Cloning, overexpression, purification, and modeling of a lectin (Rhinocelectin) with antiproliferative activity from Tiger Milk Mushroom, Lignosus rhinocerus

Abstract

A lectin gene from the Tiger Milk Mushroom Lignosus rhinocerus TM02® was successfully cloned and expressed via vector pET28a in Escherichia coli BL21(DE3). The recombinant lectin, Rhinocelectin, with a predicted molecular mass of 22.8 kDa, was overexpressed in water‐soluble form without signal peptide and purified via native affinity chromatography Ni‐NTA agarose. Blast protein analysis indicated the lectin to be homologous to jacalin‐related plant lectin. In its native form, Rhinocelectin exists as a homo‐tetramer predicted with four chains of identical proteins consisting of 11 beta‐sheet structures with only one alpha‐helix structure. The antiproliferative activity of the Rhinocelectin against human cancer cell lines was concentration dependent and selective. The IC50 values against triple negative breast cancer cell lines MDA‐MB‐231 and breast cancer MCF‐7 are 36.52 ± 13.55 μg mL−1 and 53.11 ± 22.30 μg mL−1, respectively. Rhinocelectin is only mildly cytotoxic against the corresponding human nontumorigenic breast cell line 184B5 with IC50 value at 142.19 ± 36.34 μg mL−1. The IC50 against human lung cancer cell line A549 cells is 46.14 ± 7.42 μg mL−1 while against nontumorigenic lung cell line NL20 is 41.33 ± 7.43 μg mL−1. The standard anticancer drug, Doxorubicin exhibited IC50 values mostly below 1 μg mL−1 for the cell lines tested. Flow cytometry analysis showed the treated breast cancer cells were arrested at G0/G1 phase and apoptosis induced. Rhinocelectin agglutinated rat and rabbit erythrocytes at a minimal concentration of 3.125 μg mL−1 and 6.250 μg mL−1, respectively.

Comparative Analysis of the Metabolites and Biological Activity of Cultivated and Wild Lignosus rhinocerotis

“…These biochemicals with pharmacological effects play a preventive and therapeutic role in disease. Polysaccharides are one of the therapeutic compounds of L. rhinocerotis, and numerous studies have shown that PLR is related to antitumor and immunomodulatory effects [26][27][28][29]. Phenolic compounds, as natural antioxidants, was able to scavenge free radicals, and the quantification of total phenolic content and total flavonoid content can be used as indicators to evaluate antioxidant capacity.…”

Mushroom extracts and compounds with suppressive action on breast cancer: evidence from studies using cultured cancer cells, tumor-bearing animals, and clinical trials

“…Cordycepin (3-deoxyadenosine) from C. sinensis expressed cytotoxicity on cultured MDA-MB-231 and MCF-7 human breast cancer cells (Choi et al 2011;Wang et al 2016). Under the influence of cordycepin, the cell viability plummeted, cellular proliferation declined, cellular liberation of lactate dehydrogenase increased and reactive oxygen species generation rose, mitochondrial function was disrupted, and nuclear apoptosis took place in human breast cancer cells.…”

“…Rhinocelectin, 22.8-kDa recombinant homotetrameric lectin with a single alpha-helix and eleven beta-sheets, displayed IC50 values of 53.11, 36.52, and 142.19 μg/ml, respectively, against MCF-7 and MDA-MB-231 triple-negative human breast cancer cells and 184B5 human nontumorigenic breast cells. Rhinocelectin treatment caused the breast cancer cells to stay in G0/G1 phase of the cell cycle and undergo apoptosis (Cheong et al 2019).…”