The mushroom Ganoderma lucidum suppresses breast-to-lung cancer metastasis through the inhibition of pro-invasive genes

Abstract

Breast cancer metastasis is one of the major reasons for the high morbidity and mortality of breast cancer patients. In spite of surgical interventions, chemotherapy, radiation therapy and targeted therapy, some patients are considering alternative therapies with herbal/natural products. In the present study, we evaluated a well-characterized extract from the medicinal mushroom Ganoderma lucidum (GLE) for its affects on tumor growth and breast-to-lung cancer metastasis. MDA-MB-231 human breast cancer cells were implanted into the mammary fat pads of nude mice. GLE (100 mg/kg/every other day) was administered to the mice by an oral gavage for 4 weeks, and tumor size was measured using microcalipers. Lung metastases were evaluated by hematoxylin and eosin (H&E) staining. Gene expression in MDA-MB-231 cells was determined by DNA microarray analysis and confirmed by quantitative PCR. Identified genes were silenced by siRNA, and cell migration was determined in Boyden chambers and by wound-healing assay. Although an oral administration of GLE only slightly suppressed the growth of large tumors, the same treatment significantly inhibited the number of breast-to-lung cancer metastases. GLE also downregulated the expression of genes associated with invasive behavior (HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1) in MDA-MB-231 cells. Gene silencing of HRAS, VIL2, S100A4, I2PP2A and FN1 by siRNA suppressed migration of MDA-MB‑231 cells. Our study suggests that an oral administration of GLE can inhibit breast-to-lung cancer metastases through the downregulation of genes responsible for cell invasiveness. The anti-metastatic benefits of GLE warrant further clinical studies.

Figure 1.

Effect of GLE on tumor growth and breast-to-lung cancer metastases. MDA-MB-231 breast cancer cells were implanted in the mammary fat pads of nude mice and treated with GLE as described in Materials and methods. (A) The size and (B) weight of tumors were measured after 28 days (n=16–18). (C) Representative H&E stained lungs from control and GLE-treated groups; black arrows indicate metastasis. (D) Lung metastases were quantified as described in Materials and methods (P<0.001, n=6).

Figure 2.

Effect of GLE on human tumor metastasis genes. MDA-MB-231 cells were treated with vehicle or GLE for 24 h and the gene expression was determined by Oligo GEArray Human Tumor Metastasis Microarray and confirmed by qRT-PCR as described in Materials and methods. The data are averages from 2 (microarray) and 4 (qRT-PCR) experiments. The fold change is relative to vehicle-treated cells.

Figure 3.

Effect of genetic silencing of GLE downregulated genes on cell migration. MDA-MB-231 cells were untransfected (control) or transfected with scrambled (sc-siRNA) or specific gene siRNA and cell migration and proper gene silencing was evaluated as described in Materials and methods. (A) HRAS, (B) VIL2 (ezrin), (C) S100A4, (D) MCAM, (E) I2PP2A (SET), (F) FN1 (fibronectin). The data are mean ± SD (n=3), P≤0.05 by ANOVA. Western blots show representative experiment.

Figure 4.

Pooled siRNA inhibits cell migration. MDA-MB-231 cells were untransfected (control) or transfected with scrambled (sc-siRNA) or a pool of siRNAs, (pooled-siRNA, containing HRAS-siRNA, VIL2-siRNA, S100A4-siRNA, MCAM-siRNA, I2PP2A-siRNA, and FN1-siRNA). Cell migration was evaluated by (A) the wound healing assay and (B) cell migration assay described in Materials and methods. The data are mean ± SD (n=3), p≤0.05 by ANOVA. (C) Representative western blots from MDA-MB-231 cells transfected with scrambled siRNA (SC) or pooled-siRNA (PO), or treated with GLE (GLE) or control (C).