Lac Z adenovirus and TGFβRII dominant negative (TGFβRII DN) adenovirus were kindly provided by Dr. Tae Jun Park (Ajou University, School of Medicine). Huh7-V cells and Huh7-AR cells at 2.5 x 103 cells were plated into 24-well plates. After 24hr, the Huh7-V cells and Huh7-AR cells were found to be infected with 100MOI Lac Z adenovirus or TGFβRII DN adenovirus, and they were then further cultured for 7 days. At indicated times, cells were harvested and counted with hemocytometer; culture medium was changed ever 3 days during experiment. The average counts from triplicate experiments are presented.
M. Statistics
All bars represent mean ± standard deviations of determinants. Experiments were repeated at least three times. Statistical analysis was performed by t-test.
Ⅲ
Ⅲ Ⅲ
Ⅲ. RESULTS
Androgen receptor expression and androgen responsiveness of Huh7-AR cells
To investigate the phisiological role of male sex hormone and its receptor in hepatocarcinogenesis, AR expression was analyzed in human livers by RT-PCR.
Interestingly, more AR expression was observed in non-cancer region than cancer (Fig. 3).
Next, AR overexpressing Huh7 cells, Huh7-AR, and its control, Huh7-V cells, were established, and AR expression was measured by RT-PCR (Fig. 4A) and immunoblot (Fig.
4B) analyses. As seen in the figures, the vector transfected Huh7 cells (Huh7-V) failed to express AR at both the mRNA and protein levels, however, the expression was variable, depending on the levels of AR expression, e.g. single clones (Huh7-AR3) and Huh7-AR mixture (Huh7-AR). To test androgen response in the estabilished cells, Huh7-AR cells were transfected with MMTV-Luc plasmid, using the murine mammary tumor virus (MMTV) promoter sequence as a positive control of androgen responsiveness. In the presence of 10nM DHT, AR expressing cells increased luciferase activity more than that of the Huh7-V (Fig. 4C). To investigate the response of AR to DHT treatment, immunocytochemistry was performed; When treated with 10nM DHT for 2~8 hr, AR was found in the nuclei of Huh7-AR cells, however, it was diffused over the cytoplasm in 8hr (Fig. 4D).
Reduced tumorigenesis of Huh7-AR cells in vivo and in vitro
To evaluate the effect of AR on the growth of HCC cells, the growth rates of Huh7-V and Huh7-AR cells were measured by cell counting. Reduced growth rate of Huh7-AR cells was observed, as compared with that of Huh7-V cells (Fig. 5). To investigate the degree of tumorigenecity of the Huh7-AR cells, clonogenic assay, soft agar colony formation and Matrigel invasion assaies were performed, and the results showed reduced tumorigenicity of the Huh7-AR cells, as compared with that of the Huh7-V cells (Fig. 6A, 6B, 6C).
As shown in Fig. 7, tumor volume of Huh7-V was significantly larger than that of Huh7-AR cells 31days after inoculation. HCC growth of Huh7-AR cells in nude mice was inhibited, in contrast to the HCC of Huh7-V cells. This finding is in good agreement with recent reports that AR induces apoptosis in prostate cancer cells and likely reduces in vivo prostate cancer growth.
Immunoblot analyses revealed that Bcl-XL and cyclin D1 expressions in the Huh7-AR HCC were inhibited, whereas the expression of E-cadherin was increased (Fig. 8).
The above results are well accordant with the suggestion that overexpression of AR may result in the reduced tumorigenesis.
Fig. 3. AR expression in human livers was analyzed by RT-PCR. To investigate the role of male sex hormone and its receptor in hepatocarcinogenesis, AR expression was measured by RT-PCR in human livers. (N; normal liver, C; cancer)
Fig. 4. Expression of androgen receptor in Huh7-AR cells. To investigate the effect of male sex hormone, Huh7-V or Huh7-AR cells after transfection were cultured in DMEM plus charcoal stripped serum (CSS) for 24 hr before adding 10nM DHT. AR expression in Huh-V and Huh7-AR cells was measured by RT-PCR (A) and Western blot analysis (B). (C) Androgen response of Huh7-AR cells by MMTV- promoter assay. Huh7-V and Huh7-AR cells were transfected by using LipofecAMINE with 2㎍ of MMTV-Luc plasmid and 0.5㎍
of pRL-TK (Promega) as an internal control. After washing with PBS, the cells were then incubated for 28 hr in medium containing 10% CSS in the absence or presence of 5 α-dihydrotestosterone (10nM; DHT). The cells were lysed, and luciferase activity was measured according to the Dual-Luciferase Reporter Assay System, and represented as relative light units. All transfection experiments and luciferase assays were carried out in triplicate and also repeated at least twice. (D) The localization of AR was examined by immunocytochemistry after treatment with 10nM DHT. Huh7-AR cells were cultured in DMEM plus 10% charcoal stripped serum (CSS) for 24 hr before adding 10nM DHT.
Fig. 5. Growth rate of Huh7-AR cells was decreased. Huh7-V and Huh7-AR cells (2.5 x 103 cells) were seeded into 24-well plates, and cultured further for 12 days. At the indicated times, cells were harvested and counted with hemocytometer. Culture medium was changed every 3 days during experiment. Mean ± S.D. from two independent experiments. *, **, p value by t-test.
Fig. 6. Reduced tumorigenesis of Huh7-AR cells in vitro. (A) Tumorigenicity of the Huh7-AR cells was determined by clonogenic assay. Huh7-V and Huh7-AR cells (4 x 102/35-mm plate) were maintained at 37°C for 9 days with medium which was changed every other day. Grown colones were stained with crystal violet. CM; DMEM+10% FBS, CS; DMEM+10% CSS, CD; DMEM+10% CSS+ 10nM DHT. Treated with 10nM DHT every day. (B) For soft agar assay, Huh7-V and Huh7-AR cells were plated subconfluently in six-well plates in 0.6% agarose on a 1.0% agarose bed. Every 3 days, the culture medium was added. After 2 weeks, grown colonies were counted by an optical microscope. (C) Invasion assay into Matrigel - Invasive ability of cells was tested through the basement membrane Matrigel in Transwell chambers with 8-㎛ porosity polycarbonate filters. DMEM medium supplemented with 5% FBS was placed in the lower well. Cells suspended in DMEM medium containing 1% FBS were added to the upper chambers (2.5 x 104 cells/well) and incubated for 24 hr at 37°C in 5% CO2 .
Fig. 7. Reduced tumor volumes of Huh7-AR cells in nude mice. Cells (5 x 106 cells per mouse) were subcutaneously injected at the left thigh of six week-old nude mice. (A) The number and volume of HCC developed at the injection site were measured with vernier caliper every 2 days until 31 days. (B) After 31days, the volume of tumors was measured.
Fig. 8. Difference of protein expression in nude mice injected with V and Huh7-AR cells. Tumors were excised when the volume became over 1000 to 3000 mm3 (A) Proteins were extracted from each animal in order to evaluate the expression of AR, E-cadherin, cyclinD1, Bcl-XL, Bax and actin by Western blot analysis, (B) β-Catenin expression by immunohistochemistry.
Dihydrotestosterone induced senescence of Huh7-AR cells, negative regulation of tumorigenesis
When cultured longer in the complete medium (CM), Huh7-AR cells became larger, and stress fibers were significantly expressed. When the expression of SA-β-gal was examined to confirm the senescence-like changes in the Huh7-AR cells, the number of positive cells was significantly higher in the AR maintained in CM than that of Huh7-V in CM (Fig. 9A). When other senescence markers, such as nuclear actin accumulation and cytoplasmic pErk1/2 accumulation, were examined by immunocytochemistry, the Huh7-AR cells cultured in CM revealed actin accumulation in nucleus (Fig. 9B) and pErk1/2 accumulation in cytoplasm (Fig. 9C). As shown in Fig. 9D, telomerase activity was found to be reduced in the Huh7-AR cells, as compared with Huh7-V cells which had been maintained in CM, but not DMEM+10% charcoal stripped serum (CS). Therefore, overexpression of AR seems to be able to induce senescence phenotypes.
Increased TGF-ββββ expression in HCCs by Huh7-AR cells in nude mice
Previously, we reported that androgen regulates transcription of TGF-β1 through direct binding of DHT-AR complex to the potential ARE within TGF-β1 promoter region.
Therefore, we selected TGF-β1 as a regulating factor of tumor formation in the HCC of Huh7-AR cells (Yoon et al., 2006). When TGF-β1 expression was measured by RT-PCR, it
also similar in HCCs developed in nude mice; all HCC tissues developed from the Huh7-AR cells (lanes 5–8, Fig. 10B) expressed higher level of TGF-β1 than the HCC of Huh7-V cells (lanes 1–4, Fig. 10B). The expression of TGF-β1 was also increased in human livers with the increased AR expression (Fig. 10C).
Increase of TGF- β1 secretion in Huh7-AR cells
To further confirm increased TGF-β1 expression in AR- expressing cells, we measured the secretion of TGF-β1 by MTT assay. TGF-β1 secretion was much higher in the Huh7-AR cells than that in the Huh7-V cells (Fig. 11).
These results suggest that, when AR was overexpressed in Huh7 cells, androgen receptor can inhibit tumorigenesis by induction of senescence phenotypes via the increased TGF-β1 expression.
Fig. 9. AR induced senescence phenotype. (A) Huh7-AR cells were stained for detection of SA-β gal activity. Nuclear actin accumulation (B) and cytoplasmic p-Erk1/2 accumulation (C) were examined by immunocytochemistry. (D) Telomerase activity was reduced in the Huh7-AR cells, compared with Huh7-V cells. Huh7-V and Huh7-AR cells were maintained with CM (; DMEM+10% FBS) or CS (DMEM+10% CSS) medium.
Fig. 10. TGF-β1 expression was measured by RT-PCR. (A) TGF-β1 mRNA expression was increased in Huh7-AR cells. (B) TGF-β1expression was increased in HCCs developed by Huh7-AR cells in nude mice. Each lane indicates the data for different animals. (C) TGF-β1expression was increased in AR- expressing human livers (N; Normal liver, C; cancer).
Fig. 11. Secretion of TGF-ββββ1 in Huh7–AR cells. Mv1Lu cells (5.0 x 103 cells per 96-well plate) were cultured in DMEM plus 10% FBS. After 18 hr, the medium was changed with serum free medium containing 0 ~ 1000 pg/ml recombinant hTGF-β1 or the same volume of the conditioned medium harvested from Huh7-V or Huh7-ARcells. After 4 days of incubation, TGF- β1 secretion was measured by MTT assay based on the cell toxicity induced by treatment of the cells with the known amount of recombinant hTGF-β1.
Increased p27KIP1 expression in Huh7-AR cells, but decreased its phosphorylation in cytopalsm of Huh7-AR cells after treatment with DHT
To confirm whether TGF- β1 could reduce the tumorigenicity of AR expressing cells, p27KIP1, downstream target of TGF-β1, expression was examined.
TGF- β1-induced growth arrest of Huh7 cells was accompanied with p27KIP1 expression.
When the related CDKI was examined, p27KIP1 expression in Huh7-AR cells was increased (Fig. 12A), suggesting the TGF- β1 associated growth arrest. When Huh7-V cells were treated with 10nM DHT, p-p27 KIP1 translocation to the cytoplasm was slightly increased for 4 hr, whereas the translocation in Huh7-AR cells was the same regardless of DHT treatment or not (Fig. 12B). These data strongly suggest that AR can induce senescence of Huh7-AR cells when testosterone is present.
Decreased c-myc, β-Catenin and cyclin D1 expression in Huh7-AR cells after DHT treatment
To investigate the effect of TGF- β1 downstream, cells were treated with 10nM DHT, and expression of myc was determined by immunoblot analysis. DHT treatment reduced c-myc expression of Huh7-AR cells in 4 hr, as compared with untreated control, but not in Huh7-V cells (Fig. 13A). To further elucidate the effect, these cells were treated with DHT with or without cyproterone acetate, the anti - androgen. As shown in Fig. 13B, CPA could
cyclin D1 might be involved in tumorigenicity of AR-expressing cells, the levels of β-catenin and c-myc proteins were analysed by Western blotting. Expressions of β-Catenin and cyclin D1, related to HCC development in AR cells, were also reduced with DHT treatment.
These results also strongly support that AR expression can inhibit HCC formation through increasing TGF-β and p27KIP1 expressions and reducing the c-myc, β-Catenin and cyclin D1 expressions.
Fig. 12. Increased p27KIP1 expression in Huh7-AR cells, but decreased its phosphorylation in cytoplasm of Huh7-AR cells after treatment with DHT. (A) Empty vector or AR – transient transfected cells were treated with 10nM DHT for 1 hr and AR and p27KIP1 expressions were analyzed by Western blot analysis. (B) Huh7-V and Huh7-AR cells were treated with 10nM DHT or EtOH. After 4 hr, cells were harvested and fractionated to nuclear and cytoplasmic fractions for analysis of phosphorylated p27KIP1 and
Fig. 13. Decreased c-myc expression in Huh7-AR cells after DHT treatment. (A) Huh7-V and Huh7-AR cells were treated with 10nM DHT for indicated times, and c-myc expression was determined by immunoblotting. Jurkat cell lysates were used for positive control. (B) The cells were treated with DHT (as androgen) and cyproterone acetate [(CPA), as anti-androgen] or not. After 4 hr, AR and c-myc expressions were determined. (-); Not treated, (+); treated
Fig. 14. Decreased cyclin D1 and β-catenin expression in Huh7-AR cells after DHT treatment. Huh7-V and Huh7-AR cells were treated with 10nM DHT for indicated times, and protein levels of AR, β-catenin, and cyclin D1 were analyzed by Western blot analysis.
Recovery of senescence phenotypes of Huh7-AR cells by adenovirus infection of dominant negative TGFβRII construct
To further elucidate the reduced tumorigenesis of Huh7-AR cells by TGF-β1 expression, we used a TGFβRII dominant negative adenovirus to block the TGF-β1 signal. Thus, Huh7-V and Huh7-AR cells were infected with β-galactosidase expressing adenovirus (LacZ -Ad) and dominant negative TGFβRII expressing adenovirus (TGFβRIIDN-Ad) (Fig. 15A).
Infection efficiency of adenovirus was determined by X-gal staining or TGFβRII immunocytochemistry. It was almost 100 percent infection efficiency of Lac Z-Ad or TGFβRIIDN-Ad in Huh7-V cells and Huh7-AR cells (Fig. 15B). When Lac Z-Ad was infected, the growth of AR cells was reduced, as compared with that of Huh7-V cells.
Blocking of TGF-β1 signal with TGFβRIIDN-Ad recovered the reduced growth of Huh7-AR cells (Fig. 15C), and nuclear actin accumulation (Fig. 15D) as well as cytoplasmic pErk1/2 accumulations in Huh7-AR cells (Fig. 15E). These results led us to suggest that AR overexpression could reduce the tumorigenicity of AR overexpressing cells through TGF-β1 induced cellular senescence phenotypes.
Fig. 15. Recovery of senescence phenotype in Huh7-AR cells after dominant negative TGFβRII adenovirus infection (A) Dominant negative TGFβRII adenovirus Scheme (B) Infection efficiency was determined by X-gal staining or immunocytochemistry. (C) Huh7-V and Huh7-AR cells were infected with LacZ-Ad or TGFβRIIDN-Ad and then further cultured for 7 days. At indicated times, growth rates were determined by cells counting, and culture medium was changed every 3 days during experiment. Mean ± S.D. from two independent experiments. *, **, p value by t-test. (D, E) Under the same condition as in (C), actin localization and p-Erk1/2 expression were determined by immunostaining and immunoblotting.
Ⅳ
Ⅳ
Ⅳ Ⅳ. DISCUSSION
In the present study, the potential role of androgen and AR in hepatoma development was investigated after establishment of Huh7-AR and Huh7-V cell lines by transfection of human androgen receptor (AR) in pCMV5 and its empty vector to Huh7 hepatocelluar carcinoma (HCC) cells. Previously, Boix et al. reported that the presence of androgen receptors within the tumor was significantly related to a smaller tumor size: 22 of the 29 nodules < or = 3 cm contained androgen receptors, while only six of the 14 tumors larger than 3 cm (p < 0.05) contained the receptor (Boix et al., 1995).
Reduced growth rate and tumorigenicity of Huh7-AR cells were observed, compared with the Huh7-V cells (Fig. 5, 6A, 6B, 6C). When we injected Huh7-AR or Huh7-V (5 x 106 cells/mouse) cells to control and orchiectomized (OX) nude mice, the volume of Huh7-V tumor was decreased in OX mice (Data not shown). It is well known that development of HCC, including latency, frequency and multiplicity of tumors, in OX animal is significantly decreased compared with those in intact animals (Tsubura et al., 1990). Orchiectomy resulted in a significant reduction of hepatocellular carcinomas (15% in castrated rats versus 81% in control rats) (Rao and Kashireddy, 2002). Tumor volume of Huh7-V was larger than that in Huh7-AR cells 31 days after inoculation. HCC growth of Huh7-AR cells in control nude mice was inhibited, as opposed in the HCC of Huh7-V cells (Fig.7). And the voulum of Huh7-AR induced tumor was significantly smaller than that of Huh7-V tumor in control mice, whereas Huh7-AR tumor was large in the orchiectomized mice (Data not shown). In
vivo study, we suggested that the Huh7-AR tumor was androgen-independent, in contrast to
androgen-dependent in the Huh7-V tumors. Immunohistochemical analyses revealed that E-cadherin expression was inhibited in the Huh7-AR tumors of the control nude mice (Fig.
8A), however, in vivo expression of β-catenin, Bcl-XL and cyclin D1 in the Huh7-AR tumor was decreased (Fig. 8B).
When cultured longer, the Huh7-AR cells became larger and formed significant stress fibers in complete medium (CM). When the expression of SA-β-gal, nuclear actin accumulation, and cytoplasmic p-Erk1/2 accumulation were examined to confirm senescence change in the Huh7-AR cells, the number of positive cells was significantly higher in the Huh7-AR in CM than that of Huh7-V in CM (Fig. 9A). As shown in Fig. 9D, telomerase activity was found to be reduced in the Huh7-AR cells, compared with Huh7-V cells which were maintained with CM, but not DMEM+10% charcoal stripped serum (CS).
Therefore, AR overexpression seems to be able to induce senescence phenotype and androgen induced senescence of Huh7-AR cells, which is a negative regulation of tumor progression.
We earlier reported that androgen regulates transcription of TGF-β1 through direct binding of DHT-AR complex to the potential ARE within TGF-β1 promoter region.
Therefore, we selected TGF-β1 as a factor of tumor formation by AR overexpression. When TGF-β1 expression was measured by RT-PCR, the mRNA expression of TGF- β1 was higher in the Huh7-AR cells and tumors than those in Huh7-V cells, as seen in Fig 10A and 10B.
TGF- β1 has been reported as a tumor-suppressor, and TGF- β1-induced growth arrest
dual functions: to induce growth inhibition and also topromote malignant transformation. In summary, in spite of higher expression of TGF-β1, Huh7-AR cells failed to undergo epithelial mesenchymal transition, but rather became senescent with concomitant expression of p27KIP1 in the presence of androgen.
To further elucidate reduced tumorigenesis and induced senescence phenotype by TGF-β1, we used a TGFβRII dominant negative adenovirus to block the TGF-β1 signal. When
infected with Lac Z- Ad was, the growth of AR cells was reduced, compared to Huh7-V cells.
Blocking of TGF-β1 signal with TGFβRIIDN-Ad could recover the reduced growth of Huh7-AR cells (Fig. 15C), and nuclear actin accumulation (Fig. 15D) and p-Erk1/2 accumulation were not observed in Huh7-AR cells (Fig. 15E).
Therefore, all of the above results strongly indicate that induction of senescence in AR expressing HCC may act as a negative regulator of tumor progression in the presence of testosterone.
Fig. 16. Mechanism to decrease tumorigenesis of AR overexpression in Huh7 cells.
Huh7-AR cells were established by transfection of human AR in Huh7 HCC cells. When Huh7-AR cells were cultured with DHT, TGF-β1 expression was increased. Because of increased TGF-β1 expression, tumorigenicity was decreased and growth rates were reduced.
Also, when cultured in the presence of DHT, cellular senescence phenotypes was induced in Huh7-AR cells, such as SA-β-galactosidase activity, nuclear actin translocation, cytoplasmic p-Erk1/2 sequestration and reduced telomerase activity,
Ⅴ
Ⅴ
Ⅴ Ⅴ. CONCLUSION
Steroid hormones and their receptors play a major role in the development of many types of human and animal cancers. It has been known that androgen and its receptor (AR) are significantly related with hepatocarcinogenesis both in human and animals, however, the level of AR in hepatoma is variable, and their exact effects are still poorly explained.
To investigate the potential role of AR in hepatoma, Huh7-AR and Huh7-V cell lines were established by transfection of human AR in pCMV5 and its empty vector to Huh7
To investigate the potential role of AR in hepatoma, Huh7-AR and Huh7-V cell lines were established by transfection of human AR in pCMV5 and its empty vector to Huh7