- Email: [email protected]
Rottlerin upregulates DDX3 expression in hepatocellular carcinoma
Biochemical and Biophysical Research Communications xxx (2017) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Rottlerin upregulates DDX3 expression in hepatocellular carcinoma Zhong Wang a, b, Gen-hai Shen a, Jia-ming Xie b, Bin Li a, *, Quan-gen Gao a, ** a b
Department of General Surgery, The First People's Hospital of Wu Jiang, Suzhou, Jiangsu, 215200, China Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2017 Accepted 29 November 2017 Available online xxx
Rottlerin has been reported to exert its anti-tumor activity in various types of human cancers. However, the underlying molecular mechanism has not been fully elucidated. In the current study, we explored whether rottlerin exhibits its tumor suppressive function in hepatocellular carcinoma cells. Our MTT assay results showed that rottlerin inhibited cell growth in hepatocellular carcinoma cells. Moreover, we found that rottlerin induced cell apoptosis and caused cell cycle arrest at G1 phase. Furthermore, our wound healing assay result demonstrated that rottlerin retarded cell migration in hepatocellular carcinoma cells. Additionally, rottlerin suppressed cell migration and invasion. Notably, we found that rottlerin upregulated DDX3 expression and subsequently downregulated Cyclin D1 expression and increased p21 level. Importantly, down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study suggests that rottlerin exhibits its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells. © 2017 Elsevier Inc. All rights reserved.
Keywords: DDX3 Hepatocellular carcinoma Rottlerinn Proliferation Cell cycle Invasion
1. Introduction DEAD-box proteins include the amino acid motif D-E-A-D (AspGlu-Ala-Asp), which are members of the largest family of RNA helicases. Initially, DEAD-box proteins were found to be involved in rearranging RNA-RNA and RNA-protein interactions as ATPdependent RNA binding proteins and RNA-dependent ATPases [1]. DEAD-box RNA helicase 3 (DDX3) belongs to DEAD-box family proteins. As a RNA helicase, DDX3 protein regulates several steps of RNA metabolisms, such as RNA splicing, mRNA export, transcription, and translation initiation. Therefore, DDX3 is involved in multiple cellular processes including apoptosis, cell cycle regulation, and cell stress response. Depletion of DDX3 leads to an early embryonic lethality in mice. Recently, DDX3 is reported to play an essential role in cancer development and progression [2,3]. Interestingly, DDX3 promotes cancer progression or exhibits tumor suppressive function in various types of human cancers. This concept suggests that DDX3 is a ‘double-edges sword’ gene. For example, oncogenic role of DDX3
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Q.-g. Gao).
(B.
Li),
[email protected]
in breast cancer biogenesis was reported. In line with this, overexpression of DDX3 induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays in breast cancer cells [4]. Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells [5]. Moreover, DDX3 is considered as a biomarker for metastasis and poor prognosis of squmous cells or adenocarcinoma of gallbladder [6]. Similarly, DDX3 could be a strongest prognosis marker and its downregulation enhanced metastasis in colorectal cancer [7]. However, low or negative expression of DDX3 might predict poor prognosis in non-smoker patients with oral cancer [8]. Accumulating studies suggested that DDX3 plays tumor suppressive role in hepatocellular carcinoma. However, one study reported that DDX3 is overexpressed in hepatocellular carcinoma tissues [9]. Thus, the role of DDX3 in hepatocellular carcinoma needs to be clarified. Rottlerin is a natural plant ployphenol compound, which is derived from the kalmala tree [10]. Rottelrin has been known to exert its antitumor activity in a variety of human cancers. However, the functions of rottelrin and underlying molecular mechanism in hepatocellular carcinoma have not been fully elucidated. In the current study, we explored whether rottlerin could exhibit it tumor suppressive function in hepatocellular carcinoma cells. We also determined whether rottlerin could govern cell apoptosis, migration and invasion in hepatocellular carcinoma cells. We further
https://doi.org/10.1016/j.bbrc.2017.11.198 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
2
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
investigated the mechanism of rottlerin-mediated anti-tumor activity in hepatocellular carcinoma cells. We found that rottlerin exerts its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells. Our findings suggest that rottlerin could be a useful agent for the treatment of human hepatocellular carcinoma.
cells were cultured for 20 h, the upper cells in chamber were removed by the cotton buds. The bottom surface cells were stained with 4 mg/ml Calcein AM at 37 C for 1 h. These invasive cells were photographed by a fluorescent microscope.
2. Materials and methods
After liver cancer cells grew to more than 90% confluence, a rectangular lesion on monolayers was generated using a sterile 100 ml pipette tip. Cells were treated with rottlerin for 20 h. Photographic images were taken at the lesion border using an inverted microscope (Olympus, IX71).
2.1. Reagents Anti-DDX3, anti-tubulin, anti-p21, and anti-Cyclin D1 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) Rottlerin and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Transwell inserts and Matrigel were obtained from BD Biosciences. Annexin apoptosis assay kit was bought from Beyotime Biotechnology (Shanghai, China). DDX3 shRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV6-DDX3 (NM_001356) human clone was purchased from OriGene (Rockville, MD, USA). 2.2. Cell culture The human liver cancer QGY7703 and SMMC7721 cells were cultured in RPMI 1640 medium (Gibro Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 mg/ml streptomycin and 100 U/ml penicillin. These cells were cultured in a 5% CO2 atmosphere at 37 C.
2.7. Wound healing assay
2.8. Real-time reverse transcription PCR analysis The total RNA was extracted with Trizol and reversed transcribed into cDNA by cDNA synthesis Kit. PCR analysis was performed using SYBR Green PCR Master Mix as described before [11]. 2.9. Western blotting analysis The rottlerin-treated cells were harvested and lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (EMD Millipore, Billerica, MA, USA). The equal quantity of proteins (30 mg) were loaded on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequently transferred to PVDF (polyvinylidene fluoride) membranes for Western blotting as described earlier [12]. 2.10. Transfection
2.3. MTT assay Cells were cultured in 6-well plates, and transfected with DDX3 cDNA or DDX3 shRNA or empty vector using lipofectamine 2000 following the manufacturer's instructions.
QGY7703 and SMMC7721 cells were seeded in 96-well plates. After overnight, cells were treated with different concentrations of rottlerin for 24, 48, and 72 h. MTT assay was used to measure the cell viability in liver cancer cells after different periods. Specifically, 10 ml MTT solution (0.5 mg/ml) was added in cell culture medium and incubated for 3 h in 5% CO2 atmosphere at 37 C. The supernatant in each well was discarded and added 100 ml DMSO. The absorption of each well was measured at 490 nm.
The data were presented as the mean values ± SE. The differences between groups were evaluated by Student's t-test. p < 0.05 was considered to be statistically significant.
2.4. Cell apoptosis assay
3. Results
Liver cancer cells were cultured in 6-well plates. After overnight, cells were treated with rottlerin for 48 h. Cells were then harvested and washed by PBS and suspended in 500 ml binding buffer including 5 ml annexin V-FITC and 5 ml Propidium iodide (PI) for 15 min at 37 C in the dark. Cell apoptosis was determined by a FACScalibur flow cytometer.
3.1. Rottlerin inhibited liver cancer cell growth
2.5. Cell cycle analysis Liver cells were cultured in 6-well plates for overnight and then treated with rottlerin for 48 h in RPMI 1640 medium. Then, cells were harvested and washed with PBS, and added 70% cold alcohol for overnight at 4 C. Cells were then incubated with 100 mg/ml RNase and 40 mg/ml PI for 30 min at 4 C. Cell cycle was measured by a FACScalibur flow cytometer (BD, USA). 2.6. Transwell migration and invasion assay The rottlerin-treated cells were seeded into an upper chamber of inserts with serum-free medium in a 24-well plate. The complete medium was added in bottom chamber of inserts. For cell invasion assay, the Transwell inserts were precoated with Matrigel. After
2.11. Statistical analysis
Rottlerin has been reported to suppress cell growth in various types of human cancer cells. Here, we explored whether rottlerin could inhibit liver cancer cell growth. QGY7703 and SMMC7721 cells were treated with different doses of rottlerin for 24 h, 48 h, and 72 h. We observed that rottlerin inhibited cell growth in a time and dose-dependent manner (Fig. 1A and Supplementary Fig. 1). Briefly, 1 mM rottlerin treatment for 72 h led to about 55% of cell growth inhibition in QGY7703 cells (Fig. 1A). Similarly, 2 mM rottlerin caused about 55% of cell growth inhibition in SMMC7721 cells (Fig. 1A). This data suggests that rottlerin inhibited liver cancer cell growth. 3.2. Rottlerin enhanced cell apoptosis in liver cancer cells We next investigated whether rottlerin could affect cell apoptosis in liver cancer cells. We used Annexin V-FITC/PI assay to measure cell apoptosis in QGY7703 and SMMC7721 cells with rottlerin treatment. We found that rottlerin treatment for 48 h led to induction of cell apoptosis (Fig. 1B and Supplementary Fig. 2A). Briefly, 1 mM and 2 mM rottlerin treatments enhanced cell apoptotic
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
3
Fig. 1. Rottlerin exerts its anti-tumor activities in liver cancer cells. A. Liver cancer cell viability was detected after rottlerin treatment for 72 h using MTT assay. *P < 0.05, **P < 0.01, compared to the control groups. B. PF-induced liver cancer cell apoptosis was accessed by Flow cytometry. C. Cell cycle analysis was performed by Flow cytometry in liver cancer cells treated with rottlerin. D. Cell motility was measured using wound healing assay in liver cancer cells.
death from 3.84% of control group to 15.25% and 29.44% in QGY7703 cells (Fig. 1B). In addition, 1 mM and 2 mM rottlerin treatments induced cell apoptosis from 3.52% of control group to 11.3% and 20.69% in SMMC7721 cells (Supplementary Fig. 2A). Thus, rottlerin induced cell apoptosis in liver cancer cells. 3.3. Rottlerin induced cell cycle arrest in liver cancer cells Next, we explored whether rottlerin regulated cell cycle in liver
cancer cells. PI staining and flow cytometry were used to measure the cell cycle in liver cancer cells after rottlerin treatment. Our results showed that rottlerin treatment caused cell cycle arrest at G0/ G1 phase in both liver cancer cells (Fig. 1Cand and Supplementary Fig. 2B). Clearly, 1 mM and 2 mM rottlerin treatments resulted in G0/ G1 phase from 48.88% in control group to 57.82% and 63.63% in QGY7703 cells, respectively (Fig. 2B). In line with this, 1 mM and 2 mM rottlerin treatment led to G0/G1 phase from 46.27% in control group to 56.65% and 73.84% in SMMC7721 cells, respectively
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
4
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 2. Rottlerin inhibited migration and invasion and increased DDX3 expression. A. The inhibitory effect of PF on cell migration and invasion was detected using Transwell chambers assay in QGY7703 cells. B. Cell migration and invasion was measured using Transwell chambers assay in SMMC7721 cells. C. The mRNA expression of DDX3 was measured by real-time RT-PCR in rottlerin-treated cells. D. The expression of DDX3, p21 and Cyclin D1 was measured by Western immunobloting analysis in liver cancer cells after rottlerin treatments.
(Fig. 2B and Supplementary Fig. 2B). Our data indicated that rottlerin induced cell cycle arrest at G0/G1 phase in liver cancer cells. 3.4. Rottlerin inhibited cell motility in liver cancer cells To explore whether rottlerin could affect cell motility in liver cancer cells, we performed would healing assay in QGY7703 and SMMC7721 cells. We found that rottlerin significantly retarded cell motility in both liver cancer cell lines (Fig. 3A and B). Briefly, 1 mM and 2 mM rottlerin treatment for 20 h led to 50% and 80% of
inhibition of cell motility in QGY7703 (Fig. 1D and Supplementary Fig. 3). Similar result was observed in SMMC7721 cells after rottlerin treatment for 20 h (Supplementary Fig. 3). Our findings demonstrated that rottlerin inhibited cell motility in liver cancer cells. 3.5. Rottlerin retarded cell migration and invasion in liver cancer cells To further validate whether rottlerin could retard cell motility in
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
5
Fig. 3. Down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function. A. The expression of DDX3 was detected by Western blotting analysis in QGY7703 cells after DDX3 down-regulation plus rottlerin treatment. Rot: Rottlerin; Both: Rottlerin plus DDX3 shRNA. B. MTT assay was performed to detect the effect of DDX3 down-regulation in combination with rottlerin treatment on QGY7703 cell growth. *P < 0.05, vs control; #P < 0.05 vs DDX3 shRNA or Rot treatment. C. Apoptotic cells were detected by Flow cytometry in QGY7703 cells after DDX3 shRNA infection and rottlerin treatment. D. Left panel: Cell invasion were detected by Transwell chambers assay in QGY7703 cells after DDX3 down-regulation in combination with rottlerin treatment. Right panel: Quantitative results were illustrated for left panel. *P < 0.05, vs control; #P < 0.05 vs DDX3 shRNA.
liver cancer cells, Transwel assay was performed in liver cancer cells after rottlerin treatments. Our migration assay results showed that rottlerin remarkably retarded cell migration in both QGY7703 and SMMC7721 cells (Fig. 2A and B). Moreover, our matrigel invasion assay data showed that rottlerin inhibited cell invasion in both liver cancer cell lines (Fig. 2A and B). Our findings revealed that rottlerin retarded cell migration and invasion in liver cancer cell lines. 3.6. Rottlerin increased DDX3 expression in liver cancer cells DX3 has been reported to play a tumor suppressive role in liver cancer cells. We explored whether DDX3 was involved in rottlerinmediated cell growth inhibition in liver cancer cells. Real-time RTPCR was used to measure the mRNA level of DDX3 in liver cancer cells after rottlerin treatment. We found that rottlerin increased DDX3 mRNA level in QGY7703 and SMMC7721 cells (Fig. 2C). Furthermore, Western blotting analysis was performed to measure the DDX3 protein level in liver cancer cells with rottlerin treatments. We found that rottlerin promoted the expression of DDX3 in both liver cancer cell lines (Fig. 2D). We also measured the expression of Cyclin D1 and p21, two downstream targets of DDX3, in liver cancer cells after rottlerin treatments. Indeed, rottlerin treatment led to downregulation of Cyclin D1 and upregulation of
p21 in liver cancer cells (Fig. 2C). 3.7. Down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function To further explore whether rottlerin exerted its anti-tumor activity via regulation of DDX3, SMMC7721 cells were infected with DDX3 shRNA. We found that DDX3 was significantly downregulated in SMMC7721 cells after DDX3 shRNA infection (Fig. 3A). Moreover, we found that down-regulation of DDX3 promoted cell growth in SMMC7721 cells (Fig. 3B). Down-regulation of DDX3 abrogated rottlerin-mediated cell growth inhibition in SMMC7721 cells (Fig. 3B). Furthermore, downregulation of DDX3 inhibited cell apoptosis and enhanced cell invasion (Fig. 3C and D). Strikingly, down-regulation of DDX3 abrogated rottlerin-trigged apoptosis and cell invasion inhibition (Fig. 3C and D). 3.8. Overexpression of DDX3 promoted the anti-tumor activity of rottlerin To determine whether DDX3 plays a role in anti-tumor activity of rottlerin in liver cancer cells, QGY7703 cells were transfected with DDX3 cDNA. We found that DDX3 expression was increased in
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
6
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 4. Overexpression of DDX3 promoted the anti-tumor activity of rottlerin. A. The expression of DDX3 was measured by Western blotting analysis in SMMC7721 cells after DDX3 overexpression plus rottlerin treatment. Rot: Rottlerin; Both: Rottlerin plus DDX3 cDNA. B. MTT assay was used to determine the effect of DDX3 overexpression in combination with rottlerin treatment on QGY7703 cell growth. *P < 0.05, vs control; #P < 0.05 vs DDX3 cDNA or Rot treatment. C. Apoptotic cells were detected by Flow cytometry in QGY7703 cells after DDX3 cDNA infection and rottlerin treatment. D. Left panel: Cell invasion were detected by Transwell chambers assay in QGY7703 cells after DDX3 overexpression in combination with rottlerin treatment. Right panel: Quantitative results were illustrated for left panel. *P < 0.05, vs control; #P < 0.05 vs DDX3 cDNA or rottlerin treatment.
QGY7703 cells after its cDNA transfection (Fig. 4A). DDX3 overexpression inhibited cell growth (Fig. 4B) and induced cell apoptosis (Fig. 4C), but retarded cell invasion (Fig. 4D). Moreover, overexpression of DDX3 enhanced rottlerin-induced inhibition of cell growth and invasion (Fig. 4B and D). DDX3 overexpression enhanced rottlerin-triggered cell apoptosis in QGY7703 cells (Fig. 4C). These findings indicated that DDX3 was involved in rottlerin-mediated anti-tumor functions. 4. Discussion In the present study, we determined the effects of rottlerin on hepatocellular carcinoma cells. Our results demonstrated that rottlerin suppressed cell growth and induced cell apoptosis, and caused cell cycle arrest in hepatocellular carcinoma cells. Moreover, rottlerin inhibited cell migration and invasion in hepatocellular carcinoma cells. Mechanistically, rottlerin increased DDX3 expression in hepatocellular carcinoma, indicating that rottlerin exerts its tumor suppressive function in part due to upregulation of DDX3. Thus, DDX3 could be a potential target of rottlerin in hepatocellular carcinoma. Accumulating evidence dissects the role of DDX3 in hepatocellular carcinoma. One study showed that DDX3 is necessary for the
replication of HBV (hepatitis B virus) and HCV (hepatitis C virus) [13,14]. Due to that hepatocellular carcinoma is often happened in patients with HBC and HCV, DDX3 could be involved in hepatocellular carcinoma development. Moreover, down-regulation of DDX3 was reported in HBV-positive HCC patients, but not in the HCV-positive ones [13]. Furthermore, down-regulation of DDX3 increased Cyclin D1 and decreased p21 level, leading to enhancement of cell growth in hepatocellular carcinoma cells [13]. Another study revealed that DDX3 suppressed cell colony formation ability in hepatocellular carcinoma via upregulation of p21 expression [14]. Recent study demonstrated that DDX3 down-regulation promoted stem cell-like properties and tumorigenesis through silencing several tumor suppressive miRNAs in hepatocellular carcinoma cells [15]. Notably, lower expression of DDX3 is associated with poor prognosis of hepatocellular carcinoma patients. These studies suggest that DDX3 could play a tumor suppressive role in hepatocellular carcinoma. Several RNA helicase inhibitors against DDX3 have been developed such as PLGA nanoparticle formulation of RK-33 [16]. NZ51, a ring-expanded nucleoside analog, inhibited cell motility and viability in breast cancer cells by targeting DDX3 [17]. Inhibition of DDX3 by RK-33 led to cell cycle arrest, induced apoptosis, and promoted radiation sensitization in lung cancer [18]. Doxorubicin
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
was also reported to inhibit the expression of DDX3 [19]. Rottelrin has been reported to exert its antitumor activity via inhibition f Skp2 in breast cancer cells and pancreatic cancer cells [20,21]. Moreover, rottlerin inhibited cell growth abnd invasion via downregulation of Cdc20 in glioma cells [22]. Recently, rottlerin was found to inhibit the expression of TAZ in non-small cell lung cancer cells [11]. Hou et al. reported that rottlerin induced tumor suppressive function due to suppression of Notch-1 pathway in nasopharyngeal carcinoma cells [23]. Our study showed that rottlerin increased the expression of DDX3 in hepatocellular carcinoma cells. Importantly, down-regulation of DDX3 abrogated the rottlerinmediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study indicated that rottlerin exerts its anti-tumor function via upregulation of DDX3. Further deeper investigation is required to determine the function and mechanism of rottlerin in hepatocellular carcinoma in the near future.
[6]
[7]
[8]
[9]
[10] [11]
[12]
Conflicts of interest [13]
There is no conflict of interest. Acknowledgement This work was supported by grants from Jiangsu Provincial Commission of Health and Family Planning (No. YG201402, No. YG201503), Jiangsu Provincial Medical Youth Talent, (No. QNRC2016249), Suzhou Science and Technology Bureau (No. SYSD2013041, SYSD2016044, SYS201788), Wujiang District Science and Technology Bureau (No. WS201301), and Wujiang District Commission of Health and Family Planning (No. WWK201607, WWK201609). Appendix A. Supplementary data
[14]
[15]
[16]
[17]
[18]
Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.198. References [1] R. Franca, A. Belfiore, S. Spadari, G. Maga, Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity, Proteins 67 (2007) 1128e1137. [2] B.A. Wilky, C. Kim, G. McCarty, E.A. Montgomery, K. Kammers, L.R. DeVine, R.N. Cole, V. Raman, D.M. Loeb, RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma, Oncogene 35 (2016) 2574e2583. [3] M.R. Heerma van Voss, F. Vesuna, K. Trumpi, J. Brilliant, C. Berlinicke, W. de Leng, O. Kranenburg, G.J. Offerhaus, H. Burger, E. van der Wall, P.J. van Diest, V. Raman, Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer, Oncotarget 6 (2015) 28312e28326. [4] M. Botlagunta, F. Vesuna, Y. Mironchik, A. Raman, A. Lisok, P. Winnard Jr., S. Mukadam, P. Van Diest, J.H. Chen, P. Farabaugh, A.H. Patel, V. Raman, Oncogenic role of DDX3 in breast cancer biogenesis, Oncogene 27 (2008) 3912e3922. [5] M. Botlagunta, B. Krishnamachary, F. Vesuna, P.T. Winnard Jr., G.M. Bol,
[19]
[20]
[21]
[22]
[23]
7
A.H. Patel, V. Raman, Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells, PLoS One 6 (2011) e17563. X. Miao, Z.L. Yang, L. Xiong, Q. Zou, Y. Yuan, J. Li, L. Liang, M. Chen, S. Chen, Nectin-2 and DDX3 are biomarkers for metastasis and poor prognosis of squamous cell/adenosquamous carcinomas and adenocarcinoma of gallbladder, Int. J. Clin. Exp. Pathol. 6 (2013) 179e190. C.Y. Su, T.C. Lin, Y.F. Lin, M.H. Chen, C.H. Lee, H.Y. Wang, Y.C. Lee, Y.P. Liu, C.L. Chen, M. Hsiao, DDX3 as a strongest prognosis marker and its downregulation promotes metastasis in colorectal cancer, Oncotarget 6 (2015) 18602e18612. C.H. Lee, S.H. Lin, S.F. Yang, S.M. Yang, M.K. Chen, H. Lee, J.L. Ko, C.J. Chen, K.T. Yeh, Low/negative expression of DDX3 might predict poor prognosis in non-smoker patients with oral cancer, Oral Dis. 20 (2014) 76e83. J.S. Huang, C.C. Chao, T.L. Su, S.H. Yeh, D.S. Chen, C.T. Chen, P.J. Chen, Y.S. Jou, Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma, Biochem. Biophysic. Res. Commun. 315 (2004) 950e958. M.J. Tuorkey, Cancer therapy with phytochemicals: present and future perspectives, Biomed. Environ. Sci. 28 (2015) 808e819. Z. Zhao, N. Zheng, L. Wang, Y. Hou, X. Zhou, Z. Wang, Rottlerin exhibits antitumor activity via down-regulation of TAZ in non-small cell lung cancer, Oncotarget 8 (2017) 7827e7838. Y. Huang, M. Zhao, H. Xu, K. Wang, Z. Fu, Y. Jiang, Z. Yao, RASAL2 downregulation in ovarian cancer promotes epithelial-mesenchymal transition and metastasis, Oncotarget 5 (2014) 6734e6745. P.C. Chang, C.W. Chi, G.Y. Chau, F.Y. Li, Y.H. Tsai, J.C. Wu, Y.H. Wu Lee, DDX3, a DEAD box RNA helicase, is deregulated in hepatitis virus-associated hepatocellular carcinoma and is involved in cell growth control, Oncogene 25 (2006) 1991e2003. C.H. Chao, C.M. Chen, P.L. Cheng, J.W. Shih, A.P. Tsou, Y.H. Lee, DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor, Cancer Res. 66 (2006) 6579e6588. H.K. Li, R.T. Mai, H.D. Huang, C.H. Chou, Y.A. Chang, Y.W. Chang, L.R. You, C.M. Chen, Y.H. Lee, DDX3 represses stemness by epigenetically modulating tumor-suppressive miRNAs in hepatocellular carcinoma, Sci. Rep. 6 (2016) 28637. G.M. Bol, R. Khan, M.R. Heerma van Voss, S. Tantravedi, D. Korz, Y. Kato, V. Raman, PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3, Cancer Chemother. Pharmacol. 76 (2015) 821e827. M. Xie, F. Vesuna, M. Botlagunta, G.M. Bol, A. Irving, Y. Bergman, R.S. Hosmane, Y. Kato, P.T. Winnard Jr., V. Raman, NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3, Oncotarget 6 (2015) 29901e29913. G.M. Bol, F. Vesuna, M. Xie, J. Zeng, K. Aziz, N. Gandhi, A. Levine, A. Irving, D. Korz, S. Tantravedi, M.R. Heerma van Voss, K. Gabrielson, E.A. Bordt, B.M. Polster, L. Cope, P. van der Groep, A. Kondaskar, M.A. Rudek, R.S. Hosmane, E. van der Wall, P.J. van Diest, P.T. Tran, V. Raman, Targeting DDX3 with a small molecule inhibitor for lung cancer therapy, EMBO Mol. Med. 7 (2015) 648e669. M. Botlagunta, B. Kollapalli, L. Kakarla, S.P. Gajarla, S.P. Gade, C.L. Dadi, A. Penumadu, S. Javeed, In vitro anti-cancer activity of doxorubicin against human RNA helicase, DDX3, Bioinformation 12 (2016) 347e353. X. Yin, Y. Zhang, J. Su, Y. Hou, L. Wang, X. Ye, Z. Zhao, X. Zhou, Y. Li, Z. Wang, Rottlerin exerts its anti-tumor activity through inhibition of Skp2 in breast cancer cells, Oncotarget 7 (2016) 66512e66524. J. Su, L. Wang, X. Yin, Z. Zhao, Y. Hou, X. Ye, X. Zhou, Z. Wang, Rottlerin exhibits anti-cancer effect through inactivation of S phase kinase-associated protein 2 in pancreatic cancer cells, Am. J. cancer Res. 6 (2016) 2178e2191. L. Wang, Y. Hou, X. Yin, J. Su, Z. Zhao, X. Ye, X. Zhou, L. Zhou, Z. Wang, Rottlerin inhibits cell growth and invasion via down-regulation of Cdc20 in glioma cells, Oncotarget 7 (2016) 69770e69782. Y. Hou, S. Feng, L. Wang, Z. Zhao, J. Su, X. Yin, N. Zheng, X. Zhou, J. Xia, Z. Wang, Inhibition of Notch-1 pathway is involved in rottlerin-induced tumor suppressive function in nasopharyngeal carcinoma cells, Oncotarget 8 (2017) 62120e62130.
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Rottlerin upregulates DDX3 expression in hepatocellular carcinoma Zhong Wang a, b, Gen-hai Shen a, Jia-ming Xie b, Bin Li a, *, Quan-gen Gao a, ** a b
Department of General Surgery, The First People's Hospital of Wu Jiang, Suzhou, Jiangsu, 215200, China Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2017 Accepted 29 November 2017 Available online xxx
Rottlerin has been reported to exert its anti-tumor activity in various types of human cancers. However, the underlying molecular mechanism has not been fully elucidated. In the current study, we explored whether rottlerin exhibits its tumor suppressive function in hepatocellular carcinoma cells. Our MTT assay results showed that rottlerin inhibited cell growth in hepatocellular carcinoma cells. Moreover, we found that rottlerin induced cell apoptosis and caused cell cycle arrest at G1 phase. Furthermore, our wound healing assay result demonstrated that rottlerin retarded cell migration in hepatocellular carcinoma cells. Additionally, rottlerin suppressed cell migration and invasion. Notably, we found that rottlerin upregulated DDX3 expression and subsequently downregulated Cyclin D1 expression and increased p21 level. Importantly, down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study suggests that rottlerin exhibits its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells. © 2017 Elsevier Inc. All rights reserved.
Keywords: DDX3 Hepatocellular carcinoma Rottlerinn Proliferation Cell cycle Invasion
1. Introduction DEAD-box proteins include the amino acid motif D-E-A-D (AspGlu-Ala-Asp), which are members of the largest family of RNA helicases. Initially, DEAD-box proteins were found to be involved in rearranging RNA-RNA and RNA-protein interactions as ATPdependent RNA binding proteins and RNA-dependent ATPases [1]. DEAD-box RNA helicase 3 (DDX3) belongs to DEAD-box family proteins. As a RNA helicase, DDX3 protein regulates several steps of RNA metabolisms, such as RNA splicing, mRNA export, transcription, and translation initiation. Therefore, DDX3 is involved in multiple cellular processes including apoptosis, cell cycle regulation, and cell stress response. Depletion of DDX3 leads to an early embryonic lethality in mice. Recently, DDX3 is reported to play an essential role in cancer development and progression [2,3]. Interestingly, DDX3 promotes cancer progression or exhibits tumor suppressive function in various types of human cancers. This concept suggests that DDX3 is a ‘double-edges sword’ gene. For example, oncogenic role of DDX3
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Q.-g. Gao).
(B.
Li),
[email protected]
in breast cancer biogenesis was reported. In line with this, overexpression of DDX3 induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays in breast cancer cells [4]. Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells [5]. Moreover, DDX3 is considered as a biomarker for metastasis and poor prognosis of squmous cells or adenocarcinoma of gallbladder [6]. Similarly, DDX3 could be a strongest prognosis marker and its downregulation enhanced metastasis in colorectal cancer [7]. However, low or negative expression of DDX3 might predict poor prognosis in non-smoker patients with oral cancer [8]. Accumulating studies suggested that DDX3 plays tumor suppressive role in hepatocellular carcinoma. However, one study reported that DDX3 is overexpressed in hepatocellular carcinoma tissues [9]. Thus, the role of DDX3 in hepatocellular carcinoma needs to be clarified. Rottlerin is a natural plant ployphenol compound, which is derived from the kalmala tree [10]. Rottelrin has been known to exert its antitumor activity in a variety of human cancers. However, the functions of rottelrin and underlying molecular mechanism in hepatocellular carcinoma have not been fully elucidated. In the current study, we explored whether rottlerin could exhibit it tumor suppressive function in hepatocellular carcinoma cells. We also determined whether rottlerin could govern cell apoptosis, migration and invasion in hepatocellular carcinoma cells. We further
https://doi.org/10.1016/j.bbrc.2017.11.198 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
2
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
investigated the mechanism of rottlerin-mediated anti-tumor activity in hepatocellular carcinoma cells. We found that rottlerin exerts its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells. Our findings suggest that rottlerin could be a useful agent for the treatment of human hepatocellular carcinoma.
cells were cultured for 20 h, the upper cells in chamber were removed by the cotton buds. The bottom surface cells were stained with 4 mg/ml Calcein AM at 37 C for 1 h. These invasive cells were photographed by a fluorescent microscope.
2. Materials and methods
After liver cancer cells grew to more than 90% confluence, a rectangular lesion on monolayers was generated using a sterile 100 ml pipette tip. Cells were treated with rottlerin for 20 h. Photographic images were taken at the lesion border using an inverted microscope (Olympus, IX71).
2.1. Reagents Anti-DDX3, anti-tubulin, anti-p21, and anti-Cyclin D1 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) Rottlerin and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Transwell inserts and Matrigel were obtained from BD Biosciences. Annexin apoptosis assay kit was bought from Beyotime Biotechnology (Shanghai, China). DDX3 shRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV6-DDX3 (NM_001356) human clone was purchased from OriGene (Rockville, MD, USA). 2.2. Cell culture The human liver cancer QGY7703 and SMMC7721 cells were cultured in RPMI 1640 medium (Gibro Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 mg/ml streptomycin and 100 U/ml penicillin. These cells were cultured in a 5% CO2 atmosphere at 37 C.
2.7. Wound healing assay
2.8. Real-time reverse transcription PCR analysis The total RNA was extracted with Trizol and reversed transcribed into cDNA by cDNA synthesis Kit. PCR analysis was performed using SYBR Green PCR Master Mix as described before [11]. 2.9. Western blotting analysis The rottlerin-treated cells were harvested and lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors (EMD Millipore, Billerica, MA, USA). The equal quantity of proteins (30 mg) were loaded on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequently transferred to PVDF (polyvinylidene fluoride) membranes for Western blotting as described earlier [12]. 2.10. Transfection
2.3. MTT assay Cells were cultured in 6-well plates, and transfected with DDX3 cDNA or DDX3 shRNA or empty vector using lipofectamine 2000 following the manufacturer's instructions.
QGY7703 and SMMC7721 cells were seeded in 96-well plates. After overnight, cells were treated with different concentrations of rottlerin for 24, 48, and 72 h. MTT assay was used to measure the cell viability in liver cancer cells after different periods. Specifically, 10 ml MTT solution (0.5 mg/ml) was added in cell culture medium and incubated for 3 h in 5% CO2 atmosphere at 37 C. The supernatant in each well was discarded and added 100 ml DMSO. The absorption of each well was measured at 490 nm.
The data were presented as the mean values ± SE. The differences between groups were evaluated by Student's t-test. p < 0.05 was considered to be statistically significant.
2.4. Cell apoptosis assay
3. Results
Liver cancer cells were cultured in 6-well plates. After overnight, cells were treated with rottlerin for 48 h. Cells were then harvested and washed by PBS and suspended in 500 ml binding buffer including 5 ml annexin V-FITC and 5 ml Propidium iodide (PI) for 15 min at 37 C in the dark. Cell apoptosis was determined by a FACScalibur flow cytometer.
3.1. Rottlerin inhibited liver cancer cell growth
2.5. Cell cycle analysis Liver cells were cultured in 6-well plates for overnight and then treated with rottlerin for 48 h in RPMI 1640 medium. Then, cells were harvested and washed with PBS, and added 70% cold alcohol for overnight at 4 C. Cells were then incubated with 100 mg/ml RNase and 40 mg/ml PI for 30 min at 4 C. Cell cycle was measured by a FACScalibur flow cytometer (BD, USA). 2.6. Transwell migration and invasion assay The rottlerin-treated cells were seeded into an upper chamber of inserts with serum-free medium in a 24-well plate. The complete medium was added in bottom chamber of inserts. For cell invasion assay, the Transwell inserts were precoated with Matrigel. After
2.11. Statistical analysis
Rottlerin has been reported to suppress cell growth in various types of human cancer cells. Here, we explored whether rottlerin could inhibit liver cancer cell growth. QGY7703 and SMMC7721 cells were treated with different doses of rottlerin for 24 h, 48 h, and 72 h. We observed that rottlerin inhibited cell growth in a time and dose-dependent manner (Fig. 1A and Supplementary Fig. 1). Briefly, 1 mM rottlerin treatment for 72 h led to about 55% of cell growth inhibition in QGY7703 cells (Fig. 1A). Similarly, 2 mM rottlerin caused about 55% of cell growth inhibition in SMMC7721 cells (Fig. 1A). This data suggests that rottlerin inhibited liver cancer cell growth. 3.2. Rottlerin enhanced cell apoptosis in liver cancer cells We next investigated whether rottlerin could affect cell apoptosis in liver cancer cells. We used Annexin V-FITC/PI assay to measure cell apoptosis in QGY7703 and SMMC7721 cells with rottlerin treatment. We found that rottlerin treatment for 48 h led to induction of cell apoptosis (Fig. 1B and Supplementary Fig. 2A). Briefly, 1 mM and 2 mM rottlerin treatments enhanced cell apoptotic
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
3
Fig. 1. Rottlerin exerts its anti-tumor activities in liver cancer cells. A. Liver cancer cell viability was detected after rottlerin treatment for 72 h using MTT assay. *P < 0.05, **P < 0.01, compared to the control groups. B. PF-induced liver cancer cell apoptosis was accessed by Flow cytometry. C. Cell cycle analysis was performed by Flow cytometry in liver cancer cells treated with rottlerin. D. Cell motility was measured using wound healing assay in liver cancer cells.
death from 3.84% of control group to 15.25% and 29.44% in QGY7703 cells (Fig. 1B). In addition, 1 mM and 2 mM rottlerin treatments induced cell apoptosis from 3.52% of control group to 11.3% and 20.69% in SMMC7721 cells (Supplementary Fig. 2A). Thus, rottlerin induced cell apoptosis in liver cancer cells. 3.3. Rottlerin induced cell cycle arrest in liver cancer cells Next, we explored whether rottlerin regulated cell cycle in liver
cancer cells. PI staining and flow cytometry were used to measure the cell cycle in liver cancer cells after rottlerin treatment. Our results showed that rottlerin treatment caused cell cycle arrest at G0/ G1 phase in both liver cancer cells (Fig. 1Cand and Supplementary Fig. 2B). Clearly, 1 mM and 2 mM rottlerin treatments resulted in G0/ G1 phase from 48.88% in control group to 57.82% and 63.63% in QGY7703 cells, respectively (Fig. 2B). In line with this, 1 mM and 2 mM rottlerin treatment led to G0/G1 phase from 46.27% in control group to 56.65% and 73.84% in SMMC7721 cells, respectively
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
4
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 2. Rottlerin inhibited migration and invasion and increased DDX3 expression. A. The inhibitory effect of PF on cell migration and invasion was detected using Transwell chambers assay in QGY7703 cells. B. Cell migration and invasion was measured using Transwell chambers assay in SMMC7721 cells. C. The mRNA expression of DDX3 was measured by real-time RT-PCR in rottlerin-treated cells. D. The expression of DDX3, p21 and Cyclin D1 was measured by Western immunobloting analysis in liver cancer cells after rottlerin treatments.
(Fig. 2B and Supplementary Fig. 2B). Our data indicated that rottlerin induced cell cycle arrest at G0/G1 phase in liver cancer cells. 3.4. Rottlerin inhibited cell motility in liver cancer cells To explore whether rottlerin could affect cell motility in liver cancer cells, we performed would healing assay in QGY7703 and SMMC7721 cells. We found that rottlerin significantly retarded cell motility in both liver cancer cell lines (Fig. 3A and B). Briefly, 1 mM and 2 mM rottlerin treatment for 20 h led to 50% and 80% of
inhibition of cell motility in QGY7703 (Fig. 1D and Supplementary Fig. 3). Similar result was observed in SMMC7721 cells after rottlerin treatment for 20 h (Supplementary Fig. 3). Our findings demonstrated that rottlerin inhibited cell motility in liver cancer cells. 3.5. Rottlerin retarded cell migration and invasion in liver cancer cells To further validate whether rottlerin could retard cell motility in
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
5
Fig. 3. Down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function. A. The expression of DDX3 was detected by Western blotting analysis in QGY7703 cells after DDX3 down-regulation plus rottlerin treatment. Rot: Rottlerin; Both: Rottlerin plus DDX3 shRNA. B. MTT assay was performed to detect the effect of DDX3 down-regulation in combination with rottlerin treatment on QGY7703 cell growth. *P < 0.05, vs control; #P < 0.05 vs DDX3 shRNA or Rot treatment. C. Apoptotic cells were detected by Flow cytometry in QGY7703 cells after DDX3 shRNA infection and rottlerin treatment. D. Left panel: Cell invasion were detected by Transwell chambers assay in QGY7703 cells after DDX3 down-regulation in combination with rottlerin treatment. Right panel: Quantitative results were illustrated for left panel. *P < 0.05, vs control; #P < 0.05 vs DDX3 shRNA.
liver cancer cells, Transwel assay was performed in liver cancer cells after rottlerin treatments. Our migration assay results showed that rottlerin remarkably retarded cell migration in both QGY7703 and SMMC7721 cells (Fig. 2A and B). Moreover, our matrigel invasion assay data showed that rottlerin inhibited cell invasion in both liver cancer cell lines (Fig. 2A and B). Our findings revealed that rottlerin retarded cell migration and invasion in liver cancer cell lines. 3.6. Rottlerin increased DDX3 expression in liver cancer cells DX3 has been reported to play a tumor suppressive role in liver cancer cells. We explored whether DDX3 was involved in rottlerinmediated cell growth inhibition in liver cancer cells. Real-time RTPCR was used to measure the mRNA level of DDX3 in liver cancer cells after rottlerin treatment. We found that rottlerin increased DDX3 mRNA level in QGY7703 and SMMC7721 cells (Fig. 2C). Furthermore, Western blotting analysis was performed to measure the DDX3 protein level in liver cancer cells with rottlerin treatments. We found that rottlerin promoted the expression of DDX3 in both liver cancer cell lines (Fig. 2D). We also measured the expression of Cyclin D1 and p21, two downstream targets of DDX3, in liver cancer cells after rottlerin treatments. Indeed, rottlerin treatment led to downregulation of Cyclin D1 and upregulation of
p21 in liver cancer cells (Fig. 2C). 3.7. Down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function To further explore whether rottlerin exerted its anti-tumor activity via regulation of DDX3, SMMC7721 cells were infected with DDX3 shRNA. We found that DDX3 was significantly downregulated in SMMC7721 cells after DDX3 shRNA infection (Fig. 3A). Moreover, we found that down-regulation of DDX3 promoted cell growth in SMMC7721 cells (Fig. 3B). Down-regulation of DDX3 abrogated rottlerin-mediated cell growth inhibition in SMMC7721 cells (Fig. 3B). Furthermore, downregulation of DDX3 inhibited cell apoptosis and enhanced cell invasion (Fig. 3C and D). Strikingly, down-regulation of DDX3 abrogated rottlerin-trigged apoptosis and cell invasion inhibition (Fig. 3C and D). 3.8. Overexpression of DDX3 promoted the anti-tumor activity of rottlerin To determine whether DDX3 plays a role in anti-tumor activity of rottlerin in liver cancer cells, QGY7703 cells were transfected with DDX3 cDNA. We found that DDX3 expression was increased in
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
6
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 4. Overexpression of DDX3 promoted the anti-tumor activity of rottlerin. A. The expression of DDX3 was measured by Western blotting analysis in SMMC7721 cells after DDX3 overexpression plus rottlerin treatment. Rot: Rottlerin; Both: Rottlerin plus DDX3 cDNA. B. MTT assay was used to determine the effect of DDX3 overexpression in combination with rottlerin treatment on QGY7703 cell growth. *P < 0.05, vs control; #P < 0.05 vs DDX3 cDNA or Rot treatment. C. Apoptotic cells were detected by Flow cytometry in QGY7703 cells after DDX3 cDNA infection and rottlerin treatment. D. Left panel: Cell invasion were detected by Transwell chambers assay in QGY7703 cells after DDX3 overexpression in combination with rottlerin treatment. Right panel: Quantitative results were illustrated for left panel. *P < 0.05, vs control; #P < 0.05 vs DDX3 cDNA or rottlerin treatment.
QGY7703 cells after its cDNA transfection (Fig. 4A). DDX3 overexpression inhibited cell growth (Fig. 4B) and induced cell apoptosis (Fig. 4C), but retarded cell invasion (Fig. 4D). Moreover, overexpression of DDX3 enhanced rottlerin-induced inhibition of cell growth and invasion (Fig. 4B and D). DDX3 overexpression enhanced rottlerin-triggered cell apoptosis in QGY7703 cells (Fig. 4C). These findings indicated that DDX3 was involved in rottlerin-mediated anti-tumor functions. 4. Discussion In the present study, we determined the effects of rottlerin on hepatocellular carcinoma cells. Our results demonstrated that rottlerin suppressed cell growth and induced cell apoptosis, and caused cell cycle arrest in hepatocellular carcinoma cells. Moreover, rottlerin inhibited cell migration and invasion in hepatocellular carcinoma cells. Mechanistically, rottlerin increased DDX3 expression in hepatocellular carcinoma, indicating that rottlerin exerts its tumor suppressive function in part due to upregulation of DDX3. Thus, DDX3 could be a potential target of rottlerin in hepatocellular carcinoma. Accumulating evidence dissects the role of DDX3 in hepatocellular carcinoma. One study showed that DDX3 is necessary for the
replication of HBV (hepatitis B virus) and HCV (hepatitis C virus) [13,14]. Due to that hepatocellular carcinoma is often happened in patients with HBC and HCV, DDX3 could be involved in hepatocellular carcinoma development. Moreover, down-regulation of DDX3 was reported in HBV-positive HCC patients, but not in the HCV-positive ones [13]. Furthermore, down-regulation of DDX3 increased Cyclin D1 and decreased p21 level, leading to enhancement of cell growth in hepatocellular carcinoma cells [13]. Another study revealed that DDX3 suppressed cell colony formation ability in hepatocellular carcinoma via upregulation of p21 expression [14]. Recent study demonstrated that DDX3 down-regulation promoted stem cell-like properties and tumorigenesis through silencing several tumor suppressive miRNAs in hepatocellular carcinoma cells [15]. Notably, lower expression of DDX3 is associated with poor prognosis of hepatocellular carcinoma patients. These studies suggest that DDX3 could play a tumor suppressive role in hepatocellular carcinoma. Several RNA helicase inhibitors against DDX3 have been developed such as PLGA nanoparticle formulation of RK-33 [16]. NZ51, a ring-expanded nucleoside analog, inhibited cell motility and viability in breast cancer cells by targeting DDX3 [17]. Inhibition of DDX3 by RK-33 led to cell cycle arrest, induced apoptosis, and promoted radiation sensitization in lung cancer [18]. Doxorubicin
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198
Z. Wang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
was also reported to inhibit the expression of DDX3 [19]. Rottelrin has been reported to exert its antitumor activity via inhibition f Skp2 in breast cancer cells and pancreatic cancer cells [20,21]. Moreover, rottlerin inhibited cell growth abnd invasion via downregulation of Cdc20 in glioma cells [22]. Recently, rottlerin was found to inhibit the expression of TAZ in non-small cell lung cancer cells [11]. Hou et al. reported that rottlerin induced tumor suppressive function due to suppression of Notch-1 pathway in nasopharyngeal carcinoma cells [23]. Our study showed that rottlerin increased the expression of DDX3 in hepatocellular carcinoma cells. Importantly, down-regulation of DDX3 abrogated the rottlerinmediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study indicated that rottlerin exerts its anti-tumor function via upregulation of DDX3. Further deeper investigation is required to determine the function and mechanism of rottlerin in hepatocellular carcinoma in the near future.
[6]
[7]
[8]
[9]
[10] [11]
[12]
Conflicts of interest [13]
There is no conflict of interest. Acknowledgement This work was supported by grants from Jiangsu Provincial Commission of Health and Family Planning (No. YG201402, No. YG201503), Jiangsu Provincial Medical Youth Talent, (No. QNRC2016249), Suzhou Science and Technology Bureau (No. SYSD2013041, SYSD2016044, SYS201788), Wujiang District Science and Technology Bureau (No. WS201301), and Wujiang District Commission of Health and Family Planning (No. WWK201607, WWK201609). Appendix A. Supplementary data
[14]
[15]
[16]
[17]
[18]
Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.198. References [1] R. Franca, A. Belfiore, S. Spadari, G. Maga, Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity, Proteins 67 (2007) 1128e1137. [2] B.A. Wilky, C. Kim, G. McCarty, E.A. Montgomery, K. Kammers, L.R. DeVine, R.N. Cole, V. Raman, D.M. Loeb, RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma, Oncogene 35 (2016) 2574e2583. [3] M.R. Heerma van Voss, F. Vesuna, K. Trumpi, J. Brilliant, C. Berlinicke, W. de Leng, O. Kranenburg, G.J. Offerhaus, H. Burger, E. van der Wall, P.J. van Diest, V. Raman, Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer, Oncotarget 6 (2015) 28312e28326. [4] M. Botlagunta, F. Vesuna, Y. Mironchik, A. Raman, A. Lisok, P. Winnard Jr., S. Mukadam, P. Van Diest, J.H. Chen, P. Farabaugh, A.H. Patel, V. Raman, Oncogenic role of DDX3 in breast cancer biogenesis, Oncogene 27 (2008) 3912e3922. [5] M. Botlagunta, B. Krishnamachary, F. Vesuna, P.T. Winnard Jr., G.M. Bol,
[19]
[20]
[21]
[22]
[23]
7
A.H. Patel, V. Raman, Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells, PLoS One 6 (2011) e17563. X. Miao, Z.L. Yang, L. Xiong, Q. Zou, Y. Yuan, J. Li, L. Liang, M. Chen, S. Chen, Nectin-2 and DDX3 are biomarkers for metastasis and poor prognosis of squamous cell/adenosquamous carcinomas and adenocarcinoma of gallbladder, Int. J. Clin. Exp. Pathol. 6 (2013) 179e190. C.Y. Su, T.C. Lin, Y.F. Lin, M.H. Chen, C.H. Lee, H.Y. Wang, Y.C. Lee, Y.P. Liu, C.L. Chen, M. Hsiao, DDX3 as a strongest prognosis marker and its downregulation promotes metastasis in colorectal cancer, Oncotarget 6 (2015) 18602e18612. C.H. Lee, S.H. Lin, S.F. Yang, S.M. Yang, M.K. Chen, H. Lee, J.L. Ko, C.J. Chen, K.T. Yeh, Low/negative expression of DDX3 might predict poor prognosis in non-smoker patients with oral cancer, Oral Dis. 20 (2014) 76e83. J.S. Huang, C.C. Chao, T.L. Su, S.H. Yeh, D.S. Chen, C.T. Chen, P.J. Chen, Y.S. Jou, Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma, Biochem. Biophysic. Res. Commun. 315 (2004) 950e958. M.J. Tuorkey, Cancer therapy with phytochemicals: present and future perspectives, Biomed. Environ. Sci. 28 (2015) 808e819. Z. Zhao, N. Zheng, L. Wang, Y. Hou, X. Zhou, Z. Wang, Rottlerin exhibits antitumor activity via down-regulation of TAZ in non-small cell lung cancer, Oncotarget 8 (2017) 7827e7838. Y. Huang, M. Zhao, H. Xu, K. Wang, Z. Fu, Y. Jiang, Z. Yao, RASAL2 downregulation in ovarian cancer promotes epithelial-mesenchymal transition and metastasis, Oncotarget 5 (2014) 6734e6745. P.C. Chang, C.W. Chi, G.Y. Chau, F.Y. Li, Y.H. Tsai, J.C. Wu, Y.H. Wu Lee, DDX3, a DEAD box RNA helicase, is deregulated in hepatitis virus-associated hepatocellular carcinoma and is involved in cell growth control, Oncogene 25 (2006) 1991e2003. C.H. Chao, C.M. Chen, P.L. Cheng, J.W. Shih, A.P. Tsou, Y.H. Lee, DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor, Cancer Res. 66 (2006) 6579e6588. H.K. Li, R.T. Mai, H.D. Huang, C.H. Chou, Y.A. Chang, Y.W. Chang, L.R. You, C.M. Chen, Y.H. Lee, DDX3 represses stemness by epigenetically modulating tumor-suppressive miRNAs in hepatocellular carcinoma, Sci. Rep. 6 (2016) 28637. G.M. Bol, R. Khan, M.R. Heerma van Voss, S. Tantravedi, D. Korz, Y. Kato, V. Raman, PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3, Cancer Chemother. Pharmacol. 76 (2015) 821e827. M. Xie, F. Vesuna, M. Botlagunta, G.M. Bol, A. Irving, Y. Bergman, R.S. Hosmane, Y. Kato, P.T. Winnard Jr., V. Raman, NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3, Oncotarget 6 (2015) 29901e29913. G.M. Bol, F. Vesuna, M. Xie, J. Zeng, K. Aziz, N. Gandhi, A. Levine, A. Irving, D. Korz, S. Tantravedi, M.R. Heerma van Voss, K. Gabrielson, E.A. Bordt, B.M. Polster, L. Cope, P. van der Groep, A. Kondaskar, M.A. Rudek, R.S. Hosmane, E. van der Wall, P.J. van Diest, P.T. Tran, V. Raman, Targeting DDX3 with a small molecule inhibitor for lung cancer therapy, EMBO Mol. Med. 7 (2015) 648e669. M. Botlagunta, B. Kollapalli, L. Kakarla, S.P. Gajarla, S.P. Gade, C.L. Dadi, A. Penumadu, S. Javeed, In vitro anti-cancer activity of doxorubicin against human RNA helicase, DDX3, Bioinformation 12 (2016) 347e353. X. Yin, Y. Zhang, J. Su, Y. Hou, L. Wang, X. Ye, Z. Zhao, X. Zhou, Y. Li, Z. Wang, Rottlerin exerts its anti-tumor activity through inhibition of Skp2 in breast cancer cells, Oncotarget 7 (2016) 66512e66524. J. Su, L. Wang, X. Yin, Z. Zhao, Y. Hou, X. Ye, X. Zhou, Z. Wang, Rottlerin exhibits anti-cancer effect through inactivation of S phase kinase-associated protein 2 in pancreatic cancer cells, Am. J. cancer Res. 6 (2016) 2178e2191. L. Wang, Y. Hou, X. Yin, J. Su, Z. Zhao, X. Ye, X. Zhou, L. Zhou, Z. Wang, Rottlerin inhibits cell growth and invasion via down-regulation of Cdc20 in glioma cells, Oncotarget 7 (2016) 69770e69782. Y. Hou, S. Feng, L. Wang, Z. Zhao, J. Su, X. Yin, N. Zheng, X. Zhou, J. Xia, Z. Wang, Inhibition of Notch-1 pathway is involved in rottlerin-induced tumor suppressive function in nasopharyngeal carcinoma cells, Oncotarget 8 (2017) 62120e62130.
Please cite this article in press as: Z. Wang, et al., Rottlerin upregulates DDX3 expression in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.198