiation, and phosphorylated Src and STAT3 peaked at 2 h after irradiation and then decreased with the passage of time. These findings suggested that the phosphorylation of Src was related to the phosphorylation of STAT3 through JAK2 over time (Fig. 2B). To confirm the effects of radiation on the expression of Bcl-xL by irradiation-mediated activation of the Src pathway, additional experiments were performed using the Src inhibitor dasatinib (Fig. 2C). A549 and H460 cells were exposed to 1-Gy radiation and/or 50-nM dasatinib for 2 h. The levels of phospho (p)-Src, p-JAK2, p-STAT3, and p-H2AX (Ser139) were evaluated by immunoblotting at 2 h after treatment. Pretreatment with dasatinib suppressed the elevation of p-STAT3 by irradiation in both cell lines.Additionally, to identify the effects of posttranscriptional modification of antiapoptotic members of the Bcl-2 protein, we also examined the changes in proteins associated with irradiation and autophagy. The expression levels of LC3A/B, p62, and Atg7 were evaluated by immunoblotting at 24 h after 2 Gy of irradiation. In A549 cells, the expression of LC3A/B, mainly the expression of the 14 kD band LC3A/B-II, increased with a rise in the radiation dose. The expression of LC3A/B-II approached a peak at 4 Gy of radiation and then gradually diminished. The expression of ATG7 approached a maximum at 2 Gy of radiation and steadily declined. The p62 expression was partially augmented after 1 Gy of radiation and decreased with an increase in the radiation dose. In H460 cells, the expression of LC3A/B and LC3A/B-II approached a peak at 2 Gy of radiation and then gradually diminished. The expression of ATG7 was augmented in the radiation groups compared to the control group, although dose-specific changes were not constant. The expression of p62 increased partially after 1 Gy of radiation and was reduced with increasing doses of radiation (Fig. 2D). In immunofluorescence staining of A549 and H460 cells after 4 Gy of radiation, the expression of LC3A/B and p62 (CTCF) was increased after 4 Gy of irradiation in both cell lines (p<0.05) (Fig. 2E). To confirm the effects of radiation on the expression of antiapoptotic proteins by irradiation-mediated activation of autophagy, additional experiments were performed using the autophagy inhibitor wortmannin (Fig. 2F). A549 and H460 cells were exposed to 2 Gy of radiation and/or 2.5 µM wortmannin for 24 hours. The levels of LC3A/B, Atg 7, p62, p-H2AX (Ser139), and antiapoptotic proteins were evaluated by immunoblotting 24 hours after treatment. In A549 and H460 cells, the expression of LC3A/B-II increased after irradiation but decreased when the cells were treated with radiation and wortmannin. The expression of ATG7 also increased after irradiation and decreased when treated with wortmannin and radiation together. The expression of p62 diminished after 2 Gy of radiation but increased slightly when the cells were treated with radiation and wortmannin. The expression of the antiapoptotic proteins Bcl-xL and Bcl-w was augmented after 2 Gy of radiation alone but reduced when the cells were treated with radiation and wortmannin together. Collectively, radiation treatment induced the activation of autophagy and augmented the expression of antiapoptotic proteins, but cotreatment with wortmannin suppressed this elevation in the expression of antiapoptotic proteins in both cell lines (Fig. 2F). In conclusion, these data demonstrated that the elevation of apoptotic molecules was mediated in part by the JAK/STAT transcriptional pathway and in part by induction of the autophagic pathway.
ABT-737 enhanced the radiosensitivity of A549 and H460 cells
To confirm the radiosensitizing effects of ABT-737, clonogenic assays were performed with A549 and H460 cell lines (Fig. 3A). The cells were treated with 1 µM ABT-737 or DMSO for 1 h and exposed to 2 Gy of radiation. The number of surviving clones in the combination treatment group was significantly lower than that in the group treated with radiation or ABT-737 alone. In A549 cells, the number of surviving clones in the combination treatment group was reduced to 32.6% compared to the group treated with either ABT-737 alone (80.4%, p=0.001) or radiation alone (52.2%, p=0.021). In H460 cells, for the combination treatment group, the number of surviving clones was decreased to 9.8% compared to that in the group treated with either ABT-737 alone (75.6%, p<0.001) or radiation alone (30.8%, p=0.003) (Fig. 3B). These results indicated that exposure of A549 and H460 cell lines to concurrent treatment with ABT-737 and radiation therapy enhanced the radiosensitivity compared to radiation alone.
Fig. 3
ABT-737 enhanced the radiosensitivity of A549 and H460 cells. (A) After treatment with 1 µM ABT-737 or DMSO for 1 h, the cells were exposed to 2 Gy of radiation, and clonogenic assays were performed. (B) The number of surviving clones in each group was counted using a cutoff of 50 viable cells.
Combination of ABT-737 and irradiation showed increased antitumor efficacy in the KrasG12D:p53fl/fl lung cancer mouse model
To investigate whether ABT-737 could enhance radiosensitizing effects in NSCLC in vivo, we used a KrasG12D:p53fl/fl lung cancer mouse model (Fig. 4A). Responses were measured by micro-CT (Fig. 4B). Statistically significant reductions in tumor size were observed in the combination treatment group, in which the mice were pretreated with ABT-737 and irradiated (p=0.044; Mann-Whitney U test) (Fig. 4C). Lungs were harvested after the second round of micro-CT imaging and analyzed by H&E and IHC (Fig. 4D). In H&E staining, disrupted tumor structures with apoptotic bodies were most frequently observed in the irradiated lungs of ABT-737-pretreated mice. To quantify the apoptotic effects of this combination treatment, IHC analysis of activated caspase-3 was performed. Activated caspase-3 was more abundant in lungs irradiated with ABT-737 pretreatment than in the other groups (Fig. 4E). To determine the effects of combination treatment with ABT-737 and irradiation on survival and toxicity, lung cancer-induced LSL K-ras G12D and p53fl/fl mice pretreated with vehicle or ABT-737 and subjected to irradiation of both lungs were monitored, and their body weights were regularly determined for 80 days after treatment. The median survival times were 95 and 82 days for the vehicle and ABT-737 groups, respectively. Kaplan-Meier survival curves showed that survival was improved in the combination group, although there were no significant differences between the two groups (Fig. 4F). Moreover, body weights in the two groups decreased after approximately 10 days of treatment, and showed a tendency to recover thereafter. Body weight changes in the combination group were not significantly different compared to those in the monotherapy group. Therefore, these data suggested that combination treatment with irradiation and ABT-737 caused no obvious systemic toxicity (Fig. 4F).
Fig. 4
Combination of ABT-737 and irradiation increased antitumor efficacy in a KrasG12D:p53fl/fl lung cancer mouse model. (A) Treatment schedule for lung cancer-induced LSL K-ras G12D and p53fl/fl mice. Eight-week-old heterozygous LSL K-ras G12D and p53fl/fl mice were treated with 5×107 PFU AdenoCre virus by inhalation. At 12±2 weeks, the mice were randomized and pretreated with either vehicle or ABT-737 for 3 days. Then, mice were irradiated only in the left lung. Micro-CT was performed twice: before irradiation and 2 weeks after irradiation. (B) The treatment response was evaluated by comparing micro-CT images taken before and after treatment. Yellow arrow refers to the representative tumor mass observed in micro-CT images. H; heart, L; liver. (C) Waterfall plot showing tumor responses after treatment. Each column represents one individual lung for each mouse. P values were obtained by Mann-Whitney U tests. (D) H&E staining and activated caspase-3 immunohistochemical analysis in the lungs of mice. For immunohistochemistry, DAB was used as a chromogen (magnification: 400×). Positively stained cells with activated caspase-3 are marked with red arrows. (E) The number of apoptotic bodies per high-power field (400×) is presented as a histogram; apoptotic bodies were counted in five fields. (F) Effects of combination treatment with ABT-737 and irradiation on overall survival and changes in body weights in mice. Changes in body weights were compared by Mann-Whitney U tests.
In summary, we observed significant tumor reduction in lung cancer-induced LSL K-ras G12D and p53fl/fl mice following combined treatment with ABT-737 and irradiation compared to vehicle or radiation treatment alone.
DISCUSSION
Overexpression of antiapoptotic Bcl-2 proteins has been observed in various tumor types, and is associated with resistance to radiotherapy.171819 In this report, we also showed that irradiation induced the overexpression of antiapoptotic Bcl-2 proteins. However, the underlying molecular mechanisms of overexpression of antiapoptotic molecules after radiation remain unclear.20Ho, et al.18 demonstrated that both the RNA and protein levels of Bcl-xL were elevated after irradiation in A549 cells, and that radiation enhanced STAT3 phosphorylation. Additionally, Li, et al.21 demonstrated that irradiation activated the phosphorylation of STAT3 and increased the invasion of A549 cells. In the present study, the mRNA levels of antiapoptotic molecules were augmented in A549 cells. Additionally, the phosphorylation of Src was related to the phosphorylation of STAT3 through JAK2; p-Src, p-JAK2, and p-STAT3 levels were diminished after treatment with the Src inhibitor dasatinib. Overall, our findings demonstrated that the elevation of apoptotic molecules was mediated by the JAK/STAT transcriptional pathway.Unlike A549 cells, the increase in mRNA expression was not clear in H460 cells. This suggested that mechanisms other than transcriptional regulation may be involved in mediating the expression of antiapoptotic proteins. Autophagy is another factor affecting protein expression at the posttranscriptional level. The expression levels of LC3A/B and Atg7 were augmented after irradiation with increasing doses of radiation up to 2 or 4 Gy and then diminished, similar to the pattern of γ-H2A.X and anti-apoptotic protein expression. This phenomenon might be related to cell viability after radiation. In other words, up to a certain dose of radiation, the expression of the related protein is increased. However, if the radiation dose increased up to the lethal dose, the cell viability would be decreased significantly, and the protein expression would also be decreased accordingly.We showed increased LC3-II protein levels together with a slight reduction in p62 protein levels and intense LC3A/B expression by confocal microscopy, indicating increased autophagic flux.22 According to previous studies, irradiation can induce “early” expression of p62 mRNA, and intensification of p62 transcription can lead to overexpression in the context of increased autophagy flux in lung cancer cell lines.2324 We considered that this discrepancy in the blotting and confocal microscopy seems to be due to differences in the time-point. Additionally, the increased autophagy and expression of antiapoptotic proteins after irradiation were reduced when A549 and H460 cell lines were treated with wortmannin, an autophagy inhibitor. Some studies have shown intensified autophagic flux after radiation treatment in NSCLC cell lines.2325 Jo, et al.26 showed that autophagy appeared earlier than apoptosis after irradiation, and that a portion of apoptosis was autophagy-dependent in malignant glioma cells. Therefore, our findings demonstrated that radiation induced an increased expression of autophagy, indicating that the autophagic pathway induced by irradiation was associated with the elevation of apoptotic proteins and radioresistance in NSCLC.Although the mechanisms of cancer radioresistance are complicated and affected by numerous factors, it is clear that the overexpression of antiapoptotic proteins is closely related to the radioresistance of cancer cells.27 Our study showed overexpression of antiapoptotic proteins after irradiation, and targeting these proteins could be a potential method to enhance the effects of irradiation treatment. ABT-737 binds Bcl-2 and Bcl-xL with high affinity, and many Bcl-2 family inhibitors, including ABT-737, have shown radiosensitizing effects in a variety of tumor types.282930 Additionally, we tried to investigate the molecular mechanism of the radiosensitization effect of ABT-737 by showing the increase of activated caspase-3 and cleaved PARP through immunoblotting. Unfortunately, since its expressions were not evident in the cell lines used in these experiments, we were not able to further investigate the significant relationships of ABT-737 and the radiosensitization effect. Additional experiments should be performed to determine exactly how ABT-737 affects radiosensitization.ABT-737 and ABT-263 (the oral form of ABT-737), as single agents in clinical trials, showed efficacy in hematologic malignancies but demonstrated limited success in solid tumors.3132 Although a few studies have demonstrated the radiosensitizing effects of ABT-737 in some solid tumors, including breast, head and neck, and cervical cancer,20303334 there are limited data describing the radiosensitizing effects of ABT-737 in lung cancer as a single agent. Only one study showed the radiosensitizing effect of combination treatment with ABT-737 and a mTOR inhibitor in NSCLC cells;35 however, these effects were only demonstrated in an in vitro study and lung cancer xenograft model. The cancer stroma is distinct from the normal stroma and provides a supportive microenvironment for tumor growth; xenografts do not reflect this tumor microenvironment.36 We used genetically engineered mouse models, which mimic the mechanisms of human malignancies and are thought to be more clinically relevant animal models than xenograft models.37 We first evaluated the radiosensitizing effects of ABT-737 using micro-CT image analysis, which enabled quantitative analysis, in a real-time, noninvasive manner. Furthermore, in most studies regarding the radiosensitizing effects of ABT-737, researchers showed the efficacy of the drug, but did not perform an assessment of the toxicity of the drug. In the radiation treatment of mice in our study, body weight changes were not significant between the ABT-737 and control groups. However, additional assessment of drug toxicity is needed. Survival analysis of the mice over 80 days showed that the ABT-737 combination group survived longer compared to the control group, although a significant difference was not observed between the two groups. In a study of the natural course of LSL K-ras G12D and p53fl/fl mouse models using AdCre virus inhalation, the median survival of mice was 76 days.38 In that study, the mice were infected with 2.5×107 PFU AdCre virus, which was half the level of our research. Considering that the amount of inhaled virus increased the tumor burden, our experimental data showed the prolonged survival of mice with a higher tumor burden. That is, radiation therapy or combination treatment might increase the survival of mice compared to nonirradiated, nontreated mice. The lack of difference between the radiation group and combination group in our research may be due to the small number of animals and the short observation period. Additional studies are needed to supplement these findings.Another study reported that selective inhibitors of Bcl-xL, BXI-61, and BXI-72 exhibit more potent efficacy in lung cancer compared to ABT-737.39 The study demonstrated that BXI-72 showed a better anticancer effect compared to BXI-61 and ABT-737, and BXI-72 showed a radiosensitizing effect in a NSCLC xenograft model. However, our study has its own significant advantage of using GEMM, which could reflect the tumor microenvironment. Additionally, BXI-72 did not show a significant difference from ABT-737 in terms of side effects.In conclusion, this preclinical study supported the therapeutic potential of ABT-737 treatment to sensitize lung cancer to radiotherapy. The rational therapeutic targeting of Bcl-2 family proteins by ABT-737 is a promising strategy to overcome possible resistance in K-ras mutant NSCLC to standard radiotherapy. Clinical trials are warranted to determine whether this novel strategy may benefit patients with NSCLC.
Authors: M M Borner; P Brousset; B Pfanner-Meyer; M Bacchi; S Vonlanthen; M A Hotz; H J Altermatt; D Schlaifer; J C Reed; D C Betticher Journal: Br J Cancer Date: 1999-02 Impact factor: 7.640
Authors: Ilias V Karagounis; Dimitra Kalamida; Achilleas Mitrakas; Stamatia Pouliliou; Maria V Liousia; Alexandra Giatromanolaki; Michael I Koukourakis Journal: Br J Cancer Date: 2016-07-05 Impact factor: 7.640
Fig. 1
Fig. 2
Fig. 3
Fig. 4