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Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells
Author’s Accepted Manuscript Cathepsin L knockdown enhances curcuminmediated inhibition of growth, migration, and invasion of glioma cells Yao Fei, Yajie Xiong, Yifan Zhao, Wenjuan Wang, Meilin Han, Long Wang, Caihong Tan, Zhongqin Liang www.elsevier.com/locate/brainres
PII: DOI: Reference:
S0006-8993(16)30471-1 http://dx.doi.org/10.1016/j.brainres.2016.06.046 BRES44997
To appear in: Brain Research Received date: 1 May 2016 Revised date: 24 June 2016 Accepted date: 30 June 2016 Cite this article as: Yao Fei, Yajie Xiong, Yifan Zhao, Wenjuan Wang, Meilin Han, Long Wang, Caihong Tan and Zhongqin Liang, Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells, Brain Research, http://dx.doi.org/10.1016/j.brainres.2016.06.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells Yao Feia, Yajie Xionga, Yifan Zhaoa, Wenjuan Wanga, Meilin Hana, Long Wanga, Caihong Tana,b, Zhongqin Liang* a
Department of pharmacology, College of pharmaceutical Sciences, Soochow University, Suzhou,
China b
Department of Pharmacy, Affiliated Hospital of Jiangsu University, Zhenjiang, China
*
Corresponding author: Zhongqin Liang. Department of pharmacology, College of pharmaceutical
Sciences, Soochow University, Suzhou, China. Tel.:+86 051265882071; fax:+86 051265882089. [email protected]
Abstract Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers.
Keywords: Cathepsin L; curcumin; glioma cell; apoptosis; cell cycle arrest; invasion and migration
1. Introduction Glioma is a common and aggressive malignant human brain tumor with poor prognosis. This tumor seriously harms human health and accounts for an incidence of 3 cases per 100,000 individuals (Westermark B, 2012). Malignant gliomas are treated through surgical resection, radiation, and chemotherapy. Despite various treatments, most patients with gliomas die within 1 year after diagnosis (Mangiola A et al., 2010; Van Meir EG, 2010). Gliomas are also characterized by critical prognostic factors, namely, rapid growth rate and highly diffuse infiltration; however, these factors contribute to the failure of current therapies (Gagliano N et al., 2010; An Claes et al., 2007). Furthermore, many genes associated with an increased risk of glioma are often mutated or correlated with specifically acquired mutations (Melin B et al., 2013). Therefore, the pathogenesis of gliomas should be elucidated to develop new therapeutic targets and to improve therapeutic effects. Cathepsin L (CTSL), a lysosomal cysteine protease, is a potential therapeutic target in cancer treatment (Lankelma JM et al., 2010; Navab R et al., 2008). CTSL is overexpressed in several types of human carcinomas, including breast, lung, gastric, colon, melanoma, and glioma (Qin G et al., 2016; Chen Q et al., 2011; Miyamoto K et al., 2011; Chauhan SS et al., 1991; Stabuc B et al., 2006; Strojnik T et al., 2005). The level of CTSL expression is also associated with the degree of malignancy (Skrzydlewska E et al., 2005). CTSL upregulation promotes angiogenesis (Rebbaa A et al., 2009), transformation (Goulet B et al., 2007), and differentiation (Duncan EM et al.,2008). Furthermore, CTSL is associated with the growth, survival (Primon M et al., 2013), cycle (Goulet B,Nepveu A et al., 2004), migration, and invasion (Strojnik T et al., 1999; Kirschke H et al., 2000) of tumor cells. CTSL inhibition with radiotherapy significantly impedes the growth of glioma stem cells, promotes apoptosis, improves the radiosensitivity (Wang W et al., 2016) and downregulation of CTSL, and suppresses cancer invasion and migration by inhibiting EMT (Qingqing Zhang, 2015). Therefore, CTSL knockdown is necessary to regulate cell growth, migration, and invasion. The vulnerability of glioma cells to drugs can be enhanced by targeting the increased CTSL levels in glioma, by administering CTSL inhibitors,
or by genetically manipulating CTSL expression. Thus, CTSL may be a potential therapeutic target for glioma treatment. Curcumin(Cur) is a natural compound present in turmeric (Curcuma longa Linn), a rhizomatous plant commonly used as a spice(Prasad S et al., 2014). Curcumin possesses
potent
anti-inflammatory,
antioxidant,
chemopreventive,
and
chemotherapeutic activities. It also exhibits a broad spectrum of suppressive activities in various tumors, including glioblastoma, lung cancer, ovarian cancer, and prostate cancer (Jordan BC et al., 2016; Ravindran J et al., 2009; Lin YG et al., 2007). This substance is involved in different anti-tumor mechanisms, such as apoptosis, cell cycle arrest, and migration and invasion inhibition (Wang L et al., 2015; Zhang YP et al., 2015; Ma J et al., 2014; Qian chen et al., 2015). So far the most commonly recognized mode of curcumin action on cancer cells is the induction of apoptosis as was revealed by Ewa Sikora group (Kamila Wolanin et al., 2006; Grazyna Mosieniak et al., 2016). Several proteins are also implicated in curcumin-related mechanisms. For instance, Bcl-2 protein decreases in response to curcumin. Therefore, this protein may play a key role in curcumin-induced apoptosis of Caki cells (Karunagaran D et al., 2005; Woo JH et al., 2003). Nevertheless, the precise molecular mechanisms of curcumin-mediated inhibition of tumor growth, migration, and invasion should be elucidated. Our laboratory studies have revealed that curcumin activates autophagy and triggers the differentiation cascade in GICs isolated from human GBMs (Wenzhuo Zhuang et al., 2011). Autophagy and apoptosis also contribute to curcumin-induced death of K562 cells (Yanli Jia et al., 2009). The upstream signal regulation of autophagy and apoptosis has been proposed as a new strategy to enhance the anti-tumor activities of curcumin. The activation of autophagy and apoptosis is accompanied by an increase in the activity of lysosomal cathepsins (Boland B et al., 2004; Allen Kaasik et al., 2005); this phenomenon suggests that lysosomal hydrolases are involved in cell death pathways. As a lysosomal cathepsin, CTSL mediates autophagy and apoptosis (Lingyun Li et al., 2016). Our study aimed to determine whether CTSL participates in the regulation of apoptosis and cycle arrest. This study
also aimed to investigate whether CTSL is involved in curcumin-induced inhibition of migration and invasion and to reveal the underlying mechanisms. We found that the inhibition of CTSL may represent a novel therapeutic target to reinforce the efficacy of cancer chemotherapy.
2. Results 2.1. Curcumin inhibits U87 and U251 cell proliferation and induces apoptosis We investigated the cytotoxic effect of curcumin on different malignant glioma cells through a CCK8 assay. U87 and U251 cells were treated with curcumin at increasing doses (10, 20, 40, 60, and 80 μM) and time intervals (24, 48, and 72 h). Curcumin inhibited the growth of U87 and U251 cells in a dose- and time-dependent manner (Fig. 1A). Similarly, colony formation was suppressed by curcumin in both glioma cell lines (Fig. 3C). This finding suggested that the effect of curcumin on tumor cells was irreversible. We examined whether curcumin-induced apoptosis contributes to the growth inhibitory effects. U87 and U251 cells were stained with Hoechst 33258, and morphological changes were examined through fluorescence microscopy. Typical apoptotic morphological features, such as chromatin condensation and nuclear fragmentation, were detected in curcumin-treated U87 and U251 cells (Fig. 1B). We also performed Annexin V/PI staining to verify whether curcumin treatment can induce apoptosis. Flow cytometric analysis revealed that curcumin induced the apoptosis of glioma cells in a time-dependent manner (Fig. 1C). U87 and U251 cells were treated with 20 μM curcumin and harvested after 24, 48, and 72 h to confirm the apoptotic route of curcumin-dependent inhibition activation. We investigated the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins, two of the most important proteins associated with apoptosis. We found that both of them were altered in a time-dependent manner, but their changes were inversely proportional to each other. After the cells were treated with curcumin, the expression level of Bcl-2 decreased as the expression level of Bax gradually increased. The Bax/Bcl-2 ratio (Fig. 1D) also gradually increased.
Therefore, curcumin induced the apoptosis of U87 and U251 cells.
2.2. Curcumin upregulates CTSL expression in U87 and U251 cells To investigate the association between curcumin and CTSL expression, we determined the activity of this protease in curcumin-treated U87 and U251 cells. The active protein band of CTSL is 26 kDa. Western blot analyses revealed a time- and dose-dependent change in the CTSL protein level was detected in response to different treatment durations and curcumin concentrations (Fig. 2A). We confirmed the increase in protein levels through immunofluorescence. Our results demonstrated a dose-dependent effect (Fig. 2B). Therefore, curcumin increased the CTSL expression.
2.3. Inhibition of CTSL sensitizes U87 and U251 cells in response to curcumin treatment CTSL expression is exclusively increased in U87 and U251 cells. To verify whether the inhibition of CTSL contributes to the death of curcumin-treated U87 and U251 cells, we suppressed the CTSL expression through siRNA transfection. After conducting Western blot analysis, we screened the interference chains of two cell lines with a high interference efficiency in the following experiments: U251, CTS-homo-994 (3) and U87, CTS-homo-391 (1). The interference efficiency was further confirmed through an immunofluorescence assay (Fig. 3A). We examined the effect of CTSL siRNA on the curcumin-induced death of U87 and U251 cells. CCK-8 and clonogenic survival assays indicated that the siRNA-induced downregulation of CTSL effectively enhances the sensitivity of glioma cells to curcumin-induced death (Figs. 3B and 3C).
2.4. Knockdown of CTSL enhances curcumin-induced apoptosis Curcumin induced the apoptosis of U87 and U251 cells. The levels of apoptosis in U87 and U251 cells were determined through flow cytometry to confirm the role of CTSL knockdown on curcumin-induced apoptosis. We found that the percentage of
apoptotic U87 and U251 cells increased from 41.12%±1.90% to 79.64%±11.02% and from 84.86%±2.38% to 99.74%±0.11% (Curcumin vs. CTSL siRNA+Curcumin; Fig. 4A). Likewise, the pretreatment with CTSL siRNA could increase the expression of Bax and decrease the expression of Bcl-2 in the curcumin-treated cells (Fig. 4B). These
results
indicated
that
CTSL
knockdown
significantly
enhanced
curcumin-induced apoptosis.
2.5. Knockdown of CTSL promotes the curcumin-induced G2/M phase arrest To understand the mechanism of the growth inhibitory effect of curcumin in human glioma cells and to determine whether CTSL inhibition can promote the curcumin-induced cell cycle arrest, we confirmed that curcumin induced the G2/M phase arrest of U87 and U251 cells (Fig. 5A). We then treated CTSL siRNA-transfected cells with curcumin and found that the percentage of the cells in the G2/M phase increased (Fig. 5A). In order to prevent the interference of CTSL siRNA to the cell cycle, the cells were synchronized by serum starvation for 20 h to synchronize in the G0/G1 phase. We observed that the cell cycle distribution of the control siRNA-transfected cells remained unchanged. Cyclin B1, which is closely related to G2/M arrest, was subjected to Western blot analysis to examine the expression of cell cycle-associated molecules. During the G2/M phase transition, cyclin B1 binds to cell division cycle 2, which is also called cyclin-dependent kinase 1, to form a mitosis-promoting factor that facilitates the transition from G2 phase to M phase of the cell cycle (Zhou L et al., 2014; Pandey JP et al., 2014; Bostrom P et al., 2009). Cyclin B1 was decreased in the CTSL-knocked down cells treated with curcumin compared with the cells treated with curcumin alone (Fig. 5B). These data suggested that CTSL suppression promotes curcumin-induced G2/M phase arrest.
2.6. CTSL inhibition aggravates the inhibitory effect of curcumin on cell migration and invasion
In curcumin-treated tumor cell lines, the effects of CTSL knockdown on migration and invasion were determined through wound healing and invasion assays. The width of the injured line increased. Invasion assay also revealed that the number of colonies decreased. This finding suggested that the suppression of CTSL aggravated the inhibitory effect of curcumin on the migration and invasion of U251 and U87 cells (Fig. 6). Tumor invasion and migration were decreased by inhibiting CTSL in the curcumin-treated cells. This finding suggested an important role of CTSL in the curcumin‑ induced reduction of cell migration and invasion.
3. Discussion This study revealed a possible mechanistic function by which CTSL inhibition contributes to the enhanced sensitivity of curcumin-mediated inhibition of the growth, migration, and invasion of glioma cells. Curcumin treatment activated the expression of CTSL in glioma cells (Fig. 2); CTSL is remarkably upregulated in malignant gliomas (Strojnik T et al., 2005). We further demonstrated that the curcumin-induced apoptosis and cell cycle arrest of the glioma cells were enhanced through CTSL knockdown. The curcumin-induced inhibition of the migration and invasion of glioma cells was also increased through CTSL knockdown. Therefore, CTSL inhibition and curcumin treatment could be an effective approach to treat gliomas. In vivo and in vitro studies have demonstrated the ability of curcumin to induce tumor apoptosis effectively (Dorai T et al., 2001; Lv ZD et al., 2014; Zhou X et al., 2016). This effect is partially mediated by the mitochondrial pathway, which involves the increase in Bax/Bcl-2 ratio and the release of cytochrome c (Lin Zhu et al., 2015). Consistent with these findings, our data confirmed that curcumin can induce apoptosis and increase Bax/Bcl-2 ratio in glioma cells. Navab (Navab R et al., 2008) revealed that CTSL inhibition reduces IGF-1 receptor responsiveness; as a consequence, this phenomenon enhances apoptosis. This finding further confirmed that CTSL mediates an anti-apoptotic effect. Meanwhile, cell apoptosis analysis revealed that glioma cells with CTSL knockdown underwent more cell apoptosis and Bax/Bcl-2 ratio after curcumin was administered (Fig. 4). On the basis of our experimental results, we
speculated that curcumin upregulated the CTSL expression in U87 and U251 cells and CTSL plays an anti-apoptotic role in glioma cells. Thus, cells could be protected from the toxic effect of curcumin. The silenced CTSL weakened the protective effect; as a result, the sensitivity of cells to curcumin was enhanced. Curcumin possesses an antitumor activity, which is associated with its ability to induce G2/M cell cycle arrest (Sa G et al., 2008). Mechanistically, curcumin downregulated cyclin B1 and thus induced G2/M arrest (Cheng Liang et al., 2012). Cell cycle regulation may be affected by CTSL because its active isoform possibly participates in the degradation of nuclear transcription factors (Brix K et al., 2008). The cells were subjected to serum starvation to prevent the effects of CTSL on cell cycle. Our experiments demonstrated that the curcumin-induced inhibition of CTSL enhanced the sensitivity to cell cycle arrest (Fig. 5). This phenomenon could occur possibly because the nuclear isoform of CTSL could regulate the proteolytic processing of CUX1 to generate p75 and p110 CUX1, which are the active forms of full-length p200 CUX1 (Goulet B, Baruch A et al., 2004; Fei XF et al., 2007). The target genes of CUX1 not only participate in cell cycle progression (Vadnais C et al., 2012) but also regulate tumor cell apoptosis (Kirschke H et al., 2000). This finding may also help explain why cell cycle arrest and apoptosis are increased by CTSL inhibition in curcumin-treated cells. Migration and invasion are typical hallmarks of tumors. Curcumin can suppress tumor migration and invasion (Chiang IT et al., 2015; Wang S et al., 2011). The overexpression of CTSL may switch the melanoma cell phenotype from non-metastatic to highly metastatic. As a consequence, tumor migration and invasion are increased (Rousselet N et al., 2004; Yang Z et al., 2007; Wang SM et al., 2010). Our study indicated that CTSL knockdown sensitizes to curcumin-suppressed migration and invasion (Fig. 6). We also previously revealed that downregulation of CTSL suppresses cancer invasion and migration by inhibiting the EMT process (Qingqing Zhang. 2015). CTSL also decreases cell-to-cell adhesion to enhance tumor
invasion and migration through the direct cleavage of E-cadherin, which is a marker protein of EMT (Gocheva V et al., 2006). Nevertheless, further research should be performed to determine whether curcumin suppresses tumor migration and invasion through CTSL-induced EMT. In summary, our experiments revealed that curcumin can effectively induce the apoptosis and cell cycle arrest of glioma cells. Curcumin can also suppress migration and invasion. We further demonstrated that CTSL inhibition can enhance the sensitivity of glioma cells to curcumin-mediated apoptosis and cell cycle arrest. CTSL inhibition can also impede the migration and invasion of glioma cells. Therefore, the inhibition of CTSL may be an effective strategy to treat glioma with curcumin. Further studies should investigate CTSL as a novel therapeutic target for malignant tumors.
4. Experimental procedures 4.1. Cell culture and reagents U87 and U251 human glioma cells were purchased from the Shanghai Institute of Cell Biology, the Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco Life Technologies) and incubated in an incubator humidified with 5% CO2 at 37 °C. The cells in the mid-logarithmic phase were used in the experiments. Curcumin (Sigma) was prepared as a 20 mM stock solution in dimethylsulfoxide (DMSO, Sigma) and stored at −20 °C. In each experiment, curcumin was diluted with the cell culture medium to obtain the desired final concentrations. 4.2. Antibodies The following antibodies were used in this study: cyclin B1 (1:500, Santa Cruz, USA), CTSL (1:1000, Abcam, MA, USA), β-actin (1:1000, MultiSciences, Nanjing, China), Bcl-2 (1:200, Millipore, MA, USA), and Bax (1:1000, Abcam, Cambridge, UK).
4.3. siRNA transfection Human CTSL siRNA and control siRNA were purchased from GenePharma. U251 cells were seeded in 6-well plates and grown in DMEM supplemented with 10% FBS 1 day before transfection. After 24 h in culture, siRNA plasmid DNA was transfected into the cells by using a siRNA transfection reagent kit in accordance with the manufacturer’s protocol. After 24 h, the transfected cells were subjected to different treatments and then collected. The following siRNA sequences were used. Table1.siRNA for CTSL siRNA Negtive control(NC)
base sequence sense
5’-UUCUCCGAACGUGUCACGUTT-3’
antisense
5’-ACGUGACACGUUCGGAGAATT-3’
sense
5’-GCCUCAGCUACUCUAACAUTT-3’
antisense
5’-AUGUUAGAGUAGCUGAGGCTT-3’
sense
5’-CGAUGCACAACAGAUUAUATT-3’
antisense
5’-UAUAAUCUGUUGUGCAUCGTT-3’
sense
5’-CCAAGUAUUCUGUUGCUAATT-3’
antisense
5’-UUAGCAACAGAAUACUUGGTT-3’
sense
5’-CCUUCCUGUUCUAUAAAGATT-3’
antisense
5’-UCUUUAUAGAACAGGAAGGTT-3’
CTS-homo-391(1)
CTS-homo-449(2)
CTS-homo-994(3)
CTS-homo-1112(4) 4.4. Clonogenic survival assay
The cells were seeded in 6-well plates at a density of 4×102 cells per well, incubated overnight, pretreated with CTSL siRNA and control siRNA for 24 h, and exposed to different doses of curcumin. Afterward, the treated cells were incubated at 37 °C for 10 d. Colonies were fixed and stained with 0.5% crystal violet (Sigma Aldrich). 4.5. Cell viability assay Cell viability was assessed through a CCK-8 (Sigma, USA) assay. In brief, U87 and U251 cells were plated into 96-well plates. Curcumin (10, 20, 40, 60, 80 μM) was added to the culture medium and further incubated for 24, 48, and 72 h. U87 and
U251 cells were transfected with CTSL siRNA 24 h before they were treated with various doses of curcumin to examine the contribution of CTSL to the curcumin-induced death of these cells. A CCK-8 reagent was added to each well 2 h before the endpoint of incubation. Optical density (OD) at 450 nm in each well was determined by using a microplate reader. The percentage of cell viability was calculated as follows: cell viability (%) = (OD of experimental well / OD of positive control well) × 100%. 4.6. Hoechst 33258 staining The treated cells were analyzed using a Hoechst staining kit (Sigma). Staining was performed in accordance with the manufacturer’s protocol. The cells were washed with PBS, treated with a fixing solution for 10 min, and stained with Hoechst 33258 fluorescent dye for 10 min at room temperature. Morphological nuclear changes were observed and captured using an inverted fluorescence microscope. 4.7. Immunofluorescence detection of CTSL Exponentially growing cells were transferred to 24-well plates and incubated with curcumin. After the treatments were administered, the cells were fixed by incubating in 4% freshly prepared paraformaldehyde solution for 10 min at 4 °C. The cells were washed with PBS and blocked with a blocking buffer (1% bovine serum albumin and 0.1% Triton X-100) for 30 min at 37 °C. The cells were then incubated overnight with mouse anti-human CTSL antibody diluted with 0.1% Triton X-100. The cells were incubated with a primary antibody and washed four times with PBST. Afterward, the cells were incubated with a secondary antibody conjugated to Alexa 488 (Invitrogen) for 1 h at room temperature. The cells were then washed twice with PBST and incubated with DAPI for 10 min to counterstain the DNA. The coverslips were mounted on microscope slides by using an anti-fade mounting medium (Dako) to permit examination. Images were captured with a fluorescence microscope (LEICA TCS SP2, Germany) and imported into Adobe Photoshop. 4.8. Protein preparation and Western blot analysis The cells were harvested and rinsed with ice-cold PBS twice. Five volumes of Western blot lysing buffer was added to each volume of the cell pellets, and the
mixture was sonicated in an ice bath at 1 s/mL per sonicate with a 30 sec pause between intervals in 5 cycles. The sonicated mixture was microcentrifuged at 12,000 × g at 4 °C for 10 min, and the supernatant was preserved at −80 °C for later use. Protein concentration was determined by using a BCA kit (Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with cyclin B1, CTSL, β-actin, Bcl-2, and Bax at 4 °C overnight. After the membranes were washed thrice with TBST buffer (20 mmol/L Tris-buffered saline and 0.1% Tween 20), they were incubated with rabbit or mouse secondary antibodies for 1 h. Immunoblots were detected using an Odyssey infrared imaging system (LI-COR, NE, USA). Protein β-actin was used as a loading control. 4.9. Flow cytometry analysis The phase distribution of the cell cycle was determined using a cell apoptosis propidium iodide (PI) detection kit (KeyGen, Nanjing, China) and a FACSCalibur flow cytometer. Staining was performed in accordance with the manufacturer’s protocol. The cells were seeded in 6-well plates, incubated overnight, pretreated with CTSL siRNA and control siRNA for 24 h. Cells were serum-deprived for 20 h to synchronize in the G0/G1 phase. Then, cells were treated with curcumin for 24 h. The cells were harvested and fixed in 70% ethanol at −20 °C overnight. Then, the samples were centrifuged, re-suspended in 500 µl Buffer A with 250 μg/ml RNase A at 37 °C for 30 min, and stained with 5 µl of PI in the dark at room temperature for 30 min. The percentage of apoptotic cells was measured using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (KeyGen, Nanjing, China). A total of 10,000 cells per sample were examined by using a FACSCalibur flow cytometer. 4.10. Wound healing and invasion assays The cells were grown in 6-well plates for the wound-healing assay. After confluency was obtained, the cells were scratched with a pipette tip, rinsed to remove debris, and incubated in a fresh medium in the presence or absence of curcumin for 24 h. Cell migration images were captured at 0 and 24 h. The wound healing index was determined as a percentage and quantitatively analyzed as follows: five randomly selected distances were selected across the wound at 0 and 24 h and divided by the
distance measured at 0 h. A cell invasion assay was performed using 24-well Matrigel invasion chambers (BD Biosciences). The cells were trypsinized and re-seeded in the upper chamber at a concentration of 1×105/ml in 200 µl of the medium in the presence or absence of curcumin. The lower chamber contained 700 µl of the medium supplemented with 10% FBS. After 24 h, the cells on the upper surface of the filters were removed, and the cells on the lower surface were fixed with methanol and stained with crystal violet. 4.11. Statistical analysis Three independent experiments were performed. Data were presented as mean ± SD and statistically evaluated with ANOVA and Tukey’s multiple range tests in GraphPad Prism 4. P<0.05 was regarded as significant and P<0.01 was considered as highly significant.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 30873052, 81072656,81102466, and 81373430), the Outstanding Medical Academic Leader Program of Jiangsu Province (Grant No. LJ201139).
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Fig.1. Curcumin inhibits the proliferation of U87 and U251 cells and induces their apoptosis. (A) Dose- and time-dependent curcumin cytotoxicity determined through CCK-8 assay. Cells were treated with 0, 10, 20, 40, 60, and 80 μM curcumin for 24, 48, and 72 h. (B) Cells were treated with 20 μM curcumin for 48 h and stained with Hoechst 33258; nuclear condensation was observed through Hoechst staining (the arrowhead indicates an apoptotic nucleus, 40× magnification). (C) Annexin V-FITC/PI staining and flow cytometric determination of the apoptosis of U87 and U251 cells treated with 20 μM curcumin for 24, 48, and 72 h demonstrated a remarkable increase in the percentage of apoptotic cells compared with the untreated control group. (D) U87 and U251 cells were treated with curcumin (20 μM) for 24, 48, and 72 h. Cell lysates were prepared, and protein level was determined through Western blot analysis. β-actin was used to normalize and verify protein loading. Results are expressed as mean ± SD. Statistical significance is indicated by *p<0.05, ** p<0.01 vs. control group.
Fig.2. Expression of CTSL protein in curcumin-treated U87 and U251 cells. (A) Western blot analysis of CTSL in U87 and U251 cells treated with different curcumin concentration or 20 μM curcumin at different time points. β-actin was used as an internal control. * P<0.05, **p<0.01 vs. control group. (B) Glioma cells were treated with different curcumin concentrations and then subjected to immunofluorescence assay. Green fluorescence in the cytoplasm and co-expression with DAPI were observed (630× magnification).
Fig.3. Inhibition of CTSL sensitized U87 and U251 cells to curcumin. (A) Cells were transfected with control siRNA or CTSL-specific siRNA. Cells were prepared and subjected to Western blot analysis and immunofluorescence assay 24 h after transfection to determine the CTSL knockdown efficiency. Green fluorescence in the cytoplasm and co-expression with DAPI were observed (630× magnification). ***p<0.001 vs. NC. (B) Cell viability was analyzed by CCK-8 assay. Statistical significance is indicated by *P<0.05, **p<0.01 vs. control siRNA. (C) Clonogenic assay and survival curves for U87 and U251 cells treated with 5–20 μM curcumin or curcumin with siRNA. Statistical significance is indicated by *P<0.05, **p<0.01 vs. control.
Fig.4. Curcumin-induced apoptosis was enhanced by CTSL downregulation. (A) Annexin
V-FITC/PI staining and flow cytometric determination of the apoptosis of U87 and U251 cells treated with 20 μM curcumin for 48 h or curcumin combined with siRNA demonstrated a remarkable increase in the percentage of apoptotic cells compared with the curcumin-treated group. (B) Cells were transfected with NC or CTSL siRNA. After 24 h, cells were treated with curcumin (20 μM) for 48 h and Western blot analysis was performed. β-actin was used as a loading control. * P<0.05, **p<0.01 vs. curcumin group.
Fig.5. CTSL knockdown promotes curcumin-induced G2/M phase arrest.(A) DNA flow cytometry analysis of U87 and U251 cells treated with 20 μM curcumin or curcumin combined with siRNA for CTSL. # P<0.05 vs. curcumin group, **p<0.01 vs. control group.(B) Cells were transfected with NC or CTSL siRNA. After 24 h, cells were treated with 20 μM curcumin for 24 h. Whole-cell extracts were prepared and probed for cyclin B1. β-actin was used as a loading control. * P<0.05 vs. curcumin group.
Fig.6. Effect of CTSL inhibition on the curcumin-mediated inhibition of migration and invasion. (A) U251 and U87 cells were transfected with CTSL siRNA or control siRNA. As the cells reached confluency, they were scratched and further incubated in the media with 20 μM curcumin for 24 h. Cell migration images were collected at 0 and 24 h. (B) Cells were pretreated with CTSL siRNA or control siRNA, trypsinized, and re-seeded in the upper chamber of the media at 20 μM curcumin. After 24 h, the cells on the upper surface of the filters were removed, and those on the lower surface were fixed with methanol and stained with crystal violet. Images were captured using a 100× objective lens. **P<0.01 vs. curcumin group, ##p<0.01 vs. control group.
Highlights
Curcumin treatment increased cathepsin L activity in glioma cells.
Cathepsin L knockdown could enhance curcumin-mediated apoptosis, cell cycle arrest, inhibition of migration and invasion of glioma cells.
Inhibition of cathepsin L may be exploited in adjuvant clinical glioblastoma therapy.
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PII: DOI: Reference:
S0006-8993(16)30471-1 http://dx.doi.org/10.1016/j.brainres.2016.06.046 BRES44997
To appear in: Brain Research Received date: 1 May 2016 Revised date: 24 June 2016 Accepted date: 30 June 2016 Cite this article as: Yao Fei, Yajie Xiong, Yifan Zhao, Wenjuan Wang, Meilin Han, Long Wang, Caihong Tan and Zhongqin Liang, Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells, Brain Research, http://dx.doi.org/10.1016/j.brainres.2016.06.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells Yao Feia, Yajie Xionga, Yifan Zhaoa, Wenjuan Wanga, Meilin Hana, Long Wanga, Caihong Tana,b, Zhongqin Liang* a
Department of pharmacology, College of pharmaceutical Sciences, Soochow University, Suzhou,
China b
Department of Pharmacy, Affiliated Hospital of Jiangsu University, Zhenjiang, China
*
Corresponding author: Zhongqin Liang. Department of pharmacology, College of pharmaceutical
Sciences, Soochow University, Suzhou, China. Tel.:+86 051265882071; fax:+86 051265882089. [email protected]
Abstract Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers.
Keywords: Cathepsin L; curcumin; glioma cell; apoptosis; cell cycle arrest; invasion and migration
1. Introduction Glioma is a common and aggressive malignant human brain tumor with poor prognosis. This tumor seriously harms human health and accounts for an incidence of 3 cases per 100,000 individuals (Westermark B, 2012). Malignant gliomas are treated through surgical resection, radiation, and chemotherapy. Despite various treatments, most patients with gliomas die within 1 year after diagnosis (Mangiola A et al., 2010; Van Meir EG, 2010). Gliomas are also characterized by critical prognostic factors, namely, rapid growth rate and highly diffuse infiltration; however, these factors contribute to the failure of current therapies (Gagliano N et al., 2010; An Claes et al., 2007). Furthermore, many genes associated with an increased risk of glioma are often mutated or correlated with specifically acquired mutations (Melin B et al., 2013). Therefore, the pathogenesis of gliomas should be elucidated to develop new therapeutic targets and to improve therapeutic effects. Cathepsin L (CTSL), a lysosomal cysteine protease, is a potential therapeutic target in cancer treatment (Lankelma JM et al., 2010; Navab R et al., 2008). CTSL is overexpressed in several types of human carcinomas, including breast, lung, gastric, colon, melanoma, and glioma (Qin G et al., 2016; Chen Q et al., 2011; Miyamoto K et al., 2011; Chauhan SS et al., 1991; Stabuc B et al., 2006; Strojnik T et al., 2005). The level of CTSL expression is also associated with the degree of malignancy (Skrzydlewska E et al., 2005). CTSL upregulation promotes angiogenesis (Rebbaa A et al., 2009), transformation (Goulet B et al., 2007), and differentiation (Duncan EM et al.,2008). Furthermore, CTSL is associated with the growth, survival (Primon M et al., 2013), cycle (Goulet B,Nepveu A et al., 2004), migration, and invasion (Strojnik T et al., 1999; Kirschke H et al., 2000) of tumor cells. CTSL inhibition with radiotherapy significantly impedes the growth of glioma stem cells, promotes apoptosis, improves the radiosensitivity (Wang W et al., 2016) and downregulation of CTSL, and suppresses cancer invasion and migration by inhibiting EMT (Qingqing Zhang, 2015). Therefore, CTSL knockdown is necessary to regulate cell growth, migration, and invasion. The vulnerability of glioma cells to drugs can be enhanced by targeting the increased CTSL levels in glioma, by administering CTSL inhibitors,
or by genetically manipulating CTSL expression. Thus, CTSL may be a potential therapeutic target for glioma treatment. Curcumin(Cur) is a natural compound present in turmeric (Curcuma longa Linn), a rhizomatous plant commonly used as a spice(Prasad S et al., 2014). Curcumin possesses
potent
anti-inflammatory,
antioxidant,
chemopreventive,
and
chemotherapeutic activities. It also exhibits a broad spectrum of suppressive activities in various tumors, including glioblastoma, lung cancer, ovarian cancer, and prostate cancer (Jordan BC et al., 2016; Ravindran J et al., 2009; Lin YG et al., 2007). This substance is involved in different anti-tumor mechanisms, such as apoptosis, cell cycle arrest, and migration and invasion inhibition (Wang L et al., 2015; Zhang YP et al., 2015; Ma J et al., 2014; Qian chen et al., 2015). So far the most commonly recognized mode of curcumin action on cancer cells is the induction of apoptosis as was revealed by Ewa Sikora group (Kamila Wolanin et al., 2006; Grazyna Mosieniak et al., 2016). Several proteins are also implicated in curcumin-related mechanisms. For instance, Bcl-2 protein decreases in response to curcumin. Therefore, this protein may play a key role in curcumin-induced apoptosis of Caki cells (Karunagaran D et al., 2005; Woo JH et al., 2003). Nevertheless, the precise molecular mechanisms of curcumin-mediated inhibition of tumor growth, migration, and invasion should be elucidated. Our laboratory studies have revealed that curcumin activates autophagy and triggers the differentiation cascade in GICs isolated from human GBMs (Wenzhuo Zhuang et al., 2011). Autophagy and apoptosis also contribute to curcumin-induced death of K562 cells (Yanli Jia et al., 2009). The upstream signal regulation of autophagy and apoptosis has been proposed as a new strategy to enhance the anti-tumor activities of curcumin. The activation of autophagy and apoptosis is accompanied by an increase in the activity of lysosomal cathepsins (Boland B et al., 2004; Allen Kaasik et al., 2005); this phenomenon suggests that lysosomal hydrolases are involved in cell death pathways. As a lysosomal cathepsin, CTSL mediates autophagy and apoptosis (Lingyun Li et al., 2016). Our study aimed to determine whether CTSL participates in the regulation of apoptosis and cycle arrest. This study
also aimed to investigate whether CTSL is involved in curcumin-induced inhibition of migration and invasion and to reveal the underlying mechanisms. We found that the inhibition of CTSL may represent a novel therapeutic target to reinforce the efficacy of cancer chemotherapy.
2. Results 2.1. Curcumin inhibits U87 and U251 cell proliferation and induces apoptosis We investigated the cytotoxic effect of curcumin on different malignant glioma cells through a CCK8 assay. U87 and U251 cells were treated with curcumin at increasing doses (10, 20, 40, 60, and 80 μM) and time intervals (24, 48, and 72 h). Curcumin inhibited the growth of U87 and U251 cells in a dose- and time-dependent manner (Fig. 1A). Similarly, colony formation was suppressed by curcumin in both glioma cell lines (Fig. 3C). This finding suggested that the effect of curcumin on tumor cells was irreversible. We examined whether curcumin-induced apoptosis contributes to the growth inhibitory effects. U87 and U251 cells were stained with Hoechst 33258, and morphological changes were examined through fluorescence microscopy. Typical apoptotic morphological features, such as chromatin condensation and nuclear fragmentation, were detected in curcumin-treated U87 and U251 cells (Fig. 1B). We also performed Annexin V/PI staining to verify whether curcumin treatment can induce apoptosis. Flow cytometric analysis revealed that curcumin induced the apoptosis of glioma cells in a time-dependent manner (Fig. 1C). U87 and U251 cells were treated with 20 μM curcumin and harvested after 24, 48, and 72 h to confirm the apoptotic route of curcumin-dependent inhibition activation. We investigated the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins, two of the most important proteins associated with apoptosis. We found that both of them were altered in a time-dependent manner, but their changes were inversely proportional to each other. After the cells were treated with curcumin, the expression level of Bcl-2 decreased as the expression level of Bax gradually increased. The Bax/Bcl-2 ratio (Fig. 1D) also gradually increased.
Therefore, curcumin induced the apoptosis of U87 and U251 cells.
2.2. Curcumin upregulates CTSL expression in U87 and U251 cells To investigate the association between curcumin and CTSL expression, we determined the activity of this protease in curcumin-treated U87 and U251 cells. The active protein band of CTSL is 26 kDa. Western blot analyses revealed a time- and dose-dependent change in the CTSL protein level was detected in response to different treatment durations and curcumin concentrations (Fig. 2A). We confirmed the increase in protein levels through immunofluorescence. Our results demonstrated a dose-dependent effect (Fig. 2B). Therefore, curcumin increased the CTSL expression.
2.3. Inhibition of CTSL sensitizes U87 and U251 cells in response to curcumin treatment CTSL expression is exclusively increased in U87 and U251 cells. To verify whether the inhibition of CTSL contributes to the death of curcumin-treated U87 and U251 cells, we suppressed the CTSL expression through siRNA transfection. After conducting Western blot analysis, we screened the interference chains of two cell lines with a high interference efficiency in the following experiments: U251, CTS-homo-994 (3) and U87, CTS-homo-391 (1). The interference efficiency was further confirmed through an immunofluorescence assay (Fig. 3A). We examined the effect of CTSL siRNA on the curcumin-induced death of U87 and U251 cells. CCK-8 and clonogenic survival assays indicated that the siRNA-induced downregulation of CTSL effectively enhances the sensitivity of glioma cells to curcumin-induced death (Figs. 3B and 3C).
2.4. Knockdown of CTSL enhances curcumin-induced apoptosis Curcumin induced the apoptosis of U87 and U251 cells. The levels of apoptosis in U87 and U251 cells were determined through flow cytometry to confirm the role of CTSL knockdown on curcumin-induced apoptosis. We found that the percentage of
apoptotic U87 and U251 cells increased from 41.12%±1.90% to 79.64%±11.02% and from 84.86%±2.38% to 99.74%±0.11% (Curcumin vs. CTSL siRNA+Curcumin; Fig. 4A). Likewise, the pretreatment with CTSL siRNA could increase the expression of Bax and decrease the expression of Bcl-2 in the curcumin-treated cells (Fig. 4B). These
results
indicated
that
CTSL
knockdown
significantly
enhanced
curcumin-induced apoptosis.
2.5. Knockdown of CTSL promotes the curcumin-induced G2/M phase arrest To understand the mechanism of the growth inhibitory effect of curcumin in human glioma cells and to determine whether CTSL inhibition can promote the curcumin-induced cell cycle arrest, we confirmed that curcumin induced the G2/M phase arrest of U87 and U251 cells (Fig. 5A). We then treated CTSL siRNA-transfected cells with curcumin and found that the percentage of the cells in the G2/M phase increased (Fig. 5A). In order to prevent the interference of CTSL siRNA to the cell cycle, the cells were synchronized by serum starvation for 20 h to synchronize in the G0/G1 phase. We observed that the cell cycle distribution of the control siRNA-transfected cells remained unchanged. Cyclin B1, which is closely related to G2/M arrest, was subjected to Western blot analysis to examine the expression of cell cycle-associated molecules. During the G2/M phase transition, cyclin B1 binds to cell division cycle 2, which is also called cyclin-dependent kinase 1, to form a mitosis-promoting factor that facilitates the transition from G2 phase to M phase of the cell cycle (Zhou L et al., 2014; Pandey JP et al., 2014; Bostrom P et al., 2009). Cyclin B1 was decreased in the CTSL-knocked down cells treated with curcumin compared with the cells treated with curcumin alone (Fig. 5B). These data suggested that CTSL suppression promotes curcumin-induced G2/M phase arrest.
2.6. CTSL inhibition aggravates the inhibitory effect of curcumin on cell migration and invasion
In curcumin-treated tumor cell lines, the effects of CTSL knockdown on migration and invasion were determined through wound healing and invasion assays. The width of the injured line increased. Invasion assay also revealed that the number of colonies decreased. This finding suggested that the suppression of CTSL aggravated the inhibitory effect of curcumin on the migration and invasion of U251 and U87 cells (Fig. 6). Tumor invasion and migration were decreased by inhibiting CTSL in the curcumin-treated cells. This finding suggested an important role of CTSL in the curcumin‑ induced reduction of cell migration and invasion.
3. Discussion This study revealed a possible mechanistic function by which CTSL inhibition contributes to the enhanced sensitivity of curcumin-mediated inhibition of the growth, migration, and invasion of glioma cells. Curcumin treatment activated the expression of CTSL in glioma cells (Fig. 2); CTSL is remarkably upregulated in malignant gliomas (Strojnik T et al., 2005). We further demonstrated that the curcumin-induced apoptosis and cell cycle arrest of the glioma cells were enhanced through CTSL knockdown. The curcumin-induced inhibition of the migration and invasion of glioma cells was also increased through CTSL knockdown. Therefore, CTSL inhibition and curcumin treatment could be an effective approach to treat gliomas. In vivo and in vitro studies have demonstrated the ability of curcumin to induce tumor apoptosis effectively (Dorai T et al., 2001; Lv ZD et al., 2014; Zhou X et al., 2016). This effect is partially mediated by the mitochondrial pathway, which involves the increase in Bax/Bcl-2 ratio and the release of cytochrome c (Lin Zhu et al., 2015). Consistent with these findings, our data confirmed that curcumin can induce apoptosis and increase Bax/Bcl-2 ratio in glioma cells. Navab (Navab R et al., 2008) revealed that CTSL inhibition reduces IGF-1 receptor responsiveness; as a consequence, this phenomenon enhances apoptosis. This finding further confirmed that CTSL mediates an anti-apoptotic effect. Meanwhile, cell apoptosis analysis revealed that glioma cells with CTSL knockdown underwent more cell apoptosis and Bax/Bcl-2 ratio after curcumin was administered (Fig. 4). On the basis of our experimental results, we
speculated that curcumin upregulated the CTSL expression in U87 and U251 cells and CTSL plays an anti-apoptotic role in glioma cells. Thus, cells could be protected from the toxic effect of curcumin. The silenced CTSL weakened the protective effect; as a result, the sensitivity of cells to curcumin was enhanced. Curcumin possesses an antitumor activity, which is associated with its ability to induce G2/M cell cycle arrest (Sa G et al., 2008). Mechanistically, curcumin downregulated cyclin B1 and thus induced G2/M arrest (Cheng Liang et al., 2012). Cell cycle regulation may be affected by CTSL because its active isoform possibly participates in the degradation of nuclear transcription factors (Brix K et al., 2008). The cells were subjected to serum starvation to prevent the effects of CTSL on cell cycle. Our experiments demonstrated that the curcumin-induced inhibition of CTSL enhanced the sensitivity to cell cycle arrest (Fig. 5). This phenomenon could occur possibly because the nuclear isoform of CTSL could regulate the proteolytic processing of CUX1 to generate p75 and p110 CUX1, which are the active forms of full-length p200 CUX1 (Goulet B, Baruch A et al., 2004; Fei XF et al., 2007). The target genes of CUX1 not only participate in cell cycle progression (Vadnais C et al., 2012) but also regulate tumor cell apoptosis (Kirschke H et al., 2000). This finding may also help explain why cell cycle arrest and apoptosis are increased by CTSL inhibition in curcumin-treated cells. Migration and invasion are typical hallmarks of tumors. Curcumin can suppress tumor migration and invasion (Chiang IT et al., 2015; Wang S et al., 2011). The overexpression of CTSL may switch the melanoma cell phenotype from non-metastatic to highly metastatic. As a consequence, tumor migration and invasion are increased (Rousselet N et al., 2004; Yang Z et al., 2007; Wang SM et al., 2010). Our study indicated that CTSL knockdown sensitizes to curcumin-suppressed migration and invasion (Fig. 6). We also previously revealed that downregulation of CTSL suppresses cancer invasion and migration by inhibiting the EMT process (Qingqing Zhang. 2015). CTSL also decreases cell-to-cell adhesion to enhance tumor
invasion and migration through the direct cleavage of E-cadherin, which is a marker protein of EMT (Gocheva V et al., 2006). Nevertheless, further research should be performed to determine whether curcumin suppresses tumor migration and invasion through CTSL-induced EMT. In summary, our experiments revealed that curcumin can effectively induce the apoptosis and cell cycle arrest of glioma cells. Curcumin can also suppress migration and invasion. We further demonstrated that CTSL inhibition can enhance the sensitivity of glioma cells to curcumin-mediated apoptosis and cell cycle arrest. CTSL inhibition can also impede the migration and invasion of glioma cells. Therefore, the inhibition of CTSL may be an effective strategy to treat glioma with curcumin. Further studies should investigate CTSL as a novel therapeutic target for malignant tumors.
4. Experimental procedures 4.1. Cell culture and reagents U87 and U251 human glioma cells were purchased from the Shanghai Institute of Cell Biology, the Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco Life Technologies) and incubated in an incubator humidified with 5% CO2 at 37 °C. The cells in the mid-logarithmic phase were used in the experiments. Curcumin (Sigma) was prepared as a 20 mM stock solution in dimethylsulfoxide (DMSO, Sigma) and stored at −20 °C. In each experiment, curcumin was diluted with the cell culture medium to obtain the desired final concentrations. 4.2. Antibodies The following antibodies were used in this study: cyclin B1 (1:500, Santa Cruz, USA), CTSL (1:1000, Abcam, MA, USA), β-actin (1:1000, MultiSciences, Nanjing, China), Bcl-2 (1:200, Millipore, MA, USA), and Bax (1:1000, Abcam, Cambridge, UK).
4.3. siRNA transfection Human CTSL siRNA and control siRNA were purchased from GenePharma. U251 cells were seeded in 6-well plates and grown in DMEM supplemented with 10% FBS 1 day before transfection. After 24 h in culture, siRNA plasmid DNA was transfected into the cells by using a siRNA transfection reagent kit in accordance with the manufacturer’s protocol. After 24 h, the transfected cells were subjected to different treatments and then collected. The following siRNA sequences were used. Table1.siRNA for CTSL siRNA Negtive control(NC)
base sequence sense
5’-UUCUCCGAACGUGUCACGUTT-3’
antisense
5’-ACGUGACACGUUCGGAGAATT-3’
sense
5’-GCCUCAGCUACUCUAACAUTT-3’
antisense
5’-AUGUUAGAGUAGCUGAGGCTT-3’
sense
5’-CGAUGCACAACAGAUUAUATT-3’
antisense
5’-UAUAAUCUGUUGUGCAUCGTT-3’
sense
5’-CCAAGUAUUCUGUUGCUAATT-3’
antisense
5’-UUAGCAACAGAAUACUUGGTT-3’
sense
5’-CCUUCCUGUUCUAUAAAGATT-3’
antisense
5’-UCUUUAUAGAACAGGAAGGTT-3’
CTS-homo-391(1)
CTS-homo-449(2)
CTS-homo-994(3)
CTS-homo-1112(4) 4.4. Clonogenic survival assay
The cells were seeded in 6-well plates at a density of 4×102 cells per well, incubated overnight, pretreated with CTSL siRNA and control siRNA for 24 h, and exposed to different doses of curcumin. Afterward, the treated cells were incubated at 37 °C for 10 d. Colonies were fixed and stained with 0.5% crystal violet (Sigma Aldrich). 4.5. Cell viability assay Cell viability was assessed through a CCK-8 (Sigma, USA) assay. In brief, U87 and U251 cells were plated into 96-well plates. Curcumin (10, 20, 40, 60, 80 μM) was added to the culture medium and further incubated for 24, 48, and 72 h. U87 and
U251 cells were transfected with CTSL siRNA 24 h before they were treated with various doses of curcumin to examine the contribution of CTSL to the curcumin-induced death of these cells. A CCK-8 reagent was added to each well 2 h before the endpoint of incubation. Optical density (OD) at 450 nm in each well was determined by using a microplate reader. The percentage of cell viability was calculated as follows: cell viability (%) = (OD of experimental well / OD of positive control well) × 100%. 4.6. Hoechst 33258 staining The treated cells were analyzed using a Hoechst staining kit (Sigma). Staining was performed in accordance with the manufacturer’s protocol. The cells were washed with PBS, treated with a fixing solution for 10 min, and stained with Hoechst 33258 fluorescent dye for 10 min at room temperature. Morphological nuclear changes were observed and captured using an inverted fluorescence microscope. 4.7. Immunofluorescence detection of CTSL Exponentially growing cells were transferred to 24-well plates and incubated with curcumin. After the treatments were administered, the cells were fixed by incubating in 4% freshly prepared paraformaldehyde solution for 10 min at 4 °C. The cells were washed with PBS and blocked with a blocking buffer (1% bovine serum albumin and 0.1% Triton X-100) for 30 min at 37 °C. The cells were then incubated overnight with mouse anti-human CTSL antibody diluted with 0.1% Triton X-100. The cells were incubated with a primary antibody and washed four times with PBST. Afterward, the cells were incubated with a secondary antibody conjugated to Alexa 488 (Invitrogen) for 1 h at room temperature. The cells were then washed twice with PBST and incubated with DAPI for 10 min to counterstain the DNA. The coverslips were mounted on microscope slides by using an anti-fade mounting medium (Dako) to permit examination. Images were captured with a fluorescence microscope (LEICA TCS SP2, Germany) and imported into Adobe Photoshop. 4.8. Protein preparation and Western blot analysis The cells were harvested and rinsed with ice-cold PBS twice. Five volumes of Western blot lysing buffer was added to each volume of the cell pellets, and the
mixture was sonicated in an ice bath at 1 s/mL per sonicate with a 30 sec pause between intervals in 5 cycles. The sonicated mixture was microcentrifuged at 12,000 × g at 4 °C for 10 min, and the supernatant was preserved at −80 °C for later use. Protein concentration was determined by using a BCA kit (Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with cyclin B1, CTSL, β-actin, Bcl-2, and Bax at 4 °C overnight. After the membranes were washed thrice with TBST buffer (20 mmol/L Tris-buffered saline and 0.1% Tween 20), they were incubated with rabbit or mouse secondary antibodies for 1 h. Immunoblots were detected using an Odyssey infrared imaging system (LI-COR, NE, USA). Protein β-actin was used as a loading control. 4.9. Flow cytometry analysis The phase distribution of the cell cycle was determined using a cell apoptosis propidium iodide (PI) detection kit (KeyGen, Nanjing, China) and a FACSCalibur flow cytometer. Staining was performed in accordance with the manufacturer’s protocol. The cells were seeded in 6-well plates, incubated overnight, pretreated with CTSL siRNA and control siRNA for 24 h. Cells were serum-deprived for 20 h to synchronize in the G0/G1 phase. Then, cells were treated with curcumin for 24 h. The cells were harvested and fixed in 70% ethanol at −20 °C overnight. Then, the samples were centrifuged, re-suspended in 500 µl Buffer A with 250 μg/ml RNase A at 37 °C for 30 min, and stained with 5 µl of PI in the dark at room temperature for 30 min. The percentage of apoptotic cells was measured using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (KeyGen, Nanjing, China). A total of 10,000 cells per sample were examined by using a FACSCalibur flow cytometer. 4.10. Wound healing and invasion assays The cells were grown in 6-well plates for the wound-healing assay. After confluency was obtained, the cells were scratched with a pipette tip, rinsed to remove debris, and incubated in a fresh medium in the presence or absence of curcumin for 24 h. Cell migration images were captured at 0 and 24 h. The wound healing index was determined as a percentage and quantitatively analyzed as follows: five randomly selected distances were selected across the wound at 0 and 24 h and divided by the
distance measured at 0 h. A cell invasion assay was performed using 24-well Matrigel invasion chambers (BD Biosciences). The cells were trypsinized and re-seeded in the upper chamber at a concentration of 1×105/ml in 200 µl of the medium in the presence or absence of curcumin. The lower chamber contained 700 µl of the medium supplemented with 10% FBS. After 24 h, the cells on the upper surface of the filters were removed, and the cells on the lower surface were fixed with methanol and stained with crystal violet. 4.11. Statistical analysis Three independent experiments were performed. Data were presented as mean ± SD and statistically evaluated with ANOVA and Tukey’s multiple range tests in GraphPad Prism 4. P<0.05 was regarded as significant and P<0.01 was considered as highly significant.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 30873052, 81072656,81102466, and 81373430), the Outstanding Medical Academic Leader Program of Jiangsu Province (Grant No. LJ201139).
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Fig.1. Curcumin inhibits the proliferation of U87 and U251 cells and induces their apoptosis. (A) Dose- and time-dependent curcumin cytotoxicity determined through CCK-8 assay. Cells were treated with 0, 10, 20, 40, 60, and 80 μM curcumin for 24, 48, and 72 h. (B) Cells were treated with 20 μM curcumin for 48 h and stained with Hoechst 33258; nuclear condensation was observed through Hoechst staining (the arrowhead indicates an apoptotic nucleus, 40× magnification). (C) Annexin V-FITC/PI staining and flow cytometric determination of the apoptosis of U87 and U251 cells treated with 20 μM curcumin for 24, 48, and 72 h demonstrated a remarkable increase in the percentage of apoptotic cells compared with the untreated control group. (D) U87 and U251 cells were treated with curcumin (20 μM) for 24, 48, and 72 h. Cell lysates were prepared, and protein level was determined through Western blot analysis. β-actin was used to normalize and verify protein loading. Results are expressed as mean ± SD. Statistical significance is indicated by *p<0.05, ** p<0.01 vs. control group.
Fig.2. Expression of CTSL protein in curcumin-treated U87 and U251 cells. (A) Western blot analysis of CTSL in U87 and U251 cells treated with different curcumin concentration or 20 μM curcumin at different time points. β-actin was used as an internal control. * P<0.05, **p<0.01 vs. control group. (B) Glioma cells were treated with different curcumin concentrations and then subjected to immunofluorescence assay. Green fluorescence in the cytoplasm and co-expression with DAPI were observed (630× magnification).
Fig.3. Inhibition of CTSL sensitized U87 and U251 cells to curcumin. (A) Cells were transfected with control siRNA or CTSL-specific siRNA. Cells were prepared and subjected to Western blot analysis and immunofluorescence assay 24 h after transfection to determine the CTSL knockdown efficiency. Green fluorescence in the cytoplasm and co-expression with DAPI were observed (630× magnification). ***p<0.001 vs. NC. (B) Cell viability was analyzed by CCK-8 assay. Statistical significance is indicated by *P<0.05, **p<0.01 vs. control siRNA. (C) Clonogenic assay and survival curves for U87 and U251 cells treated with 5–20 μM curcumin or curcumin with siRNA. Statistical significance is indicated by *P<0.05, **p<0.01 vs. control.
Fig.4. Curcumin-induced apoptosis was enhanced by CTSL downregulation. (A) Annexin
V-FITC/PI staining and flow cytometric determination of the apoptosis of U87 and U251 cells treated with 20 μM curcumin for 48 h or curcumin combined with siRNA demonstrated a remarkable increase in the percentage of apoptotic cells compared with the curcumin-treated group. (B) Cells were transfected with NC or CTSL siRNA. After 24 h, cells were treated with curcumin (20 μM) for 48 h and Western blot analysis was performed. β-actin was used as a loading control. * P<0.05, **p<0.01 vs. curcumin group.
Fig.5. CTSL knockdown promotes curcumin-induced G2/M phase arrest.(A) DNA flow cytometry analysis of U87 and U251 cells treated with 20 μM curcumin or curcumin combined with siRNA for CTSL. # P<0.05 vs. curcumin group, **p<0.01 vs. control group.(B) Cells were transfected with NC or CTSL siRNA. After 24 h, cells were treated with 20 μM curcumin for 24 h. Whole-cell extracts were prepared and probed for cyclin B1. β-actin was used as a loading control. * P<0.05 vs. curcumin group.
Fig.6. Effect of CTSL inhibition on the curcumin-mediated inhibition of migration and invasion. (A) U251 and U87 cells were transfected with CTSL siRNA or control siRNA. As the cells reached confluency, they were scratched and further incubated in the media with 20 μM curcumin for 24 h. Cell migration images were collected at 0 and 24 h. (B) Cells were pretreated with CTSL siRNA or control siRNA, trypsinized, and re-seeded in the upper chamber of the media at 20 μM curcumin. After 24 h, the cells on the upper surface of the filters were removed, and those on the lower surface were fixed with methanol and stained with crystal violet. Images were captured using a 100× objective lens. **P<0.01 vs. curcumin group, ##p<0.01 vs. control group.
Highlights
Curcumin treatment increased cathepsin L activity in glioma cells.
Cathepsin L knockdown could enhance curcumin-mediated apoptosis, cell cycle arrest, inhibition of migration and invasion of glioma cells.
Inhibition of cathepsin L may be exploited in adjuvant clinical glioblastoma therapy.
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