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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

Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. (A) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; (B) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; (C) E6 and (D) CDKN1A mRNA expression as determined by qRT-PCR; and (E) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.
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ijms-16-12243-f003: Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. (A) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; (B) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; (C) E6 and (D) CDKN1A mRNA expression as determined by qRT-PCR; and (E) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.

Mentions: Based on our results, HPV18-type lead siRNAs (426 and 450) and HPV16-type lead siRNAs (366, 448 and 497) were chemically modified to enhance the nuclease stability. In our first line investigation, we examined the stability of siRNAs and the silencing efficiency by introducing 2ʹ-OMe and 2ʹ-F modifications. As shown in Figure 3A, the modified derivatives of siRNA 426 or 497 (Table 2) decreased the proliferation of HeLa or SiHa cells. In particular, cells receiving siRNA 426_d5 (derivative 5) and siRNA 497_d2 (derivative 2) showed significant cell growth inhibition compared to the other derivatives. We further assessed the efficiency of modified E6/E7-specific siRNAs by investigating the changes in TP53 and E7 protein levels. siRNA 426_d5 and siRNA 497_d2 were shown to affect the expression levels of TP53 and E7 (Figure 3B). Interestingly, their derivatives also significantly decreased E6 mRNA levels (Figure 3C) and increased CDKN1A mRNA levels (Figure 3D). We next evaluated the serum stability of chemically-modified siRNAs by using gel electrophoresis analysis. The chemically-modified derivatives were comparatively more stable than the unmodified siRNAs (Figure 3E). We simultaneously determined the efficacy of other HPV16 E6/E7-specific modified siRNAs (366, 448) and HPV16 E6/E7-specific siRNA 450 (Supplementary Table S3). Among the modified derivatives, siRNA 450_d4 (derivative 4), siRNA 366_d2 (derivative 2) and siRNA 448_d2 (derivative 2) were selected as candidates based on the experimental results (Figures S3–S5). Similarly, in flow cytometric analysis, modified siRNAs transfection resulted in an increased percentage of apoptotic cells (Figure S6).


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)

Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. (A) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; (B) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; (C) E6 and (D) CDKN1A mRNA expression as determined by qRT-PCR; and (E) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12243-f003: Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. (A) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; (B) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; (C) E6 and (D) CDKN1A mRNA expression as determined by qRT-PCR; and (E) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.
Mentions: Based on our results, HPV18-type lead siRNAs (426 and 450) and HPV16-type lead siRNAs (366, 448 and 497) were chemically modified to enhance the nuclease stability. In our first line investigation, we examined the stability of siRNAs and the silencing efficiency by introducing 2ʹ-OMe and 2ʹ-F modifications. As shown in Figure 3A, the modified derivatives of siRNA 426 or 497 (Table 2) decreased the proliferation of HeLa or SiHa cells. In particular, cells receiving siRNA 426_d5 (derivative 5) and siRNA 497_d2 (derivative 2) showed significant cell growth inhibition compared to the other derivatives. We further assessed the efficiency of modified E6/E7-specific siRNAs by investigating the changes in TP53 and E7 protein levels. siRNA 426_d5 and siRNA 497_d2 were shown to affect the expression levels of TP53 and E7 (Figure 3B). Interestingly, their derivatives also significantly decreased E6 mRNA levels (Figure 3C) and increased CDKN1A mRNA levels (Figure 3D). We next evaluated the serum stability of chemically-modified siRNAs by using gel electrophoresis analysis. The chemically-modified derivatives were comparatively more stable than the unmodified siRNAs (Figure 3E). We simultaneously determined the efficacy of other HPV16 E6/E7-specific modified siRNAs (366, 448) and HPV16 E6/E7-specific siRNA 450 (Supplementary Table S3). Among the modified derivatives, siRNA 450_d4 (derivative 4), siRNA 366_d2 (derivative 2) and siRNA 448_d2 (derivative 2) were selected as candidates based on the experimental results (Figures S3–S5). Similarly, in flow cytometric analysis, modified siRNAs transfection resulted in an increased percentage of apoptotic cells (Figure S6).

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