Asparagus racemosus and Geodorum densiflorum lectins induce apoptosis in cancer cells by altering proteins and genes expression

https://doi.org/10.1016/j.ijbiomac.2021.09.101Get rights and content

Highlights

  • ARL (Mr. 14 kDa), a thermostable glycoprotein was purified from A. racemosus root.

  • ARL & GDL inhibited HCT-116 cell growth and arrested G2/M cell cycle phase.

  • Apoptosis was confirmed by Hoechst-33342, caspase inhibitors and ROS for ARL.

  • The lectins altered several genes involved in multiple apoptotic pathways.

  • ARL inhibited EAC cells growth in vivo in mice and arrested S cell cycle phase.

Abstract

A lectin (designated as ARL) was purified first time from the Asparagus racemosus root with the molecular weight of 14.0 kDa containing about 4.8% carbohydrate. ARL showed hemagglutination activity in both mice and human erythrocytes that were inhibited by three complex sugars among the 26 sugars tested. ARL was thermostable that mostly preserved activity at its optimum pH 8.0. Around 48% and 52.5% human colorectal cancer (HCT-116) cells growth was inhibited by 160 μg/ml of ARL and 256 μg/ml of previously purified Geodorum densiflorum rhizome lectin (GDL). Induction of apoptosis in HCT-116 cells was confirmed by Hoechst 33342 staining, caspase inhibitors, but ROS generation was only observed for ARL. The expression level of BAX and p53 genes increased with a decrease of PARP gene expression for both lectins. The expression of FAS and FADD were increased with the decrease of WNT after treatment with GDL. ARL inhibited 68% and 26% of Ehrlich ascites carcinoma cell growth in vivo in mice after treating with 3.0 and 1.5 mg/kg/day doses for five consecutive days. ARL increased the expression level of NFκB and arrested S cell cycle phase in EAC cells, in contrast, G2/M phase was arrested by ARL and GDL in HCT-116.

Introduction

Lectins are a group of proteins that bind specifically to certain sugars and cause agglutination of particular cell types, mainly lack of enzymatic activity and occur ubiquitously in nature. Several lectins have been isolated from different sources such as plants, fungi, viruses, bacteria, animals and algae [1]. In plants, lectin is identified in roots, bulbs, tubers, bark, rhizomes, stems, seeds, fruits, and leaves. Plant lectins were classified in different ways and more recently subdivided into twelve families [2]. Although thousands of lectins were purified, the anticancer mechanism was studied by using few lectins.

Asparagus racemosus Wild (Family: Asparagaceae) commonly known as ‘Satamuli’ in Bangladesh and ‘Shatavari’ in India. The root powder was used for the development of bread and biscuit in India and the addition of Satamuli extract in animal feed causes an increase in milk production [3]. Satamuli is one of the most beneficial medicinal plants, regarded as a “Queen of herbs” in the Ayurvedic healthcare system. This plant provides a broad range of medical applications against several diseases, including cancer treatment. Several bioactive compounds isolated from the root those shown anticancer activity e.g. Sar sapogenin and Diosgenin-derived steroidal constituents, induced death of colon carcinoma cells [4] and shatavarin IV showed anticancer activity against different cancer cell lines [5]. Besides those, the root is used for snakebite treatment in southwestern parts of Bangladesh [6]. Recently, several plant lectins were purified with anticancer properties at our laboratory [7], [8], [9], [10], [11]. Geodorum densiflorum rhizome lectin (GDL) is one of them that has a molecular weight of 12.0 kDa, agglutinated mice & human red blood cells and inhibited by 4-nitrophenyl-β-D-glucopyranoside. The lectin inhibited Ehrlich ascites carcinoma (EAC) cells growth by the induction of apoptosis and unable to inhibit HeLa cells growth [10]. After treatment of EAC cells with the lectin BAX, BAK and p53 were increased consequently with the decrease of BCL-X and NFκB genes [10].

In the present study, for the first time, we are reporting the purification of a lectin from A. resmosas root and evaluating the anticancer mechanism of this lectin and recently purified GDL against the human colon cancer cell line (HCT-116) in vitro. Beside the in vitro study, antitumor properties of the purified lectin against EAC cells in vivo in mice were also stated in this investigation.

Section snippets

Chemicals and reagents

SYBR green RT master mix from Applied Biosystem, marker protein and 100 bp DNA ladder from Takara Bio Inc., Japan, QA-cellulose from WAKO, Japan, DMEM medium, trypsin-EDTA, fetal bovine serum from Gibco. Streptomycin and neomycin from Amresco. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNase A from Carl Roth, Germany, sephadex G-50, Hoechst 33342, propidium iodide from Sigma, primer from Beijing TsingKe Biological Technology, China.

Preparation of crude protein extract and purification of lectins

A. racemosus roots were collected

Purification of A. racemosus root lectin

For the purification of lectin, A. racemosus roots were homogenized and the homogenate was centrifuged twice and finally loaded on a QA-cellulose column (Fig. 1A). The protein fractions were eluted with the linear gradient of NaCl. The unbound fraction, bound fraction F-1 and F-2 did not show any hemagglutination activity while bound fraction F-3 agglutinated mice RBC strongly. This fraction was then concentrated and loaded on a G-50 column (Fig. 1B) and the eluted fraction showed strong

Discussion

By using ion exchange and gel filtration chromatography, 14.0 kDa lectin was purified first time from the A. racemosus root. The lectin agglutinated human (A and O) and mouse erythrocyte and did not agglutinate chicken erythrocytes. The hemagglutination activity was inhibited only in the presence of 25 mM of 4-nitrophenyl-α-D-galactopyranoside, methyl-β-D-glucopyranoside and 4-nitrophenyl-β-D-galactopyranoside sugars. In the early experiment, it was reported that the hemagglutination activity

Author statement

Conceptualization; S. R. Kabir.

Data curation; Formal analysis; Funding acquisition; S. R. Kabir.

Investigation; S. R. Kabir, J. Islam, M. S. Ahamed, M. T. Alam,

Methodology; Project administration; S. R. Kabir.

Resources; S. R. Kabir, M. T. Alam.

Software; Supervision; S. R. Kabir.

Validation; Visualization; Roles/Writing – original draft: S. R. Kabir.

Writing – review & editing. S. R. Kabir.

Declaration of competing interest

Authors declare no conflict of interest.

Acknowledgment

This research work was funded by the Faculty of Science, University of Rajshahi, Bangladesh (Grant ID No. 1225/5/52/RU/Science-25/2019-2020) and the Ministry of Science and Technology, People's Republic of Bangladesh (Grant ID No. 39.00.0000.009.06.024.19/MEDI's-505). HCT-116 cell line was kindly given by Dr. Zhao XuDong, KIZ, CAS, China.

Experimental animals and ethical clearance

Swiss albino mice were produced at our university and the in vivo experiment was approved by the Institutional Animal,Medical Ethics, Bio-safety and Bio-security Committee (IAMEBBC) for Experimentations on Animals, Human, Microbes and Living Natural Sources (102(6)/320-IAMEBBC/IBSc) Institute of Biological Sciences (IBSc), University of Rajshahi, Bangladesh.

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