Overexpression of Hypermethylated Homeobox A11 (HOXA11) Inhibits Tumor Cell Growth and Induces Apoptosis in Cervical Cancer

Abstract

This study aimed to elucidate the potential of Homeobox A11 (HOXA11) as a therapeutic target and a diagnostic methylation marker for cervical cancer. Gene expression analysis using cDNA microarray in cervical cancer cell lines revealed significantly reduced expression of the HOXA11 gene. Subsequent investigation of HOXA11 promoter methylation in samples from normal individuals and invasive cervical cancer patients showed over 53.2% higher methylation in cancer scrapes compared to normal scrapes. Furthermore, overexpression of HOXA11, which is downregulated in cervical cancer, strongly suppressed cell growth in cervical cancer cell lines, HeLa and HT3. Additionally, we performed transferase dUTP nick end labeling assay and confirmed that the inhibition of cervical cancer cell proliferation occurred via apoptosis. Mechanistically, overexpression of HOXA11 led to mitochondrial apoptosis characterized by PARP cleavage due to increased c-Myc and enhanced cytochrome C secretion into the cytoplasm. These findings suggest that HOXA11 could potentially serve as a methylation marker for diagnosing cervical cancer and as a novel therapeutic target for its treatment.

Keywords: Apoptosis; Cervical cancer; Homeobox A11 (HOXA11); Methylation marker.

Fig. 1.. Homeobox A11 (HOXA11) is hypermethylated in cervical cancer.

Quantitative methylation analysis of HOXA11 promoter in cervical scrapes (n=387) representing normal, various cervical dysplasia and biopsy-confirmed invasive cervical cancer. Average methylation percentages on the HOXA11 promoter are represented by box and whisker plots.

Fig. 2.. Homeobox A11 (HOXA11) inhibits tumor cell growth.

Cervical cancer cells were seeded on 24-well culture plates and attached for 24 hours. HeLa and HT3 cancer cells were transiently transfected with expression vectors encoding HOXA11 and the number of survival cells was counted at 1 day intervals for 3 days. Results are means±SD from three samples each. The cells were observed at 40× magnification.

Fig. 3.. Homeobox A11 (HOXA11) induces apoptosis.

HeLa cells were transiently transfected with pcDNA-PACAP and a transferase dUTP nick end labeling (TUNEL) assay was performed 48 h after transfection. Images were captured on a fluorescence microscope. BF, bright field. The cells were observed at 40× magnification.

Fig. 4.. Homeobox A11 (HOXA11) induces mitochondrial apoptotic cancer cell death.

HeLa cells were transiently transfected with control pcDNA3.1 and pcDNA3.1-HOXA11. (A) Mitochondrial and cytosolic fractions were collected at 48 hrs after transfection and quantified cytochrome C concentration by western hybridization. (B) HeLa cells were transiently transfected with pcDNA3.1-HOXA11, then harvested at 24 hrs and 48 hrs after transfection and analyzed by western blot. The expression levels of HOXA11, Bax, cleaved PARP, c-Myc, VEGF, and GAPDH were measured.