Andrographolide sensitizes Ras-transformed cells to radiation in vitro and in vivo.
OBJECTIVE
Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization.


METHODS
An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-kappaB) and Akt in andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting.


RESULTS
Concurrent andrographolide treatment (10 microM, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 +/- 2.5 days) compared with radiation alone (22 +/- 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-kappaB activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-kappaB activity.


CONCLUSIONS
Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-kappaB activity. These observations indicate that andrographolide is a novel radiosensitizing agent with potential application in cancer radiotherapy.
Hung
Shih-Kai
SK
Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan.
Hung
Ling-Chien
LC
Kuo
Cheng-Deng
CD
Lee
Kuan-Yi
KY
Lee
Moon-Sing
MS
Lin
Hon-Yi
HY
Chen
Yu-Jen
YJ
Fu
Shu-Ling
SL
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D053148:Caspase 3 / Q000378:metabolism; D002461:Cell Line, Transformed; D004396:Coloring Agents; D053938:DNA Fragmentation; D004224:Diterpenes / Q000494:pharmacology*; D015536:Down-Regulation; D011905:Genes, ras; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000032:analysis; D011836:Radiation Tolerance / Q000187:drug effects* / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D051381:Rats; D013778:Tetrazolium Salts; D013844:Thiazoles; D014183:Transplantation, Heterologous; D018631:ras Proteins / Q000378:metabolism
D017304:Annexin A5; D004396:Coloring Agents; D004224:Diterpenes; D016328:NF-kappa B; D011838:Radiation-Sensitizing Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C030419:andrographolide; D051057:Proto-Oncogene Proteins c-akt; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D053148:Caspase 3; D018631:ras Proteins; C022616:thiazolyl blue