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LXR agonist regulates the proliferation and apoptosis of human T-Cell acute lymphoblastic leukemia cells via the SOCS3 pathway
The International Journal of Biochemistry & Cell Biology 78 (2016) 180–185
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
LXR agonist regulates the proliferation and apoptosis of human T-Cell acute lymphoblastic leukemia cells via the SOCS3 pathway Rong Zhang ∗ , Zhuogang Liu, Yingchun Li, Bin Wu Department of Hematology, Shengjing hospital of China Medical University, Shenyang City 110021, China
a r t i c l e
i n f o
Article history: Received 31 March 2016 Received in revised form 13 June 2016 Accepted 11 July 2016 Available online 12 July 2016 Keywords: Liver X receptors Suppressor of cytokine signaling-3 E2F
a b s t r a c t Background: Recent studies show that Liver X receptors (LXR) activation is involved in the regulation of tumor cell death in solid cancer via E2 factor (E2F) transcription factor or suppressor of cytokine signaling-3 (SOCS3) pathway. However, the effect of LXR activation on leukemic cell fate has not been tested. Methods: Two human acute lymphoblastic leukemia (ALL) cell lines, Jurkat and SupT1, were cultured. Cells were transfected with small-interfering RNA (si-RNA) against SOCS3 and E2F family members (including E2F1, E2F2 and E2F3a) followed by treatment with LXR activator GW3965. The cellular biological behaviors, including proliferation, colony-forming ability and apoptosis were tested afterward. Results: Activation of LXR by GW3965 significantly decreased the cell proliferation rates and colonyforming abilities in the Jurkat and SupT1 cells, but increased their apoptosis rates. Western blot assay show that GW3965 treatment dramatically up-regulated the SOCS3 protein in both cell lines, without affecting E2F1, E2F2 and F2F3a expression levels. SOCS3 inhibition by si-RNA transfection, instead of E2F1, E2F2 and F2F3a pathway inhibition, abolished the aforementioned effects of LXR activation on Jurkat and SupT1 cells. Conclusion: Our finding suggests that LXR activation regulates leukemic cell fate and biological behavior via SOCS3 pathway, rather than E2F family members. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Liver X receptor (LXR) belongs to the nuclear receptor family of ligand-dependent transcription factors and regulats cholesterol, glucose, and fatty acid metabolism and inflammatory responses in mammal cells (Baranowski, 2008). LXR activation also plays an important role in regulating the proliferation or apoptosis in cancer cells (Mehrotra et al., 2011; El Roz et al., 2012; Lo Sasso et al., 2013; Chuu and Lin, 2010) . In breast cancer cell lines, LXR ligand treatment down-regulates a member of E2F transcription factor, namely, E2F2, and decrease its binding to cis-regulatory regions of target genes, leading to a significant disruption of breast cancer cell proliferation (Nguyen-Vu et al., 2013). In prostate cancer cells LXR activator GW3965 enhances suppressor of cytokine signaling 3 (SOCS3), accompanied by dramatically reduced cellular proliferation rate, immigration and invasion of cultured cells (Fu et al.,
2014). These results show that E2F2 or SOCS3 pathways are critical for effect of LXR on tumor biology. To date, the effect of LXR was reported only in solid tumors; its effect on non-solid tumor, such as leukemia, remains unknown. In this study, we cultured two human acute lymphoblastic leukemia (ALL) cell lines, Jurkat and SupT1, to study the role of LXR activation on the cellular biological behaviors, including proliferation, colony-forming ability and apoptosis in Jurkat and SupT1 cell lines. In addition, we illustrated the signal pathway mediating the effect of LXR on leukemic T lymphocytes.
2. Method 2.1. Cell culture
∗ Corresponding author. Tel.: +86 24 96615 24111; fax: +86 24 96615 24112. E-mail addresses: dr [email protected], [email protected] (R. Zhang), oncology [email protected] (Z. Liu), zhangjin1 [email protected] (y. Li), [email protected] (B. Wu). http://dx.doi.org/10.1016/j.biocel.2016.07.007 1357-2725/© 2016 Elsevier Ltd. All rights reserved.
The human T-ALL cell lines, namely, Jurkat and SupT1, were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM l-glutamine. Cells were seeded into 6-well plates and were grown until >75% confluent.
R. Zhang et al. / The International Journal of Biochemistry & Cell Biology 78 (2016) 180–185
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Fig. 1. (a) and (b) show that LXR activator GW3965 decreased the proliferation rates of Jurkat and SupT1 cells in time-dependent manners. (c) and (b) show data from Flow Cytometric Analysis of cell cycle: in both two cell lines, the numbers of cell with G2/M phrase are significantly reduced by GW3965 (both P < 0.05, vs. without GW3965). (e) and (d) show that LXR activator reduced the numbers of colonies in the colony formation assay in Jurkat and SupT1 cells. (g) and (h0 show that LXR activator dramatically increased the apoptosis rates in both Jurkat and SupT1 cell lines. GW3965 further potentiates the pro-apoptostic effect of Dexamethasone in both cell lines.
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R. Zhang et al. / The International Journal of Biochemistry & Cell Biology 78 (2016) 180–185
2.2. SOCS3 and E2F1, E2F2 and E2F3a small-interfering RNA transfection The cells were transiently transfected with 25 nmol/L of SOCS3siRNA, E2F1, E2F2 (both from Santa Cruz, USA) and F2F3a siRNA (Origen, USA) as well as their respective control siRNA according to the manufacturer’s instructions for 8 h. cells were further cultured for 48 h and the protein expression of SOCS3 and E2F family members were detected by Western blotting assay. 2.3. Cell treatment After successful si-RNA transfection, cells were treated with LXR activator GW3965 (20 uM for 2 h, Invitrogen, Carlsbad, CA). Cells with GW3965 solvent DMSO treatment were used as controls. 2.4. Proliferation assay The effect of hypoxia on the viability of cultured cells was evaluated by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, a monosodium salt (WST-8) assay (Dojindo Molecular Technologies, Tokyo, Japan). Briefly, cells were seeded at a density of 5 × 103 cells per well in 96-well microplates and placed in the hypoxic incubator for 24 h, 48 h or 72 h, 10 ul WST-8 solution was applied to each well and they were incubated for 4 h at 37 ◦ C. Absorbance was measured at 450 nm using a microplate reader (Benchmark Microplate Reader, BIORAD) with a reference wavelength of 490 nm.
2.8. Statistical analysis Results are expressed as means ± standard deviation. Statistical analyses were performed using SPSS statistical software. A Student t test and 1-way ANOVA followed by Dunnett multiple comparison tests were adopted. Values of P < 0.05 were considered significant.
3. Results 3.1. LXR activator decreases the proliferation and colony formation, but increased apoptosis of Jurkat and SupT1 cells Compared to untreated cells, the cell proliferation curve showed that LXR activator notably decreased the proliferation of Jurkat (Fig. 1a) and SupT1 (Fig. 1b) cells in time-dependent manners. To further confirm this results, our data from Flow Cytometric Analysis of cell cycle showed that in both two cell lines, the numbers of cell with G2/M phrase are significantly reduced by GW3965 (Fig. 1c and 1d, respectively). In the colony formation assay, the numbers of colonies were significantly reduced by GW3965 treatment in Jurkat (Fig. 1e) and SupT1 (Fig. 1f). As shown in Fig. 1e and f, in the absence of dexamethasone, the apoptosis rates in two cell lines were increased by GW3965 treatment (Fig. 1g). Dexamethasone along induced a robust increase in apoptosis rates in both cell lines, which were further increased by GW3965 treatment (Fig. 1h).
2.5. Soft agar colony-forming assay
3.2. The effects of LXR activator on SOCS3 and E2F transcription factor family member expressions in on Jurkat and SupT1 cells
Cells were plated in 24 well plates at a density of 200 cells/well in the complete medium containing 0.3% agar (Sigma-Aldrich) overlying an 0.5% agar layer. Cells were cultured at 37 ◦ C in 5% CO2 for 7 days. Colonies (>50 cells) were counted under an inverted microscope. Each experiment was repeated at least 3 times.
Compared to untreated cells, the application of GW3965 dramatically increased the SOCS3 expression in both cell lines, but did not change the E2F1, E2F2 and F2F3a expressions (Fig. 2).
2.6. Flow cytometric analysis of cell cycle progression and apoptosis We performed Flow cytometric analysis of cell cycle with propidium iodide (PI) staining. Cells were seeded at an initial concentration of 1 × 104 cells/ml and treated with 0.5 g/ml of PI for 24 h. To induce apoptosis, Jurkat and Sup-T1 cells were treated with 1 M dexamethasone (Sigma-Aldrich, St. Louis, USA) for 48 h. Cells were collected for the apoptosis assay. An annexin V-fluorescein isothiocyanate apoptosis detection kit (Zymed, Invitrogen Corporation) was used to detect cell apoptosis according to the manufacturer’s instructions. 2.7. Western blot Cells were harvested in ice-cold RIPA lysis buffer in the presence of the protease inhibitor phenylmethylsulfonyl fluoride for 30 min, and lysates were cleared by centrifugation. Proteins (10 ug) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with anti-SOCS3, antiE2F1, E2F2 and F2F3a and anti-GAPDH (all from Millipore, 1:1000 dilutions, USA) overnight at 4 ◦ C, followed by incubation with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG at room temperature for 1 h. After washing, immunoreactive bands were detected by enhanced chemiluminescence (Millipore, Billerica, MA).
Fig. 2. show that the addition of GW3965 to cells dramatically increased the SOCS3 expression in Jurkat and SupT1 cell lines, but did not change the E2F1, E2F2 and E2F3a expressions.
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Fig. 3. (a) show the SOCS3 protein expression after SOCS3 si-RNA transfection in Jurkat and SupT1 cells. SOCS3 was almost completely inhibited by western blot detection, while the E2F1, E2F2 and F2F3a expressions were not affected by SOCS3 si-RNA transfection. (b) and (c) show that SOCS3 inhibition induced restorations in proliferation rate in two cell lines, suggesting SOCS3 is involved in the anti-survival effect of LXR. (d) and (e) show SOCS3 siRNA transfection increased colony formation abilities than cells receiving scramble RNA transfection. (f) and (g) show the pro-apoptotic effect of GW3965 was totally abolished by SOCS3 inhibition in these two cell lines, while scramble RNA transfection did not change the effect of GW3965.
3.3. SOCS3 inhibition abolished the effects of LXR activator on Jurkat and SupT1 cells Fig. 3a shows the E2F and SOCS3 expression status after SOCS3 si-RNA transfection. As expected, SOCS3 protein expressions in
Jurkat and SupT1 cells were dramatically suppressed compared to scramble RNA transfection, while the E2F1, E2F2 and F2F3a expressions were not affected by SOCS3 si-RNA transfection. As shown in Fig. 3, when these cells were treated with GW3965, we observed restorations of reduced proliferation rate in SOCS3 si-RNA
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transfected Jurkat (Fig. 3b) and SupT1(Fig. 3c) cell lines. Scramble RNA transfection did not change the effects of LXR activator on leukemia cell lines. Likewise, with the presence of GW3965, the colony-forming assay showed that cells underwent SOCS3 siRNA transfection had increased colony formation abilities than cells receiving scramble RNA transfection (Fig. 3d–e). Meanwhile, the pro-apoptotic effect of GW3965 was totally abolished by SOCS3 inhibition in these two cell lines, while scramble RNA transfection did not change the effect of GW3965 (Fig. 3f–g). 3.4. E2F family member inhibition did not affect the effect of LXR activator on t cells Fig. 4 show the E2F, E2F2 and E2F3a expressions in two cell lines after their siRNA inhibition. Specifically, E2F 1siRNA inhibit did not change the E2F2, E2F3a and SOCS3 expression levels. Similarly, E2F2 and E2F3a siRNA transfection did not affect the other proteins in two cell lines. Then these cells were all treated with GW6954 for the proliferation and apoptosis assays. Our data show that specific E2F inhibition by si-RNA did not restore the reduced proliferation rate and colony-forming abilities induced by LXR activator in Jurkat and SupT1 cell lines. E2F1 specific knockdown did not affect the pro-apoptotic effect of GW3965 as well (Supplementary Fig. S1a–f in the online version at: 10.1016/j.biocel.2016.07. 008). Similarly,the E2F2 and E2F3a specific inhibition had no interference with the effects of LXR activator in Jurkat and SupT1 cell lines (Supplementary Fig. S2a–f and Fig. S3a–f in the online version at: 10.1016/j.biocel.2016.07.008). 4. Discussion In this study, we found that LXR activation significantly inhibits the proliferation and colony formation ability and increases the dexamethasone induced apoptosis rate in Jurkat and SupT1 cell lines. All these effects of LXR activator were abolished by the SOCS3 inhibition, but the inhibition of E2F family members did not change the effects of LXR activator on cultured cell lines. Collectively, these data suggest that SOCS3, instead of E2F family members, mediates the effects of GW3965 on Jurkat and SupT1 cell lines. LXR is a member of the nuclear receptor family of ligand-dependent transcription factors and has established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. The anti-proliferative effects of LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment (Nguyen-Vu et al., 2013). The association between LXR and solid tumor had been previously
reported. Lack of LXR leads to reduced cell proliferation rate in a model of mouse dorsal prostate epithelial cell (Dufour et al., 2013). LXR agonists were reported to suppress the proliferation of prostate cancer, breast cancer and ovarian cancer (El Roz et al., 2012; de Boussac et al., 2013; Rough et al., 2010). Modulating LXR signaling is therefore a potential adjuvant therapy for cancer (Mehrotra et al., 2011). To the best of our knowledge, this is the first study to investigate the effect of LXR pathway on the biological behaviors of leukemia cells. Our results were consistent with the previous studies in other types of blood tumor. For instance, LXR is involved in the induced differentiation of acute myeloid leukemia (AML) cells. LXR agonists, T0901317 or GW3965, induced potent differentiation and cytotoxicity in AML cell lines and primary human AML cells, but not in normal progenitor cells (Sanchez et al., 2014). LXR also interferes with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes. LXR activation suppresses cell proliferation and cell cycle progression of human T-cell blasts (Geyeregger et al., 2009). SOCS3 is one of the most important inhibitory molecules of inflammatory signal transduction pathways (Vegran et al., 2013; White et al., 2011). SOCS3 is an inducible cytokine and its expression is regulated by a variety of nuclear receptor, such as peroxisome-proliferator-activated receptor ˛ (PPAR) ˇ/ı. LXR is also a nuclear receptor that controls the expression of genes involved in glucose and lipid homoeostasis. Previous study suggests LXR regulate the carcinogenesis of prostate cancer cells via SOCS3 pathway (Fu et al., 2014). In hematological tumor, previous studies have shown that SOCS3 is highly expressed in anaplastic lymphoma (Zhang et al., 2011) and anaplastic large cell lymphoma (ALCL) cell lines (Cho-Vega et al., 2004). SOCS3 also regulates of sensitivity to interferon-alpha in chronic myelogenous leukemia (CML) cell lines (Sakai et al., 2002). Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by interferon-alpha. In our study, we found that SOCS3 is essential to LXR activation induced proliferation and colony formation ability and apoptosis, suggesting the involvement of SOCS3 pathway in the anti-tumor effects of LXR activator. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1), which has been implicated in many human malignancies (Zhang, 2008). E2F1, E2F2 and E2F3a are three major members of E2F transcription factor family. Previous studies showed that all the three transcription factor mRNAs are associated with gastric cancer cell invasion capacity and tumor differentiation (Muller and Helin, 2000; Fang and Han, 2006). E2F1 is a transcription factor that stimulates cellular proliferation and
Fig. 4. (a) show expression expressions after E2F si-RNA transfection in Jurkat and SupT1 cells. (b) show expression expressions after E2F2 si-RNA transfection in Jurkat and SupT1 cells. (c) show expression expressions after E2F3a si-RNA transfection in Jurkat and SupT1 cells.
R. Zhang et al. / The International Journal of Biochemistry & Cell Biology 78 (2016) 180–185
cell cycle progression. E2F-1 alone is sufficient to stimulate cells to initiate DNA synthesis and trigger of apoptosis. In breast cancer, Patients with high E2F1 activity were shown to have worse outcomes such as relapse free survival and distant metastasis free survival (Johnson et al., 2016). In breast cancer cells, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of breast cancer cell proliferation (Nguyen-Vu et al., 2013). However, in our current study, we did not observe the E2F1,E2F2 and E2F3a inhibition by si-RNA transfection affect the proliferation, colony formation abilities and apoptosis response to dexamethasone in cultured ALL cell lines. Our study suggests the mechanism of anti-tumor effect of LXR activation is cell type specific. 5. Conclusion In the present study, for the first time we observed that LXR activation by GW3965 inhibits proliferation and colony formation ability and increases dexamethasone induced apoptosis in cultured Jurkat and SupT1 cell lines. These effects are mediated by SOCS3 pathway, instead of E2F family, indicating SOCS3 may be a possible therapeutic target in leukemia treatment. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this article. Contribution ZR conceived this study and drafted the manuscript. ZR, ZL and LC carried out the cell culture, apoptosis assay and signal path study. ZR and WB performed the statistical analysis. All authors read and approved the final manuscript Acknowledgement We thank Dr Xuwei Hou for his help in statistic. References Baranowski, M., 2008. Biological role of liver X receptors. J. Physiol. Pharmacol. 59 (Suppl 7), 31–55.
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Mehrotra, A., Kaul, D., Joshi, K., 2011. LXR-alpha selectively reprogrammes cancer cells to enter into apoptosis. Mol. Cell. Biochem. 349 (1–2), 41–55. El Roz, A., Bard, J.M., Huvelin, J.M., Nazih, H., 2012. LXR agonists and ABCG1-dependent cholesterol efflux in MCF-7 breast cancer cells: relation to proliferation and apoptosis. Anticancer Res. 32 (7), 3007–3013. Lo Sasso, G., Bovenga, F., Murzilli, S., Salvatore, L., Di Tullio, G., Martelli, N., et al., 2013. Liver X receptors inhibit proliferation of human colorectal cancer cells and growth of intestinal tumors in mice. Gastroenterology 144 (7), 1497–1507, 507, e1–13. Chuu, C.P., Lin, H.P., 2010. Antiproliferative effect of LXR agonists T0901317 and 22(R)-hydroxycholesterol on multiple human cancer cell lines. Anticancer Res. 30 (9), 3643–3648. Nguyen-Vu, T., Vedin, L.L., Liu, K., Jonsson, P., Lin, J.Z., Candelaria, N.R., et al., 2013. Liver x receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism. Breast Cancer Res. 15 (3), R51. Fu, W., Yao, J., Huang, Y., Li, Q., Li, W., Chen, Z., et al., 2014. LXR agonist regulates the carcinogenesis of PCa via the SOCS3 pathway. Cell. Physiol. Biochem. 33 (1), 195–204. Dufour, J., Pommier, A., Alves, G., De Boussac, H., Lours-Calet, C., Volle, D.H., et al., 2013. Lack of liver X receptors leads to cell proliferation in a model of mouse dorsal prostate epithelial cell. PLoS One 8 (3), e58876. de Boussac, H., Pommier, A.J., Dufour, J., Trousson, A., Caira, F., Volle, D.H., et al., 2013. LXR, prostate cancer and cholesterol: the good, the bad and the ugly. Am. J. Cancer. Res. 3 (1), 58–69. Rough, J.J., Monroy, M.A., Yerrum, S., Daly, J.M., 2010. Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells. J. Ovarian Res. 3, 13. Sanchez, P.V., Glantz, S.T., Scotland, S., Kasner, M.T., Carroll, M., 2014. Induced differentiation of acute myeloid leukemia cells by activation of retinoid X and liver X receptors. Leukemia 28 (4), 749–760. Geyeregger, R., Shehata, M., Zeyda, M., Kiefer, F.W., Stuhlmeier, K.M., Porpaczy, E., et al., 2009. Liver X receptors interfere with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes. J. Leukoc. Biol. 86 (5), 1039–1048. Vegran, F., Berger, H., Ghiringhelli, F., Apetoh, L., 2013. Socs3 induction by PPARgamma restrains cancer-promoting inflammation. Cell Cycle 12 (14). White, G.E., Cotterill, A., Addley, M.R., Soilleux, E.J., Greaves, D.R., 2011. Suppressor of cytokine signalling protein SOCS3 expression is increased at sites of acute and chronic inflammation. J. Mol. Histol. 42 (2), 137–151. Zhang, Y., Forootan, S.S., Kamalian, L., Bao, Z.Z., Malki, M.I., Foster, C.S., et al., 2011. Suppressing tumourigenicity of prostate cancer cells by inhibiting osteopontin expression. Int. J. Oncol. 38 (4), 1083–1091. Cho-Vega, J.H., Rassidakis, G.Z., Amin, H.M., Tsioli, P., Spurgers, K., Remache, Y.K., et al., 2004. Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia 18 (11), 1872–1878. Sakai, I., Takeuchi, K., Yamauchi, H., Narumi, H., Fujita, S., 2002. Constitutive expression of SOCS3 confers resistance to IFN-alpha in chronic myelogenous leukemia cells. Blood 100 (8), 2926–2931. Zhang, A., 2008. comprehensive modular map of molecular interactions in RB/E2 F pathway. Mol. Syst. Biol. 4, 173. Muller, H., Helin, K., 2000. The E2F transcription factors: key regulators of cell proliferation. Biochim. Biophys. Acta 1470 (1), M1–12. Fang, Z.H., Han, Z.C., 2006. The transcription factor E2F: a crucial switch in the control of homeostasis and tumorigenesis. Histol. Histopathol. 21 (4), 403–413. Johnson, J., Thijssen, B., McDermott, U., Garnett, M., Wessels, L.F., Bernards, R., 2016. Targeting the RB-E2F pathway in breast cancer. Oncogene (February(29)), http://dx.doi.org/10.1038/onc.2016.32.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
LXR agonist regulates the proliferation and apoptosis of human T-Cell acute lymphoblastic leukemia cells via the SOCS3 pathway Rong Zhang ∗ , Zhuogang Liu, Yingchun Li, Bin Wu Department of Hematology, Shengjing hospital of China Medical University, Shenyang City 110021, China
a r t i c l e
i n f o
Article history: Received 31 March 2016 Received in revised form 13 June 2016 Accepted 11 July 2016 Available online 12 July 2016 Keywords: Liver X receptors Suppressor of cytokine signaling-3 E2F
a b s t r a c t Background: Recent studies show that Liver X receptors (LXR) activation is involved in the regulation of tumor cell death in solid cancer via E2 factor (E2F) transcription factor or suppressor of cytokine signaling-3 (SOCS3) pathway. However, the effect of LXR activation on leukemic cell fate has not been tested. Methods: Two human acute lymphoblastic leukemia (ALL) cell lines, Jurkat and SupT1, were cultured. Cells were transfected with small-interfering RNA (si-RNA) against SOCS3 and E2F family members (including E2F1, E2F2 and E2F3a) followed by treatment with LXR activator GW3965. The cellular biological behaviors, including proliferation, colony-forming ability and apoptosis were tested afterward. Results: Activation of LXR by GW3965 significantly decreased the cell proliferation rates and colonyforming abilities in the Jurkat and SupT1 cells, but increased their apoptosis rates. Western blot assay show that GW3965 treatment dramatically up-regulated the SOCS3 protein in both cell lines, without affecting E2F1, E2F2 and F2F3a expression levels. SOCS3 inhibition by si-RNA transfection, instead of E2F1, E2F2 and F2F3a pathway inhibition, abolished the aforementioned effects of LXR activation on Jurkat and SupT1 cells. Conclusion: Our finding suggests that LXR activation regulates leukemic cell fate and biological behavior via SOCS3 pathway, rather than E2F family members. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Liver X receptor (LXR) belongs to the nuclear receptor family of ligand-dependent transcription factors and regulats cholesterol, glucose, and fatty acid metabolism and inflammatory responses in mammal cells (Baranowski, 2008). LXR activation also plays an important role in regulating the proliferation or apoptosis in cancer cells (Mehrotra et al., 2011; El Roz et al., 2012; Lo Sasso et al., 2013; Chuu and Lin, 2010) . In breast cancer cell lines, LXR ligand treatment down-regulates a member of E2F transcription factor, namely, E2F2, and decrease its binding to cis-regulatory regions of target genes, leading to a significant disruption of breast cancer cell proliferation (Nguyen-Vu et al., 2013). In prostate cancer cells LXR activator GW3965 enhances suppressor of cytokine signaling 3 (SOCS3), accompanied by dramatically reduced cellular proliferation rate, immigration and invasion of cultured cells (Fu et al.,
2014). These results show that E2F2 or SOCS3 pathways are critical for effect of LXR on tumor biology. To date, the effect of LXR was reported only in solid tumors; its effect on non-solid tumor, such as leukemia, remains unknown. In this study, we cultured two human acute lymphoblastic leukemia (ALL) cell lines, Jurkat and SupT1, to study the role of LXR activation on the cellular biological behaviors, including proliferation, colony-forming ability and apoptosis in Jurkat and SupT1 cell lines. In addition, we illustrated the signal pathway mediating the effect of LXR on leukemic T lymphocytes.
2. Method 2.1. Cell culture
∗ Corresponding author. Tel.: +86 24 96615 24111; fax: +86 24 96615 24112. E-mail addresses: dr [email protected], [email protected] (R. Zhang), oncology [email protected] (Z. Liu), zhangjin1 [email protected] (y. Li), [email protected] (B. Wu). http://dx.doi.org/10.1016/j.biocel.2016.07.007 1357-2725/© 2016 Elsevier Ltd. All rights reserved.
The human T-ALL cell lines, namely, Jurkat and SupT1, were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM l-glutamine. Cells were seeded into 6-well plates and were grown until >75% confluent.
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Fig. 1. (a) and (b) show that LXR activator GW3965 decreased the proliferation rates of Jurkat and SupT1 cells in time-dependent manners. (c) and (b) show data from Flow Cytometric Analysis of cell cycle: in both two cell lines, the numbers of cell with G2/M phrase are significantly reduced by GW3965 (both P < 0.05, vs. without GW3965). (e) and (d) show that LXR activator reduced the numbers of colonies in the colony formation assay in Jurkat and SupT1 cells. (g) and (h0 show that LXR activator dramatically increased the apoptosis rates in both Jurkat and SupT1 cell lines. GW3965 further potentiates the pro-apoptostic effect of Dexamethasone in both cell lines.
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2.2. SOCS3 and E2F1, E2F2 and E2F3a small-interfering RNA transfection The cells were transiently transfected with 25 nmol/L of SOCS3siRNA, E2F1, E2F2 (both from Santa Cruz, USA) and F2F3a siRNA (Origen, USA) as well as their respective control siRNA according to the manufacturer’s instructions for 8 h. cells were further cultured for 48 h and the protein expression of SOCS3 and E2F family members were detected by Western blotting assay. 2.3. Cell treatment After successful si-RNA transfection, cells were treated with LXR activator GW3965 (20 uM for 2 h, Invitrogen, Carlsbad, CA). Cells with GW3965 solvent DMSO treatment were used as controls. 2.4. Proliferation assay The effect of hypoxia on the viability of cultured cells was evaluated by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, a monosodium salt (WST-8) assay (Dojindo Molecular Technologies, Tokyo, Japan). Briefly, cells were seeded at a density of 5 × 103 cells per well in 96-well microplates and placed in the hypoxic incubator for 24 h, 48 h or 72 h, 10 ul WST-8 solution was applied to each well and they were incubated for 4 h at 37 ◦ C. Absorbance was measured at 450 nm using a microplate reader (Benchmark Microplate Reader, BIORAD) with a reference wavelength of 490 nm.
2.8. Statistical analysis Results are expressed as means ± standard deviation. Statistical analyses were performed using SPSS statistical software. A Student t test and 1-way ANOVA followed by Dunnett multiple comparison tests were adopted. Values of P < 0.05 were considered significant.
3. Results 3.1. LXR activator decreases the proliferation and colony formation, but increased apoptosis of Jurkat and SupT1 cells Compared to untreated cells, the cell proliferation curve showed that LXR activator notably decreased the proliferation of Jurkat (Fig. 1a) and SupT1 (Fig. 1b) cells in time-dependent manners. To further confirm this results, our data from Flow Cytometric Analysis of cell cycle showed that in both two cell lines, the numbers of cell with G2/M phrase are significantly reduced by GW3965 (Fig. 1c and 1d, respectively). In the colony formation assay, the numbers of colonies were significantly reduced by GW3965 treatment in Jurkat (Fig. 1e) and SupT1 (Fig. 1f). As shown in Fig. 1e and f, in the absence of dexamethasone, the apoptosis rates in two cell lines were increased by GW3965 treatment (Fig. 1g). Dexamethasone along induced a robust increase in apoptosis rates in both cell lines, which were further increased by GW3965 treatment (Fig. 1h).
2.5. Soft agar colony-forming assay
3.2. The effects of LXR activator on SOCS3 and E2F transcription factor family member expressions in on Jurkat and SupT1 cells
Cells were plated in 24 well plates at a density of 200 cells/well in the complete medium containing 0.3% agar (Sigma-Aldrich) overlying an 0.5% agar layer. Cells were cultured at 37 ◦ C in 5% CO2 for 7 days. Colonies (>50 cells) were counted under an inverted microscope. Each experiment was repeated at least 3 times.
Compared to untreated cells, the application of GW3965 dramatically increased the SOCS3 expression in both cell lines, but did not change the E2F1, E2F2 and F2F3a expressions (Fig. 2).
2.6. Flow cytometric analysis of cell cycle progression and apoptosis We performed Flow cytometric analysis of cell cycle with propidium iodide (PI) staining. Cells were seeded at an initial concentration of 1 × 104 cells/ml and treated with 0.5 g/ml of PI for 24 h. To induce apoptosis, Jurkat and Sup-T1 cells were treated with 1 M dexamethasone (Sigma-Aldrich, St. Louis, USA) for 48 h. Cells were collected for the apoptosis assay. An annexin V-fluorescein isothiocyanate apoptosis detection kit (Zymed, Invitrogen Corporation) was used to detect cell apoptosis according to the manufacturer’s instructions. 2.7. Western blot Cells were harvested in ice-cold RIPA lysis buffer in the presence of the protease inhibitor phenylmethylsulfonyl fluoride for 30 min, and lysates were cleared by centrifugation. Proteins (10 ug) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with anti-SOCS3, antiE2F1, E2F2 and F2F3a and anti-GAPDH (all from Millipore, 1:1000 dilutions, USA) overnight at 4 ◦ C, followed by incubation with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG at room temperature for 1 h. After washing, immunoreactive bands were detected by enhanced chemiluminescence (Millipore, Billerica, MA).
Fig. 2. show that the addition of GW3965 to cells dramatically increased the SOCS3 expression in Jurkat and SupT1 cell lines, but did not change the E2F1, E2F2 and E2F3a expressions.
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Fig. 3. (a) show the SOCS3 protein expression after SOCS3 si-RNA transfection in Jurkat and SupT1 cells. SOCS3 was almost completely inhibited by western blot detection, while the E2F1, E2F2 and F2F3a expressions were not affected by SOCS3 si-RNA transfection. (b) and (c) show that SOCS3 inhibition induced restorations in proliferation rate in two cell lines, suggesting SOCS3 is involved in the anti-survival effect of LXR. (d) and (e) show SOCS3 siRNA transfection increased colony formation abilities than cells receiving scramble RNA transfection. (f) and (g) show the pro-apoptotic effect of GW3965 was totally abolished by SOCS3 inhibition in these two cell lines, while scramble RNA transfection did not change the effect of GW3965.
3.3. SOCS3 inhibition abolished the effects of LXR activator on Jurkat and SupT1 cells Fig. 3a shows the E2F and SOCS3 expression status after SOCS3 si-RNA transfection. As expected, SOCS3 protein expressions in
Jurkat and SupT1 cells were dramatically suppressed compared to scramble RNA transfection, while the E2F1, E2F2 and F2F3a expressions were not affected by SOCS3 si-RNA transfection. As shown in Fig. 3, when these cells were treated with GW3965, we observed restorations of reduced proliferation rate in SOCS3 si-RNA
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transfected Jurkat (Fig. 3b) and SupT1(Fig. 3c) cell lines. Scramble RNA transfection did not change the effects of LXR activator on leukemia cell lines. Likewise, with the presence of GW3965, the colony-forming assay showed that cells underwent SOCS3 siRNA transfection had increased colony formation abilities than cells receiving scramble RNA transfection (Fig. 3d–e). Meanwhile, the pro-apoptotic effect of GW3965 was totally abolished by SOCS3 inhibition in these two cell lines, while scramble RNA transfection did not change the effect of GW3965 (Fig. 3f–g). 3.4. E2F family member inhibition did not affect the effect of LXR activator on t cells Fig. 4 show the E2F, E2F2 and E2F3a expressions in two cell lines after their siRNA inhibition. Specifically, E2F 1siRNA inhibit did not change the E2F2, E2F3a and SOCS3 expression levels. Similarly, E2F2 and E2F3a siRNA transfection did not affect the other proteins in two cell lines. Then these cells were all treated with GW6954 for the proliferation and apoptosis assays. Our data show that specific E2F inhibition by si-RNA did not restore the reduced proliferation rate and colony-forming abilities induced by LXR activator in Jurkat and SupT1 cell lines. E2F1 specific knockdown did not affect the pro-apoptotic effect of GW3965 as well (Supplementary Fig. S1a–f in the online version at: 10.1016/j.biocel.2016.07. 008). Similarly,the E2F2 and E2F3a specific inhibition had no interference with the effects of LXR activator in Jurkat and SupT1 cell lines (Supplementary Fig. S2a–f and Fig. S3a–f in the online version at: 10.1016/j.biocel.2016.07.008). 4. Discussion In this study, we found that LXR activation significantly inhibits the proliferation and colony formation ability and increases the dexamethasone induced apoptosis rate in Jurkat and SupT1 cell lines. All these effects of LXR activator were abolished by the SOCS3 inhibition, but the inhibition of E2F family members did not change the effects of LXR activator on cultured cell lines. Collectively, these data suggest that SOCS3, instead of E2F family members, mediates the effects of GW3965 on Jurkat and SupT1 cell lines. LXR is a member of the nuclear receptor family of ligand-dependent transcription factors and has established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. The anti-proliferative effects of LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment (Nguyen-Vu et al., 2013). The association between LXR and solid tumor had been previously
reported. Lack of LXR leads to reduced cell proliferation rate in a model of mouse dorsal prostate epithelial cell (Dufour et al., 2013). LXR agonists were reported to suppress the proliferation of prostate cancer, breast cancer and ovarian cancer (El Roz et al., 2012; de Boussac et al., 2013; Rough et al., 2010). Modulating LXR signaling is therefore a potential adjuvant therapy for cancer (Mehrotra et al., 2011). To the best of our knowledge, this is the first study to investigate the effect of LXR pathway on the biological behaviors of leukemia cells. Our results were consistent with the previous studies in other types of blood tumor. For instance, LXR is involved in the induced differentiation of acute myeloid leukemia (AML) cells. LXR agonists, T0901317 or GW3965, induced potent differentiation and cytotoxicity in AML cell lines and primary human AML cells, but not in normal progenitor cells (Sanchez et al., 2014). LXR also interferes with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes. LXR activation suppresses cell proliferation and cell cycle progression of human T-cell blasts (Geyeregger et al., 2009). SOCS3 is one of the most important inhibitory molecules of inflammatory signal transduction pathways (Vegran et al., 2013; White et al., 2011). SOCS3 is an inducible cytokine and its expression is regulated by a variety of nuclear receptor, such as peroxisome-proliferator-activated receptor ˛ (PPAR) ˇ/ı. LXR is also a nuclear receptor that controls the expression of genes involved in glucose and lipid homoeostasis. Previous study suggests LXR regulate the carcinogenesis of prostate cancer cells via SOCS3 pathway (Fu et al., 2014). In hematological tumor, previous studies have shown that SOCS3 is highly expressed in anaplastic lymphoma (Zhang et al., 2011) and anaplastic large cell lymphoma (ALCL) cell lines (Cho-Vega et al., 2004). SOCS3 also regulates of sensitivity to interferon-alpha in chronic myelogenous leukemia (CML) cell lines (Sakai et al., 2002). Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by interferon-alpha. In our study, we found that SOCS3 is essential to LXR activation induced proliferation and colony formation ability and apoptosis, suggesting the involvement of SOCS3 pathway in the anti-tumor effects of LXR activator. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1), which has been implicated in many human malignancies (Zhang, 2008). E2F1, E2F2 and E2F3a are three major members of E2F transcription factor family. Previous studies showed that all the three transcription factor mRNAs are associated with gastric cancer cell invasion capacity and tumor differentiation (Muller and Helin, 2000; Fang and Han, 2006). E2F1 is a transcription factor that stimulates cellular proliferation and
Fig. 4. (a) show expression expressions after E2F si-RNA transfection in Jurkat and SupT1 cells. (b) show expression expressions after E2F2 si-RNA transfection in Jurkat and SupT1 cells. (c) show expression expressions after E2F3a si-RNA transfection in Jurkat and SupT1 cells.
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cell cycle progression. E2F-1 alone is sufficient to stimulate cells to initiate DNA synthesis and trigger of apoptosis. In breast cancer, Patients with high E2F1 activity were shown to have worse outcomes such as relapse free survival and distant metastasis free survival (Johnson et al., 2016). In breast cancer cells, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of breast cancer cell proliferation (Nguyen-Vu et al., 2013). However, in our current study, we did not observe the E2F1,E2F2 and E2F3a inhibition by si-RNA transfection affect the proliferation, colony formation abilities and apoptosis response to dexamethasone in cultured ALL cell lines. Our study suggests the mechanism of anti-tumor effect of LXR activation is cell type specific. 5. Conclusion In the present study, for the first time we observed that LXR activation by GW3965 inhibits proliferation and colony formation ability and increases dexamethasone induced apoptosis in cultured Jurkat and SupT1 cell lines. These effects are mediated by SOCS3 pathway, instead of E2F family, indicating SOCS3 may be a possible therapeutic target in leukemia treatment. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this article. Contribution ZR conceived this study and drafted the manuscript. ZR, ZL and LC carried out the cell culture, apoptosis assay and signal path study. ZR and WB performed the statistical analysis. All authors read and approved the final manuscript Acknowledgement We thank Dr Xuwei Hou for his help in statistic. References Baranowski, M., 2008. Biological role of liver X receptors. J. Physiol. Pharmacol. 59 (Suppl 7), 31–55.
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