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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

Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. (A) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; (B) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; (C) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and (D) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.
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ijms-16-12243-f004: Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. (A) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; (B) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; (C) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and (D) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.

Mentions: To assess whether the E6/E7-specific pooled siRNAs promote cell death and senescence, we first tested and compared the cytotoxicity of cervical cancer cells. Irradiated (2 Gy) HeLa or SiHa cells were transfected with pooled HPV E6/E7-specific siRNAs and analyzed after three-days’ exposure. SP-transfected HeLa or SiHa cells without irradiation served as the controls. As shown in Figure 4A, HPV18 E6/E7-specific SP (426_d5 and 450_d4) led to significantly higher growth inhibition than either siRNA 426_d5 or 450_d4 alone did. Pooled siRNAs in combination with irradiation significantly enhanced growth inhibition to levels greater than observed with other treatments. Similar results were observed in SiHa cells transfected with HPV16 E6/E7-specific SP (366_d2, 448_d2 and 497_d2).


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)

Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. (A) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; (B) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; (C) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and (D) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12243-f004: Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. (A) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; (B) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; (C) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and (D) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.
Mentions: To assess whether the E6/E7-specific pooled siRNAs promote cell death and senescence, we first tested and compared the cytotoxicity of cervical cancer cells. Irradiated (2 Gy) HeLa or SiHa cells were transfected with pooled HPV E6/E7-specific siRNAs and analyzed after three-days’ exposure. SP-transfected HeLa or SiHa cells without irradiation served as the controls. As shown in Figure 4A, HPV18 E6/E7-specific SP (426_d5 and 450_d4) led to significantly higher growth inhibition than either siRNA 426_d5 or 450_d4 alone did. Pooled siRNAs in combination with irradiation significantly enhanced growth inhibition to levels greater than observed with other treatments. Similar results were observed in SiHa cells transfected with HPV16 E6/E7-specific SP (366_d2, 448_d2 and 497_d2).

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