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Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer.

Cho H, Chung JY, Song KH, Noh KH, Kim BW, Chung EJ, Ylaya K, Kim JH, Kim TW, Hewitt SM, Kim JH - BMC Cancer (2014)

Bottom Line: Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004).In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival.API5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, 146-92 Dogok-Dong, Gangnam-Gu, Seoul 135-720, South Korea. genejock@helix.nih.gov.

ABSTRACT

Background: The apoptosis inhibitor-5 (API5), anti-apoptosis protein, is considered a key molecule in the tumor progression and malignant phenotype of tumor cells. Here, we investigated API5 expression in cervical cancer, its clinical significance, and its relationship with phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in development and progression of cervical cancer.

Methods: API5 effects on cell growth were assessed in cervical cancer cell lines. API5 and pERK1/2 immunohistochemical staining were performed on a cervical cancer tissue microarray consisting of 173 primary cervical cancers, 306 cervical intraepithelial neoplasias (CINs), and 429 matched normal tissues.

Results: API5 overexpression promoted cell proliferation and colony formation in CaSki cells, whereas API5 knockdown inhibited the both properties in HeLa cells. Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004). API5 expression levels were positively associated with pERK1/2 in cervical cancer (P < 0.001) and high grade CIN (P = 0.031). In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival.

Conclusions: API5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.

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API5 expression and its localization in various cervical cancer cell lines. (A) Characterization of API5 expression in various cervical cancer cell lines by western blot analysis. (B) Nuclear and cytoplasmic fractions from HeLa cells were analyzed by western blot analysis. Calnexin and Lamin B1 were used as an index for cytosolic or nuclear fraction, respectively. (C) Confocal fluorescent microscopy was used to further evaluate the distribution of API5 in HeLa cells 24 hrs after transfection of pEGFP-API5. DAPI fluorescent dye was used for a nuclear counterstaining. Magnified images of boxed areas are shown in the lower panels. Arrowheads indicate cytoplasmic EGFP-API5 in the transfected HeLa cells.
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Fig1: API5 expression and its localization in various cervical cancer cell lines. (A) Characterization of API5 expression in various cervical cancer cell lines by western blot analysis. (B) Nuclear and cytoplasmic fractions from HeLa cells were analyzed by western blot analysis. Calnexin and Lamin B1 were used as an index for cytosolic or nuclear fraction, respectively. (C) Confocal fluorescent microscopy was used to further evaluate the distribution of API5 in HeLa cells 24 hrs after transfection of pEGFP-API5. DAPI fluorescent dye was used for a nuclear counterstaining. Magnified images of boxed areas are shown in the lower panels. Arrowheads indicate cytoplasmic EGFP-API5 in the transfected HeLa cells.

Mentions: The expression of human API5 was investigated in human cervical cancer cell lines using western blot analysis. HEK 293 human embryonic kidney epithelial cells were used as a control cell line representing non-tumorigenic cells. As shown in Figure 1A, API5 was detected as doublet bands, as has been reported in mammals [13]. Expression of API5 was most profound in HeLa and C33A while that in CaSki and SiHa was similar to non-turmorigenic HEK293 cells. We further analyzed the expression of API5 protein in cytoplasmic and nuclear fractions of the HeLa cells which have the highest expression of API5 among the cervical cancer cell lines examined by western blot analysis. As shown in Figure 1B, API5 was exclusively detected in the nuclear fraction. To further confirm the nuclear/cytosolic localization of API5, HeLa cells were transfected with pEGFP-Api5 DNA, and, in turn, examined with confocal laser scanning microscopy after counterstaining nuclear with DAPI. As shown in Figure 1C, we observed the dominant localization of API5 in nucleus although cytoplasmic API5 (indicated by arrowheads) was observed in small population of the transfected HeLa cells (less than 8%). We also observed a similar localization pattern of endogenous API5 in CaSki cells after immunofluorescence staining (Additional file 1: Figure S1). Taken together, these results demonstrate that API5 expresses in cervical cancer cell lines and is primarily localized in nucleus.Figure 1


Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer.

Cho H, Chung JY, Song KH, Noh KH, Kim BW, Chung EJ, Ylaya K, Kim JH, Kim TW, Hewitt SM, Kim JH - BMC Cancer (2014)

API5 expression and its localization in various cervical cancer cell lines. (A) Characterization of API5 expression in various cervical cancer cell lines by western blot analysis. (B) Nuclear and cytoplasmic fractions from HeLa cells were analyzed by western blot analysis. Calnexin and Lamin B1 were used as an index for cytosolic or nuclear fraction, respectively. (C) Confocal fluorescent microscopy was used to further evaluate the distribution of API5 in HeLa cells 24 hrs after transfection of pEGFP-API5. DAPI fluorescent dye was used for a nuclear counterstaining. Magnified images of boxed areas are shown in the lower panels. Arrowheads indicate cytoplasmic EGFP-API5 in the transfected HeLa cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125689&req=5

Fig1: API5 expression and its localization in various cervical cancer cell lines. (A) Characterization of API5 expression in various cervical cancer cell lines by western blot analysis. (B) Nuclear and cytoplasmic fractions from HeLa cells were analyzed by western blot analysis. Calnexin and Lamin B1 were used as an index for cytosolic or nuclear fraction, respectively. (C) Confocal fluorescent microscopy was used to further evaluate the distribution of API5 in HeLa cells 24 hrs after transfection of pEGFP-API5. DAPI fluorescent dye was used for a nuclear counterstaining. Magnified images of boxed areas are shown in the lower panels. Arrowheads indicate cytoplasmic EGFP-API5 in the transfected HeLa cells.
Mentions: The expression of human API5 was investigated in human cervical cancer cell lines using western blot analysis. HEK 293 human embryonic kidney epithelial cells were used as a control cell line representing non-tumorigenic cells. As shown in Figure 1A, API5 was detected as doublet bands, as has been reported in mammals [13]. Expression of API5 was most profound in HeLa and C33A while that in CaSki and SiHa was similar to non-turmorigenic HEK293 cells. We further analyzed the expression of API5 protein in cytoplasmic and nuclear fractions of the HeLa cells which have the highest expression of API5 among the cervical cancer cell lines examined by western blot analysis. As shown in Figure 1B, API5 was exclusively detected in the nuclear fraction. To further confirm the nuclear/cytosolic localization of API5, HeLa cells were transfected with pEGFP-Api5 DNA, and, in turn, examined with confocal laser scanning microscopy after counterstaining nuclear with DAPI. As shown in Figure 1C, we observed the dominant localization of API5 in nucleus although cytoplasmic API5 (indicated by arrowheads) was observed in small population of the transfected HeLa cells (less than 8%). We also observed a similar localization pattern of endogenous API5 in CaSki cells after immunofluorescence staining (Additional file 1: Figure S1). Taken together, these results demonstrate that API5 expresses in cervical cancer cell lines and is primarily localized in nucleus.Figure 1

Bottom Line: Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004).In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival.API5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, 146-92 Dogok-Dong, Gangnam-Gu, Seoul 135-720, South Korea. genejock@helix.nih.gov.

ABSTRACT

Background: The apoptosis inhibitor-5 (API5), anti-apoptosis protein, is considered a key molecule in the tumor progression and malignant phenotype of tumor cells. Here, we investigated API5 expression in cervical cancer, its clinical significance, and its relationship with phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in development and progression of cervical cancer.

Methods: API5 effects on cell growth were assessed in cervical cancer cell lines. API5 and pERK1/2 immunohistochemical staining were performed on a cervical cancer tissue microarray consisting of 173 primary cervical cancers, 306 cervical intraepithelial neoplasias (CINs), and 429 matched normal tissues.

Results: API5 overexpression promoted cell proliferation and colony formation in CaSki cells, whereas API5 knockdown inhibited the both properties in HeLa cells. Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004). API5 expression levels were positively associated with pERK1/2 in cervical cancer (P < 0.001) and high grade CIN (P = 0.031). In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival.

Conclusions: API5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.

Show MeSH
Related in: MedlinePlus