TY - JOUR
T1 - Napabucasin (BBI 608), a potent chemoradiosensitizer in rectal cancer
AU - Nagaraju, Ganji Purnachandra
AU - Farran, Batoul
AU - Farren, Matthew
AU - Chalikonda, Gayathri
AU - Wu, Christina
AU - Lesinski, Gregory B.
AU - El-Rayes, Bassel F.
N1 - Funding Information:
The florescence images were supported in part by the Integrated Cellular Imaging Microscope Core of Emory University and the National Institutes of Health/National Cancer Institute (award 2P30CA138292-04). The contents are the responsibility of the authors and do not necessarily reflect the official views of the National Institutes of Health. Nude mice were purchased from Envigo (Indianapolis, Indiana) and maintained according to Institutional Animal Care and Use Committee protocols at Emory University. HCT116 cells (1 × 106) were mixed with 15% Matrigel and subcutaneously inserted into the mice. The animals were randomized into 4 groups when the tumor reached 100 mm3 in size. Group 1 received water orally and served as a control group. Group 2 received napabucasin orally (100 mg/kg) for 11 days daily. Group 3 received 5-FU (30 mg/kg intravenously) plus a single fraction of radiation at 4 Gy once a week for 2 weeks. Finally, group 4 received napabucasin, 5-FU, and radiation. The tumor volumes were measured once every 4 days with Vernier caliper. Mice were euthanized via CO2 asphyxiation, which was followed by dissection to peel the skin covering tumors over the inserted Matrigel. This was then was photographed under visible light to evaluate angiogenesis. Human CRC cell lines (HCT116 and HT29) were procured from the American Type Culture Collection. Napabucasin was obtained from Boston Biomedical, Inc. 5-Fluorouacil (5-FU), N-acetylcysteine (NAC), and interleukin 6 (IL-6) were bought from Sigma-Aldrich. The bromodeoxyuridine (BrdU) assay kit was purchased from Roche (Indianapolis, Indiana). Antibodies against pATM (Ser1981; D6H9; no. 5883), ATM (no. sc-28901), pATR (Ser428; no. 2853), Rad51 (no. sc-8349), pγH2AX (pS139; no. ab26350), MDM2 (no. sc-965), Chk2 (no. sc-5278), p53 (no. sc-9282), NQO1 (no. sc-32793), STAT3 (no. sc-8019), pSTAT3 (Tyr705; no. 9138), VEGF (no. sc-7269), and β-actin (no. sc-8432) were obtained from Cell Signaling, Santa Cruz, and Abcam. The VEGF and IL-6 quantification kits were obtained from R&D Systems (catalog no. DY293B). Eggs were purchased from Charles River (North Franklin, Connecticut). All human CRC cell lines were grown in McCoy's 5A medium and were maintained according to American Type Culture Collection guidelines and to our previously published protocol.14 HCT116 and HT29 cell lines were cultured in 96-well plates and then treated with or without napabucasin in a concentration-dependent fashion (range, 0.3-2.4 µM). After 36 hours, CRC cell proliferation was evaluated with the BrdU assay kit according to the manufacturer's instructions (no. 11647 229 001; Roche).15 A microplate reader was used to evaluate absorbance at 450 nm. These experiments were performed in triplicate. Equal numbers of both types of CRC cells (100 ± 10) were plated in 6-well plates containing culture media and were maintained overnight at 37 °C according to a published protocol.16 The CRC cell lines were then treated with napabucasin (1 µM) alone or in combination with 5-FU (4 µM) and subjected to different fractions of radiation (0, 2, 4, or 6 Gy). The medium containing the treatment was discarded after 36 hours, and fresh medium was replaced once every 4 days. On day 12 after radiation exposure, the colonies were marked with a crystal violet solution for 10 minutes and washed with water. The number of stained colonies was counted with a microscope (a DP20 Olympus camera at a magnification of ×1.5). For the purposes of this quantification, a clone of 50 or more cells was considered a colony. The survival fraction was calculated according to our previously published protocol.16 Treated or untreated CRC cell lines were attained and lysed with a radioimmunoprecipitation assay buffer comprising phosphatase and protease inhibitors (Sigma-Aldrich). Protein levels for each sample were then estimated with a bicinchoninic acid quantification assay. Equal amounts of protein (100 µg) were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 2.5% bovine serum albumin (BSA) or 5% milk according to the antibody type. Membranes were next probed with selected primary antibodies for 4 hours at room temperature (RT). The membranes were then washed 3 times with a phosphate-buffered saline with Tween 20 (PBST) buffer for 10-minute intervals each and were probed with specific horseradish peroxidase–conjugated secondary antibodies for 45 minutes at RT. The blots were washed again 3 times with PBST for 10-minute intervals and developed with an enhanced chemiluminescence reagent. The signal was developed with X-ray films. CRC cells were plated in 6- or 96-well plates and treated as indicated in the previous sections for 24 hours, after which the cells were pretreated with the ROS inhibitor NAC (5 mM) for 2 hours before staining. After 2 hours, the CRC cells were stained with CellROX Deep Red (Thermo Fisher Scientific) for 1 hour at 37 °C. 4′,6-Diamidino-2-phenylindole was then added for 10 minutes to estimate cell viability. ROS levels were measured with BD FACSCalibur flow cytometry analysis (BD Biosciences, Heidelberg, Germany). The results were analyzed with FlowJo software (Tree Star, Inc, San Carlos, California). The microplate reader evaluated absorbance at 640 and 665 nm (96-well plate). These experiments were performed in triplicate. Immunofluorescence was performed according to our previously published protocol.17 CRC cell lines were incubated in an 8-well chamber slide overnight; afterward, the cell lines were treated as indicated in the preceding section. After 24 hours, the medium was removed, and they were fixed with 4% paraformaldehyde for 20 minutes. Then, the cell lines were blocked with 2.5% BSA with 0.02% Triton-X100 for 1 hour. Subsequently, the blocking buffer was replaced with 2.5% BSA containing an antibody against pγH2AX for 2 hours in RT. After this step, the buffer containing the antibody was removed, and the cells were washed with phosphate-buffered saline. Then, 2.5% BSA containing a goat–anti-mouse Alexa Fluor–conjugated secondary antibody was supplemented, and the cells were incubated for 45 minutes. Cells were later washed with phosphate-buffered saline to remove the unbounded secondary antibody, and this allowed the cell lines to be stained and covered with a slip with a 4′,6-diamidino-2-phenylindole mounting medium. Photographs were taken under a fluorescence microscope. CRC cells were treated with or without napabucasin, 5-FU for 36 hours, and a single fraction of radiation (4 Gy) at 24 hours. The treatment-containing medium was removed and replaced with fresh medium. This conditioned medium was collected after 16 hours for VEGF quantification and egg chorioallantoic membrane (CAM) assays. The secretion of VEGF was measured with an enzyme-linked immunosorbent assay per the manufacturer's instructions (R&D Systems). Fertilized eggs were kept in the incubator in a humidified environment at 37 °C for 5 days. The eggs were then perforated to form small holes at their tops and were injected with a conditioned medium (as per the previous section) or a serum-free medium (control). The eggs were then maintained for 10 days in a humidified environment at 37 °C. On day 16, the eggs were opened, and CAMs were pictured with a microscope (a DP20 Olympus camera at a magnification of ×1.5) to estimate vascularization with AngioQuant software.18 Nude mice were purchased from Envigo (Indianapolis, Indiana) and maintained according to Institutional Animal Care and Use Committee protocols at Emory University. HCT116 cells (1 × 106) were mixed with 15% Matrigel and subcutaneously inserted into the mice. The animals were randomized into 4 groups when the tumor reached 100 mm3 in size. Group 1 received water orally and served as a control group. Group 2 received napabucasin orally (100 mg/kg) for 11 days daily. Group 3 received 5-FU (30 mg/kg intravenously) plus a single fraction of radiation at 4 Gy once a week for 2 weeks. Finally, group 4 received napabucasin, 5-FU, and radiation. The tumor volumes were measured once every 4 days with Vernier caliper. Mice were euthanized via CO2 asphyxiation, which was followed by dissection to peel the skin covering tumors over the inserted Matrigel. This was then was photographed under visible light to evaluate angiogenesis. Statistical significance between untreated and treated cell lines or animals was assessed with a One-way analysis of variance (ANOVA) tailed by a Student paired t test with InStat software for Windows. Results are shown as mean ± SD. Variances between means were considered significant if P <.05.
Funding Information:
The florescence images were supported in part by the Integrated Cellular Imaging Microscope Core of Emory University and the National Institutes of Health/National Cancer Institute (award 2P30CA138292‐04). The contents are the responsibility of the authors and do not necessarily reflect the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 American Cancer Society
PY - 2020/7/15
Y1 - 2020/7/15
N2 - Background: The induction of reactive oxygen species (ROS) represents a viable strategy for enhancing the activity of radiotherapy. The authors hypothesized that napabucasin would increase ROS via its ability to inhibit NAD(P)H:quinone oxidoreductase 1 and potentiate the response to chemoradiotherapy in rectal cancer via distinct mechanisms. Method: Proliferation studies, colony formation assays, and ROS levels were measured in HCT116 and HT29 cell lines treated with napabucasin, chemoradiation, or their combination. DNA damage (pγH2AX), activation of STAT, and downstream angiogenesis were evaluated in both untreated and treated cell lines. Finally, the effects of napabucasin, chemoradiotherapy, and their combination were assessed in vivo with subcutaneous mouse xenograft models. Results: Napabucasin significantly potentiated the growth inhibition of chemoradiation in both cell lines. Napabucasin increased ROS generation. Inhibition of ROS by N-acetylcysteine decreased the growth inhibitory effect of napabucasin alone and in combination with chemoradiotherapy. Napabucasin significantly increased pγH2AX in comparison with chemoradiotherapy alone. Napabucasin reduced the levels of pSTAT3 and VEGF and inhibited angiogenesis through an ROS-mediated effect. Napabucasin significantly potentiated the inhibition of growth and blood vessel formation by chemoradiotherapy in mouse xenografts. Conclusion: Napabucasin is a radiosensitizer with a novel mechanism of action: increasing ROS production and inhibiting angiogenesis. Clinical trials testing the addition of napabucasin to chemoradiotherapy in rectal cancer are needed.
AB - Background: The induction of reactive oxygen species (ROS) represents a viable strategy for enhancing the activity of radiotherapy. The authors hypothesized that napabucasin would increase ROS via its ability to inhibit NAD(P)H:quinone oxidoreductase 1 and potentiate the response to chemoradiotherapy in rectal cancer via distinct mechanisms. Method: Proliferation studies, colony formation assays, and ROS levels were measured in HCT116 and HT29 cell lines treated with napabucasin, chemoradiation, or their combination. DNA damage (pγH2AX), activation of STAT, and downstream angiogenesis were evaluated in both untreated and treated cell lines. Finally, the effects of napabucasin, chemoradiotherapy, and their combination were assessed in vivo with subcutaneous mouse xenograft models. Results: Napabucasin significantly potentiated the growth inhibition of chemoradiation in both cell lines. Napabucasin increased ROS generation. Inhibition of ROS by N-acetylcysteine decreased the growth inhibitory effect of napabucasin alone and in combination with chemoradiotherapy. Napabucasin significantly increased pγH2AX in comparison with chemoradiotherapy alone. Napabucasin reduced the levels of pSTAT3 and VEGF and inhibited angiogenesis through an ROS-mediated effect. Napabucasin significantly potentiated the inhibition of growth and blood vessel formation by chemoradiotherapy in mouse xenografts. Conclusion: Napabucasin is a radiosensitizer with a novel mechanism of action: increasing ROS production and inhibiting angiogenesis. Clinical trials testing the addition of napabucasin to chemoradiotherapy in rectal cancer are needed.
KW - angiogenesis
KW - colorectal cancer
KW - DNA damage
KW - napabucasin (BBI 608)
KW - reactive oxygen species (ROS)
KW - STAT3
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U2 - 10.1002/cncr.32954
DO - 10.1002/cncr.32954
M3 - Article
C2 - 32383803
AN - SCOPUS:85085133487
VL - 126
SP - 3360
EP - 3371
JO - Cancer
JF - Cancer
SN - 0008-543X
IS - 14
ER -
