Induction of apoptosis on K562 cell line and double strand breaks on colon cancer cell line expressing high affinity receptor for granulocyte macrophage-colony stimulating factor (GM-CSF).
Immunotoxins are comprised of both the cell targeting and the cell killing moieties. We previously established a new immunotoxin, i.e. Shiga toxin granulocyte macrophage-colony stimulating factor (StxA1-GM-CSF), comprises of catalytic domain of Stx, as a killing moiety and GM-CSF, as a cell targeting moiety. In this study, the ability of the immunotoxin to induce apoptosis and double strand breaks (DSB) on different cell lines was investigated.



The recombinant hybrid protein was expressed in bacterial expression system and purified with nickel-nitrilotriacetate acid resin. The K562 (erythroid leukemia) cell line and LS174 (colon carcinoma) were used in this study. The neutral comet assay was carried out for the detection of DSB and Hoechst staining was performed for apoptosis.



StxA1-GM-CSF effectively induced apoptosis on K562 cell line and DNA Double Strand Break (DSB) were observed on colon cancer cell line treated with StxA1-GM-CSF.



This novel action i.e. DNA damage might be a relevant mechanism of action for StxA1-GM-CSF that is designed to act as immunotoxin, although further investigation is required.
Roudkenar
Mehryar Habibi
MH
Research Center, Iranian Blood Transfusion Organization, Tehran, Iran.
Bouzari
Saeid
S
Molecular Biology Unit, Pasteur Institute of Iran, Pasteur Ave., Tehran 13164, Iran.
Kuwahara
Yoshikazu
Y
Dept. of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-Cho 4-1, Aoba-Ward, Sendai-City, Miyagi-Prefecture 980-8575, Japan.
Roushandeh
Amaneh Mohammadi
AM
Dept. of Anatomy, Faculty of Medicine, Medical University of Tabriz, Tabriz, Iran.
Baba
Taisuke
T
Dept. of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-Cho 4-1, Aoba-Ward, Sendai-City, Miyagi-Prefecture 980-8575, Japan.
Oloomi
Mana
M
Molecular Biology Unit, Pasteur Institute of Iran, Pasteur Ave., Tehran 13164, Iran.
Fukumoto
Manabu
M
Dept. of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-Cho 4-1, Aoba-Ward, Sendai-City, Miyagi-Prefecture 980-8575, Japan.
D017209:Apoptosis / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000737:chemistry / Q000188:drug therapy*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006657:Histones / Q000032:analysis; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D020014:K562 Cells; D021122:Protein Subunits / Q000494:pharmacology*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis*; D022621:Shiga Toxin / Q000494:pharmacology*
C468783:H2AX protein, human; D006657:Histones; D000922:Immunotoxins; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; C493898:Shiga toxin subunit A; D022621:Shiga Toxin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor