|Year : 2015 | Volume
| Issue : 1 | Page : 28-36
Valproic acid exerts an anti-tumor effect on tongue cancer sas cells in vitro and in vivo
Gu-Jiun Lin1, Shu-Sheng Kao Chen2, Shing-Hwa Huang3, I-Hsun Li4, Li-Chen Yen5, Jang-Yi Chen1, Huey-Kang Sytwu6, Yuan-Wu Chen7
1 Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan
2 Department of Dentistry, Taoyuan General Hospital, Taoyuan, Taiwan
3 Department of Biology and Anatomy, National Defense Medical Center, Taipei; ] Department of General Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
4 Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei; School of Pharmacy, National Defense Medical Center, Taipei, Taiwan
5 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
6 Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
7 School of Dentistry, National Defense Medical Center, Taipei; Department of Oral and Maxillofacial Surgery, Tri-Service General Hospital, Taipei, Taiwan
|Date of Submission||01-Sep-2014|
|Date of Decision||15-Oct-2014|
|Date of Acceptance||15-Dec-2014|
|Date of Web Publication||12-Feb-2015|
School of Dentistry, National Defense Medical Center, Taiwan 11490
Source of Support: None, Conflict of Interest: None
Background: Valproic acid (VPA) is a drug approved by the Food and Drug Administration for epilepsy and bipolar disorders. It is also a known histone deacetylase inhibitor and has been evaluated as an anti-cancer agent. However, the in vitro and in vivo anti-tumor effect of VPA on human tongue cancer has not been evaluated. Materials and Methods: We tested VPA for its anti-tumor activity on the human tongue cancer (SAS) cell line in vitro and in vivo in a tumor xenograft model in mice. The effect of VPA on the cell cycle and apoptosis was examined. Results: Growth inhibition was noted when SAS, squamous cell carcinoma 25 and OECM-1 cells were treated with various doses of VPA for 24-72 h, and it was found that VPA treatment caused G1 arrest and apoptosis in SAS cells. VPA also inhibited the phosphorylation of Akt and ERK in SAS cells in vitro. Tumor growth inhibition was observed in NOD/SCID mice bearing xenografts of human tongue cancer that were treated with a VPA dose of 400 mg/kg/day. Conclusions: This study demonstrates that VPA can inhibit the growth of human tongue cancer cells in vitro and in vivo without causing any significant adverse effects.
Keywords: Valproic acid, oral squamous cell carcinoma, human tongue cancer, cell cycle, apoptosis
|How to cite this article:|
Lin GJ, Chen SSK, Huang SH, Li IH, Yen LC, Chen JY, Sytwu HK, Chen YW. Valproic acid exerts an anti-tumor effect on tongue cancer sas cells in vitro and in vivo
. J Med Sci 2015;35:28-36
|How to cite this URL:|
Lin GJ, Chen SSK, Huang SH, Li IH, Yen LC, Chen JY, Sytwu HK, Chen YW. Valproic acid exerts an anti-tumor effect on tongue cancer sas cells in vitro and in vivo
. J Med Sci [serial online] 2015 [cited 2020 Jul 10];35:28-36. Available from: http://www.jmedscindmc.com/text.asp?2015/35/1/28/151289
| Introduction|| |
Oral squamous cell carcinoma (OSCC) accounts for the majority of all oral malignancies and is a subtype of head and neck squamous cell carcinoma (HNSCC). HNSCC is the sixth most prevalent malignancy worldwide and the third most common cancer in developing countries.  Patients with OSCC typically receive a standard therapeutic regimen used for solid tumors, which includes surgery, radiotherapy, and/or chemotherapy. However, treatment outcomes for end-stage oral cancer patients are uniformly poor. For this reason, new therapeutic modalities are urgently needed to control this cancer.
Valproic acid (VPA) is an eight-carbon, branched-chain fatty acid used for the treatment of epileptic seizures and bipolar disorder. , VPA alters the expression of numerous genes and their corresponding proteins that are believed to play important roles in tumor reprogramming, apoptosis, terminal differentiation, and cell cycle arrest. ,, A recent study also demonstrated that VPA inhibits the growth of head and neck cancer cell lines by inducing terminal differentiation and senescence in these cells.  VPA has been shown to inhibit class I and II histone deacetylases (HDACs) and has anti-tumor activity against a broad spectrum of cancer cells.  These effects are thought to occur primarily by inhibition of HDACs. 
While in vitro anti-cancer effects of VPA in some solid tumors have been reported, , the in vitro inhibitory effects of VPA in tongue cancer have not been well-characterized. Furthermore, the in vivo anti-tumor effect of VPA on oral cancer has not been evaluated. In the present study, we investigated the growth inhibitory effects of VPA in a tongue cancer cell line and an in vivo xenograft tumor model. We also studied the molecular mechanisms underlying the anti-tumor effects of VPA and found that VPA exerts inhibitory effects in vitro and in vivo by inducing apoptosis and G1 arrest in this type of cancer.
| Materials and Methods|| |
Cells and chemicals
SAS cells (a gift from Jeng-Fan Lo, Institute of Oral Biology, Department of Dentistry, National Yang-Ming University, Taipei) are a poorly differentiated human squamous cell carcinoma (SCC) cell line.  OECM-1 was obtained from gingival epidermoid carcinoma of a Taiwanese patient. SCC 25 was obtained from tongue squamous cancer cells. All cell lines were maintained in RPMI medium containing 10% fetal calf serum, 100 u/ml penicillin, and 100 mg/ml streptomycin in tissue culture flasks in a humidified atmosphere of 5% CO 2 and a temperature of 37°C. VPA was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Growth inhibition assay
Cells in the logarithmic growth phase were cultured at a density of 10,000 cells/well in 24-well plates. The cells were exposed to various concentrations of VPA for 24, 48, or 72 h. The methylene blue dye assay was used to evaluate the effect of VPA on cell growth.  The IC 50 value resulting from 50% inhibition of cell growth was calculated graphically in comparison with the growth observed in controls.
Cell cycle analysis
Cells were harvested at the time points mentioned above, washed with ice-cold phosphate-buffered saline (PBS), and fixed overnight with 70% ethanol at 4°C; this was followed by resuspension in 500 μl of PBS. After addition of 500 μl of propidium iodide (PI/RNase Staining Buffer, BD Pharmingen, San Diego, CA, USA), the cells were incubated for 15 min at room temperature and then analyzed by flow cytometry. Flow cytometric analysis was performed with a fluorescence-activated cell sorting (FACS) Caliber using CellQuest software (Becton Dickinson, San Jose, CA, USA).
SAS cells were treated with various concentrations of VPA. The treated cells were harvested at 24 and 48 h after VPA was added and washed twice with PBS. The washed cells were resuspended in 1X binding buffer (BD Pharmingen, San Diego, CA, USA). Resuspended cells were stained with phycoerythrin (PE)-conjugated annexin V and 7-amino-actinomycin D (7-AAD, BD Pharmingen) at room temperature for 15 min and then analyzed by flow cytometry. Flow cytometric analysis was performed with an FACS Caliber using the CellQuest software (Becton Dickinson).
Protein extraction and western blot analysis
Cells were lysed in a modified RIPA buffer containing 50 mM Tris (pH 7.8), 0.15 M NaCl, 5 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 0.5% NP-40, 0.1% sodium deoxycholate, and a protease inhibitor mixture. The relative protein concentration in the supernatants was determined using a bicinchoninic acid protein assay kit (Pierce) and calibrated against standard bovine serum albumin concentrations. Forty micrograms of cell lysate protein per lane was loaded on an 8-10% SDS-PAGE gradient gel, electrophoresed, and transferred onto Immobilon-P transfer membrane (Millipore). The membranes were probed with antibodies against b-actin (BioVision), pERK (cell signaling), ERK (cell signaling), pAkt (cell signaling) and Akt (cell signaling). After incubation for 2 h at room temperature, the membrane was washed in 1X phosphate-buffered saline with Tween 20. The polyvinylidene difluoride membrane was then incubated with peroxidase-linked anti-mouse or anti-rabbit IgG antibodies for 1 h, developed using an enhanced chemiluminescence detection kit (Millipore), and analyzed with a Las-3000 imaging system (Fujifilm).
Xenograft tumor mouse model
Eight-week-old NOD/SCID mice (NOD.CB17 Prkdc scid /J, National Laboratory Animal Center, Taiwan) were maintained in microisolators under specific pathogen-free conditions and were fed sterile chow and sterile chlorinated water. Fourteen mice were injected subcutaneously with 2 × 10 6 SAS cells. Three days after tumor cell injection, the mice were randomized into two treatment groups. One treatment group was administered VPA (400 mg/kg/day), and a control group was treated with PBS by intraperitoneal (i.p.) injection. The size of the transplanted tumors was determined by calipers measurements every 3 days, and the tumor volume was calculated using the following formula: Volume (V) =1/2× (length × width  ). At the end of treatment, the mice were sacrificed, and the tumors were removed, weighed, and photographed.
Tumors were harvested from NOD/SCID recipients and then fixed with formalin. Sections (6 μm in thickness) were cut and stained with hematoxylin and eosin staining and then analyzed via light microscopy.
Paraffin-embedded tissue sections were deparaffinized and rehydrated. The slides were rinsed twice with PBS, and endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide in PBS for 1 h, rinsed three times with PBS, and incubated with a protein-blocking solution consisting of PBS (pH, 7.5) containing 5% normal horse serum for 20 min at room temperature. Tissue samples were incubated overnight at 4°C with a 1:50 dilution of monoclonal mouse anti-human Ki67 antigen (Dako, Glostrup, Denmark). The slides were rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution according to the manufacturer's recommendation of peroxidase-conjugated anti-rabbit immunoglobulin G. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine. The slides were then rinsed three times with distilled water and assessed for Ki67 staining using light microscopy. Brown staining indicated a positive reaction.
All data were expressed as the mean ± standard deviation of at least three determinations unless otherwise stated. The statistical differences between two groups were determined using the two-sample Student's t-test or single-factor ANOVA.
| Results|| |
Valproic acid inhibits oral cancer cells growth invitro
To examine the influence of VPA treatment on OSCC, we treated SAS cells with various concentrations of VPA for 24 h and observed their morphological changes by phase-contrast microscopy. VPA treatment at concentrations ranging from 1 mM to 4 mM significantly decreased the densities of cultured cells compared to that of the untreated cells [Figure 1]a]. To evaluate the growth inhibitory effect of VPA, SAS cells were treated with various doses of VPA, and the number of the surviving cells was measured at different time points and compared to that of the untreated cells as a control in the growth inhibition assay. Following VPA treatment, the number of surviving SAS cells significantly reduced in time- and dose-dependent manner [Figure 1]b-d]. The IC 50 of VPA was approximately 2 mM for SAS cells after 72 h of treatment [Figure 1]d]. These results demonstrated that VPA had dose-dependent growth inhibitory activity against SAS cells. Furthermore, we also examined the growth inhibitory effect in other oral cancer cell lines, including SCC 25 [Figure 2]a] and OECM-1 [Figure 2]b]. We found that VPA also exhibited a significant growth inhibitory effect in these two oral cancer cell lines at the dosages of 2 mM and 4 mM. These results indicated that VPA has an in vitro anti-oral cancer effect.
|Figure 1. Valproic acid (VPA) inhibits SAS cell growth in vitro. (a) Morphological changes in SAS cells treated with 0, 1, 2, 4 mM VPA for 24 h. The density of cultured cells reduced with VPA treatment. The survival of VPA-treated SAS cells was assessed at (b) 24 h (c) 48 h (d) 72 h with various concentrations. There were significant differences in the survival of SAS cells at the indicated VPA concentrations. After 72 h of treatment, the IC50 of VPA was approximately 2 mM for SAS cells. Survival proportions for SAS cells showed statistically significant differences in values when compared with the vehicle control groups (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001)|
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|Figure 2. Valproic acid (VPA) treatment exhibits an anti-oral cancer cell effect. Oral cancer cells (squamous cell carcinoma [SCC] 25 and OECM-1) were treated with VPA for 72 h with indicated concentrations. The viability of these two cell lines was assessed by MTT assay. The results indicated that 2 mM VPA induced (a) SCC 25 and (b) OECM-1 cell deaths (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001)|
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Valproic acid treatment leads to G1 arrest and apoptosis in SAS cells
A previous study indicated that VPA inhibits cell proliferation by inducing cell cycle arrest.  We evaluated the effect of VPA on cell cycle progression in SAS cells. Cells were exposed to VPA at the indicated concentrations for 48 h, and cell cycle distribution was analyzed by flow cytometry [Figure 3]a]. The percentage of cells in the G1 phase increased in a dose-dependent manner [Figure 3]b]. In contrast, the percentage of cells in the S and G2/M phases decreased with high-dose VPA treatment [Figure 3]c and d]. These results show that VPA treatment causes G1 arrest in SAS tongue cancer cells. We also found that the percentage of sub-G1 phase cells significantly increased with VPA treatment at a concentration of 4 mM [Figure 3]e], suggesting induction of apoptosis due to treatment with VPA.
|Figure 3. In vitro valproic acid (VPA) treatment induces G1 phase arrest in SAS cell. SAS cells were treated with the indicated concentrations of VPA for 48 h. (a) Flow cytometric analysis of the cell cycle at the indicated concentrations was carried out at 48 h. The percentages of cells in the (b) G1 phase, (c) S phase, (d) G2/M phase, and (e) sub G1 phase are presented in the plots. Statistical significance was determined by comparisons between VPA-treated and vehicle control-treated groups (n = 3; *P < 0.05; **P < 0.01)|
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We further examined whether VPA treatment induced apoptosis in SAS cells by performing annexin V and 7-AAD double staining, and analyzed the effects on these markers by using flow cytometry. The percentage of annexin V-positive cells slightly increased after treatment with a high concentration VPA for 24 or 48 h [Figure 4]a and b]. These results indicate that VPA treatment can induce apoptosis in SAS cells.
|Figure 4. Valproic acid (VPA) treatment induces apoptosis in vitro. SAS cells were treated with various concentrations of VPA and stained with phycoerythrin -conjugated annexin V and amino-actinomycin D (7-AAD) at (a) 24 or (b) 48 h. The percentage of annexin V-positive (X-axis) and/or 7-AAD-positive (Y-axis) cells was determined by performing flow cytometry. The percentages of annexin V-positive cells are presented in the plots. Statistical significance was determined by comparisons between VPA-treated and vehicle control-treated groups (n = 3; *P < 0.05; **P < 0.01)|
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Valproic acid treatment suppresses the phosphorylation of Akt and ERK
Most OSCCs can become dependent on the aberrant activation of multiple signaling pathways, including RAS/ERK and PI3K/Akt. , To study VPA's effect on cellular signaling pathways, we evaluated the protein expression and phosphorylation of Akt and ERK by performing western blot analysis [Figure 5]a]. We noted a dose-dependent increase in phosphorylated Akt [Figure 5]b] and ERK [Figure 5]c] with VPA treatment. These results show that VPA treatment suppresses Akt and ERK phosphorylation.
|Figure 5. Valproic acid (VPA) treatment suppresses the phosphorylation of Akt and ERK in vitro. (a) SAS cells were treated with different VPA concentrations (0, 1, 2, and 4 mM) for 48 h; immunoblotting was carried out for endogenous total and phosphorylated Akt and ERK proteins in control-and VPA-treated SAS cells. (b) Akt phosphorylation significantly decreased in a dose-dependent manner. (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001). (c) ERK phosphorylation significantly decreased in a dose-dependent manner, (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001)|
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Valproic acid treatment inhibits tumor growth in vivo
Because of the inhibitory effect of VPA on SAS cell growth, the anti-tumor effects of VPA were evaluated in vivo. SAS cells were inoculated into NOD/SCID mice, and VPA (400 mg/kg/day) or vehicle control dosing was started injected on day 3 after tumor inoculation and continued daily until study day 13. The average tumor volume reduced in the VPA treatment group compared to that in the vehicle control treatment group [Figure 6]a]. The mean tumor weights of vehicle control- and VPA-treated mice were 137.6 mg ± 18.3 mg and 36.7 mg ± 10.8 mg, respectively [Figure 6]b]. The tumor volumes in vehicle control- and VPA-treated mice were also determined at indicated time points. Significant differences were noted in the tumor volumes between these two groups at each time point [Figure 6]c], demonstrating that VPA treatment inhibited tumor growth in the SAS tongue cancer xenograft model. Of note, VPA treatment in tumor-bearing NOD/SCID mice did not cause a significant loss in body weight compared to that in the vehicle control group [Figure 6]d], suggesting that no severe VPA-mediated toxicity in the treated mice.
|Figure 6. Valproic acid (VPA) treatment inhibits tumor growth in vivo. (a) SAS xenograft-bearing NOD/SCID mice were treated daily with VPA (400 mg/kg/ day) or vehicle, and the tumors were examined on day 13. (b) Mean tumor weights were compared between VPA-treated and vehicle control-treated groups (n = 7; ***P < 0.001) (c) Tumor volumes of SAS xenografts in the mice treated with VPA or vehicle were measured at indicated times (n = 7). (d) Body weights in VPA-treated (400 mg/kg/day) and vehicle control-treated mice were monitored daily|
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The morphological change in tumors from vehicle control [Figure 7]a] and VPA-treated [Figure 7]b] mice were analyzed by HE staining. Both of these tumors showed central necrosis. However, the tumor size was much smaller in VPA-treated group. To characterize the in vivo inhibitory effect of VPA treatment on tumor growth, we evaluated the expression of Ki67 in the tumors from vehicle control - [Figure 7]c] or VPA-treated [Figure 7]d] mice by performing immunohistochemical staining. Ki67 staining in tumor xenografts from mice were treated with VPA was significantly lower than that observed for the vehicle control group [Figure 7]e], indicating an in vivo inhibitory effect of VPA treatment on tumor cell proliferation.
|Figure 7. Valproic acid (VPA) inhibits cancer cell proliferation in vivo. Histological analysis (×40) was performed by using hematoxylin and eosin staining to observe the morphological change in tumor sections of (a) vehicle control-treated mice and (b) VPA-treated mice. Immunohistochemical staining of Ki67 in tumor sections of (c) vehicle control-treated mice and (d) VPA-treated mice. (e) Histograms depicted compare the average scores of Ki67 protein expression in vehicle control -and VPA-treated mice (n = 3; *P < 0.05)|
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| Discussion|| |
VPA is an effective anti-epileptic drug and has been found to exhibit inhibitory effects on HDAC activity. Epigenetic changes in the status of methylation or acetylation in either CpG or histones play a critical role in the development of cancer. Epigenetic drugs target chromatin through the inhibition of HDACs and DNMTs and may act upon many tumor types since deregulation of methylation and deacetylation are common hallmarks of neoplasia. ,
A previous study demonstrated that VPA is a potent inhibitor of proliferation in some HNSCC cell lines.  Another study reported that VPA had a cytotoxic effect on HNSCC cells and showed synergistic killing activity when combined with cisplatin.  VPA was evaluated in refractory solid and CNS tumors in a phase I clinical trial.  However, the in vivo activity of VPA on oral cancer has not been evaluated in any animal models of the disease. In this study, we examined the anti-cancer effects of VPA in vitro and in vivo on SAS tongue cancer cells and tumor xenografts in mice. Our results show that VPA has anti-tongue cancer cell effects through induction of G1 arrest and apoptosis in SAS cells. VPA treatment also suppressed the in vivo growth of SAS tumor xenografts by inhibiting the proliferation of SAS tumors, as shown by decreased Ki67 expression in VPA-treated tumor-bearing mice. These data provide experimental evidence supporting the clinical evaluation of VPA for the treatment of oral cancer.
The most frequently used chemotherapy drugs include cisplatin and 5-fluorouracil (5-FU). Cisplatin exhibits an anti-tumor effect by inducing oxidative stress and apoptosis in cancer cells.  However, it has significant clinical toxicities, including myelosuppression, nausea, vomiting, mucositis, dermatitis, ototoxicity, and nephrotoxicity. , Among these, the major concern for the use of cisplatin is drug-induced nephrotoxicity.  5-FU is an antimetabolite used for the treatment of cancer; it acts by inhibiting thymidylate synthase and incorporating its metabolites into RNA and DNA.  However, it also has major toxicities, particularly myelosuppression and cardiotoxicity.  Other chemotherapeutics have also been evaluated for the treatment of OSCC. For example, doxorubicin, an anthracycline antibiotic frequently used for the treatment of acute myeloid leukemia and various solid tumors, , has been evaluated for the treatment of oral cancer.  Unfortunately, it has a significant cardiotoxicity risk, which is a major concern for clinicians regarding the use of this drug. ,, VPA has been widely used in humans for long-term treatment of epilepsy with minor side effects.  In this study, we also evaluated the safety and tolerability of VPA in a xenograft tumor mouse model. Our data showed that 400 mg/kg/day treatment with VPA, which resulted in significant tumor growth inhibition, did not lead to any deaths or reduction of body weights in VPA-treated mice. This VPA dose was also used in a previous study in a mouse model of medulloblastoma.  These data indicate that VPA is a promising candidate for the combination treatment with standard chemotherapeutics such as cisplatin, 5-FU, and doxorubicin for the treatment of oral cancers of a squamous cell origin.
| Conclusion|| |
The present study is one of the first to evaluate the in vivo anti-tumor effect of VPA in oral cancer. Our results demonstrated that VPA treatment suppresses the growth of cancer cells by inducing G1 phase arrest and apoptosis; therefore, VPA should be evaluated as an alternative or adjuvant therapy for OSCC in the clinic.
| Acknowledgments|| |
This study was supported by research grants from the National Science Council, Taiwan, Republic of China (NSC102-2314-B-016-018-MY3 and NSC102-2314-B-016-032-MY2), Tri-Service General Hospital, Republic of China (Grant No. TSGH-C102-007-009-S06 and TSGH-C103-005-007-009-S06), Ministry of National Defense, Republic of China (103-M055), Taoyuan General Hospital, Republic of China (Grant No. AFTYGH-102-29), and in part by the C. Y. Foundation for Advancement of Education, Science and Medicine.
| Disclosure|| |
All authors declare no competing financial interests.
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