Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

Abstract

Background: Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism.

Methods: HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface beta1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined.

Results: A low concentration of arecoline (<or= 100 microg/ml) caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased beta1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-XL and Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline.

Conclusions: This study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased RhoA/Rock activation and that the STAT3 and RhoA/Rock signaling pathways are connected.

Figure 1

Arecoline induces detachment of HA22T/VGH cells, followed by apoptosis. (A) HA22T/VGH cells were treated with 0 (left), 30 (center), or 100 (right) μg/ml of arecoline for 24 h or 48 h, then cell morphology was observed under a phase-contrast microscopy at 200× magnification. The black arrows indicate detached cells. (B) After treatment of arecoline for 24 h, β1-integrin expression was measured by flow cytometric analysis using RPE-conjugated antibody. The histogram of red filled area is the untreated control and the black lines the treated groups. The values shown are the mean fluorescence intensity as a percentage of the untreated control value. (C) HA22T/VGH cells or primary normal rat hepatocytes were treated with the indicated concentration of arecoline for 72 h, then viable cells were counted using Trypan blue and the results expressed as a percentage of the untreated control value. (D) After treatment of arecoline for 72 h, the cells were harvested all together or the detached and adherent cells separately and genomic DNA extracted and analyzed on an agarose gel for DNA fragmentation. The left panel shows the total cells and the right panel adherent and detached cells separately. (E) TUNEL staining of the detached cells detected by flow cytometric analysis. The green filled area is the untreated control and the black lines the treated groups. The values shown are the percentage of TUNEL-positive cells in the detached cells. All data are the mean ± S.D. for three independent experiments. *: p < 0.05 as compared to the untreated control.

Figure 2

Arecoline alters the expression of apoptosis-related proteins and caspase activity in HA22T/VGH cells. (A) HA22T/VGH cells were treated with 0, 30, or 100 μg/ml of arecoline for 24 h, then the cells were harvested and proteins extracted and used for Western blotting for Bcl-2, Bcl-X_L, Bax, cytochrome c, or procaspase-9. β-actin was used as the internal control. The values shown are the quantitative density analysis expressed as the relative density compared to that in untreated cells (control), taken as 100%. The results are expressed as the mean ± S.D. for three separate experiments. (B) Caspase-3 activity was detected using RPE-conjugated anti-active caspase-3 antibody by flow cytometric analysis. The values shown are the percentage of cells with active caspase-3 and are the mean ± S.D. of three independent experiments. The red filled area is the untreated control and the black lines the treated groups. *: p < 0.05 as compared to the untreated control.

Figure 3

Arecoline inhibits activation of STAT3, p190RhoGAP, and SHP2 and increases Rock-1 cleavage in HA22T/VGH cells. HA22T/VGH cells were treated with 0-100 μg/ml of arecoline for 24 h, then the cells were harvested and proteins extracted and used for Western blotting with antibodies against Gp130, phospho-Tyr705-STAT3 (STAT3-Tyr-p), p190RhoGAP, SHP2, or Rock-1; the Gp130, p190RhoGAP, and SHP2 blots were then stripped and re-blotted using antibodies against phospho-Tyr (Tyr-p). β-actin was used as the internal control. The values shown are the quantitative density analysis expressed as the relative density compared to that in untreated cells (control), taken as 100%. The results are expressed as the mean ± S.D. for three separate experiments. *: p < 0.05 as compared to the untreated control.

Figure 4

Arecoline increases actin stress fiber formation in HA22T/VGH cells. HA22T/VGH cells were treated with 0, 10, 30, 60, or 100 μg/ml of arecoline for 24 h, then were fixed with paraformaldehyde for actin labeling and observation by fluorescence microscopy (A) or flow cytometry (B). In A, panel AC is untreated cells incubated with second antibody alone, and C is the untreated control group incubated with primary and secondary antibodies. The values shown in B are the percentage of actin staining-cells and are the mean ± S.D. for three independent experiments. The green filled area is the untreated control and those delimited by the black lines the treated groups. *: p < 0.05 as compared to the untreated control.

Figure 5

The RhoA inhibitor, C3 exoenzyme, abolishes arecoline-induced actin stress fiber formation. (A) HA22T/VGH cells were left untreated (C), treated with 1.5 μg/ml of C3 exoenzyme for 25 h (Ri1.5), with 100 μg/ml of arecoline for 24 h (A100), or with C3 exoenzyme for 1 h, then with 100 μg/ml of arecoline in the continued presence of C3 exoenzyme for 24 h (Ri1.5+A100), then were fixed with paraformaldehyde for actin labeling and observation of stress fiber by fluorescence microscopy (A) or flow cytometry (B). In B, the values are the percentage of actin staining-cells and are the mean ± S.D. for three independent experiments. The green filled area is the untreated control and those delimited by the black lines the treated groups. *: p < 0.05 as compared to the untreated control; #: p < 0.05 as compared to the arecoline (A100) treatment.

Figure 6

The RhoA inhibitor attenuates the effects of arecoline on STAT3, p190RhoGAP, and SHP2 in HA22T/VGH cells. Cells were left untreated or were treated with arecoline alone or arecoline plus C3 exoenzyme as in Fig. 5, then were harvested and proteins were extracted preparing for Western blotting for RhoA, phospho-Tyr705-STAT3 (STAT3-Tyr-p), phospho-Tyr (Tyr-p) p190RhoGAP, and phospho-Tyr (Tyr-p) SHP2, as described in the legend to Fig. 3. β-actin was used as the internal control. The values shown are the quantitative density analysis expressed as the relative density compared to that in untreated cells (control), taken as 100%. The results are expressed as the mean ± S.D. for three separate experiments. *: p < 0.05 as compared to the untreated control; #: p < 0.05 as compared to the arecoline (A100) treatment.

Figure 7

Addition of IL-6 does not alter the effects of arecoline. HA22T/VGH cells were left untreated (C) or were incubated with 100 μg/ml of arecoline for 24 h (A100), with 100 ng/ml of IL-6 for 25 h (IL-6), or with IL-6 for 1 h, then with arecoline in the continued presence of IL-6 for 24 h (IL-6+A100). The cells were then harvested and proteins extracted and used for Western blotting for phospho-Tyr705-STAT3 (STAT3-Tyr-p), Bcl-2, phospho-Tyr (Tyr-p) p190RhoGAP, phospho-Tyr (Tyr-p) SHP2, and Rock-1, as described in the legend to Fig. 3. β-actin was used as the internal control. The values shown are the quantitative density analysis expressed as the relative density compared to that in untreated cells (control), taken as 100%. The results are expressed as the mean ± S.D. for three separate experiments. *: p < 0.05 as compared to the untreated control.

Figure 8

Arecoline inhibits hepatoma cell colony growth in soft agar. The growth of HA22T/VGH cells in soft agar was measured in 35 mm diameter dishes with a lower layer of 0.7% agar solution and an upper layer of 0.35% agar solution in which 1 × 10⁵ cells were resuspended. The soft agar was covered with culture medium alone or containing the indicated concentration of arecoline. After 3 weeks, colonies larger than 0.1 mm in diameter were scored. (A) Representative photomicrographs of soft agar colonies at 200× magnification. The black arrows indicate colonies. (B) Number of colonies in the presence or absence of arecoline. The results are the mean ± S.D. for three independent experiments. *: p < 0.05; #: p < 0.001 as compared to the untreated control.

Figure 9

Schematic representation of the arecoline-stimulated signaling pathways for detachment and apoptosis of HA22T/VGH cells. Arecoline treatment decreases IL-6 levels, but does not change gp130 of IL-6 receptor. In addition, phosphorylation/activation of STAT3, which provides protection against anoikis, is inhibited and levels of its downstream signals IL-6, Bcl-2, and Bcl-X_L are decreased, while Bax levels, mitochondrial cytochrome c release, caspase-9 levels, and caspase-3 activity are increased. Phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and its upstream regulator, SHP2, are inhibited, while the activation/cleavage of Rock-1, a RhoA downstream kinase, and actin stress fiber formation are increased, contributing to anoikis.