The Ibr-7 Derivative of Ibrutinib Exhibits Radiosensitivity in Pancreatic Cancer Cells via Targeting EGFR

Background: Radiotherapy is one of the main therapeutic methods for pancreatic cancer, but radiation resistance limits the clinical application. As a result, novel therapeutic agents to improve the radiosensitivity is urgent. This study aimed to investigate the effect of Ibr-7 (the derivative of ibrutinib) on radiosensitivity of human pancreatic cancer cells. Methods: The effect of Ibr-7 on pancreatic cancer cell’s proliferation were detected by CCK-8 assay. Radiosensitivity was assessed by clonogenic formation assay. Cell cycle, cell apoptosis were analyzed by ow cytometry. DNA damage was detected by immunouorescence analysis. The expression of p-EGFR, EGFR were determined by western blot. Results: Ibr-7 showed anti-proliferative effect in PANC-1 and Capan2 cells in a dose- and time-dependent manner. Ibr-7 (2 µmol/L) enhanced the effect of radiation in PANC-1 and Capan2 cells. Further ndings showed that this combination enhanced G2/M phase arrest and increased cell apoptosis. Additional molecular mechanism studies revealed that the expression of p-EGFR was decreased by Ibr-7 alone or combined with radiation. Overexpression of EGFR reversed the cell apoptosis induced by Ibr-7 combined with radiation. Moreover, the expression of γ-H2AX was signicantly decreased in Ibr-7 combined with radiation group. Conclusions: Our study indicated that the potential application of Ibr-7 as a highly effective radiosensitizer for the treatment of pancreatic cancer cells. did not suppress the the AKT/mTOR signaling pathway. These offer a effective strategy for enhancing effect radiation it will provide revealing insights into the development of ibrutinib 1.07%) compared ± results ± 4.36% vs 5.93 ± It Ibr-7


Background
Pancreatic cancer is a highly invasive and fatal disease, with a low 5-year survival rate of only 5% [1]. It is diagnosed in the medium or terrminal stage, and 80% of patients are ineligible for surgery. The treatment options for them are chemotherapy and radiation therapy [2,3]. Approximately 70% of pancreatic cancer patients receive radiotherapy, either alone or in combination with other treatments [4,5]. However, pancreatic cancer cells inevitably develop radioresistance, which is one of the main cause for poor prognosis of patients in clinical treatment [6][7][8]. Recent progress in radiation combination with chemotherapy has raised concerns. Exploring new targeted agents to improve the radiosensitivity of pancreatic cancer is urgent.
Ibrutinib is a small molecule inhibitor that targets Bruton's tyrosine kinase [9]. It exhibited potent antitumor effect against various cancers, including lymphoma and solid tumors (such as non-small cell lung cancer and pancreatic cancer) [10][11][12][13][14]. However, in our previous research, it was demonstrated that ibrutinib's anti-tumor effect was limited to blood cancer cells [15]. The clinical trials of ibrutinib in solid tumors are facing dilemma. Based on these, we synthesized a series of ibrutinib derivatives, of which Ibr-7 exhibited great anti-tumor toward NSCLC and pancreatic cancer [15,16]. Besides, in our previous research ibrutinib (10 µmol/L) has been shown to enhance the effects of radiation therapy [17]. Therefore, it is of great interest to explore the radio-sensitivity of Ibr-7 towards pancreatic cancer cells. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js In the present study, we sought to evaluate the radiosensitizing effect of Ibr-7 in pancreatic cancer cell lines. Our results suggested that Ibr-7 sensitized pancreatic cancer cell lines PANC-1, Capan2. Further study indicated that Ibr-7 induced G2/M cell cycle arrest and cell apoptosis. Ibr-7 e ciently inhibited the p-EGFR, just as ibrutinib. However, as distinct from ibrutinib, Ibr-7 did not suppress the phosphorylation of the AKT/mTOR signaling pathway. These results may offer a effective strategy for enhancing the effect of radiation therapy, and it will provide revealing insights into the development of ibrutinib derivatives. The pancreatic cancer cells were plated in six-well plates and pretreated with Ibr-7 or DMSO as control for 24 h. Then the cells were exposed to the indicated doses (0, 2, 4, 6 Gy) of radiation respectively. The cells were then incubated with DMEM or RPMI-1640 medium supplementing 10% FBS at 37℃ in a 5% CO2 atmosphere. After 14 days, the colonies were stained with 0.1% crystal violet (Sigma-Aldrich) in absolute methanol for 30 min. Colonies more than 50 cells were counted. The survival fraction (SF) was calculated as described previously [18]. SF = mean number of colonies / (plating e ciency × number of cells inoculated) in treated groups, the plating e ciency= (mean number of colonies / number of cells inoculated) in untreated groups. A multi-target click mathematical model was applied to simulate the cell survival fraction. We then calculated the the sensitization enhancement ratio (SER) as the dose (2 Gy) for the radiation divided by the dose (2 Gy) for radiation plus Ibr-7 (SER = SFIR/SFIR + Ibr-7). The mean lethal dose of cells (D0), extrapolation number (N) values, the quasithreshold dose (Dq) were also calculated according to the curve. Error bars were calculated as SD via the results of three independent experiments.

Materials And
Cell cycle assay PANC-1 and Capan2 cells were divided into four groups: The control group (DMSO), the Ibr-7 (2 µM) group, the radiation (2 Gy) group, The combination group. 5 × 10 5 cells were plated into 6-well plates, exposed to Ibr-7 for 24 h, then the cells exposed to 6-MV X-ray. After another 24 h, the cells were trypsinized and xed with precooled 70% ethanol at 20℃ overnight. Then the cells were added with PI (supplemented with RNase A) and incubated for 30 min at room temperature. Then the treated cells were detected by ow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Error bars were calculated as SD via the results of three independent experiments.

Apoptosis assay
Cells were divided into four groups as mentioned above. Cells were plated into 6-well plates and pretreated with Ibr-7 for 24 h, then the cells were exposed at 2 Gy of radiation. After another 24 h, the cells were trypsinized and washed with PBS for twice. Then cell suspension was stained with 5 µL Annexin V-FITC and 5 µL PI solution and analyzed by owcytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
Error bars were calculated as SD via the results of three independent experiments. Immuno uorescence 3 × 10 4 cells were plated on coverslips in 24-well plates and pretreated with DMSO or Ibr-7 for 24 h. Then the cells were exposed to radiation (6 Gy). The cells were then incubated for 24 h and collected. The cells were washed in PBS for 3 times and xed with 4% paraformaldehyde for 30 min. After permeabilizing cells with 0.5% Triton-100 for 15 min, the cells were blocked with 1% BSA, then incubated with anti-γ-H2AX antibody overnight at 4 °C. The cells were incubated with goat anti-rabbit IgG (FITC-labeled) Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js antibody (Beyotime) for 60 min, followed by incubation with DAPI for 5 min. Finally, the cells were photographed with immuno uorescence microscopy Western blot analysis Cultured cells were collected and washed with PBS thrice. Total proteins extraction was conducted using RIPA lysing buffer containing phosphatase inhibitors and protease according to manufacturer instructions. A total of 20-40 µg of proteins were submitted to 10% SDS/PAGE and transferred to PVDF membrane. The membranes were then blocked with 5% skim milk for 1 h in room temperature. The membranes were incubated with primary antibodies against EGFR, p-EGFR and β-actin respectively overnight at 4 °C, and then incubated with secondary antibodies for 1 h at room temperature. The membranes were washed with TBST 3 times, the proteins were visualized by adding ECL.

Plasmid transfection
The pcDNA3.1-EGFR plasmid and empty vector were purchased from GenePharma. The plasmid was transfected into PANC-1 and Capan2 cells by jetPRIME according to the manufacturer's protocol.

Statistical analysis
The results came from at least three independent experiments. All quantitative values are given as mean ± SD (standard deviation). Student's t-test was used to determine the signi cance and statistically signi cant was de ned when p < 0.05. Graphs were performed using GraphPad Software.

Ibr-7 induced cell growth inhibition in pancreatic cancer cells
Ibr-7 is a newly synthesized derivative of ibrutinib that showed anti-tumor activity against various cancer cells compared with its the parental compound [15]. To determine the anti-proliferation effect of Ibr-7 in pancreatic cancer cells, both two pancreatic cancer cells were treated with increasing doses of ibrutinib or Ibr-7 (1.56-25 µmol/L) for 24 h, 48 h or 72 h respectively and changes in cell proliferation were examined by CCK-8 assay. Our data demonstrated that cell viability was markedly reduced by Ibr-7 in both doseand time-dependent ways. The IC50 values of Ibr-7 at 48 h of treatment were 1.7 µmol/L, 2.5 µmol/L, while the IC50 values of ibrutinib were 20.8 µmol/L and 29.6 µmol/L for PANC-1 and Capan2 respectively.
For both two cells, the IC50 values of Ibr-7 treatments were approximately one-tenth of ibrutinib's IC50, indicating more effective anti-tumor activity of Ibr-7 compared to ibrutinib. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Ibr-7 pretreatment enhanced the radiosensitivity of pancreatic cancer cells Based on Ibr-7's effect in pancreatic cancer cells, we further investigated the radiosensitizing effect of Ibr-7 by clonegenic survival assay. Cells were seeded into 6-well plates and treated with 2 µmol/L Ibr-7, and then exposed to IR (2, 4, or 6 Gy). The survival curve was derived from the single-hit multi-target model [ ]. The sensitization enhancement ratio (SER) was measured according to the model. As shown in Fig. 2, pretreatment with Ibr-7 enhanced radiosensitivity of both PANC-1 (SER=1.63) and Capan2 cells (SER = 1.59). these results suggests that Ibr-7 enhanced radiosensitivity in pancreatic cancer cells. These results suggested that pretreatment with Ibr-7 enhanced radiosensitivity in pancreatic cancer cells.

Ibr-7 promoted G2/M cell cycle arrest of pancreatic cancer cells after radiation
To identify whether Ibr-7 could alter radiation induced cell cycle distribution, PI staining assay in PANC-1 and Capan2 cells was evaluated. PANC-1 and Capan2 cells were treated with 2 µmol/L Ibr-7 for 24 h, then irradiated for 6 Gy, the cells were collected after 24 h. As shown in Fig. 3, pretreatment with Ibr-7 showed little effect (16.12% ± 0.88% vs 8.32% ± 0.48%) while exposed to 6 Gy radiation in PANC-1 cells compared with controls in G2/M phase. Same results were detected in Capan2 cells (11.43% ± 2.07% vs 11.54% ± 1.87%). Cell exposed to radiation alone accumulated G2/M phase arrest compared with control.

Ibr-7 increased IR-induced apoptosis in pancreatic cancer cells
To further investigate the potential mechanisms of radiation enhancement by Ibr-7, we next conducted the apoptosis assay by Annexin V-conjugated FITC and propidium iodide (PI) staining. The total apoptosis rate was calculated. The results indicated that Ibr-7 (11.67 ± 0.84%) and radiation (25.56 ± 1.07%) could increase apoptosis at 48 h and ibrutinib combined with radiation (50.2 ± 1.33%) increased the apoptosis ratio signi cantly (Fig. 4A, C), compared with the control group (5.57 ± 0.30%) in PANC-1 cells. The results were the same in Capan2 cells (31.37 ± 4.36% vs 5.93 ± 0.52%). It indicates that Ibr-7 enhances apoptosis which is induced by radiation.

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Ibr-7 combined with radiation increases the DNA damage in pancreatic cancer cells Since the major impact of radiation in cells is to induce DNA double strand breaks (DSBs) and stimulate DNA damage repair. Meanwhile, γ-H2AX is a rapid and sensitive cellular biomarker of the DSBs [19]. γ-H2AX (phosphorylated at C-terminal serine residue 139) forms nuclear foci in the region of the nascent DSBs, and subsequently endures de-phosphorylation after the repair of DSBs. Therefore, the numbers of γ-H2AX foci are used as a standard of the relative amount of DSBs and repair. To determine whether Ibr-7 could enhance radiation induced DNA damage, we conducted immuno uorescence to evaluate foci of γ-H2AX. As shown in Fig. 5, Ibr-7 signi cantly increased the number of γ-H2AX foci per cell at 24 h following 6 Gy radiation than that in radiation alone (12.0 ± 2.0 compared to 6.67 ± 1.53 in PANC-1, and 10.0 ± 1.0 compared to 6.0 ± 1.0 in Capan2, *p < 0.05). These results suggested that Ibr-7 combined radiation increased DNA damage.

Enhanced radiosensitivity of pancreatic cancer cells by Ibr-7 is p-EGFR dependent
Down-regulation of p-EGFR was considered to play a major role in the effect of ibrutinib. We therefore asked whether p-EGFR was the major target of ibrutinib derivative Ibr-7 in PANC-1 and Capan2 cells after radiation. As we expected, the expression of p-EGFR was decreased markedly in response to Ibr-7 alone or together with radiation (Fig. 6A), indicating the involvement of EGFR in Ibr-7 combined with radiation in PANC-1 and Capan2 cells.

Discussion
Our ndings demonstrate that Ibr-7 have the strong ability to enhance the radiosensitivity of pancreatic cancer cells. These effects of Ibr-7 is attributable to activate G2/M arrest and induce apoptosis, which resulted from the decrease of the expression of p-EGFR.
To explore the underlying molecular mechanisms responsible for effect of Ibr-7, we rst analyzed the molecular structure of Ibr-7 and the relationship between ibrutinib and Ibr-7. Due to the similar structure of BTK, ibrutinib forms complex with BTK and then inhibits the BTK activity. Data also showed ibrutinib inhibits the expression of EGFR, HER2/neu, Her4/ErbB4. As the derivative of ibrutinib, Ibr-7 could also inhibit EGFR in NSCLC [28]. Whether Ibr-7 could exchange the expression of EGFR in pancreatic cancer cells is still unknown. In our study, Ibr-7 inhibited the expression of EGFR in pancreatic cancer cell lines as we expected. EGFR inhibition could increase the radiosensitivity in various tumors. Inhibition of EGFR/HER2 enhances radiosensitivity in pancreatic cancer [29,30]. Targeting of EGFR and β1 integrin receptor showed e cient radiosensitization in head and neck cancers.....based on these results, we further investigated the necessary of EGFR in Ibr-7's radiosensitivity in pancreatic cancer. Overexpression Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js of EGFR decreased the apoptosis induced by Ibr-7 and radiation, which demonstrated the important role of EGFR in radiosensitivity of Ibr-7.
AKT signaling pathway showed e ciency in many tumor progress. Ibrutinib (10 µmol/L) was capable of decreasing the p-AKT (S473) and its downstream genes, including mTOR, p-p70s6. However, Ibr-7 (2 µmol/L) did not showed signi cantly decrease the phosphorylation of AKT, mTOR and p70s6 (data not shown). As a result, the intrinsic mechanism remains to be further studied.
Radiation therapy leads to DNA lesions, DSBs (DNA double strand breaks) are regarded as the major impact of radiation [31]. The radiosensitivity largely depends on the ability to repair radiation-induced DNA damage. γ-H2AX (the phosphorylated form of H2AX) induced by radiation is involved in the event of DNA damage, and showed closely interralated with DSBs, which served as a sensitive and classic cellular indicator of DSBs [32,33]. The number of γ-H2AX indicated the relative amount of DSBs. In our study, we have found that Ibr-7 enhanced the foci of γ-H2AX induced by radiation. In response to DNA damage, the cell cycle progression is arrested. Our study found that Ibr-7 prolonged radiation induced G2/M phase arrest, which is consistent with the higher level of γ-H2AX at 24 h in Ibr-7 + radiation group that that in radiation group alone. Based on these results, it indicated that Ibr-7 may affect the expression of p-EGFR, leading to DNA damage in pancreatic cancer cells in response to radiation.
In conclusion, this study showed that the Ibr-7 sensitized pancreatic cancer cells to radiation in vitro, and that the effect was likely attributable, at least in part, to the induction of DNA damage, subsequently stimulation of G2/M phase arrest and cell apoptosis that result from the downregulation of p-EGFR. To our knowledge, this is the rst evidence showing that the radiosensitivity of Ibr-7 in pancreatic cancer cells. Our preclinical results not only suggested an effective strategy to improve the radiosensitivity in pancreatic cancer, but also provide a meaningful insights into the investigation of Ibr-7 in cancer treatment.

Conclusions
In summary, Ibr-7 enhanced radiation sensitivity in pancreatic cancer cells by promoting cell cycle arrest at G2/M phase, inducing cell apoptosis, and increasing DSBs induced by radiation. The mechanism investigation revealed that decrease of p-EGFR played an important role in its radiosensitizing effects.
This study indicate that Ibr-7 may be a novel potential radiosensitizer for pancreatic cancer.

Declarations
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