The inhibition of mRFP Ub E formation
The inhibition of mRFP-Ub–E1 formation by ginsenosides Re was not in time-dependent in vitro (Fig. 4B). Fifty micrometres ginsenoside Rg1 decreased E1 activity to 0.24- to 0.36-fold over 30min. This finding suggests that ginsenoside Rg1 may irreversibly inhibit mRFP-Ub–E1 formation or a tight-binding substrate. This observation will help us to further investigate the E1 mechanism using ginsenoside Rg1.
To address whether ginsenosides Re and Rg1 directly inhibited mRFP-Ub substrate without E1, the reaction mixture was separated by 8% SDS–PAGE under nondenaturing conditions. An electropherogram revealed significant differences between the control and E1-untreated samples. There were no differences between mRFP-Ub-only and mRFP-Ub with inhibitor ginsenosides Re or Rg1 (Supplemental Fig. 1). Therefore, ginsenosides Re and Rg1 cannot directly interact to inhibit mRFP-Ub.
We proposed a model for the ginsenoside Rb1, Rb2, Rc, Rd, Re, and Rg1 inhibition of the ubiquitin–proteasome pathway (Fig. 5). Ginsenosides Re and Rg1 induced protein accumulation by blocking the first step of ubiquitination, which is ubiquitin-E1 activation. Ginsenosides Rb1 and Rd inhibited the chymotrypsin-like activity of the 26S proteasome but increased E1 activity, resulting in the accumulation of ubiquitinated proteins. Ginsenosides Rb2 and Rc did not inhibit the chymotrypsin-like activity of the 26S proteasome; moreover, they (+)-Bicuculline increased E1 activity, and therefore, ginsenosides Rb2 and Rc could lead to more protein degradation.
Declaration of conflicting interests
Acknowledgments The authors thank Dr. Tai-Sheng Cheng, Yu-Wen Huang and Ting-Wei Gau for technical assistance. This work was supported by the National Science Council of TaiwanNSC 98-2320-B-024-002-MY3 and National University of TainanAB102-216. This information is available free of charge via the Internet at http://pubs.acs.org.
Introduction Cervical cancer is the fourth most frequent cancer in women worldwide  with high-risk human papillomavirus (HPV) as its etiologic agent . According to their oncogenic potential, HPVs are classified as high- (HR-HPV) or low-risk (LR-HPV), being HPV16 and HPV18 the most common viral types found in cervical cancer patients , . Persistent infection with high-risk HPV can lead to cervical intraepithelial neoplasia (CIN) that, without treatment, could progress to in situ carcinoma . During an HPV infection, viral genes are differentially and sequentially expressed along the various layers of the cervical epithelium . HPV proteins E1 and E2 regulate viral replication , , of which the E1 helicase is necessary for DNA double-strand separation during viral replication  and is the most conserved among early expressed HPV proteins , . Malignant transformation is not a common occurrence of an HPV infection, as only a minor number of cervical lesions infected with HR-HPV types evolve into cervical cancer . However, for unclear reasons the HPV genome, in some cases, integrates randomly into the host DNA leading to cervical transformation, which is mainly due to overexpression of the major HPV viral oncoproteins, E6 and E7 , . Although the adaptive immune system plays an important role in the elimination of HPV infected cells , , an estimated 15% of women infected with HR-HPV fail to develop an effective response against the virus due to viral immune evasion mechanisms , leading to HPV persistent infections . HPV prophylactic vaccines activate the adaptive immune system and induce neutralizing antibodies that prevent infections by viral types covered by the vaccines but cannot eliminate established HPV infections. On the other hand, therapeutic vaccine candidates must be directed at inducing cell-mediated adaptive immune responses through the induction of HPV-specific CD8+ cytotoxic T lymphocytes (CTL) , . An ideal therapeutic vaccine candidate should be able to induce cross-reactivity among different HPV types with activation of tumor-specific CD8+ cytotoxic T cells, as well as CD4 + helper T cells , . A number of lines of evidence have shown that HPV-specific T cells and tumor infiltration by CD8+ T cells are associated with a better outcome in patients ,  and tumor regression in experimental models . Although the most common HPV antigens employed for vaccination have been the major oncoproteins E6 and E7, which have shown some benefit against HPV and cervical cancer , these oncoproteins differ substantially among HPV types, affecting their potential as wide spectrum HPV therapeutic immunogens. On the other hand, as the HPV-E1 sequence is highly conserved among all known HPV types , it could constitute a good target for a universal HPV vaccine.