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Ablation of EIF5A2 induces tumor vasculature remodeling and improves tumor response to chemotherapy via regulation of matrix metalloproteinase 2 expression.

Wang FW, Cai MY, Mai SJ, Chen JW, Bai HY, Li Y, Liao YJ, Li CP, Tian XP, Kung HF, Guan XY, Xie D - Oncotarget (2014)

Bottom Line: In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients.Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU).Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Sun Yat-sen University Cancer Center, Guangzhou, China. These authors contributed equally to this work.

ABSTRACT

Unlabelled: Hepatocellular carcinoma (HCC) is a highly vascularized tumor with poor clinical outcome. Our previous work has shown that eukaryotic initiation factor 5A2 (EIF5A2) over-expression enhances HCC cell metastasis. In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients. Both in vitro and in vivo assays indicated that ablation of endogenous EIF5A2 inhibited tumor angiogenesis by reducing matrix metalloproteinase 2 (MMP-2) expression. Given that MMP-2 degrades collagen IV, a main component of the vascular basement membrane (BM), we subsequently investigated the effect of EIF5A2 on tumor vasculature remodeling using complementary approaches, including fluorescent immunostaining, transmission electron microscopy, tumor perfusion assays and tumor hypoxia assays. Taken together, our results indicate that EIF5A2 silencing increases tumor vessel wall continuity, increases blood perfusion and improves tumor oxygenation. Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU). Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.

Conclusion: This study highlights an important role of EIF5A2 in HCC tumor vessel remodeling and indicates that EIF5A2 represents a potential therapeutic target in the treatment of HCC.

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Related in: MedlinePlus

EIF5A2 ablation increases tumor blood perfusion, reduces tumor hypoxia and enhances sensitivity to chemotherapy(A) Representative images of Doppler laser imaging of intratumoral blood flow. Left side: Doppler imaging; Right side: the corresponding position of the Doppler imaging on the mice. The mean value of the blood flow is the mean number of the blood flow of six tumors in one group mice. Bar graphs show quantification of corresponding perfusions. P=0.011. (B) Increased tumor vessel perfusion and reduced vessel leakiness in the PLC8024-sh#3 group following injection with Texas Red-conjugated Dextran (red) and FITC-conjugated Lectin (green). White arrowheads on merged images indicated sites of leakage. (C) Reduced staining for pimonidazole in tumors derived from PLC8024 cells transfected with NC or sh#3. Arrowheads indicated necrosis regions. Cells peripheral to necrotic areas were stained most intensely, indicating severe hypoxia. (D) EIF5A2 silencing sensitizes xenografts to 5-fluorouracil. Representative images of xenograft tumors induced by PLC8024-NC or PLC8024-sh#3 in nude mice. Left column, before treatment; middle column, 5 weeks after treatment with 5-FU (intraperitoneal injection, 40mg/kg body weight, once weekly for 5 weeks); right column, representative tumors excised from mice after 5 weeks of treatment. E. Tumor growth curves (N=6 mice per group); tumor volumes were measured on a weekly basis.
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Figure 5: EIF5A2 ablation increases tumor blood perfusion, reduces tumor hypoxia and enhances sensitivity to chemotherapy(A) Representative images of Doppler laser imaging of intratumoral blood flow. Left side: Doppler imaging; Right side: the corresponding position of the Doppler imaging on the mice. The mean value of the blood flow is the mean number of the blood flow of six tumors in one group mice. Bar graphs show quantification of corresponding perfusions. P=0.011. (B) Increased tumor vessel perfusion and reduced vessel leakiness in the PLC8024-sh#3 group following injection with Texas Red-conjugated Dextran (red) and FITC-conjugated Lectin (green). White arrowheads on merged images indicated sites of leakage. (C) Reduced staining for pimonidazole in tumors derived from PLC8024 cells transfected with NC or sh#3. Arrowheads indicated necrosis regions. Cells peripheral to necrotic areas were stained most intensely, indicating severe hypoxia. (D) EIF5A2 silencing sensitizes xenografts to 5-fluorouracil. Representative images of xenograft tumors induced by PLC8024-NC or PLC8024-sh#3 in nude mice. Left column, before treatment; middle column, 5 weeks after treatment with 5-FU (intraperitoneal injection, 40mg/kg body weight, once weekly for 5 weeks); right column, representative tumors excised from mice after 5 weeks of treatment. E. Tumor growth curves (N=6 mice per group); tumor volumes were measured on a weekly basis.

Mentions: We further assessed the effect of EIF5A2 ablation on blood perfusion and the tumor microenvironment. Tumor blood vessel flow, as measured by laser Doppler imaging, was significantly increased in the EIF5A2-silenced group compared to the NC control group (Fig.5A). To further examine intratumoral blood perfusion and leakage, a mouse model was created by injecting FITC-Lectin and Dextran into the tail vein of tumor bearing nude mice. Fluorescent immunostaining of tumor sections indicated that EIF5A2 downregulation increased tumor perfusion while reducing blood leakage (Fig. 5B). Since supplying oxygen is an ancestral function of vessels, we hypothesized that EIF5A2 inhibition would improve tumor oxygenation. IHC staining of the hypoxia-marker pimonidazole revealed a reduction in hypoxia in PLC8024-sh#3 cells (Fig. 5C). We then investigated whether endogenous EIF5A2 ablation could increase the sensitivity of HCC cells to chemotherapy. Twelve nude mice were injected subcutaneously with either PLC8024-NC (n=6) or PLC8024-sh#3 (n=6) cells. When the xenograft tumors reached ~5 mm3 in size, mice were injected intraperitoneally with 5-FU (40 mg/kg body weight once per week for 5 weeks). In comparison with the PLC8024-NC group, tumor growth rates and tumor volumes were significantly reduced in the PLC8024-sh#3 group (p<0.05, Fig. 5D and 5E). These data suggested that silencing of endogenous EIF5A2 expression could reduce tumor hypoxia, thus increase the chemosensitivity to 5-FU by remodeling tumor vessels.


Ablation of EIF5A2 induces tumor vasculature remodeling and improves tumor response to chemotherapy via regulation of matrix metalloproteinase 2 expression.

Wang FW, Cai MY, Mai SJ, Chen JW, Bai HY, Li Y, Liao YJ, Li CP, Tian XP, Kung HF, Guan XY, Xie D - Oncotarget (2014)

EIF5A2 ablation increases tumor blood perfusion, reduces tumor hypoxia and enhances sensitivity to chemotherapy(A) Representative images of Doppler laser imaging of intratumoral blood flow. Left side: Doppler imaging; Right side: the corresponding position of the Doppler imaging on the mice. The mean value of the blood flow is the mean number of the blood flow of six tumors in one group mice. Bar graphs show quantification of corresponding perfusions. P=0.011. (B) Increased tumor vessel perfusion and reduced vessel leakiness in the PLC8024-sh#3 group following injection with Texas Red-conjugated Dextran (red) and FITC-conjugated Lectin (green). White arrowheads on merged images indicated sites of leakage. (C) Reduced staining for pimonidazole in tumors derived from PLC8024 cells transfected with NC or sh#3. Arrowheads indicated necrosis regions. Cells peripheral to necrotic areas were stained most intensely, indicating severe hypoxia. (D) EIF5A2 silencing sensitizes xenografts to 5-fluorouracil. Representative images of xenograft tumors induced by PLC8024-NC or PLC8024-sh#3 in nude mice. Left column, before treatment; middle column, 5 weeks after treatment with 5-FU (intraperitoneal injection, 40mg/kg body weight, once weekly for 5 weeks); right column, representative tumors excised from mice after 5 weeks of treatment. E. Tumor growth curves (N=6 mice per group); tumor volumes were measured on a weekly basis.
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Related In: Results  -  Collection

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Figure 5: EIF5A2 ablation increases tumor blood perfusion, reduces tumor hypoxia and enhances sensitivity to chemotherapy(A) Representative images of Doppler laser imaging of intratumoral blood flow. Left side: Doppler imaging; Right side: the corresponding position of the Doppler imaging on the mice. The mean value of the blood flow is the mean number of the blood flow of six tumors in one group mice. Bar graphs show quantification of corresponding perfusions. P=0.011. (B) Increased tumor vessel perfusion and reduced vessel leakiness in the PLC8024-sh#3 group following injection with Texas Red-conjugated Dextran (red) and FITC-conjugated Lectin (green). White arrowheads on merged images indicated sites of leakage. (C) Reduced staining for pimonidazole in tumors derived from PLC8024 cells transfected with NC or sh#3. Arrowheads indicated necrosis regions. Cells peripheral to necrotic areas were stained most intensely, indicating severe hypoxia. (D) EIF5A2 silencing sensitizes xenografts to 5-fluorouracil. Representative images of xenograft tumors induced by PLC8024-NC or PLC8024-sh#3 in nude mice. Left column, before treatment; middle column, 5 weeks after treatment with 5-FU (intraperitoneal injection, 40mg/kg body weight, once weekly for 5 weeks); right column, representative tumors excised from mice after 5 weeks of treatment. E. Tumor growth curves (N=6 mice per group); tumor volumes were measured on a weekly basis.
Mentions: We further assessed the effect of EIF5A2 ablation on blood perfusion and the tumor microenvironment. Tumor blood vessel flow, as measured by laser Doppler imaging, was significantly increased in the EIF5A2-silenced group compared to the NC control group (Fig.5A). To further examine intratumoral blood perfusion and leakage, a mouse model was created by injecting FITC-Lectin and Dextran into the tail vein of tumor bearing nude mice. Fluorescent immunostaining of tumor sections indicated that EIF5A2 downregulation increased tumor perfusion while reducing blood leakage (Fig. 5B). Since supplying oxygen is an ancestral function of vessels, we hypothesized that EIF5A2 inhibition would improve tumor oxygenation. IHC staining of the hypoxia-marker pimonidazole revealed a reduction in hypoxia in PLC8024-sh#3 cells (Fig. 5C). We then investigated whether endogenous EIF5A2 ablation could increase the sensitivity of HCC cells to chemotherapy. Twelve nude mice were injected subcutaneously with either PLC8024-NC (n=6) or PLC8024-sh#3 (n=6) cells. When the xenograft tumors reached ~5 mm3 in size, mice were injected intraperitoneally with 5-FU (40 mg/kg body weight once per week for 5 weeks). In comparison with the PLC8024-NC group, tumor growth rates and tumor volumes were significantly reduced in the PLC8024-sh#3 group (p<0.05, Fig. 5D and 5E). These data suggested that silencing of endogenous EIF5A2 expression could reduce tumor hypoxia, thus increase the chemosensitivity to 5-FU by remodeling tumor vessels.

Bottom Line: In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients.Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU).Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Sun Yat-sen University Cancer Center, Guangzhou, China. These authors contributed equally to this work.

ABSTRACT

Unlabelled: Hepatocellular carcinoma (HCC) is a highly vascularized tumor with poor clinical outcome. Our previous work has shown that eukaryotic initiation factor 5A2 (EIF5A2) over-expression enhances HCC cell metastasis. In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients. Both in vitro and in vivo assays indicated that ablation of endogenous EIF5A2 inhibited tumor angiogenesis by reducing matrix metalloproteinase 2 (MMP-2) expression. Given that MMP-2 degrades collagen IV, a main component of the vascular basement membrane (BM), we subsequently investigated the effect of EIF5A2 on tumor vasculature remodeling using complementary approaches, including fluorescent immunostaining, transmission electron microscopy, tumor perfusion assays and tumor hypoxia assays. Taken together, our results indicate that EIF5A2 silencing increases tumor vessel wall continuity, increases blood perfusion and improves tumor oxygenation. Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU). Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.

Conclusion: This study highlights an important role of EIF5A2 in HCC tumor vessel remodeling and indicates that EIF5A2 represents a potential therapeutic target in the treatment of HCC.

Show MeSH
Related in: MedlinePlus