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Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

Jung HS, Rajasekaran N, Song SY, Kim YD, Hong S, Choi HJ, Kim YS, Choi JS, Choi YL, Shin YK - Int J Mol Sci (2015)

Bottom Line: In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model.Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice.In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 151-742, Korea. hunsoonjung@abionbio.com.

ABSTRACT
The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

No MeSH data available.


Related in: MedlinePlus

Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. (A) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); (B) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; (C) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; (D) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and (E) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.
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ijms-16-12243-f001: Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. (A) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); (B) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; (C) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; (D) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and (E) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.

Mentions: In a previous study, we revealed that E6/E7-specific siRNA, silencing both E6 and E7 mRNA, was more efficacious than E6-specific siRNA [17]. Moreover, the combination of E6/E7-specific siRNA and CDDP had a greater therapeutic efficacy in cervical cancer cells. The aim of this study was to identify siRNAs that have the potential to silence both HPV18- and 16-type E6/E7 mRNA and simultaneously decrease E6/E7 protein-mediated degradation of TP53 in cervical cancer cells. A list of HPV18- and 16-type E6/E7 siRNA target sequences was generated (Tables S1 and S2). Ten library HPV-siRNAs were generated and screened for their silencing effects on HPV18, as well as HPV16-type E6/E7, respectively. The inhibition efficiency of siRNAs (103, 426, 450, 456 and 458) was shown by their significant anti-proliferative effect, unlike that of the other siRNAs (Figure S1a). To identify potential therapeutic siRNA, five out of 10 siRNAs were subjected to further analysis. As shown in Figure 1A, the inhibition of HeLa cell growth by at least 70% was achieved even at a low concentration. We also determined the cellular protein expression levels of E7, as well as TP53 in HeLa cells after E6/E7 silencing by siRNAs. With regard to TP53 and E7 protein levels, we found that siRNA 426 or 450 was able to silence E6/E7 expression more effectively than the other siRNAs (Figure 1B). Our results indicate that siRNA 426 and 450 showed a more robust effect than the other siRNAs did, in a dose-dependent manner (Figure S1b,c). After systematic screening of the library in triplicate, these results demonstrate that new, highly potent HPV18 siRNAs termed 426 and 450 are capable primary lead siRNAs. Similarly, on our screening analysis in SiHa cells (Figure S1c), HPV16-type-specific lead siRNAs termed 366 and 448 were selected along with siRNA 497 [16] for further studies.


Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

Jung HS, Rajasekaran N, Song SY, Kim YD, Hong S, Choi HJ, Kim YS, Choi JS, Choi YL, Shin YK - Int J Mol Sci (2015)

Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. (A) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); (B) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; (C) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; (D) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and (E) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490442&req=5

ijms-16-12243-f001: Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. (A) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); (B) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; (C) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; (D) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and (E) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.
Mentions: In a previous study, we revealed that E6/E7-specific siRNA, silencing both E6 and E7 mRNA, was more efficacious than E6-specific siRNA [17]. Moreover, the combination of E6/E7-specific siRNA and CDDP had a greater therapeutic efficacy in cervical cancer cells. The aim of this study was to identify siRNAs that have the potential to silence both HPV18- and 16-type E6/E7 mRNA and simultaneously decrease E6/E7 protein-mediated degradation of TP53 in cervical cancer cells. A list of HPV18- and 16-type E6/E7 siRNA target sequences was generated (Tables S1 and S2). Ten library HPV-siRNAs were generated and screened for their silencing effects on HPV18, as well as HPV16-type E6/E7, respectively. The inhibition efficiency of siRNAs (103, 426, 450, 456 and 458) was shown by their significant anti-proliferative effect, unlike that of the other siRNAs (Figure S1a). To identify potential therapeutic siRNA, five out of 10 siRNAs were subjected to further analysis. As shown in Figure 1A, the inhibition of HeLa cell growth by at least 70% was achieved even at a low concentration. We also determined the cellular protein expression levels of E7, as well as TP53 in HeLa cells after E6/E7 silencing by siRNAs. With regard to TP53 and E7 protein levels, we found that siRNA 426 or 450 was able to silence E6/E7 expression more effectively than the other siRNAs (Figure 1B). Our results indicate that siRNA 426 and 450 showed a more robust effect than the other siRNAs did, in a dose-dependent manner (Figure S1b,c). After systematic screening of the library in triplicate, these results demonstrate that new, highly potent HPV18 siRNAs termed 426 and 450 are capable primary lead siRNAs. Similarly, on our screening analysis in SiHa cells (Figure S1c), HPV16-type-specific lead siRNAs termed 366 and 448 were selected along with siRNA 497 [16] for further studies.

Bottom Line: In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model.Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice.In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 151-742, Korea. hunsoonjung@abionbio.com.

ABSTRACT
The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

No MeSH data available.


Related in: MedlinePlus