At the end of the experiments, the mean tumor quantities were significantly smaller in lenvatinib and losartan-treated mice than in those treated by either single agent
At the end of the experiments, the mean tumor quantities were significantly smaller in lenvatinib and losartan-treated mice than in those treated by either single agent. AT-II-stimulated production of vascular endothelial growth factor-A (VEGF-A) and interleukin-8 and suppressed lenvatinib-mediated autocrine VEGF-A production in HCC cells. Moreover, it directly inhibited VEGF-mediated endothelial cell growth. Notably, the combination of lenvatinib and losartan augmented the cytostatic and angiostatic effects of the former at a low-dose, reaching those accomplished with a conventional dose. Correspondingly, a HCC tumor xenograft assay showed that the oral administration of losartan combined with lenvatinib reduced the subcutaneous tumor burden and intratumor vascularization in BALB/c nude mice. These findings support that this regimen could be a viable option for individuals intolerant to standard lenvatinib dose. = 10 in each group): vehicle, Los (losartan, 30?mg/kg), Lv-LD (lenvatinib, 3?mg/kg), Lv-HD (lenvatinib, 10?mg/kg), Lv-LD (3?mg/kg) + Los (30?mg/kg), and Lv-HD (10?mg/kg) + Los (30?mg/kg), and were administered treatment through daily dental gavage while monitoring their tumor quantities . The dosages of losartan and lenvatinib for mice were defined relating to earlier reports [18,23,24]. All mice were sacrificed 21 days after administration and their subcutaneous tumors were then collected. Serum biological markers were measured using routine laboratory methods. All animal methods complied with the recommendations of the Guideline for Care and Use of Laboratory Animals (National Study Council of Japan), and the study was authorized by the ethics committee of Nara Medical University or college, Kashihara, Japan (Authorization No. 12767). 2.8. Histological and Immunohistochemical Analyses Resected tumor cells were fixed over night at 4 C in 10% formalin and inlayed in paraffin. Subsequently, 5 m paraffin sections were regularly stained with hematoxylin and eosin. Immunohistochemical analyses were also performed using paraffin-embedded tumor sections as explained . As the primary antibodies, rabbit anti-Ki67 (Abcam, Cambridge, England; 1:100 dilution) and rabbit anti-CD34 (Abcam; 1:2500 dilution) were used, with staining performed according to the suppliers recommendations. TdT-mediated dUTP Nick End Labeling (TUNEL)-positive cells in subcutaneous tumor sections were detected by using an In-Situ Cell Death Detection Kit (SigmaCAldrich, St. Louis, MO, USA), as recommended for tissue sections from the supplier. Ki67-positive cells and TUNEL-positive cells were counted in high-power fields (HPF) at 400-fold magnification. MIM1 CD34-positive areas were quantified in HPFs at 400-fold magnification using the ImageJ software. All quantitative analyses were performed for five fields per each section. 2.9. Measurement of AT1R Protein Levels After equalizing the protein concentration from freezing subcutaneous tumor samples to 5 mg/mL, AT1R protein levels were measured using the Human being Angiotensin II Receptor 1 ELISA Kit (MyBioSource, Inc., San Diego, CA, USA) MIM1 according to the manufacturers instructions. Quantitative ideals were relatively indicated as fold switch to the value of total protein from human being adult liver cells (BioChain Institute Inc., Newark, CA, USA). 2.10. Statistical Analyses Statistical analyses were performed using Prism, version 9 (GraphPad Software, La Jolla, CA, USA). Data are indicated as the mean standard deviation. Statistical variance between each experimental group was analyzed using an analysis of variance test. Bartletts test was used to determine the homogeneity of variances. All checks were two-tailed and and improved pro-apoptotic Bcl-2-connected X protein (manifestation). (Number 1F). Open in a separate windows Number 1 In vitro cytostatic effects of lenvatinib and losartan on liver malignancy cells. (A) Cell proliferation of human being liver malignancy cells (Huh-7, HLE, and JHH-6) incubated with lenvatinib (0C10-5 M) for 0C6 days. (B) Cell proliferation of human being liver malignancy cells pre-treated with different concentrations of angiotensin-II (AT-II) (0C1 M) for 12 h and consequently treated with losartan (Los) (1 M) for 12 MIM1 h. (CCF) Cell proliferation of human being liver malignancy cells (C), relative mRNA expression levels of cell cycle-related markers in Huh-7 (D), the levels of cleaved caspase-3 in human being liver cancer cells tradition extract assessed by ELISA (E), relative mRNA expression levels of apoptosis-related markers in Huh-7 (F), cells were pre-treated with AT-II (1 Rabbit Polyclonal to HCFC1 M) for 12 h and consequently treated with Los (1 M) and lenvatinib (Lv) (1 or 3 M) for 12 h. The mRNA manifestation levels were measured by qRT-PCR, and was used as MIM1 internal control (D and F). Quantitative ideals are relatively indicated as fold changes to the ideals of (A) group at the start of treatment with lenvatinib in each dose, (B) group MIM1 of AT-II (0.