Drugs targeting the mitochondrial pore act as cytotoxic and cytostatic agents in temozolomide-resistant glioma cells.
BACKGROUND
High grade gliomas are one of the most difficult cancers to treat and despite surgery, radiotherapy and temozolomide-based chemotherapy, the prognosis of glioma patients is poor. Resistance to temozolomide is the major barrier to effective therapy. Alternative therapeutic approaches have been shown to be ineffective for the treatment of genetically unselected glioma patients. Thus, novel therapies are needed. Mitochondria-directed chemotherapy is an emerging tool to combat cancer, and inner mitochondrial permeability transition (MPT) represents a target for the development of cytotoxic drugs. A number of agents are able to induce MPT and some of them target MPT-pore (MPTP) components that are selectively up-regulated in cancer, making these agents putative cancer cell-specific drugs.


OBJECTIVE
The aim of this paper is to report a comprehensive analysis of the effects produced by selected MPT-inducing drugs (Betulinic Acid, Lonidamine, CD437) in a temozolomide-resistant glioblastoma cell line (ADF cells).


METHODS
EGFRvIII expression has been assayed by RT-PCR. EGFR amplification and PTEN deletion have been assayed by differential-PCR. Drugs effect on cell viability has been tested by crystal violet assay. MPT has been tested by JC1 staining. Drug cytostatic effect has been tested by mitotic index analysis. Drug cytotoxic effect has been tested by calcein AM staining. Apoptosis has been assayed by Hoechst incorporation and Annexine V binding assay. Authophagy has been tested by acridine orange staining.


RESULTS
We performed a molecular and genetic characterization of ADF cells and demonstrated that this line does not express the EGFRvIII and does not show EGFR amplification. ADF cells do not show PTEN mutation but differential PCR data indicate a hemizygous deletion of PTEN gene. We analyzed the response of ADF cells to Betulinic Acid, Lonidamine, and CD437. Our data demonstrate that MPT-inducing agents produce concentration-dependent cytostatic and cytotoxic effects in parallel with MPT induction triggered through MPTP. CD437, Lonidamine and Betulinic acid trigger apoptosis as principal death modality.


CONCLUSIONS
The obtained data suggest that these pharmacological agents could be selected as adjuvant drugs for the treatment of high grade astrocytomas that resist conventional therapies or that do not show any peculiar genetic alteration that can be targeted by specific drugs.
Lena
Annalisa
A
Dipartimento di Morfologia Umana e Biologia Applicata, University of Pisa, Via Volta 4, 56126 Pisa, Italy. annalisa.lena@inwind.it
Rechichi
Mariarosa
M
Salvetti
Alessandra
A
Bartoli
Barbara
B
Vecchio
Donatella
D
Scarcelli
Vittoria
V
Amoroso
Rosina
R
Benvenuti
Lucia
L
Gagliardi
Rolando
R
Gremigni
Vittorio
V
Rossi
Leonardo
L
D000165:Acridine Orange; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D054697:Cytostatic Agents / Q000494:pharmacology*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D066246:ErbB Receptors / Q000378:metabolism; D005910:Glioma / Q000235:genetics / Q000473:pathology*; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D000083162:Mitochondrial Permeability Transition Pore; D053978:Pentacyclic Triterpenes; D016133:Polymerase Chain Reaction; D012176:Retinoids / Q000494:pharmacology; D000077204:Temozolomide; D014315:Triterpenes / Q000494:pharmacology; D000094062:Betulinic Acid
C099555:CD 437; D054697:Cytostatic Agents; D007191:Indazoles; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D053978:Pentacyclic Triterpenes; D012176:Retinoids; D014315:Triterpenes; C410723:epidermal growth factor receptor VIII; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003606:Dacarbazine; D016572:Cyclosporine; D066246:ErbB Receptors; D000165:Acridine Orange; C016371:lonidamine; D000077204:Temozolomide; D000094062:Betulinic Acid