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Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression
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Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cellcycle progression ⁎
Yasuhiko Okuboa, Atsushi Kasamatsub, , Masanobu Yamatojib, Kazuaki Fushimic, Takashi Ishigamid, Toshihiro Shimizue, Hiroki Kasamac, Masashi shiibaf, Hideki Tanzawaa,b, ⁎ Katsuhiro Uzawaa,b, a
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan c Division of Oral Surgery, Eastern Chiba Medical Center, Chiba, Japan d Department of Dentistry and Oral Surgery, Asahi General Hospital, Asahi, Chiba, Japan e Division of Oral Surgery, Kashima Rosai Hospital, Ibaraki, Japan f Department of Medical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: DAGLA OSCC Tumoral progression Cell-cycle arrest at G1 phase Orlistat In vivo
Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.
1. Introduction Diacylglycerol lipase (DAGL) catalyzes the hydrolysis of diacylglycerol (DAG) to 2-arachidonoylglycerol (2-AG) and free fatty acid (FFA) [1,2]. DAGL activity, the initial reaction for arachidonic acid release, regulates development of the central nervous system, such as axonal growth and generation and migration of new neurons [3]. 2-AG, a calcium-dependent endocannabinoid, increases intracellular calcium concentrations in neuroblastomas [4]. Both 2-AG and FFA have been implicated in lipid homeostasis and adiposity [5,6]. Therefore, recent findings have suggested that inhibition of DAGL activity may be beneficial in the treatment for metabolic disorders, such as obesity, diabetes, and metabolic syndrome [7]. However, little is known about the role of DAGL in cancer development and progression. Two isoforms of DAGL, DAGL-alpha (DAGLA) and DAGL-beta (DAGLB), have been identified in mammals [8]. DAGLA-deficient mice
have less 2-AG in the spinal cord, brain, liver, and adipose tissue, although the spinal cords of DAGLB-deficient mice are unaffected and the brains of these mice had less of a reduction of DAGLB [8]. Among the two isoforms, DAGLA was up-regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs) in our previous microarray data [9]. In the current study, we found that DAGLA is overexpressed in OSCC cells and primary OSCCs and that high DAGLA expression in OSCCs was associated closely with tumoral growth. We also identified a new mechanism of tumoral growth using a DAGLA knockdown model and its specific inhibitor.
Abbreviations: DAGLA, diacylglycerol lipase alpha; OSCC, oral squamous cell carcinoma; HNOKs, human normal oral keratinocytes ⁎ Correspondence to: Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail addresses: [email protected] (A. Kasamatsu), [email protected] (K. Uzawa). https://doi.org/10.1016/j.yexcr.2018.03.041 Received 8 December 2017; Received in revised form 28 March 2018; Accepted 30 March 2018 0014-4827/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Okubo, Y., Experimental Cell Research (2018), https://doi.org/10.1016/j.yexcr.2018.03.041
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2. Materials and methods
2.7. Functional assay
2.1. Ethics statement
In order to evaluate the effect of DAGLA knockdown on cellular proliferation and migratory activities, we analyzed these two functions. The protocol has been described previously [13,14,27–29].
The ethics committee of Chiba University approved the protocol of the current research (protocol number, 680). All patients provided written informed consent for inclusion in the study.
2.8. Cell-cycle analysis The transfectants were treated with 200 ng/ml nocodazole (SigmaAldrich, St. Louis, MO, USA) for 16 h to synchronize cells at G2/M transition [13,21,30–34]. After treatment, the cells were probed with the CycleTEST Plus DNA reagent kit (Becton-Dickinson, Franklin Lakes, NJ, USA). The BD Accuri™ C6 Flow Cytometer (Becton-Dickinson) was used to determine the DNA content.
2.2. Cells and clinical OSCC tissue samples Nine OSCC cells, including HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KOSC-2, SAS, Ho-1-u-1, and Ho-1-N-1 were obtained from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and the JCRB cell bank (Ibaraki, Osaka, Japan). We used primary cultured HNOKs as a normal control cells and clinical OSCC tissue samples as described previously [10–19].
2.9. Orlistat treatment Orlistat (Cayman Chemical, Ann Arbor, MI, USA), a drug for weight management used to treat obese patients, inhibited the hydrolase activity of DAGLA. We challenged the OSCC cells (KOSC-2 and Ho-1-N-1) with orlistat using the indicated doses for cellular proliferation, migration, and cell-cycle assays.
2.3. mRNA expression analysis Extraction of total RNA, generation of cDNA, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were carried out as described previously [10,11,20]. The primer sequences were: DAGLA, 5′-AAGCACCAAGCCCAAATG-3′ and 5′-CTCAGGCAGCTCCGA CTT-3′ and universal probe #49.
2.10. In vivo tumor xenograft study To investigate the cellular proliferation inhibitory effect of orlistat, we used xenograft models (KOSC-2 and Ho-1-N-1). The cells (1 × 107 cells) were injected independently subcutaneously. Female nude mice (6-week-old; BALB/cA-nu [nu/nu]) were purchased from Oriental Yeast Co. (Tokyo, Japan). The volume of the implanted tumor into the dorsal side of the mice was measured with a digital caliper every 2–3 days after injection. We used the formula 4π/3 × (width/2)2 × (length/2) to calculate tumoral volume. After the tumors reached approximately 100 mm3, daily orlistat intraperitoneal injections (240 mg/kg/day) started for 14 days (4 mice/group). Orlistat was dissolved in 33 µl of ethanol, and diluted with 66 µl of saline just before injection. As a control, the equivalent amount of vehicle was injected in the same manner. After 14 days, the mice were sacrificed and tumoral tissues were collected for further analyses [35].
2.4. Immunoblot analysis In order to investigate the DAGLA protein expression levels in oral cancer cells and HNOKS, protein was extracted and immunoblot analysis was carried out as described previously [17,21]. The antibodies were affinity-purified goat anti-DAGLA polyclonal antibody (dilute concentration 1:200, Abcam, Cambridge, UK, ab81984), rabbit antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (dilute concentration 1:200, Santa Cruz Biotechnology, Dallas, TX, USA, sc-25778), rabbit anti-p21 (dilute concentration 1:1000, Cell Signaling Technology, Danvers, MA, USA, #2947), rabbit anti-cyclin D1 (dilute concentration 1:1000, Cell Signaling Technology, #2978), and rabbit anti-CDK2 (dilute concentration 1:1000, Cell Signaling Technology, #2546).
2.11. Statistical analysis 2.5. Immunohistochemistry (IHC)
The statistical differences in DAGLA expression were analyzed using the Mann-Whitney U test, Student's t-test, χ2 test, and Fisher's exact test.
IHC was carried out as described previously [17,22–24]. We used the IHC scoring systems described previously to quantify the status of the DAGLA protein expression in those components [21,22]. The mean percentages of positive tumoral cells were determined in three random fields in each section. The intensities of the stained cells were classified into four levels as described previously [25]. The cellular numbers and the staining intensities were multiplied to produce DAGLA IHC scores. The ± 3-standard deviation (SD) cutoff, which statistically is just 0.2% of the measurement and expected to fall outside this range, was used because it was unlikely to be affected by random experimental errors produced by sample manipulation. Cases with a score exceeding 143.5 (+ 3 SD score for normal oral tissue) were defined as DAGLA-positive. Two pathologists working in Chiba University Hospital made these judgments [26].
3. Results 3.1. Up-regulation of DAGLA in OSCC cells In order to evaluate the status of DAGLA expression as a cancerrelated gene, we conducted qRT-PCR and immunoblot analysis in nine OSCC cells and HNOKs. DAGLA mRNA expression was clearly (P < 0.05) up-regulated in all OSCC cells compared with the HNOKs (Fig. 1A). Fig. 1B shows representative results of immunoblot analysis. The DAGLA protein also increased in all OSCC cells compared with the HNOKs. 3.2. Evaluation of DAGLA status in primary OSCCs
2.6. Transfection with siRNA We evaluated the DAGLA expression in primary OSCCs using an IHC scoring system [22]. The IHC scores in primary OSCCs were clearly (P < 0.05) greater than in normal oral tissues (Fig. 1C). Representative IHC data for DAGLA in normal oral tissue and primary OSCC are shown in Fig. 1D. Intense DAGLA immunoreactivity was observed in the cell membrane of the OSCC specimens, whereas the normal oral tissues
DAGLA siRNA (siDAGLA) and control siRNA (siControl) (Santa Cruz Biotechnology, sc-96964, sc-37007) were transfected into KOSC-2 and Ho-1-N-1 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Knockdown of DAGLA was verified by qRT-PCR and immunoblot analyses. The protocol has been described previously [10,11]. 2
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Fig. 1. Up-regulation of DAGLA in OSCC cells. (A) DAGLA mRNA expression in OSCC cells by qRT-PCR. Significant (*P < 0.05, Mann-Whitney U test) upregulation of DAGLA mRNA is seen in nine OSCC cells compared with the HNOKs. The data are expressed as the mean ± standard error of the mean (SEM) from triplicate results. (B) Representative immunoblot analysis of DAGLA protein expression. Densitometric DAGLA protein data are normalized to GAPDH protein levels. (C) The status of DAGLA protein expression in primary OSCCs (n = 100) and normal oral tissues based on the IHC scoring system. The DAGLA IHC scores of OSCCs and normal oral tissues range from 9.5 to 129.4 (median, 53.8) and 69.1–257.1 (median, 165.0), respectively. The DAGLA protein expression levels in OSCCs are markedly (*P < 0.05, MannWhitney U test) higher than in normal oral tissues. (D) Representative IHC results for DAGLA protein in normal oral tissues and primary OSCCs. Original magnification, × 400. Scale bars, 50 µm. DAGLA is observed in the cell membrane of the OSCC cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04864.
showed almost negative immunostaining.
Fig. 2. Establishment of DAGLA knockdown cells. (A) Expression of DAGLA mRNA in siControl and siDAGLA cells (KOSC-2 and Ho-1-N-1 derived transfectants). DAGLA mRNA expression in siDAGLA cells is clearly (*P < 0.05, Mann-Whitney U test) lower than in siControl cells. (B) Immunoblot analysis of DAGLA protein levels in siControl cells and siDAGLA cells. The DAGLA protein levels in the siDAGLA cells are decreased markedly compared with siControl cells. (C) Cellular proliferation assays of DAGLA knockdown cells. The results are expressed as the mean ± SEM of the values from three assays. The cellular growth of siDAGLA cells is inhibited clearly (*P < 0.05, Mann-Whitney U test) compared with the siControl cells after 120 h. (D) Migration assay of DAGLA knockdown cells. The mean value is calculated from data obtained from three separate chambers. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04865.
3.3. Establishment of DAGLA knockdown cells Because overexpression of DAGLA was seen frequently in OSCC in vitro and in clinical samples (Fig. 1), we transfected DAGLA siRNA or siControl into the OSCC cells (KOSC-2 and Ho-1-N-1). To investigate the efficiency of the transfection, we carried out qRT-PCR and immunoblot analyses. The DAGLA mRNA expression levels in the siDAGLA cells were clearly (P < 0.05) lower than in the siControl cells (Fig. 2A). Similarly, the DAGLA protein level in the siDAGLA cells decreased markedly compared with the siControl cells (Fig. 2B). 3.4. Functional assays
area of uniform wounds in confluent cell culture (Fig. 2D), indicating that siDAGLA cells had decreased cellular migratory abilities.
In order to investigate the effect of DAGLA knockdown on cellular proliferation, we carried out a cell growth assay that showed clearly (P < 0.05, Student's t-test) lower cellular growth in siDAGLA cells compared with siControl cells after 120 h (Fig. 2C). We also carried out a migration assay in order to evaluate the effect of DAGLA knockdown on cellular migratory abilities. In a migration assay, the wounds in the siDAGLA cells closed clearly (P < 0.05; Student's t-test) later than those in the siControl cells after 12 h, when we visually monitored the
3.5. Cell-cycle analysis of siDAGLA cells The percentage of the siDAGLA cells at G1 phase was clearly (P < 0.05) higher than the siControl cells (Fig. 3A). We also assessed the expression levels of p21Cip1, cyclin D1, and CDK2. As expected, p21Cip1 expression was up-regulated, and cyclin D1 and CDK2 3
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Fig. 3. Cell-cycle analysis of siDAGLA cells. (A) Flow cytometric analysis shows the cell-cycle distribution in siControl and siDAGLA cells after synchronization at G2/ M phase using nocodazole. The percentage of cells at G1 phase in siDAGLA cells is increased markedly (*P < 0.05, Mann-Whitney U test) compared with the siControl cells. The percentage G1 phase in KOSC-2 transfectants: siControl, 59.9%; siDAGLA, 74.1%. The percentage G1 phase in Ho-1-N-1 transfectants: siControl, 66.8%; siDAGLA, 76.7%. (B) Immunoblot analysis shows up-regulation of p21Cip1 and down-regulation of cyclin D1 and CDK2 in siDAGLA cells compared with the siControl cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04866.
expression levels were clearly (P < 0.05) down-regulated in siDAGLA (Fig. 3B). These data indicated that siDAGLA inhibited cellular proliferation by cell-cycle arrest at G1 phase. 3.6. Orlistat treatment In order to investigate the efficacy of orlistat, we assessed the functional analyses after the treatments (concentration, 10 μM) for 48 h. The cellular growth of the orlistat-treated cells was inhibited clearly (P < 0.05, Student's t-test) compared with the control (Fig. 4A). In the migration assay, the wounds in the orlistat-treated cells closed clearly (P < 0.05, Student's t-test) later than those in the control cells after 12 h (Fig. 4B), indicating that the orlistat-treated cells had decreased cellular proliferation and cellular migratory abilities. 3.7. Cell-cycle analysis of orlistat-treated cells The percentage of the orlistat-treated cells at G1 phase was clearly (P < 0.05) higher than of the control cells (Fig. 5A). We also assessed the expression levels of p21Cip1, cyclin D1, and CDK2. As expected, p21Cip1 expression was up-regulated, and cyclin D1 and CDK2 expression levels were clearly (P < 0.05) down-regulated in the orlistattreated cells (Fig. 5B). These data indicated that the inhibition of DAGLA activity decreased cellular proliferation in the orlistat-treated cells by cell-cycle arrest at G1 phase. 3.8. Orlistat inhibited tumoral growth in vivo Fig. 4. Functional assay of the orlistat-treated cells. (A) A cellular proliferation assay of the control and orlistat-treated cells. The cellular growth of the orlistattreated cells is inhibited clearly (*P < 0.05, Student's t-test) compared with the control. (B) Migration assay of the orlistat-treated cells. The wounds in the orlistat-treated cells close clearly (*P < 0.05, Student's t-test) later than those in the control cells after 12 h. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04867.
We appraised the effect of orlistat on tumoral growth in vivo using target tumor xenografts in nude mice. OSCC cells (KOSC-2 and Ho-1-N1) were injected subcutaneously into the backs of female nude mice (four mice in each group). The tumoral volume of the orlistat-treated group was clearly (P < 0.05) smaller than that of the control group (Fig. 6A). Orlistat did not affect the body weight of the mice compared with the control group (data not shown).
4. Discussion 3.9. Relationship between DAGLA expression and clinical classification in OSCCs
We found that overexpression of DAGLA controlled cellular proliferation in OSCC cells and clinical samples through cell-cycle progression. Furthermore, DAGLA-positive OSCC was correlated highly with the primary tumoral size. DAGLA involves hydrolysis of DAG to generate 2-AG and FFA [3]. Dramatic losses of 2-AG in the brain and spinal cord are observed in DAGLA-deficient mice [3], indicating that DAGLA is responsible for the
The relationships between the clinicopathologic features of OSCC cases and their DAGLA protein levels using an IHC scoring system are shown in Table 1. Among the clinical classifications, patients who were DAGLA-positive exhibited clearly (P < 0.05) more tumoral progression compared with patients who were DAGLA-negative. 4
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Fig. 5. Cell-cycle analysis of the orlistat-treated cells. (A) Cell-cycle progression in the orlistat-treated cells is investigated by flow cytometric analysis. The percentage of cells at G1 phase in the orlistat-treated cells is increased markedly (*P < 0.05, Mann-Whitney U test) compared with the control cells. The percentage of G1 phase in KOSC-2 with/without orlistat: control, 59.4%; orlistat (+), 76.9%. The percentage G1 phase in Ho-1-N-1 with/ without orlistat: control, 59.2%; orlistat (+), 82.7%. (B) Immunoblot analysis shows up-regulation of p21Cip1 and down-regulation of cyclin D1 and CDK2 in the orlistattreated cells compared with the control cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04868.
Table 1 Relationship between DAGLA expression and clinical classification in OSCCs. Clinical classification
Age at surgery (years) ≦ 70 > 70 Gender Male Female T-primary tumor T1 + T2 T3 + T4 N-regional lymph node Negative Positive Vascular invasion Negative Positive Stage I Ⅱ III IV Histopathologic type Well Moderately Poorly
Total
Immunostaining No. patients
P value
DAGLA-negative
DAGLA-positive
61 39
22 14
39 25
0.986*
58 42
23 13
35 29
0.371†
58 42
26 10
32 32
0.031†‡
65 35
26 10
39 25
0.256†
68 32
27 9
41 23
0.260†
17 27 14 42
8 12 5 11
9 15 9 31
0.322†
66 27 7
25 10 1 36
41 17 6 64
0.425†
* χ2 test. † Fisher’s exact test. ‡ P < 0.05.
Fig. 6. Orlistat inhibits tumoral growth in vivo. (A) Female nude mice (6 weeks old; BALB/cA-nu [nu/nu]) with tumor xenografts were injected daily intraperitoneally with control or orlistat for 14 days (n = 4). Orlistat was dissolved in 33 µl of ethanol, and diluted with 66 µl of saline just before injection. As a control, the equivalent amount of vehicle was injected in the same manner. Tumoral growth in the orlistat-treated group is inhibited significantly (P < 0.05) compared with the control group. (B) Tumor size was measured every 2–3 days, and there was a significant reduction in the relative tumor volume of the orlistat-treated group when compared to the control group. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04869.
inhibitor-1 and SMAD4 [37]. The enzymes for FFA synthesis are upregulated in cancer cells to play a critical role in cancer progression [38]. Previous studies have tried to use lipase inhibitors, such as orlistat, to identify a new anticancer drug [39] as well as the current study. Orlistat, a serine esterase inhibitor, has a covalent binding domain for mammalian lipases and inhibits their activity [39]. Orlistat deactivates intestinal lipase and intestinal fat lipolysis and is approved for weight management in obese patients [40]. The drug also inhibits cellular proliferation and induces cell-cycle arrest at G1 phase by inhibiting the AKT/mTOR pathway in several types of cancers [41]. These results suggested that orlistat has anti-tumorigenic functions and that inhibition of lipid metabolism may serve as a promising therapeutic strategy for treating cancers. In addition to our data from orlistattreatment, DAGLA silencing had an anticancer effect in OSCCs. Therefore, our data showed that DAGLA would be a critical target for cancer therapy.
2-AG level and its activity is dependent on synapse-rich regions [3]. Our knockdown cells were likely to have low concentrations of 2-AG; however, several reports have shown that high levels of 2-AG had anticancer effects [36], suggesting that 2-AG might have pivotal roles in several cellular types. We then focused on FFA among the products of the DAGLA reaction. FFA enhances breast cancer invasion by plasminogen activator 5
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We speculated that DAG, the substrate of the DAGLA reaction, accumulates in DAGLA knockdown cells. Consistent with that hypothesis, the DAG levels decreased in colon cancers compared to paired adjacent normal mucosa samples [42]. While further experiments are needed to investigate the DAG concentration in cancer, DAG might be a key molecule in the treatment of OSCCs. In conclusion, the current study is the first to verification that DAGLA plays a crucial role in primary tumoral growth in OSCCs by cellcycle progression. These altered expressions promoted environmental changes in cellular proliferation, migratory abilities, and cell cycles, suggesting that the DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs.
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cellcycle progression ⁎
Yasuhiko Okuboa, Atsushi Kasamatsub, , Masanobu Yamatojib, Kazuaki Fushimic, Takashi Ishigamid, Toshihiro Shimizue, Hiroki Kasamac, Masashi shiibaf, Hideki Tanzawaa,b, ⁎ Katsuhiro Uzawaa,b, a
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan c Division of Oral Surgery, Eastern Chiba Medical Center, Chiba, Japan d Department of Dentistry and Oral Surgery, Asahi General Hospital, Asahi, Chiba, Japan e Division of Oral Surgery, Kashima Rosai Hospital, Ibaraki, Japan f Department of Medical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: DAGLA OSCC Tumoral progression Cell-cycle arrest at G1 phase Orlistat In vivo
Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.
1. Introduction Diacylglycerol lipase (DAGL) catalyzes the hydrolysis of diacylglycerol (DAG) to 2-arachidonoylglycerol (2-AG) and free fatty acid (FFA) [1,2]. DAGL activity, the initial reaction for arachidonic acid release, regulates development of the central nervous system, such as axonal growth and generation and migration of new neurons [3]. 2-AG, a calcium-dependent endocannabinoid, increases intracellular calcium concentrations in neuroblastomas [4]. Both 2-AG and FFA have been implicated in lipid homeostasis and adiposity [5,6]. Therefore, recent findings have suggested that inhibition of DAGL activity may be beneficial in the treatment for metabolic disorders, such as obesity, diabetes, and metabolic syndrome [7]. However, little is known about the role of DAGL in cancer development and progression. Two isoforms of DAGL, DAGL-alpha (DAGLA) and DAGL-beta (DAGLB), have been identified in mammals [8]. DAGLA-deficient mice
have less 2-AG in the spinal cord, brain, liver, and adipose tissue, although the spinal cords of DAGLB-deficient mice are unaffected and the brains of these mice had less of a reduction of DAGLB [8]. Among the two isoforms, DAGLA was up-regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs) in our previous microarray data [9]. In the current study, we found that DAGLA is overexpressed in OSCC cells and primary OSCCs and that high DAGLA expression in OSCCs was associated closely with tumoral growth. We also identified a new mechanism of tumoral growth using a DAGLA knockdown model and its specific inhibitor.
Abbreviations: DAGLA, diacylglycerol lipase alpha; OSCC, oral squamous cell carcinoma; HNOKs, human normal oral keratinocytes ⁎ Correspondence to: Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail addresses: [email protected] (A. Kasamatsu), [email protected] (K. Uzawa). https://doi.org/10.1016/j.yexcr.2018.03.041 Received 8 December 2017; Received in revised form 28 March 2018; Accepted 30 March 2018 0014-4827/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Okubo, Y., Experimental Cell Research (2018), https://doi.org/10.1016/j.yexcr.2018.03.041
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2. Materials and methods
2.7. Functional assay
2.1. Ethics statement
In order to evaluate the effect of DAGLA knockdown on cellular proliferation and migratory activities, we analyzed these two functions. The protocol has been described previously [13,14,27–29].
The ethics committee of Chiba University approved the protocol of the current research (protocol number, 680). All patients provided written informed consent for inclusion in the study.
2.8. Cell-cycle analysis The transfectants were treated with 200 ng/ml nocodazole (SigmaAldrich, St. Louis, MO, USA) for 16 h to synchronize cells at G2/M transition [13,21,30–34]. After treatment, the cells were probed with the CycleTEST Plus DNA reagent kit (Becton-Dickinson, Franklin Lakes, NJ, USA). The BD Accuri™ C6 Flow Cytometer (Becton-Dickinson) was used to determine the DNA content.
2.2. Cells and clinical OSCC tissue samples Nine OSCC cells, including HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KOSC-2, SAS, Ho-1-u-1, and Ho-1-N-1 were obtained from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and the JCRB cell bank (Ibaraki, Osaka, Japan). We used primary cultured HNOKs as a normal control cells and clinical OSCC tissue samples as described previously [10–19].
2.9. Orlistat treatment Orlistat (Cayman Chemical, Ann Arbor, MI, USA), a drug for weight management used to treat obese patients, inhibited the hydrolase activity of DAGLA. We challenged the OSCC cells (KOSC-2 and Ho-1-N-1) with orlistat using the indicated doses for cellular proliferation, migration, and cell-cycle assays.
2.3. mRNA expression analysis Extraction of total RNA, generation of cDNA, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were carried out as described previously [10,11,20]. The primer sequences were: DAGLA, 5′-AAGCACCAAGCCCAAATG-3′ and 5′-CTCAGGCAGCTCCGA CTT-3′ and universal probe #49.
2.10. In vivo tumor xenograft study To investigate the cellular proliferation inhibitory effect of orlistat, we used xenograft models (KOSC-2 and Ho-1-N-1). The cells (1 × 107 cells) were injected independently subcutaneously. Female nude mice (6-week-old; BALB/cA-nu [nu/nu]) were purchased from Oriental Yeast Co. (Tokyo, Japan). The volume of the implanted tumor into the dorsal side of the mice was measured with a digital caliper every 2–3 days after injection. We used the formula 4π/3 × (width/2)2 × (length/2) to calculate tumoral volume. After the tumors reached approximately 100 mm3, daily orlistat intraperitoneal injections (240 mg/kg/day) started for 14 days (4 mice/group). Orlistat was dissolved in 33 µl of ethanol, and diluted with 66 µl of saline just before injection. As a control, the equivalent amount of vehicle was injected in the same manner. After 14 days, the mice were sacrificed and tumoral tissues were collected for further analyses [35].
2.4. Immunoblot analysis In order to investigate the DAGLA protein expression levels in oral cancer cells and HNOKS, protein was extracted and immunoblot analysis was carried out as described previously [17,21]. The antibodies were affinity-purified goat anti-DAGLA polyclonal antibody (dilute concentration 1:200, Abcam, Cambridge, UK, ab81984), rabbit antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (dilute concentration 1:200, Santa Cruz Biotechnology, Dallas, TX, USA, sc-25778), rabbit anti-p21 (dilute concentration 1:1000, Cell Signaling Technology, Danvers, MA, USA, #2947), rabbit anti-cyclin D1 (dilute concentration 1:1000, Cell Signaling Technology, #2978), and rabbit anti-CDK2 (dilute concentration 1:1000, Cell Signaling Technology, #2546).
2.11. Statistical analysis 2.5. Immunohistochemistry (IHC)
The statistical differences in DAGLA expression were analyzed using the Mann-Whitney U test, Student's t-test, χ2 test, and Fisher's exact test.
IHC was carried out as described previously [17,22–24]. We used the IHC scoring systems described previously to quantify the status of the DAGLA protein expression in those components [21,22]. The mean percentages of positive tumoral cells were determined in three random fields in each section. The intensities of the stained cells were classified into four levels as described previously [25]. The cellular numbers and the staining intensities were multiplied to produce DAGLA IHC scores. The ± 3-standard deviation (SD) cutoff, which statistically is just 0.2% of the measurement and expected to fall outside this range, was used because it was unlikely to be affected by random experimental errors produced by sample manipulation. Cases with a score exceeding 143.5 (+ 3 SD score for normal oral tissue) were defined as DAGLA-positive. Two pathologists working in Chiba University Hospital made these judgments [26].
3. Results 3.1. Up-regulation of DAGLA in OSCC cells In order to evaluate the status of DAGLA expression as a cancerrelated gene, we conducted qRT-PCR and immunoblot analysis in nine OSCC cells and HNOKs. DAGLA mRNA expression was clearly (P < 0.05) up-regulated in all OSCC cells compared with the HNOKs (Fig. 1A). Fig. 1B shows representative results of immunoblot analysis. The DAGLA protein also increased in all OSCC cells compared with the HNOKs. 3.2. Evaluation of DAGLA status in primary OSCCs
2.6. Transfection with siRNA We evaluated the DAGLA expression in primary OSCCs using an IHC scoring system [22]. The IHC scores in primary OSCCs were clearly (P < 0.05) greater than in normal oral tissues (Fig. 1C). Representative IHC data for DAGLA in normal oral tissue and primary OSCC are shown in Fig. 1D. Intense DAGLA immunoreactivity was observed in the cell membrane of the OSCC specimens, whereas the normal oral tissues
DAGLA siRNA (siDAGLA) and control siRNA (siControl) (Santa Cruz Biotechnology, sc-96964, sc-37007) were transfected into KOSC-2 and Ho-1-N-1 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Knockdown of DAGLA was verified by qRT-PCR and immunoblot analyses. The protocol has been described previously [10,11]. 2
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Fig. 1. Up-regulation of DAGLA in OSCC cells. (A) DAGLA mRNA expression in OSCC cells by qRT-PCR. Significant (*P < 0.05, Mann-Whitney U test) upregulation of DAGLA mRNA is seen in nine OSCC cells compared with the HNOKs. The data are expressed as the mean ± standard error of the mean (SEM) from triplicate results. (B) Representative immunoblot analysis of DAGLA protein expression. Densitometric DAGLA protein data are normalized to GAPDH protein levels. (C) The status of DAGLA protein expression in primary OSCCs (n = 100) and normal oral tissues based on the IHC scoring system. The DAGLA IHC scores of OSCCs and normal oral tissues range from 9.5 to 129.4 (median, 53.8) and 69.1–257.1 (median, 165.0), respectively. The DAGLA protein expression levels in OSCCs are markedly (*P < 0.05, MannWhitney U test) higher than in normal oral tissues. (D) Representative IHC results for DAGLA protein in normal oral tissues and primary OSCCs. Original magnification, × 400. Scale bars, 50 µm. DAGLA is observed in the cell membrane of the OSCC cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04864.
showed almost negative immunostaining.
Fig. 2. Establishment of DAGLA knockdown cells. (A) Expression of DAGLA mRNA in siControl and siDAGLA cells (KOSC-2 and Ho-1-N-1 derived transfectants). DAGLA mRNA expression in siDAGLA cells is clearly (*P < 0.05, Mann-Whitney U test) lower than in siControl cells. (B) Immunoblot analysis of DAGLA protein levels in siControl cells and siDAGLA cells. The DAGLA protein levels in the siDAGLA cells are decreased markedly compared with siControl cells. (C) Cellular proliferation assays of DAGLA knockdown cells. The results are expressed as the mean ± SEM of the values from three assays. The cellular growth of siDAGLA cells is inhibited clearly (*P < 0.05, Mann-Whitney U test) compared with the siControl cells after 120 h. (D) Migration assay of DAGLA knockdown cells. The mean value is calculated from data obtained from three separate chambers. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04865.
3.3. Establishment of DAGLA knockdown cells Because overexpression of DAGLA was seen frequently in OSCC in vitro and in clinical samples (Fig. 1), we transfected DAGLA siRNA or siControl into the OSCC cells (KOSC-2 and Ho-1-N-1). To investigate the efficiency of the transfection, we carried out qRT-PCR and immunoblot analyses. The DAGLA mRNA expression levels in the siDAGLA cells were clearly (P < 0.05) lower than in the siControl cells (Fig. 2A). Similarly, the DAGLA protein level in the siDAGLA cells decreased markedly compared with the siControl cells (Fig. 2B). 3.4. Functional assays
area of uniform wounds in confluent cell culture (Fig. 2D), indicating that siDAGLA cells had decreased cellular migratory abilities.
In order to investigate the effect of DAGLA knockdown on cellular proliferation, we carried out a cell growth assay that showed clearly (P < 0.05, Student's t-test) lower cellular growth in siDAGLA cells compared with siControl cells after 120 h (Fig. 2C). We also carried out a migration assay in order to evaluate the effect of DAGLA knockdown on cellular migratory abilities. In a migration assay, the wounds in the siDAGLA cells closed clearly (P < 0.05; Student's t-test) later than those in the siControl cells after 12 h, when we visually monitored the
3.5. Cell-cycle analysis of siDAGLA cells The percentage of the siDAGLA cells at G1 phase was clearly (P < 0.05) higher than the siControl cells (Fig. 3A). We also assessed the expression levels of p21Cip1, cyclin D1, and CDK2. As expected, p21Cip1 expression was up-regulated, and cyclin D1 and CDK2 3
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Fig. 3. Cell-cycle analysis of siDAGLA cells. (A) Flow cytometric analysis shows the cell-cycle distribution in siControl and siDAGLA cells after synchronization at G2/ M phase using nocodazole. The percentage of cells at G1 phase in siDAGLA cells is increased markedly (*P < 0.05, Mann-Whitney U test) compared with the siControl cells. The percentage G1 phase in KOSC-2 transfectants: siControl, 59.9%; siDAGLA, 74.1%. The percentage G1 phase in Ho-1-N-1 transfectants: siControl, 66.8%; siDAGLA, 76.7%. (B) Immunoblot analysis shows up-regulation of p21Cip1 and down-regulation of cyclin D1 and CDK2 in siDAGLA cells compared with the siControl cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04866.
expression levels were clearly (P < 0.05) down-regulated in siDAGLA (Fig. 3B). These data indicated that siDAGLA inhibited cellular proliferation by cell-cycle arrest at G1 phase. 3.6. Orlistat treatment In order to investigate the efficacy of orlistat, we assessed the functional analyses after the treatments (concentration, 10 μM) for 48 h. The cellular growth of the orlistat-treated cells was inhibited clearly (P < 0.05, Student's t-test) compared with the control (Fig. 4A). In the migration assay, the wounds in the orlistat-treated cells closed clearly (P < 0.05, Student's t-test) later than those in the control cells after 12 h (Fig. 4B), indicating that the orlistat-treated cells had decreased cellular proliferation and cellular migratory abilities. 3.7. Cell-cycle analysis of orlistat-treated cells The percentage of the orlistat-treated cells at G1 phase was clearly (P < 0.05) higher than of the control cells (Fig. 5A). We also assessed the expression levels of p21Cip1, cyclin D1, and CDK2. As expected, p21Cip1 expression was up-regulated, and cyclin D1 and CDK2 expression levels were clearly (P < 0.05) down-regulated in the orlistattreated cells (Fig. 5B). These data indicated that the inhibition of DAGLA activity decreased cellular proliferation in the orlistat-treated cells by cell-cycle arrest at G1 phase. 3.8. Orlistat inhibited tumoral growth in vivo Fig. 4. Functional assay of the orlistat-treated cells. (A) A cellular proliferation assay of the control and orlistat-treated cells. The cellular growth of the orlistattreated cells is inhibited clearly (*P < 0.05, Student's t-test) compared with the control. (B) Migration assay of the orlistat-treated cells. The wounds in the orlistat-treated cells close clearly (*P < 0.05, Student's t-test) later than those in the control cells after 12 h. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04867.
We appraised the effect of orlistat on tumoral growth in vivo using target tumor xenografts in nude mice. OSCC cells (KOSC-2 and Ho-1-N1) were injected subcutaneously into the backs of female nude mice (four mice in each group). The tumoral volume of the orlistat-treated group was clearly (P < 0.05) smaller than that of the control group (Fig. 6A). Orlistat did not affect the body weight of the mice compared with the control group (data not shown).
4. Discussion 3.9. Relationship between DAGLA expression and clinical classification in OSCCs
We found that overexpression of DAGLA controlled cellular proliferation in OSCC cells and clinical samples through cell-cycle progression. Furthermore, DAGLA-positive OSCC was correlated highly with the primary tumoral size. DAGLA involves hydrolysis of DAG to generate 2-AG and FFA [3]. Dramatic losses of 2-AG in the brain and spinal cord are observed in DAGLA-deficient mice [3], indicating that DAGLA is responsible for the
The relationships between the clinicopathologic features of OSCC cases and their DAGLA protein levels using an IHC scoring system are shown in Table 1. Among the clinical classifications, patients who were DAGLA-positive exhibited clearly (P < 0.05) more tumoral progression compared with patients who were DAGLA-negative. 4
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Fig. 5. Cell-cycle analysis of the orlistat-treated cells. (A) Cell-cycle progression in the orlistat-treated cells is investigated by flow cytometric analysis. The percentage of cells at G1 phase in the orlistat-treated cells is increased markedly (*P < 0.05, Mann-Whitney U test) compared with the control cells. The percentage of G1 phase in KOSC-2 with/without orlistat: control, 59.4%; orlistat (+), 76.9%. The percentage G1 phase in Ho-1-N-1 with/ without orlistat: control, 59.2%; orlistat (+), 82.7%. (B) Immunoblot analysis shows up-regulation of p21Cip1 and down-regulation of cyclin D1 and CDK2 in the orlistattreated cells compared with the control cells. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04868.
Table 1 Relationship between DAGLA expression and clinical classification in OSCCs. Clinical classification
Age at surgery (years) ≦ 70 > 70 Gender Male Female T-primary tumor T1 + T2 T3 + T4 N-regional lymph node Negative Positive Vascular invasion Negative Positive Stage I Ⅱ III IV Histopathologic type Well Moderately Poorly
Total
Immunostaining No. patients
P value
DAGLA-negative
DAGLA-positive
61 39
22 14
39 25
0.986*
58 42
23 13
35 29
0.371†
58 42
26 10
32 32
0.031†‡
65 35
26 10
39 25
0.256†
68 32
27 9
41 23
0.260†
17 27 14 42
8 12 5 11
9 15 9 31
0.322†
66 27 7
25 10 1 36
41 17 6 64
0.425†
* χ2 test. † Fisher’s exact test. ‡ P < 0.05.
Fig. 6. Orlistat inhibits tumoral growth in vivo. (A) Female nude mice (6 weeks old; BALB/cA-nu [nu/nu]) with tumor xenografts were injected daily intraperitoneally with control or orlistat for 14 days (n = 4). Orlistat was dissolved in 33 µl of ethanol, and diluted with 66 µl of saline just before injection. As a control, the equivalent amount of vehicle was injected in the same manner. Tumoral growth in the orlistat-treated group is inhibited significantly (P < 0.05) compared with the control group. (B) Tumor size was measured every 2–3 days, and there was a significant reduction in the relative tumor volume of the orlistat-treated group when compared to the control group. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04869.
inhibitor-1 and SMAD4 [37]. The enzymes for FFA synthesis are upregulated in cancer cells to play a critical role in cancer progression [38]. Previous studies have tried to use lipase inhibitors, such as orlistat, to identify a new anticancer drug [39] as well as the current study. Orlistat, a serine esterase inhibitor, has a covalent binding domain for mammalian lipases and inhibits their activity [39]. Orlistat deactivates intestinal lipase and intestinal fat lipolysis and is approved for weight management in obese patients [40]. The drug also inhibits cellular proliferation and induces cell-cycle arrest at G1 phase by inhibiting the AKT/mTOR pathway in several types of cancers [41]. These results suggested that orlistat has anti-tumorigenic functions and that inhibition of lipid metabolism may serve as a promising therapeutic strategy for treating cancers. In addition to our data from orlistattreatment, DAGLA silencing had an anticancer effect in OSCCs. Therefore, our data showed that DAGLA would be a critical target for cancer therapy.
2-AG level and its activity is dependent on synapse-rich regions [3]. Our knockdown cells were likely to have low concentrations of 2-AG; however, several reports have shown that high levels of 2-AG had anticancer effects [36], suggesting that 2-AG might have pivotal roles in several cellular types. We then focused on FFA among the products of the DAGLA reaction. FFA enhances breast cancer invasion by plasminogen activator 5
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We speculated that DAG, the substrate of the DAGLA reaction, accumulates in DAGLA knockdown cells. Consistent with that hypothesis, the DAG levels decreased in colon cancers compared to paired adjacent normal mucosa samples [42]. While further experiments are needed to investigate the DAG concentration in cancer, DAG might be a key molecule in the treatment of OSCCs. In conclusion, the current study is the first to verification that DAGLA plays a crucial role in primary tumoral growth in OSCCs by cellcycle progression. These altered expressions promoted environmental changes in cellular proliferation, migratory abilities, and cell cycles, suggesting that the DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs.
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