- Email: [email protected]
survivin axis in human hepatocellular carcinoma cells
Journal Pre-proof β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cell Daisuke Mikami, Mamiko Kobayashi, Junsuke Uwada, Takashi Yazawa, Kazuko Kamiyama, Kazuhisa Nishimori, Yudai Nishikawa, Sho Nishikawa, Seiji Yokoi, Takanobu Taniguchi, Masayuki Iwano PII:
S1347-8613(19)35725-1
DOI:
https://doi.org/10.1016/j.jphs.2019.10.007
Reference:
JPHS 696
To appear in:
Journal of Pharmacological Science
Received Date: 18 August 2019 Revised Date:
4 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Mikami D, Kobayashi M, Uwada J, Yazawa T, Kamiyama K, Nishimori K, Nishikawa Y, Nishikawa S, Yokoi S, Taniguchi T, Iwano M, β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cell, Journal of Pharmacological Science, https://doi.org/10.1016/j.jphs.2019.10.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.
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β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cell
Daisuke Mikami1,*, Mamiko Kobayashi1,*, Junsuke Uwada2, Takashi Yazawa2, Kazuko Kamiyama1, Kazuhisa Nishimori1, Yudai Nishikawa1, Sho Nishikawa1, Seiji Yokoi1, Takanobu Taniguchi2, and Masayuki Iwano1
1
Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
2
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical
University, Asahikawa, Japan
*These authors contributed equally to this work.
Address correspondence to: Daisuke Mikami, MD, PhD Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan 23-3 Matsuoka-shimoaizuki, Eiheiji, Yoshida, Fukui 910-1193, Japan Tel.: +81-776-61-3111 (Ext. 3456) Fax.: +81-776-61-8120
2
E-mail: [email protected]
Keywords: ketone body, β-hydroxybutyrate, histone deacetylase, apoptosis, survivin
3367 words used
3
ABSTRACT Ketone bodies, including acetoacetate and β-hydroxybutyrate (βOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. βOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, βOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of βOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether βOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that βOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, βOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, βOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that βOHB could be a new adjuvant agent for cisplatin chemotherapy.
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1. Introduction Hepatocellular carcinoma (HCC) is the fifth-most frequent type of cancer and second leading cause of cancer death 1. In addition, it has a dismal prognosis and high mortality2. The leading cause of HCC is viral hepatitis, which has recently become possible to control well. However, HCC is associated with lifestyle syndromes, such as diabetes, obesity, and excessive alcohol intake, so its prevalence is increasing in many countries and this trend is expected to continue in the future 3. Therefore, new therapies are needed for patients with HCC. Ketone bodies (KBs), including acetoacetic acid, acetone, and β-hydroxybutyrate (βOHB), are molecules produced mainly from acetyl coenzyme A in the liver and then released into the blood. βOHB has been recently reported to function as a ligand for G protein-coupled receptors GPR41 4 and GPR109A 5. It also regulates metabolism by suppressing sympathetic nerve activity and lipolysis. In addition, βOHB has been reported to inhibit histone deacetylase (HDAC) activity, such as that of HDACs 1, 3 and 4 (classes I and IIa), and prevents their involvement in gene transcriptional regulation 6. Therefore, KBs appear to have diverse physiological roles. Several studies have reported that administrating βOHB or inducing a physiological state of ketosis has an antitumor effect. Surendra et al. showed that treatment with KBs inhibited growth and induced apoptosis in pancreatic cell lines, including capan1 and S2-013 7. In addition, a ketogenic diet decreased tumor growth in
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an S2-013 xenograft model and mitigated the cachectic phenotype. Poff et al. reported that tumor growth and metastatic spread tended to be slower in mice on a ketone supplement diet than in controls. As a result, survival time was extended by 50%-70% in the mice on the ketone supplementation diet, compared with in controls 8. Skinner et al. showed that acetoacetate and βOHB decreased the viability and increased the apoptosis rate in human neuroblastoma cells, despite no such effects being noted in normal fibroblasts 9. Cisplatin is widely used in many standard chemotherapy regimens for solid malignant tumors, including inoperable HCC. It inhibits DNA replication by forming interstrand and intrastrand crosslinks between purine bases are a major obstacle to its clinical use
11
10
. However, dose-limiting toxicities of cisplatin
. The most common is nephrotoxicity, with acute
renal failure occurring in approximately 10% of patients following the initial dose of cisplatin. Therefore, it may be necessary to develop a drug of low toxicity and a synergistic effect with cisplatin to improve chemotherapy. We previously showed that βOHB decreased cisplatin-induced renal damage by modulating HDAC5 in normal human renal cortical epithelial cells
12
. However, few studies have explored how βOHB combined with cisplatin
affects malignant tumors. Therefore, in the present study, we aimed to determine whether βOHB enhances cisplatin-induced apoptosis in HCC cells and to elucidate the underlying mechanism of the synergistic effect.
6
2. Materials and methods 2.1. Materials We used DL-3-hydroxybutyric acid (Tokyo Chemical Industry, Tokyo, Japan); cisplatin (Sigma-Aldrich, St. Louis, MO); polyclonal rabbit antibodies against cleaved caspase-3 (Asp175), cleaved caspase-9, acetyl-histone H3 (Lys9/Lys14) (all Cell Signaling Technology, Boston, MA), human β-actin (Abcam, Cambridge, UK); monoclonal rabbit antibodies against survivin, HDAC8 (Abcam), HDACs 1, 2, 4, 5, 6, and 7 (Cell Signaling Technology); monoclonal mouse antibodies against caspase-8, HDAC3 (Cell Signaling Technology); and horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulins (Dako, Glostrup, Denmark). 2.2. In vivo studies Six-week-old male SHO nude mice (Charles River, Japan) were kept in appropriate pathogen-free conditions. The mice were subcutaneously inoculated in their hind flanks with 2.0×106 HepG2 cells. When tumors had grown to >500 mm3, the mice were randomly distributed into 2 groups (n = 5 per group) and intraperitoneally injected with 8 mg/kg cisplatin alone or 8 mg/kg cisplatin plus 100 mg/kg βOHB in saline suspensions. The tumors were measured with calipers on days 1, 2, 3, 5, 7, and 10 of treatment. Tumor mass was estimated according to tumor volume (mm3) = (length × width2)/2. All experimental designs were approved by the Regulations for Animal Research at the University of Fukui and were
7
reviewed by the Animal Research Committee of the University of Fukui. Mice were kept under standard conditions with a 12-h light-dark cycle. 2.3. Cell culture HepG2 and HLE cells were purchased from the Japanese Collection of Research Bioresources Cell Bank. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. 2.4. Knockdown of HDAC3/5/6 in HepG2 cells The HDAC3/5/6 genes in HepG2 cells were silenced using a previously reported procedure 13. Small interfering RNA (siRNA) against HDAC 3/5/6 (ON-TARGET plus Human), and control siRNA (ON-TARGET plus Human) were purchased from GE Healthcare Dharmacon (Lafayette, CO). HepG2 cells (70% confluence) were transfected with siRNA against HDAC 3/5/6 or control siRNA using a transfection reagent (DharmaFECT; Dharmacon) at a final concentration of 50 nmol/L. After incubation for 48 h, HepG2 cells were treated for immunoblotting. 2.5. Immunoblotting Cells were lysed in radioimmunoprecipitation assay buffer with phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Lysates (5 µg protein) were then subjected to immunoblot analysis, first with antibodies targeting β-actin (1:1000), acetyl-H3 (1:500), cleaved caspase-3
8
(1:1000), caspase-8 (1:500), cleaved caspase-9 (1:1000), survivin (1:1000), HDAC1 (1:1000), HDAC2 (1:1000), HDAC3 (1:1000), HDAC4 (1:1000), HDCA5 (1:500), HDAC6 (1:1000), HDAC7 (1:500), and HDAC8 (1:5000) for 24 h. Second, analysis was carried out with appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000). Both analyses were conducted at room temperature for 1 h. Visualization of immunoreactive bands was performed as previously reported 13. 2.6. Cell proliferation assays The viability of the HCC cells was estimated by MTS assay (3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium; Promega, Madison, WI). HCC cells were plated onto 96-well plates (5×103 cells/mL), and then treatment was administered, comprising either cisplatin (25 µM) alone, βOHB (10 mM) alone, or βOHB (10 mM) combined with cisplatin (25 µM) for 24, 48, and 72 h. After each period, each well was administered 20 µL MTS reagent supplied in the CellTiter 96 Aqueous Assay. The plates were incubated for an additional 4 h at 37 °C. Product absorbance was measured with a microplate reader at 490 nm, with a reference wavelength of 650 nm. 2.7. Quantification of apoptosis We used an annexin V kit (MBL, Nagoya, Japan) to quantify apoptosis in HCC cells. After cells were resuspended in binding buffer (85 µL), they were incubated with Annexin V-FITC (10 µL) and propidium iodide (PI; 5 µL) for 15 min in the dark at room temperature. Next,
9
binding buffer (400 µL) was added to each tube. Fluorescence activated cell sorting (FACS; BD FACSCanto™ II) was used to quantify the percentage of apoptotic cells. 2.8. Statistical analyses Data from in vivo experiments are expressed as the mean ± standard error (SE) and data from in vitro experiments are expressed as the mean ± standard deviation (SD). We evaluated statistical significance (p<0.05) using Student’s t-test or analysis of variance (ANOVA) with Tukey-Kramer’s multiple comparison test.
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3. Results 3.1. Efficacy of βOHB combined with cisplatin in HepG2 xenografts First, we tested the combination of βOHB with cisplatin using HepG2 (an HCC cell line) xenografts to evaluate therapeutic efficacy in vivo. Tumor growth in the HepG2 xenograft was significantly suppressed by βOHB (100 mg/kg) + cisplatin (8 mg/kg) compared with cisplatin (8 mg/kg) alone (Fig. 1). No significant differences were observed in daily food intake or body weight changes between the cisplatin alone and βOHB + cisplatin groups (data not shown), and no deaths occurred in either group. 3.2. Effects of βOHB combined with cisplatin on the proliferation and apoptosis of HCC cells To clarify the molecular mechanism of tumor growth inhibition by βOHB combined with cisplatin, we performed an in vitro analysis using HepG2 and HLE cells and examined the cytotoxic effects of βOHB (10 mM), cisplatin (25 µM), and cisplatin (25 µM) + βOHB (10 mM) on HCC cells at 24 h, 48 h and 72 h. HCC cell proliferation was not inhibited by βOHB alone but was inhibited by cisplatin alone. HCC cell proliferation was inhibited to a greater extent by treatment with βOHB + cisplatin than by cisplatin alone at 24, 48, and 72 h (Fig. 2A, B). FACS analysis revealed a significantly higher apoptotic rate with βOHB + cisplatin than with cisplatin alone in HCC cells at 48 h (Fig. 3A, B), demonstrating that βOHB (10 mM) increased the sensitivity of HCC cells sensitivity to cisplatin. 3.3. βOHB enhanced cisplatin-induced apoptosis via caspase-3 and caspase-8 dependent
11
pathways Next, to evaluate the effect of βOHB and cisplatin or the combination of the two on the activation of caspases, we used Western blotting to measure the cleaved, active form of caspases (caspase-3, caspase-8, and caspase-9) in HCC cells (Fig. 4A, B). We found significantly higher protein levels of the cleaved caspases for all 3 caspases with cisplatin alone than in the control group. In addition, we observed significantly higher protein levels of activated caspase-3 and caspase-8 with cisplatin + βOHB than with cisplatin alone in HCC cells (Fig. 4A, B). 3.4. βOHB combined with cisplatin downregulated HDACs in HCC cells We previously reported that cisplatin-induced apoptosis in HepG2 cells was enhanced by the pan-HDAC inhibitor, trichostatin A 14. Therefore, we hypothesized that βOHB-induced enhancement was due to the modulation of activity and/or expression of HDACs in HCC cells. Hence, we, measured the expression level of histone H3 acetylation in HCC cells by Western blotting (Fig. 5A and 5B). The expression level of histone H3 acetylation was significantly increased by cisplatin alone and by the combination treatment. Further, the combination treatment was also found to significantly increase acetylation level in comparison with cisplatin treatment alone in HCC cells (Fig. 5A and 5B). We also investigated the protein expression of HDACs 1-8 in HCC cells. The protein levels of HDACs 3-8 were significantly lower with cisplatin alone compared with the control group. In
12
addition, the protein levels of HDACs 3/5/6 were significantly lower with cisplatin + βOHB than with cisplatin alone in HepG2 cells (Fig. 5C and 5D). We also found a significant decrease in protein levels of HDACs 1/3-8 with cisplatin alone compared with the control group. Moreover, the protein levels of HDACs 4/6 were significantly lower with cisplatin + βOHB than with cisplatin alone in HLE cells (Fig. 5C and 5E). 3.5. βOHB combined with cisplatin decreased survivin expression in HepG2 cells Knockdown of HDAC3/6 has been recently reported to significantly decrease survivin levels and cell viability in MCF7 breast cancer cells
15
. In addition, YM155, a selective survivin
antagonist, has been shown to enhance cisplatin-induced apoptosis by increased activation of caspase-3 and caspase-8 in HCC cells
16
. We therefore investigated survivin expression in
HepG2 cells. The expression level of survivin was significantly lower with cisplatin alone compared with the control group. In addition, the expression level of survivin was significantly lower with cisplatin + βOHB than with cisplatin alone in HepG2 cells (Fig. 6A). Next, we performed HDAC 3/5/6 gene silencing using siRNA to examine whether survivin expression was decreased by the knockdown of HDACs 3/5/6 in HepG2 cells. In the HDAC 3/5/6 gene silencing condition (Fig. 6B), we found significant downregulation of survivin with HDAC 3/6 silencing but not with HDAC5 silencing (Fig. 6B). Furthermore, in the HDACs 3/6 gene silencing condition, we found significant enhancement in cisplatin-induced cleavage of caspase-3 compared with the control group (Fig. 6C). These results combined
13
showed that βOHB enhanced cisplatin-induced apoptosis of HepG2 cells, at least partially, by decreasing survivin levels via downregulation of HDACs 3/6.
4. Discussion In this study, we showed that βOHB, a ketone body, enhances cisplatin-induced apoptosis via the HDACs inhibition/survivin axis in HepG2 cells. To our knowledge, our results are the first to demonstrate that antitumor cytotoxicity of cisplatin may be enhanced by the addition of βOHB in HCC cells. A key finding of this study is that βOHB enhanced cisplatin-induced apoptosis by modulating HDACs. There is an association between aberrant expression of HDACs and poor prognosis in many cancers
17
upregulated in many HCC cells
18
. Kim et al. reported that HDACs 1, 2, 3, 4, 5, and 8 are . Wu et al. found that overexpression of HDAC3 could
predict poor prognosis in hepatitis B virus-associated HCC and that inhibition of HDACs 2 and 3 suppressed the invasiveness of HCCLM3 cells, which have high metastatic potential 19. Fan et al. showed that proliferation was inhibited by knockdown of HDAC5 in Hep3B, Huh7, and HepG2 cells via an increase in p53 stability and promotion of its nuclear localization 20. Kanno et al. reported that knockdown of HDAC6 decreased the invasiveness and migration of HLF, Hep3B, and PLC cells 21. Furthermore, overexpression of HDAC6 was observed in 20% of primary HCCs and was significantly correlated with an advanced clinical stage, intrahepatic metastasis, and vascular invasion
21
. Thus, HDAC suppression is potentially
14
effective for treatment of HCC. In this study, we observed a significant decrease in protein levels of HDACs 3-8 in HepG2 cells with cisplatin alone and a further decrease in the protein levels of HDACs 3/5/6 with the addition of βOHB in HepG2 cells. Another key finding is the significant decrease in survivin protein level by treatment with βOHB plus cisplatin in HCC cells. Survivin, an anti-apoptotic protein, is an inhibitor of apoptosis protein pancreatic tumors
22
. It is highly expressed in cancers, such as lung, breast, liver, colon, and
23, 24
, but it is not detected or is detected at low levels in normal tissue 23.
According to previous reports, survivin expression is associated with poor prognosis and drug resistance
25-28
. Furthermore, knockdown of survivin enhances the cytotoxicity of
chemotherapy regimens including cisplatin in bladder and prostate cancer cells 29, 30. Lee et al. reported that the knockdown of HDAC3/6 significantly decreased the level of survivin and decreased cell viability in MCF7 breast cancer cells
15
. Consistent with these findings, we
found that knockdown of HDAC 3/6 decreased survivin protein levels in HepG2 cells. In addition, Yu et al. reported enhancement of cisplatin-induced apoptosis with YM155, a selective survivin antagonist, by increased activation of caspase-3 and caspase-8 in HepG2 and HuH-6 cells
16
. In line with these results, we demonstrated that βOHB enhanced
cisplatin-induced apoptosis by increasing the activation of caspase-3 and caspase-8 in HepG2 cells. These findings indicate that HDAC 3/6 expression was decreased by βOHB combined with cisplatin, leading to downregulation of survivin and then enhanced cisplatin-induced
15
cleaved caspase-3 and caspase-8 in HepG2 cells. Based on these results, we proposed a mechanism in which the combination of βOHB and cisplatin decreased HDAC 3/6 expression and caused downregulation of survivin, resulting in cisplatin-induced cleavage of caspase-3 and caspase-8 in HepG2 cells.
5. Conclusions The administration of βOHB significantly enhances cisplatin-induced cytotoxicity and apoptosis of HCC cells, at least in part, by decreasing HDAC 3/6 expression, leading to a decrease in survivin. Our data suggest that the administration of βOHB or a ketogenic diet with chemotherapeutic regimens including cisplatin may result in improved curative effects in HCC patients.
ABBREVIATIONS βOHB, β-hydroxybutyrate; KB, ketone body; HDAC, histone deacetylase; HCC, hepatocellular carcinoma
Acknowledgements This study was supported in part by JSPS KAKENHI grant number 19K07117 and grant number 18K06946 (Grant-in-Aid for Scientific Research (C)), grant numbers 18K15971,
16
19K17735, 19K17702, and 19K17395 (Grant-in-Aid for Young Scientists) from the Japan Society for the Promotion of Science and the National Center for Child Health and Development (29-1).
Conflicts of Interest The authors declare no competing interests.
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FIGURE LEGENDS Figure 1. Therapeutic efficacy of βOHB + cisplatin in HCC HepG2 xenografts. Six-week-old male SHO nude mice were subcutaneously injected with 1.0 × 106 cells. When the tumor size reached >500 mm3, mice were treated by intraperitoneal injection of 8 mg/kg cisplatin or 8 mg/kg cisplatin + 100 mg/kg βOHB suspended in saline. Tumor size was measured as described in the Materials and Methods. Data are expressed as the mean ± SE (n=5). *P<0.05, **P<0.01, by Student’s t-test.
Fig. 2. Effects of βOHB combined with cisplatin on proliferation rate of HCC cell lines. MTS assay was used to examine the effects of βOHB (10 mM) alone, cisplatin (25 µM) alone, or both agents on the proliferation of HepG2 cells (A) and HLE cells (B) at 24, 48, and 72 h. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01 by one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Figure 3. Effects of βOHB combined with cisplatin on apoptotic rate of HCC cell lines. HepG2 cells (A) and HLE cells (B) were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 48 h. Cells were then stained with annexin V and PI, followed by FACS analysis. Data are shown as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01 by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
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Fig. 4. βOHB elicited an extrinsic apoptotic pathway in HCC cells. HepG2 cells (A) and HLE cells (B) were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h. Western blotting was performed to detect the apoptosis-related proteins (cleaved caspase-3, caspase-8, and caspase-9) in cell lysates. The levels of cleaved caspases (all relative to β-actin) were normalized to 1.0 in cells treated with cisplatin alone. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01, NS not significant by Tukey-Kramer’s multiple comparison test.
Fig. 5. βOHB combined with cisplatin decreased HDAC expression in HCC cells. HepG2 and HLE cells were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h. Western blotting was performed to detect acetyl-histone H3 and HDACs 1-8 in cell lysates. The levels of acetyl-H3 (all relative to β-actin) were normalized to 1.0 in cells treated with cisplatin alone. The levels of HDACs (all relative to β-actin) were normalized to 1.0 in cells treated with control. Data are expressed as the mean ± SD of 3 independent experiments. *P<0.05, **P<0.01 by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Fig. 6. βOHB combined with cisplatin decreased survivin expression in HepG2 cells. HepG2 cells were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h.
24
(A) Western blotting was performed to detect survivin in cell lysates. (B) HepG2 cells were treated with control siRNA or HDAC3 siRNA, or HDAC5 siRNA, and HDAC6 siRNA. Western blotting was performed to detect HDAC3/5/6 or survivin in siRNA-mediated control and HDAC3/5/6 knockdown cells in cell lysates. (C) HepG2 cells were treated with vehicle or cisplatin (25 µM) in siRNA-mediated control and HDAC3/6 knockdown cells. The cleaved caspase-3 level relative to β-actin in cisplatin-induced cells treated with control siRNA was set at 1.0. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01, by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Relative mean tumor weight
Fig.1
cisplatin 8 mg/kg cisplatin 8 mg/kg + βOHB 100 mg/kg
2.5
2.0
1.5
1.0
* *
0.5
**
**
**
day3
day5
day7
0.0 day0
day1
day2
day10
Fig.2
A
B
Fig.3
A
HepG2
B
HLE
Fig.4
HepG2
A
B
HepG2
HLE
HLE
Fig.5
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HepG2
HepG2
B
HLE
D
HepG2
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E
HLE
Fig.6 A
B
C
S1347-8613(19)35725-1
DOI:
https://doi.org/10.1016/j.jphs.2019.10.007
Reference:
JPHS 696
To appear in:
Journal of Pharmacological Science
Received Date: 18 August 2019 Revised Date:
4 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Mikami D, Kobayashi M, Uwada J, Yazawa T, Kamiyama K, Nishimori K, Nishikawa Y, Nishikawa S, Yokoi S, Taniguchi T, Iwano M, β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cell, Journal of Pharmacological Science, https://doi.org/10.1016/j.jphs.2019.10.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.
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β-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cell
Daisuke Mikami1,*, Mamiko Kobayashi1,*, Junsuke Uwada2, Takashi Yazawa2, Kazuko Kamiyama1, Kazuhisa Nishimori1, Yudai Nishikawa1, Sho Nishikawa1, Seiji Yokoi1, Takanobu Taniguchi2, and Masayuki Iwano1
1
Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
2
Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical
University, Asahikawa, Japan
*These authors contributed equally to this work.
Address correspondence to: Daisuke Mikami, MD, PhD Department of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan 23-3 Matsuoka-shimoaizuki, Eiheiji, Yoshida, Fukui 910-1193, Japan Tel.: +81-776-61-3111 (Ext. 3456) Fax.: +81-776-61-8120
2
E-mail: [email protected]
Keywords: ketone body, β-hydroxybutyrate, histone deacetylase, apoptosis, survivin
3367 words used
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ABSTRACT Ketone bodies, including acetoacetate and β-hydroxybutyrate (βOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. βOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, βOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of βOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether βOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that βOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, βOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, βOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that βOHB could be a new adjuvant agent for cisplatin chemotherapy.
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1. Introduction Hepatocellular carcinoma (HCC) is the fifth-most frequent type of cancer and second leading cause of cancer death 1. In addition, it has a dismal prognosis and high mortality2. The leading cause of HCC is viral hepatitis, which has recently become possible to control well. However, HCC is associated with lifestyle syndromes, such as diabetes, obesity, and excessive alcohol intake, so its prevalence is increasing in many countries and this trend is expected to continue in the future 3. Therefore, new therapies are needed for patients with HCC. Ketone bodies (KBs), including acetoacetic acid, acetone, and β-hydroxybutyrate (βOHB), are molecules produced mainly from acetyl coenzyme A in the liver and then released into the blood. βOHB has been recently reported to function as a ligand for G protein-coupled receptors GPR41 4 and GPR109A 5. It also regulates metabolism by suppressing sympathetic nerve activity and lipolysis. In addition, βOHB has been reported to inhibit histone deacetylase (HDAC) activity, such as that of HDACs 1, 3 and 4 (classes I and IIa), and prevents their involvement in gene transcriptional regulation 6. Therefore, KBs appear to have diverse physiological roles. Several studies have reported that administrating βOHB or inducing a physiological state of ketosis has an antitumor effect. Surendra et al. showed that treatment with KBs inhibited growth and induced apoptosis in pancreatic cell lines, including capan1 and S2-013 7. In addition, a ketogenic diet decreased tumor growth in
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an S2-013 xenograft model and mitigated the cachectic phenotype. Poff et al. reported that tumor growth and metastatic spread tended to be slower in mice on a ketone supplement diet than in controls. As a result, survival time was extended by 50%-70% in the mice on the ketone supplementation diet, compared with in controls 8. Skinner et al. showed that acetoacetate and βOHB decreased the viability and increased the apoptosis rate in human neuroblastoma cells, despite no such effects being noted in normal fibroblasts 9. Cisplatin is widely used in many standard chemotherapy regimens for solid malignant tumors, including inoperable HCC. It inhibits DNA replication by forming interstrand and intrastrand crosslinks between purine bases are a major obstacle to its clinical use
11
10
. However, dose-limiting toxicities of cisplatin
. The most common is nephrotoxicity, with acute
renal failure occurring in approximately 10% of patients following the initial dose of cisplatin. Therefore, it may be necessary to develop a drug of low toxicity and a synergistic effect with cisplatin to improve chemotherapy. We previously showed that βOHB decreased cisplatin-induced renal damage by modulating HDAC5 in normal human renal cortical epithelial cells
12
. However, few studies have explored how βOHB combined with cisplatin
affects malignant tumors. Therefore, in the present study, we aimed to determine whether βOHB enhances cisplatin-induced apoptosis in HCC cells and to elucidate the underlying mechanism of the synergistic effect.
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2. Materials and methods 2.1. Materials We used DL-3-hydroxybutyric acid (Tokyo Chemical Industry, Tokyo, Japan); cisplatin (Sigma-Aldrich, St. Louis, MO); polyclonal rabbit antibodies against cleaved caspase-3 (Asp175), cleaved caspase-9, acetyl-histone H3 (Lys9/Lys14) (all Cell Signaling Technology, Boston, MA), human β-actin (Abcam, Cambridge, UK); monoclonal rabbit antibodies against survivin, HDAC8 (Abcam), HDACs 1, 2, 4, 5, 6, and 7 (Cell Signaling Technology); monoclonal mouse antibodies against caspase-8, HDAC3 (Cell Signaling Technology); and horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulins (Dako, Glostrup, Denmark). 2.2. In vivo studies Six-week-old male SHO nude mice (Charles River, Japan) were kept in appropriate pathogen-free conditions. The mice were subcutaneously inoculated in their hind flanks with 2.0×106 HepG2 cells. When tumors had grown to >500 mm3, the mice were randomly distributed into 2 groups (n = 5 per group) and intraperitoneally injected with 8 mg/kg cisplatin alone or 8 mg/kg cisplatin plus 100 mg/kg βOHB in saline suspensions. The tumors were measured with calipers on days 1, 2, 3, 5, 7, and 10 of treatment. Tumor mass was estimated according to tumor volume (mm3) = (length × width2)/2. All experimental designs were approved by the Regulations for Animal Research at the University of Fukui and were
7
reviewed by the Animal Research Committee of the University of Fukui. Mice were kept under standard conditions with a 12-h light-dark cycle. 2.3. Cell culture HepG2 and HLE cells were purchased from the Japanese Collection of Research Bioresources Cell Bank. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. 2.4. Knockdown of HDAC3/5/6 in HepG2 cells The HDAC3/5/6 genes in HepG2 cells were silenced using a previously reported procedure 13. Small interfering RNA (siRNA) against HDAC 3/5/6 (ON-TARGET plus Human), and control siRNA (ON-TARGET plus Human) were purchased from GE Healthcare Dharmacon (Lafayette, CO). HepG2 cells (70% confluence) were transfected with siRNA against HDAC 3/5/6 or control siRNA using a transfection reagent (DharmaFECT; Dharmacon) at a final concentration of 50 nmol/L. After incubation for 48 h, HepG2 cells were treated for immunoblotting. 2.5. Immunoblotting Cells were lysed in radioimmunoprecipitation assay buffer with phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Lysates (5 µg protein) were then subjected to immunoblot analysis, first with antibodies targeting β-actin (1:1000), acetyl-H3 (1:500), cleaved caspase-3
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(1:1000), caspase-8 (1:500), cleaved caspase-9 (1:1000), survivin (1:1000), HDAC1 (1:1000), HDAC2 (1:1000), HDAC3 (1:1000), HDAC4 (1:1000), HDCA5 (1:500), HDAC6 (1:1000), HDAC7 (1:500), and HDAC8 (1:5000) for 24 h. Second, analysis was carried out with appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000). Both analyses were conducted at room temperature for 1 h. Visualization of immunoreactive bands was performed as previously reported 13. 2.6. Cell proliferation assays The viability of the HCC cells was estimated by MTS assay (3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium; Promega, Madison, WI). HCC cells were plated onto 96-well plates (5×103 cells/mL), and then treatment was administered, comprising either cisplatin (25 µM) alone, βOHB (10 mM) alone, or βOHB (10 mM) combined with cisplatin (25 µM) for 24, 48, and 72 h. After each period, each well was administered 20 µL MTS reagent supplied in the CellTiter 96 Aqueous Assay. The plates were incubated for an additional 4 h at 37 °C. Product absorbance was measured with a microplate reader at 490 nm, with a reference wavelength of 650 nm. 2.7. Quantification of apoptosis We used an annexin V kit (MBL, Nagoya, Japan) to quantify apoptosis in HCC cells. After cells were resuspended in binding buffer (85 µL), they were incubated with Annexin V-FITC (10 µL) and propidium iodide (PI; 5 µL) for 15 min in the dark at room temperature. Next,
9
binding buffer (400 µL) was added to each tube. Fluorescence activated cell sorting (FACS; BD FACSCanto™ II) was used to quantify the percentage of apoptotic cells. 2.8. Statistical analyses Data from in vivo experiments are expressed as the mean ± standard error (SE) and data from in vitro experiments are expressed as the mean ± standard deviation (SD). We evaluated statistical significance (p<0.05) using Student’s t-test or analysis of variance (ANOVA) with Tukey-Kramer’s multiple comparison test.
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3. Results 3.1. Efficacy of βOHB combined with cisplatin in HepG2 xenografts First, we tested the combination of βOHB with cisplatin using HepG2 (an HCC cell line) xenografts to evaluate therapeutic efficacy in vivo. Tumor growth in the HepG2 xenograft was significantly suppressed by βOHB (100 mg/kg) + cisplatin (8 mg/kg) compared with cisplatin (8 mg/kg) alone (Fig. 1). No significant differences were observed in daily food intake or body weight changes between the cisplatin alone and βOHB + cisplatin groups (data not shown), and no deaths occurred in either group. 3.2. Effects of βOHB combined with cisplatin on the proliferation and apoptosis of HCC cells To clarify the molecular mechanism of tumor growth inhibition by βOHB combined with cisplatin, we performed an in vitro analysis using HepG2 and HLE cells and examined the cytotoxic effects of βOHB (10 mM), cisplatin (25 µM), and cisplatin (25 µM) + βOHB (10 mM) on HCC cells at 24 h, 48 h and 72 h. HCC cell proliferation was not inhibited by βOHB alone but was inhibited by cisplatin alone. HCC cell proliferation was inhibited to a greater extent by treatment with βOHB + cisplatin than by cisplatin alone at 24, 48, and 72 h (Fig. 2A, B). FACS analysis revealed a significantly higher apoptotic rate with βOHB + cisplatin than with cisplatin alone in HCC cells at 48 h (Fig. 3A, B), demonstrating that βOHB (10 mM) increased the sensitivity of HCC cells sensitivity to cisplatin. 3.3. βOHB enhanced cisplatin-induced apoptosis via caspase-3 and caspase-8 dependent
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pathways Next, to evaluate the effect of βOHB and cisplatin or the combination of the two on the activation of caspases, we used Western blotting to measure the cleaved, active form of caspases (caspase-3, caspase-8, and caspase-9) in HCC cells (Fig. 4A, B). We found significantly higher protein levels of the cleaved caspases for all 3 caspases with cisplatin alone than in the control group. In addition, we observed significantly higher protein levels of activated caspase-3 and caspase-8 with cisplatin + βOHB than with cisplatin alone in HCC cells (Fig. 4A, B). 3.4. βOHB combined with cisplatin downregulated HDACs in HCC cells We previously reported that cisplatin-induced apoptosis in HepG2 cells was enhanced by the pan-HDAC inhibitor, trichostatin A 14. Therefore, we hypothesized that βOHB-induced enhancement was due to the modulation of activity and/or expression of HDACs in HCC cells. Hence, we, measured the expression level of histone H3 acetylation in HCC cells by Western blotting (Fig. 5A and 5B). The expression level of histone H3 acetylation was significantly increased by cisplatin alone and by the combination treatment. Further, the combination treatment was also found to significantly increase acetylation level in comparison with cisplatin treatment alone in HCC cells (Fig. 5A and 5B). We also investigated the protein expression of HDACs 1-8 in HCC cells. The protein levels of HDACs 3-8 were significantly lower with cisplatin alone compared with the control group. In
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addition, the protein levels of HDACs 3/5/6 were significantly lower with cisplatin + βOHB than with cisplatin alone in HepG2 cells (Fig. 5C and 5D). We also found a significant decrease in protein levels of HDACs 1/3-8 with cisplatin alone compared with the control group. Moreover, the protein levels of HDACs 4/6 were significantly lower with cisplatin + βOHB than with cisplatin alone in HLE cells (Fig. 5C and 5E). 3.5. βOHB combined with cisplatin decreased survivin expression in HepG2 cells Knockdown of HDAC3/6 has been recently reported to significantly decrease survivin levels and cell viability in MCF7 breast cancer cells
15
. In addition, YM155, a selective survivin
antagonist, has been shown to enhance cisplatin-induced apoptosis by increased activation of caspase-3 and caspase-8 in HCC cells
16
. We therefore investigated survivin expression in
HepG2 cells. The expression level of survivin was significantly lower with cisplatin alone compared with the control group. In addition, the expression level of survivin was significantly lower with cisplatin + βOHB than with cisplatin alone in HepG2 cells (Fig. 6A). Next, we performed HDAC 3/5/6 gene silencing using siRNA to examine whether survivin expression was decreased by the knockdown of HDACs 3/5/6 in HepG2 cells. In the HDAC 3/5/6 gene silencing condition (Fig. 6B), we found significant downregulation of survivin with HDAC 3/6 silencing but not with HDAC5 silencing (Fig. 6B). Furthermore, in the HDACs 3/6 gene silencing condition, we found significant enhancement in cisplatin-induced cleavage of caspase-3 compared with the control group (Fig. 6C). These results combined
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showed that βOHB enhanced cisplatin-induced apoptosis of HepG2 cells, at least partially, by decreasing survivin levels via downregulation of HDACs 3/6.
4. Discussion In this study, we showed that βOHB, a ketone body, enhances cisplatin-induced apoptosis via the HDACs inhibition/survivin axis in HepG2 cells. To our knowledge, our results are the first to demonstrate that antitumor cytotoxicity of cisplatin may be enhanced by the addition of βOHB in HCC cells. A key finding of this study is that βOHB enhanced cisplatin-induced apoptosis by modulating HDACs. There is an association between aberrant expression of HDACs and poor prognosis in many cancers
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upregulated in many HCC cells
18
. Kim et al. reported that HDACs 1, 2, 3, 4, 5, and 8 are . Wu et al. found that overexpression of HDAC3 could
predict poor prognosis in hepatitis B virus-associated HCC and that inhibition of HDACs 2 and 3 suppressed the invasiveness of HCCLM3 cells, which have high metastatic potential 19. Fan et al. showed that proliferation was inhibited by knockdown of HDAC5 in Hep3B, Huh7, and HepG2 cells via an increase in p53 stability and promotion of its nuclear localization 20. Kanno et al. reported that knockdown of HDAC6 decreased the invasiveness and migration of HLF, Hep3B, and PLC cells 21. Furthermore, overexpression of HDAC6 was observed in 20% of primary HCCs and was significantly correlated with an advanced clinical stage, intrahepatic metastasis, and vascular invasion
21
. Thus, HDAC suppression is potentially
14
effective for treatment of HCC. In this study, we observed a significant decrease in protein levels of HDACs 3-8 in HepG2 cells with cisplatin alone and a further decrease in the protein levels of HDACs 3/5/6 with the addition of βOHB in HepG2 cells. Another key finding is the significant decrease in survivin protein level by treatment with βOHB plus cisplatin in HCC cells. Survivin, an anti-apoptotic protein, is an inhibitor of apoptosis protein pancreatic tumors
22
. It is highly expressed in cancers, such as lung, breast, liver, colon, and
23, 24
, but it is not detected or is detected at low levels in normal tissue 23.
According to previous reports, survivin expression is associated with poor prognosis and drug resistance
25-28
. Furthermore, knockdown of survivin enhances the cytotoxicity of
chemotherapy regimens including cisplatin in bladder and prostate cancer cells 29, 30. Lee et al. reported that the knockdown of HDAC3/6 significantly decreased the level of survivin and decreased cell viability in MCF7 breast cancer cells
15
. Consistent with these findings, we
found that knockdown of HDAC 3/6 decreased survivin protein levels in HepG2 cells. In addition, Yu et al. reported enhancement of cisplatin-induced apoptosis with YM155, a selective survivin antagonist, by increased activation of caspase-3 and caspase-8 in HepG2 and HuH-6 cells
16
. In line with these results, we demonstrated that βOHB enhanced
cisplatin-induced apoptosis by increasing the activation of caspase-3 and caspase-8 in HepG2 cells. These findings indicate that HDAC 3/6 expression was decreased by βOHB combined with cisplatin, leading to downregulation of survivin and then enhanced cisplatin-induced
15
cleaved caspase-3 and caspase-8 in HepG2 cells. Based on these results, we proposed a mechanism in which the combination of βOHB and cisplatin decreased HDAC 3/6 expression and caused downregulation of survivin, resulting in cisplatin-induced cleavage of caspase-3 and caspase-8 in HepG2 cells.
5. Conclusions The administration of βOHB significantly enhances cisplatin-induced cytotoxicity and apoptosis of HCC cells, at least in part, by decreasing HDAC 3/6 expression, leading to a decrease in survivin. Our data suggest that the administration of βOHB or a ketogenic diet with chemotherapeutic regimens including cisplatin may result in improved curative effects in HCC patients.
ABBREVIATIONS βOHB, β-hydroxybutyrate; KB, ketone body; HDAC, histone deacetylase; HCC, hepatocellular carcinoma
Acknowledgements This study was supported in part by JSPS KAKENHI grant number 19K07117 and grant number 18K06946 (Grant-in-Aid for Scientific Research (C)), grant numbers 18K15971,
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19K17735, 19K17702, and 19K17395 (Grant-in-Aid for Young Scientists) from the Japan Society for the Promotion of Science and the National Center for Child Health and Development (29-1).
Conflicts of Interest The authors declare no competing interests.
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FIGURE LEGENDS Figure 1. Therapeutic efficacy of βOHB + cisplatin in HCC HepG2 xenografts. Six-week-old male SHO nude mice were subcutaneously injected with 1.0 × 106 cells. When the tumor size reached >500 mm3, mice were treated by intraperitoneal injection of 8 mg/kg cisplatin or 8 mg/kg cisplatin + 100 mg/kg βOHB suspended in saline. Tumor size was measured as described in the Materials and Methods. Data are expressed as the mean ± SE (n=5). *P<0.05, **P<0.01, by Student’s t-test.
Fig. 2. Effects of βOHB combined with cisplatin on proliferation rate of HCC cell lines. MTS assay was used to examine the effects of βOHB (10 mM) alone, cisplatin (25 µM) alone, or both agents on the proliferation of HepG2 cells (A) and HLE cells (B) at 24, 48, and 72 h. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01 by one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Figure 3. Effects of βOHB combined with cisplatin on apoptotic rate of HCC cell lines. HepG2 cells (A) and HLE cells (B) were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 48 h. Cells were then stained with annexin V and PI, followed by FACS analysis. Data are shown as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01 by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
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Fig. 4. βOHB elicited an extrinsic apoptotic pathway in HCC cells. HepG2 cells (A) and HLE cells (B) were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h. Western blotting was performed to detect the apoptosis-related proteins (cleaved caspase-3, caspase-8, and caspase-9) in cell lysates. The levels of cleaved caspases (all relative to β-actin) were normalized to 1.0 in cells treated with cisplatin alone. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01, NS not significant by Tukey-Kramer’s multiple comparison test.
Fig. 5. βOHB combined with cisplatin decreased HDAC expression in HCC cells. HepG2 and HLE cells were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h. Western blotting was performed to detect acetyl-histone H3 and HDACs 1-8 in cell lysates. The levels of acetyl-H3 (all relative to β-actin) were normalized to 1.0 in cells treated with cisplatin alone. The levels of HDACs (all relative to β-actin) were normalized to 1.0 in cells treated with control. Data are expressed as the mean ± SD of 3 independent experiments. *P<0.05, **P<0.01 by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Fig. 6. βOHB combined with cisplatin decreased survivin expression in HepG2 cells. HepG2 cells were treated with cisplatin (25 µM), βOHB (10 mM), or both agents for 24 h.
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(A) Western blotting was performed to detect survivin in cell lysates. (B) HepG2 cells were treated with control siRNA or HDAC3 siRNA, or HDAC5 siRNA, and HDAC6 siRNA. Western blotting was performed to detect HDAC3/5/6 or survivin in siRNA-mediated control and HDAC3/5/6 knockdown cells in cell lysates. (C) HepG2 cells were treated with vehicle or cisplatin (25 µM) in siRNA-mediated control and HDAC3/6 knockdown cells. The cleaved caspase-3 level relative to β-actin in cisplatin-induced cells treated with control siRNA was set at 1.0. Data are expressed as the mean ± SD of 3 independent experiments. **P<0.01, by a one-way ANOVA with Tukey-Kramer’s multiple comparison test.
Relative mean tumor weight
Fig.1
cisplatin 8 mg/kg cisplatin 8 mg/kg + βOHB 100 mg/kg
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Fig.6 A
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