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Baicalein-induced apoptosis of human HCT116 colon cancer cells was mediated by DEPP/Gadd45a upregulation and MAPKs activation Running title: Baicalein induces DEPP and Gadd45a resulting in apoptosis Abstract Decidual protein induced by progesterone (DEPP), a transcriptional target of FOXO, was originally found from the human endometrial stromal cells cDNA library. However, the expression and physiological functions of DEPP in human colon cancer cells remains largely unknown. Baicalein with its efficient antitumor properties has been reported for promoting apoptosis of several human cancer cells. Here, we reported that baicalein could stimulate apoptosis and morphology changes of HCT116, A549, Panc-1 cells in a dose-dependent manner. Meanwhile, we found for the first time that baicalein could up-regulated the mRNA and protein levels of decidual protein induced by progesterone (DEPP) and growth arrest and DNA damage-inducible 45 alpha (Gadd45a). To further investigate the role of DEPP and Gadd45a in baicalein-induced apoptosis, we used HCT116 cells which were transfected with siRNA against DEPP or Gadd45a as in vitro models. Through the Annexin V/PI double staining assay, we observed that baicalein-induced apoptosis was strongly impaired by DEPP or Gadd45a inactivation. Then western blotting results revealed that DEPP and Gadd45a deficiency also restricted baicalein-induced activation of caspase-3, -9 and phosphorylation of MAPKs. In line with these results, the overexpression of DEPP promoted the MAPKs phosphorylation. Moreover, the inhibition of JNK/p38 activities with SP600125/SB203580 decreased the expression of Gadd45a in turn while the inactivation of ERK with SCH772984 had no effects on the expression of Gadd45a. Taken together, our results demonstrated that the expression of both DEPP and Gadd45a which was baicalein induced resulted in the increased apoptosis response in human colon cancer cells. Meanwhile, DEPP and Gadd45a up-regulated by baicalein promoted the activation of MAPKs with a positive feedback loop between Gadd45a and JNK/p38.Key Words: DEPP; Baicalein; Gadd45a; colon cancer; MAPKs. IntroductionDEPP (decidual protein induced by progesterone) was originally found from the human endometrial stromal cells cDNA library as progesterone-induced gene8. It is mainly expressed in multiple tissues included placenta, ovary, kidney, white adipose and liver9, 10. In our previous study, DEPP was markedly up-regulated in baicalein treated human colon cancer HCT116 cells by microarray. Additionally, some reports suggested the relationship between DEPP and tumor cell death8. Thus, we speculated that DEPP may play a potential role in baicalein induced cell death in HCT116 cells.Natural products isolated from plants have raised great interest in recent years due to their potent biological and pharmaceutical activities1. Baicalein (5, 6, 7-trihydroxyflavone) extracted from the dry root of Scutellaria baicalensis Georgi (also known as Baikal skullcap) with a wide range of pharmacological functions has been reported possessed a potential antitumor capability in human liver cancer2, breast cancer3, 4, 5, 6, lung cancer cells7. Multiple mechanisms are related to its anti-tumor activity but detailed mechanisms on baicalein-induced apoptosis in human colon cancer HCT116 cells are still not well known.Gadd45a (the growth arrest and DNA damage-inducible 45 alpha) is one member of the Gadd45 family which could interact with pivotal cell effectors, such as p21, p38, PCNA, Cdc2/Cyclin B1 and MEKK411. Besides, Gadd45a as a vital regulator of cell cycle arrest, apoptosis, and differentiation is strictly regulated by p5312. Moreover, Gadd45a has been shown to play a crucial role in suppression of tumor cell growth13-14. Therefore, it is interesting to investigate whether Gadd45a also participate in baicalein-stimulated apoptosis in human colon cancer cells and explore the further mechanism in it.In this study, we verified that baicalein significantly stimulated apoptosis of HCT116, A549 and Panc-1 cells and up-regulated both DEPP and Gadd45a as well as notably triggered the phosphorylation of MAPKs. Further experiments revealed that as DEPP increased the protein and mRNA levels of Gadd45a, both DEPP and Gadd45a expression contributed to baicalein-induced apoptosis and MAPKs activation. Intriguingly, JNK/p38 inhibitions could also restrict Gadd45a expression while there was no such change with ERK inactivation. Above all, our findings identified the essential role of DEPP and Gadd45a in baicalein-induced apoptosis and highlight that baicalein might be a promising anti-tumor agent for the treatment of human colon cancer. ResultsBaicalein induced apoptosis of HCT116, A549 and Panc-1 cellsTo detected the effect of baicalein on human cancer cells, light microscope was used to observe the morphology changes of HCT116, A549 and Panc-1 cells after treated with 0, 10, 20, 40 ?M baicalein and 1 ?M Gemcitabine as positive control. As shown in Figure 1A, cells treated with Baicalein (10, 20, 40 ?M) or Gemcitabine (1 ?M) were in a flattened, blebby and shrinking appearance consistent with cell death whereas cells in negative control adopted an intact and polygonal morphology. And baicalein inhibited the proliferation of HCT116 (Figures 1B), A549 (Figures 1C), Panc-1 (Figures 1D) in a dose-dependent manner. Furthermore, the data indicated that baicalein could induce apoptosis in HCT116 cells in a dose-dependent manner by the Annexin V/PI double staining assay (Figures 2A) and a significant increase in cleaved caspase-3, -9 (Figures 2B) after the treatment for indicated time. Additionally, similar results were also observed in A549 (Figures 2C, 2D) and Panc-1 (Figures 2E, 2F), suggesting that baicalein could induce apoptosis of HCT116, A549 and Panc-1 cells.DEPP and Gadd45a were elevated in baicalein-induced apoptosis of cancer cells In order to verify the potential role of DEPP and Gadd45a played in baicalein-induced apoptosis cells, this study was initiated by investigating whether the expression of DEPP and Gadd45a are altered in baicalein treated cells. Western blot analysis and qRT-PCR showed that DEPP and Gadd45a were markedly elevated in baicalein treated HCT116 cells. The same results in both protein (Figures 3A) and mRNA expression (Figures 3B) could observed. These data in HCT116 cells were similar to existence in other human cancer cell lines, such as A549 (Figures 3C) and Panc-1 (Figures 3E). However, the mRNA expression of DEPP and Gadd45a in A549 (Figures 3D) and Panc-1 (Figures 3F) had distinctly increased after 6 h other than 24 h with baicalein treatment, possibly attributable to asynchronism between transcription and translation. These results confirmed that baicalein up-regulated DEPP and Gadd45a in three distinct types of human cancer cell lines.DEPP and Gadd45a deficiency restricted baicalein-induced apoptosis of HCT116 cellsTo address whether DEPP and Gadd45a were necessary for baicalein-induced apoptosis, the siRNA against DEPP and Gadd45a were transfected into HCT116 cells, respectively. Western blotting analysis showed that the accumulations of cleaved caspase-3, -9 that baicalein-induced were dramatically reduced by silencing of DEPP and Gadd45a (Figure 5A, 5C). These results were further confirmed by the Annexin V/PI double staining assay. As shown in Figures 5B and 5D, there was a dramatical decline between the cells transfected with nonspecific siRNA and the cells transfected with DEPP siRNA or Gadd45a siRNA after treated with 40 ?M Baicalein in apoptosis rate. Taken together, our data showed that baicalein up-regulated DEPP and Gadd45a which participated in the apoptotic response in HCT116 cells through the activation of caspase-3 and caspase-9.Baicalein-induced HCT116 cell apoptosis by DEPP/Gadd45a upregulation was mediated with phosphorylation of MAPKsTo further address the mechanism between baicalein-induced apoptosis and the expression of DEPP and Gadd45a, the MAPK signalings was examined by immunoblot assay in HCT116 cells for 0, 6, 12, 24 h after baicalein treatment. Notably, a significant increase in the phosphorylation of JNK, ERK and p38 was observed in western blotting analysis (Figures 5A). Then the expression of DEPP was stably inhibited by using siRNA to test whether DEPP is required for baicalein-induced phosphorylation of MAPKs. Following treatment, the absence of DEPP (Figures 5B, C) had a negative impact on the protein level of p-JNK, p-ERK, p-p38, Gadd45a as well as the mRNA level of p21, p53 and Gadd45a which were induced by baicalein. In addition, DEPP overexpression plasmid was used to further validate these findings (Figures 5D). Likewise, p-JNK, p-ERK, p-p38, p21, p53 and Gadd45a were strongly induced only if there was the overexpression of DEPP (Figures 5D and E). Together, these results suggested that DEPP is required for baicalein-induced phosphorylation of MAPKs and up-reguleted Gadd45a in both protein and mRNA levels. We next study whether Gadd45a also regulated the baicalein-induced phosphorylation of MAPKs and similar results were observed in p-JNK, p-ERK, p-p38 using a siRNA against Gadd45a (Figures 5F). In sum, these data suggested the essential role DEPP and Gadd45a played in baicalein-induced apoptosis through MAPK signalings.Baicalein induced Gadd45a upregulation was attenuated by the inhibition of MAPKs phosphorylationThe aforementioned results showed that Gadd45a up-regualted the phosphorylation of JNK, ERK and p38 in baicalein-induced apoptosis in HCT116 cells. Intriguingly, there were other reports which had shown that MAPK signalings mediate the expression of Gadd45a. Therefore, to test whether MAPK signalings played roles in baicalein-mediated upregulation of GADD45a, the specific JNK inhibitor SP600125, ERK inhibitor SCH772084 and p38 inhibitor SB203580 were used to prevent the activation of JNK, ERK and p38. Interstingly, inhibition of JNK and p38 signalings were accompanied by a obvious decrease in baicalein-induced Gadd45a expression (Figures 6A, B) but there was no such a change in the presence of ERK inhibitor SCH772084 (Figures 6C). These results suggested that there was a positive feedback loop between Gadd45a and JNK/p38 when baicalein triggered the apoptosis response in human colon cancer cells whereas ERK suppression had no limit impact on Gadd45a expression. Therefore, the data clearly revealed a critical role of MAPK signalings activation in the induction of Gadd45a in baicalein-induced apoptosis (Figures 7).  DiscussionIn this study, we reported for the first time that Baicalein up-regulated DEPP and Gadd45a which could activate caspase-3,-9 and JNK/ERK/p38 MAPK pathways leading to apoptosis of human colon cancer HCT116 cells. DEPP as a progesterone-induced gene is regulated by progesterone in endometrial stromal cells and by insulin level in adipose tissue and is induced in malignant glioma cells under hypoxic condition9, 10,15. But there are rare reports about its physiological function. Stefan Salcher and his colleagues found that the expression of DEPP contributes to FOXO3-induced apoptosis because DEPP-knockdown significantly reduced FOXO3-induced cell death8 and another study demonstrated that the upregulation of DEPP activated MAPK signaling pathway to stimulate the transcription factor ELK115. Hence, our finding that DEPP promotes baicalein induced apoptosis by activation of Gadd45a, JNK/ERK/p38 and caspase-3, -9 is both initiation and extension to further investigate the physiological function of DEPP. But there remains several questions of how baicalein increases the mRNA and protein levels of DEPP, how DEPP induces the phosphorylation of MAPKs that need thoroughly exploring.As a natural compound extracted from Scutellaria baicalensis Georgi, baicalein has been widely researched for its potent anti-tumor property. Most of them revealed that baicalein exerts its anti-tumor ability via the induction of apoptosis such as in human lung cancer6, breast cancer7 and osteosarcoma cells16. Moreover, it also has been demonstrated that the MAPK signaling pathway results in the baicalein-induced apoptosis of cancer cells7. Therefore, we initiated our study to validate these findings and observed not only strong induction of apoptosis by baicalein in human colon cancer HCT116, lung carcinoma A549 and pancreatic cancer Panc-1, but also the prominent activation of caspase-3, -9 and JNK/ERK/p38 in baicalein treated HCT116 cells.However, it is still unknown about the effectors which worked as MAPKs upstream and downstream to mediate baicalein-induced apoptosis. Thus, on the basis of our previous results that DEPP and Gadd45a were markedly up-regulated in baicalein treated human colon cancer HCT116 cells by microarray, we proposed an important role for DEPP and Gadd45a in baicalein-induced apoptosis and the activation of caspase-3,-9 and MAPKs in HCT116 cells. Consistent with our envision, the absence of DEPP notably decreased the apoptosis rate and the level of pro-apoptotic proteins such as cleaved caspase-3, -9, phosphorylated JNK/ERK/p38, p21, p53 and Gadd45a which were induced by baicalein. Similar phenomena were also observed in Gadd45a siRNA transfected HCT116 cells. Furthermore, overexpression of DEPP provoked the phosphorylation of MAPKs and the expression of p21, p53 and Gadd45a. These results together indicated that as DEPP up-regulated Gadd45a in protein and mRNA levels, both of them contributed to the activation of JNK/ERK/p38 during baicalein induced apoptosis. Nevertheless, further in vitro and in vivo studies are needed to expound whether baicalein has a direct impact on the expression of DEPP or not. In contrast to DEPP, it has been sufficiently explored between the induction of Gadd45a and the apoptosis via MAPK signaling pathway13, 14. Nevertheless, detailed conclusions about the loop between Gadd45a and MAPKs are less clear as many reports are conflict, including some reports show that Gadd45a is upstream of MAPKs signaling and activate JNK pathway17, whereas others suggest that Gadd45a is downstream of MAPKs pathway and is positively regulated by JNK and was negatively regulated by ERK/p3814. Accordingly, our further analysis about the mediation between the expression of Gadd45a and baicalein-induced the activation of JNK/ERK/p38 in human colon cancer cells is desirable as a supplement to the work for the former research. Indeed, our work in vitro suggests the existence of a positive feedback loop between Gadd45a and JNK/p38. However, further studies on the detailed role of Gadd45a and JNK/p38 on this loop and the role of ERK outside this loop in baicalein-induced apoptosis are still necessary to be performed.In summary, we provided a new machenism to do the research about baicalein induced apoptosis in human colon cancer cells and for the first time that we found baicalein up-regulated both DEPP and Gadd45a, leading to apoptosis via MAPK signaling pathway and caspase-3, -9 in HCT116 cells. In this context, we also validate the existence of a positive feedback loop between Gadd45a and JNK/p38. In general, our findings may encourage the development of new options both to target DEPP and Gadd45a-JNK/p38 feedback loop in the treatment of colon cancer. Materials and MethodsCell Cultures and Drug TreatmentsHCT116, A549, Panc-1 cell lines were obtained from American Type Culture Collection and were grown in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (CellMax), penicillin-streptomycin mixed antibiotics (Beyotime). The cells were cultured at 37? and 5% CO2 in a humidified incubator and were exposed to the indicated concentrations of Baicalein (Jiangsu Institute for Food and Drug Control), SP600125 (Selleck), SB203580 (Selleck) or SCH772984 (Selleck).Western blot analysisCell lysates were prepared with RIPA buffer containing PMSF (Beyotime). An equal amount of cellular protein was loaded into 12% SDS-PAGE gel and then was transferred to a PVDF membrane (Millipore). The following primary antibodies were used: rabbit anti-DEPP (ProteinTech), mouse anti-?-actin (SAB), rabbit anti-Gadd45a, rabbit anti-PARP, rabbit anti-Cleaved-PARP, rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Cleaved-capase-9, mouse anti-JNK, rabbit anti-p-JNK, rabbit anti-ERK, rabbit anti-p-ERK, rabbit anti-p38, rabbit anti-p-p38 (Cell Signaling Technology). The secondary antibodies employed were goat anti-rabbit IgG or goat anti-mouse IgG (SAB). The signal was visualized by the Immobilon Western Chemiluminescent HRP Substrate (Millipore).Apoptosis detectionApoptosis induced by Baicalein was determined using the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme). Cells were planted into 12-well plate at 5×104 cells/mL and treated with different concentrations of Baicalein for 48h. Cells were detached with 0.25% trypsin without EDTA, washed twice with cold PBS and resuspended in 100 ?L binding buffer. After that, the cells were incubated with 5 ?L Annexin V-FITC and 5 ?L PI for 10 min at room temperature in the dark and analyzed by Accuri C6 (BD).Quantitative RT-PCRTotal RNA from cultured cells were extracted using TRIzol (Vazyme) and chloroform/isopropanol as specified by the manufacturer and then reverse transcribed using HiScript Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme). qPCR was subsequently performed using ChamQ SYBR qPCR Master Mix Kit (Vazyme). The PCR primers used as the following: GAPDH: F: 5′-GGGAAACTGTGGCGTGAT-3′, R: 5′-GAGTGGGTGTCGCTGTTG A-3′; DEPP: F: 5′-ATACGTCCTGTGGTGGCATTG-3′, R: 5′-CCTGATTCCCGTTCCCTGAT -3′; Gadd45a: F: 5′-TTGCAATATGACTTTGGAGGAA-3′, R: 5′-CATCCCCCACCTTATCCAT -3′; p21: F: 5′-AGCAGAGGAAGACCATGTGGA-3′, R: 5′-AATCTGTCATGCTGGTCTGCC- 3′; p53: F: 5′-CTCTCCCCAGCCAAAGAAGAA-3′, R: 5′-TCCAAGGCCTCATTCAGCTCT-3′. The relative mRNA expression was normalized to GAPDH and was determined by the comparative Ct method (2-??Ct).siRNA transfectionThe siRNA targeting DEPP, Gadd45a and nonspecific siRNA were synthesized by GenScript. (DEPP: 5′-GCAGUGUCCUCAGAACACU-3′; Gadd45a: 5′-AAAGUCGCUACAUGGAUCAA U-3′; NC: 5′-UUCUCCGAACGUGUCACGU-3′) Cells were transfected siDEPP, siGadd45a or nonspecific siRNA control using EntransterTM-R4000 (Engreen Biosystem) according to the manufacturer’s instructions.DEPP overexpressionHCT116 cells were transfected with 0.82 ?g/?l pcDNA3.1 either blank or loaded the cDNA of DEPP using Lipo 2000 transfection reagent (Thermo). Cells were harvested after 72h.Statistical AnalysisAll of the data are showed as mean ± standard deviation at least three independent experiment. Statistical analyses were performed using GraphPad Prism 5 and data were analyzed using the Student’s t-test. *P < 0.05 was considered to be significant.AcknowledgmentsThis work was supported by National Natural Science Foundation of China (No. 81472233) and the Jiangsu National Natural Science Foundation of China (No.BK20150700).Conflicts of InterestThe authors declare no conflict of interest.

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