Response rate of fibrosarcoma cells to cytotoxic drugs on the expression level correlates to the therapeutic response rate of fibrosarcomas and is mediated by regulation of apoptotic pathways.
BACKGROUND
Because of the high resistance rate of fibrosarcomas against cytotoxic agents clinical chemotherapy of these tumors is not established. A better understanding of the diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new chemotherapeutic strategies. In this study we elucidated the response of a fibrosarcoma cell line to clinically used cytostatic agents on the level of gene expression.


METHODS
HT1080 fibrosarcoma cells were exposed to the chemotherapeutic agents doxorubicin, actinomycin D or vincristine. Total RNA was isolated and the gene expression patterns were analyzed by microarray analysis. Expression levels for 46 selected candidate genes were validated by quantitative real-time PCR.


RESULTS
The analysis of the microarray data resulted in 3.309 (actinomycin D), 1.019 (doxorubicin) and 134 (vincristine) probesets that showed significant expression changes. For the RNA synthesis blocker actinomycin D, 99.4% of all differentially expressed probesets were under-represented. In comparison, probesets down-regulated by doxorubicin comprised only 37.4% of all genes effected by this agent. Closer analysis of the differentially regulated genes revealed that doxorubicin induced cell death of HT1080 fibrosarcoma cells mainly by regulating the abundance of factors mediating the mitochondrial (intrinsic) apoptosis pathway. Furthermore doxorubicin influences other pathways and crosstalk to other pathways (including to the death receptor pathway) at multiple levels. We found increased levels of cytochrome c, APAF-1 and members of the STAT-family (STAT1, STAT3), while Bcl-2 expression was decreased. Caspase-1, -3, -6, -8, and -9 were increased indicating that these proteases are key factors in the execution of doxorubicin mediated apoptosis.


CONCLUSIONS
This study demonstrates that chemotherapy regulates the expression of apoptosis-related factors in fibrosarcoma cells. The number and the specific pattern of the genes depend on the used cytotoxic drug. The response rates on the gene expression level, i.e. the number of genes regulated by the drugs actinomycin D, doxorubicin and vincristine, correlate to the clinical effectiveness of the drugs. Doxorubicin seems to exert its cytotoxic mechanism by regulating genes, which are involved in several different apoptosis regulating pathways. The exact knowledge of the genes affected by the drugs will help to understand the diverse modes of soft tissue sarcoma cell death in response to cytotoxic therapies.
Department of Plastic Surgery, Burn Center, Hand surgery, Sarcoma Reference Center, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bürkle de la Camp Platz 1, 44789 Bochum, Germany. marcus.lehnhardt@rub.de
Marcus
M
Lehnhardt
Ludger
L
Klein-Hitpass
Cornelius
C
Kuhnen
Heinz Herbert
HH
Homann
Adrien
A
Daigeler
Hans Ulrich
HU
Steinau
Sonja
S
Roehrs
Laura
L
Schnoor
Lars
L
Steinstraesser
Oliver
O
Mueller
D000903:Antibiotics, Antineoplastic / Q000627:therapeutic use; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use; D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019295:Computational Biology; D003609:Dactinomycin / Q000627:therapeutic use; D004317:Doxorubicin / Q000627:therapeutic use; D005354:Fibrosarcoma / Q000188:drug therapy*; D015972:Gene Expression Regulation, Neoplastic; D023281:Genomics; D006801:Humans; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016896:Treatment Outcome; D014750:Vincristine / Q000627:therapeutic use
D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D009841:Oligonucleotides; D012333:RNA, Messenger; D003609:Dactinomycin; D014750:Vincristine; D012313:RNA; D004317:Doxorubicin