Fig 2: (-)-Oleocanthal treatment suppresses tumor growth in human tumor xenograft model.MDA-MB-231/GFP human breast cancer cells were cultured and resuspended in serum-free DMEM medium (20 µl). After anesthesia, cell suspensions (1×106 cells/20 µl) were inoculated subcutaneously into the second mammary gland fat pad just beneath the nipple of each athymic nude mouse to generate orthotopic breast tumors. At 48 h post-inoculation, the mice were randomly divided into two groups: i) the vehicle-treated control group (n = 5), ii) the (-)-oleocanthal-treated group (n = 5). Treatment (3X/week) started 5 days postinoculation with intraperitoneal (i.p.) administered vehicle control or 5 mg/kg (-)-oleocanthal. (A) Left panel; tumor size was evaluated periodically during treatment at indicated days postinoculation. Tumor volume (V) was calculated by V = L/2 x W2, where L was the length and W was the width of tumors. Points, mean of tumor volume in mm3 of several tumors (n = 5) during the course of the treatment period; bars ± SEM. *P<0.05 as compared to vehicle-treated control. Right panel; shown are two mice harboring human breast cancer. The mouse on the right shows suppression of tumor growth with (-)-oleocanthal treatment (5 mg/kg/day) compared to vehicle treated control mouse on the left. (B) No significant change in body weight was observed among treated animals, indicating the safety of (-)-oleocanthal treatment. Error bars indicate SEM for n = 5. (C) Vertical bars indicate mean tumor weight at the end of the experiment (left panel). *P<0.05 compared to vehicle-treated controls. Right panel shows primary breast tumors from mice with vehicle-treated cancer (left), and cancer treated with (-)-oleocanthal (5 mg/kg/day) (right). (D) Protein expression of c-Met, phospho-c-Met and Cleaved PARP in breast tumors detected by Western blot. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs beside their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane ± SEM, *P<0.05 as compared with vehicle-treated controls. (E) Immunostaining of sections (left panel) obtained from vehicle-treated or (-)-oleocanthal-treated (5 mg/kg/day) mice against Ki-67 (mitosis marker), CD31 (endothelial marker). Right panel shows quantification of Ki-67 positive cells and microvessel density (MVD). Ki-67+ cells in breast cancer tissues were examined in 5 areas at a magnification of ×200. Microvessel density (MVD) of breast tumor tissue sections was evaluated. Any CD31+stained endothelial cell or endothelial cell cluster was counted as one microvessel. The mean microvessel count of the five most vascular areas was taken as the MVD, which was expressed as the absolute number of microvessels per 1.485 mm2 (×200 field). Vertical indicate the average of 5 readings ± SEM, *P<0.05 as compared with vehicle-treated controls.

Fig 3: VNP20009 inhibited tumor growth and induced tumor apoptosis in vivo. (A) Representative immunohistochemical microphotographs of Ki67-stained xenograft CFPAC-1 pancreatic tumor sections. Proliferative nuclei were stained brown by Ki67. Scale bars, 50 µm. (B) Quantified data were determined by the number of Ki67-positive cells dividing the total number of cells in 5 randomly selected fields under light microscopy. (C) Western bolt analyses of Bax and caspase-3 in the tumors at the end of treatment of a single intratumoral injection of 2 × 106 cfu of VNP20009-luc or PBS. The expression of (D) Bax and (E) Cleaved caspase-3 were quantified and compared. n = 5 mice per group. **P < 0.01, ***P < 0.001. Bars, mean ± SEM. VNP-luc is abbreviation for VNP20009-luc.

Fig 4: HOMER3-AS1 promoted HCC tumor progression in vivo.a Bioluminescence imaging of liver tumors in mice at day 14 after inoculation with small pieces of subcutaneous tumors formed by luciferase labelled SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. b Bioluminescence imaging of liver tumors in mice at day 14 after inoculation with small pieces of subcutaneous tumors formed by luciferase labelled SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. c Ki67 IHC staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. Scale bars, 50 µm. d Ki67 IHC staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. Scale bars, 50 µm. e Cleaved caspase-3 IHC staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. Scale bars, 50 µm. f Cleaved caspase-3 IHC staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. Scale bars, 50 µm. g F4/80 IF staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. Scale bars, 50 µm. h F4/80 IF staining of liver tumors formed by SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. Scale bars, 50 µm. Results are presented as mean ± SD based on n = 5 mice in each group. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test (a, c, e, g) or one-way ANOVA followed by Dunnett’s multiple comparisons test (b, d, f, h).

Fig 5: PD-CDDP treatment effectively delayed the proliferation of MDA-MB-231 xenografts in vivo. a Sample dosing schedule for PD and CDDP. b Randomly grouped nude mice bearing MDA-MB-231 tumours were treated with vehicle (PBS), PD (150 mg/kg), CDDP (5 mg/kg), or PD-CDDP treatment for three cycles. Tumour volume was determined every 3 days after the onset of treatment. c Harvested tumours were subsequently collected, and representative immunohistochemistry results show Ki-67 expression in MDA-MB-231 tumours. The percentage of Ki-67-positive tumour cells and their staining intensities were assessed by ImageJ