Recently, we developed a simple and potent therapeutic liposome cancer vaccine 
consisting of a peptide antigen and a cationic lipid. The molecular mechanism of 
the adjuvanticity of cationic liposome was studied and described in the current 
report. First, cationic DOTAP liposome, but not the neutral liposome DOPC, was 
shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived 
dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38 
activation and downstream chemokine/cytokine induction. Furthermore, ROS were 
shown to be involved in the expression of the co-stimulatory molecules CD86/CD80 
induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800 
microM, the apoptotic marker Annexin V and ROS double positive cells increased, 
suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo, 
optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV 
positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7 
oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP 
were attenuated by incorporating DOPC in the formulation, suggesting that ROS 
are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol 
DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor 
regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the 
same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and 
this formulation showed tumor regression activity. In conclusion, DOTAP is an 
active DC stimulator resulting in the activation of ERK and p38 and induction of 
chemokines, cytokines and co-stimulatory molecules mediated by appropriate 
amount of ROS. Our data elucidated an important mechanism of adjuvant activity 
of cationic liposome and could facilitate rational design of synthetic lipid 
based adjuvants and vaccine formulation.