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Role of autophagy in high linear energy transfer radiation-induced cytotoxicity to tumor cells.

Jin X, Liu Y, Ye F, Liu X, Furusawa Y, Wu Q, Li F, Zheng X, Dai Z, Li Q - Cancer Sci. (2014)

Bottom Line: Heavy-ion radiotherapy has a potential advantage over conventional radiotherapy due to improved dose distribution and a higher biological effectiveness in cancer therapy.Autophagy, as a novel important target to improve anticancer therapy, has recently attracted considerable attention.Our data imply that targeting autophagy might enhance the effectiveness of heavy-ion radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China; Key Laboratory of Heavy Ion Radiation Biology and Medicine, Chinese Academy of Sciences, Lanzhou, China.

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Effect of Beclin 1 and Atg5 siRNA knockdown on the sensitivity of HeLa cells to high linear energy transfer radiation. (a) Western blot analysis of Beclin 1 and Atg5 levels in double knockdown (KD) HeLa cells. The relative level of proteins in comparison to β-actin is indicated below each immunoblot image. (b) Inhibition of siRNA on autophagy induced by the carbon ions after 24 h, KD of Beclin 1 together with Atg5 significantly attenuated the high linear energy transfer radiation-induced formation of acidic vesicular organelles (AVOs). Beclin 1 or Atg5 KD alone had only marginal effects. The white and gray columns indicate the control groups and the irradiation groups with 75 keV/μm carbon ions at 2 Gy, respectively. *P > 0.05 and **P < 0.05, compared with 2 Gy radiation alone. (c) Effect of inhibiting autophagy on the clonogenic survival of HeLa cells. Square, round, triangular, and diamond symbols represent the radiation groups, radiation + siRNA Beclin 1 groups, radiation + siRNA Atg5 groups, and radiation + siRNA Beclin 1 and Atg5 groups, respectively.
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fig04: Effect of Beclin 1 and Atg5 siRNA knockdown on the sensitivity of HeLa cells to high linear energy transfer radiation. (a) Western blot analysis of Beclin 1 and Atg5 levels in double knockdown (KD) HeLa cells. The relative level of proteins in comparison to β-actin is indicated below each immunoblot image. (b) Inhibition of siRNA on autophagy induced by the carbon ions after 24 h, KD of Beclin 1 together with Atg5 significantly attenuated the high linear energy transfer radiation-induced formation of acidic vesicular organelles (AVOs). Beclin 1 or Atg5 KD alone had only marginal effects. The white and gray columns indicate the control groups and the irradiation groups with 75 keV/μm carbon ions at 2 Gy, respectively. *P > 0.05 and **P < 0.05, compared with 2 Gy radiation alone. (c) Effect of inhibiting autophagy on the clonogenic survival of HeLa cells. Square, round, triangular, and diamond symbols represent the radiation groups, radiation + siRNA Beclin 1 groups, radiation + siRNA Atg5 groups, and radiation + siRNA Beclin 1 and Atg5 groups, respectively.

Mentions: To assess whether autophagy contributes to the resistance or sensitivity of tumor cells to high-LET radiation, HeLa cells were used in our experiment. Two key autophagy regulators, Beclin 1 and Atg5, were depleted with siRNA. Beclin 1 and Atg5 are required at vesicle nucleation and elongation steps, respectively.(27) These two genes were knocked down separately and together. The double KD result is shown in Figure 3(a). The degree of KD achieved for both genes was >50% at 24 and 48 h. Twenty-four hours after transfection, HeLa cells were irradiated with the carbon ions of 75 keV/μm. The inhibitory effect of siRNA on the proportion of AVO-positive cells was analyzed with flow cytometry. As shown in Figure 4(b), the KD of Beclin 1 together with Atg5 depressed the high-LET radiation-induced AVO formation significantly, whereas Beclin 1 or Atg5 KD alone had only a marginal effect. Shown in Figure 4(c) are the survival curves for HeLa cells under the conditions of irradiation, irradiation + siRNA Beclin 1, irradiation + siRNA Atg5, and irradiation + siRNA Beclin 1 and Atg5. The survival fractions of the cells treated with siRNA of both genes were significantly lower than those of the other three groups (P < 0.05 at the various doses).


Role of autophagy in high linear energy transfer radiation-induced cytotoxicity to tumor cells.

Jin X, Liu Y, Ye F, Liu X, Furusawa Y, Wu Q, Li F, Zheng X, Dai Z, Li Q - Cancer Sci. (2014)

Effect of Beclin 1 and Atg5 siRNA knockdown on the sensitivity of HeLa cells to high linear energy transfer radiation. (a) Western blot analysis of Beclin 1 and Atg5 levels in double knockdown (KD) HeLa cells. The relative level of proteins in comparison to β-actin is indicated below each immunoblot image. (b) Inhibition of siRNA on autophagy induced by the carbon ions after 24 h, KD of Beclin 1 together with Atg5 significantly attenuated the high linear energy transfer radiation-induced formation of acidic vesicular organelles (AVOs). Beclin 1 or Atg5 KD alone had only marginal effects. The white and gray columns indicate the control groups and the irradiation groups with 75 keV/μm carbon ions at 2 Gy, respectively. *P > 0.05 and **P < 0.05, compared with 2 Gy radiation alone. (c) Effect of inhibiting autophagy on the clonogenic survival of HeLa cells. Square, round, triangular, and diamond symbols represent the radiation groups, radiation + siRNA Beclin 1 groups, radiation + siRNA Atg5 groups, and radiation + siRNA Beclin 1 and Atg5 groups, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317928&req=5

fig04: Effect of Beclin 1 and Atg5 siRNA knockdown on the sensitivity of HeLa cells to high linear energy transfer radiation. (a) Western blot analysis of Beclin 1 and Atg5 levels in double knockdown (KD) HeLa cells. The relative level of proteins in comparison to β-actin is indicated below each immunoblot image. (b) Inhibition of siRNA on autophagy induced by the carbon ions after 24 h, KD of Beclin 1 together with Atg5 significantly attenuated the high linear energy transfer radiation-induced formation of acidic vesicular organelles (AVOs). Beclin 1 or Atg5 KD alone had only marginal effects. The white and gray columns indicate the control groups and the irradiation groups with 75 keV/μm carbon ions at 2 Gy, respectively. *P > 0.05 and **P < 0.05, compared with 2 Gy radiation alone. (c) Effect of inhibiting autophagy on the clonogenic survival of HeLa cells. Square, round, triangular, and diamond symbols represent the radiation groups, radiation + siRNA Beclin 1 groups, radiation + siRNA Atg5 groups, and radiation + siRNA Beclin 1 and Atg5 groups, respectively.
Mentions: To assess whether autophagy contributes to the resistance or sensitivity of tumor cells to high-LET radiation, HeLa cells were used in our experiment. Two key autophagy regulators, Beclin 1 and Atg5, were depleted with siRNA. Beclin 1 and Atg5 are required at vesicle nucleation and elongation steps, respectively.(27) These two genes were knocked down separately and together. The double KD result is shown in Figure 3(a). The degree of KD achieved for both genes was >50% at 24 and 48 h. Twenty-four hours after transfection, HeLa cells were irradiated with the carbon ions of 75 keV/μm. The inhibitory effect of siRNA on the proportion of AVO-positive cells was analyzed with flow cytometry. As shown in Figure 4(b), the KD of Beclin 1 together with Atg5 depressed the high-LET radiation-induced AVO formation significantly, whereas Beclin 1 or Atg5 KD alone had only a marginal effect. Shown in Figure 4(c) are the survival curves for HeLa cells under the conditions of irradiation, irradiation + siRNA Beclin 1, irradiation + siRNA Atg5, and irradiation + siRNA Beclin 1 and Atg5. The survival fractions of the cells treated with siRNA of both genes were significantly lower than those of the other three groups (P < 0.05 at the various doses).

Bottom Line: Heavy-ion radiotherapy has a potential advantage over conventional radiotherapy due to improved dose distribution and a higher biological effectiveness in cancer therapy.Autophagy, as a novel important target to improve anticancer therapy, has recently attracted considerable attention.Our data imply that targeting autophagy might enhance the effectiveness of heavy-ion radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China; Key Laboratory of Heavy Ion Radiation Biology and Medicine, Chinese Academy of Sciences, Lanzhou, China.

Show MeSH
Related in: MedlinePlus