Ocoxin oral solution® as a complement to irinotecan chemotherapy in the metastatic progression of colorectal cancer to the liver

Abstract

Colorectal cancer (CRC) is an aggressive disease in which patients usually die due to its metastatic progression to the liver. Up to date, irinotecan is one of the most used chemotherapeutic agents to treat CRC metastasis with demonstrated efficacy. However, the severity of the side effects constitute the main limitation to its use in the treatment. Consequently, new complementary therapies are being developed to avoid these adverse effects while maintaining the efficacy of the antitumoral drugs. Ocoxin oral solution (OOS®) is a nutritional mixture containing biologically active compounds with demonstrated antitumoral and immunomodulatory effects. Thus, we aimed to analyze the effect of OOS® as a suitable complement to irinotecan therapy in the treatment of CRC metastasis to the liver. First, the effect of OOS®, irinotecan and the combination of both on the viability of C26 cells was tested in vitro and in vivo. Second, the expression of caspase-3, Ki67 and the macrophage infiltration by F4/80 marker was quantified in liver tissue sections by immunohistochemistry. Finally, mRNA microarray study was carried out on tumor cells isolated from tumor-bearing livers collected from mice subjected to the above treatments. Our results show that OOS® administered as a complementary therapy to irinotecan reduced tumor cell viability in vitro. Moreover, irinotecan administered either alone or in combination with 100 µl OOS® from the 7th day after tumor cell inoculation decreased the metastatic growth in the liver. Besides, several genes with binding and catalytic activities showed to be deregulated by RNA microarray analysis. In conclusion, OOS®, when administered as a complement to irinotecan, reduced the metastatic development of colorectal cancer to the liver. Additionally, the overall health state of the animals improved. These results point out OOS® as a potential supplement to the anti-tumoral treatments used in clinical settings in patients suffering from disseminated colorectal cancer.

Keywords: colorectal cancer; liver; metastasis; nutrient mixture; ocoxin oral solution.

Figures

Figure 1.

Experimental animal groups treatment pattern. Seven days after tumor cell inoculation, at least 6 mice per group were divided into 5 experimental groups as follows: group I, untreated mice; group II, mice treated with a daily oral administration of 100 µl of OOS®; group III, mice treated with a i.p. dose of 20 µg/ml of irinotecan every two days; group IV, mice treated with both, a daily oral dose of 100 µl of OOS® and 20 µg/ml of irinotecan every two days; group V, untreated mice used to control the metastatic development. All mice were treated for a period of 14 days.

Figure 2.

Effect of the combined treatment of irinotecan and OOS® in the viability of C26 cells. The viability of C26 cells was tested in the presence of OOS® (1:100), irinotecan (50 µM) or the combination of OOS® and irinotecan (1:100 and 50 µM) for 48 h. Then, the viability was quantified in untreated (black) and treated (grey) cells. Data are mean values ± SD from three different experiments. 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.

Figure 3.

Effect of the combined treatment of irinotecan and OOS® in the development of CRC metastasis to the liver. C26 cells were i.s. inoculated into mice and seven days later they were divided into the five groups shown in Fig. 1 with at least 6 mice per group. Mice were either untreated or treated with OOS® (100 µl), irinotecan (20 mg/kg) or with the combined therapy (100 µl OOS® and 20 mg/kg irinotecan) as described in Material and Methods. (A) Images showing tumor foci grown in the liver of untreated and treated mice. Image original magnification was ×4. (B) The total tumoral burden was quantified and represented as the percentage of liver area occupied by the tumor. Differences were considered statistically significant at *P<0.05.

Figure 4.

Effect of the combined treatment of irinotecan and OOS® in the proliferation and apoptotic markers. The expression of Ki67 and caspase-3 in livers collected from tumor-bearing mice either untreated or treated with OOS® (100 µl), irinotecan (20 mg/kg) or the complementary therapy (100 µl OOS® and 20 mg/kg irinotecan) was analyzed by immunohistochemistry with at least 6 mice per group. (A) Ki67 expression was quantified in livers collected from untreated and treated C26-bearing mice as the percentage of the area positive for Ki67 expression within tumor foci respect to the total metastatic tumor area. (B) Caspase-3 expression was quantified in livers collected from untreated and treated C26-bearing mice as the percentage of the area positive for caspase-3 expression respect to the total metastatic tumor area. (C) The ratio between Ki67 and caspase-3 in livers was calculated from results shown in A and B. Differences were considered statistically significant at *P<0.05.

Figure 5.

Effect of the combined treatment of irinotecan and OOS® on the tumor infiltration of macrophages in vivo. Expression level of F4/80 was analyzed in liver tissue by immunohistochemistry. (A) Images showing F4/80 expression (brown) and hematoxylin (purple) in liver tissue collected from untreated and treated mice. Image magnification was ×20. (B) F4/80 expression was quantified in livers collected from untreated and treated C26-bearing mice. Data are calculated as % of F4/80 expression per tumor foci area. At least 6 mice per group were used and differences were considered statistically significant at *P<0.05.

Figure 6.

Metastatic tumor explant based mRNA microarray study. A microarray assay was carried out to detect differences in gene expression levels between tumor cells collected from mice treated under different protocols. Four tumor explants per experimental group were collected, each of them from one tumor bearing mouse. (A) A molecular functional gene analysis was carried out with the PANTHER (v.10.0) (9,10) analysis system for the untreated Control vs. irinotecan treatments. (B) A molecular functional gene analysis was carried out with the PANTHER (v.10.0) (9,10) analysis system for the OOS®+irinotecan vs. irinotecan treatments.