There is a compelling need to develop anticancer therapies that target cancer cells and tissues. Arising from innovative femtomedicine studies, a new class of non–platinum-based halogenated molecules (called FMD molecules) that selectively kill cancer cells and protect normal cells in treatments of multiple cancers has been discovered. This article reports the first observation of the radiosensitizing effects of such compounds in combination with ionizing radiation for targeted radiotherapy of a variety of cancers. We present in vitro and in vivo studies focused on combination with radiotherapy of cervical, ovarian, head and neck, and lung cancers. Our results demonstrate that treatments of various cancer cells in vitro and in vivo mouse xenograft models with such compounds led to enhanced efficiencies in radiotherapy, while the compounds themselves induced no or little radiotoxicity toward normal cells or tissues. These compounds are therefore effective radiosensitizers that can be translated into clinical trials for targeted radiotherapy of multiple types of cancer. This study also shows the potential of femtomedicine to bring breakthroughs in understanding fundamental biologic processes and to accelerate the discovery of novel drugs for effective treatment or prevention of a variety of cancers. Mol Cancer Ther; 15(4); 640–50. ©2016 AACR.

This article is featured in Highlights of This Issue, p. 531

Radiotherapy remains a major curative therapy for cancer (1–4). A number of chemical compounds, termed radiosensitizers, have been tested in clinical trials to enhance the radiosensitivity (2, 4). The presence of hypoxia in tumors has important impacts on tumor progression and treatment outcome (2, 5); hypoxic cells are refractory to radiation treatment. Thus, discovery of hypoxic radiosensitizers that target tumor hypoxia is in large demand.

In liquid water, a free electron resulting from ionizing radiation (IR) is rapidly solvated by surrounding H2O molecules to form the well-known hydrated electron (ehyd), which was first discovered in the 1960s, and its reactivity was well determined (6, 7). With the advent in the 1980s of femtosecond (fs; 1 fs = 10−15 seconds) time-resolved laser spectroscopy (fs-TRLS), which is a direct technique to visualize molecular reactions in real time (8), the ultrashort-lived precursor to ehyd (the so-called prehydrated/solvated electron, epre) was directly observed (9–11). Our careful fs-TRLS measurements (12) led to resolving the long-standing controversies about the lifetime and physical properties of epre. After identifying and removal of the coherent “spike” effect in fs-TRLS measurements, we demonstrated that epre has a lifetime of about 500 fs and is a weakly bound excited state of the well-bound ehyd (12). Moreover, our fs-TRLS studies demonstrated the high reactivity of epre via direct real-time observations of the formation and dissociation kinetics of the intermediate state AB* of dissociative electron transfer (DET) reactions: epre+AB→AB*→A+B (12–18). Previous reviews on ultrafast DET reactions of epre with various biologic and environmental molecules can be found in refs.19–21.

Through fs-TRLS studies, we found that weakly bound epre plays a key role in IR-induced DNA damage in an aqueous environment. Particularly, we discovered that the ultrafast DET reaction of epre leads to chemical bond breaks at the guanine base (17) and DNA strand breaks (22). Our results challenged the conventional notion that damage to the genome by IR is mainly induced by an oxidizing OH radical and might lead to improved strategies for radiotherapy of cancer and radioprotection (19,23). Moreover, we have recently demonstrated a reductive damaging mechanism in living cells, which may be related to the pathology of diseases, especially cancer (24).

We also discovered the DET reaction mechanisms of cisplatin as both a widely used chemotherapeutic drug and a radiosensitizer (14, 15) and of halopyrimidines (XdUs, X = Cl, Br, or I) as potential radiosensitizers (13, 16, 18). Our findings have provided a molecular basis for the success and failure in clinical use/trials of cisplatin and halopyrimidines, respectively.

The integration of fs-TRLS technique with molecular biology and cell biology methods to advance fundamental understanding and therapies of human diseases, notably cancer, might lead to a new transdisciplinary frontier called femtomedicine (FMD) and advances in cancer therapy (19). Our mechanistic studies in FMD (12–19, 22) provide remarkable opportunities to improve the therapies of existing drugs and to develop new effective drugs. Indeed, our studies have led to the development of novel cisplatin-based combination therapies that could significantly improve the therapeutic efficacy of cisplatin against human ovarian, cervical, and lung cancers (25). On the basis of our new understanding of differential intracellular environments between normal and cancerous cells, we have also discovered a new family of non–platinum-based molecules (called FMD molecules) that selectively kill cancer cells and protect normal cells, effective for targeted chemotherapy of multiple cancers (26).

The newly discovered FMD compounds, such as 4,5-dichloro/dibromo/diiodo-1,2-diaminobenzene (benzenediamine/phenylenediamine) and 4(3)-chloro/bromo/iodo-1,2-diaminobenzene (benzenediamine/phenylenediamine; shortened as FMD-nX-DABs or B(NH2)2Xn, with X = Cl, Br, or I and n = 1, 2), act essentially as cisplatin analogues, whose molecular structures were reported previously (26). They are expected to be highly effective in DET reactions, with either weakly bound electrons intrinsic in the more reductive intracellular environment of abnormal (cancer) cells or ultrashort-lived epre produced by IR of biologic systems. The resultant radical [B(NH2)2Xn-1] is highly reactive and can effectively lead to DNA damage and cell death. An advantage of these FMD compounds over cisplatin and XdUs is that the DET reaction of FMDs is sufficiently effective, but they are far less toxic due to the absence of the heavy metal (Pt). Such FMD compounds alone have shown highly desirable targeting properties as antitumor agents for targeted chemotherapy of multiple types of cancer (26). Moreover, the DET reaction with an FMD molecule will be more effective in the microenvironment of tumor due to its hypoxia, which reduces the competitive attachment of epre to oxygen. Thus, the FMD compounds are expected to be effective hypoxic radiosensitizers. This article presents results of in vitro and in vivo studies of the FMD compounds in combination with IR to achieve targeted radiotherapy of human cervical, ovarian, head and neck, and lung cancers.

Chemicals and reagents

Details on all the chemicals and reagents used in this study have been given previously (26). FMD-nX-DABs have excellent solubility of >40 mmol/L in ethanol (EtOH) and limited solubility ranging from 0.1 to 10 mmol/L in MEM (cell culture medium). Thus, their stock solutions in 20 or 40 mmol/L were prepared by dissolving the compounds in pure EtOH. For in vitro cell line experiments, small volumes of the compound stock solutions were added into the culture wells, while the vehicle (EtOH) concentration was kept below 1.5%. For in vivo mouse experiments, our studies were focused on FMD-2Br-DAB, and its solubility in MEM was well determined to be 0.85 ± 0.07 mmol/L by spectrophotometric measurements. The stock solution of FMD-2Br-DAB was diluted in MEM to have 10% ETOH, and 100 μL of the diluted solution was injected into each mouse (∼20 g) to make a concentration at 7 mg/kg of FMD-2Br-DAB in mice, corresponding to an estimated in vivo concentration of 0.35 ± 0.03 mmol/L.

Cell lines and culture conditions

In the Waterloo laboratory (24–26), a human skin diploid fibroblast (GM05757 cell line) was directly obtained from the Coriell Cell Repositories (CCR); human cervical cancer cell line [HeLa, ATCC CCL-2; or ME-180 (ATCC HTB-33)], human ovarian cancer cell line (NIH:OVCAR-3, ATCC HTB-161), and human lung cancer cell line (A549, ATCC CCL-185), together with RPMI1640, F-12K, McCoy 5A, and L-15 culture media, which were directly obtained from the ATCC. FBS was obtained from HyClone Laboratories. The GM05757 normal cells and HeLa cells were cultivated with MEM (HyClone Laboratories) supplemented with 10% FBS, 100 U/mL penicillin G, and 100 μg/mL streptomycin (HyClone Laboratories). ME-180, NIH:OVCAR-3, and A549 cells were cultured with the ATCC-formulated McCoy 5A medium supplemented with 10% FBS, RPMI1640 medium with 20% FBS, F-12K medium with 10% FBS, and L-15 Medium (Leibovitz) with 10% FBS, respectively. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. In the STTARR facility in Toronto, 74B cells were acquired through a generous donation by Dr. Brad Wouters (Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada) and were cultured in Iscove MDM media with 10% FBS, whereas ME-180 and A549 cells were obtained from the ATCC directly.

The above listed cells obtained directly from the CCR or ATCC were passaged in our laboratories for fewer than 6 months after receipt or resuscitation. The CCR performed cell line characterizations by karyotypic and chromosome analyses, whereas the ATCC performed cell line characterizations by karyotypic analysis, morphology check, isoenzymology, and short tandem repeat analysis (DNA fingerprinting). The 74B cells obtained from Dr. Brad Wouters were tested by mycoplasma testing using PCR and genotyping within 6 months after receipt.

In vitro cell viability and clonogenic assays

The cell growth and survival rates with various treatments were respectively determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay (Invitrogen), one of the most commonly used cell viability assays, and the clonogenic assay. The details have been given previously (24–26). Briefly, cells were plated at a density of 7 × 103 cells/well in 96-well plates. Following overnight incubation, the culture medium was replaced by fresh culture medium. For in vitro radiotoxicity tests, the cells were incubated for 12 hours with varying drug concentrations, then irradiated by 225 kV X-ray (Precision, X-RAD IR 225) at a dose rate of 2 Gy/minute with various X-ray doses, and after irradiation, the cells were incubated for 12 days. The MTT assays of cell viability were then conducted. For in vitro tests of the radiosensitizing effects of the FMD compounds, similar MTT assays were conducted at 6 days postirradiation. For clonogenic assays (24, 27), various numbers of the cells ranging from 200 up to 2 × 105 were seeded to 100-mm tissue culture dishes (Thermo Scientific BioLite), depending on types of cancer cells and treatments. After X-ray irradiation, the cells were incubated for 15 to 17 days to form colonies. The cell colonies were then were fixed with glutaraldehyde (6.0%), stained with crystal violet (0.5% w/v), and counted.

In vitro DNA DSB assay

The phosphorylated H2AX foci are a biomarker of DNA double-strand break (DSB). The HCS DNA Damage Kit (Invitrogen) was developed to quantify the population of apoptotic cells by specific antibody-based detection of phosphorylated H2AX (γH2AX) in the nucleus. In addition, the kit included Hoechst 33342, which is a DNA-binding dye (blue fluorescence) and allows the observation of nuclear morphology of all normal and damaged cells. Briefly, the cells were treated with different concentrations of the compound for 8 hours, followed by IR (225 kV X-rays). At 12 hours after X-ray irradiation, the cells were fixed and stained. Following the detailed protocol provided by the manufacturer, we performed the HCS DNA Damage Assay of the treated cells, as described previously (24–26). The images of cells were acquired with a Nikon Eclipse TS100 fluorescence microscope; quantitative analyses of activated γH2AX (DNA DBS yield) in the cells were performed using an Image J software.

Xenograft mouse cancer models

ME-180 cervix, 74B head and neck, and A549 lung xenografts were derived at STTARR (University Health Network, Toronto, Ontario, Canada) for in vivo growth delay studies involving a FMD compound in combination with a large single dose IR (X-rays). Seven- to 8-week-old female SCID mice were injected subcutaneously in the flank with 1.5 × 106 cells; the treatments were started when xenografts reached a volume of approximately 100 mm3. Mice were randomly divided into four groups with 5 mice per group: (i) injection vehicle only; (ii) FMD-2Br-DAB (7 mg/kg); (iii) IR 15 Gy; and (iv) the FMD compound plus IR. The FMD compound at 7 mg/kg was administrated by intraperitoneal injection into the mice, and at 1 hour after injection, the mice received given doses of X-rays from a X-RAD 225 kV Irradiator (Precision X-Ray), either targeted to the tumor or the gut using a collimator. Animals were weighed and flank tumors measured every 2 to 5 days with calipers; measurements were converted into tumor volume. Growth delay was calculated as the time difference (in days) between treatment and control groups to grow to 1,000 mm3.

Similar mouse xenograft models (26) of ME-180 cervical cancer and A549 lung cancer were established in the Central Animal Facility of the University of Waterloo (Waterloo, Ontario, Canada) to investigate tumor growth delays caused by a FMD compound in combination with multiple small-dose fractionated IR (X-rays). Namely, 6- to 8-week-old female SCID mice were injected subcutaneously in the flank with 1.5 × 106 ME-180 cells or 5 × 106 A549 cells. The treatments were started when xenografts reached a volume of approximately 100 mm3. For each treatment, mice were randomly divided into four groups with 5 mice per group: (i) control (vehicle only); (ii) FMD-2Br-DAB alone at 7 mg/kg was administrated for totally 3 intraperitoneal injections at days 1, 3, and 5 (7 mg/kg × 3); (c) IR alone (2 Gy × 3); and (d) for the combination of a FMD with radiation. The FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and at 1 hour after injection, the targeted tumor in mice received radiation (2 Gy, 225 kV X-rays), with totally 3 treatments at days 1, 3, and 5 (7 mg/kg FMD × 3 + 2 Gy × 3). Mice were weighed, and flank tumors were measured every 2 to 5 days with calipers, and tumor volumes were calculated.

All animal experiments were conducted in accordance with the guidelines of the University Health Network Animal Care Committee or the University of Waterloo Animal Care Committee.

IHC

All serial sections were cut from paraffin-embedded tumor tissue and stained with hematoxylin and eosin (H&E). Sections were also stained for EF5 (provided by Dr. Cameron Koch, University of Pennsylvania, Philadelphia, PA), CD31 (Dr. Cameron Koch), γH2AX (Bethyl Laboratories, rabbit polyclonal), 53BP1 (Bethyl Laboratories, rabbit polyclonal), CC3 (Cell Signaling Technology, rabbit polyclonal), and Ki67 (Thermo Scientific, rabbit monoclonal). Secondary antibodies were used alone to control for nonspecific background. Sections were counterstained with 1 μg/mL DAPI to outline the nuclear area. Images were scanned on the TS4000 (Huron Technologies) at 0.5 μm/pixel. Regions of tumor, necrosis, stroma, and folds were specified, creating a training rule set for tissue recognition. Cellular analyses included nucleus identification and separation, objects < 10 μm2 being excluded.

In vivo radiotoxicity assays in gut, kidney, and liver, overall and general toxicity assays

For gut/kidney/liver radiotoxicity assay, the experimental details were given previously (27). Briefly, SCID and nude mice were irradiated with targeted small-intestine radiation at doses of 5, 10, 12, or 15 Gy with and without compound FMD-2Br-DAB at 7 mg/kg. Mice were sacrificed after 72 hours, and the small intestine was removed for Ki67 staining. After image staining and scanning, surviving crypts in the intestine were measured compared with those that did not show a positive Ki67 stain. The overall radiotoxicity was assessed through analysis of proteins, enzymes, metabolites, and plasma electrolytes in blood. The mice were treated with 0, 5, 7 mg/kg FMD-2Br-DAB daily for 10 days. In addition, to evaluate radiation-induced toxicity therapy, mice were also injected with 0, 5, or 7 mg/kg of FMD and underwent 15 Gy of X-ray radiation at 1 hour after injection, in accordance with the pharmacokinetics data obtained. At the end of treatments, blood samples were collected from the tail vein; if this was not technically possible the saphenous vein was used as an appropriate substitute. These samples were then centrifuged at 13,000 rpm, and serum was removed from the mixture. The hepatotoxicity (ALT, ALP, and AST), nephrotoxicity (blood BUN and creatinine), and electrolytes (Na, K, etc.) were measured from the samples using an AutoAnalyzer (Pulse Instrumentation). Moreover, mice were observed for any general toxicity over the whole period of treatment. These physical indicators included body weight changes, dull sunken eyes, rapid/shallow breathing, hunched back, and lethargy.

Dynamic contrast enhanced MRI

All MR imaging was performed on the Bruker ICON high-performance 1T MRI Icon system (Aspect Imaging/Bruker). Tumors were visualized 19 days after treatment, using a T2 weighted sequence: RARE (TE/TR = 15/2319, FOV = 30 mm, Matrix = 130, Rare Factor 8, Averages = 6, resolution 233 μm, acquisition time 3 minutes:28 seconds). Dynamic contrast enhanced (DCE) MRI was performed, on the same day, by injecting 30 μL of Gd-HP-DO3A (ProHance, Bracco Diagnostics) via tail vein cannulation. T1-FLASH (TE/TR = 3.17/41.7, FOV =3 0 mm, Matrix = 120, resolution 250 μm, 3 slices acquired every 5 seconds, continued over 300 seconds). Tumor volume was quantified using the semiautomated tools within the VivoQuant software (inviCRO). DCE-MRI analysis was completed by inviCRO using the Kety–Tofts equation, assuming a fixed T1 value and a model input function. Ktrans and AUC were tabulated along with parametric color maps for both.

Pharmacokinetic studies

SCID mice were injected with 7 mg/kg of FMD-2Br-DAB through intraperitoneal injection (100 μL injection volume). At previously determined endpoints, mice were sacrificed through cervical dislocation, and tumor tissue was removed for analysis. Blood was also collected in heparinized tubes using the cardiac puncture technique. The blood was then centrifuged at 5,000 rpm for 5 minutes at 4°C. The tissue (tumor) or plasma concentration of FMD-2Br-DAB was determined by HPLC-MS (Applied Biosystems). Data analysis, including peak integration, and calculations were performed using Applied Biosystems MDS Analyst 1.4.2 software. The elimination rate constant ke of the compound removed from the body was determined by the measured elimination half-life (t1/2): ke = ln2/t1/2, and the clearance (CL) was calculated by: CL = Vd× ke, where Vd is the plasma volume of a mouse.

Radiation treatment

Irradiations of cells and mice were performed at 225 kV. The X-ray unit at Waterloo (XRAD 225, Precision X-ray) was used for cell and animal irradiations at a dose rate of 2 Gy/minute. For in vivo studies at STTARR (University Health Network, Toronto, Ontario, Canada), image-guided irradiations were targeted using cone-beam CT guidance (XRAD225Cx, Precision X-Ray) and a 1.0 cm circular collimator (dose rate: 2.88 Gy/minute; parallel-opposed beam geometry). All mice at the University Health Network (Toronto, Ontario, Canada) received a large single dose of 15 Gy, either targeted to the tumor or the gut/kidney/liver using the 1 cm collimator, whereas the cells and mice in the University of Waterloo (Ontario, Canada) were treated with various X-ray doses and multiple fractionated radiation doses (2 Gy × 3), respectively.

Statistical analysis

When two groups were compared, the appropriate Student t test was used. Multiple groups were compared by ANOVA, extended with an HSD post hoc test. Statistical analyses were performed using STATISTICA 8 (StatSoft). A P value < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

In vitro radiotoxicity tests of FMD-nX-DABs as novel radiosensitizers

The in vitro radiotoxicity of FMD-nX-DABs in combination with IR (225 kV X-rays) was investigated in human skin fibroblasts (GM05757). The GM05757 cell line has been widely used as human normal cells in cancer research, particularly in testing new radiosensitizing agents (27). The cells were treated by FMD-nX-DABs at various concentrations of 0 to 300/400 μmol/L for 12 hours, followed by various doses of X-ray irradiation. At 12 days postirradiation, the viability of the cells in 96-well plates was measured by MTT assay. First, the data plotted in Fig. 1 show that at zero radiation dose (0 Gy), FMD compounds appeared to have significant “toxicity”, but this was to be expected when the normal cells were incubated with the compounds for 12.5 days (12 hour-preirradiation plus 12 days postirradiation). It was not surprising that the viability of normal cells decreased with such a long compound incubation time. This is different from the results for normal cells incubated with FMD compounds alone for up to 72 hours, exhibiting little or no effect on the cell viability (26). Second, Fig. 1 interestingly shows that the viability of normal cells pretreated with FMD compounds up to 400 μmol/L was independent of radiation dose, that is, no radiotoxicity was observed on normal cells pretreated with FMD compounds. This is in marked contrast to the results for radiosensitizing effects of FMD compounds on cancer cells (see Fig. 2 below).

Figure 1.

A–C, in vitro radiotoxicity assays of FMD compounds (FMD-nX-DABs). Cell viabilities of human normal cells (GM05757) after the 12-hour pretreatment of FMD compounds with various concentrations at 0–300/400 μmol/L, followed by 225 kV X-ray irradiation. At 12 days postirradiation, the viability of the cells in 96-well plates was measured by MTT assay.

Figure 1.

A–C, in vitro radiotoxicity assays of FMD compounds (FMD-nX-DABs). Cell viabilities of human normal cells (GM05757) after the 12-hour pretreatment of FMD compounds with various concentrations at 0–300/400 μmol/L, followed by 225 kV X-ray irradiation. At 12 days postirradiation, the viability of the cells in 96-well plates was measured by MTT assay.

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Figure 2.

A–F, in vitro radiosensitizing assays of FMD compounds (FMD-nX-DABs). Cell viabilities or clonogenic survival rates of human cancer cells after the treatment of the FMD compounds at various concentrations for 12 hours, followed by 0–10 Gy of 225 kV X-ray irradiation. At 6 days postirradiation, MTT assays of the cells were performed. For clonogenic assays, the cells were incubated for 15–17 days postirradiation to form colonies. The ER, defined as the ratio of the radiation dose without FMD to that with FMD for the same level of biologic effect, is shown for the clonogenic assay results in D–F (see the text).

Figure 2.

A–F, in vitro radiosensitizing assays of FMD compounds (FMD-nX-DABs). Cell viabilities or clonogenic survival rates of human cancer cells after the treatment of the FMD compounds at various concentrations for 12 hours, followed by 0–10 Gy of 225 kV X-ray irradiation. At 6 days postirradiation, MTT assays of the cells were performed. For clonogenic assays, the cells were incubated for 15–17 days postirradiation to form colonies. The ER, defined as the ratio of the radiation dose without FMD to that with FMD for the same level of biologic effect, is shown for the clonogenic assay results in D–F (see the text).

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In vitro radiosensitizing effect tests of FMD-nX-DABs

The in vitro radiosensitizing effects of FMD-nX-DABs in combination with IR (225 kV x-rays) were investigated in cisplatin-sensitive human cervical cancer cell line (HeLa or ME-180), cisplatin-resistant human ovarian cancer (NIH:OVCAR-3, HTB-161) and lung cancer (A549) cell lines. The cells were treated by FMD-nX-DABs at various concentrations for 12 hours, followed by various doses of X-ray irradiation. The cell viability at 6 days postirradiation was measured by MTT, whereas the cell survival rates at 15 to 17 days postirradiation were measured by the clonogenic assay. As plotted in Fig. 2A–F, together with Supplementary Fig. S1, the results clearly show that FMD-nX-DABs enhanced the antiproliferative effects of IR in cancer cells in an IR dose-dependent manner (**, P < 0.01). The enhancement ratio (ER) was calculated as the ratio of the radiation dose without FMD to that with FMD for the same level of biologic effect for the clonogenic survival curves in Fig. 2D–F. The ERs for these cancer cell lines under air conditions (with an oxygen concentration typically of a few mmol/L) were determined to be 1.2 to 1.5 only. However, the ERs of FMD compounds are expected to be significantly larger for cells under hypoxic conditions or in the hypoxic tumor tissue due to the lack of the competitive attachment of epre to oxygen. Further confirmation of this property by in vitro cell experiments using a hypoxic chamber will be interesting, but this will be examined by in vivo tests of the radiosensitizing effects of FMDs in normal and cancer tissues in mice by combination with both a large single-dose radiation and multiple small-dose fractionated radiation (see Results below).

In vitro DNA DSB measurements in cancer cells

Given the observed results shown in Fig. 2 and Supplementary Fig. S1, we further measured DNA DSBs in cancer cells treated by FMD-2Br-DAB in combination with IR (225 kV X-rays), using the HCS DNA Damage (γH2AX) Kit (24–26). Figure 3 shows the fluorescence images of cervical cancer (ME-180) cells with/without the treatment of 25/200 μmol/L FMD and the yield of DNA DSBs detected by γH2AX foci with various X-ray doses, respectively. A significant increase in DNA DSB yield was observed with the presence of the FMD compound. These data clearly show that the radiosensitizing effect of FMD-2Br-DAB resulted in significant enhancements in DNA DSBs in cancer cells (**, P < 0.01), consistent with the cell viability and clonogenic survival results shown above. We therefore conclude that FMD-2Br-DAB can effectively induce DNA DSBs in cancer cells under IR.

Figure 3.

In vitro DSB assay of DNA in cancer cells treated by a FMD compound combined with X-ray radiation. ME-180 cancer cells were treated with/without 25/200 μmol/L FMD-2Br-DAB for 8 hours, followed by IR (225 kV X-rays) with various IR doses. At 12 hours postirradiation, the cells were fixed and stained, and γH2AX assays were performed. A, images show γH2AX foci in the cells treated with the FMD compound at 25 and 200 μmol/L in combination with IR at 0, 1, 2, and 4 Gy; Hoechst 33342 was used to map nuclei (a–l). B, the graph shows the γH2AX intensity in ME-180 cells versus X-ray dose.

Figure 3.

In vitro DSB assay of DNA in cancer cells treated by a FMD compound combined with X-ray radiation. ME-180 cancer cells were treated with/without 25/200 μmol/L FMD-2Br-DAB for 8 hours, followed by IR (225 kV X-rays) with various IR doses. At 12 hours postirradiation, the cells were fixed and stained, and γH2AX assays were performed. A, images show γH2AX foci in the cells treated with the FMD compound at 25 and 200 μmol/L in combination with IR at 0, 1, 2, and 4 Gy; Hoechst 33342 was used to map nuclei (a–l). B, the graph shows the γH2AX intensity in ME-180 cells versus X-ray dose.

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The in vitro results presented in Figs. 1–3 and Supplementary Fig. S1 demonstrate that the presence of a FMD compound enhanced the effectiveness of IR, leading to tumor cell death while the compound itself induced no radiotoxicity for normal cells. Thus, the FMD compounds are expected to enhance the effect of IR while inducing minimal or no radiotoxicity in vivo animals.

In vivo pharmacokinetic and radiotoxicity studies of FMD-2Br-DAB

The pharmacokinetic characteristics and in vivo radiotoxicity of a representative FMD agent, FMD-2Br-DAB, in 6- to 8-week-old SCID or nude mice are presented in Fig. 4. Figure 4A demonstrates pharmacokinetic profiles of the compound at both the tumor and blood plasma. Significant levels of FMD-2Br-DAB were detected in the plasma, and the highest concentration was observed at about 30 minutes after intraperitoneal injection and then dropped exponentially with time. For the tumor, the maximum uptake of the compound was observed at approximately 60 minutes. We measured t1/2 of the FMD compound in plasma to be about 60 minutes, and given that the plasma volume of a mouse is typically approximately 50 mL/kg, our estimated CL is approximately 0.7 mL/hour. These results indicate that the compound has promising pharmacokinetic properties.

Figure 4.

In vivo pharmacokinetics and (radio)toxicity of FMD-2Br-DAB in mice. A, the levels of the FMD in blood plasma and tumor over 150 minutes postinjection (note that the y-axis is plotted with a log scale). B, gut radiotoxicity of both SCID and nude mice treated by the vehicle only (control) and FMD at 7 mg/kg was measured as the number of surviving and proliferating crypts, determined through Ki67 staining. C, kidney and liver (radio)toxicity. H&E and Ki67 staining were conducted for 4 treatment groups: vehicle only, FMD, radiation therapy (15 Gy), and radiation in combination with FMD. Cell proliferation was presented through the pixel count positivity of Ki67, a ratio of the number of positive cells to the total cell number.

Figure 4.

In vivo pharmacokinetics and (radio)toxicity of FMD-2Br-DAB in mice. A, the levels of the FMD in blood plasma and tumor over 150 minutes postinjection (note that the y-axis is plotted with a log scale). B, gut radiotoxicity of both SCID and nude mice treated by the vehicle only (control) and FMD at 7 mg/kg was measured as the number of surviving and proliferating crypts, determined through Ki67 staining. C, kidney and liver (radio)toxicity. H&E and Ki67 staining were conducted for 4 treatment groups: vehicle only, FMD, radiation therapy (15 Gy), and radiation in combination with FMD. Cell proliferation was presented through the pixel count positivity of Ki67, a ratio of the number of positive cells to the total cell number.

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FMD-2Br-DAB acute radiotoxicity was first studied in both SCID and nude mouse models through a gut toxicity assay, as described previously (27). The nude mouse model is more resistant to radiotherapy and therefore has a larger radioresistance toward gut toxicity. Figure 4B shows the results of these tests in both SCID and nude mice. The crypt survival was determined through a histologic marker Ki67, which marks proliferating cells. First, the results elucidate a decrease in crypt survival in the small intestine (jejunum and ileum) as the radiation doses increased at 0 to 15 Gy, consistent with those reported previously (27). Second, the results show that there was no significant difference in crypt survival rate between irradiated mice in the control and irradiated mice pretreated with 7 mg/kg FMD-2Br-DAB, indicating that the FMD compound did not enhance radiation-induced gut toxicity (P > 0.05). The histologic images representing the gut tissues from the irradiated SCID mice are also shown in Supplementary Fig. S2.

In addition, we studied the acute radiotoxicity of FMD-2Br-DAB in kidney and liver normal tissues. The results are shown in Fig. 4C and Supplementary Fig. S3, which demonstrates no significant difference in Ki67 levels between mice injected with FMD and those with vehicle only for both 0 and 15 Gy irradiation. Although the level of Ki67 increased with radiation, this was expected as after radiation, cells began to repair and proliferate in response to injury. H&E images in Supplementary Fig. S3 show that there was very little or no radiobiologic toxicity associated with the FMD compound with and without 15 Gy radiation. These data indicate that FMD as a novel radiosensitizer is specific to the tumor tissue.

Direct drug (radio)toxicity was also studied in mice through the overall drug toxicity and general toxicity. FMD-2Br-DAB was administered by intraperitoneal injection into mice at 0, 5, and 7 mg/kg daily for 10 days, due to the nontoxicity observed in vitro cell line experiments (26). The results displayed in Supplementary Table S1 show that the FMD compound (even up to 7 mg/kg/day × 10 days) induced no observable overall toxicity, that is, no hepatotoxicity, no nephrotoxicity, and no changes in electrolytes. Moreover, Supplementary Table S1 also shows that there were no considerable changes in the overall drug radiotoxicity induced by 15 Gy X-rays between the groups pretreated with 0, 5, or 7 mg/kg of FMD. Nearly all the measured values were within the normal ranges, except the lower level of globulin. The latter was however also observed in the control group, which received no FMD compound/IR. This is attributed to the phenotype of SCID mice employed in the study. Moreover, the survival rate of mice was 100% over a 60-day period, with no change in body weight observed (Supplementary Fig. S4A), demonstrating that the FMD agent exhibited no general toxicity.

Overall, the substantial in vivo data for toxicity assays in normal tissues and blood shown in Fig. 4B and C and Supplementary Figs. S2–S4, together with Supplementary Table S1, demonstrate no significant overall, acute, and general radiotoxicities (P > 0.05), consistent with the in vitro results in Fig. 1, at the FMD dose levels tested.

In vivo DNA DSBs and apoptosis measurements in IR of a cervical cancer xenograft model

The in vivo DNA damage and cell death induced by FMD-2Br-DAB were investigated in a xenograft mouse tumor model of human cervical cancer (ME-180). As shown in Fig. 5, we measured DSBs and apoptosis in tumor tissue, including hypoxic and nonhypoxic regions, at 1 and 24 hours post-IR of 15 Gy. Figure 5A shows that higher levels of DNA DSBs (γH2AX foci) were observed at 1 hour after the combination treatment of the FMD compound at 7 mg/kg, followed by IR (applied 1 hour after drug injection corresponding to the time of highest FMD concentration in the tumor) in the whole tissue, including both hypoxic and nonhypoxic regions. It is seen from Fig. 5B that there was a significant (***, P < 0.001) enhancement in DSBs detected by γH2AX of DNA in hypoxic regions identified by the EF5 hypoxia marker (4) at both 1 and 24 hours posttreatment for the FMD + IR combination. Similarly, Fig. 5C shows that there was also an enhancement in the level of the DNA DSB repair marker at both time points in the hypoxic areas (53BP1/EF5 double positivity) for the combination treatment (*, P < 0.05). Moreover, a similar enhancement in apoptosis measured by the CC3 marker was observed, as shown in Fig. 5D. Histologic images corresponding to the results shown in Fig. 5A–D are shown in Supplementary Figs. S5 and S6. These results indicate that the presence of FMD-2Br-DAB enhanced radiosensitivity by increasing DNA DSBs and apoptosis in the tumor, especially in hypoxic regions. As a result, FMD-2Br-DAB can be used as an effective hypoxic radiosensitizer.

Figure 5.

A–D, in vivo DNA DSBs and apoptosis caused by FMD-2Br-DAB plus 15 Gy IR in mouse xenograft cervical (ME-180) cancer model. The FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and, at 1 hour after injection, the mice received IR (15 Gy, 225 kV X-rays) targeted to the tumor. The measurements were performed at both 1 and 24 hours posttreatment. A, DNA DSBs (γH2AX foci) in the whole tissue including both hypoxic and nonhypoxic regions. B, DNA DSBs in hypoxic regions identified by the hypoxia marker EF5. C, DNA damage repair marker 53BP1 for hypoxic regions. D, apoptosis in hypoxic regions. Histologic images corresponding to these results (A–D) are given in Supplementary Figs. S5 and S6. E–H, histologic changes in three xenograft models after the combination treatment of IR with FMD-2Br-DAB. All tissues were collected at 21 days posttreatment. E, necrosis fraction in the xenograft cervical cancer (ME-180) model. F, average vessel density in the ME-180 model. G, hypoxia in all three tumor models with treatments of the FMD compound, IR, and the FMD plus IR. H, Hoechst vessel perfusion in the ME180 model. Rad, radiation. **, P < 0.01 and ***, P < 0.001.

Figure 5.

A–D, in vivo DNA DSBs and apoptosis caused by FMD-2Br-DAB plus 15 Gy IR in mouse xenograft cervical (ME-180) cancer model. The FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and, at 1 hour after injection, the mice received IR (15 Gy, 225 kV X-rays) targeted to the tumor. The measurements were performed at both 1 and 24 hours posttreatment. A, DNA DSBs (γH2AX foci) in the whole tissue including both hypoxic and nonhypoxic regions. B, DNA DSBs in hypoxic regions identified by the hypoxia marker EF5. C, DNA damage repair marker 53BP1 for hypoxic regions. D, apoptosis in hypoxic regions. Histologic images corresponding to these results (A–D) are given in Supplementary Figs. S5 and S6. E–H, histologic changes in three xenograft models after the combination treatment of IR with FMD-2Br-DAB. All tissues were collected at 21 days posttreatment. E, necrosis fraction in the xenograft cervical cancer (ME-180) model. F, average vessel density in the ME-180 model. G, hypoxia in all three tumor models with treatments of the FMD compound, IR, and the FMD plus IR. H, Hoechst vessel perfusion in the ME180 model. Rad, radiation. **, P < 0.01 and ***, P < 0.001.

Close modal

Histological changes in xenograft models treated by FMD-2Br-DAB plus 15 Gy IR

Histologic changes in the tumor tissue at 21 days posttreatment were also measured, as shown in Fig. 5E–H. Figure 5E shows that in the ME-180 xenograft model, there was a significant increase in the necrotic fraction for the treatment of IR in combination with FMD-2Br-DAB (***, P < 0.001). Figure 5F shows that a decrease in vessel density was observed for the combination treatment, compared with the IR alone. Figure 5G shows the relationship between levels of hypoxia following treatment in all 3 tumor models, whereas Fig. 5H shows that Hoechst vessel perfusion was decreased after treatment with the FMD compound plus IR when compared with IR alone. This was also confirmed by DCE MRI AUC analysis. Detailed histologic images related to Fig. 5E–H are also shown in Supplementary Figs. S7–S9. These results indicate that the FMD + IR treatment led to a decrease in vessel density and an increase in tumor cell death (necrosis).

Tumor growth inhibition/regrowth delay caused by 15 Gy IR combined with FMD-2Br-DAB

Tumor growth inhibition and regrowth delay by FMD-2Br-DAB combined with a larger single IR dose (15 Gy) were investigated in the three xenograft mouse tumor models of human cervical cancer (ME-180), head/neck (74b) cancer, and lung (A549) cancer through tumor size measurements using DCE MRI and calipers. As shown in Fig. 6A–C, for the three xenograft models, significant growth delays of 41, 35, and 20 days between the IR alone and the combination of 7 mg/kg FMD-2Br-DAB followed by IR were observed for the ME-180, 74B, and A549 models, respectively (***, P < 0.001). This significant growth delay, which was observed for all the three tumor models, demonstrates that this novel FMD compound can act as an effective radiosensitizer with a significant effect and impact on tumor growth. For the ME-180 xenograft model, more detailed analyses of relative tumor volume changes and the drug treatment efficacy are also given in Supplementary Figs. S10A and S10B. It is shown that a ≥40% increase in radiation treatment efficacy was observed when combined with the FMD. Moreover, Fig. 6D illustrates the results of DCE-MRI measurements of the tumor size and perfusion characteristics. These show that the combination of FMD with IR resulted in more tumor perfusion and a much smaller tumor region.

Figure 6.

A–D, tumor growth delays in three mouse xenograft cancer (ME-180, 74B, and A549) models treated by FMD-2Br-DAB combined with large single-dose IR (15 Gy, 225 kV X-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and at 1 hour after injection, the mice received 15 Gy IR (225 kV X-rays) targeted to the tumor. Tumor growth delays were measured through tumor volume change over time. D, DCE MRI images illustrate the differences between the combination and the 3 control groups at 19 days posttreatment in the ME180 cancer; the color bar represents vessel perfusion in the tumor area. E and F, tumor growth delays in two mouse xenograft cancer models of human cervical (ME-180) cancer and lung (A549) cancer treated by FMD-2Br-DAB combined with fractional IR (2 Gy × 3, 225 kV X-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and, at 1 hour after injection, the targeted tumor in mice received radiation (2 Gy, 225 kV X-rays), with in total 3 treatments done at days 1, 3, and 5. **, P < 0.01 and ***, P < 0.001.

Figure 6.

A–D, tumor growth delays in three mouse xenograft cancer (ME-180, 74B, and A549) models treated by FMD-2Br-DAB combined with large single-dose IR (15 Gy, 225 kV X-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and at 1 hour after injection, the mice received 15 Gy IR (225 kV X-rays) targeted to the tumor. Tumor growth delays were measured through tumor volume change over time. D, DCE MRI images illustrate the differences between the combination and the 3 control groups at 19 days posttreatment in the ME180 cancer; the color bar represents vessel perfusion in the tumor area. E and F, tumor growth delays in two mouse xenograft cancer models of human cervical (ME-180) cancer and lung (A549) cancer treated by FMD-2Br-DAB combined with fractional IR (2 Gy × 3, 225 kV X-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by intraperitoneal injection into the mice, and, at 1 hour after injection, the targeted tumor in mice received radiation (2 Gy, 225 kV X-rays), with in total 3 treatments done at days 1, 3, and 5. **, P < 0.01 and ***, P < 0.001.

Close modal

Tumor growth inhibition/regrowth delay caused by fractionation IR combined with FMD-2Br-DAB

Conventionally, fractionated radiotherapy uses multiple small doses of 1.8 to 3 Gy to achieve local tumor control for acceptable normal tissue toxicity. However, the reoxygenation effect that occurs during multiple fractionation schemes is not expected to benefit the radiosensitizing effect of FMD compounds being hypoxic radiosensitizers, suggested by the observed results described above. Here, tumor growth inhibition by fractionated radiotherapy using FMD-2Br-DAB was investigated in the xenograft mouse models of human cervical cancer (ME-180) and lung cancer (A549). As shown in Fig. 6E and F, certain tumor regrowth delays between the control group, the (7 mg/kg × 3) FMD-2Br-DAB alone, the fractionated radiation (2 Gy × 3) alone and the combination of FMD (7 mg/kg × 3) with radiation (2 Gy × 3) were observed, respectively. The chemotherapeutic effect of the FMD at 7 mg/kg × 3 alone was observed, in accordance with the previous study (26). The combination of FMD with fractionated irradiation enhanced the tumor regrowth delays compared with the radiation alone (**, P < 0.01), but it is not as significant as the large single dose 15 Gy irradiation (Fig. 6A–C). No changes in body weight over a 90-day period for all four groups of mice were observed (Supplementary Fig. S4B). These results are consistent with the radiosensitizing properties of FMD compounds acting as effective hypoxic radiosensitizers, which may particularly benefit the stereotactic ablative radiotherapy (SABR) using large single doses.

A practical way to improve the efficacy of radiotherapy is to combine it with a chemical compound (radiosensitizer) (2, 4, 27, 28). Our femtomedicine approach and the discovery of the key role in causing damage to the DNA of epre, a novel electron species produced in the radiolysis of water under IR, have offered unique opportunities to improve existing drugs and to develop new effective drugs for high-performance therapy of cancer. Enabled by this innovative strategy and based on the DET mechanism established in our previous studies (12–18, 19, 22), we have successfully found a series of non–platinum-based halogenated compounds (FMD compounds) as potent anticancer agents (26) and now as hypoxic radiosensitizers.

This study offers several important observations. First, our in vitro results show that the FMD compounds caused essentially no (radio)toxicity toward normal cells. These compounds are in contrast to the clinically used platinum-based anticancer drugs, which are highly toxic. Second, our in vitro results show that a FMD compound significantly enhanced the antiproliferative effects of IR in both cisplatin-sensitive and cisplatin-resistant cancer cells. Enhancements in DNA DSBs in the treated cancer cells were also observed. Thus, these FMD compounds have significant potential as anticancer agents that achieve radiosensitization while inducing no or little systemic toxicity and radiotoxicity.

Correspondingly, our in vivo results have clearly demonstrated that FMD-2Br-DAB as an exemplary FMD compound combined with IR exhibited minimal (radio)toxicity in mice, that is, no overall, acute, and general toxicities in normal tissues (gut, kidney, and liver) and blood. These results are in good agreement with the in vitro results observed in human normal cells. The observed results also show that the FMD compound is no longer detectable in the blood after 3 to 4 hours, exhibiting interesting pharmacokinetic properties that could be exploited for improved clinical efficacy.

Furthermore, the in vivo results from the xenograft mouse tumor models of human cervical, head/neck, and lung cancers exhibited significant tumor growth inhibition and regrowth delay due to the radiosensitizing effect of FMD-2Br-DAB combined with a large single-dose radiation (15 Gy). Our in vivo results also show that the compound induced significant enhancements in DNA DSBs and apoptosis especially in hypoxic regions, indicating that it is an effective hypoxic radiosensitizer. Moreover, our histologic and MRI data also show that FMD in combination with IR led to a decrease in vessel density and an increase in tumor cell death (necrosis).

The radiosensitizing effect of FMD-2Br-DAB combined with multiple small-dose fractionated radiation (2 Gy × 3) was also observed, but it was less significant compared with the large single dose of 15 Gy. This is consistent with the properties of FMD compounds as hypoxic radiosensitizers, where reoxygenation between multiple fractionated irradiations reduces the DET reaction efficiency of FMD and the radiosensitizing effect. These results demonstrate that the FMD compound is indeed a novel and effective (hypoxic) radiosensitizer, with significant radiosensitizing effect and impact on tumor growth, especially in hypoxic tumor areas. This is of special significance, as there is growing interest in the use of SABR using large single IR doses in the clinic (28). The presence of tumor hypoxia is a major negative factor in limiting the curability of tumors by SABR at radiation doses that are tolerable to surrounding normal tissues. It has been suggested that this negative effect of hypoxia could be overcome by the addition of a hypoxic cell radiosensitizer at clinically tolerable doses (28). Compared with clinically available hypoxic cell radiosensitizers, the FMD compounds have a significant advantage that they are essentially nontoxic even at very high doses, inducing no systemic toxicity in chemotherapy (26) and no radiotoxicity in combination with radiation, as observed in this study.

The results observed in this study can well be explained by the DET mechanism established in our previous studies (12–19, 22): the DET reaction of FMD-nX-DABs as highly reactive halogenated molecules with epre is expected to be much more effective under the hypoxic conditions due to the absence of the competing attachment of epre to O2. In other words, there are a larger number of epre available for the DET reaction with FMD-nX-DABs in the tumor tissue characteristic of hypoxia. Thus, these FMD compounds can be used as effective hypoxic radiosensitizers.

Finally, it is believed that the in vitro and in vivo results reported in this study have significant potential to be extended to other cancer cells and models beyond those exemplified here. More broadly, our efforts in advancing the femtomedicine approach have identified a new and promising family of anticancer compounds and represent an efficient and powerful alternative to random screening approaches for drug development. As one of the main outcomes of our efforts in femtomedicine, the current results have demonstrated its potential as an exciting new frontier to bring breakthroughs in fundamental understandings of major human diseases, such as cancer, and to design more effective drugs.

R.G. Bristow and D.A. Jaffray have ownership interests (including patents) in Gashu Femtomedicine Inc. Q.-B. Lu is the President of and has ownership interest (including patents) in Gashu Femtomedicine Inc. No potential conflicts of interest were disclosed by the other authors.

Conception and design: R.G. Bristow, D.A. Jaffray, Q.-B. Lu

Development of methodology: C.-R. Wang, J. Warrington, R. G. Bristow, D.A. Jaffray, Q.-B. Lu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-R. Wang, J. Mahmood, Q.-R. Zhang, A. Vedadi, J. Warrington, N. Ou, D.A. Jaffray

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-R. Wang, J. Mahmood, Q.-R. Zhang, A. Vedadi, J. Warrington, N. Ou, R.G. Bristow, D.A. Jaffray, Q.-B. Lu

Writing, review, and/or revision of the manuscript: C.-R. Wang, J. Mahmood, Q.-R. Zhang, A. Vedadi, J. Warrington, N. Ou, R.G. Bristow, D.A. Jaffray, Q.-B. Lu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Mahmood, D.A. Jaffray

Study supervision: D.A. Jaffray, Q.-B. Lu

Other [discovered the DET reaction mechanism and identified the new anticancer (radiosensitizing) compounds]: Q.-B. Lu

The authors thank Drs. Cameron Koch (University of Pennsylvania, Philadelphia, PA), Brad Wouters [Princess Margaret Cancer Centre, University Health Network (PMH, UHN), Toronto, Ontario, Canada], and Ming Tsao (PMH, UHN) for generously providing of biomarkers EF5 and CD31, 74B cells, and A549 cells, respectively, to our experiments carried out at PMH/OCI/UHN in Toronto. The authors also particularly thank Dr. Richard Hill for his very helpful comments on an earlier version of this manuscript.

This study was supported by an operating grant #102483 (to Q.-B. Lu, D.A. Jaffray, and R.G. Bristow), a New Investigator Award #112642 (to Q.-B. Lu) from the Canadian Institutes of Health Research, a Discovery grant #299089 (to Q.-B. Lu) from Natural Science and Engineering Research Council of Canada, and an Early Researcher Award # 41477 (to Q.-B. Lu) from the Ontario Ministry of Research and Innovation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Bristow
R
,
Hill
RP
. 
Molecular and Cellular Radiobiology
. In:
Tannock
IF
,
Hill
RP
,
Bristow
R
,
Harrington
L
, editors.
The basic science of oncology
. 4th ed.
New York, NY
:
McGraw-Hill
; 
2005
.
p. 261
88
.
2.
Lehnert
S
.
Biomolecular action of ionizing radiation
.
London, United Kingdom
:
Taylor & Francis Ltd
; 
2008
.
3.
Dawson
LA
,
Jaffray
DA
. 
Advances in image-guided radiation therapy
.
J Clin Oncol
2007
;
25
:
938
46
.
4.
Koch
CJ
,
Parliament
MB
,
Brown
JM
,
Urtasun
RC
. 
Chemical modifiers of radiation response
. In:
Phillips
TL
,
Hoppe
RT
,
Roach
M
 III
, editors.
Leibel and Phillips textbook of radiation oncology
. 3rd ed.
New York, NY
:
Elsevier
; 
2010
. p.
55
68
.
5.
Bagley
RG
, editor. 
The tumor microenvironment, cancer drug discovery and development
.
New York, NY
:
Springer
; 
2010
.
6.
Hart
E
,
Anbar
M
.
The hydrated electron
.
New York, NY
:
John Wiley & Sons, Inc.
; 
1970
.
7.
Buxton
GV
,
Greenstock
CL
,
Helman
WP
,
Ross
AB
. 
Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (OH/O−•) in aqueous solution
.
J Phys Chem Ref Data
1988
;
17
:
513
886
.
8.
Zewail
AH
. 
Femtochemistry: atomic-scale dynamics of the chemical bond using ultrafast lasers (Nobel lecture)
.
Angew Chem Int Ed
2000
;
39
:
2586
631
.
9.
Migus
A
,
Gauduel
Y
,
Martin
JL
,
Antonetti
A
. 
Excess electrons in liquid water: First evidence of a prehydrated state with femtosecond lifetime
.
Phys Rev Lett
1987
;
58
:
1559
62
.
10.
Long
FH
,
Lu
H
,
Eisenthal
KB
. 
Femtosecond studies of the presolvated electron–an excited-state of the solvated electron
.
Phys Rev Lett
1990
;
64
:
1469
72
.
11.
Laenen
R
,
Roth
T
,
Laubereau
A
. 
Novel precursors of solvated electrons in water: Evidence for a charge transfer process
.
Phys Rev Lett
2000
;
85
:
50
3
.
12.
Wang
CR
,
Luo
T
,
Lu
QB
. 
On the lifetimes and physical nature of prehydrated electrons in liquid water
.
Phys Chem Chem Phys
2008
;
10
:
4463
70
.
13.
Wang
CR
,
Hu
A
,
Lu
QB
. 
Direct observation of the transition state of ultrafast electron transfer reaction of a radiosensitizing drug bromodeoxyuridine
.
J Chem Phys
2006
;
124
:
241102
.
14.
Lu
QB
. 
Molecular reaction mechanisms of combination treatments of low-dose cisplatin with radiotherapy and photodynamic therapy
.
J Med Chem
2007
;
50
:
2601
4
.
15.
Lu
QB
,
Kalantari
S
,
Wang
CR
. 
Electron transfer reaction mechanism of cisplatin with DNA at the molecular level
.
Mol Pharm
2007
;
4
:
624
8
.
16.
Wang
CR
,
Lu
QB
. 
Real-time observation of a molecular reaction mechanism of aqueous 5-halo-2′-deoxyuridines under UV/ionizing radiation
.
Angew Chem Intl Ed Engl
2007
;
46
:
6316
20
.
17.
Wang
CR
,
Nguyen
J
,
Lu
QB
. 
Bond breaks of nucleotides by dissociative electron transfer of nonequilibrium prehydrated electrons: a new molecular mechanism for reductive DNA damage
.
J Am Chem Soc
2009
;
131
:
11320
2
.
18.
Wang
CR
,
Lu
QB
. 
Molecular mechanism of the DNA sequence selectivity of 5-halo-2′-deoxyuridines as potential radiosensitizers
.
J Am Chem Soc
2010
;
132
:
14710
3
.
19.
Lu
QB
. 
Effects of ultrashort-lived prehydrated electrons in radiation biology and their applications for radiotherapy of cancer
.
Mutat Res
2010
;
704
:
190
9
.
20.
Lu
QB
. 
Dissociative electron transfer reactions of halogenated molecules adsorbed on ice surfaces: implications for atmospheric ozone depletion and global climate change
.
Phys Rep
2010
;
487
:
141
67
.
21.
Lu
QB
.
New theories and predictions on the ozone hole and climate change
.
New Jersey
:
World Scientific Publishing Co.
; 
2015
.
22.
Nguyen
J
,
Ma
Y
,
Luo
T
,
Bristow
RG
,
Jaffray
DA
,
Lu
QB
. 
Direct Observation of Ultrafast Electron Transfer Reactions Unravels High Effectiveness of Reductive DNA Damage
.
Proc Natl Acad Sci U S A
2011
;
108
:
11778
83
.
23.
Sanche
L
. 
Beyond radical thinking
.
Nature
2009
;
461
:
358
9
.
24.
Lu
LY
,
Ou
N
,
Lu
QB
. 
Antioxidant induces DNA damage, cell death and mutagenicity in human lung and skin normal cells
.
Sci Rep
2013
;
3
:
3169
.
25.
Luo
T
,
Yu
JQ
,
Nguyen
J
,
Wang
CR
,
Bristow
RG
,
Jaffray
DA
, et al
Electron transfer-based combination therapy of cisplatin with tetramethyl-p-phenylenediamine for ovarian, cervical, and lung cancers
.
Proc Natl Acad Sci U S A
2012
;
109
:
10175
80
.
26.
Lu
QB
,
Zhang
QR
,
Ou
N
,
Wang
CR
,
Warrington
J
. 
In Vitro and In Vivo studies of non-platinum-based halogenated compounds as potent antitumor agents for natural targeted chemotherapy of cancers
.
EBioMedicine
2015
;
2
:
544
53
.
27.
Choudhury
A
,
Zhao
H
,
Jalali
F
,
Rashid
S
,
Ran
J
,
Supiot
S
, et al
Targeting homologous recombination using imatinib results in enhanced tumor cell chemosensitivity and radiosensitivity
.
Mol Cancer Ther
2009
;
8
:
203
13
.
28.
Brown
JM
,
Diehn
M
,
Loo
BW
 Jr
. 
Stereotactic ablative radiotherapy should be combined with a hypoxic cell radiosensitizer
.
Int J Radiat Oncol Biol Phys
2010
;
78
:
323
7
.