MCF7 human breast cancer cell line (American Type Culture Collection, Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin (Invitrogen Life Technologies). MCF7 constitutively active (CA)-Akt cells were established by stable transfection with pcDNA3 Myr HA Akt1 (#9008; Addgene, Cambridge, MA, USA) into MCF7 cells. Cycloheximide (CHX), doxorubicin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased from Biomol Research Labs (Farmingdale, NY, USA). All other reagents were obtained from Sigma-Aldrich, unless otherwise specified.

Human Aven cDNA was cloned into the pEGFP-C1 vector to generate GFP-Aven. The cells on 60 mm plates were transfected using TransIT-2020 (Mirus, Madison, WI, USA) according to the manufacturer’s instructions. The cells were subcultured for 24 h prior to transfection on 60 mm plates. For use in transfection, 15 μl TransIT-2020 was mixed with 5 μg of plasmid and incubated for 30 min at room temperature. The TransIT-2020 reagent:DNA complexes were added dropwise into the wells and then incubated for 72 h. Following transfection, cell lysates were collected and analyzed further.

Cells were lysed with 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 5 mM EGTA, 15 mM sodium pyrophosphate, 30 mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride and 1% Nonidet P-40 for 20 min at 4°C and subsequently centrifuged at 15,000 × g for 15 min at 4°C. Cell lysates were boiled in Laemmli sample buffer for 3 min, and 30 μg protein was subjected to either 10 or 12% SDS-PAGE (Mini-Protean Electrophoresis system; Bio-Rad, Hercules, CA, USA) depending on the molecular weight of the target proteins. Proteins were then transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA) according to standard procedures. Membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 (M. Biotech, Hanam, South Korea) and 5% non-fat dry milk for 1 h. Following blocking, membranes were probed overnight at 4°C with primary antibodies in antibody dilution buffer (Tris-buffered saline containing 0.05% Tween 20 and 1% nonfat dry milk), followed by 1 h incubation with secondary antibodies at room temperature. Antibody detection was accomplished with WesternBright™ ECL (Advansta Inc., Menlo Park, CA, USA) and exposed on Hyperfilm (Eastman Kodak Co., Rochester, NY, USA). The following primary antibodies were used: rabbit polyclonal immunoglobulin G (IgG) anti-green fluorescent protein (GFP; sc-8334; 1:1,000), rabbit polyclonal IgG anti-poly ADP ribose polymerase (PARP; sc-7150; 1:1,000) and rabbit polyclonal IgG anti-p21 (sc-756; 1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); rabbit polyclonal anti-pAkt (#9271s; 1:1,000), rabbit polyclonal anti-Akt (#9272s; 1:1,000), rabbit polyclonal anti-Aven (#2300s; 1:1,000) and mouse monoclonal IgG_2a anti-pS6 (#9206s; 1:1,000) obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA); mouse monoclonal anti-cathepsin D (#610800; 1:1,000) purchased from BD Biosciences (San Diego, CA, USA) and mouse monoclonal IgG₁ anti-β-actin (A1978; 1:3,000) purchased from Sigma-Aldrich. Peroxidase-conjugated secondary antibodies polyclonal goat anti-rabbit IgG (#111-035-003; 1:1,000) and polyclonal goat anti-mouse IgG (#115-035-003; 1:1,000) were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, USA).

Single-stranded cDNA was made with reverse transcriptase (M. Biotech, Hanam, South Korea) from 1 μg of RNA using an oligo-(dT)₁₈ primer. Subsequently, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the relative expression levels of the Aven and GAPDH genes. PCR was performed for 35 cycles with an annealing temperature of 55°C. PCR products were analyzed by electrophoresis on agarose gels.

For the protein stability analysis, the cells were treated with 10 μg/ml CHX and then whole cell lysates were prepared at 0, 0.25, 0.5, 1, 3, 6, 12 and 24 h. The lysates (50 μg) were then subjected to western blot analysis to identify the Aven and β-actin proteins.

The MTT viability assay was performed with slight modifications as previously described (15,16). MTT was first prepared as a stock solution of 5 mg/ml in phosphate-buffered saline (PBS; pH 7.2) and was filtered. Briefly, cells (200 μl/well) were seeded at 3×10⁴ cells/ml into 96 well plates in the presence of various concentrations (0.005–1 μM) of doxorubicin (or vehicle control) for 72 h. MTT was added to the cultures during the last 4 h of incubation and then the media was removed with a needle and syringe. The blue formazan crystals trapped in cells were dissolved in sterile dimethyl sulfoxide (100 μl) by incubating at room temperature for 30 min. The well plate was read using the Epoch Microplate Spectrophotometer™ (BioTek Instruments, Inc., Winooski, VT, USA) with absorbance at a wavelength of 570 nm with a reference wavelength of 630 nm.

To identify the molecular mechanism involved in Aven expression, the expression level of Aven in several cell lines was analyzed. Notably, the expression level of Aven in MCF7 CA-Akt cell lines was significantly higher than that of its parental MCF7 cells (Fig. 1A). To confirm the possible involvement of the Akt signaling pathway in Aven expression, LY294002, an upstream kinase inhibitor of Akt, was administered to MCF7 CA-Akt cells. As shown in Fig. 1B, Aven expression was significantly abrogated by LY294002 treatment, suggesting that the expression level of Aven is associated with the Akt signaling pathway. In addition, this hypothesis was further supported by the observation that ectopic expression of the constitutively active form of Akt significantly increases the level of Aven in MCF7 cells, but its dominant negative form does not (Fig. 1C).

To elucidate whether the level of Aven may be regulated by a transcriptional process, the mRNA level of Aven was analyzed in MCF7 and MCF7 CA-Akt cells. As shown in Fig. 2A, no significant difference was observed in the mRNA levels of Aven between these two cells, which was further confirmed by qPCR (data not shown). This finding indicates that accumulation of Aven in MCF7 CA-Akt cells is regulated in a transcription-independent manner. Subsequently, the degradation rate of the Aven protein in MCF7 cells was determined using western blot analysis along a time course following addition of the protein synthesis inhibitor, cycloheximide. As shown in Fig. 2B, Aven protein stability was not significantly altered up to 24 h (Fig. 2B), indicating that Aven is a stable protein in cellular systems. In addition, the protein level of Aven in MCF7 CA-Akt cells was not altered during the 24 h time period (data not shown). All these results suggest that accumulation of Aven in MCF7 CA-Akt cells may not be affected by its stability.

A previous study revealed that full-length Aven may be cleaved by cathepsin D to unleash its anti-apoptotic potential (10). There are two cleavage sites between amino acid 144/145 and between amino acid 196/197 in the Aven protein. Therefore, it was investigated whether cathepsin D regulates the level of Aven. As shown in Fig. 3A, the expression level of cathepsin D in MCF7 CA-Akt cells was lower than that of MCF7 cells, which was inversely correlated with Aven levels, indicating that the expression of cathepsin D may be a major determinant for full-length Aven level in MCF7 CA-Akt cells. To confirm this hypothesis, Aven levels were compared in the absence or presence of pepstatin A, an inhibitor of cathepsin D. As shown in Fig. 3B, Aven levels were markedly increased following treatment with pepstatin A, supporting the possible role of cathepsin D in the regulation of Aven levels. Subsequently, the present study aimed to detect cleaved Aven fragment, which is generated by cathepsin D activity in MCF7 cells, however, the Aven antibody was not able to detect the fragment (data not shown). Thus, a GFP-Aven overexpression system was used. When GFP-Aven was overexpressed in MCF7 cells, full-length GFP Aven and cleaved GFP-Aven were clearly detected. However, the only form of GFP-Aven in MCF7 CA-Akt cells was full-length and the cleaved form was not detected (Fig. 3C). In addition, pepstatin A treatment resulted in an evident increase in the full-length form and a decrease in the cleaved form (Fig. 3C). These findings support the hypothesis that the cellular level of full-length Aven is determined by cathepsin D activity, which may be regulated through the Akt signaling pathway.

It has previously been demonstrated that ΔN-Aven cleaved by cathepsin D is the active form for the anti-apoptotic functions of Aven (10). Thus, the present study aimed to determine whether the expression levels of Aven affect the sensitivity of cancer cells to chemotherapeutic agents. The present data demonstrate that MCF7 CA-Akt cells are more sensitive to doxorubicin than the parental MCF7 cells, which was evidenced by a cell proliferation assay (Fig. 4A) and PARP cleavage (Fig. 4B). These results suggest that higher levels of ΔN-Aven may contribute to doxorubicin resistance. To further clarify this theory, the effect of pepstatin A on doxorubicin-induced apoptosis in MCF7 cells was determined. As shown in Fig. 4C, pepstatin A treatment led to an increase in doxorubicin-induced PARP cleavage. These results indicate that inhibition of ΔN-Aven formation by pepstatin A is associated with increased apoptosis in MCF7 cells and full-length Aven may be insufficient to inhibit apoptosis.