Photochemically enhanced delivery of a cell-penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells.
OBJECTIVE
Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function.


METHODS
Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status.


RESULTS
After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX.


CONCLUSIONS
Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.
Folini
M
M
Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Bandiera
R
R
Millo
E
E
Gandellini
P
P
Sozzi
G
G
Gasparini
P
P
Longoni
N
N
Binda
M
M
Daidone
M G
MG
Berg
K
K
Zaffaroni
N
N
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016503:Drug Delivery Systems; D004705:Endocytosis / Q000187:drug effects; D015696:Gene Products, tat / Q000378:metabolism; D006801:Humans; D008297:Male; D008677:Metaphase / Q000187:drug effects; D020135:Peptide Nucleic Acids / Q000378:metabolism / Q000494:pharmacology*; D010777:Photochemistry / Q000379:methods*; D011471:Prostatic Neoplasms / Q000473:pathology*; D019098:Telomerase / Q000037:antagonists & inhibitors*; D016615:Telomere / Q000187:drug effects* / Q000235:genetics
D015696:Gene Products, tat; D020135:Peptide Nucleic Acids; C509186:TERT protein, human; D019098:Telomerase