Scutellarin Enhances Antitumor Effects and Attenuates the Toxicity of Bleomycin in H22 Ascites Tumor-Bearing Mice

Abstract

Bleomycin (BLM) is a broad spectrum anti-tumor drug and inducing pulmonary fibrosis. As an anti-tumor drug without immunosuppression, it is urgent to find a drug that reduces the side effects of BLM. Scutellarin (SCU), a flavone extracted from Erigeron breviscapus (Vant.) Hand-Mazz, has anti-inflammatory activity and ability to inhibit tumor cell growth, migration, and invasion. However, the combined role of SCU and BLM treatment in tumor is unclear. This study aimed to investigate the possible effect and related mechanisms of BLM combined with SCU in the treatment of tumor through in vivo and in vitro experiments. In vivo experiments showed that BLM combined with SCU in the treatment of mice bearing H22 ascites tumor prolonged the survival time, alleviated BLM-induced pulmonary fibrosis, reduced the production of TNF-α; IL-6, and the levels of MDA and MPO. BLM combined with SCU increased the apoptotic rate of H22 ascites cells and the levels of cleaved-caspases-3 and -8. Furthermore, BLM combined with SCU increased the protein expression of p53 and gene expression of miR-29b, and decreased the expression of TGF-β1. In vitro experiment results showed that BLM combined with SCU inhibited the viability of H22 cells and MRC-5 cells, promoted H22 cell apoptosis, up-regulated the protein expression of p53 and down-regulated the protein expression of α-SMA and collagen-I in MRC-5 cells. These experimental results suggested that SCU could enhance the anti-tumor effect of BLM and reduce BLM-induced pulmonary fibrosis, indicating SCU as a potential adjuvant for BLM in the future.

Keywords: anti-tumor; bleomycin; combined administration; pulmonary fibrosis; scutellarin.

FIGURE 1

Effects of SCU, BLM, and their combination on tumor-bearing mice. (A) The survival curves of mice. The survival rate was followed up to 22 days after inoculation. (B) The weight of mouse. (C) The abnormal diameter of mouse. (D) The ascites volume of mouse. Each point represents the mean ± SD. (n = 8). ^#p < 0.05 compared with model group; ^##p < 0.01 compared with model group; ^∗p < 0.05 compared with BLM group; a < 0.05 compared with control group.

FIGURE 2

Effects of BLM, SCU, and their combination on the H22 cells. (A) The inhibitory rate of BLM on H22 cells after 24 and 48 h. (B) The inhibitory rate of SCU on H22 cells after 24 and 48 h. (C) The inhibitory rate of BLM combined with SCU on H22 cells after 24 and 48 h. The inhibitory rate = 1 – (OD _Experimental – OD _Blank/OD _Control – OD _Blank × 100%). (D) The combination index (CI) values were determined. CI vs. fraction affected (Fa) plots were analyzed with CompuSyn 2.0 software. Data are expressed as the mean ± SD. (n = 5). a < 0.05 compared with control group.

FIGURE 3

Effects of BLM, SCU, and their combination induce apoptosis of H22 ascites cells and the H22 cells cultured in vitro. The apoptosis rate of (A) H22 ascites cell and (B) the H22 cells cultured in vitro were analyzed by Flow cytometry. (C) H22 ascites cell and (D) in vitro H22 cell apoptotic rates were calculated. The apoptotic cells in the Q2 region are late apoptotic cells stained with FITC+/PI+ and the apoptotic cells in the Q4 region are early apoptotic cells stained with FITC+/PI–. The total apoptotic rate of cells was the sum of the apoptotic rates of Q2 and Q4. Data are expressed as the mean ± SD. (n = 4). ^#p < 0.05 compared with model group; ^##p < 0.01 compared with model group; ^∗p < 0.05 compared with BLM group.

FIGURE 4

SCU enhanced the anti-tumor effect of BLM by modulating the activities of cleaved-caspase-3 and cleaved-caspase-8 in tumor-bearing mice. The activities of cleaved-caspase-3 (A) and cleaved-caspase-8 (B) were measured. Data are presented as the mean ± SD of the changes compared to the model group. (n = 8). ^#p < 0.05 compared with model group; ^∗p < 0.05 compared with BLM group.

FIGURE 5

Effects of SCU attenuated BLM-induced lung fibrosis. Lung tissue sections were stained with hematoxylin–eosin (HE) staining (A) for pathological observation (×200), with Masson (B) for collagen deposition (×200). (C) The lung injury scores; the slides were histopathological evaluated using a semi quantitative scoring method. (a) Interstitial inflammation (score: 0–4), (b) inflammatory cell infiltration (score: 0–4), (c) congestion (score: 0–4), and (d) edema (score: 0–4). The total lung injury score was calculated by adding up the individual scores of each category. Scale bar indicates 50 μm. Data are shown as the mean ± SD. (n = 4). ^##p < 0.01 compared with model group; ^∗p < 0.05 compared with BLM group.

FIGURE 6

Effects of BLM, SCU, and their combination on the viability of MRC-5 cells. (A) The viability of MRC-5 cells after 24 and 48 h treatment of BLM alone. (B) The viability of MRC-5 cells after 24 and 48 h treatment of SCU alone. (C) The viability of MRC-5 cells after 24 and 48 h treatment of BLM combined with SCU. The data of the control group were pegged as 0.0%, whereas other data were calculated relative to it. Data are shown as the mean ± SD. (n = 5). a < 0.05 compared with control group.

FIGURE 7

Effects of SCU, BLM, and their combination on cytokine production in lung tissues of H22 tumor-bearing mice. (A) The levels of TNF-α in lung tissues. (B) The levels of IL-6 in lung tissues. The data are shown as the mean ± SD. (n = 8). ^#p < 0.05 compared with model group; ^∗p < 0.05 compared with BLM group.

FIGURE 8

Effects of SCU, BLM, and their combination on the MPO (A), MDA (B) levels in lung tissue of H22 tumor-bearing mice. Data are shown as the mean ± SD. (n = 8). ^#p < 0.05 compared with model group; ^∗p < 0.05 compared with BLM group.

FIGURE 9

Effects of BLM, SCU, and their combination on the expression of α-SMA. (A, ×200) Immunofluorescence staining was performed to measure α-SMA protein expression (red) in MRC-5 cells. (B, ×200) The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Representative images of each group are shown. Scale bar indicates 200 μm.

FIGURE 10

Effects of BLM, SCU, and their combination on the expression p53, TGF-β1 collagen-I and α-SMA. The representative images about protein expressions of p53, TGF-β1, collagen-I, α-SMA, and β-actin were measured by Western blot. (A) The expression of p53 in ascites. (B) The expression of TGF-β1 in lung tissues. (C) The expression of p53 and TGF-β1 in H22 cells. (D) The expression of p53, TGF-β1, collagen-I, and α-SMA in MRC-5 cells. Data are shown as the mean ± SD. (n = 3). ^#p < 0.05 compared with model group; ^∗p < 0.05 compared with BLM group; a < 0.05 compared with control group.

FIGURE 11

Effects of BLM, SCU, and BLM + SCU on the miR-29b levels in the lung tissues (A) and ascetic cells (B). The miR-29b levels was detected by real-time quantitative PCR. Data are shown as the mean ± SD. (n = 4). ^#p < 0.05 compared with model group; ^∗p < 0.05 compared with BLM group.