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Terameprocol (Tetra-O-Methyl Nordihydroguaiaretic Acid), an Inhibitor of Sp1-Mediated Survivin Transcription, Induces Radiosensitization in Non-small Cell Lung Carcinoma

      Introduction:

      Survivin, an inhibitor of apoptosis protein and key regulator of mitosis, is up-regulated in a variety of cancers and is often associated with a worse prognosis. Terameprocol down-regulates the Sp1-mediated transcription of survivin and Cdk1, which is important for cell cycle progression and many other proteins. Survivin inhibition has previously been shown to result in the induction of apoptosis and radiosensitization.

      Methods:

      This study examined the effects of terameprocol administration on survivin transcription and expression in HCC2429 and H460 lung cancer cells. We also examined the combined effects of radiation and terameprocol on apoptosis and radiosensitivity.

      Results:

      Using immunoblot analysis and luciferase assays, we confirmed that terameprocol decreases survivin transcription and protein expression. Ultimately, however, decreases in survivin expression failed to correlate with an increase in apoptosis. Nonetheless, clonogenic assay revealed that terameprocol induces increased radiosensitization in HCC2429 (dose enhancement ratio = 1.26, p = 0.019) and H460 (dose enhancement ratio = 1.18, p = 0.001) cells. Additionally, the data show no effect of terameprocol on cell cycle in either HCC2429 or H460 cells.

      Conclusions:

      Terameprocol significantly enhances the sensitivity of non-small cell lung carcinoma cell lines to radiation therapy, although the mechanism of action remains unclear. Further study is warranted to assess the potential of terameprocol as an agent that may enhance the therapeutic ratio of radiotherapy in lung cancer.

      Key Words:

      Lung cancer continues to be the leading cause of cancer mortality in the United States.
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      Approximately 80% of all lung cancer cases are non-small cell lung carcinoma (NSCLC), and the 5-year overall survival is 15%. Radiation therapy plays an integral role in the multimodality treatment approach of locally advanced NSCLC. The efficacy of radiotherapy, however, is hindered partly by defects in the apoptotic machinery of cancer cells. Mechanisms of apoptosis, therefore, have become promising targets for the development of novel therapeutic agents that enhance radiation-induced cell death and lead to improved clinical outcomes.
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      Radiation-induced apoptosis.
      Apoptosis, or programmed cell death, involves the release of caspase proteases from the mitochondria and is regulated by members of the Bcl-2 proteins family and the inhibitor of apoptosis proteins (IAPs) family.
      • Altieri DC
      Validating survivin as a cancer therapeutic target.
      At 16.5-kDa, survivin is the smallest member of the IAP family and has been shown to play critical roles in inhibiting apoptosis and promoting angiogenesis and cellular proliferation.
      • Ryan BM
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      • Duffy MJ
      Survivin: a new target for anti-cancer therapy.
      Survivin is selectively expressed at G2/M and localizes to the mitotic spindle, playing an integral role in several steps of cell division including centrosome functions and spindle formation.
      • Altieri DC
      Targeted therapy by disabling crossroad signaling networks: the survivin paradigm.
      Inhibition of apoptosis may involve a collection of survivin segregated in mitochondria and subsequently released into the cytoplasm in response to apoptotic stimuli.
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      Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.
      Similar to most members of the IAP family, survivin does not directly inhibit caspases. Survivin's effects on cell death inhibition seem to be mediated by interactions with other proteins, including another IAP, X-linked IAP (XIAP),
      • Dohi T
      • Beltrami E
      • Wall NR
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      Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.
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      Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection.
      and the hepatitis B X-interacting protein.
      • Marusawa H
      • Matsuzawa S
      • Welsh K
      • et al.
      HBXIP functions as a cofactor of survivin in apoptosis suppression.
      The relative balance of proapoptotic factors such as Bax, Bak, and Bid and antiapoptotic factors such as survivin, Bcl-2, and Bcl-XL regulates the level of apoptosis. Thus, overexpression of antiapoptotic survivin promotes cell survival in cancer cells. This also affects the ability of cancer cells to avoid treatment-induced apoptosis and contributes to therapeutic resistance to various agents, including radiation.
      Indeed, overexpression of survivin has been noted in virtually all cancers, including lung,
      • Monzo M
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      A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers.
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      Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.
      breast,
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      Expression of survivin and its relationship to loss of apoptosis in breast carcinomas.
      colon,
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      Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer.
      pancreas,
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      Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors.
      and hematological malignancies,
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      Aberrant expression of caspase cascade regulatory genes in adult T-cell leukaemia: survivin is an important determinant for prognosis.
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      Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays.
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      Relationship between expression of genes involved in cell cycle control and apoptosis in diffuse large B cell lymphoma: a preferential survivin-cyclin B link.
      whereas survivin expression in most terminally differentiated tissues is undetectable.
      • Ambrosini G
      • Adida C
      • Altieri DC
      A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.
      Additionally, survivin has been shown to confer resistance to several anticancer agents,
      • Asanuma K
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      Survivin as a radioresistance factor in pancreatic cancer.
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      A role for survivin in chemoresistance of endothelial cells mediated by VEGF.
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      and high expression of survivin correlates with poorer prognosis in a variety of cancers, including NSCLC.
      • Monzo M
      • Rosell R
      • Felip E
      • et al.
      A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers.
      • Altieri DC
      Survivin, versatile modulation of cell division and apoptosis in cancer.
      Given its importance in cancer, elucidating how survivin is implicated in NSCLC apoptosis may promote the development of novel therapeutic strategies to sensitize NSCLC tumors to treatment and improve clinical outcome.
      In this study, we investigated the role for survivin inhibition in NSCLC radiosensitization. We tested the use of terameprocol (tetra-O-methyl nordihydroguaiaretic acid, formerly known as EM-1421 and M4N), a DNA major groove binder that was previously shown to inhibit survivin expression and induce apoptosis in cancer cells with high levels of survivin in vitro and in vivo,
      • Chang CC
      • Heller JD
      • Kuo J
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression.
      • Park R
      • Chang CC
      • Liang YC
      • et al.
      Systemic treatment with tetra-O-methyl nordihydroguaiaretic acid suppresses the growth of human xenograft tumors.
      as a radiosensitizing agent in NSCLC. The lignan terameprocol targets and inhibits the Sp1-mediated transactivation of survivin transcription. The anticancer activity of terameprocol also stems from its ability to inhibit Sp1-mediated Cdk1 (Cdc2) expression, another protein frequently up-regulated in human cancer that is involved in the phosphorylation of several proteins involved in the G2/M transition.
      • Chang CC
      • Heller JD
      • Kuo J
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression.
      • Huang RC
      • Chang CC
      • Mold D
      Survivin-dependent and -independent pathways and the induction of cancer cell death by tetra-O-methyl nordihydroguaiaretic acid.
      Several studies have already shown that inhibition of survivin enhances apoptosis and sensitizes cancer cells to anticancer therapies.
      • Lu B
      • Mu Y
      • Cao C
      • et al.
      Survivin as a therapeutic target for radiation sensitization in lung cancer.
      • Kim KW
      • Mutter RW
      • Willey CD
      • et al.
      Inhibition of survivin and aurora B kinase sensitizes mesothelioma cells by enhancing mitotic arrests.
      • Kami K
      • Doi R
      • Koizumi M
      • et al.
      Downregulation of survivin by siRNA diminishes radioresistance of pancreatic cancer cells.
      • Mita AC
      • Mita MM
      • Nawrocki ST
      • et al.
      Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics.
      YM155, a small molecule survivin suppressant, has been shown to enhance apoptosis and tumor regression in hormone-refractory prostate tumors in vivo
      • Nakahara T
      • Takeuchi M
      • Kinoyama I
      • et al.
      YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts.
      and to radiosensitize NSCLC cell lines in vitro.
      • Iwasa T
      • Okamoto I
      • Suzuki M
      • et al.
      Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines.
      To date, however, no studies have shown the effect of terameprocol on sensitizing NSCLC to radiation. Our results suggest that there is value in using terameprocol to sensitize NSCLC to radiation, although this effect is likely independent of survivin inhibition.

      MATERIALS AND METHODS

      Cell Culture and Reagents

      Human NSCLC cells were obtained from the following sources: NCI-H460 (H460) from the American Type Culture Collection (Manassas, VA), and HCC2429 was kindly provided by Dr. Thao P. Dang (Vanderbilt University, Nashville, TN). All cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) at 37°C and humidified 5% CO2. Terameprocol (tetra-O-methyl nordihydroguaiaretic acid) was provided by Erimos Pharmaceuticals (Raleigh, NC). A stock solution was prepared in 100% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) and stored at −20°C. The drug was diluted in fresh media before each experiment.

      Plasmids, Transfection, and Dual-Luciferase Assay

      The pLuc and pLuc2931 plasmids were obtained from Health Research, Inc. (Buffalo, NY). The pLuc2931 plasmid contains 2931 base pairs of human survivin promoter, whereas pLuc is a nonpromoter control. HCC2429 and H460 cells were plated at 2 × 104/well in a 24-well format, and 200 ng total DNA was transfected with 0.6 μL Lipofectamine 2000 (Invitrogen), and the pRL Renilla-Thymidine kinase reporter vector (Promega, Madison, WI) was used as a transfection efficiency control. Luciferase activity was detected by the Dual-Luciferase Assay System (Promega).

      Immunoblotting

      H460 and HCC2429 cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed in M-PER mammalian lysis buffer (Thermo Scientific, Waltham, MA). The protein concentration of lysates was quantified using the Bradford reagent (Bio-Rad, Hercules, CA), and equal amounts of protein were loaded into each well and separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Perkin Elmer Life Science), which was then exposed to 5% nonfat dried milk in 0.1% tris-buffered saline Tween 20 for 1 hour at room temperature. The blots were then incubated overnight at 4°C with the following rabbit polyclonal antibodies: anti-survivin (1:2000 dilution; R&D Systems, Minneapolis, MN); anti-cellular inhibitor of apoptosis protein 1 (cIAP1) and anti-cellular inhibitor of apoptosis protein 2 (cIAP2) (1:1000 dilution; MBL International, Woburn, MA); anti-XIAP, anti-Bcl-2, anti-Bcl-XL, anti-Mcl-1 (1:1000 dilution; Cell Signaling Technology, Danvers, MA); anti-Bax and anti-Bak (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase-3 (1:1000 dilution; Cell Signaling Technology); anti-tubulin (1:1000 dilution; Sigma); and anti-β-actin (1:8000 dilution; Sigma). The membranes were then washed with tris-buffered saline Tween 20 before incubation for 1 hour at room temperature with horseradish peroxidase-conjugated goat antirabbit IgG secondary (1:5000; Santa Cruz Biotechnology). Immune complexes were finally detected with enhanced chemiluminescence reagents (Perkin-Elmer Life Science, Waltham, MA).

      In Vitro Clonogenic Assay

      H460 and HCC2429 cells in 100-mm dishes were harvested by exposure to trypsin (0.05% trypsin-ethylenediaminetetraacetic acid (EDTA), GIBCO, Grand Island, NY) and counted. They were diluted serially to appropriate densities and plated in triplicate in 60-mm dish containing 5 ml of complete medium in the presence of 10 μM terameprocol or DMSO (final DMSO concentration of 0.1%; we confirmed that this DMSO concentration did not affect the proliferation of NSCLC cell lines). After incubation for 24 hours, the cells were irradiated using a 137Cs irradiator (J.L. Shepherd and Associates, Glendale, CA) at room temperature. The dose rate was 1.8 Gy/min, and the dose range was 0 to 6 Gy. Fourty-eight hours after irradiation, the cells were washed with PBS, cultured in drug-free medium for 7 to 8 days, fixed with 70% ethanol, and stained with 0.5% crystal violet (Sigma). After staining, colonies were counted using a cutoff of 50 viable cells. Surviving fraction was calculated as (mean colony counts)/(cells inoculated) × (plating efficiency), where plating efficiency was defined as (mean colony counts)/(cells inoculated for nonirradiated controls). The radiation dose enhancement ratio (DER) was calculated as the dose (Gy) of radiation plus vehicle (DMSO) divided by the dose (Gy) for radiation plus terameprocol (normalized for terameprocol toxicity) necessary for a surviving fraction of 0.2. Experiments were conducted in triplicate, and mean, standard deviation (SD), and p values (using Student's t test) were calculated.

      Cell Cycle Analysis

      Cells (2 × 106) were seeded in 100-mm dishes and treated with DMSO, 10 μM terameprocol, or 30 μM terameprocol for 24 hours or 48 hours. The cells were then collected by trypsinization (0.05% trypsin-EDTA, GIBCO), fixed with 70% ethanol, and stored overnight at −20°C. Cells were then collected by centrifugation and were resuspended in 1 ml of PBS with 40 μg/ml DNase-free RNase A (Clontech Laboratories, Inc., Mountain View, CA) and then incubated at 37°C for 30 minutes. Propidium iodide (50 μg/ml) (Sigma) was then added, and the cells were incubated at room temperature for 5 minutes. Cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population. The analysis was repeated three times using flow cytometry, and the mean and SD were calculated and graphed.

      Statistic Analysis

      The data presented represent mean ± SD for all measured biologic parameters. Student's t test was used to determine the significance between groups. Statistical analysis was performed with Microsoft Excel. Significance was defined at the level of p < 0.05. Asterisks (*) represent statistical significance.

      RESULTS

      Terameprocol Induces Transcriptional Down-Regulation and Decreased Expression of Survivin Protein in HCC2429 and H460 NSCLC Cells

      To evaluate the capacity of terameprocol to down-regulate survivin, HCC2429 and H460 lung cancer cells were transfected with pLuc2931, a luciferase reporter under the control of a human survivin promoter fragment, and pLuc control. As shown in Figure 1A, survivin transcription was down-regulated at 24 hours and significantly so after 48 hours of treatment with 10 μM terameprocol in HCC2429 cells (p < 0.05). Treatment with 10 μM terameprocol in H460 lung cancer cells resulted in significant down-regulation of survivin transcription at both 24 (p < 0.05) and 48 hours (p < 0.05).
      Figure thumbnail gr1
      FIGURE 1Terameprocol down-regulates survivin transciption and protein expression in HCC2429 and H460 cells. A, HCC2429 and H460 cells were transfected with 200 ng pLuc2931 or pLuc control and 0.6 μL lipofectamine 2000. Luciferase activity was detected 24 and 48 hours posttransfection and plotted. The luciferase assay was performed in triplicate, and error bar is shown as mean ± standard deviation (SD). B, Immunoblotting was used to determine survivin expression in HCC2429 and H460 cell lysates 24 and 48 hours after 0, 10, or 30 μM terameprocol treatment. Actin was probed to demonstrate equal loading.
      To further examine survivin expression after treatment with terameprocol in vitro, levels of survivin were determined by Western blot analysis (Figure 1B). Analysis revealed that baseline expression of survivin protein was comparable in the two cell lines. As expected, HCC2429 and H460 cells exhibit inhibition of survivin expression in both a time- and dose-dependent manner after terameprocol treatment.

      Decreased Levels of Survivin Expression do Not Necessarily Correspond with Increased Levels of Apoptosis

      To test the relationship between survivin expression and apoptosis, HCC2429 and H460 cells were treated with radiation (3Gy) for 0, 24, 48, and 72 hours. Immunoblotting revealed increased levels of survivin, with a decline in survivin expression 72 hours after radiation in each cell line (Figure 2A). Notably, cleaved caspase-3 levels increased in HCC2429 NSCLC cells after 48 and 72 hours. In contrast, no cleaved caspase-3 was observed in H460 cells, even at 72 hours when survivin expression was decreased. These data demonstrate that H460 cells are apoptosis-resistant relative to HCC2429 cells.
      Figure thumbnail gr2
      FIGURE 2Radiation induces increased cleaved caspase-3 in HCC2429 cells but not H460 cells. A, HCC24249 and H460 cells were irradiated with 3 Gy. The expression of survivin, total caspase-3, and cleaved caspase-3 in HCC2429 and H460 cell lysates was determined 0, 24, 48, and 72 hours after radiation treatment by immunoblotting. Actin was probed to demonstrate equal loading. B, Immunoblotting was used to determine the expression of Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, cIAP-1, cIAP-2, XIAP, and survivin in HCC2429 and H460 cell lysates. Tubulin was probed to demonstrate equal loading.

      Differential Expression of Apoptotic Proteins in HCC2429 and H460 Lung Cancer Cell Lines

      Protein expression levels of members of the Bcl-2 protein family and IAP family in lung cancer cell lines were determined by Western blot analysis, as shown in Figure 2B. Both HCC2429 and H4260 NSCLC cells express high, comparable levels of survivin. H460 cells, an apoptotic-resistant cell line, were found to express higher levels of Bcl-2, Bcl-XL, and Mcl-1 compared with HCC2429 cells. In contrast, the proapoptotic proteins Bax and Bak were expressed in greater quantities in HCC2429 cells relative to H460 lung cancer cells. Notably, expression of cIAP-1 and cIAP-2 was greater in HCC2429 cells, whereas XIAP exhibited slightly higher expression in H460 cells. These findings suggest differential regulation in the expression of Bcl-2 family and IAP family member proteins in these two lung cancer cell lines and may help explain the different levels of apoptosis exhibited in HCC2429 and H460 cells in Figure 2A.

      Terameprocol Enhances Radiosensitivity in Both HCC2429 and H460 Lung Cancer Cells

      We used in vitro clonogenic assays to determine the effect of terameprocol on the radiosensitivity of HCC2429 and H460 cell lines (Figure 3A). Both cell lines were treated with 10 μM terameprocol or DMSO control and irradiated with 0 to 6 Gy. Clonogenic survival assay demonstrates a significant decrease in cell survival in both HCC2429 and H460 cells, with DERs of 1.26 (p = 0.019) and 1.18 (p = 0.001), respectively, when compared with control.
      Figure thumbnail gr3
      FIGURE 3Terameprocol induces increased radiosensitivity in HCC2429 and H460 cells but enhances apoptosis in only HCC2429 cells when used with radiation. A, HCC2429 and H460 cells were treated with 10 μM terameprocol or dimethyl sulfoxide (DMSO) for 24 hours, followed by irradiation with 0 to 6 Gy at 1.8 Gy/min. Forty-eight hours after irradiation, cells were cultured in drug-free media and incubated for 7 to 8 days. At this point, the surviving fraction and the drug enhancement ratio (DER) were determined. Each experiment was performed in triplicate, and data are shown as mean ± standard deviation (SD). B, HCC2429 and H460 cells were treated with 10 μM terameprocol or DMSO for 24 hours followed by radiation treatment with 0 or 3 Gy. Expression of survivin, total caspase-3, and cleaved caspase-3 was determined by immunoblotting 48 hours postradiation treatment. Actin was probed to demonstrate equal loading.

      Administration of Terameprocol and Radiation Results in Increased Apoptosis in HCC2429 Cells but Not H460 Cells

      To investigate the effects of survivin inhibition on radiation-induced apoptosis, HCC2429 and H460 cells were pretreated with 10 μM terameprocol for 24 hours followed by administration of 3 Gy radiation and subsequent incubation for 48 hours (Figure 3B). Western blot analysis showed that in both HCC2429 and H460 cells, the expression of survivin in nonirradiated cells was decreased after terameprocol treatment, when compared with DMSO controls. Nevertheless, after administration of radiation HCC2429 cells exhibited increased levels of survivin in terameprocol-treated cells. In contrast, irradiated H460 cells pretreated with terameprocol saw slightly decreased expression of survivin. Despite the increase in survivin expression, HCC2429 cells treated with radiation and terameprocol exhibited a synergistic increase in apoptosis relative to radiation alone, whereas no cleaved caspase-3 was detected in H460 cells under any conditions. Although terameprocol seems to be more effective at decreasing survivin expression in H460 cells, it is not sufficient to produce apoptosis in this relatively apoptosis-resistant cell line. Thus, these data suggest that even though terameprocol does not increase levels of apoptosis in H460 cells, the drug is sufficient for radiosensitization of this cell line (Figure 3A), suggesting an important role for terameprocol in radiation-induced cell death in these cells.

      Terameprocol has No Effect on Cell Cycle in Either HCC2429 or H460 Lung Cancer Cells

      Survivin expression is increased in actively dividing cells and is expressed predominately during the G2/M transition, when it plays a key role in regulating mitosis.
      • Altieri DC
      Targeted therapy by disabling crossroad signaling networks: the survivin paradigm.
      To test whether inhibition of survivin affects the cell cycle in NSCLC cell lines, terameprocol-treated HCC2429 and H460 cells were fixed, stained with propidium iodide, and cell cycle distribution determined using flow cytometry (Figure 4). Neither 10 μM nor 30 μM terameprocol resulted in significant alterations in the distribution of cells in G1, S, or G2 phases at either time point. These data suggest that mitotic arrest does not play a significant role in the radiosensitization of HCC2429 or H460 cells.
      Figure thumbnail gr4
      FIGURE 4Terameprocol treatment does not induce cell cycle changes in HCC2429 or H460 cells. HCC2429 and H460 cells were cultured with dimethyl sulfoxide (DMSO), 10 μM terameprocol, or 30 μM terameprocol for 24 or 48 hours, followed by ethanol fixation and staining with propidium iodide. Flow cytometry was used to determine percentage of HCC2429 or H460 cells in G1, S, or G2 phases of the cell cycle. The analysis was repeated three times using flow cytometry and the data graphed as a function of mean ± standard deviation (SD).

      DISCUSSION

      In this study, we have shown that terameprocol treatment results in decreased transcription and expression of survivin in HCC2429 and H460 lung cancer cell lines, though this decrease was not shown to be associated with an increase in apoptosis. Nonetheless, terameprocol induces a significant increase in the radiosensitization of both cell lines.
      Terameprocol is an inhibitor of Sp1-mediated transcription, and has been shown to decrease transcription and protein expression of several genes, including survivin and CDK1.
      • Chang CC
      • Heller JD
      • Kuo J
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression.
      • Park R
      • Chang CC
      • Liang YC
      • et al.
      Systemic treatment with tetra-O-methyl nordihydroguaiaretic acid suppresses the growth of human xenograft tumors.
      In this study, we have validated that terameprocol effectively down-regulates transcription of survivin in both HCC2429 and H460 cells. Interestingly, we found that suppression of survivin transcription by terameprocol treatment was greater in H460 cells compared with HCC2429 cells at 24 hours. Terameprocol treatment also induced decreased survivin protein expression in a time- and dose-dependent manner in both HCC2429 and H460 cell lines. Nevertheless, subsequent data showed that although the terameprocol-induced decrease in survivin transcription was greater in H460 cells, these results did not correlate with increased levels of apoptosis relative to HCC2429 cells. Indeed, our data show that only HCC2429 cells exhibited measurable levels of apoptosis, despite a less profound down-regulation of survivin transcription after terameprocol treatment.
      HCC2429 and H460 cell lines differ in their susceptibility to apoptosis, as evidenced by their response to radiation. HCC2429 cells expressed cleaved caspase-3, a marker for apoptosis, in response to radiation treatment in a time-dependent manner. H460 cells, however, showed no detectable levels of cleaved caspase-3 at any time point. Notably, radiation resulted in little to no change in survivin levels during the first 48 hours of treatment. We have previously demonstrated that normal cells will down-regulate survivin expression in response to radiation. Malignant cells, however, fail to use this down-regulation.
      • Lu B
      • Mu Y
      • Cao C
      • et al.
      Survivin as a therapeutic target for radiation sensitization in lung cancer.
      • Shinohara ET
      • Hallahan DE
      • Lu B
      The use of antisense oligonucleotides in evaluating survivin as a therapeutic target for radiation sensitization in lung cancer.
      Our current data show that HCC2429 and H460 cells behave similarly to other malignant cells in response to radiation, although there is noticeable down-regulation of survivin protein expression after 72 hours in both cell lines.
      Because we saw a difference in levels of apoptosis between the two cell lines, the protein expression levels of antiapoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, and Mcl-1), antiapoptotic XIAP family proteins (cIAP-1, cIAP-2, survivin, and XIAP), and proapoptotic Bcl-2 family proteins (Bax and Bak) were determined in both HCC2429 and H460 cell lines. Overall, the levels of the antiapoptotic members of the Bcl-2 family were increased and the levels of proapoptotic members of the Bcl-2 family were decreased in H460 cells relative to HCC2429 cells. Expression of cIAP-1 and cIAP-2 was slightly increased in HCC2429 cells compared with H460 cells, and both cell lines showed high expression of XIAP and survivin. We should note that H460 cells express wild-type p53, so the mutational status of this gene does not explain the relative resistance of this cell line to radiation. On the basis of these results, we suggest that H460 cells have a greater block for undergoing apoptosis because of their high expression of antiapoptotic Bcl-2 proteins and weak expression of proapoptotic Bcl-2 members. One can speculate, therefore, that the differences in Bcl-2 family protein expression may contribute to the observed resistance to radiation-induced apoptosis in the H460 cell line.
      In this study, the administration of 10 μM terameprocol significantly enhanced the sensitivity of both HCC2429 (DER = 1.26, p = 0.019) and H460 (DER = 1.18, p = 0.001) NSCLC cell lines to radiation treatment in clonogenic assays. When survivin is inhibited, radiation results in increased levels of apoptosis.
      • Kami K
      • Doi R
      • Koizumi M
      • et al.
      Downregulation of survivin by siRNA diminishes radioresistance of pancreatic cancer cells.
      • Iwasa T
      • Okamoto I
      • Suzuki M
      • et al.
      Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines.
      • Rodel F
      • Frey B
      • Leitmann W
      • et al.
      Survivin antisense oligonucleotides effectively radiosensitize colorectal cancer cells in both tissue culture and murine xenograft models.
      This has previously been demonstrated in H460 cells with the use of the small molecule inhibitor YM155
      • Iwasa T
      • Okamoto I
      • Suzuki M
      • et al.
      Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines.
      and antisense oligonucleotides.
      • Cao C
      • Mu Y
      • Hallahan DE
      • et al.
      XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer.
      Nevertheless, survivin inhibition by terameprocol yields different results. The enhanced radiosensitization demonstrated in both cell lines was only associated with an increase in apoptosis in HCC2429 cells and not H460 cells, which exhibited no measurable level of cleaved caspase-3. H460 cells again show a larger decrease in survivin expression after radiation and terameprocol treatment, when compared with HCC2429 cells, similar to data showing the effect of terameprocol treatment on survivin transcription levels in H460 cells (Figure 1A). Also, HCC2429 cells actually see an increase in survivin expression with the combination of terameprocol treatment and 3 Gy radiation compared with terameprocol alone. This is especially puzzling because the terameprocol and radiation combination induces increased levels of cleaved caspase-3 relative to radiation alone. Thus, apoptosis is occurring in HCC2429 cells even though survivin levels remain high. It can be suggested then that terameprocol must exert effects not explained by survivin levels alone. Mak et al.
      • Mak DH
      • Schober WD
      • Chen W
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid inhibits growth and induces death of leukemia cells independent of Cdc2 and survivin.
      demonstrated a similar phenomenon in leukemic cells. Administration of 0 to 40 μM terameprocol promoted cell death but failed to result in suppression of survivin or Cdk1 expression.
      Previous studies have shown that terameprocol induces G2 cell cycle arrest by inhibiting expression of Cdk1 (Cdc2).
      • Chang CC
      • Heller JD
      • Kuo J
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression.
      • Heller JD
      • Kuo J
      • Wu TC
      • et al.
      Tetra-O-methyl nordihydroguaiaretic acid induces G2 arrest in mammalian cells and exhibits tumoricidal activity in vivo.
      Terameprocol may also induce G2 cell cycle arrest through suppression of survivin levels, as survivin is a key regulator of the mitotic spindle checkpoint along with INCENP and Aurora Kinase B.
      • Mita AC
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      • et al.
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      • Wheatley SP
      • Carvalho A
      • Vagnarelli P
      • et al.
      INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis.
      Our data show, however, that terameprocol treatment did not induce any changes in cell cycle distribution, including G2 arrest, in either HCC2429 or H460 cells relative to vehicle controls. These results suggest that G2 cell cycle arrest does not contribute to the increased radiosensitivity demonstrated in terameprocol-treated lung cancer cell lines.
      Terameprocol has been demonstrated to suppress survivin and Cdk1 transcription by incorporating into GC-rich sequences and interfering with the binding of the Sp1 protein to promoter regions.
      • Hwu JR
      • Tseng WN
      • Gnabre J
      • et al.
      Antiviral activities of methylated nordihydroguaiaretic acids. 1. Synthesis, structure identification, and inhibition of tat-regulated HIV transactivation.
      Sp1 is ubiquitously expressed and has been shown to regulate the expression of thousands of genes.
      • Tan NY
      • Khachigian LM
      Sp1 phosphorylation and its regulation of gene transcription.
      As such, Sp1 is implicated in nearly all facets of cellular function, including cell growth, differentiation, apoptosis, and angiogenesis. It would be extremely difficult to test the effect of terameprocol on the expression of each Sp1-mediated gene and to determine each gene's role in radioresistance in NSCLC. Nevertheless, further DNA and proteomic analysis may lend to our understanding of the mechanism of action of terameprocol in enhanced radiosensitization of lung cancer.
      In conclusion, this study supports the potential of terameprocol as a radiosensitizing agent in lung cancer, although the mechanism of action is unclear. Initial phase I clinical trials have already shown that terameprocol is well tolerated and has shown promising antitumor activity. Further study is needed to assess the clinical efficacy of terameprocol as a drug that may raise the therapeutic ratio of radiation therapy in lung cancer.

      ACKNOWLEDGMENTS

      Supported by NCI grant 1R01 CA125842-01A1.
      The authors thank Lauren Mitchell for aiding in the initial drafting of this manuscript.

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