Grape Seed Proanthocyanidins play the roles of radioprotection on Normal Lung and radiosensitization on Lung Cancer via differential regulation of the MAPK Signaling Pathway

Abstract

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 these 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.

Keywords: MAPK; Proanthocyanidins; lung cancer; radioprotection; radiosensitization.

Figure 1

Grape seed proanthocyanidins (GSP) sensitizes lung cancer cells LLC and A549 to ionizing radiation (IR) while alleviates radiation damage to normal lung cells MLE-12 and BEAS-2B. (A) Cytotoxicity of different concentrations of GSP on lung cancer cells and normal lung cells. In CCK8 assay, no toxicity is found in both cancer cells (LLC and A549) and normal cells (MLE-12 and BEAS-2B) treated with GSP at the concentration less than 20 ug/ml. (B) Cytotoxicity of GSP on lung cancer cells and normal lung cells at different administration times. At the concentration of 20 ug/ml, no toxicity is found until 24 hours after administration. (C) In flow cytometry, compared with IR group, GSP treatment significantly decreases apoptosis in MLE-12 cells and BEAS-2B cells, while increases apoptosis in A549 cells. (D) The colony formation assay shows that GSP treatment significantly improves the viability of MLE-12 cells and BEAS-2B cells after irradiation, and reduces the viability of A549 after irradiation. LLC, A549, MLE-12 and BEAS-2B cells were exposed to ⁶⁰Co with a dose of 8Gy at a dose rate of 1Gy/min. Data were presented as mean ± SD (n =3). *P<0.05, **P <0.01, ***P < 0.001, **** P < 0.0001.

Figure 2

Suppressive and radiosensitive potential of GSP on tumor in vivo. (A) Representative images of HE stained sections of lung tissues with tumor. The largest cross section of the tumor is taken as the experimental result. IR, GSP, and IR+GSP reduce tumor size in tumor-bearing mice. (B) The cross-sectional area of the tumor on the 4^th, 7^th, 11^th, 14^th, 15^th, 16^th and 18^th day after irradiation is compared with the cross-sectional area of the tumor on the 1st day after irradiation in each group, and the value obtained is used to compare the groups. The results show that IR + GSP has the most obvious inhibitory effect on tumor. (C) In the same way, the weight changes of the upper left lung lobe between the groups are compared. IR, GSP, and IR+GSP reduce the weight of the lung lobe where the tumor is located. (D) IR, GSP, and IR+GSP improve the adverse effect of lung cancer on body weight, most significant in IR+GSP group. (E) IR, GSP, and IR+GSP improve the survival rate of tumor-bearing mice, most significant in IR+GSP group. Local chest of all radiated mice were exposed to ⁶⁰Co with a dose of 25Gy at a dose rate of 1 Gy/min. GSP (2 mg/ml) was delivered through drinking after LLC cells injected. Data were presented as mean ± SD (n=3). *P<0.05, **P <0.01, ***P < 0.001, **** P < 0.0001.

Figure 3

GSP attenuates inflammation of normal lung tissues and promotes apoptosis of tumor after irradiation. (A) Representative images of HE stained sections of lung tissues with tumor. GSP treatment markedly attenuates the inflammation in normal lung tissue surrounding the tumor. After local chest ionizing radiation, a large number of inflammatory cells accumulate in normal lung tissue, and the extracellular matrix is excessively deposited. The normal structure of the alveoli is destroyed, with thickened alveolar wall. However, GSP treatment significantly alleviates these changes. (B) Representative images and a quantification of Ki67 and P53 expressions in normal lung tissues by immunohistochemical staining. Ionizing radiation increases the expression of P53 in normal lung tissues, while GSP reduces the increment of P53 expression. GSP has no effect on the expression of Ki67 in normal lung tissues. (C) Representative images and a quantification of Ki67 and P53 expressions in tumor by immunohistochemical staining. Ionizing radiation increases the expression of P53 in lung cancer tissues, and GSP further increases the expression of P53. In addition, GSP down-regulates the expression of Ki67 in lung cancer tissues. Data were presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001, **** P < 0.0001.

Figure 4

GSP regulates secretion of cytokines IL-6 and IFN-γ after ionizing radiation. Compared with the normal mice, tumor-bearing mice have significantly higher IL-6 levels (A) and lower IFN-γ levels (B). At 12 hours after irradiation, the serum IL-6 level in the IR+GSP mice is significantly lower than that of IR mice (C), while IFN-γ levels are significantly increased (D). Data were presented as mean ± SD (n=3). *P<0.05, **P <0.01, ***P < 0.001, **** P < 0.0001.

Figure 5

GSP reduces ROS levels in both cancer cells A549 and normal cells BEAS-2B. 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 (8Gy, 1Gy/min), GSP were given at a concentration of 20 ug/ml and NAC at a concentration of 10 mmol/L. The intracellular ROS level of A549 and BEAS-2B cells were detected after irradiation by Reactive Oxygen Species Fluorogenic Probe and were observed with confocal microscopy. GSP treatment significantly reduces ROS levels in both cancer cells A549 (A) and normal cells BEAS-2B (B). Data were presented as mean ± SD (n=3). *P<0.05, **P <0.01, ***P < 0.001, **** P < 0.0001.

Figure 6

GSP differentially regulates MAPK signaling pathways in lung cancer cells and normal lung cells respectively. (A) In lung cancer cells A549, GSP significantly increases the expression of p-JNK, p-P38 protein before and after irradiation, and the value of Bax/Bcl-2 after irradiation, and has less effect on p-ERK protein. (B) In normal lung epithelial cells BEAS-2B, GSP reduces the expression of p-JNK, p-P38, and p-ERK proteins of MAPK family after irradiation, and reduces the expression of Bax and value of Bax/ Bcl-2. The raw density of Western blot figures was quantified. Data were presented as mean ± SD (n=3). *P<0.05. **P <0.01, ***P < 0.001, **** P < 0.0001.

Figure 7

GSP differentially regulates MAPK signaling pathways in lung cancer tissues and normal lung tissues. (A) In tumor-bearing mice, ionizing radiation increases expression of p-JNK, p-P38, and p-ERK proteins of MAPK family in normal lung tissues, while GSP reduces the increments. In lung cancer tissues, GSP activates the expression of p-JNK protein. (B) In normal mice, the expression of p-JNK, p-P38, and p-ERK proteins of MAPK family in the lung tissues is significantly reduced when GSP is given in diet, but the protein expression gradually increases over the feeding time. The raw density of Western blot figures was quantified. Data were presented as mean ± SD (n=3). *P<0.05. **P <0.01, ***P < 0.001, **** P < 0.0001.