Mol Biol Rep DOI 10.1007/s11033-014-3846-6

Adenovirus-mediated IL-24 confers radiosensitization to human lung adenocarcinoma in vitro and in vivo Shi-Ying Zheng • Jin-Feng Ge • Jun Zhao Dong Jiang • Fang Li

•

Received: 11 May 2012 / Accepted: 19 November 2012 Ó Springer Science+Business Media Dordrecht 2014

Abstract The current paper aims to study the effect of adenovirus-mediated IL-24 (Ad-IL-24) on human lung adenocarcinoma in vitro and in vivo and determine its possible mechanism of action. The growth-suppressing and apoptosis-inducing effects of Ad-IL-24, radiotherapy, and Ad-IL-24? radiotherapy (hereinafter referred to as the joint group) on SPC-A1 lung carcinoma cells were assessed by using 3-(4,5-dimethyliazolyl-2)-2,5-diphnyltetrazolium bromide and flow cytometry. A human lung model was established with SPC-A1 cells in nude mice. Groups of mice were subjected to multi-point injections to their tumors. Gross tumor volumes were measured dynamically. The ratios of tumor suppression and radiosensitization effect were evaluated according to the method of probability sum Q values. The expressions of Bax, Bcl-2, Survivin, and Caspase-3 in tumor samples were detected by immunohistochemistry. The ratios of inhibition and apoptosis in the joint group were higher than those in the individual Ad-IL-24 and radiotherapy groups. In vitro, the joint group suppressed tumor growth conspicuously, showing a weight inhibition rate of about 64 %. The expressions of FasL, Bax and Caspase-3 were upregulated in the joint group, while the expressions of Cox,Bcl-

Dong Jiang and Shi-Ying Zheng have contributed equally to this work. S.-Y. Zheng (&)  J.-F. Ge  J. Zhao  D. Jiang Department of Thoracocardiac Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, China e-mail: [email protected] F. Li (&) Department of Human Anatomy, Histology and Embryology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215007, China e-mail: [email protected]

2,VEGF,CD34 and Survivin were downregulated. The current study proves that Ad-IL-24 suppresses growth of SPC-A1 cells both in vitro and in vivo. Its functions appear to be related to cell apoptosis and antiangiogenesis. Keywords IL-24  Radiotherapy  Adenocarcinoma  Radiosensitization Introduction Lung cancer has emerged as a main disease that threatens human health, ranking first in fatality among other malignant tumors. Although traditional treatments for lung cancer (e.g., surgical treatment, chemotherapy, and radiotherapy) have undergone significant development, the overall survival rate of patients with lung cancer has not improved satisfactorily. The five-year survival rate remains less than 15 % [1]. The occurrence and development of lung cancer is a multi-step and multi-gene process, including many related gene mutations, such as the activation of oncogenes and the deactivation of tumor suppressor genes. Such mutations are involved in every stage of cancer development [2], including priming, development, and metastasis. Gene therapy is currently a hot topic of tumor research because of the molecular biological features of lung cancer. Through gene transfer techniques, exogenous target genes have been transferred into tumor cells to correct or compensate for endogenous gene defects in the cells. Such techniques allow the achievement of treatment goals [3]. Over the last 20–30 years, great accomplishments have been made in gene therapy such that it is now regarded as an effective treatment for inborn and acquired gene diseases [4]. As a kind of tumor suppressor gene, IL-24 was obtained first through the induction in human melanoma cells [5, 6].

123

Mol Biol Rep

The encoding gene of IL-24 is located at 1q32.2–q41; it is a single copy gene located at a 195 kb gene cluster. This gene cluster also includes the encoding genes of IL-10, IL19, and IL-20, all of which belong to the IL-10 family. Thus, the encoding gene of IL-24 has some structural similarities with and belongs to the IL-10 family [7, 8]. The DNA fragment length of IL-24 is 6.33 kb, and its encoding product is a protein with 206 amino acids [9]. Mhashilkar et al. [10] observed that the IL-24 gene can inhibit the growth of lung cancer in a time- and dosage-independent manner without inhibition of the growth of normal cells. Animal experiments revealed that IL-24 [11] transfected by adenovirus has significant antitumor activity in a lung cancer rat model. In this model, the tumor volume decreased and the angiogenesis of tumor tissues was inhibited. Using immunohistochemical analysis, Saeki et al. [12] found that Caspase-3 was overexpressed by IL24 in lung cancer cells to induce apoptosis. Thus, IL-24 can inhibit tumor growth through many ways and is considered a safe and effective tumor suppressor gene. While IL-24 significantly inhibits the growth of lung cancer cells, introduction of the gene via an adenovirus vector (AdV) may further enhance its activity. The current experiment provides experimental basis for the use of IL24 gene therapy to treat lung cancer through observation of the influence of IL-24 gene expression on the growth of NCI-H460 lung cancer cells.

Materials and methods

The number of QBI-293A cells in good growth condition was calculated after trypsin digestion. After the cell concentration was diluted to 105 mL, cells were inoculated on a 96-well plate at about 100 lL/well. After 24 h of culture, the reconstructed virus obtained was diluted as 10-4, 10-5, 10-6, l0-7, 10-8. All wells in a line were inoculated at 100 lL/well for each dilution. Cells were then cultured in a 5 % CO2 cell incubator at 37 °C for 18 h. Cell counts were conducted afterwards under a fluorescence microscope. The virus potency was calculated according to the formula: virus potency (pfu/ mL) = (fluorescence number 9 10)/dilution. AdV infection In the log phase, the SPC-A1 cell line was dispensed into the cell suspension with RPMI 1640 after digestion by 0.25 % trypsin. The cell concentration was adjusted to 1 9 105/mL after the count. The cells were inoculated to a 96-well plate at 100 lL/well and 37 °C with 5 % CO2 and cultured overnight. The next day, the SPC-A1 cells were infected with free AdV according to the multiplicity of infection (MOI) of (1, 10, 25, 50, 100, and 200) to determine the best infection dosage. The SPC-A1 cells were divided into three groups: the PBS cell control group, the free AdV group, and the Ad-IL-24 single gene group. After 72 h of infection, the growth morphology of the cells was observed under a common light microscope and the expression of GFP green fluorescence was observed. Thus, the effects and infection efficiency of adenovirus infection dosage on the growth of SPC-A1 cells were obtained.

Cells and animals RT-PCR Lung adenocarcinoma cell strain SPC-A1, QBI-293A cells were provided by the Cellular and Molecular Biology Institute of the College of Medicine, Soochow University, China. SPC-A1 cells were cultured in complete medium (10 % FCS) at 37 °C with 5 % CO2. One passage was obtained after (2–3) days. Male BALB/c nu/nu nude rats (3–4 years old),weigh approximately 20 g were purchased from Shanghai Slack Laboratory Animal Center, Chinese Academy of Sciences [License number: SCXK (Shanghai) 2007-0005].

Adenovirus amplification and titer determination Approximately 70 % of the adherent QBI-293A cells were infected by AdV and Ad-IL-24. The cells were collected after 48 h, and the cell suspension was frozen and thawed 4 times at -80 and 37 °C, respectively. The supernatant was then collected and amplified repeatedly. Finally, the virus was stored at -80 °C.

123

SPC-A1 cells were infected by 50 MOI Ad-IL-24 and AdV, and the uninfected SPC-A1 cells were used as control. After 72 h, cells were subjected to centrifugation at 1,500 r/min and then washed twice or thrice using PBS. Total RNA was extracted using an RNA extraction kit according to the manufacturer’s instructions. First chain cDNA was obtained through reverse transcription. The primers used were shown in Table 1. The cDNA template and P1 and P2 upstream and downstream primers were amplified in the PCR instrument. The PCR conditions were as follows: 94 °C for 4 min, 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min for 30 cycles, and 72 °C for 10 min. Finally, 10 lL of the product and the DNA marker were subjected to agarose gel electrophoresis. Western blot analysis The AdV and Ad-IL-24 infected SPC-A1 cells and the uninfected SPC-A1 cells were collected after 72 h and cell

Mol Biol Rep Table 1 The primer and its sequence used by PCR amplification

IL-24 b-Actin

Primer

Primer sequence

P1:

50 -GCACTCGAGACCATGAATTTTCAACAGAGGCTGCA-30

P2:

50 -GCTTCTAGATCAGAGCTTGTAGAATTTCTG-30

P3:

50 -TGCGTGACATTAAGGAGAAG-30

P4:

50 -CTGCATCCTGTCGGCAATG-30

lysis buffer (including the protease inhibitor at a final concentration of 1 mM PMSF) was added to achieve a concentration of 107/mL for cracking. After full cracking, the cells were centrifugated at 12,000 r/min for 5 min. The total protease supernatant was then collected and mixed with 5 9 sodium dodecyl sulphate (SDS) buffer solution at a ratio of 4:1. The liquid was boiled at 100 °C for 5 min, centrifuged for 5 min at 12,000 r/min, and then subjected to SDS-PAGE (100 V, 2 h) using polyacrylamide gel with a separation rate of 12 %. The protein was transferred onto a nitrocellulose (NC) membrane at 300 mA for 2 h. The NC membrane was first blocked by skimmed milk powder at 37 °C for 1 h., incubated by the mouse anti-human antibody (IL-24 antibody) at 37 °C for 1 h, and washed by TBST for three times, then it was incubated by horseradish peroxidase (HRP)-marked goat anti-mouse IgG at 37 °C for another 1 h. after that the NC membrane was again washed by TBST for three times. Finally, the NC membrane was incubated with the emulsion working liquid containing A and B solutions were mixed with the same volume) and incubated at room temperature for 3 min. Tableting, exposure, development, and fixation were conducted in a darkroom. MTT The following groups were prepared and processed: PBS group: 1 9 105/mL SPC-A1 ? 0.1 mol/L PBS; AdV group: 1 9 105/mL SPC-A1 ? 50 MOI AdV; Radiotherapy group: 1 9 105/mL SPC-A1 ? Radiation after 48 h (4 Gy); AdIL-24 group: 1 9 105/mL SPC-A1 ? 50 MOI Ad-IL-24; Ad-IL-24? Radiotherapy group: 1 9 105/mL SPCA1 ? 50 MOI Ad-IL-24? Radiation after 48 h (4 Gy). In the log phase, the SPC-A1 cell line was dispensed into a single cell suspension using complete medium after trypsinization. The cell concentration was adjusted after the number of cells was calculated. The cells were inoculated on a 96-well plate at concentrations of 1 9 104/ 50 lL. After cell attachment, the cells were divided into groups and processed according to the above experiment. Three parallel wells were set in each group and the cells were cultured in the incubator with 5 % CO2 at 37 °C for (1–4) d. Morphological changes, growth, and reproduction of cells at different time periods were observed, and 10 lL

of MTT (5 mg/mL) was added to each well on a daily basis. After continuous culture at 37 °C for 4 h, 10 % SDSHCl stop buffer was added at about 100 lL/well. After complete dissolution at 37 °C, the light absorption value (OD value) was measured using a microplate reader at 570 nm. A growth curve was drawn with the OD value as the ordinate and the time (D) as the abscissa. The growth inhibition rate was calculated according to the formula: cell growth inhibition rate (%) = (1–OD value in experimental group/OD value in negative control group) 9 100 %. Flow cytometry SPC-A1 cells (at exponential growth phase, 70 % cell density) were divided into five groups: 0, 2, 4, 6, and 8 Gy. 60 Co c radiation exposure with an absorption dose rate of 1 Gy/min was provided by the Radiation Center of Radiology of the First Affiliated Hospital of Soochow University at room temperature. After 72 h of culture at 37 °C under 5 % CO2, the cells were collected and subjected to PBS twice. Cells were then adjusted to 1 9 106/mL by 1 9 binding buffer. Approximately 100 lL of the cell suspension was placed into a flow tube, fully mixed with 10 lL Annexin-V-PE on ice, kept from light for 15 min, and then successively mixed with 380 lL 1 9 binding buffer and 10 lL 7-AAD. Cells were subjected to flow cytometry. The experiment was repeated thrice to select the best exposure dose to use as a reference during research on the biological functions of Ad-IL-24 combined with radiotherapy. Grouping and processing were conducted according to MTT procedures. The cells were collected after 72 h of culture at 37 °C under 5 % CO2 and washed twice with PBS. The cells were then fixed using 70 % ice ethanol, stained using propidium iodide (PI) for 30 min, and washed thrice with PBS. Finally, the cell cycle was detected via flow cytometry. All procedures were repeated thrice. To determine apoptosis rates, grouping and processing were conducted according to MTT procedures. Cells were collected after 72 h, and the same procedures were performed as mentioned above. Apoptosis rates were determined by flow cytometry, and experimental procedures were repeated thrice.

123

Mol Biol Rep

Establishment of nude mice xenograft model SPC-A1 human lung adenocarcinoma cells were cultured in RPMI 1640 complete medium (10 % FCS) under conventional culture condition (5 % CO2, 37 °C). Adenocarcinoma cells in the log phase were washed using PBS and digested using 0.25 % trypsin. After culturing was terminated by a serum-free solution, cells were obtained by centrifugation for 5 min at 1,000 r/min. The cell number was calculated, and the cell concentration was adjusted to yield a cell suspension of 3 9 107/mL. Twenty-five 4-yearold male BALB/c nude rats of SPF grade were used. Each rat was disinfected with anerdian administered to the right forelimb armpit and was administered a subcutaneous injection of 3.0 9 106/100 lL cell suspension. Observations of subcutaneous growth and tumor formation of SPCA1 lung adenocarcinoma cells in nude rats followed. Anti-tumor experiment Subcutaneous inoculation of SPC-A1 lung adenocarcinoma cells was performed on nude rats. When the tumor diameter was approximately 5 mm, the rats were randomly grouped and subjected to anti-tumor experiments. The rats were randomly divided into five groups (5 rats 9 5 groups): PBS group, AdV group, simple radiotherapy (10 Gy) group, Ad-IL-24 group, and Ad-IL-24? radiotherapy group. The anti-tumor plan of each group was as follows: (1) PBS group: The PBS dosage was 50 lL/rat. Multi-site injections were performed inside the tumor. Injection was done five times on a daily basis. (2) AdV group and Ad-IL-24 group: The reconstructed virus dosage was 1.5 9 108 pfu/50 lL/rat. Multi-site injections were performed inside the tumor. Injection was done five times on a daily basis. (3) Simple radiotherapy group: On the fifth day after the anti-tumor experiment, nude rats were anaesthetized using 10 % chloral hydrate (3 lL/g) and fixed on a special device. Local tumor radiation was conducted with the other parts of the rat shielded by a lead plate. The following parameters were used: voltage of the linear accelerator = 5 MeV, SSD = 100 cm, dosage = 10 Gy/rat, frequency = once. (4) Ad-IL24? radiotherapy group: The dosage and method of AdIL-24 were similar to that of the Ad-IL-24 group; local tumor radiation was conducted on the day of the third injection. The dosage and method were similar to the simple radiotherapy group.

(V = a 9 b2/2) and the volume-time curve of the tumor was drawn. The tumor inhibition effect of IL-24 and combined radiotherapy on the SPC-A1 lung adenocarcinoma transplantable tumor was analyzed further. After 15 d of treatment, the rats were killed by decapitation. The local skin of the tumor was conventionally disinfected with anerdian and then slit. The tumor was removed and weighed using an electronic balance. The tumor inhibition effect of IL-24 and combined radiotherapy on the SPC-A1 lung adenocarcinoma transplantable tumor was then analyzed. The tumor inhibition rate (E) was calculated according to the tumor weight. Tumor inhibition rate (%) = (1 - the average tumor weight in experiment group/the average tumor weight in control group) 9 100 %. The radiosensitization effect of IL-24 and combined radiotherapy was evaluated according to the Jin zheng-jun method based on the method of probability sum Q values reflected the nature of the experimental results: the role of simply adding (0.85 \ Q \ 1.15), enhanced significantly (Q [ 1.15), enhanced antagonistic (0.55 \ Q \ 0.85) or significantly antagonized (Q \ 0.55) and it was calculated according to the formula: Q value = E(A?B)/[EA ? (1 - EA) 9 EB]. HE and immunohistochemical staining The tumor tissue in each group was fixed with 10 % neutral formalin. The slice was subjected to conventional paraffin embedding, hematoxylin and eosin (HE) staining, and immunohistochemical staining. HE staining was used to observe morphological changes in tumor tissue cells, such as reductions in cell volume, cytoplasm concentration, pyknosis, fragmentation, and dissolution of the nucleus; and also a large amount of bubble formation between the tissues. Immunohistochemical staining was used to detect the growth-related factor expression of SPC-A1 lung adenocarcinoma transplantable tumors: (1) pro-apoptotic factors, such as Bax, Caspase-3, and FasL; (2) apoptosis inhibitors, such as Bcl-2, Cox-2, and Survivin; and (3) tumor angiogenesis factors, such as vascular endothelial growth factor (VEGF). Each slice was observed under a 400 9 light microscope; the positive cell number of ten visual fields was counted (positive cells were those brown particles scattered or diffused into the cytoplasm). Data were further analyzed and the molecular mechanism of the tumor inhibition of IL24 and combined radiotherapy on SPC-A1 lung adenocarcinoma transplantable tumors was explored.

Tumor inhibition effect

Statistical analysis

Before the first treatment and every other day after treatment, the long (a) and short (b) diameters of the tumor were measured. The tumor volume was calculated

The SPSS16.0 statistical software package was used and all the data were expressed as x  s. Data were processed using analysis of variance. P \ 0.05 signified statistical

123

Mol Biol Rep

difference and P \ 0.01 indicates significant statistical difference. The Jin zheng-jun method (calculation of the Q value) was used to evaluate the apoptosis rate, tumor inhibition effect and radiosensitization effect of Ad-IL-24 combined with radiotherapy. In the formula Q = E (A?B)/[EA ? (1 EA) 9 EB], numerator E(A?B) represents the actual combined effect, and EA, EB were the effects of Ad-IL-24 and radiotherapy alone. When the Q value = 1 ± 0.15, the additive effect between A and B exists. When the Q value is [1.15, the synergistic effect between A and B is observed, and when the Q value is\0.85, antagonism between A and B can be observed.

Results SPC-A1 lung adenocarcinoma cells infected by adenovirus SPC-A1 cells were infected by the free AdV according to different MOI: (1, 10, 25, 50, 100, and 200). Almost all groups of AdV-infected SPC-A1 cells, except those infected at a dosage of 200 MOI, showed normal morphologies and good growth conditions. No difference was observed between infected cells and those in the control. However, in the 200 MOI-infected group, SPC-A1 cells became rounded and abscission was observed, indicating obvious cytotoxicity caused by the adenovirus itself. Under the fluorescence microscope, over 90 % SPC-A1 cells infected with free AdV at dosages of (50, 100, and 200) MOI could be observed under green fluorescence. Based on the virus infection dosage principle, when the effect of infection caused by free AdV is highest, toxicity is lowest. The best infection dosage of adenovirus for SPC-A1 cells was 50 MOI. As shown in Fig. 1, 72 h after SPC-A1 cells were infected by Ad-IL-24 (50 MOI), they produced obvious cytotoxicity caused by adenovirus-mediated IL-24 gene expression. SPC-A1 cells infected by free AdV at the same infection dosage grew well after 72 h, similar to the cells in the control group without infection. The results primarily indicate that adenovirus-mediated IL-24 gene expression in vitro had obvious inhibition effects on the growth of cancer cells. Ad-I L-24 expression In Ad-IL-24 group, an IL-24 specific band was detected at 650 bp; this band did not appear in the PBS and AdV control groups. RT-PCR results showed that the Ad-IL-24 reconstructed adenovirus can mediate the successful transcription of exogenous IL-24 genes in SPC-A1 cells (Fig. 2a).

Western blot results showed that Ad-IL-24 featured a 24 kD strip that combined with the specificity of the antihuman IL-24 antibody. The free AdV infection group and the cell control group did not show this strip at the corresponding position, indicating that Ad-IL-24 can mediate the successful IL-24 protein expression of exogenous IL-24 genes in SPC-A1 cells (Fig. 2b). Cell apoptosis of SPC-A1 cells The apoptosis rates of the 0, 2, 4, 6, and 8 Gy groups were (1.83 ± 0.11), (12.51 ± 0.95), (24.13 ± 0.60), (38.46 ± 2.13), and (57.75 ± 0.83) %, respectively. To observe the radiosensitization effect of Ad-IL-24 clearly, 4 Gy was selected as the radiation dosage in subsequent experiments (Fig. 4a). Growth of SPC-A1 cells In Fig. 3, Ad-IL-24 was shown to have varying degrees of growth inhibition effects on SPC-A1 cells; these effects appeared to be time-dependent. Compared with the AdV and PBS groups, a significant difference (P \ 0.01) was observed. The growth inhibition effects of the Ad-IL-24? radiotherapy group on SPC-A1 cells was significantly different from those of the simple AdIL-24 and radiotherapy groups (P \ 0.01), moreover it was also better than those of the simple Ad-IL-24 and radiotherapy groups, and the synergistic effect of radiosensitization between Ad-IL-24 and radiotherapy was observed (Q = 1.17). Cell cycle and apoptosis rate of SPC-A1 cells The G2/M stage cells in Ad-IL-24 group was (15.25 ± 0.67) % and in the radiotherapy group was (23.98 ± 2.31) %, both values were significantly higher than those of the control [(7.64 ± 0.98) %] and Adv (11.38 ± 1.14) % groups (P \ 0.01). Moreover, G2/M arrest in the Ad-IL-24? radiotherapy group (53.87 ± 1.33) % was significantly higher than those in the simple Ad-IL-24 and radiotherapy groups (P \ 0.01). The G1-stage cells in the AdIL-24 group was (53.87 ± 1.33) %, while those in the Ad-IL24? radiotherapy and simple radiotherapy groups were (17.31 ± 1.65) and (37.81 ± 1.77) %, respectively. Comparing the PBS (81.51 ± 2.98) % and Adv (70.75 ± 1.06) % groups, cell ratios at the G1 stage significantly decreased (P \ 0.05) (Fig. 4b). Figure 4c showed flow cytometry results obtained via the Annexin V-PE/7 AAD double-staining method. After application of Ad-IL-24 and AdV, the apoptosis rates of

123

Mol Biol Rep

Fig. 1 The transcription identify of the reconstructed adenovirus -mediated IL-24 in SPC-A1 cells. a PBS group; b Ad-GFP group; c Ad-IL-24 group

SPC-A1 cells were (18.38 ± 1.41) and (3.93 ± 1.17) %, respectively. The apoptosis rates of the PBS and radiotherapy groups were (1.94 ± 0.65) and (24.27 ± 2.02) %, respectively. Compared with the AdV and PBS groups, the apoptosis rate of the Ad-IL-24 group was obviously

123

increased (P \ 0.01). The apoptosis rate of the Ad-IL24? radiotherapy group [(40.57 ± 3.30) %] was significantly higher than those in the simple Ad-IL-24 and radiotherapy groups (P \ 0.01), and synergistic effects were observed (Q = 1.16).

Mol Biol Rep

Fig. 2 The expression of reconstructed adenovirus mediated IL-24 in SPC-A1 cells. a RT-PCR; b Western blot. 1 PBS; 2 AdV; 3 Ad-IL24

The tumor volume was calculated according to the formula, and a tumor volume-time curve was drawn (Fig. 5a). The Ad-IL-24, simple radiotherapy, and Ad-IL24? radiotherapy groups showed varying degrees of tumors volume 15 d after treatment. Comparing the PBS and Adv groups, a significant difference (P \ 0.01) was observed. The tumor volume decrease more obviously in the Ad-IL-24? radiotherapy group than those of the AdIL-24 and simple radiotherapy groups. Fifteen days after treatment, nude rats were killed and tumor tissues were obtained. Tumor weights were determined on an electronic balance (precise to 0.001 g). The average tumor quality of the Ad-IL-24, simple radiotherapy, and Ad-IL-24? radiotherapy groups (x  s) were 1.335 ± 0.051, 1.232 ± 0.042, and 0.683 ± 0.033, respectively. Comparing the PBS (1.907 ± 0.103) and AdV (1.882 ± 0.128) groups, a significant difference (P \ 0.01) was observed. The tumor inhibition effect of the Ad-IL-24? radiotherapy group was better than those of the Ad-IL-24 and simple radiotherapy groups with significant statistical difference (P \ 0.01) (Fig. 5b). The tumor inhibition rate was calculated according to the formula, and a histogram of the tumor inhibition rate was drawn (Fig. 5c). The Ad-IL-24, simple radiotherapy, and Ad-IL-24? radiotherapy groups had obvious tumor inhibition effects on SPC-A1 lung adenocarcinoma cells in nude rats. Inhibition rates were 30.00, 35.40, and 64.18 %, respectively. Compared with the Ad-IL-24 and simple radiotherapy groups, a significant difference was observed in the Ad-IL-24? radiotherapy group (P \ 0.01) and synergistic effects (Q = 1.25) were found. HE staining

Fig. 3 The growth inhibition effect of Ad-IL-24 and others SPC-A1 lung adenocarcinoma cells. Compared with PBS group and Adv group, *P \ 0.01; Compared with Ad-IL-24 group and radiotherapy group, 4P \ 0.01

The above tumor tissue was subjected to HE conventional staining and observed under a light microscope (9400). A large number of cells in the simple radiotherapy and AdIL-24? radiotherapy groups showed karyopyknosis, cracking, and dissolution of the nucleolus, cytoplasmic concentration, incomplete membranes, and a large quantity of bubbles between the tissues (Fig. 6).

Anti-tumor effects

Expression of VEGF and CD34

About (2–3) d after the subcutaneous inoculation of SPCA1 lung adenocarcinoma cells in nude rats, the mound around the local skin shrank and gradually disappeared. Soon after, the tumor volume continuously increased and the diameter became approximately 5 mm after 10 d. In the present experiment, the tumor formation rate of SPCA1 lung adenocarcinoma cells inoculated into nude rats was 100 % (25/25).

The tissue slice was given to the tumor in the above five groups. The expression of the pro-apoptosis factors, such as Bax, Caspase-3, and FasL, were respectively detected via immunohistochemical staining (Fig. 6). Under a light microscope (9400), the number of positive cells was calculated. The results showed that the number of Fas-L, Bax, and Caspase-3 positive cells in each experimental group was significantly higher than that in the PBS and

123

Mol Biol Rep Fig. 4 a The apoptosis rates of SPC-A1 lung adenocarcinoma cell treated by different exposure dose. b The comparison of SPC-A1 lung adenocarcinoma cell cycle with the influence of Ad-IL-24 and others detected by PI staining method. The Ad-IL24? radiotherapy group has most significant growth inhibition effects on SPC-A1 cells compare with other groups.(Compared respectively with PBS group and AdV group, *P \ 0.01; Compared respectively with Ad-IL-24 group and radiotherapy group, 4 P \ 0.01; Compared respectively with PBS group and AdV group, qP \ 0.05.) c The results of apoptosis rate of the SPC-A1 lung adenocarcinoma cell with the influence of Ad-IL-24 and others detected by flow cytometry. The apoptosis rate of the Ad-IL-24? radiotherapy group was significantly higher than any other group

123

Mol Biol Rep Fig. 5 a The tumor volumetime curve of the each group. The tumor volume decrease more obviously in the Ad-IL24? radiotherapy group than any other group. (Compared respectively with PBS group and Adv group, *P \ 0.01, compared respectively with AdIL-24 group and simple radiotherapy group, 4P \ 0.01.) b Tumor weight of each group. The tumor weight decrease more obviously in the Ad-IL24? radiotherapy group than any other group. (Compared respectively with PBS group and Adv group, *P \ 0.01; Compared respectively with Ad-IL-24 group and simple radiotherapy group, 4P \ 0.01.) c The tumor inhibition rate in each group. The tumor inhibition rate in the Ad-IL24? radiotherapy group was most obvious (compared respectively with Ad-IL-24 group and simple radiotherapy group, *P \ 0.01, Q = 1.25)

AdV groups (P \ 0.01). The Ad-IL-24? radiotherapy group performed better than the Ad-IL-24 and simple radiotherapy groups with significant difference (P \ 0.01). The numbers of Cox-2, Bcl-2, VEGF, and CD34 (MVD) positive cells in each experimental group were all significantly lower than those in the PBS and

AdV groups (P \ 0.01), except that the difference of the number of VEGF positive cells between simple radiotherapy and PBS groups was not obvious. The Ad-IL24? radiotherapy group performed better than the AdIL-24 and simple radiotherapy groups with significant difference (P \ 0.01, Tables 2).

123

Mol Biol Rep Fig. 6 HE staining of the tumor tissue in each group. In the simple radiotherapy and AdIL-24? radiotherapy groups, a large number of cells showed karyopyknosis, cracking, and dissolution of the nucleolus, and a large quantity of bubbles among tissues was also observed

Discussion Currently, apoptosis occurs via two pathways: extrinsic (death receptor pathway) and intrinsic (mitochondria). The extrinsic pathway activates caspase in the cells mainly through the combination of extracellular signal and death receptors in the surface of the cells. The intrinsic pathway activates caspase through the caspase activating factor released by the mitochondria. Caspase belongs to the cysteine protease family and Caspase-3 is the key enzyme that activates apoptosis in this family. Most of the apoptosis-inducing factors finally leads to the cell apoptosis through the Caspase-3-mediated signal [13]. In the present experiment, the number of cells with caspase-3 positive expression in the Ad-IL-24? radiotherapy group was significantly higher than those in the Ad-IL-24 and simple radiotherapy groups with obvious difference (P \ 0.01). These findings indicate that apoptosis mediated by Caspase-3 is one of the more important mechanisms for tumor apoptosis induced by Ad-IL-24 combined with radiotherapy. Fas are an important death receptor. FasL, the ligand of Fas, plays an important role in the apoptosis course induced by radiation injury and its DNA damaging agent. The literature reports that increases in FasL expression can help increase sensitivity to and the efficacy of radiotherapy [14]. When Fas is combined with FasL, Caspases-3, -6, and -7 can be activated in turn to induce apoptosis [15]. The present experiment showed that the Ad-IL-24? radiotherapy group

123

performed significantly better than the Ad-IL-24 and simple radiotherapy groups with significant difference (P \ 0.01). The Bcl-2 family is divided into two types. One type includes the anti-apoptosis factors, such as Bcl-2, Bcl-xl, Bclw, and Mcl-1. The other type includes pro-apoptotic factors, including Bax, Bak, and Bad. Bcl-2 and Bax are two main members of the Bcl-2 (the apoptosis-regulatory gene) family, having the function of regulating and controlling the release of cytochrome C. They can adjust mitochondrial apoptotic pathway through the formation of homo- or heterodimers [16]. The ratio of Bax and Bcl-2 in cells determines whether or not the cells will undergo apoptosis. In the present experiment, Bax and Bcl-2 were detected in different groups after treatment by immunocytochemistry. The results showed that the number of cells with positive Bcl-2 expression in the Ad-IL-24? radiotherapy group was obviously lower than those in the Ad-IL-24 and simple radiotherapy groups with significant difference (P \ 0.01). The number of cells that positively expressed Bax in the Ad-IL-24? radiotherapy group was significantly higher than those in the Ad-IL-24 and simple radiotherapy groups with obvious difference (P \ 0.01). The experimental results indicate that the obvious increase of the pro-apoptotic geneBax and the obvious decrease of the anti-apoptotic gene-Bcl-2 led to the obvious increase of the ratio of Bax/Bcl-2, thereby accelerating tumor apoptosis. Many recent studies have shown that the COX-2 inhibitor can improve tumor radiosensitivity without causing

Mol Biol Rep Table 2 The related-factor expression of the tumor tissue in each treatment group (/HP) Group/factor

Bax

Caspase-3

Cox-2

Bcl-2

Survivin

VEGF

CD34

PBS

22 ± 4.45

13 ± 3.69

113 ± 11.2

94 ± 6.3

95 ± 5.04

95 ± 5.93

54 ± 4.79

AdV

24 ± 3.29

15 ± 3.04

110 ± 9.57

92 ± 7.27

91 ± 6.11

96 ± 6.51

52.3 ± 3.95

FasL 14 ± 3.87 16 ± 4.8

Ad-IL-24

49 ± 4.96*

38 ± 3.3*

75 ± 7.00*

65 ± 5.87*

66 ± 6.61*

63 ± 5.55*

29 ± 4.54*

36 ± 5.86*

Radiotherapy

57 ± 5.71*

47 ± 3.74*

67 ± 5.92*

57 ± 6.44*

59 ± 5.99*

89 ± 4.78

48 ± 4.61*

41 ± 4.63*

Ad-IL-24? Radiotherapy

39 ± 6.31*4

75 ± 5.29*4

43 ± 6.31*4

39 ± 5.50*4

47 ± 4.88*4

50 ± 4.01*4

17 ± 3.16*4

65 ± 6.31*4

* Meant that each treatment group compared respectively with PBS group and AdV group * P \ 0.05; D meant that each treatment group compared with Ad-IL-24 group and radiotherapy group, D P \ 0.05

radiation injury to normal tissues; some studies have even shown it to protect normal tissues from radiation [17–21]. Immunocytochemical examination showed that the number of cells positive for COX-2 expression in the Ad-IL24? radiotherapy group was significantly lower than those in the Ad-IL-24 and simple radiotherapy groups with significant difference (P \ 0.01). The Survivin gene is one of the strongest apoptosis inhibitors uncovered thus far [22]. The high expression of Survivin in the cytoplasm can deactivate inhibitors of the apoptosis protein (IAP), whose release was induced by radiotherapy leading to tolerance of tumor cells to radiation. Thus, the inhibition of Survivin expression has important significance in improving the radiosensitivity of tumor cells [23]. In the present experiment, the number of cells with positive Survivin expression in the Ad-IL-24? radiotherapy group was obviously lower than those in the Ad-IL-24 and simple radiotherapy groups with significant difference (P \ 0.01). VEGF is the angiogenesis factor with the strongest activity and highest specificity. It is also one of the most important factors in tumor angiogenesis [24]. The CD34 gene positions on clql2-qt, is the glucoprotein on the surface of the vascular endothelial cell, and is an important marker for tumor angiogenesis [25]. As such, VEGF and CD34 were subjected to immunocytochemical examination. The reconstructed Ad-IL-24 adenovirus can obviously decrease the expression of VEGF in SPC-A1 lung cancer cells. In summary, the Ad-IL-24 reconstructed adenovirus expression vector combined radiotherapy has significant anti-tumor and radiosensitization effects on SPC-A1 lung cancer cells and transplantable tumors of nude rats with lung cancer. Its significant effect on cell cycle control and apoptosis-related protein expression may be one of its more important mechanisms. The current article provides experimental basis for the treatment efficacy of Ad-IL-24 gene therapy in combination with radiotherapy.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

References 13. 1. Swisher SG, Roth JA (2002) Clinical update of Ad-p53 gene therapy for lung cancer. Surg Oncol Clin N Am 11:521–535 2. Sainz-Perez A, Gary-Gouy H, Gaudin F, Maarof G, MarfaingKoka A, de Revel T, Dalloul A (2008) IL-24 induces apoptosis of

chronic lymphocytic leukemia B cells engaged into the cell cycle through dephosphorylation of STAT3 and stabilization of p53 expression. J Immunol 181:6051–6060 Sarkar D, Lebedeva IV, Gupta P, Emdad L, Sauane M, Dent P, Curiel DT, Fisher PB (2007) Melanoma differentiation associated gene-7 (mda-7)/IL-24: a magic bullet for cancer therapy? Expert Opin Biol Ther 7:577–586 Robson S, Pelengaris S, Khan M (2006) c-Myc and downstream targets in the pathogenesis and treatment of cancer. Recent Pat Anticancer Drug Discov 1:305–326 Pan XT, Sheng WH, Yang JC, Xie Y, Ye Z, Xiang J, Li D, Yang J (2008) Inhibition of pancreatic carcinoma growth by adenovirus-mediated human interleukin-24 expression in animal model. Cancer Biother Radio 23:425–434 Roy I, Holle L, Song W, Holle E, Wagner T, Yu X (2002) Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro. Anti-cancer Res 22:3185–3189 Xie YF, Sheng WH, Yang JC, Ye Z, Zhu Y, Chen X, Yang J (2008) Recombinant human IL-24 suppresses lung carcinoma cell growth via induction of cell apoptosis and inhibition of tumor angiogenesis. Cancer Biother Radio 23:310–320 Lebedeva IV, Su ZZ, Chang Y, Kitada S, Reed JC, Fisher PB (2002) The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21:708–718 Caudell EG, Mumm JB, Poiindexter N, Ekmekcioglu S, Mhashilkar AM, Yang XH, Retter MW, Hill P, Chada S, Grimm EA (2002) The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immuno-stimulatory activity and is designated IL-24. J Immunol 168:6041–6046 Mhashilkar AM, Schrock RD, Hindi M, Liao J, Sieger K, Kourouma F, Zu-Yang XH, Onishi E, Takh O, Vedvick TS, Fanger G, Stewart L, Watson GJ, Snary D, Fisher PB, Saeki T, Roth JA, Ramesh R, Chada S (2001) Melanoma differentiation associated gene-7 (mda-7): a novel antitumor gene for cancer gene therapy. Mol Med 7:271–282 Su ZZ, Lebedeva IV, Sarkar D, Gopalkrishnan RV, Sauane M, Sigmon C, Yacoub A, Valerie K, Dent P, Fisher PB (2003) Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 22:1164–1180 Saeki T, Mhashilkar A, Swanson X, Zou-Yang XH, Sieger K, Kawabe S, Branch CD, Zumstein L, Meyn RE, Roth JA, Chada S, Ramesh R (2002) Inhibition of human lung cancer growth following adenovirus-mediated Mda-7 gene expression in vivo. Oncogene 21:4558–4566 Yuan SS, Chang HL, Chen HW, Kuo FC, Liaw CC, Su JH, Wu YC (2006) Selective cytotoxicity of squamocin on T24 bladder cancer cells at the S-phase via a Bax, Bad, and caspase-3 related pathways. Life Sci 78:869–874

123

Mol Biol Rep 14. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776 15. Mercurio AM, Bachelder RE, Bates RC, Chung J (2004) Autocrine signaling in carcinoma: VEGF and the alpha6beta4 integrin. Semin Cancer Biol 14:115–122 16. Swamy MV, Herzog CR, Rao CV (2003) Inhibitor of COX-2 in colon cancer cell lines by celecoxib increases the nuclear localization of active P53. Cancer Res 63:5239–5242 17. Zhou XM, Wang BC, Fan XM, Zhang HB, Lin MC, Kung HF, Fan DM, Lam SK (2001) Non-steroidal anti-inflammatory drug induce apoptosis in gastric cancer cells through up-regulation of bax and bak. Carcinogenesis 22:1393–1397 18. Narayanan BA, Condon MS, Bosland MC, Narayanan NK, Reddy BS (2003) Suppression of N-methy-N-nitrosourea/testosterone-induced rat prostate cancer growth by celecoxib: effects on cyclooxygenase-2, cell cycle regulation, and apoptosis mechanism. Clin Cancer Res 9:3503–3513 19. Krysan K, Merhant FH, Zhu L, Dohadwala M, Luo J, Lin Y, Heuze-Vourc’h N, Po˜ld M, Seligson D, Chia D, Goodglick L, Wang H, Strieter R, Sharma S, Dubinett S (2004) COX-2-

123

20.

21.

22.

23.

24.

dependent stabilization of survivin in non-small cell lung cancer. FASEB J 18:206–208 Nix P, Lind M, Greenman J, Stafford N, Cawkwell L (2004) Expression of COX-2 protein in radioresistant laryngeal cancer. Ann Oncol 15:797–801 Hou JQ, HE J, Wen DG, Chen ZX, Zeng J (2006) Surviving mRNA expression in urine as a biomarker for patients with transitional cell carcinoma of bladder. Chin Med J 119:1118–1120 Suzuki Y, Oka K, Yoshida D, Shirai K, Ohno T, Kato S, Tsujii H, Nakano T (2007) Correlation between survivin expression and locoregional control in cervical squamous cell carcinomas treated with radiation therapy. Gynecol Oncol 104:642–646 El-kott AF, El-baz MA, Mokhtar AA (2006) Proliferating cell nuclear antigen (PCNA) overexpression and microvessel density predict survival in the urinary bladder carcinoma. Int Urol Nephrol 38:237–242 Khmhoff M, Guan Y, Shappell HW, Davis L, Jack G, Shyr Y, Koch MO, Shappell SB, Breyer MD (2000) Enhanced expression of cyslooxygenase-2 in higher grade human transitional cell bladder carcinomas. Am J Pathol 157:29–35