Sorafenib

Curcumin enhanced antitumor effects of sorafenib via regulating metabolism and tumor microenvironment

Shuli Man a1, Jingwen Yao a1, Panpan Lv a, Yu Liu a, Li Yang a, Long Ma a*

a State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.

Curcumin, the main active ingredient of turmeric, was widely used as a kind of food additives and processed pharmacological activities such as anti-inflammation, anti-tumor, liver and kidney protection and so forth. Sorafenib was the first targeted agent against hepatocellular carcinoma (HCC), whose intolerance was related to the promotion of lipid synthesis and epithelial-to-mesenchymal transition (EMT) formation. In this study, biochemical analysis, immune cells composition, tumor microenvironment, metabolomics and relative metabolic enzymes and transporters were detected in H22-bearing mice treated with curcumin combining with sorafenib vs. control groups. As a result, curcumin protected against liver cancer progression through reducing the level of alpha fetoprotein in liver tissues, increasing the number of immune cells like NK cells, inhibiting EMT via the regulation of IL-6/JAK/STAT3 and IL-1β/NF-κB pathways, suppressing anaerobic glycolysis through inhibition of LDH and HIF-1α, and decreasing the lipid synthesis via down-regulation of FASN and up-regulated serum HDL-C and mRNA level of apoA1 in sorafenib-treated mice. Furthermore, curcumin regulating the disorder of glycolipid metabolism and EMT also based on PI3K/AKT pathway. Docking study proved the strong affinity between curcumin and protein of STAT3, FASN and AKT. All in all, this experiment provided evidences for the addition of curcumin in diet enhancing the antitumor efficacy of sorafenib through activating immune function, down-regulating EMT and reversing the disorder of metabolism.

Keywords: Curcumin; Sorafenib; microenvironment; metabolism; hepatoDcOeI:l1l0u.1l0a3r9/C9FO01901D

Sorafenib was the first targeted agent to prolong survival time of patients with advanced hepatocarcinoma (HCC) by blocking kinases such as Raf serine/threonine and VEGF1. However, long-term use of sorafenib led to tolerance with EMT which was one of the most important mechanisms in progression of various carcinomas including HCC2. Besides, many pathways related with sorafenib resistance contained PI3K/AKT, HIF-1α and so forth3.

Curcumin, the main bioactive ingredients of turmeric, was widely used as a spice and colorant in food. It possessed multiple pharmacological activities including anti-inflammation4, anti-oxidant5, anti-carcinogen6 and so forth. In our previous research, curcumin attenuated diethylnitrosamine-induced liver cancer through regulating the oxidant stress enzymes, inflammatory factors and attenuating metabolic disorder 7. In addition, it could inhibit epithelial-to-mesenchymal transition (EMT) and angiogenesis by blocking PI3K/AKT pathway and up-regulating inflammatory factors like lnterleukin-6 (IL-6), lnterleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα) and nuclear factor-κ-gene binding (NF-κB) 8-10.

Notably, curcumin analogue11, 12 and its self-assembled nanoparticles 3, 13 displayed to increase the cytotoxicity of sorafenib in liver cancer. Our purpose in this study was to investigate whether the addition of curcumin in diet could strengthen the antitumor efficacy of sorafenib in HCC and to examine whether curcumin could overcome the sorafenib limitation through inhibiting inflammation, EMT and disorder of metabolism and regulating immune function in mice models.

Methods

Sorafenib was supplied from Energy Chemical (Shanghai, China, D050333, MV: 637.03). Curcuminoid (90% of curcumin) was obtained from Zhongda Co. (Henan, China, C160516, MV: 368.38). Curcumin is a water-insoluble polyphenol with potential beneficial health effects for humans. Carboxymethyl cellulose as a biodegradable polymer possesses many kinds of emulsifying properties to improve curcumin release rate14. Drug suspension was freshly prepared with 0.5% sodium carboxymethyl cellulose which was a food additive generally used as a viscosity modifier15. Incorporating curcumin into a CMC-Na can improve its water solubility. The structures of carboxymethyl cellulose (CMC-Na), curcumin (CUR) and putative schematic structures of CMC-Na-curcumin were shown in Figure 1 animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Tianjin University of Science and Technology and approved by the Ministry of Science and Technology of the People’s Republic of China. In experiment, 1×105 of H22 tumor cells were inoculated in axillae of 24 mice. Then, mice were allocated to five treatment groups (n=6) randomly when tumors grew nearly 300 mm3. Group I: normal group without inoculation of H22 cells (Normal). Group II: administering 0.5% sodium carboxymethyl cellulose every day (Model). Group III: administering oral medication with sorafenib in 0.5% sodium carboxymethyl cellulose (60 mg/kg/d) (Sorafenib). Group IV: administering oral medication with curcumin in 0.5% sodium carboxymethyl cellulose (25 mg/kg/d) (Curcumin). Group V: administering oral medication with sorafenib (60 mg/kg/d) and curcumin in 0.5% sodium carboxymethyl cellulose (25 mg/kg/d) (Sora+Cur). After 3 weeks, blood samples (0.5 mL) were collected by puncturing retro-orbital sinus and centrifugated for 10 min at 4000 g. Serum samples were maintained at -20℃. After sacrificed, major organs were dissected, weighed and maintained at -80℃. The viscera index was calculated using the formula: Viscera index (mg/g) = weight of viscera (mg)/body weight (g).

Figure 1 Curcumin (CUR) was dissolved in 0.5% sodium carboxymethyl cellulose. (A) The structures of curcumin, and (B) carboxymethyl cellulose (CMC-Na). (C1) Curcumin was dissolved in water. (C2) CMC-Na-curcumin was dissolved in water. (D) Curcumin dispersed in CMC-Na.

Animals

Biochemical Analyses

Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total cholesterol (T-CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and lactate dehydrogenase (LDH) were measured by the detection kits which were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

Immune function analyses

Total leukocytes isolated from the spleens were suspended in PBS at a concentration of 1 ×106 cells/mL and then labeled with the antibodies CD3e, CD4, CD8 and NK1.1 (Sungene, China) for 30 minutes at 4°C in the dark condition. Next, 400 μL of binding buffer was added to each sample and then analyzed by flow cytometry.

Measurement of malondialdehyde (MDA)

100 mg of liver tissue was homogenized in 1 mL of PBS at 4℃. The suspension was measured according to the manufacturer’s instructions using commercially available kits (Nanjing Jiancheng Bioengineering Institute). The content of MDA in liver tissue homogenates was determined by reacting with thiobarbituric acid (TBA) as an index of lipid peroxidation.

Measurement of fatty acid synthase (FASN) levels

Serum samples were analyzed by FASN mouse ELISA kits according to the kit instructions (SenBeiJia Biological Technology Co., Ltd. China). Values of samples were measured within the standard curve.

Sample preparation for GC/MS

250 μL of acetonitrile was added in 100 μL of serum samples and whirled for 3 min at 4℃. The suspension was centrifugated for 10 min at 10, 000 g. Then, the supernatant was removed into the centrifuge tube in which supernatant was dried under nitrogen gas. Subsequently, 50 μL of pyridine with 15 mg/mL of methoxylamine hydrochloride was added to the dried sample and incubated for 1 h at 70℃. Next, 50 μL of derivatization reagent (BSTFA: TMCS = 100:1 v/v) was added and kept for 1 h. 150 μL of heptane with 0.1 mg/mL of docosane as internal standard was added to each sample, whirled for 2 min and centrifugated for another 5 min at 10,000 g. Finally,the supernatant was transferred to GC vial and analyzed by GC-MS.

According to previous methods16, serum samples were analyzed by Agilent 7890A gas chromatography coupled to an Agilent 5975C mass selective detector (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) and performed in the electron ionization (EI) mode.

Reverse transcription PCR (RT-PCR)

Total RNA of mouse tissue was obtained with Trizol (Sangon Biotech Co., China) reagent according to the manufacturer instructions. Polymerase chain reaction products were electrophore on 3.0% agarose gel in which GelRed Nucleic Acid Stain (Biotium, USA) was added. The expression degree of gene was quantified by Image J software.

Western Blot Analyses

Protein samples from liver and tumor tissues of mice were separated by Sodium Dodecyl Sulfonate-polyacrylamide gel and transferred onto a Polyvinylidene Fluoride (PVDF) membrane. Then, PVDF membrane was blocked in 5% skim milk for 1 h and incubated for 4 h with primary antibodies AFP (Bioworld Technology, USA, MB9042, 1:500), AKT (Boster, China, BM4400, 1:200), E-Cad (Cell signaling, USA, 14472, 1:1000), Vim (Santa cruz, USA, sc-6260, 1:200), HIF-1α (Cell signaling, USA, 14179S, 1:1000), TNFα (Proteintech, USA, 17590-I-AP, 1:1000),
NF-κB (Santa cruz, USA SC-372, 1:200), IL-6 (Proteintech, USA, 21865-I-AP, 1:1000),Phospho-JAK1/2 (Cell signaling, USA, 66245S, 1:1000), Phospho-STAT3 (Cell signaling, USA, 9134T, 1:1000), IL-1β (Santa cruz, USA, SC-52012 1:200), GLUT1 (Boster, China, BM4235,1:200), p53 (Proteintech, USA, 10442-I-AP, 1:1000), CPT1A (Proteintech, USA, 66039-I-Ig,1:1000), PI3K (Boster, China, BM5187, 1:200), Phospho-PI3- kinase (Cell signaling, USA, 4228S,SC-47778, 1:1000). After that, PVDF membrane was washed with PBST for three times and incubated for 2 h with second antibody. Finally, the protein expression was examined by the Odyssey infrared imaging system (LI-COR Biotechnology, USA).

Molecular docking

Molecular Docking were simulated by Discovery Studio 2.5. Dimensional structures of protein were downloaded from RCSB Protein Date Bank (https://www.rcsb.org) and prepared by MGLTools-1.4.6. Interaction visualization between them was showed off by Pymol.

Statistical analysis

SPSS 19.0 was used to analyze the data. Multi-group comparisons were carried out through One-way ANOVA test, while couple comparisons based on q-test. The metabolite data were analyzed by SIMCA-P + software (version 11.5, Umetrics, Sweden) using principal component analysis (PCA). Besides, the hierarchical clustering analysis (HCA) of metabolite date was carried out with the Multi Experiment Viewer software (Version 4.8.1).

Results

Curcumin enhanced antitumor effects of sorafenib

In this experiment, curcumin enhanced the inhibition of the tumor growth in sorafenib-treated mice (Figure 2 C). Levels of ALT and AST as liver function markers, AFP as a hepatocarcinoma marker and MDA as a product of lipid peroxidation were remarkably increased in the model group (p<0.05). Sora+Cur significantly reduced the level of ALT in serum and MDA concentration in liver tissues compared with model group (Figure 2 D, E, F&G). Curcumin especially remarkably decreased AFP level in sorafenib-treated liver tissues. These indicated that curcumin inhibited the tumor growth and protected liver function in curcumin, pathological analysis of tumor tissues and liver tissues were performed by H&E assay. As shown in Figure 2 H, the tumor necrosis was not obvious in the model group. However, combination group enhanced the necrosis area. In the liver group, there were no obvious pathological changes among these groups. Figure 2 Inhibition rate of tumor growth and evaluation of liver function. (A) The body weight of different groups. (B) The tumor volume of different groups. (C) Organ indexes of AST in serum. (F) Activity of ALT in serum. (G) Concentration of MDA in liver tissues. Different letters meant significant differences between two groups. (H) Representative sections (HE staining) of the tumors and livers in mice. Letters shared in common indicated no significant differences between or among the groups (p <0.05). Curcumin strengthened immune function in sorafenib-treated liver cancer As Figure 2C shown, organ indexes of spleen and thymus in curcumin or combination group were slightly increased compared with model and sorafenib groups. Then the total leukocytes were isolated from the spleens and analyzed by flow cytometry. As a result, the proportion of CD4+ T cells was significantly increased in curcumin group and comparably increased in combination group compared with the model and sorafenib groups. In addition, curcumin remarkably increased the proportion of NK cells in sorafenib-treated mice (Figure 3 A&B). These indicated curcumin-activated host immune responses. Curcumin inhibited EMT of tumor in sorafenib-treated liver cancer EMT played an important role in the progression of HCC. As we known, the decrease of E-Cad and increase of Vim indicated the occurrence of EMT. In this research, the dual treatment significantly increased the protein level of E-Cad and decreased the level of Vim compared with the single sorafenib-treated mice, which indicated that curcumin down-regulated the EMT formation. To identify its mechanisms, we analyzed EMT related proteins. As Figure 3C shown, curcumin remarkably down-regulated protein levels of IL-1β, NF-κB, p-JAK1/2 and p-STAT3,ELISA analysis of IL-6, IL-1β, NF-kβ and IL-4 in the serum as shown in Figure 3D. As a result, ELISA analysis of NF-κB in the serum was coincided with that protein change in the tumor and curcumin remarkably down-regulated levels of IL-6 in the serum of sorafenib-treated mice. Besides, the addition of curcumin up-regulated the anti-inflammatory cytokines expression of IL-4 and remarkably decreased the level of HIF-1α in sorafenib-treated mice (Figure 3 C). Figure 3 Immune analysis and related protein levels in EMT. (A1, 2&3) The ratio of CD4+ CD3e+ cells was 17.07±0.50% in model group, 15.13±1.05% in sorafenib group, 21.33±1.31% in curcumin group and 17.57±2.07% in Sora+Cur group. The ratio of CD8+ CD3e+ cells was 7.3±0.75% in model group, 6.10±0.26% in Sorafenib group, 8.20±1.15% in was 4.8±0.83% in model group, 4.1±0.58% in Sorafenib group, 9.97±0.20% in Curcumin group and 9.76±0.99% in Sora+Cur group. (C) Protein expression of E-Cad, Vim, HIF-1α, TNFα, NF-κB, IL-1β, IL-6, p-JAK1/2 and p-STAT3 in tumor tissues. (D) ELISA analysis of NF-κB, IL-6, IL-1β and IL4 in the serum. Different letters meant significant differences between two groups. Letters shared in common indicated no significant differences between or among the groups (p <0.05). Curcumin reversed metabolic disorder in sorafenib-treated liver cancer Different letters meant significant differences between two groups. Letters shared in common indicated no significant differences between or among the groups (p<0.05). Metabolic disorder could enhance invasive potential of cancer cells by up-regulating EMT17. As Table 1 indicated, curcumin significantly decreased serum concentration of LDH, TG, FASN and increased level of HDL-C in sorafenib-treated mice. Meanwhile, lactate, glycerol, ribitol, D-fructose, galactose, D-glucose, D- mannose, hexadecanoic acid and octadecanoic acid were detected by GC/MS analysis. As a result, the dual treatment significantly decreased the concentration of lactate, D-fructose, D-glucose, and hexadecanoic acid compared with the single sorafenib-treated mice (p<0.05) (Table 2). All the GC-MS data obtained from serum samples were analyzed by PCA scores plot which was used widely to detect the biologic effects of drugs using multivariate analysis of endogenous metabolites (Figure 4D). As a result, PCA scores plot showed an obvious discrimination among different groups. Figure 4 RT-PCR analysis of metabolism related genes in liver (A) and tumor (B) tissues from different groups. (C) Hierarchical clustering heat maps of metabolic related genes in tumor tissues from different groups. (D) PCA score plot (t[Comp.1]/t[Comp.2]) based on data of serum for GC-MS integral regions from H22 mice (N: Normal group; M: Model group; S Sorafenib group; C: Curcumin group; SC: Sora+Cur group). (E) Protein expression of GLUT1, p53, CPT1A, p-PI3K and p-AKT (Ser473) in liver tissues. Different letters meant significant differences between or among the groups (p<0.05). Figure 5 Intermolecular interaction of curcumin with (A) STAT3, (B) FASN, and (C) AKT in 3D space. Orange stick models presented the residues of the protein. Green stick models presented the curcumin. Red dash lines were hydrogen bonds. Discussion Curcumin, a kind of natural products used widely in food, showed multiple pharmacological activities such as anti-inflammation, anti-oxidant, metabolic regulation and inhibition of EMT18. Sorafenib was the most popular drug available for the treatment of advanced HCC, whose intolerance was related to its promotion of lactate production, lipid synthesis and EMT formation 2, 19. For curcumin analogue 11, 12 and its self-assembled nanoparticles 3, 13, 20, 21 could increase the cytotoxicity of drug in cancer, we wanted to know whether and how addition of curcumin in diet enhanced the antitumor efficacy of sorafenib or overcame the sorafenib limitation. As a result, curcumin increased the anticancer effects of sorafenib-treated H22 mice and protected against liver injury by down-regulating level of AFP in liver tissues. Meanwhile, curcumin remarkably increased the number of NK cells and slightly increased organ indexes of spleen and thymus in sorafenib-treated group which indicated its activation of host immune responses22. As previous reports, EMT played an important role in the progress of tumor in model and sorafenib groups indicated the occurrence of EMT2. Curcumin significantly reversed the protein levels of E-Cad and Vim in sorafenib-treated mice, which indicated curcumin down-regulated the EMT formation in sorafenib-treated mice. Sorafenib remarkably increased protein levels of IL-1β, p-JAK1/2, p-STAT3 and NF-κB indicated that sorafenib resistance was related to the inflammation inducing EMT24, 25. As previous reported, STAT3 could promote the pro-oncogenic inflammatory pathways, such as NF-κB and IL-6/JAK pathways, and suppress NF-κB-mediated T helper 1 anti-tumor immune responses 26. Curcumin remarkably down-regulated protein levels of IL-1β, p-JAK1/2, p-STAT3 and NF-κB and showed well binding energy with STAT3 in sorafenib-treated mice, which indicated the possibly control of the sorafenib limitation via inhibiting IL-6/JAK/STAT3 and IL-1β/NF-κB pathways. Glycolipid metabolic disorder was associated with EMT promoting tumor growth and sorafenib resistance. Lactate as a waste product of glycolysis was regarded as a critical regulator of cancer metastasis and tumor invasion induced by up-regulation of EMT17 27. In the experiment, curcumin significantly decreased concentration of lactate in sorafenib treatment. LDH, as a necessary to maintain the aerobic glycolysis providing the energy in cancer cells28, and HIF-1α, whose overexpression could up-regulate aerobic glycolysis and EMT29, 30 were remarkably inhibited by curcumin in sorafenib-treated group. All these indicated that curcumin enhanced antitumor effect of sorafenib in HCC via the inhibition of aerobic glycolysis. which promoted the tumor regrowth and metastasis19. In this study, curcumin significantly decreased serum concentration of TG and hexadecanoic acid in sorafenib-treated mice, which may be related to its down-regulation of FASN, a key enzyme involved in neoplastic lipogenesis31, and up-regulation of serum concentration of HDL-C, a protein carrying the tissues cholesterol and promoting lipid excretion, and mRNA level of apoA1, a major gene in the synthesis of HDL32. Meanwhile, curcumin had a great binding force with FASN based on Discovery Studio 2.5 program analysis. These indicated that curcumin could overcome the limitation of sorafenib by regulating the disorder of lipid metabolism25. Activation of PI3K/AKT pathways was correlated with multiple metabolisms in HCC, such as glycolipid metabolism and EMT9, 33. In this study, addition of curcumin significantly down-regulated protein phosphorylation of AKT in liver tissues. Besides, curcumin showed strong appetency with AKT protein as Discovery Studio 2.5 program displayed. These indicated that curcumin could enhance the antitumor effect and overcome the sorafenib limitation in HCC by regulating PI3K/AKT pathway (Figure 6). Figure 6 Schematic diagram showed the mechanisms of curcumin synergizes with sorafenib to suppress the tumor growth and overcoming the tolerance of sorafenib. Heat maps showed the expression levels of genes, while the histograms showing the expression of proteins. N, Normal group；M, Model group; S, Sorafenib group; C, Curcumin group; SC, Sora+Cur group. *p<0.05, significant differences between two groups. Conclusion All in all, curcumin enhanced the antitumor effects and overcame the limitation of sorafenib in HCC. Curcumin activated the inherent immune cells, inhibited EMT via the regulation of IL-6/JAK/STAT3 and IL-1β/NF-κB pathways, suppressed anaerobic glycolysis through inhibition of LDH and HIF-1α, and decreased the lipid synthesis via down-regulation of FASN, and up-regulated serum HDL-C and mRNA level of apoA1 in sorafenib-treated mice. Furthermore, curcumin regulated PI3K/AKT metabolic and immunological methods were used to analyze the changes of tumor microenvironment and provide more evidence for the addition of curcumin in diet strengthening antitumor efficacy and overcoming the limitation of sorafenib. In the future, the beverage containing curcumin and CMC-Na can be used as an adjuvant chemotherapy agent. 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