Sanghuangporus sanghuang extract inhibits the proliferation and invasion of lung cancer cells in vitro and in vivo

Abstract

Background/objectives: Sanghuangporus sanghuang (SS) has various medicinal effects, including anti-inflammation and anticancer activities. Despite the extensive research on SS, its molecular mechanisms of action on lung cancer are unclear. This study examined the impact of an SS alcohol extract (SAE) on lung cancer using in vitro and in vivo models.

Materials/methods: Different concentrations of SAE were used to culture lung cancer cells (A549 and H1650). A cell counting kit-8 assay was used to detect the survival ability of A549 and H1650 cells. A scratch assay and transwell cell invasion assay were used to detect the migration rate and invasive ability of SAE. Western blot analysis was used to detect the expression of B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax), cyclin D1, cyclin-dependent kinases 4 (CDK4), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3). Lung cancer xenograft mice were used to detect the inhibiting ability of SAE in vivo. Hematoxylin and eosin staining and immunohistochemistry were used to detect the effect of SAE on the structural changes to the tumor and the expression of Bcl-2, Bax, cyclin D1, CDK4, STAT3, and p-STAT3 in lung cancer xenograft mice.

Results: SAE could inhibit lung cancer proliferation significantly in vitro and in vivo without cytotoxicity. SAE suppressed the viability, migration, and invasion of lung cancer cells in a dose and time-dependent manner. The SAE treatment significantly decreased the proapoptotic Bcl-2/Bax ratio and the expression of pro-proliferative proteins Cyclin D1 and CDK4 in vitro and in vivo. Furthermore, SAE also inhibited STAT3 expression.

Conclusions: SAE reduced the cell viability and suppressed cell migration and invasion in human lung cancer cells. Moreover, SAE also exhibited anti-proliferation effects in vivo. Therefore, SAE may have benefits in cancer therapy.

Keywords: Sanghuangporus sanghuang; cell proliferation; neoplasm invasion; nonsmall cell lung cancer; plant extracts.

Fig. 1. SAE significantly suppress the proliferation and DNA synthesis of lung cancer cells. (A) The morphology of A549 and H1650 cells treated with SAE at the concentrations of 0, 3, 6 and 9 mg/mL for 48 h were observed. Representative images are presented (200×). (B) The viability of A549 and H1650 cells were inhibited by SAE in a dose and time-dependent manner. (C) EdU staining of lung cancer cells treated with various concentrations of SAE for 40 h. (D) Relative EdU-positive cell ratios for lung cancer cells treated with indicated concentration of SAE.

SAE, Sanghuangporus sanghuang alcohol extract; EdU, 5-ethynyl-2'-deoxyuridine. ^*P < 0.05, ^**P < 0.01 vs. untreated control.

Fig. 2. SAE inhibit the migration of A549 and H1650 cells with the increased dose. (A) Effect of SAE on migration of lung cancer cell were assessed by scratch assay. The wound area were measured at 24 after treatment. Scale bar: 200 µm. (B) The data were calculated from 3 independent experiments and the results show the distance of the migration.

SAE, Sanghuangporus sanghuang alcohol extract. ^*P < 0.05, ^**P < 0.01 vs. untreated control.

Fig. 3. SAE inhibit the invasion of A549 and H1650 cells in transwell assay. (A) Effect of SAE on invasion of lung cancer cell were assessed by transwell assay. (B) Transwell results showed that SAE inhibited cell invasion of A549 and H1650 in a dose-dependent manner. Magnification 200×.

SAE, Sanghuangporus sanghuang alcohol extract. ^**P < 0.01 vs. untreated control.

Fig. 4. SAE affect the protein expression of STAT3, Bcl-2, Bax, cyclin D1, and CDK4 in lung cancer cells. (A, B) The protein expression of STAT3 and p-STAT3 in A549 and H1650 were determined by western blotting. GAPDH was used as an internal control. (C, D) The protein levels of Bcl-2, Bax, cyclin D1 and CDK4 in A549 and H1650 treated by SAE were determined by western blotting. GAPDH was used as an internal control.

SAE, Sanghuangporus sanghuang alcohol extract; STAT3, signal transducer and activator of transcription 3; Bcl-2, B-cell lymphoma-2; Bax, Bcl2-associated X; CDK4, cyclin-dependent kinases 4; p-STAT3, phosphorylated signal transducer and activator of transcription 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^*P < 0.05, ^**P < 0.01 vs. untreated control.

Fig. 5. SAE suppress the growth of subcutaneous tumor in mice. (A) The transplanted subcutaneous tumors. (B) Tumor weight of 2 groups. (C) Tumor volume changes in mice of 2 groups. (D) Body weight changes in mice of 2 groups.

SAE, Sanghuangporus sanghuang alcohol extract. ^*P < 0.05, vs. control.

Fig. 6. Effect of SAE on the structural changes of tumor in lung cancer xenograft mice. Histopathological diversity were detected under a light microscope. Images are representative photographs taken at a magnification of 200×.

SAE, Sanghuangporus sanghuang alcohol extract.

Fig. 7. Effect of SAE on the expression of Bcl-2, Bax, cyclin D1, CDK4, STAT3, and p-STAT3 in lung cancer xenograft mice. (A) SAE down-regulation of phosphorylation STAT3 in lung cancer xenograft mice. Total STAT3 and p-STAT3 were analyzed via immunohistochemical staining. The photographs are representative images taken at a magnification of 200×. (B) The protein expression levels of Bcl-2, Bax, cyclin D1, and CDK4 in tumor tissues were analyzed via immunohistochemical staining. The photographs are representative images taken at a magnification of 200×.

SAE, Sanghuangporus sanghuang alcohol extract; Bcl-2, B-cell lymphoma-2; Bax, Bcl2-associated X; CDK4, cyclin-dependent kinases 4; STAT3, signal transducer and activator of transcription 3; p-STAT3, phosphorylated signal transducer and activator of transcription 3. ^*P < 0.05, ^**P < 0.01 vs. control.