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Rational Design of Cancer-targeted Selenadiazole Derivative as Efficient Radiosensitizer for Precise Cancer Therapy Delong Zeng, Shulin Deng, Chengcheng Sang, Jianfu Zhao Zhao, and Tianfeng Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00247 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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Bioconjugate Chemistry

Rational Design of Cancer-targeted Selenadiazole Derivative as Efficient Radiosensitizer for Precise Cancer Therapy Delong Zeng#, Shulin Deng#, Chengcheng Sang, Jianfu Zhao*, Tianfeng Chen*

The First Affiliated Hospital, and Department of Chemistry, Jinan University, Guangzhou 510632, China. E-mail addresses: [email protected], [email protected]. # These authors contribute equally to this study. * Corresponding author: Tel: +86 20 85223393.

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ABSTRACT: Chemical drug design based on the biochemical characteristics of cancer cells has become an important strategy for discovery of targeted therapies for personalized cancer medicine. Herein, cancer targeting RGD peptide has been covalently conjugated to selenadiazole derivative (RGD-SeD) to improve its cancer selectivity. The RGD decoration significantly enhances the anticancer efficacy of RGD-SeD in αVβ3 integrin-overexpressing HepG2 liver cancer cells, but not in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of RGD-SeD to integrin overexpressing cancer cells. RGD-SeD strongly sensitizes HepG2 cells to clinically-used X-ray radiotherapy through ROS overproduction, which triggers DNA damage-mediated apoptosis and G2/M cell cycle arrest. This X-ray responsive DNA damage activates p53 signaling pathways by phosphorylation of ATM/ATR and γ-H2A.X. Furthermore, in HepG2 nude mice xenograft model, RGD-SeD combined with X-ray demonstrates potent in vivo antitumor efficacy via induction of apoptotic cell death, but shows no toxicity on the functions of major organs. In summary, this study provides a strategy to design selenium-based cancer targeting radiosensitizer for precise cancer therapy.

Key words: Cancer targeting; Drug design; Selenadiazole derivative; Radiosensitizer

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INTRODUCTION Radiotherapy is one of the major therapeutic strategies to cure cancer, which is received by approximately 50% of all cancer patients 1, 2. Using high-energy ionizing radiation, such as X-ray or γ-ray, to irradiate cancer tissue, radiotherapy causes DNA damages as well as lesions of plasma membrane in cancer cells to kill them or slow down their growth 3. Radiotherapy is effective in cancer treatment and has several advantages over surgery and chemotherapy, such as noninvasiveness and low systemic toxicity. However, a large proportion of cancer patients still suffer from local recurrence of disease after radiotherapy 4. The effectiveness of radiotherapy is limited by the maximum tolerated dose of the normal tissues around tumors. In addition, cancer cells may resist to radiation inherently or acquire resistance during therapy 4. Therefore, chemotherapy is widely used in combination with radiotherapy to gain additive of synergistic effect; e.g. cisplatin, 5-fluorouracil, mitomycin C are commonly used as radiosensitizers in head and neck cancers, lung cancers and gastrointestinal cancers 5. Tirapazamine and nitroimidazoles can sensitize hypoxic tumors to radiation. EGFR antibodies and inhibitors cetuximab, gefitinib, and erlotinib show enhanced radiosensitivity in vitro and in vivo 6, 7. The inhibitor of PARP-1, which is required for the DNA base excision repair and single strand breaks (SSB) repair, has entered clinical trial 8. Despite the progress in radiosensitizer research that has been made in recent years, the clinical outcome of many of these radiosensitizers is not quite satisfactory. For example, although platinum complexes, including cisplatin, oxaliplatin and carboplatin, are the most successful and best-known metal complex radiosensitizers, their radiosensitization effects are not selective for tumor cells, enhancing the damage to normal tissue during radiation 9. Therefore, it is of great significance to develop new radiosensitizers with high efficacy and low toxicity. Selenium compounds may be one kind of candidates that meet these criteria. Selenium (Se) is an 3



essential trace element, which plays important roles in metabolism, inflammation and reproduction, as well as in maintaining the functions of immune cells, thyroid and brain 10, 11. Selenium compounds supplied in nutritional level can work as antioxidants and prevent the incidences of cancer and cancer motility 12-14. Although higher level intake will shift selenium compounds into pro-oxidants and exhibit high toxicity, the fact that cancer cells are more vulnerable to selenium,15 due to their increased cellular reactive oxygen species (ROS) levels, endows the selenium compounds with selective cancer-cell killing capacity. Various selenium compounds, both inorganic and organic, has been reported to show anticancer activity against different types of cancer and to augment the efficacy of chemo- and radiotherapy 12, 16. In our previous studies, we have designed and synthesized several selenadiazole derivatives and selenium-containing metal complexes, which showed high activities on tumor growth inhibition 17-20

, anti-angiogenesis 21, 22 and anti-invasion 23, as well as reversion of drug resistance 24, 25 and

synergism with chemo- and radiotherapy 26, 27. However, cancer selectivity of these selenium compound was limited. Therefore, to further improve the selectivity of selenadiazole derivatives to cancer cells in radiotherapy, we decorated a novel selenadiazole derivative (SeD) with RGD peptide that could bind to αVβ3 integrin frequently overexpressed on various types of cancer cells 28. The in vitro and in vivo radiosensitization effects of RGD-SeD, and the underlying action mechanisms were also elucidated (Figure 1A). Taken together, this study demonstrated an effective way to improve the cancer selectivity of selenium-based radiosensitizer for personalized cancer medicine.

RESULTS Rational design of cancer-targeted RGD-SeD We first examined the expression levels of integrin in HepG2 liver cancer cells and L02 normal 4


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liver cells. Western blot analysis showed about 1.7 folds higher of integrin expression in HepG2 cells than that in L02 cells (Figure 1B and C), which indicate that integrin may be an effective target for anticancer drugs selectively recognizing liver cancer cells. In order to enhance the effect of radiotherapy, we designed and synthesis a new selenadiazole derivative as a radiosensitizer, and further decorated it with a circular RGD peptide, which can highly specifically bind to αVβ3 integrin. The synthesized selenadiazole derivatives were characterized by UV/vis spectroscopy, ESI mass spectrometry, elemental analysis and HPLC (see the Experimental Section for further details and Figures S1 and S2 in the Supporting Information).

Figure 1. Scheme of the present work and cytotoxic and radiosensitization effects of SeD and RGD-SeD to HepG2 and L02 cells expressing different levels of integrin. (A) Schematic illustration of the design SeD and RGD-SeD and the synergistic mechanism of X-ray and RGD to selectively kill cancer cells. Expression of Integrin in HepG2 and L02 cells were detected by Western blot (B), and the relative intensity of the bands were analyzed by ImageJ software (C). (D-E) Cytotoxic and radiosensitization effect of SeD and RGD-SeD against HepG2 (D) and L02 (E) cells. Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA).

In vitro antitumor and radiosensitization effects of RGD-SeD 5



MTT assay was used to assess the in vitro antitumor activity and radiosensitization effects of SeD and RGD-SeD. As shown in Figure 1 D and E, the IC50 values of SeD and RGD-SeD against HepG2 cells were 50.18 µM and 31.22 µM, respectively; while in L02 cells the IC50 value were 93.69 µM and 98.07 µM, respectively. As a positive control, cisplatin showed the highest antitumor activity with IC50 value at 3.9 µM against HepG2. However, its cytotoxicity was not selective toward tumor cells, as evidenced by the comparable IC50 of L02 cells (4.3 µM) to that of HepG2 cells. Although, to some extent, SeD showed selectivity to cancer cells compared with normal cells, the selectivity was much higher after decoration with RGD peptide. Similarly, the radiosensitization effect of SeD on HepG2 cells was enhanced by RGD decoration; The IC50 of SeD in combination with radiation (8 Gy) was 20.79 µM, and it reduced to 10.26 µM when decorated with RGD. In L02 cells, the radiosensitization effects of both SeD and RGD-SeD were remarkably weaker.

Cellular uptake and localization of RGD-SeD To evaluate whether the enhanced antitumor and radiosensitization effects of RGD-SeD were due to its increase of cellular uptake by HepG2 cells mediated by binding of RGD group to integrin on the cell surface, we measured the intracellular Se concentration in cells treated with SeD and RGD-SeD for different time periods. We found that the uptake of RGD-SeD by HepG2 cells was significantly higher than that of SeD at all the tested time points (Figure 2A and C). In L02 cells, the uptake of SeD was comparable with RGD-SeD (Figure 2B and D). The different uptake properties between RGD-SeD and SeD in HepG2 and L02 cells further confirmed the tumor targeting ability of RGD-SeD.

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Figure 2. Cellular uptake of SeD and RGD-SeD by HepG2 and L02 cells. HepG2 (A) and L02 (B) cells were treated with SeD (50 µM) and RGD-SeD (50 µM) for the indicated time before harvested. Then the cells were digested and subjected to ICP-MS for Se measurement. HepG2 (C) and L02 (D) cells were treated as A and B and the fluorescent images were taken at the indicated time points to visualize the intake of the compounds (green fluorescence). DAPI (blue) was used to stain cell nucleus. Scale bar represents 100 µm.

We next used fluorescence imaging technique to explore the intracellular trafficking of RGD-SeD in HepG2 cells. As shown in Figure 3, green fluorescent RGD-SeD accumulated in HepG2 cells in a time-dependent manner. RGD-SeD appeared inside HepG2 cells as early as 2 h, and the fluorescent intensity increased in 4 h and 8 h. We also used the LysoTracker® Red to label lysosomes, and found that the intracellular RGD-SeD was co-localized with lysosome. The results indicated that the uptake of RGD-SeD by HepG2 cells might be through endocytosis after binding to integrin.

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Figure 3. Localization of RGD-SeD in HepG2 cells detected by fluorescent imaging. HepG2 cells were treated with RGD-SeD (50 µM) and labeled by DAPI for nucleus and LysoTracker® Red for lysosome. Images were taken in different time points by fluorescent microscopy. Scale bar represents 100 µm.

RGD-SeD synergizes with X-ray to inhibit colony formation of HepG2 cells The decrease of IC50 when RGD-SeD combined with X-ray to treat HepG2 cells, implied the radiosensitization effect of RGD-SeD. To further confirm the synergism between RGD-SeD and X-ray, we measured the colony formation of HepG2 cells after treatment with RGD-SeD combined with or without X-ray. As shown in Figure 4A, treatment of 2 Gy X-ray alone did not significantly inhibit the colony formation ability of HepG2, compared with control, implying the capability of HepG2 cell to repair DNA damage caused by low dose of X-ray. However, treatment with RGD-SeD, even in a concentration much lower than IC50, greatly reduced the formation of colonies. The inhibition were further enhanced by combining RGD-SeD with X-ray. Only several colonies 8


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were found after combination treatment; the survival fraction was less than 10% (Figure 4B). The results demonstrated that colony inhibition effect on HepG2 cells of X-ray could be enhanced by RGD-SeD.

Figure 4. RGD-SeD synergizes with X-ray to attenuate the colony formation and cell cycle of HepG2 cells. (A and B) HepG2 cells were seeded in low density (1000 cells/mL) and treated with RGD-SeD (8 µM) and X-ray (2 Gy), and then continue to culture for 2 weeks. The colonies were visualized by staining with crystal violet and imaged (A), colonies contained >50 cells in each well were counted and shown as percentage to untreated (control) group (B). Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA). (C and D) HepG2 cells were treated with RGD-SeD (20 µM) and X-ray (8 Gy). The cells were harvested, fixed and stained with PI for cell cycle analysis by flow cytometry (C) and stacked bar chart of each cell cycle phase were shown (D). 9



RGD-SeD enhances X-ray-caused G2/M arrest and apoptosis of HepG2 cells Cell cycle arresting on G2/M phase and apoptosis are two main consequences of cells exposed to radiation. We therefore examined the cell cycle distribution of HepG2 cells after treatment with RGD-SeD and X-ray. Cell cycle analysis by flow cytometry showed that X-ray (8 Gy) significantly caused G2/M arrest of HepG2 cells; the percentage increase from 15.27% to 30.51% (Figure 5). G0/G1 phase was modestly increased, while apoptosis, which was indicated by sub-G1 population, was not changed. RGD-SeD treatment alone was able to arrest the cell cycle on G2/M phase and caused apoptosis. Co-treatment with RGD-SeD and X-ray caused more profound G2/M arrest and apoptosis; nearly half of the cells were arrested on G2/M phase and sub-G1 proportion reached about 20% (Figure 4C and D). These data suggested that RGD-SeD inhibited HepG2 cell growth by causing cell cycle arrest on G2/M phase and killed the cells by induction of apoptosis, and these effects might contribute to the synergism with X-ray radiation.

RGD-SeD enhances X-ray-induced cellular ROS overproduction Reactive oxygen species (ROS) can be induced by organic selenium compounds 24 and radiation 29 which then play an important role in mediating cell death. In order to explore whether ROS is produced in HepG2 cells treated with RGD-SeD and X-ray, we labeled the cells with dihydroethidium (DHE), a fluorescent ROS probe, and recorded the fluorescent intensities in different time points. As shown in Figure 5A, ROS level was increased to about 120% after X-ray irradiation, compared with untreated control. RGD-SeD treatment alone did not changed the ROS level significantly. When combined with X-ray, however, RGD-SeD strongly enhanced the ROS produced by X-ray. The fluorescent images taken by fluorescent microscope also clearly showed 10


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the enhancement of ROS production in the combination treatment group (Figure 5B).

Figure 5. RGD-SeD enhances ROS overproduction induced by X-ray. HepG2 cells were treated with RGD-SeD (20 µM) alone or in combination with X-ray (8 Gy). DHE fluorescent probe was added to the cells to labeled ROS, fluorescent intensity (A) were measured by microplate reader in different time points. Fluorescent images (B) were taken at the end of reading by a fluorescent microscope. Scale bar represents 100 µm.

RGD-SeD enhanced X-ray-caused DNA damage in HepG2 cells Excessive ROS in irradiated cells may react with DNA in the nuclei causing DNA damage, which is the main cause of cell cycle arrest and apoptosis after radiation. Phosphorylation on serine 139 of histone H2A.X, which is then referred as γH2AX, is an early step of DNA damage response and can be served as an biomarker of DNA damage 30. As RGD-SeD enhanced the X-ray-induced intracellular ROS production, we further investigated whether this effect was translated to more DNA damage. Immunofluorescence analysis of RGD-SeD and X-ray treated HepG2 cells (Figure 6A) showed that RGD-SeD or X-ray treatment alone increased the γH2AX foci by a moderate extent. However, co-treatment of HepG2 cells with RGD-SeD and X-ray resulted in significant increase of γH2AX focus and fluorescent intensity, indicating more DNA damage produced. We then confirmed this result by examining the protein level of γH2AX using Western blot analysis 11



(Figure 6B). Quantitative analysis of integrated density of the bands in Western blot showed that RGD-SeD and X-ray treatment alone increased the γH2AX signal by about 2 folds, while the combination treatment resulted in about 10-fold increase (Figure 6C). These data indicated that RGD-SeD enhanced X-ray-caused DNA damages in HepG2 cells, which promoted cell apoptosis.

Figure 6. RGD-SeD increases γ-H2A.X foci induced by X-ray in HepG2 cells. (A) HepG2 cells were treated with RGD-SeD (20 µM) and X-ray (8 Gy), and subjected to immunofluorescent analysis for γ-H2A.X. DAPI was used to label nuclei and images were taken by fluorescent microscopy. Scale bar represents 10 µm. (B) HepG2 cells were treated with RGD-SeD (20 µM) and X-ray (8 Gy), and the cells were lysed and subjected to Western blot for γ-H2A.X. (C) The relative intensity of bands of γ-H2A.X in (B) were measured using ImageJ software. Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA).

RGD-SeD and X-ray induce mitochondria fragmentation in HepG2 cells Mitochondria play an important role in regulating apoptosis in addition to their energy-producing function. Extracellular and intracellular stresses may cause mitochondria dysfunction, which then release several apoptosis promoting factors to activate apoptosis process. Mitochondria 12


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fragmentation is the morphology change reflecting the mitochondria dysfunction. We therefore monitored the mitochondria status by labeling the cells with MitoTracker Red CMXRos. The results showed that the mitochondria in untreated control group were filamentous, interconnected as a network throughout the cytoplasm (Figure 7). Treatment with RGD-SeD or X-ray resulted in mitochondria fragmentation, breaking the network. Co-treatment with RGD-SeD and X-ray enhanced this fragmentation. The results confirmed that mitochondria were involved in mediating the apoptosis caused by RGD-SeD and X-ray.

Figure 7. RGD-SeD and X-ray causes mitochondrial fragmentation of HepG2 cells. HepG2 cells were treated with RGD-SeD (20 µM) and X-ray (8 Gy), then the cells were labeled with MitoTracker® Red CMXRos and DAPI, and images were by fluorescent microscopy. Scale bar represents 10 µm. Arrows showed the fragmented mitochondria.

DNA damage response and apoptosis signaling activated by RGD-SeD and X-ray To further confirm the apoptosis induced by RGD-SeD and X-ray, we examined the expressions of apoptosis-related proteins by Western blot. The hypothetic molecular mechanism of RGD-SeD and 13



X-ray triggering apoptosis were depicted as Figure 8A, wherein RGD-SeD synergized with X-ray to cause ROS overproduction, DNA damage and activation of ATM/ATR and p53, which then induced cell cycle arrest and apoptosis. We first measured the phosphorylation level of ATM, ATR and p53 in RGD-SeD and/or X-ray treated HepG2 cells. As shown in Figure 8B, treatment with RGD-SeD or X-ray increased the phosphorylation of ATM, ATR and p53, which was further enhanced by combination treatment. The results demonstrated the activation of DNA damage response in the treated cells. The occurrence of apoptosis was then examined by detecting the cleavages of caspase-8, -9 and -3, as well as their substrate PARP (Figure 8C). Strongest cleavage of these proteins was demonstrated in the co-treatment group, reflecting significant synergism of the two treatments. We further confirmed the activation of caspase -9 and -3 by measurement of the enzyme activities using specific fluorescent substrates. Figure 8D showed that the activities of caspase-9 and -3 were significantly increased after treatment with X-ray or RGD-SeD, and the combined treatment potently enhanced the activation.

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Figure 8. RGD-SeD synergizes with X-ray to activate DNA damage and apoptosis pathways in HepG2 cells. (A) Proposed signaling pathways underlying RGD-SeD and X-ray-mediated cancer cell killing. HepG2 cells were treated with RGD-SeD (20 µM) and/or X-ray (8 Gy). Cells were harvested, and cell lysate were subjected Western blot to determine the activation of DNA damage pathway (B) and apoptosis pathway (C). (D) Caspase-3 and -9 activities of HepG2 cells treated with RGD-SeD (20 µM) and/or X-ray (8 Gy) were determined by specific fluorescent substrates. Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA).

In vivo radiosensitization activity of RGD-SeD To further examine the in vivo radiosensitization activity of SeD and RGD-SeD, we tested their tumor inhibition effects on nude mice bearing HepG2 xenograft. As shown in Figure 9A, treatment 15



with X-ray alone significantly inhibited the growth of tumor. The mice receiving SeD or RGD-SeD alone also showed substantial retardation of tumor growth, and better activity of RGD-SeD than SeD can be observed. When combined SeD or RGD-SeD with X-ray treatment, the tumor growth were further inhibited by factors of 1.60 and 2.11, respectively. Measurement of the tumor weights shown similar results (Figure 9B and C). Treatment with SeD or RGD-SeD alone resulted in no change of mice’s body weights, while X-ray treatment alone or in combination with SeD or RGD-SeD caused considerable decrease of the mice’s body weights (Figure 9D), which might be the results of the side effects of X-ray. No further loss of body weight in the mice of co-treatment groups comparing with X-ray group demonstrated no significant toxicity of SeD and RGD-SeD. We next evaluated the apoptosis and proliferation status of cells in tumor tissues by immunohistochemical staining of cleaved-caspase-3 and Ki67. As shown in Figure 9E, lowest expression of cleaved-caspas-3 was in control group, and the expression level increased in the treatment groups, most strongly in RGD-SeD and X-ray combination group. Accordingly, the control group showed highest expression of Ki67 and treatment with X-ray or SeD or RGD-SeD resulted in decreased expression (Figure 9E). Finally, the side effects of SeD and RGD-SeD were evaluated by H&E staining of major organs sections and examination of blood biochemical indexes. No obvious toxicity of SeD and RGD-SeD to major organs was seen in H&E staining sections (Figure 10A). Blood biochemical indexes analysis showed that RGD-SeD combined with X-ray most potently alleviated the xenograft-induced changes of blood glucose (GLU), blood urea nitrogen (BUN), creatine kinase (CK), aspartate aminotransferase (AST) and high-density lipoprotein cholesterol (HDL-C) and cholesterol (CHOL) (Figure 10B).

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Figure 9. In vivo radiosensitization effect of SeD and RGD-SeD. (A) Growth curves of tumors on mice receiving the indicated treatments. (B, C) The weights (B) and image (C) of tumors dissected at the end of the experiment from the mice in different treatment groups. Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA). (D) Body weight of the mice receiving the indicated treatments. (E) Immunohistochemical analysis of the expressions of cleaved-caspase-3 (C-Casp-3) and Ki67 in tumor tissues of mice. Scale bar represents 100 µm.

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Figure 10. Histochemical and hematological analysis of in vivo radiosensitization effect and toxicity of SeD and RGD-SeD. (A) H&E staining of sections from the mice’s major organs and tumor. The black arrows denoted the sites of pulmonary hemorrhage in alveolus pulmonis. Scale bar represents 100 µm. (B) Hematological analysis of healthy (no xenograft) and treated nude mice. The biochemistry indexes included blood glucose (GLU), blood urea nitrogen (BUN), high-density lipoprotein cholesterol (HDL-C), total cholesterol (CHOL), creatine kinase (CK) and aspartate aminotransferase (AST). Bars with different characters are statistically significant (P < 0.05, Tukey’s test, one-way ANOVA).

DISCUSSION In the past decade, promising anticancer activity had been found in Se-containing species, including inorganic selenite and selenate, organic selenodiglutathione, selenoaminoacid derivatives, methylseleninic acid, selenides and diselenides, selenocyanates, se containing heterocycles, as well as selenium nanoparticles 12. Among these agents, we previously found that selenadiazole derivatives had high anticancer activity with relative low toxicity, which trigged ROS 18


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overproduction and DNA damage to induce apoptosis in various cancer cell lines 17, 18, 20, 23-25. Moreover, selenadiazole derivatives strongly sensitized cancer cells to radiotherapy by inhibition of thioredoxin reductase (TrxR) 26. In order to find novel radiosensitizer, a selenadiazole derivative SeD was synthesized in the present study. In vitro data showed high radiosensitization activity of the newly synthesized selenadiazole derivative. However, like other traditional chemotherapy agents, the selectivity of previous reported selenadiazole derivatives between cancer cells and normal cells was limited. Decoration of cancer cell-targeting groups is a useful strategy to improve the selectivity of both small molecule drugs and nanoparticle delivery systems. RGD peptide, due to its selective binding ability to ανβ3 integrin overexpressed in various types of cancer cells, has been considered to be an ideal targeting molecule for modification of anticancer drugs and nanoparticle delivery systems 28. RGD conjugates of several traditional chemotherapy drugs had been synthesized and evaluated, such as paclitaxel 31., doxorubicin,32 platinum complexes 33 and camptothecin 34. Our previous study also showed that surface decoration of nanoparticle with RGD peptide significantly increased their selectivity between cancer and normal cells 35-37. Here, a c(RGDfk) group was conjugated to the newly synthesized selenadiazole derivative, to improve its radiosensitization effect and lower its toxicity in liver cancer, as we found that ανβ3 integrin was overexpressed in HepG2 cells but not in L02 cells. After decoration, the radiosensitization effect of the conjugate was enhanced by increased cellular uptake. ROS is important in mediating the X-ray-induced cell death through damaging of DNA. Therefore, anticancer agents which could induce ROS overproduction may synergize with X-ray to kill cancer cells. Several of our previous reports had suggested that selenadiazole derivatives can induce ROS overproduction in cancer cells 18, 19, 24, 26, which may be caused by the inhibition of ROS-eliminating 19



enzyme, TrxR 26, 38. Consistent with this, RGD-SeD synthesized in the present study effectively increased production of ROS in HepG2 cells, and showed strong synergism with X-ray. As a result of ROS overproduction, the cell cycle of HepG2 cells were arrested in G2/M phase, and more DNA damages were evidenced by increased γ-H2A.X foci in the nuclei. Increased sub-G1 fraction was also observed in HepG2 cells treated with combination of RGD-SeD and X-ray, which implied the apoptosis of HepG2 cells. DNA damage and apoptosis of HepG2 cells were further confirmed by Western blot examining the phosphorylation of p53, ATM/ATR and cleavage of caspase. In addition, mitochondrion fragmentation, which may happen in the early stage of apoptosis, was also detected. Using HepG2 xenograft model we confirmed the in vivo antitumor activity of SeD and RGD-SeD, which showed similar to in vitro results. The toxicities of SeD and RGD-SeD were also evaluated by monitoring the mice body weight, blood biochemical indexes and histochemical analysis. SeD or RGD-SeD showed no evident toxicity to the mice. However, X-ray treatment alone caused obvious body weight loss of mice, which was consistent with the severe side effects of clinical radiotherapy. But no further loss of mice body weight in the combination groups, suggesting that SeD or RGD-SeD increased the therapeutic effects of X-ray without exaggerating its side effects.

CONCLUSIONS In this study, cancer targeting RGD peptide has been covalently conjugated to selenadiazole derivative to improve its cancer selectivity. The RGD decoration significantly enhances the anticancer efficacy of RGD-SeD in αVβ3 integrin-overexpressing HepG2 liver cancer cells, but not in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of RGD-SeD to integrin overexpressing cancer cells. RGD-SeD strongly sensitizes HepG2 cells to 20


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X-ray radiotherapy through ROS overproduction, which triggers DNA damage-mediated apoptosis and G2/M cell cycle arrest. This X-ray responsive DNA damage activates p53 signaling pathways by phosphorylation of ATM/ATR and γ-H2A.X. Furthermore, in HepG2 nude mice xenograft model, RGD-SeD combined with X-ray demonstrates potent in vivo antitumor efficacy via induction of apoptotic cell death, but shows no toxicity on the functions of major organs. Taken together, this study demonstrates an effective way to improve the cancer selectivity of selenium-based radiosensitizer for personalized cancer medicine.

EXPERIMENTAL PROCEDURES Synthesis and characterization Synthesis of SeD:

(1) (1,1’-Biphenyl)-3,3’,4,4’-tetraamine (30 mmol) was dissolved in 600 mL

hydrochloric acid solution (HCl/H2O=1:5) in a 1000 mL flask. Selenium dioxide (30 mmol) was dissolved in 30 mL hot distilled water and transferred to constant pressure funnel, then the solution was added drop wise to the flask. The mixture was stirred for 1.5 h at room temperature. The yellow solid precipitated during the reaction were filtered and redissolved in water. The pH of the solution was adjusted to about 7.0 with sodium hydroxide solution. The mixture was filtered again and vacuum dried to afford 4- (benzo[c][1,2,5]selenadiazol-5-yl)benzene-1,2-diamine in a yield of 95%. (2) The product of step (1) was dissolved (3 mmol) in 30 mL DMF in a 100 mL beaker. N-Acetic acid-indole-3-carboxaldehyde was added with a catalytic amount of p-methylbenzene sulfonic acid. The mixture was stirred at 80 °C for 1 h. After cooling, the mixture was added into 200 mL saturated Na2CO3 solution and stirred for 10 min at room temperature. The mixture was filtered and the filtrate was transferred in to 500 mL lithium chloride (2 M) solution. A large amount of yellow solid precipitated and the precipitate was filtered and vacuum dried to give crude product, which 21



was then purified by column chromatography on silica gel (petroleum ether/EtOAc=5:1) to obtain pure product [3-(5-Benzo[1,2,5]selenadiazol-5-yl-1H-benzoimidazol-2-yl)-indol-1-yl]-acetic acid (SeD); Yield: 60%.

Synthesis of RGD-SeD: SeD (1 mmol) was dissolved in DMF then EDC and NHS (1.2 mmol each) were added to activate the compound. After 2 h, 1 mmlo of c(RGDfK) (Gier biochemistry CO., Ltd, Shanghai, China) and a catalytic amount of riethylamine were added and the mixture was stirred for another 24 h. When the reaction completed, the mixture was centrifuged at 6000 rpm to obtain supernatant, which were then added to diethyl ether to precipitate the produce. After filtration, the cure produce was washed twice by diethyl ether then vacuum dried. The produce was further purified by HPLC using acetonitrile - water as eluent. After freeze drying, the pure product (RGD-SeD) was obtained; Yield: 80%.

SeD: Yellow solid (60%); elemental analysis calcd for C23H15N5O2Se (%): C, 58.48; H, 3.20; N, 14.83; found (%): C, 58.80; H, 3.28; N, 14.72; ESI-MS (in CH3OH): m/z calcd for C23H15N5O2Se (M+H)+473.9; found 474.9. RGD-SeD:

Yellow solid (80%); elemental analysis calcd for C50H54N14O8Se (%): C, 56.76; H,

5.14; N, 18.53; found (%): C, 56.80; H, 5.28; N, 18.12; ESI-MS (in CH3OH): m/z calcd for C50H54N14O8Se (M+H)+ 1059.9; found 1059.9.

Cell culture HepG2 human liver cancer cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). L02 human normal liver cell line was obtained from Cell Source 22


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Center, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 UmL-1), and streptomycin (50 U mL-1) at 37 °C in a humidified incubator with 5% CO2 and 95% air. All the cell lines were tested for mycoplasma by PCR and fluorescent DNA staining before used.

Cell viability assay Cells were plated in 96-well plates in a density of 2000 cells/well (HepG2) or 4000 cells/well (L02) and cell viabilities after treatment with various concentrations of compounds were evaluated by MTT assay 39, 40.

Determine of cellular uptake of compounds Quantitative analysis of cellular uptake of SeD and RGD-SeD was determined by ICP-MS as previously described 27, 41.

Clonogenic assays Colony formation ability of HepG2 cells were measured by clonogenic assay as previously described 26, 42.

Live cell imaging Localization of RGD-SeD in HepG2 cells and fragmentation of mitochondrion induced by RGD-SeD and X-ray were visualized by EVOS® FL Auto Imaging System (Life technologies). Labeling of nucleus, lysosome and mitochondrion were done by incubation of DAPI (300 nM), 23



LysoTracker® Red (50 nM) and MitoTrakcer® Red CMXRos (300 nM), respectively, according to the manufactory’s protocol. The probes were all from Molecular Probes, Life technologies.

Cell cycle analysis Cell cycle distribution of HepG2 cells were detected by flow cytometry after propidium iodide (PI) staining as previously described 43-45.

Immunofluorescent analysis of γH2AX HepG2 cells were plated on 2-cm glass dishes and treated RGD-SeD (20 µM) for 6 h then were exposed to X-ray (8 Gy). After 2 h, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 1% Triton X-100 and 0.5% NP-40 for 20 min at room temperature. After washed by PBS for 3 times and blocked by 2% BSA for 1 h at room temperature, cells were incubated with primary antibody against phospho-Histone H2A.X (Ser139) (1:500, Cell Signaling Technology) at 4 °C overnight. Cells were then washed 3 times with PBS and incubated with Alexa fluor-488 conjugated secondary antibody (1:500, Cell Signaling Technology) for 1 h at room temperature. The cells were then washed with PBS again and immediately imaged by EVOS® FL Auto Imaging System (Life technologies).

Measurement of intracellular reactive oxygen species (ROS) The intracellular ROS level of HepG2 cells was measured by using DHE staining 46, 47.

Western blotting Expression level or phosphorylated form of proteins related to DNA damage and apoptosis were 24


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determined by Western blot analysis 48, 49. Rabbit anti-human phospho-Histone H2A.X (Ser139) antibody (#2577), rabbit anti-human Integrin β3 antibody (#13166), rabbit anti-Human p53 antibody(#2527), mouse anti-human phospho-p53 (Ser15) antibody (#9286), rabbit anti-human phospho-ATM (Ser1981) antibody (#5883), rabbit anti-human phospho-ATR (Ser428) antibody (#2853), rabbit anti-human PARP antibody (#9532), rabbit anti-human caspase-8 antibody (#4790), rabbit anti-human caspase-9 antibody (#9520), rabbit anti-human caspase-3 antibody (#9662), HRP-linked horse anti-mouse IgG antibody (#7076) and HRP-linked goat anti-rabbit IgG antibody (#7074) were purchased from Cell Signaling Technology, Inc. Mouse anti-human β-Actin (A1978) antibody was from Sigma-Aldrich.

Caspase activity assay Measurement of caspase-9 and -3 activities were conducted as previously described by using specific substrates 44.

Nude mice xenograft model All nude mice experiments were approved by the Laboratory Animal Ethics Committee of Jinan University. Female Balb/c nude mice (3-4 weeks old and weighing 14–15 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The animals were acclimated to the environment for 10 days before inoculation of cancer cells. Each mouse was injected subcutaneously with 1.5 × 106 HepG2 cells in 100 µL PBS. When the tumor volume reached ~100 mm3, the mice were randomly divided into 6 groups, receiving X-ray, SeD, RGD-SeD, X-ray + SeD and X-ray + RGD-SeD treatment, and saline as control group. Every other day, the body weight and tumor volume (V = l × w2/2, l = tumor length, w = tumor width) of the 25



mice were measured, and the compounds (SeD or RGD-SeD, 2 mg/kg) and saline were administrated through intravenous injection at the same time. The mice that needed to be irradiated were then exposed to X-ray (4 Gy). After 21 days, the mice were sacrificed to collect the blood, organs, and tumors for biochemical and immunohistochemical analyses 50-52.

Hematological and histological analyses The blood samples of mice were centrifuged at 1500 rpm for 10 min, and the serum were obtained and sent to the Blood Test Center of the First Affiliated Hospital of Jinan University for hematological analysis. The tumor, heart, liver, spleen, lung and kidney were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining and immunohistochemical analysis.

Statistical analysis All experiments were carried out in triplicate and the data were expressed as mean ± standard deviation (SD). Statistical analysis was carried out in GraphPad Prism 5 software (GraphPad Software Inc.). Differences between two groups were analyzed by the two-tailed Student’s t test. One-way analysis of variance was used in multiple group comparisons. P < 0.05 was considered to be significant differences between groups.

CONFLICTS OF INTEREST The authors declare no competing financial interest.

ACKNOWLEDGEMENTS 26


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This work was supported by National Program for Support of Top-notch Young Professionals (W02070191), Yang Fan Innovative & Entepreneurial Research Team Project (201312H05), Fundamental Research Funds for the Central Universities and China Postdoctoral Science Foundation (20182018M633274).

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed methods and ESI-MS, HPLC and UV/vis absorption spectra of SeD and RGD-SeD.

ABBREVIATIONS RGD, Arginylglycylaspartic acid; ROS, reactive oxygen species; DHE, dihydroethidium; DAPI, 4',6-diamidino-2-phenylindole; GLU, blood glucose; BUN, blood urea nitrogen; CK, creatine kinase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein cholesterol; CHOL, cholesterol.

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Table of Contents Graphic

Herein we have designed and synthesized an RGD peptide-decorated selenadiazole derivative, RGD-SeD, as a selective radiosensitizer, targeting αVβ3 integrin-overexpressing cancer cells for precise cancer therapy.

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Rational Design of Cancer-Targeted ... - ACS Publications

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