The research on the mechanism of Tsoong inhibiting for colon cancer

Abstract

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer effects. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Using high throughput sequencing the apoptotic pathway was ranked as one of the most important pathways and the differential expressions of apoptosis-related genes such as Casp3, Casp6 and Apaf1 were identified. The following experiments were qRT-PCR which were used to verify the genes. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti-proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. H&E staining was performed with oxaliplatin set as a positive control. The results showed that Tsoong up-regulated apoptosis-related genes, inhibited tumor cell proliferation, promoted tumor cellapoptosis in a dose-dependent manner and restrained the growth of colon neoplasm. The effects of a high dose of Tsoong on colon cancer cells were similar to those of oxaliplatin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease. Therefore, Tsoong may be a promising Chinese herbal compound for development for use in cancer therapy.

Keywords: Apaf1; Apoptosis; Casp3; Casp6; Colon cancer; Tsoong.

Fig. 1

Experimental flow chart. CCastands for the experimental group gavaged with1/2 original dosage of Loperamide to induce intestinal tumor after the successful establishment of constipation model (n = 56); CCaT stands for Tsoong treatment mice group in CCa (n = 28); The rest mice without Tsoong treatment remained CCa (n = 28).

Fig. 2

Sarcoma morphology in the external and internal wall of the intestine (below images present visible sarcomas under naked eyes in the internal wall of the intestine under stereomicroscope after fixation by 10% neutral formalin solution).

Fig. 3

Intestinal tumors under 40X microscope (H&E staining).

Fig. 4

The significantly enriched KEGG pathways in B vs. CCa. Apoptosis pathway was one of the most significantly enriched KEGG pathways.

Fig. 5

Differentially expression of Casp3, Casp6 and Apaf1 in B group, CCa group and CCaT group were verified with qRT-PCR experiments. They are the most important apoptosis-related genes. The qRT-PCR experiments verified that the Tsoong restored the expression of apoptosis genes.

Fig. 6

Effect of Tsoong on the proliferation and cell cycle of HCT116 and SW480 cancer cells. The cells were treated with Tsoong-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 5 (low), 10 (mid) or 20 (high) g/kg of Tsoong for indicated durations. (A) cell proliferation was detected using a cell counting kit‐8 assay, and (B) cell cycle was detected using flow cytometry. (C) Cell apoptosis was analyzed using flow cytometry. Oxaliplatin was used as a control. P < 0.05 vs. control; P < 0.01 vs. control. Tsoong or cbFeD, Codonopis bulleynana Forest ex diels.

Fig. 7

Effects of Tsoong and oxaliplatin on growth of xenograft tumors. Nude mice were subcutaneously injected in the right flank with 2.0 × 10⁶ SW480 cells in 0.1 ml PBS. Once tumors had formed, tumor volume was measured. The mice were then randomly divided into three groups (n = 5): Normal control group, mice treated with normal saline via gavage; high Tsoong group, mice treated with a high dose (20 g/kg) of Tsoong via gavage; oxaliplatin group, mice with colon cancer treated with oxaliplatin (5 mg/kg) via gavage or injection. After 6 weeks, the dissected tumors were collected, and H&E were performed. (A) Tumor size. (B) tumor volume. (C) H&E staining of xenograft tumors. Magnification, x400. Oxaliplatin was used as a control. P < 0.01 vs. control; P < 0.001 vs. control. Tsoong or cbFeD, Codonopis bulleynana Forestex diels.