Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Kikkawa, Yamato; Jin, Fang; Kobuchi, Hirotsugu; Nasu, Yasutomo; Yamada, Taketo; Nishihara, Tatsuji; Ikeda, Yasuo; Kizaki, Masahiro; Yoshinouchi, Mitsuo

doi:10.1038/sj.cgt.7700891

Cited by 54 publications

(39 citation statements)

References 38 publications

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“…These results encourage further investigation of RNAi as a potential treatment for cervical cancer. Although the knockdown of HPV E6 and E7 expression in HeLa cells has been reported by a few groups using siRNA, 14,13,18 we showed here a stable knockdown of E6 or E7 in HeLa cells (at least 2 weeks) with LV-shRNA. In previous reports, Yoshinouchi et al 17 showed that synthetic HPV 16 siRNA induced a modest reduction in tumor growth of SiHa cells in NOD/ SCID mice.…”

Section: Discussionmentioning

confidence: 76%

“…[8][9][10][11][12] Previous studies have demonstrated that small interference RNA (siRNA) can suppress HPV E6 and/or E7 expression in various cancer cell lines. [13][14][15][16][17][18] However, the suppression in these studies was either transient (owing to the short half-life of siRNA), against only one target, or not all cells were transfected leading to escape. We wished to improve these results by using shRNA delivered by a third-generation lentiviral vector for a more effective, stable and long-term RNAi.…”

Section: Introductionmentioning

confidence: 97%

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Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

et al. 2006

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In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into RagÀ/À mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

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Section: Discussionmentioning

confidence: 76%

Section: Introductionmentioning

confidence: 97%

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

et al. 2006

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“…Among the promising gene approaches developed thus far to specifically target E6 and/or E7 (5), it was found that siRNA led to ablation of E6 in HPV18-positive HeLa cells leading to apoptosis, albeit under harsh conditions of cell transfection (7,10). Studies done with siE6 or short hairpin RNA showed that treated HeLa or SiHa cells did not die but stopped proliferating and became more sensitive to chemotherapy (9,22,32).…”

Section: Discussionmentioning

confidence: 99%

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Courtête¹,

Sibler²,

Zeder‐Lutz³

et al. 2007

Molecular Cancer Therapeutics

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Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPVpositive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOHterminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipidmediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

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“…5B). Inhibiting E6/E7 gene transcription in HPV-transformed cells using small interfering RNA has similarly been shown to reduce cell growth and/or induce apoptosis in HPV-transformed cells (40,41).…”

Section: Why Might This Two-step Process For Rb Degradation Havementioning

confidence: 99%

Human Papillomavirus E7 Requires the Protease Calpain to Degrade the Retinoblastoma Protein

Darnell

Schroder

Antalis

et al. 2007

Journal of Biological Chemistry

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Cervical cancers transformed by high risk human papilloma virus (HPV) express the E7 oncoprotein, which accelerates the degradation of the retinoblastoma protein (Rb). Here we show that the E7-mediated degradation of Rb requires the calciumactivated cysteine protease, calpain. E7 bound and activated -calpain and promoted cleavage at Rb 810 , with mutation of this residue preventing E7-mediated degradation. The calpain cleavage product, Rb 1-810 , was unable to mediate cell cycle arrest but retained the ability to repress E6/E7 transcription. E7 also promoted the accelerated proteasomal degradation of Infection with high risk human Papillomavirus (HPV)2 types is responsible for virtually all cases of cervical cancer, which is currently the second most common cause of death from cancer among women worldwide (1). Vaccination will likely have a substantial impact; however, current modeling suggests that it may take 40 -50 years to reach an Ϸ50% reduction in mortality (2). Integration of part of the HPV genome is believed to be central to the transformation process (3) and often results in the increased expression of the two viral oncoproteins, E6 and E7. A principle activity of E6 and E7 is promotion of the accelerated degradation of p53 and the retinoblastoma protein (Rb), respectively (4, 5). Hypophosphorylated Rb binds the E2F transcription factor, resulting in repression of E2F-dependent gene transcription and G 1 cell cycle arrest (6). The promotion of Rb degradation mediated by E7 is believed to involve a two-step process (7). First, E7 binds hypophosphorylated Rb (8), which leads to the displacement of E2F and entry into the cell cycle. How E2F is displaced by E7 remains unclear as E7 and E2F binding sites on Rb have recently been shown to be quite distinct (9). The second step involves E7 targeting Rb for accelerated degradation via the proteasome (10 -12). A new insight into the mechanism of E7-mediated degradation of Rb was recently provided by our observation that the serine protease inhibitor SerpinB2 was able to inhibit E7-mediated degradation of Rb in HeLa cells (13,14). There are no reports that SerpinB2 inhibits the proteasome, and it has recently emerged that SerpinB2 inhibits calpain-mediated degradation of Rb. 3Calpains represent a group of calcium-activated cysteine proteases, two of which, -calpain and m-calpain, are ubiquitously expressed and found both in the cytoplasm and the nucleus (15). Herein we provide evidence that the first step in the E7-mediated degradation of Rb involves calpain cleavage. E7 was shown to bind and activate -calpain and promoted cleavage of full-length Rb 1-928 to yield Rb . Rb 1-810 was unable to promote cell cycle arrest, suggesting that this cleavage event was sufficient to displace E2F from Rb. The second step involved the promotion by E7 of the proteasomal degradation of Rb . Calpain inhibitors are being developed for a number of diseases including cancer (16, 17) and were able to inhibit E7-mediated degradation of Rb. They also reduced the viability of HPV-tr...

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Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Cited by 54 publications

References 38 publications

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Human Papillomavirus E7 Requires the Protease Calpain to Degrade the Retinoblastoma Protein

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