The anti-cancer property of proteins extracted from Gynura procumbens (Lour.) Merr

Abstract

Gynura procumbens (Lour.) Merr. belongs to the Asteraceae Family. The plant is a well-known traditional herb in South East Asia and it is widely used to treat inflammation, kidney discomfort, high cholesterol level, diabetic, cancer and high blood pressure. Our earlier study showed the presence of valuable plant defense proteins, such as peroxidase, thaumatin-like proteins and miraculin in the leaf of G. procumbens. However, the effects of these defense proteins on cancers have never been determined previously. In the present study, we investigated the bioactivity of gel filtration fractionated proteins of G. procumbens leaf extract. The active protein fraction, SN-F11/12, was found to inhibit the growth of a breast cancer cell line, MDA-MB-231, at an EC50 value of 3.8 µg/mL. The mRNA expressions of proliferation markers, Ki67 and PCNA, were reduced significantly in the MDA-MB-23 cells treated with SN-F11/12. The expression of invasion marker, CCL2, was also found reduced in the treated MDA-MB-231 cells. All these findings highlight the anti-cancer property of SN-F11/12, therefore, the proteins in this fraction can be a potential chemotherapeutic agent for breast cancer treatment.

Figure 1. The chromatogram of crude protein extracted in the leaf of G. procumbens.

The gel filtration separation was performed using HiPrep 16/60 Sephacryl S-100 HR column. The protein fractions were eluted with 50 mM sodium phosphate, pH 7.2, and flow rate of 0.5 mL/min. The active protein fractions 11 and 12 were eluted out within 50 mL to 60 mL of elution buffer.

Figure 2. The SDS-PAGE analysis of protein fractions collected from gel filtration separation using HiPrep 16/60 Sephacryl S-100 HR column.

Our preliminary analysis found that fractions 11 and 12 possessed anti-proliferation activity on MDA-MB-231. Both fractions 11 and 12 showed identical protein profiles.

Figure 3. A. The dose- and time-dependent effects of SN-F11/12 on MDA-MB-231 using the LDH Cytotoxicity assay.

Each curve of % LDH release was plotted against respective treatment time point. B. The EC50 value was plotted against respective treatment time point for the determination of constant EC50 value. The constant EC50 value of SN-F11/12 on MDA-MB-231 was 3.8 µg/mL.

Figure 4. The expressions of proliferation markers on the SN-F11/12-treated MDA-MB-231 cells.

The figures show normalised mRNA expression of (A) Ki67 and (B) PCNA on the treated MDA-MB-231 cells. The target gene mRNA expression was normalised to the mRNA expression of beta-actin. Each data represents mean ± SE from three independent experiments. Statistical analysis was determined using the Student’s t test with *p<0.05 as statistical significance, compared to the non-treated cells at each time point.

Figure 5. The expressions of invasion markers on the SN-F11/12-treated MDA-MB-231 cells.

The figures show normalised mRNA expression of (A) CCL2 and (B) IL6 on the treated MDA-MB-231 cells. The target gene mRNA expression was normalised to the mRNA expression of beta-actin. Each data represents mean ± SE from three independent experiments. Statistical analysis was determined using the Student’s t test with *p<0.05 as statistical significance, compared to the non-treated cells at each time point.

Figure 6. The protein profile analysis of SN-F11/12 by SDS-PAGE.

The identified protein bands were numbered as indicated on the right. The analysis identified 15 protein bands on the gel.