Suppression of STAT3 Phosphorylation and RelA/p65 Acetylation Mediated by MicroRNA134 Plays a Pivotal Role in the Apoptotic Effect of Lambertianic Acid

As p300-mediated RelA/p65 hyperacetylation by signal transducers and activators of transcription 3 (STAT3) is critical for NF-κB activation, in the current study, the apoptotic mechanism of lambertianic acid (LA) was explored in relation to STAT3 phosphorylation and RelA/p65 acetylation in MCF-7, DU145, PC-3, and MDA-MB-453 cells. LA significantly increased the cytotoxicity, sub G 1 population, and the cleavage of poly (ADP-ribose) polymerase (PARP) in MDA-MB-453 or PC-3 cells (STAT3 mutant), more than in the MCF-7 or DU145 cells (STAT3 wild). Consistently, LA inhibited the phosphorylation of STAT3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and disrupted the interaction between p-STAT3, p300, NF-κB, and RelA/p65 acetylation (Ac-RelA/p65) in the MCF-7 and DU145 cells. Also, LA reduced the nuclear translocation of STAT3 and NF-κB via their colocalization, and also suppressed the protein expression of XIAP, survivin, Bcl-2, Bcl-xL, vascular endothelial growth factor (VEGF), Cox-2, c-Myc and mRNA expression of interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) in MCF-7 cells. Conversely, IL-6 blocked the ability of LA to suppress the cytotoxicity and PARP cleavage, while the depletion of STAT3 or p300 enhanced the PARP cleavage of LA in the MCF-7 cells. Notably, LA upregulated the level of miRNA134 and so miRNA134 mimic attenuated the expression of pro-PARP, p-STAT3, and Ac-RelA, while the miRNA134 inhibitor reversed the ability of LA to reduce the expression of Ac-RelA and pro-PARP in MCF-7 cells. Overall, these findings suggest that LA induced apoptosis via the miRNA-134 mediated inhibition of STAT3 and RelA/p65 acetylation.

Accumulating evidence has revealed that p300-mediated RelA/p65 hyperacetylation by STAT3 is essential for NF-κB activation in several cancers [9], as protein hyper-acetylation in cells is activated 2 of 14 in inflammation and cancers [10]. In the same line, acetylation, like phosphorylation, is important in regulating the nuclear translocation of NF-κB [11].

LA Induced Cytotoxicity and Sub-G1 Accumulation Increased the Cleavage of Poly (ADP-Ribose) Polymerase (PARP) in STAT3-Dependent or STAT3-Independent Cancer Cells
To evaluate the specific apoptotic effect of LA in STAT3-dependent or independent cancer cells, a cytotoxicity assay was conducted in breast cancer (MDA-MB-453; STAT3 mutant, MCF-7; STAT3 wild type) and prostate cancer (PC-3; STAT3 null, DU145; STAT3 wild type) cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Here, LA reduced the viability of the DU145, PC-3 cells, MCF-7, and MDA-MB-453 cells in a dose-dependent manner ( Figure 1b). However, the cytotoxicity of LA was better in the MDA-MB-453 and PC-3 cells than in the MCF-7 and DU145 cells. Consistently, the cell cycle analysis revealed that LA at 30 µM increased the sub-G1 population to 42.75% in MDA-MB-453, more than 6.84% of the MCF-7 cells (Figure 1c). Also, LA enhanced the cleavages of PARP in the MDA-MB-453 and PC-3 cells better than in the MCF-7 and DU145 cells (Figure 1d).

2.2.
LA Suppressed the Phosphorylation of STAT3 and NF-κB, and the Expression of p300 and RelA Acetylation in MCF-7 and DU145 Cells To determine the role of STAT3 in LA-induced apoptosis, Western blotting was conducted in MCF-7 and DU145 cells. As shown in Figure 2a, LA attenuated the phosphorylation of STAT3 and NF-κB, and also reduced the expression of p300 and RelA/p65 acetylation in MCF-7 and DU145 cells. Consistently, LA effectively suppressed the nuclear translocation of p-STAT3 and NF-κB p65 via their co-localization in MCF-7 cells (Figure 2b). The cells were distributed onto 96-well plates and were exposed to indicated concentrations of LA (0, 7.5, 10, 15, 20, 30, 40, 60, and 80 μM) for 24 h. The cell viability was calculated using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The data stand for means ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001. (c) Effect of LA on the sub-G1 population in MCF-7 and MDA-MB-453 cells, by cell cycle analysis. The MCF7 and MDA-MB-453 cells were exposed to LA (0, 15, and 30 M) for 24 h, and were stained with propidium iodide (PI) for flow cytometric analysis. The bar graphs represent the quantification of the The cells were distributed onto 96-well plates and were exposed to indicated concentrations of LA (0, 7.5, 10, 15, 20, 30, 40, 60, and 80 µM) for 24 h. The cell viability was calculated using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The data stand for means ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001. To determine the role of STAT3 in LA-induced apoptosis, Western blotting was conducted in MCF-7 and DU145 cells. As shown in Figure 2a, LA attenuated the phosphorylation of STAT3 and NF-B, and also reduced the expression of p300 and RelA/p65 acetylation in MCF-7 and DU145 cells. Consistently, LA effectively suppressed the nuclear translocation of p-STAT3 and NF-B p65 via their co-localization in MCF-7 cells (Figure 2b).
To confirm the p65 acetylation inhibition of LA, Western blotting and qRT-PCR were conducted in MCF-7 cells. Consistently, the LA attenuated the expression of the NF-B regulated genes, including Bcl-2, Bcl-xL, XIAP, survivin, (anti-apoptotic proteins), VEGF (angiogenic protein), COX-2 (inflammatory protein), and c-Myc (oncogenic genes) in the MCF-7 cells (Figure 3a). Also, STAT3 activators IL-6 and TNF-α (inflammatory factor) were downregulated at the mRNA level in the LAtreated MCF-7 cells (Figure 3b).  2.5. LA Inhibited the Expression of p300 and RelA/65 Acetylation, and Disrupted the Interaction between p-STAT3, p300, and NF-κB in MCF-7 and DU145 Cells To validate whether LA inhibits the p300-mediated RelA/p65 acetylation, immunofluorescence and co-immunoprecipitation (IP) were performed in MCF-7 and DU145 cells. As shown Figure 5a, the scores of the protein-protein interactions (PPI) between STAT3 and p300, STAT3 and p65, or p65 and p300 were 0.970, 0.820, and 0.981, respectively. IP revealed that LA disrupted the binding between p-STAT3, p300, and RelA (p65) after treatment with LA for 24 h, in the MCF-7 and DU145 cells (Figure 5b). p300, and Ac-RelA. (f) The effect of p300 depletion on p300, Ac-RelA, and cleaved PARP in the LAtreated MCF-7 cells. The cells transfected with either p300 or scrambled siRNA plasmid (40 nM) for 24 h were exposed to LA (0, 15, and 30 M) for 24 h, and subjected to Western blotting with antibodies of p300, Ac-RelA, and cleaved-PARP.

LA Inhibited the Expression of p300 and RelA/65 Acetylation, and Disrupted the Interaction between p-STAT3, p300, and NF-B in MCF-7and DU145 Cells.
To validate whether LA inhibits the p300-mediated RelA/p65 acetylation, immunofluorescence and co-immunoprecipitation (IP) were performed in MCF-7 and DU145 cells. As shown Figure 5a, the scores of the protein-protein interactions (PPI) between STAT3 and p300, STAT3 and p65, or p65 and p300 were 0.970, 0.820, and 0.981, respectively. IP revealed that LA disrupted the binding between p-STAT3, p300, and RelA (p65) after treatment with LA for 24 h, in the MCF-7 and DU145 cells (Figure 5b).  Figure 5. Effect of LA on the interaction between p-STAT3, p300, and RelA in the MCF-7 and DU145 cells. (a) Protein-protein interaction (PPI) scores between STAT3, p300, and RelA/p65 by search tool for the retrieval of interacting genes/proteins (STRING) database. (b) MCF-7 and DU145 cells were treated with LA for 24 h. Immunoprecipitation (IP) was performed with cell lysates, using antibodies of STAT3 and p300, and Western-blot analysis was conducted to detect RelA/p65, p-STAT3, STAT3, p300, and Ac-RelA in the whole cell lysates. IB-immunoblotting.

miR134 Plays a Pivotal Role in the LA-Induced Apoptotic Effect in MCF-7 Cells
To identify the role of miR134 in the LA-induced apoptotic effect in MCF-7 cells, the miR134-mimic or -inhibitor plasmid was transfected into MCF-7 cells. Here, LA increased the expression of miR134 in the MCF-7 cells by RT-PCR (Figure 6a). The miR134-mimic attenuated the expression of the pro-PARP, p-STAT3, and Ac-RelA in MCF-7 cells (Figure 6b). In contrast, the miR134 inhibitor reversed the cytotoxicity and inhibition of pro-PARP and Ac-RelA by LA in the MCF-7 cells (Figure 6c,d). for the retrieval of interacting genes/proteins (STRING) database. (b) MCF-7 and DU145 cells were treated with LA for 24 h. Immunoprecipitation (IP) was performed with cell lysates, using antibodies of STAT3 and p300, and Western-blot analysis was conducted to detect RelA/p65, p-STAT3, STAT3, p300, and Ac-RelA in the whole cell lysates. IB-immunoblotting.

miR134 Plays a Pivotal Role in the LA-Induced Apoptotic Effect in MCF-7 Cells
To identify the role of miR134 in the LA-induced apoptotic effect in MCF-7 cells, the miR134mimic or -inhibitor plasmid was transfected into MCF-7 cells. Here, LA increased the expression of miR134 in the MCF-7 cells by RT-PCR (Figure 6a). The miR134-mimic attenuated the expression of the pro-PARP, p-STAT3, and Ac-RelA in MCF-7 cells (Figure 6b). In contrast, the miR134 inhibitor reversed the cytotoxicity and inhibition of pro-PARP and Ac-RelA by LA in the MCF-7 cells (Figure  6c,d).

Discussion
The aim of the current work is to elucidate the underlying apoptotic mechanism of LA in association with STAT3 and NF-κB signaling in breast and prostate cancer cells. Herein, MDA-MB-453 and PC-3 STAT3 mutant cells were more susceptible to the cytotoxicity of LA, compared with MCF-7 and DU145 STAT3 wild-type cells. Likewise, LA significantly increased sub-G1 accumulation along with an increased cleavage of PARP in the MDA-MB-453 and PC-3 cells, more than in the MCF-7 and DU145 cells, implying the important role of STAT3 in the cytotoxic and apoptotic effect of LA.

Discussion
The aim of the current work is to elucidate the underlying apoptotic mechanism of LA in association with STAT3 and NF-κB signaling in breast and prostate cancer cells. Herein, MDA-MB-453 and PC-3 STAT3 mutant cells were more susceptible to the cytotoxicity of LA, compared with MCF-7 and DU145 STAT3 wild-type cells. Likewise, LA significantly increased sub-G1 accumulation along with an increased cleavage of PARP in the MDA-MB-453 and PC-3 cells, more than in the MCF-7 and DU145 cells, implying the important role of STAT3 in the cytotoxic and apoptotic effect of LA.
Accumulating evidence has revealed that STAT3 and NF-κB are involved in a variety of biological processes, such as inflammation, and cancer progression and growth [24,25]. Thus, STAT3 and NF-κB can be considered as potent target molecules for cancer therapy [26,27]. Our Western blotting showed that LA treatment suppressed the phosphorylation of STAT3 and NF-κB, and the nuclear translocation of STAT3 and NF-κB, indicating the involvement of STAT3 and NF-κB pathways in LA-induced apoptosis in MCF-7 and DU145 cells.
Recent evidence has demonstrated that STAT3 is upregulated in most cancers, and NF-κB enhances the STAT3-p300 interaction [9]. Also, the accumulation of RelA/p65 in the nucleus can be promoted by acetylation by p300 [28], and STAT3 is essential for p300-mediated RelA acetylation [29,30]. Through the close interaction between STAT3 and NF-κB, STAT3 increases the NF-κB activity, and also, the persistent activation of STAT3 is dependent on NF-κB signaling [9,31]. It is well documented that several inflammatory cytokines, including IL-6, COX-2, and IL-23, activate STAT3 through NF-κB regulated inflammatory responses, and RelA/p65 is maintained through p300-mediated RelA/p65 acetylation by STAT3 [29]. Here, LA attenuated the expression of p300 and RelA/p65 acetylation (Ac-RelA), and also disrupted the binding of p-STAT3 with p300 or Ac-RelA in MCF-7 and DU145 cells, indicating that LA induces the hypoacetylation of RelA/p65, leading to the downregulation of NF-κB-regulated proteins via the interrupted binding of p-STAT3 with p300 or NF-κB.
MicroRNAs are well known to mediate cell differentiation, proliferation, and apoptosis [38]. Recent evidence revealed that miR134 induces apoptosis, and inhibits proliferation and migration by targeting the STAT3 in bladder cancer cells [39], and miR134 abrogates proliferation and EMT in renal cell carcinoma and colorectal cancer cells [17]. Here, LA upregulated the miRNA level of miRNA134 in MCF-7 cells, and also, the miRNA134-mimic abrogated the expression of p-STAT3, pro-PARP, and Ac-RelA, whereas the miRNA134-inhibitor reversed the ability of LA to inhibit the expression of pro-PARP and Ac-RelA in MCF-7 cells, indicating a critical role of miRNA134 in LA-induced apoptosis.
In summary, LA significantly increased the cytotoxicity, sub-G 1 population, and cleavage of PARP in MDA-MB-453, PC-3, MCF-7, and DU145 cells. Also, LA reduced the phosphorylation of STAT3 and NF-κB, and disrupted the interaction between p-STAT3, p300, NF-κB, and RelA/p65 acetylation in the MCF-7 and DU145 cells. Furthermore, LA reduced the nuclear translocation of STAT3 and NF-κB, and suppressed several survival genes, including Bcl-2, Bcl-XL, XIAP, survivin, VEGF, Cox-2, C-Myc, and IL-6, and TNF-α in MCF-7 cells. However, IL-6 blocked the apoptotic effect of LA, and the depletion of the STAT3-or p300-enhanced PARP cleavage of LA in MCF-7 cells. Notably, LA activated miRNA134, and so the miRNA134-mimic reduced the expression of p-STAT3, pro-PARP, and Ac-RelA, while the miRNA134 inhibitor reversed the apoptotic effect of LA in the MCF-7 cells. Taken together, these findings demonstrate that LA induces apoptosis via the miRNA-134 mediated inhibition of STAT3 and RelA/p65 acetylation (Figure 7).

Cell Viability Assay
The cytotoxicity of LA was evaluated by using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, the MCF-7, MDA-MB-453, DU145, and PC-3 cells (1 × 10 4 cells/well) were exposed to indicated concentrations of LA for 24 h, and were incubated with MTT (1 mg/mL); (Sigma Chemical, St. Louis, MO, USA) for 2 h. Then, the cell viability was calculated as a percentage of the viable cells in the LA-treated group vs. the untreated control, with optical density (OD) values obtained using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 570 nm.

Co-Immunoprecipitation
The MCF-7 and DU145 cells were lyzed and immunoprecipitated with STAT3 and p300 antibodies. Thereafter, protein A/G sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were applied. The final precipitated proteins were subjected to immunoblotting with the indicated antibodies.

Statistical Analysis
All of the values were expressed as means ± standard deviation (SD). Sigmaplot version 12 software (Systat Software Inc., San jose, CA, USA) was used for the statistical analysis. Student t-test was used for comparison of two groups, and p < 0.05 was considered as statistically significant.

Conclusions
Our findings suggest that LA significantly increased the cytotoxicity, sub G 1 population, and the cleavage of PARP in MDA-MB-453, PC-3, MCF-7, and DU145 cells. Also, LA reduced the phosphorylation of STAT3 and NF-κB, and disrupted the interaction between p-STAT3, p300, NF-κB, and RelA/p65 acetylation in MCF-7 and DU145 cells. Furthermore, LA reduced the nuclear translocation of STAT3 and NF-κB, and suppressed several survival genes, including Bcl-2, Bcl-XL, XIAP, survivin, VEGF, Cox-2, C-Myc, IL-6, and TNF-α in MCF-7 cells. However, IL-6 blocked the apoptotic effect of LA and the depletion of the STAT3-or p300-enhanced PARP cleavage of LA in MCF-7 cells. Notably, LA activated miRNA134, and so the miRNA134 mimic attenuated the expression of p-STAT3, pro-PARP, and Ac-RelA, while the miRNA134 inhibitor reversed the apoptotic effect LA in the MCF-7 cells. Taken together, these findings demonstrate that LA induces apoptosis via the miRNA-134 mediated inhibition of STAT3 and RelA/p65 acetylation