The 18-kDa translocator protein, formerly known as the peripheral-type benzodiazepine receptor, confers proapoptotic and antineoplastic effects in a human colorectal cancer cell line.
OBJECTIVE
The involvement of the 18-kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, in apoptosis regulation of HT29 colorectal cancer cells was studied in-vitro. In-vivo TSPO involvement in tumor growth of HT29 cells xenografted into SCID mice was studied.


METHODS
Knockdown of TSPO expression in the human HT29 cell line was established by stable transfection with vectors containing the TSPO gene in the antisense direction. Successful TSPO knockdown was characterized by reduction of 20% in TSPO RNA levels, 50% in protein expression of the TSPO, and 50% in binding with the TSPO ligand, [3H]PK 11195. Subsequently, in-vitro cell viability and proliferation assays were applied. In addition, transient transfecton with short interfering RNA (siRNA) directed against human TSPO was studied in this way. Furthermore, we also grafted HT29 cells subcutaneously into the right thighs of SCID mice to examine the effects of the putative TSPO agonist, FGIN-1-27, on tumor growth in-vivo.


RESULTS
In-vitro TSPO knockdown established by stable transfection of TSPO antisense gene resulted in HT29 clones displaying significantly lower levels of cell death as determined with trypan blue (50% less), lower apoptotic rates (28% less), and higher proliferation rates (48% more one week after seeding and 27% more two weeks after seeding). Transient transfection with anti-human TSPO siRNA resulted in similar viability and antiapoptotic effects. In-vivo, the proapoptotic TSPO ligand, FGIN-1-27 significantly reduced the growth rate of grafted tumors (40% less), in comparison with vehicle-treated mice.


CONCLUSIONS
TSPO knockdown by genetic manipulation transforms the human HT29 cancer line to a more malignant type in-vitro. In-vivo pharmacological treatment with the putative TSPO agonist FGIN-1-27 reduces tumor growth of the HT29 cell line. These data suggest that TSPO involvement in apoptosis provides a target for anticancer treatment.
Shoukrun
Rami
R
Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel.
Veenman
Leo
L
Shandalov
Yulia
Y
Leschiner
Svetlana
S
Spanier
Ilana
I
Karry
Rachel
R
Katz
Yeshayahu
Y
Weisinger
Gary
G
Weizman
Abraham
A
Gavish
Moshe
M
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D004742:Enhancer Elements, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D007210:Indoleacetic Acids / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D016513:Mice, SCID; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018079:Receptors, GABA / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D007210:Indoleacetic Acids; D008024:Ligands; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018079:Receptors, GABA; C499229:TSPO protein, human; C081306:N,N-di-n-hexyl-2-(4-fluorophenyl)indole-3-acetamide; D015500:Chloramphenicol O-Acetyltransferase