Transiently Expressed Mistletoe Lectin II in Nicotiana benthamiana Demonstrates Anticancer Activity In Vitro

Abstract

Mistletoe (Viscum album) extracts have been used as alternative and complementary therapeutic preparations in multiple cancers for decades. Mistletoe lectins (ML-I, ML-II, and ML-III) are considered to be the main anticancer components of such preparations. In the present study, ML-II was transiently expressed in Nicotiana benthamiana using the pEAQ-HT expression system. Expression levels of up to 60 mg/kg of the infiltrated plant tissue were obtained, and a three-fold increase was achieved by adding the endoplasmic reticulum (ER) retention signal KDEL to the native ML-II sequence. The native protein containing His-tag and KDEL was purified by immobilized metal affinity chromatography (IMAC) and gel filtration. We found that the recombinant ML-II lectin was glycosylated and retained its carbohydrate-binding activity. In addition, we demonstrated that plant produced ML-II displayed anticancer activity in vitro, inhibiting non-small cell lung cancer H460 and A549 cells with EC50 values of 4 and 3.5 µg/mL, respectively. Annexin V-448A and PI double staining revealed that cell cytotoxicity occurred via apoptosis induction. These results indicate that ML-II transiently expressed in N. benthamiana plants is a promising candidate as an anticancer agent, although further optimization of production and purification methods is required to enable further in vitro testing, as well as in vivo assays.

On Overview Of Bioactive Compounds, Biological And Pharmacological Effects Of Mistletoe (Viscum Album L)

“…In terms of chemical structure, mistletoe contains three lectins that differ in their molecular weight, about 60000Da and depending on the binding of carbohydrates [71,72]. These lectins are coded as follows: ML 1, ML 2 and ML 3; (ML -Mistellectin) [73]. Lectin ML 1, is a glycoprotein that has two polypeptide chains linked together by a disulfide bond [74].…”

Mistletoe lectin inhibits growth of Myc‐amplified small‐cell lung cancer

“…For instance, ML was found to inhibit the growth of two NSCLC cell lines in vitro by inducing apoptosis. 26 In a phase II clinical trial in advanced NSCLC, the mistletoe preparation iscador was studied as a complimentary treatment to chemotherapy. It was not found to have an effect on quality of life or total adverse events but resulted in less frequent chemotherapy dose reductions, severe non‐hematological side effects, and hospitalizations.…”

“…Other studies that examined mistletoe in lung cancer were limited to non‐small‐cell lung cancer (NSLC). For instance, ML was found to inhibit the growth of two NSCLC cell lines in vitro by inducing apoptosis 26 . In a phase II clinical trial in advanced NSCLC, the mistletoe preparation iscador was studied as a complimentary treatment to chemotherapy.…”

Medicinal Plants: Guests and Hosts in the Heterologous Expression of High-Value Products

“…Cancer ML-II lectin 60 mg/kg FW [123] Rituximab 385 mg/kg FW [124] BR55-2 30 mg/kg FW [125] Chikungunya virus CHKV mAb 100 µg/g FW [126] Cholera Cholera toxin B subunit 3.1 g/kg FW [127] Cholera toxin B subunit 0.5-1.5 g/kg FW [128] Dengue virus E60 120 µg/g FW [129] cEDIII 5.2 mg/g FW [130] cEDIII-Co1 4.8 mg/g FW [130] Ebola virus 6D8 1.21 mg/g FW [131] Erythropoetin rhEPO 85 mg/kg FW [132] Fabry disease α-GAL 71 nmol/h/µg TP [133] α-NAGAL 5 nmol/h/µg TP [133] α-NAGALEL 7 nmol/h/µg TP [133] Influenza Hemagglutinin VLPs 50 mg/kg FW [134] rHA0 0.2 g/kg FW [135] Hepatitis B virus (HBV) HBV surface antigen 295 µg/g FW [136] HBV core antigen 0.2-1 mg/g FW [137] Human epidermal growth factor hEGF 15.695 µg/g FW [138] Human immunodeficiency virus HIV Env gp140 5-6 mg/kg FW [139] VRC01 Fab -Avaren 40 mg/kg FW [140] CAP256-VRC26.08 489 mg/kg FW [141] CAP256-VRC26.09 487 mg/kg FW [141] Herpes simplex virus HSV8 1.42 mg/g FW [131] Malaria CCT 2 mg/g FW [142] Rabies E559/62-71-3 mAbs 490 mg/kg FW [143] SARS-CoV-2 mAb B38 4 µg/g FW [144] mAb H4 35 µg/g FW [144] ACE2-Fc 100 µg/g FW [145] ACE2-Fc ≈ 80 µg/g FW [146] CR3022 mAb 130 µg/g FW [147] West Nile virus pE16 0.74 mg/g FW [148] pE16scFv-CH 0.77 mg/g FW [148] DIII 73 µg/g FW [149] HBcAg-wDIII 1.2 mg/g FW [150] E16 mAb 339.9 µg/g FW [151] Zika virus c2A10G6 1.47 mg/g FW …”