Antitumor and radiosensitization effect of ¹²C⁶⁺ heavy ion irradiation mediated by radiation-inducible gene therapy

Antitumor and radiosensitization effect of 12 C 6+ heavy ion irradiation mediated by radiation-inducible gene therapy 1 corresp first-author Corresponding author, hui.liu@fds.org.cn Corresponding author, hui.liu@fds.org.cn hui.liu@fds.org.cn 1 2 3 Key Laboratory of Neutronics and Radiation Safety, Institute of Nuclear Energy Safety Technology, Chinese Academy of Sciences, Hefei 230031, China Key Laboratory of Neutronics and Radiation Safety, Institute of Nuclear Energy Safety Technology, Chinese Academy of Sciences, Hefei 230031, China Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology & Agriculture Engineering, Chinese Academy of Sciences, Hefei 230031, China Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology & Agriculture Engineering, Chinese Academy of Sciences, Hefei 230031, China Radio genetic therapy which combines gene therapy with radiotherapy, has shown promising results in cancer treatment. In this study, an oncolytic adenovirus-based gene therapy system regulated by radiation was constructed to improve the cancer curative effect. This gene therapy system incorporated the radiation-inducible early growth response gene (Egr-1) promoter and the anticancer gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To confirm the antitumor effect of Ad-ET combined with 12 C 6+ ion irradiation, the survival and apoptosis fraction of tumor cells HT1080 and normal cells MRC-5 in combination treatment were detected by CCK-8 assay and FACS analysis. Then the expression levels of TRAIL gene and protein were tested by real-time PCR and western blotting. The results show that 12 C 6+ ion irradiation could induce cell growth inhibition and apoptosis by activating the TRAIL gene expression in tumor cells, while exhibiting no obvious toxicity to the normal lung cell line MRC-5. The results also demonstrate that use of an oncolytic adenovirus-based radiation-inducible gene therapy system together with 12 C 6+ ion irradiation could cause synergistic antitumor effect specifically in tumor cells but not in normal cells. The results indicate that the novel radio genetic therapy could potentiate radiation treatment by improving the safety and efficiency of monotherapy, and provide theoretical support for clinical application of combination treatment. Radio genetic therapy 12 C ion irradiation Apoptosis Egr-1 prompter Tumor necrosis factor-related apoptosis-inducing ligand 1 Introduction 1 Adenovirus-based gene therapy has been proven to be a promising candidate for cancer treatment, with excellent safety and efficacy, as demonstrated by a wealth of clinical data. In clinical research, many strategies were applied in gene therapy [ 1 1 - 3 3 1-3 ]. Oncolytic adenovirus H101, as a gene therapy vector, is modified on the basis of ONYX-015. H101 can specifically infect and lyse P53-mutated tumor cells with deletion of E1B55K and part of E3 region [ 4 4 ]. It has been demonstrated that many tumor cell lines are deficient o r mutant of P53 gene. Thus, H101 can be widely used in targeted gene therapy. The suicide gene transferred by adenovirus vector is the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) gene, which is a member of the TNF family of Type 2 transmembrane proteins. TRAIL can specifically induce apoptosis in a variety of tumor cell types by binding two pro-apoptotic receptors: TRAIL-R1 (DR4) and TRAIL-R2 (DR5) [ 5 5 ], and was reported to induce apoptosis resulting in receptor cross-linking and caspase activation [ 6 6 ]. However, TRAIL is almost nontoxic for most normal tissues due to existence of two decoy receptors: TRAIL-R1 (DcR1) and TRAIL-R2 (DcR2) [ 7 7 ]. Studies show that TRAIL is a potential method for cancer treatment when combined with conventional therapy module, such as radiotherapy and chemotherapy, because of synergistic antitumor effects, including apoptosis and tumor growth inhibition [ 8 8 - 10 10 8-10 ]. Compared with X-rays and γ -rays, heavy-ions are of high-LET (linear energy transfer) value. Also, ion beams exhibit Bragg peak effect which means most energy of an ion is deposited on the target region with little normal tissue toxicity. These make heavy-ion beams an advanced radiotherapy modality, and brings outstanding results in clinical researches [ 11 11 , 12 12 ]. Using heavy-ion beams to initiate suicide gene expression in combination gene therapy will enhance the antitumor activity of monotherapy. The activity of antitumor monotherapy will be enhanced when the advantage of heavy ion treatment is combined with gene therapy. To further control the antitumor apoptosis effect of TRAIL protein specifically acting in tumor cells within the radiotherapy target region, early growth response-1 (Egr-1) is incorporated into the adenovirus as a promoter that can be initiated in response to various stimuli including radiation. Egr-1 is a zinc finger transcription factor, which can be rapidly and transiently induced when exposed to radiation. In the presence of zinc ions, the three zinc-finger motifs included in Egr-1 protein enable Egr-1 to recognize and combine with a specific DNA sequence, 5’-CGCCCCCGC-3’, and finally regulate downstream genes’ expression by activating the Egr-1 promoter transcription [ 16 16 , 17 17 ]. Under the control of Egr-1 promoter, the therapeutic gene can be effectively regulated by irradiating the specific target region for a chosen period of time. This combination treatment of gene therapy and heavy-ion radiotherapy improve the safety and efficiency of gene therapy [ 18 18 , 19 19 ]. In this study, we used an adenoviral delivery system to construct the Egr-1 promoter/TRAIL therapeutic cassette into the backbone of an E1B55K-/E3-deleted oncolytic adenovirus. Although Wang et al . applied Ad-ET to tumor cells exposed to X rays and observed enhanced tumor targeting effect [ 20 20 ], no research has been conducted, to the authors’ knowledge, to combine Ad-ET with heavy-ion radiotherapy as a synergistic antitumor effect. In this paper, we investigate cell apoptosis in tumor and normal cells following combined treatment of Ad-ET and 12 C 6+ ion beams to evaluate whether it can cause the synergistic pro-apoptotic effect. The results strongly support the novel treatment strategy of heavy-ion radiotherapy and radiation-inducible gene therapy, and the preliminary data are useful for future clinical applications of combination treatment. 2 Materials and Methods 1 2.1 Cell lines and cell culture 2 The human fibrosarcoma cell line HT1080 and normal human lung embryonic cell line MRC-5 were kindly provided by Institute of Modern Physics, Chinese Academy of Sciences, and obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Both of the cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and cultured at 37 ℃ in a humidified 5% CO 2 incubator (Thermo Scientific). The population doubling time of MRC5 was about 24 h, and that of HT1080 was 12 h. 2.2 Heavy-ion irradiation 2 The cells were exposed to 160 MeV/u 12 C 6+ ion beams at a dose rate of 0.2 Gy/min derived from the Heavy-ion Research Facility in Lanzhou (HIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China). The LET value is 80 at peak region and 17 at plateau region. Exponentially growing HT1080 and MRC-5 cells were seeded in 96-well microtiter plate or 24-well microtiter plate, and the culture medium was replaced 24 h later before exposed to 12 C 6+ ion beams performed through the plate bottom. 2.3 Adenovirus vector and infection 2 Three kinds of recombinant adenoviruses were used: PPE3-EGR1-TRAIL (Ad-ET), PXC20- Δ E1b55Kda (Ad) and PPE3-GFP(Ad-GFP). These were commercially packaged and purified by Minghong Biotech (Shanghai, China). All the adenoviruses were constructed on the basis of the oncolytic adenovirus H101 backbone. The new antitumor adenovirus was constructed and packed as described [ 21 21 ]. After being cultured for 24 hours, the cell medium was replaced by fresh medium without FBS. The adenovirus was added into the medium for transfection. Four hours later, the same amount of medium with 20% FBS was added for the subsequent cell culturing. 2.4 Cell growth inhibition 2 The CCK-8 assay (Cell Counting Kit-8, Beyotime, Jiangsu, China) was used for analyzing the cell growth by quantitatively measuring cell viability. The action principle is that WST-8 can be deoxidized by dehydrogenases of viable cells to an orange formazan. Approximately 5d3 exponentially growing cells were seeded into the 96-well microtiter plates. Cells were irradiated by 12 C 6+ ion beams to 1, 2 and 4 Gy, so as to determine the optimal dose that could cause the maximal synergistic tumor cell killing effect. After 12 C 6+ ion irradiation, cells were transfected with the Ad or Ad-ET adenovirus at a MOI (multiplicity of infection) of 10 and incubated at 37 ℃ in a humidified atmosphere containing 5% CO 2 for 48 h. To test cell growth and viability, 20 μL of a CCK-8 reaction mixture in fresh medium was added to each well after incubation. Cells were incubated at 37 ℃ for 1 hour with gentle shaking and then absorbance was measured at 450 nm in a SpectraMax M5 multifunctional microplate reader (Molecular Devices, Sunnyvale, CA). The absorbance values in the treated groups were normalized to the values of untreated cells to calculate the survival fraction: [1-(OD cells treated/OD cells treated with PBS)] × 100. Each experiment was performed in quadruplicate and repeated at least three times. 2.5 Apoptosis assay 2 To analyze whether the synergistic apoptosis effect can be induced by Ad-ET treatment combined with 12 C 6+ heavy-ion irradiation, First HT1080 and MRC-5 cells were seeded into 96-well microtiter plates. After exposure to 12 C 6+ ion irradiation at a dose of 0.5 or 1 Gy, Ad or Ad-ET was immediately added into the medium for transfection. To detect the cell apoptosis fraction, both floating and attached cells were harvested after 48 h incubation following the treatment. After being washed two times with cold PBS, the cells were resuspended in 100 μL of 1 × binding buffer (Invitrogen, Carlsbad, CA). Following the protocol provided with the Invitrogen Apoptosis Detection Kit, total cells were stained with both annexin V-fluorescein isothiocyanate (FITC) and propidium iodide, and analyzed using a FACScan flow cytometer with the CellQuest program (Becton Dickinson) as soon as possible. 2.6 Real-time PCR 2 After treated with Ad-ET and 12 C 6+ ion irradiation, total RNA was isolated from cells using Trizol reagent (Invitrogen). First-strand cDNA was synthesized with RNA reverse transcriptase. Quantitative real-time RT-PCR amplification was carried out by using QuantiTect SYBR green (Qiagen, Valencia, CA) as described in previous article [ 20 20 ]. Primers for amplifying TRAIL and GAPDH mRNA were expressed as follows: TRAIL sense: GAGCTGAAGCAGATGCAGGAC, and TRAIL antisense: TGACGGAGTTGCCACTTGACT; GAPDH sense: CATCAAGAAGGTGGTGAAGCAGG, and GAPDH antisense: AGCGTCAAAGGTGGAGGAGTGG. The CT (threshold cycle) value of TRAIL was quantitated by Q-PCR in triplicate using an ABI Prism 7900 HT sequence detector (AB Applied Biosciences, CA) following the manufacturer’s protocol and was normalized over the CT of the GAPDH control. 2.7 Western blot analysis 2 Further detection of the TRAIL gene expression level and the relative apoptisis pathway, the TRAIL protein was quantified by western blotting. The cells were harvested and lysed in lysis buffer (Beyotime, Jiangsu, China), and analyzed using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Cell lysates containing 40 μg of soluble total cellular protein were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Roche, Basle, Switzerland). These membranes were blocked for 1 hour with a buffer containing 1% fat-free dried milk and TBST (TBS with 0.1% Tween). The membrane were washed three times with TBST, and incubated overnight with primary antibodies at 4 ℃. Primary antibodies were used against TRAIL (1:500, Cell Signaling Technology, Danvers, MA), and β-actin (1:2000 Cell Signaling Technology). After being washed with TBST three times, the membranes were incubated for 1 hour with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, 1:4000) or anti-rabbit IgG secondary antibody (Cell Signaling Technology, 1:4000). Signals were visualized by enhanced chemiluminescence (Applygen, Beijing, China) using FluorChem E (Cell Biosciences, Santa Clara, CA) according to the manufacturer’s instructions. 2.8 Statistical analysis 2 Data were presented as means and standard derivations. Significance levels were assessed using Student’s t-test. A P-Value of 0.05 or less between groups was considered significant. 3 Results 1 3.1 Cell viability of the cells following combined treatment of Ad-ET and 12 C 6+ ion irradiation 2 To test whether the combined treatment with Ad-ET and 12 C 6+ ion irradiation could cause a synergistic cell growth inhibitory effect on HT1080 tumor and MRC-5 normal cells, the cell viability was determined by the CCK-8 assay after combination treatment in vitro . To maximize the synergistic effect of combination treatment, three doses of 1, 2 and 4 Gy were used at the MOI of 10 in both tumor and normal cells. The viability of HT1080 decreased with increasing doses within 2 Gy, and it increased under the combined treatment. Comparing the treatment group of Ad plus 12 C 6+ ion irradiation with the control group of irradiation, the survival fraction of the combined treatment decreased by varying degrees at all the three doses ( Fig. 1(a) Fig. 1(a) ). This is probably due to synergistic effect of combination treatment and the efficiency of Ad-ET, which could reach the highest value when receiving the 2 Gy irradiation dose. The results indicate that 12 C 6+ ion irradiation can effectively activate the cell killing capacity of the radiation-inducible adenovirus Ad-ET in tumor cells HT1080. The MRC-5 cell line was also in combination treatment on normal tissues. The results show, however, that the survival fraction did not decrease significantly ( Fig. 1(b) Fig. 1(b) ). All these indicate that combination treatment with Ad-ET and irradiation could significantly enhance the cell growth inhibition in tumor cells and is nontoxic to normal cells. 3.2 Cell apoptosis enhanced by the combined treatment 2 For further investigation of mechanism of the cell death, an Annexin-V-FITC/PI assay was used by flow cytometry after the tumor cell line HT1080 and normal cell line MRC-5 were treated with Ad-ET plus 12 C ion irradiation. According to the results in Sec. 3.1, the 12 C ion irradiation dose of 2 Gy was adopted in the combined treatment for apoptosis detection. As shown in Fig. 2(a) Fig. 2(a) , the apoptotic fraction in the Ad-ET group and the RAD group were only 5.8 % and 5.46 %, respectively, and it increased to 20.7 % with the Ad-ET+RAD treatment. The survival fraction was much higher than that of the Ad plus RAD group, indicating that increasing apoptosis effect can be induced by 12 C 6+ ion irradiation only when combined with Ad-ET. That means 12 C 6+ ion irradiation can greatly sensitize the cells to the effect of Ad-ET by activating TRAIL-induced apoptosis in HT1080 cells. However, similar synergistic apoptosis effect was not observed in the MRC-5 cells when treated with Ad-ET+ RAD. As shown in Fig. 2(b) Fig. 2(b) , the degree of apoptosis just slightly increased in MRC-5 cells with the Ad-ET+RAD group. These demonstrate that the combined treatment of Ad-ET and 12 C 6+ ion irradiation could only induce a significant synergistic cell apoptosis effect specifically in tumor cells, but not in normal lung cells. This is probably due to the different sensitivity of TRAIL-induced apoptosis between tumor cells and normal cells. 3.3 TRAIL gene expression in HT1080 cells treated with Ad-ET+RAD 2 To elucidate mechanism of the synergistic apoptosis induced by Ad-ET combined with 12 C 6+ ion irradiation, we detected the level of TRAIL gene expression by real-time PCR. As shown in Fig. 3 Fig. 3 , the fold of increase in the RAD, Ad-GFP, Ad-GFP+RAD and Ad-ET groups was less than 20 when the TRAIL gene expression in PBS control group was set as 1, while in the group of Ad-ET plus 12 C 6+ ion irradiation, the TRAIL gene increased to 100. The results show that 12 C 6+ ion irradiation could enhance the pro-apoptotic TRAIL gene expression in tumor cells treated with Ad-ET. The increased cytotoxicity observed in apoptosis analysis was mostly due to the elevated TRAIL gene expression when activated by 12 C 6+ ion irradiation. 3.4 Expression of TRAIL proteins following the combined treatments of HT1080 cells 2 For further detection of the TRAIL protein expression following TRAIL gene activation in HT1080 cells treated with Ad-ET and 12 C 6+ ion irradiation, we detected the level of TRAIL protein by western blotting. As shown in Fig. 4 Fig. 4 , the band in Ad-ET plus 12 C 6+ ion irradiation group was much thicker than other treatment groups, showing that the expression of TRAIL protein in Ad-ET-infected HT1080 cells was dramatically enhanced following exposure to 12 C 6+ ion beams. The result indicates that the pro-apoptotic effect in Ad-ET treated tumor cells could be enhanced by 12 C 6+ ion irradiation by activating the TRAIL expression. 4 Discussion 1 Radiotherapy is a main modality of cancer treatment, though it has limitation in treatment accuracy [ 22 22 - 33 33 22-33 ]. Each dose of radiotherapy is usually below 2 Gy, so as to save the normal tissue. In clinical, radiotherapy is often used in conjunction with other treatments, such as surgery and chemotherapy. In our study, radiotherapy is applied in combination with gene therapy. While showing its promising prospect in the future for tumor therapy, this approach has limitations, too, such as the low efficiency of tumor target [ 1 1 ]. Combining radiotherapy with gene therapy, radio genetic therapy can overcome weakness of the two treatment methods. This method can constrain the therapeutic gene expression in radiotherapy target region to realize the double action of radiation and therapeutic gene in tumor cells, and improve the therapeutic efficacy. It takes full advantages of radiotherapy and gene therapy, so as to reduce the dosage of radiotherapy and gene therapy, with decreased cytotoxicity in normal tissue. Some studies reveal that radio gene therapy induces the DNA damage through different pathways, i.e. combining the two methods probably induces the synergistic effect in tumor cells [ 34 34 , 35 35 ]. In our adenovirus gene therapy system, early growth response-1 (Egr-1) is constructed as a promoter to selectively activate the therapeutic gene in the target tumor region and avoid probable cytotoxic effects in normal tissues. The Egr-1 gene acted as a promoter in radio genetic therapy and has demonstrated that it can regulate the downstream therapeutic gene expression in radiation target region effectively to improve the therapeutic gain. Weichselbaum et al . [ 36 36 ] constructed a recombinant expression plasmid pEgr-TNF- α to include Egr-1 promoter and TNF- α gene. After exposure to X-rays, the tumor cells treated with pEgr-TNF- α were significantly inhibited without increase of the whole body toxicity The tumor necrosis factor (TNF)-related apoptosis-inducing TRAIL ligand, which was inserted into the radiation-inducible gene therapy system, has been confirmed as being able to selectively induce apoptosis in a broad range of cancer cells but spares normal cells. However, some studies revealed that TRAIL has limitation in eradicating tumors when used as monotherapy. This is probably due to deficiency of TRAIL death receptors or competition of antagonistic receptor. Thus, in our research, by combining TRAIL gene with radiotherapy in radio genetic therapy, one could potentiate its antitumor activity and specificity in a variety of radio-resistant tumor cells [ 37 37 ]. In addition, without understanding the mechanisms, it has been revealed that TRAIL activated a pro-apoptotic effect in human liver cells [ 38 38 ]. This radiation-inducible strategy in our research could offer a double guarantee of specific antitumor activity. On the other hand, radiation could also enhance the sensitivity of TRAIL-resistant cells in combination treatment by up-regulating the death receptors of TRAIL or activating the caspase pathway [ 39 39 ]. Also we tested the tumor killing capacity and apoptosis fraction of radiation-inducible TRAIL gene therapy. The results demonstrate that the tumor killing capacity and apoptosis fraction is significantly increased in Ad-ET combined with 12 C 6+ ion irradiation than monotherapy of 12 C 6+ ion beams or Ad-ET. 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Sensitization of resistant lymphoma cells to irradiation-induced apoptosis by the death ligand TRAIL We thank other members of the FDS team for their assistance in this work. We thank Professors ZHOU Guang-Ming and HU Bu-Rong who kindly provide us the human fibrosarcoma cell line HT1080 and normal human lung embryonic cell line MRC-5. 10.1007/s41365-016-0021-x Supported by National Magnetic Confinement Fusion Science Program of China (No. 2014GB112006), National Natural Science Foundation of China (No. 11305204) and Natural Science Foundation of Anhui Province of China (No. 1508085SME220) 2014-12-25 2015-04-16 2015-04-18 2016-03-01 2016-02-20 2021-04-11 © China Science Publishing & Media Ltd. (Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2016 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-016-0021-x http://dx.doi.org/10.1007/s41365-016-0021-x . 2016 Nuclear Science and Techniques 01 27