Ocoxin® oral solution slows down tumor growth in an experimental model of colorectal cancer metastasis to the liver in Balb/c mice

Abstract

Liver metastatic disease is the main cause of death in colorectal cancer (CRC) patients. During metastatic spread of the disease an imbalance in the oxidative stress and inflammation plays a crucial role in tumor progression. In order to improve the efficacy of current therapies, new complementary therapeutic approaches are being analyzed including biologically active compounds with low side effects. The anti-inflammatory and anti-oxidant properties of Ocoxin® oral solution (OOS) prompt us to analyze its effect on the metastatic development of CRC to the liver. First, in vitro effect of OOS in tumor cell viability and migration was analyzed. Second, in vivo effect of different dosage patterns and concentrations in the development of hepatic metastasis was analyzed by intra-splenic inoculation of C26 colon carcinoma cells in Balb/c mice. Third, the expression of alpha smooth muscle actin, caspase-3 and Ki-67 expression was quantified by immunohistochemistry, then gene expression levels of inflammatory factors were measured by quantitative RT-PCR. According to our results, OOS reduced tumor cell viability and migration in vitro. Moreover, in vivo daily administration of OOS from the 7th day after tumor cell inoculation decreased the total area and size of metastatic foci in the liver. Furthermore, cell proliferation and fibroblast recruitment was decreased in tumor foci while a higher number of apoptotic cells were observed. Finally, RNA levels for the inflammatory mediators COX-2, IFNγ, IL1β, IL6 and TNFα were reduced in total liver. In conclusion, OOS reduced the metastatic development of colorectal cancer to the liver by increasing apoptosis, and decreasing tumor cell proliferation and fibroblast recruitment in the tumor foci, as well as the expression of inflammatory mediators in total liver. These results point out OOS as a potential supplement to be applied as complementary therapy for the treatment of liver metastasis from colorectal cancer.

Figures

Figure 1

Diagram of the experimental animal groups according to OOS dosage pattern. Animals were divided into 3 experimental groups as follows: group I, OOS untreated group; group II, mice treated with OOS 4 days prior to tumor cell inoculation; group III, mice treated with OOS from the 7th day after tumor cell inoculation. All treated groups were administered a daily dose during the period of treatment.

Figure 2

OOS reduces in vitro viability of C26 cells. (A) The viability of C26 cells was tested after 24-h incubation in the presence of OOS. C26 cells were treated with increasing concentrations of OOS ranging from 1:200 to 1:50 during 24 h before viability quantification and compared to the viability of initially cultures cells (light grey) and to that of untreated cells (black). Differences in the viability of treated cells vs. Untreated cells (*) and vs. initially cultured cells (**) were considered to be statistically significant at P<0.05. (B) The cell number in each cell phase respect to total gated cell number was analyzed after PI staining by flow cytometry after 72-h incubation with 1:100 OOS (V/Vf). Differences between treated and untreated cells were considered statistically significant at *P<0.05.

Figure 3

OOS reduces in vitro migration of C26 cells. C26 cells were cultured on top of 8 µm-pore membrane modified Boyden chambers. The number of migrated C26 cells were quantified after 18-h incubation in the presence of 1:100 OOS (V/Vf) Data are mean values ± SD from three different experiments. Differences were considered statistically significant at *P<0.05.

Figure 4

OOS slows down in vivo tumor growth. (A) C26 cells were i.s. inoculated and mice were treated with 100 µl of OOS under the administration patterns described in Materials and methods. Group I, untreated; group II, OOS administered four days prior to tumor cell inoculation; group III, OOS administered from day 7th after tumor cell inoculation until sacrifice. (B) Mice were treated with 50 and 100 µl of OOS under administration pattern of group III. (C) The amount of liver foci developed in the liver of mice untreated or treated with 50 and 100 µl of OOS was quantified in liver tissue based on tumor foci size. Image original magnification, ×20. Differences were considered statistically significant at *P<0.05.

Figure 5

OOS affects proliferation and apoptosis in vivo. The effect of OOS on Ki-67 and caspase-3 expression in livers from tumor-bearing mice was analyzed by immunohistochemistry. (A) Caspase-3 expression (brown) was quantified in livers collected from untreated, 50 and 100 µl OOS treated C26-bearing mice as the percentage of the area positive for caspase-3 expression respect to total liver section area. (B) Ki-67 expression (brown) was quantified in livers collected from untreated, 50 and 100 µl OOS treated C26-bearing mice as the percentage of the area positive for Ki-67 expression in respect to total liver section area. (C) The ratio between Ki-67 and caspase-3 was calculated as the expression of Ki-67 relative to caspase-3 expression. Image original magnification, ×20. Differences were considered statistically significant at *P<0.05.

Figure 6

In vivo HSC infiltration in the tumor is reduced by OOS. Expression levels of ASMA were analyzed in liver tissue by immunohistochemistry. ASMA was stained with specific antibodies in liver tissue collected from untreated, 50 and 100 µl OOS treated mice. Data are calculated as % of ASMA expression per tumor foci area. Image original magnification, ×20. Differences were considered statistically significant at *P<0.05.

Figure 7

OOS affects inflammatory gene expression in total liver. Gene expression of IFNγ, TNFα, IL1β, IL6 and COX-2 was analyzed by RT-PCR. Total RNA was extracted from paraffin-embedded liver tissue collected from untreated or 100 µl OOS treated C26-bearing mice. Data are expressed as the mean values ± SD from six livers from two independent experiments. Differences were considered statistically significant at *P<0.05.