Title Page Grape Seed Proanthocyanidins Play the Roles of Radioprotection on Normal Lung and Radiosensitization on Lung Cancer via Differential Regulation of the MAPK Signaling Pathway

Radiation-induced lung injury (RILI) is a common serious complication and dose-limiting factor caused by radiotherapy for lung cancer. This study was to investigate radioprotective effects of grape seed proanthocyanidins (GSP) on normal lung as well as radiosensitizing effects on lung cancer. In vitro, we demonstrated radioprotective effects of GSP on normal alveolar epithelial cells (MLE-12 and BEAS/2B) and radiosensitizing effects on lung cancer cells (LLC and A549). In vivo, we confirmed this two-way effects in tumor-bearing mice. The results showed that GSP inhibited tumor growth, and played a synergistic killing effect with radiotherapy on lung cancer. Meanwhile, GSP reduced radiation damage to normal lung tissues. The two-way effects related to the differential regulation of the MAPK signaling pathway by GSP on normal lung and lung cancer. Moreover, GSP regulated secretion of cytokines IL-6 and IFN-γ and expression of p53 and Ki67 on normal lung and lung cancer. Our findings suggest that GSP is expected to be an ideal radioprotective drug for lung cancer patients who are treated with radiotherapy. Introduction Lung cancer ranks first in morbidity and mortality among malignant tumors [1], and radiotherapy is one of the main treatments for lung cancer. The incidence of radiation-induced lung injury (RILI) in patients with lung cancer after chest radiotherapy is 15% to 40% [2]. The occurrence of RILI severely limits the dose of radiotherapy in the target area. Some patients even need to discontinue radiotherapy due to the occurrence and progressive exacerbation of RILI. Severe radiation-induced pulmonary fibrosis can even lead to death of patients [3, 4]. Therefore, the protection of RILI caused by radiotherapy has extremely important medical significance. The main reason that some currently known radioprotective agents cannot be used in the clinic is that they prevent radiation damage to normal tissues and also have a radioprotective effect on tumor tissues, reducing the effect of radiotherapy [5-8]. Proanthocyanidin has obvious free radical scavenging ability, and its antioxidant activity in the body is 20 times that of VC and 50 times that of VE [9, 10]. Free radicals play important roles in injuries induced by ionizing radiation. Our previous experiments have found that grape seed proanthocyanidins (GSP) have significant radioprotective effects on radiation pneumonitis and pulmonary fibrosis [11, 12]. In addition, proanthocyanidins also have anti-tumor effects. The study of breast cancer [13], colorectal cancer [14], liver cancer [15], gastric cancer [16], oral squamous cell carcinoma [17], bladder cancer [18], skin cancer [19], kidney cancer [20], prostate cancer [21] and lung cancer [22] reported the obvious anti-tumor effects of proanthocyanidins. Therefore, when GSP is used for lung cancer patients who are treated with radiotherapy, it protects normal lung tissue against ionizing radiation and meanwhile it may enhance the killing effect of radiotherapy for lung cancer, which means that GSP is expected to be an ideal radioprotective drug for lung cancer patients who are treated with radiotherapy. Based on different effects of proanthocyanidins on normal lung and lung cancer, this study conducted a preliminary discussion on its mechanism. Mitogen-activated protein kinase (MAPK) plays important roles in regulating proliferation, differentiation and apoptosis of cells [23]. The MAPK/ERK signaling pathway is mainly related to cell proliferation and differentiation, while the MAPK/JNK and MAPK/p38 pathways are closely related to apoptosis of cells [23]. Activation of MAPK/ERK signaling pathway promotes the growth of prostate cancer [24], cervical cancer [25], thyroid cancer[26], and breast cancer[27]. MAPK/ERK-induced epithelial-mesenchymal transition promotes migration and metastasis of lung cancer cells [28]. However, Blocking the MAPK/ ERK signaling pathway suppresses proliferation of human ovarian cancer cells [29] and prostate cancer cells [30]. Unlike the MAPK/ERK pathway, activating the MAPK/JNK or MAPK/p38 pathway induces apoptosis of tumor cells, such as prostate cancer cells [24], breast cancer cells [31, 32], gastric cancer cells [33], and lung cancer cells [34]. Anthocyanins induce apoptosis of colon cancer cells [35] and leukemic cells [36] by activating the MAPK/JNK or MAPK/p38 pathway. However, for normal cells, down-regulating the MAPK pathway protect them when they are physically or chemically damaged. Anthocyanins also protect normal cells by down-regulating the MAPK signaling pathway. Grape seed proanthocyanidins inhibit the activation of MAPK signaling pathway mediated by UV-induced oxidative stress in human epidermal keratinocytes [37]. Grape seed proanthocyanidins played a similar effect in UV-exposed mouse skin when given to animals in the diet [38]. Anthocyanins protect the retinal pigment epithelial cells from damage by suppressing the MAPK signaling pathway [39]. Moreover, anthocyanins attenuate osteoarthritis [40, 41], encephalitis [42, 43], and skin inflammation [44] by inhibiting the MAPK signaling pathway. In this study, we investigated dual role of grape seed proanthocyanidins in normal lung and lung cancer after ionizing radiation, and found that the diverse regulation of MAPK signaling pathway account for the underlying mechanism. Materials and methods Cell culture and GSP treatment All cells were from American Type Culture Collection. LLC (Mice Lewis lung cancer cells) and A549 cells (human non-small cell lung cancer cells) were cultured in DMEM medium and BEAS-2B (Human bronchial epithelial cells) and MLE-12 cells (mice lung epithelial cells) were cultured in RPMI 1640 medium. Both the DMEM and RPMI 1640 medium contained 10% fetal calf serum. Cell incubator kept at 37°C with 5% CO2 and 95% humidity. Grape seed proanthocyanidins (GSP) was obtained from Tianjin Peak Natural Products Research and Development co. LTD. (Tianjin, China). One hour before ionizing radiation, cells were pretreated with or without GSP-containing PBS. Twenty-four hours after treatment, cells were transferred to normal DMEM of RPMI 1640 medium. CCK-8 assay, colony formation assay and flow cytometric analysis Cell viability was detected by CCK-8 assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). Cell proliferation was detected by Colony formation assay as previous research [12]. Cell apoptosis was detected by flow cytometric analysis using an Apoptosis Detection Kit (Invitrogen, Carlsbad, California, USA). Intracellular ROS measurement The anti-oxidant (NAC) was used as a positive control to detect the scavenging effect of GSP on free radicals after irradiation. One hour before irradiation, GSP was given at a concentration of 20ug/ml and NAC at a concentration of 10mmol/L. Reactive Oxygen Species Fluorogenic Probe (Cat. No S0033; Beyotime; China) was used for measurement of intracellular ROS of A549 and BEAS-2B cells. Mice and GSP treatments C57BL/6 6-week-old male mice were purchased from Shanghai Ling Chang biological technology co., LTD. All the experiments associated with mice were approved by the Laboratory Animal Center of Naval Medical University, Shanghai. Mice were used and randomly divided into six groups: two groups without lung cancer and other four groups with lung cancer. We injected 1× 106 Lewis lung carcinoma cells (LLC, ATCC) mixed with Matrigel (Matrigel: Medium, a ratio of 1:1, 25μl total) into the left upper lobe of the mice to establish the lung cancer model. The lung cancer mice were divided into four groups, including a non-irradiated group, a GSP treatment group, an irradiation group and an irradiation with GSP treatment group. The lung cancer-free mice were divided into two groups, including a sham operation group and a simple Matrigel injected group. GSP (2mg/ml) was delivered through drinking after LLC cells injected. Lung extraction and pathological staining The left upper lobe lungs were resected and weighed at different times. The left upper lobe lungs, with or without cancers, were then used for pathological analysis and western blot analysis. HE staining and immunofluorescence staining was performed as previously described [45]. Anti-P53 (1:500) and anti-Ki67 (1:500) antibodies were from Cell Signaling Tech, China. After the upper lobe of the left lung of the mouse was embedded in paraffin, it was sectioned and stained every 0.5 mm with a microtome, and the largest cross section of the tumor was taken as the experimental result. The cross-sectional area of the tumor on the 4th, 7th, 11th, 14th, 15th, 16th and 18th day after irradiation was compared with the cross-sectional area of the tumor on the 1st day after irradiation in each group, and the value obtained was used to compare the groups. In the same way, the weight changes of the upper left lung lobe between the groups were compared. ELISA assay ELISA kits (Westang Tech., Shanghai, China) were used to detect the serum levels of IL-6 and IFN-γ. Western blot analysis Proteins from cells and tissues were extracted by ProtecJETTM Mammalian Cell Lysis Reagent (Fermentas, Vilnius, Baltic, Lithuania). MAPK related antibodies (1:1000) were provided by Abcam Corporation. Other antibodies including the secondary antibody (1:1000) were provided by Cell Signaling Technology Corporation. Irradiation LLC, A549, MLE-12 and BEAS-2B cells were exposed to 60Co in the radiation center (Naval Medical University, Shanghai) with a dose of 8Gy at a dose rate of 1Gy/min. Local chest of all radiated mice were exposed to 60Co with a dose of 25Gy at a dose rate of 1 Gy/min. Statistical analysis Data were expressed as mean ± SD of three independent experiments and calculated using one-way ANOVA (Prism version 6.0 software). Student-Newman-Keuls post-hoc test was used to determine variance between groups. The difference between the groups was considered statistically significant when P < 0.05. Results Grape seed proanthocyanidins (GSP) se