Vitamin K2 Inhibits Hepatocellular Carcinoma Cell Proliferation by Binding to 17β-Hydroxysteroid Dehydrogenase 4

Our previous studies have proved that 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) is a novel proliferation-promoting protein. The overexpression of HSD17B4 promotes hepatocellular carcinoma (HCC) cell proliferation. Vitamin K2 (VK2), a fat-soluble vitamin, has the function of promoting coagulation and can inhibit the progression of liver cancer. A previous study demonstrated that VK2 could bind to HSD17B4 in HepG2 cells. However, the mechanism of VK2 in inhibiting HCC cell proliferation is not clear. In this study, we investigate whether VK2 can inhibit the proliferation of HCC cell induced by HSD17B4 and the possible mechanism. We detected the effect of VK2 on HSD17B4-induced HCC cell proliferation, and the activation of STAT3, AKT, and MEK/ERK signaling pathways. We measured the effect of HSD17B4 on the growth of transplanted tumor and the inhibitory effect of VK2. Our results indicated that VK2 directly binds to HSD17B4, but does not affect the expression of HSD17B4, to inhibit the proliferation of HCC cells by inhibiting the activation of Akt and MEK/ERK signaling pathways, leading to decreased STAT3 activation. VK2 also inhibited the growth of HSD17B4-induced transplanted tumors. These findings provide a theoretical and experimental basis for possible future prevention and treatment of HCC using VK2.

Rutaecarpine Increases Anticancer Drug Sensitivity in Drug-Resistant Cells through MARCH8-Dependent ABCB1 Degradation

The overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) subfamily B member 1 (ABCB1; P-glycoprotein; MDR1) in some types of cancer cells is one of the mechanisms responsible for the development of multidrug resistance (MDR), which leads to the failure of chemotherapy. Therefore, it is important to inhibit the activity or reduce the expression level of ABCB1 to maintain an effective intracellular level of chemotherapeutic drugs. In this study, we found that rutaecarpine, a bioactive alkaloid isolated from Evodia Rutaecarpa, has the capacity to reverse ABCB1-mediated MDR. Our data indicated that the reversal effect of rutaecarpine was related to the attenuation of the protein level of ABCB1. Mechanistically, we demonstrated that ABCB1 is a newly discovered substrate of E3 ubiquitin ligase membrane-associated RING-CH 8 (MARCH8). MARCH8 can interact with ABCB1 and promote its ubiquitination and degradation. In short, rutaecarpine increased the degradation of ABCB1 protein by upregulating the protein level of MARCH8, thereby antagonizing ABCB1-mediated MDR. Notably, the treatment of rutaecarpine combined with other anticancer drugs exhibits a therapeutic effect on transplanted tumors. Therefore, our study provides a potential chemotherapeutic strategy of co-administrating rutaecarpine with other conventional chemotherapeutic agents to overcome MDR and improve therapeutic effect.

Synergistic Anticancer Effects of Cisplatin Combined with Combretastatin A4 Phosphate on Human Osteosarcoma-Xenografted Mice

This study aimed to investigate the effectiveness of anticancer therapy combining a cytotoxic chemotherapeutic agent, cisplatin (DDP), and a vascular disruptive drug, combretastatin A4 phosphate (CA4P), in osteosarcoma. First, a human osteosarcoma-xenografted mice model was established. Second, the transplanted tumor models were treated with DDP and CA4P in combination or as monotherapy. Third, the therapeutic effects and the mechanism of the drug combination in the inhibition of transplanted tumors was studied. Finally, the toxic effects of the drugs were observed and recorded. The results showed that DDP combined with CA4P significantly inhibited the growth and lung metastasis of transplanted tumors compared with the monotherapy drug group and vehicle control group. Histopathological analysis revealed that apoptotic and necrotic cell death significantly increased in the combination group, and combined treatment significantly inhibited the proliferation of osteosarcoma cells compared with either agent alone or the vehicle control. Additionally, no obvious toxic effects were observed in the combination group. These results indicate that the combined effects of DDP and CA4P on the progression of human osteosarcoma in vivo were superior to that of either agent alone. DDP combined with CA4P exerted synergistic effects at lower concentrations and promoted apoptosis and necrosis, as well as inhibited proliferation of osteosarcoma cells, but it did not increase the systemic toxic effects of chemotherapy. Our findings highlight CA4P as an effective anticancer agent candidate for combination with DDP in clinical applications to treat osteosarcoma.

Study on Molecular Mechanism of Benzo (ɑ) pyrene on CMA by HSP90ɑ and HIF-1ɑ

OBJECTIVE: The effects of benzo (α) pyrene (BaP) on molecular chaperone autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia inducible factor-1 (HIF-1) were studied by RNA interference and subcutaneous tumor formation technique in nude mice. METHODS: 40 nude mice inoculated with the shHSP90ɑ A549 cell line under the armpits of the forelimbs were divided into 4 groups, 1.80 mg/kg/d BaP-corn oil solution was intragastrically administered for 60 days (except the Control group), and the growth of nude mice and transplanted tumors was recorded curve. The size and morphological changes of tumors were observed by small animal imaging technique and HE staining method. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1μmol/L, 1μmol/L and 10μmol/L BaP for 24h, EPO, HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10μmol/L BaP and added HIF-1ɑ inhibitor 24h, qPCR, Western blot and Immunofluorescence method were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. RESULTS: BaP can reduce silence HSP90ɑ the weight of nude mice and transplanted tumors (P<0.01); BaP can reduce silence HSP90ɑ the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and protein in transplanted tumor tissues (P<0.05); BaP has no significant difference in the organization structure of silence HSP90ɑ and non-silence HSP90ɑ; BaP can reduce the total number of bioluminescence photons of silence HSP90ɑ transplanted tumors (P<0.01). 10μmol/L BaP can increase the concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells (P<0.05); and after adding HIF-1ɑ inhibitors treat A549 cells, HSP90ɑ, HSC70, Lamp-2A mRNA and protein expression were decreased (P<0.05), and the fluorescence intensity of HSP90ɑ was decreased. CONCLUSIONS: BaP can promote the growth of transplanted tumor in nude mice, while the growth of transplanted tumor in nude mice is inhibited shHSP90ɑ. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are important regulatory genes for BaP to activate CMA.

Glutaraldehyde (GLA) Loaded Albumin Nanoparticles Mediated p53 Targeting Cervical Cancer

Nano-albumin-mediated different doses of glutaraldehyde (GLA) were used in cervical cancer nude mice to observe the expression of p53, Bax protein and genes in transplanted tumors and explore the mechanism of GLA on cervical cancer. Cervical cancer Hela cells were cultured. 20 nude BALB/C-nu female mice were selected to establish transplanted tumor models and separated into control group (n = 5) and GLA group (n = 15). Grouping of glutaraldehyde (GLA)-loaded human serum albumin nano-carriers (GLA-HSANC) drugs was done and separated into GLA low-dose (0.06 µmol/mg) and medium-dose (0.12 µmol/mg) and high-dose group (0.18 µmol/mg) followed by assessing xenograft tumor apoptosis, p53 and Bax expression and Hela cell cycle. The shape and structure of various organs of nude mice were normal. The volume of transplanted tumors, tumor markers CA125 and CEA in different dose groups were lower than the control group. The tumor inhibition rate, growth inhibition rate of transplanted tumors, the number of apoptotic cells in transplanted tumors, p53, Bax protein and mRNA expression were all opposite in a dose dependent manner. Compared with the control group, the number of cells in G1 phase of other groups was higher with lower cell number in S phase dose-dependently. GLA nano-albumin particles-mediated p53 targeting cervical cancer tumors can significantly promote tumor cell apoptosis possibly via upregulating p53 and Bax, and increasing cells in G1 phase.

The Fusion Circular RNA F-circEA1 Promotes Non-small Cell Lung Carcinoma Progression through ALK Downstream Signaling

Background Circular RNA has a stable closed structure, which plays an important role in the occurrence and development of tumors. However, there are no reports on the relationship between fusion circular RNA and EML4-ALK variant 1, or their underlying molecular mechanisms in non-small cell lung carcinoma (NSCLC). Methods To test F-circEA1 in NCI-H3122 cells (carrying the EML4-ALK variant 1 gene) by RT-PCR, FISH and Sanger sequencing; To determine cell proliferation with a CCK-8 assay. Transwell experiments were used to detect cell migration and invasion. Cell cycle stage and apoptosis were detected by flow cytometry. The sensitivity of cells to crizotinib was tested using a CCK-8 assay. RT-PCR and western blots were used to measure the expression of EML4-ALK1 and downstream signaling factors associated with ALK. Subcutaneously transplanted tumors were used in nude mice to determine the effect of F-circEA1 on tumor formation and the expression of EML4-ALK1 by immunohistochemistry. Statistical analyses were carried by GraphPad Prism 8.0 and differences between groups were compared using Student's t test. Difference with p value <0.05 is statistically significant.Results F-circEA1 was present both in the cytoplasm and nucleus of NCI-H3122 cells. F-circEA1 was contributed to cell proliferation, metastasis, invasion. F-circEA1 induced cell arrest, promoted cell apoptosis and improved drug sensitivity to crizotinib. After knockdown with F-circEA1, subcutaneously transplanted tumors in nude mice grew slowly, the expression of EML4-ALK1 in tumor tissues decreased significantly. The mRNA and protein levels of EML4-ALK1 decreased after knockdown of F-circEA1 but increased after its overexpression. The protein expression of downstream ALK signaling factors increased after overexpression of F-circEA1, but not at the mRNA level.Conclusions We have confirmed a potential carcinogenenic and therapeutic role for F-circEA1 in NSCLC. Our experimental results may provide a new biomarker, treatment method, and prognostic monitoring index for use in the clinic.

Cyclopamine Reduces the Growth of Endometrial Carcinoma and Hedgehog Pathway Proteins in a Xenograft Mouse Model

Endometrial carcinoma is a frequently occurring malignancy of the female reproductive tract. Previous investigations have implicated the Hedgehog signaling pathway, including the smoothened (Smo) receptor, in tumor progression. Here, we established a nude mouse xenograft model of endometrial cancer to investigate the effect of the Smo receptor antagonist cyclopamine on the growth of endometrial carcinoma. Mice were randomly sorted into two groups receiving 0.2 mL/mouse every other day of either cyclopamine (20 mg/mL) or solvent (control). The growth of the mice was observed for 18 days. Then, mice were euthanized, tumors were removed and weighed, and tumor volume inhibition rate (VIR) and weight inhibition rate (WIR) were calculated. Smo, Gli1, and Gli2 mRNA expression was measured in tumor tissues by RT-PCR, and Vascular endothelial growth factor (VEGF) and CD31 were detected by immunohistochemistry. We found that cyclopamine treatment reduced the tumor growth rate and significantly reduced the volumes and weights of the transplanted tumors compared with those of the controls. Furthermore, the transplanted tumors of cyclopamine-treated mice possessed lower expression levels of Smo, Gli1, and Gli2, and displayed a lower positive expression of VEGF and CD31. Thus, the Smo receptor antagonist cyclopamine inhibited transplanted tumor growth in nude mice with endometrial carcinoma, which is likely connected with cyclopamine downregulation of Smo, Gli1, and Gli2 mRNA expression that promote angiogenesis.

MODL-25. REPLICATION REPAIR DEFICIENT MOUSE MODELS PROVIDE INSIGHT ON HYPERMUTANT BRAIN TUMOURS, MECHANISMS OF IMMUNE EVASION, AND COMBINATORIAL IMMUNOTHERAPY

Replication repair deficiency (RRD) is the leading cause of hypermutant brain tumours in children. RRD is caused by defects in one of four mismatch repair (MMR) genes and mutations in POLE or POLD1. Such tumours are resistant to common therapeutic agents and animal models are needed to study RRD in vivo and test novel therapies like immune checkpoint inhibitors (ICIs). To model RRD brain tumours specifically, we engineered a Pole mutant mouse model harbouring the S459F mutation (PoleS459F). We combined PoleS459F mice with conditional Msh2 knockout (Msh2LoxP) and Nestin-cre mice. All Nestin-cre+Msh2LoxP/LoxPPoleS459F/+ mice rapidly succumbed to posterior fossa brain tumours between 8.6 and 12.4 weeks. Importantly, tumours exhibited hallmark “ultrahypermutation” (~350 mutations/Mb) and the corresponding signatures characteristic of human combined MMR and POLE-proofreading glioblastoma. Interestingly, Nestin-cre+Msh2LoxP/LoxPPoleS459F/S459F mice failed to establish normal cerebella, suggesting such mutational loads may not support normal brain development. Furthermore, OLIG2-cre+Msh2LoxP/LoxPPoleS459F/+ mice failed to develop tumors. Tumors transplanted into syngeneic vs immunocompromised animals egrafted well orthotopically in the mouse hindbrain but significantly less efficiently when engrafted subcutaneously. Furthermore, immunocompromised and subcutaneous tumors revealed striking differences in mutational burden and clonal architecture, suggestive of nonautonomous immunoediting. Finally, anti-PD1 was sufficient to treat subcutaneously engrafted tumors in immunocompetent animals. This first mouse model of immunocompetent, hypermutant brain tumors can be used to uncover unique characteristics of RRD tumour evolution and allow for immune based therapeutic preclinical testing. Experiments to assess combinational ICIs and other therapeutic interventions in orthotopically transplanted tumors will also be presented.

TNFAIP8 inhibits gastric cancer proliferation by mTOR-Akt-ULK1 and autophagy signal pathway

Background: The purpose of this article was to study the role of TNFAIP8 in gastric cancer.Methods: RT-PCR was used to detect the expression of TNFAIP8 mRNA and protein level in normal gastric mucosa cells and four gastric cancer (GC) cell lines. TNFAIP8 was silenced or overexpressed in two cell lines, CCK-8 cell viability was used, transwell experiment was used to detect cell invasion capability, and flow cytometry was used to detect cell apoptosis. TNFAIP was silenced or overexpressed in a cell line, and nude mice were inoculated to form transplanted tumors. HE staining and immunohistochemistry staining were used to detect the histopathological changes of tumors. Results: The mRNA and protein expression of TNFAIP8 was significantly up-regulated in GC patients and cells. After silencing and overexpressing TNFAIP8, GC cells with high expression increased, apoptosis decreased, and cell invasion increased. Expression of mTOR-Akt-ULK1 signal pathway was inhibited and autophagy signal was activated. Conclusions: Our findings indicate that TNFAIP8 inhibited GC cells by inhibiting mTOR-Akt-ULK1 signal pathway and activating autophagy signal.