Notoginseng enhances anti-cancer effect of 5-fluorouracil on human colorectal cancer cells

Abstract

Purpose: Panax notoginseng is a commonly used Chinese herb. Although a few studies have found that notoginseng shows anti-tumor effects, the effect of this herb on colorectal cancer cells has not been investigated. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of colorectal cancer that interferes with the growth of cancer cells. However, this compound has serious side effects at high doses. In this study, using HCT-116 human colorectal cancer cell line, we investigated the possible synergistic anti-cancer effects between notoginseng flower extract (NGF) and 5-FU on colon cancer cells.

Methods: The anti-proliferation activity of these modes of treatment was evaluated by MTS cell proliferation assay. Apoptotic effects were analyzed by using Hoechst 33258 staining and Annexin-V/PI staining assays. The anti-proliferation effects of four major single compounds from NGF, ginsenosides Rb1, Rb3, Rc and Rg3 were also analyzed.

Results: Both 5-FU and NGF inhibited proliferation of HCT-116 cells. With increasing doses of 5-FU, the anti-proliferation effect was slowly increased. The combined usage of 5-FU 5 microM and NGF 0.25 mg/ml, significantly increased the anti-proliferation effect (59.4 +/- 3.3%) compared with using the two medicines separately (5-FU 5 microM, 31.1 +/- 0.4%; NGF 0.25 mg/ml, 25.3 +/- 3.6%). Apoptotic analysis showed that at this concentration, 5-FU did not exert an apoptotic effect, while apoptotic cells induced by NGF were observed, suggesting that the anti-proliferation target(s) of NGF may be different from that of 5-FU, which is known to inhibit thymidilate synthase.

Conclusions: This study demonstrates that NGF can enhance the anti-proliferation effect of 5-FU on HCT-116 human colorectal cancer cells and may decrease the dosage of 5-FU needed for colorectal cancer treatment.

Fig. 1

Chemical structures and HPLC analysis of ginsenosides in notoginseng flower extract. The structures of determined ginsenosides are shown in a. HPLC chromatogram of notoginseng extract recorded at 202 nm is shown in b. HPLC conditions are described in Materials and methods. Saponin content of selected ginsenosides is shown in c. The results showed that the main constituents of notoginseng flower extract are ginsenoside Rb3 and Rc, while notoginsenoside R1 has limited content in this extract

Fig. 2

Notoginseng flower extract and 5-fluorouracil on the cell proliferation of HCT-116 human colorectal cancer cells (a, b) and IEC-18 rat intestinal epithelial cells (c) determined by MTS method. Time and dose dependent effect of NGF on HCT-116 cells is shown in a. After treatment with NGF (0.1, 0.25 mg/ml), cell proliferation was initially measured at 12, 24, 48, 72 and 96 h. After 48 h treatment, the proliferation was stable. The effect of 5-FU on HCT-116 cells is shown in b. Cell proliferation in the presence of 5-FU (2.5–100 μM) was measured after 48 h treatment. High dose of 5-FU (100 μM) could not increase its anti-proliferation effect on HCT-116 cells. The effect of NGF on IEC-18 cells (treatment for 48 h) is shown in c (mean ± SE, n = 3). 5-FU 5-fluorouracil, NGF notoginseng flower extract

Fig. 3

Effects of 5-fluorouracil combined with notoginseng flower extract on the growth of HCT-116 human colorectal cancer cells. HCT-116 cells were exposed to 5-FU and/or NGF for 48 h and cell proliferation was determined by MTS (a) (mean ± SE, n = 3). *P < 0.05, compared to 5-FU alone. The data showed that NGF 0.25 mg/ml could enhance the anti-proliferation effect of 5-FU 5 μM on HCT-116 cells significantly. The morphological aspects were photographed with a microscope after staining with crystal violet (b). The number of cells following treatment with the combination of 5-FU and NGF decreased remarkably and cell aspects are different compared with the treatment groups of 5-FU or NGF alone. 5-FU 5-fluorouracil, NGF notoginseng flower extract

Fig. 4

Apoptosis assay of HCT-116 human colorectal cancer cells using Hoechst 33258 and Annexin V/PI staining. For the Hoechst 33258 staining assay (a), HCT-116 cells were incubated for 48 h with 5-FU and/or notoginseng flower extract (NGF). Some apoptotic cells were found in the NGF and the 5-FU + NGF groups. For the flow cytometry analysis (b), HCT-116 cells were incubated for 8 h with 5-FU and/or NGF and flow cytometry was performed after staining with Annexin V/PI. Compared with the control (apoptotic cells <1%), the amount of apoptotic cells was not increased in the group of 5-FU 20 μM (apoptotic cells <1%). Much more apoptotic cells were observed in the NGF 1 mg/ml group (39%) and the 5-FU 20 μM combined with NGF 1 mg/ml group (41%), suggesting that the apoptotic effect of the combination group is supplied mainly by NGF. 5-FU 5-fluorouracil, NGF notoginseng flower extract

Fig. 5

Anti-proliferation effects of ginsenosides Rb1, Rb3, Rc and Rg3 on HCT-116 cells assayed by MTS method. HCT-116 cells were exposed to ginsenosides for 48 h and cell proliferation was determined by MTS (mean ± SE, n = 3). Ginsenoside Rb1, Rb3 and Rc do not decrease the cell proliferation at 30–300 μM. Ginsenoside Rg3 inhibits cell growth at 100 and 300 μM significantly