RESUMO
BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Quilópodes , Peptídeos , Animais , Humanos , Masculino , Camundongos , Antineoplásicos/análise , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Quilópodes/química , Quilópodes/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Camundongos Nus , Simulação de Acoplamento Molecular , Peptídeos/análise , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Tripsina , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Injeções Intraperitoneais , Células Hep G2RESUMO
This study is to assess the efficacy of BPCBG on the decorporation of uranium (VI) and protecting human renal proximal tubular epithelial cells (HK-2) against uranium-induced damage. BPCBG at different doses was injected intramuscularly to male SD rats immediately after a single intraperitoneal injection of UO2(CH3COO)2. Twenty-four hours later uranium contents in urine, kidneys and femurs were measured by ICP-MS. After HK-2 cells were exposed to UO2(CH3COO)2 immediately or for 24 h followed by BPCBG treatment at different doses for another 24 or 48 h, the uranium contents in HK-2 cells were measured by ICP-MS, the cell survival was assayed by cell counting kit-8 assay, formation of micronuclei was determined by the cytokinesis-block (CB) micronucleus assay and the production of intracellular reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) oxidation. DTPA-CaNa3 was used as control. It was found that BPCBG at dosages of 60, 120, and 600 micromol kg(-1) resulted in 37%-61% increase in 24 h-urinary uranium excretion, and significantly decreased the amount of uranium retention in kidney and bone to 41%-31% and 86%-42% of uranium-treated group, respectively. After HK-2 cells that had been pre-treated with UO2(CH3COO)2 for 24 h were treated with the chelators for another 24 h, 55%-60% of the intracellular uranium was removed by 10-250 micromol L(-1) of BPCBG. Treatment of uranium-treated HK-2 cells with BPCBG significantly enhanced the cell survival, decreased the formation of micronuclei and inhibited the production of intracellular ROS. Although DTPA-CaNa3 markedly reduced the uranium retention in kidney of rats and HK-2 cells, its efficacy of uranium removal from body was significantly lower than that of BPCBG and it could not protect uranium-induced cell damage. It can be concluded that BPCBG effectively decorporated the uranium from UO2(CH3COO)2-treated rats and HK-2 cells, which was better than DTPA-CaNa3. It could also scavenge the uranium-induced intracellular ROS and protect against the uranium-induced cell damage. BPCBG is worth further investigation.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Células Epiteliais/citologia , Compostos Organometálicos/toxicidade , Urânio/metabolismo , Animais , Linhagem Celular , Quelantes/administração & dosagem , Quelantes/química , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Rim/metabolismo , Túbulos Renais Proximais/citologia , Masculino , Testes para Micronúcleos , Estrutura Molecular , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Urânio/urinaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by dysregulation of the immune microenvironment and the development of chemoresistance. Specifically, expression of the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis, an immune checkpoint, may lead to tumour immune escape, resulting in disease progression. The latest research shows that tumour immune escape may be caused by the upregulation of PD-L1 mediated by hypoxia-inducible factor-1 alpha (HIF-1α), and simultaneous inhibition of HIF-1α and PD-L1 has the potential to enhance the host's antitumour immunity. Moreover, inhibition of the PD-1/PD-L1 axis may mitigate tumour chemoresistance. Shuyu pills (SYPs) contain immunity-enhancing and antitumour components, making them a potential HCC treatment. AIM: To investigate the efficacy of SYPs for HCC treatment via simultaneous HIF-1α and PD-L1 inhibition and the mechanism involved. METHODS: A subcutaneous xenograft tumour model was first established in BALB/c nude mice by the subcutaneous injection of 1 × 107 SMMC-7721 cells. Male mice (male, 5 weeks old; n = 24) were then randomly divided into the following four groups (n = 6): Control (0.9% normal saline), SYP (200 mg/kg), SYP + cisplatin (DDP) (200 mg/kg + 5 mg/kg DDP weekly via intraperitoneal injection), and DDP (5 mg/kg cisplatin weekly via intraperitoneal injection). The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d via intragastric administration. The tumour volumes and body weights of the mice were measured every 2 d. The mice were euthanized by cervical dislocation after 14 d of continuous treatment, and the xenograft tissues were excised and weighed. Western blot assays were used to measure the protein expression of HIF-1α, PD-1, PD-L1, CD4+ T cells, and CD8+ T cells in HCC tumours from mice. Quantitative reverse transcription polymerase chain reaction was used for real-time quantitative detection of PD-1, PD-L1, and HIF-1α mRNA expression. An immunofluorescence assay was conducted to examine the expression of CD4+ T cells and CD8+ T cells. RESULTS: Compared to mice in the control group, those in the SYP and SYP + DDP groups exhibited reduced tumour volumes and tumour weights. Moreover, the protein and mRNA expression levels of the oncogene HIF-1α and that of the negative immunomodulatory factors PD-1 and PD-L1 were decreased in both the SYP and SYP + DDP groups, with the decrease effects being more prominent in the SYP + DDP group than in the SYP group (HIF-1α protein: Control vs SYP, P = 0.0129; control vs SYP + DDP, P = 0.0004; control vs DDP, P = 0.0152, SYP + DDP vs DDP, P = 0.0448; HIF-1α mRNA: control vs SYP, P = 0.0009; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SYP vs SYP + DDP, P = 0.0192. PD-1 protein: Control vs SYP, P = 0.0099; control vs SYP + DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0009; SYP + DDP vs DDP, P < 0.0001; PD-1 mRNA: control vs SYP, P = 0.0002; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SPY vs SYP + DDP, P = 0.0003; SYP + DDP vs DDP, P = 0.0002. PD-L1 protein: control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0040; SYP + DDP vs DDP, P = 0.0010; PD-L1 mRNA: Control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P = 0.0014). Additionally, the quantitative and protein expression levels of CD4+ T cells and CD8+ T cells were simultaneously upregulated in the SYP + DDP group, whereas only the expression of CD4+ T cells was upregulated in the SYP group. (CD4+ T cell quantitative: Control vs SYP + DDP, P < 0.0001, SYP vs SYP + DDP, P = 0.0005; SYP + DDP vs DDP, P = 0.0002. CD4+ T cell protein: Control vs SYP, P = 0.0033; Control vs SYP + DDP, P < 0.0001; Control vs DDP, P = 0.0021, SYP vs SYP + DDP, P = 0.0004; SYP + DDP vs DDP, P = 0.0006. Quantitative CD8+ T cells: Control vs SYP + DDP, P = 0.0013; SYP vs SYP + DDP, P = 0.0347; SYP + DDP vs DDP, P = 0.0043. CD8+ T cell protein: Control vs SYP + DDP, P < 0.0001; SYP vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P < 0.0001). Finally, expression of HIF-1α was positively correlated with that of PD-1/PD-L1 and negatively correlated with the expression of CD4+ T cells and CD8+ T cells. CONCLUSION: SYPs inhibit immune escape and enhance chemosensitization in HCC via simultaneous inhibition of HIF-1α and PD-L1, thus inhibiting the growth of subcutaneous xenograft HCC tumours.
RESUMO
BACKGROUND: Liver cancer is the sixth most frequently occurring cancer in the world and the fourth most common cause of cancer mortality. The pathogenesis of liver cancer is closely associated with inflammation and immune response in the tumor microenvironment. New therapeutic agents for liver cancer, which can control inflammation and restore cellular immunity, are required. Curcumin (Cur) is a natural anti-inflammatory drug, and total ginsenosides (TG) are a commonly used immunoregulatory drug. Of note, both Cur and TG have been shown to exert anti-liver cancer effects. AIM: To determine the synergistic immunomodulatory and anti-inflammatory effects of Cur combined with TG in a mouse model of subcutaneous liver cancer. METHODS: A subcutaneous liver cancer model was established in BALB/c mice by a subcutaneous injection of hepatoma cell line. Animals were treated with Cur (200 mg/kg per day), TG (104 mg/kg per day or 520 mg/kg per day), the combination of Cur (200 mg/kg per day) and TG (104 mg/kg per day or 520 mg/kg per day), or 5-fluorouracil combined with cisplatin as a positive control for 21 d. Tumor volume was measured and the protein expression of programmed cell death 1 and programmed cell death 1 ligand 1 (PD-L1), inflammatory indicators Toll like receptor 4 (TLR4) and nuclear factor-κB (NF-κB), and vascular growth-related factors nitric oxide synthases (iNOS) and matrix metalloproteinase 9 were analyzed by Western blot analysis. CD4+CD25+Foxp3+ regulatory T cells (Tregs) were counted by flow cytometry. RESULTS: The combination therapy of Cur and TG significantly inhibited the growth of liver cancer, as compared to vehicle-treated animals, and TG showed dose dependence. Cur combined with TG-520 markedly decreased the protein expression of PD-L1 (P < 0.0001), while CD4+CD25+Foxp3+ Tregs regulated by the PD-L1 signaling pathway exhibited a positive correlation with PD-L1. Cur combined with TG-520 also inhibited the cascade action mediated by NF-κB (P < 0.0001), thus inhibiting the TLR4/NF-κB signalling pathway (P = 0.0088, P < 0.0001), which is associated with inflammation and acts on PD-L1. It also inhibited the NF-κB-MMP9 signalling pathway (P < 0.0001), which is associated with tumor angiogenesis. CONCLUSION: Cur combined with TG regulates immune escape through the PD-L1 pathway and inhibits liver cancer growth through NF-κB-mediated inflammation and angiogenesis.