Abstract:

Objective To explore the anti-tumor effect of atractylenolide Ⅱ （ATL-Ⅱ） on colon cancer and its immunomodulatory mechanism. Methods A subcutaneous xenograft model of colon cancer was constructed using C57BL/6 mice. The mice were randomly divided into the model group （intraperitoneal injection of PBS）， ATL-Ⅱ low-dose （20 mg/kg） group， medium-dose （40 mg/kg） group， high-dose （60 mg/kg） group， as well as the 5-fluorouracil （5-FU 30 mg/kg） group， with 5 mice in each group， and the administration lasted for 21 days. HE staining was performed to detect the histopathological changes in the tumor tissues. The levels of expressions of Ki-67， Caspase-3， and Bcl-2 were detected by immunohistochemistry. The rates of CD8⁺ T and NK1.1 positive cells in tumor tissues were detected by immunofluorescence. The serum levels of granzyme B （GzmB） and interferon-λ （IFN-λ） were measured by ELISA. The levels of expressions of PD-L1 and proteins involved in the ERK/MAPK signaling pathway were analyzed by Western blotting. Results The tumor volumes of colon cancer xenograft mice in the model group， ATL -Ⅱ low-， medium-， high-dose groups and the 5-FU group were （1 845.17±65.72） ， （1 637.20±122.65）， （1 232.86±209.16）， （1 002.29±41.84）， and （911.59±294.71） mm³， respectively， with a statistically significant difference （F=125.61， P<0.001）. Compared with the model group， there were statistically significant differences in the ATL-Ⅱ low-， medium-， high-dose groups and the 5-FU group （all P<0.05）. Compared with the low-dose group， there were statistically significant differences in the ATL-Ⅱ medium-， high-dose groups and the 5-FU group （all P<0.05）， with a statistically significant difference also observed between the medium- and high-dose groups （P<0.05）. Compared with the ATL-Ⅱ medium- and high-dose groups， the 5-FU group demonstrated statistically significant differences （both P<0.05）. The mass of tumors of the five groups was （1.34±0.11）， （1.26±0.09）， （0.93±0.07）， （0.94±0.10）， and （0.59±0.08） g， respectively， with a statistically significant difference （F=88.88， P<0.001）. Compared with the model group， there were statistically significant differences in the ATL-Ⅱ medium-， high-dose groups and the 5-FU group （all P<0.05）. Compared with the low-dose group， there were statistically significant differences in the ATL-Ⅱ medium-， high-dose groups and the 5-FU group （all P<0.05）. Compared with the ATL-Ⅱ medium- and high-dose groups， there were statistically significant differences in the 5-FU group （both P<0.05）. The spleen indexes of the five groups were 7.42±0.88， 7.38±1.32， 8.42±0.78， 9.72±1.18， and 6.16±1.05， respectively， with a statistically significant difference （F=33.20， P<0.001）. Compared with the model group， there were statistically significant differences in the ATL-Ⅱ medium- and high-dose groups and the 5-FU group （all P<0.05）. Compared with the low-dose group， there were statistically significant differences in the ATL-Ⅱ medium-， high-dose groups and the 5-FU group （all P<0.05）， and the spleen index in the high-dose group was higher than that in the medium-dose group （P<0.05）. Compared with the ATL-Ⅱ medium- and high-dose groups， there were statistically significant differences in the 5-FU group （both P<0.05）. HE staining showed that， the tumor tissues of mice in the model group exhibited typical malignant tumor characteristics， including cellular density， large deeply stained nuclei， atypia， and reduced intercellular matrix. In tumor tissues of mice treated with ATL-Ⅱ and 5-FU， significantly reduced cell proliferation activity， loosely arranged cells， reduced mitotic activity， and markedly reduced necrotic areas were observed. In the ATL-Ⅱ medium-， high-dose groups and the 5-FU group， relatively small round or oval cells with large nuclei and deeply stained chromatin were observed. There were statistically significant differences in the percentage of positive areas for Ki-67， Caspase-3， and Bcl-2 among the five groups （F=13.86， P=0.043； F=477.63， P<0.001； F=40.48， P<0.001）. Compared with the model group， there were statistically significant differences in the ATL-Ⅱ medium-， high-dose groups and the 5-FU group （all P<0.05）. Compared with the low-dose group， there were statistically significant differences in the ATL-Ⅱ high-dose groups and the 5-FU group （both P<0.05）. Compared with the medium- and high-dose ATL-Ⅱ groups， there were statistically significant differences in the proportion of Caspase-3 positive areas in the 5-FU group （both P<0.05）. There was a statistically significant difference between the medium- and high-dose groups （P<0.05）. The positive cell rates for CD8⁺ T cells in the tumor tissues of the five groups were （10.33±3.53）%， （15.00±5.65）%， （30.33±10.51）%， （59.33±9.04）%， and （33.62±9.11）%， respectively， with a statistically significant difference （F=96.33， P<0.001）. Compared with the model group， there were statistically significant differences in the ATL-Ⅱ low-， medium-， high-dose groups and the 5-FU group （all P<0.05）. The ATL-Ⅱ medium-， high-dose groups and the 5-FU group were significantly different from the low-dose group （all P<0.05）， and a statistically significant difference was found between the medium- and high-dose groups （P<0.05）. The 5-FU group was significantly different from the ATL-Ⅱ high-dose group （P<0.05）. The positive cell rates for NK1.1 cells in the tumor tissues of the five groups were （12.33±6.52）%， （13.00±7.00）%， （35.33±9.51）%， （43.67±12.21）%， and （14.50±7.05）%， respectively， with a statistically significant difference （F=283.17， P<0.001）. The ATL-Ⅱ medium-， high-dose ATL-II groups and the 5-FU group showed statistically significant differences compared to the model group （all P<0.05）. The ATL-Ⅱ high-dose group was significantly different from the low-dose group （P<0.05）. The 5-FU group was significantly different from the ATL-Ⅱ medium- and high-dose groups （both P<0.05）. The serum GzmB levels in the five groups were （5.00±1.00）， （5.27±0.76）， （8.27±0.61）， （10.00±1.21）， （6.15±0.69） ng/L， respectively， with a statistically significant difference （F=21.45， P<0.001）. The ATL-Ⅱ medium-， high-dose groups and the 5-FU group showed statistically significant differences compared to the model group （all P<0.05）. The ATL-Ⅱ medium- and high-dose groups were significantly different from the low-dose group （both P<0.05）， and a statistically significant difference was found between the medium- and high-dose groups （P<0.05）. The 5-FU group were significantly differences from the ATL-Ⅱ medium- and high-dose groups （both P<0.05）. The serum IFN-λ levels in the five groups were （617.33±65.06）， （743.33±40.41）， （910.00±36.06）， （1 009.00±35.54）， （703.62±56.00） ng/L， respectively， with a statistically significant difference （F=43.08， P<0.001）. The ATL-Ⅱ low-， medium-， and high-dose groups showed statistically significant differences compared to the model group （all P<0.05）. The ATL-Ⅱ medium- and high-dose groups were significantly differences from the low-dose group （both P<0.05）， and a statistically significant difference was found between the medium- and high-dose groups （P<0.05）. The 5-FU group was significantly different from the ATL-Ⅱ medium- and high-dose groups （both P<0.05）. There were statistically significant differences in the expression levels of PD-L1， p-ERK/ERK， and p-MEK/MEK proteins among the five groups of tumor tissues （F=125.34， P<0.001； F=89.63， P<0.001； F=35.33， P=0.002）. Statistically significant differences in PD-L1 expression levels were found among the low-， medium-， high-dose ATL-Ⅱ groups and the 5-FU group compared to the model group （all P<0.05）. The ATL-Ⅱ high-dose group and the 5-FU group showed statistically significant differences compared to the low-dose group （both P<0.05）. A statistically significant difference was found between the ATL-Ⅱ medium- and high-dose groups （P<0.05）. The ATL-Ⅱ medium-dose group was significantly different from the 5-FU group （P<0.05）. Statistically significant differences in p-ERK/ERK expression levels were observed among the ATL-Ⅱ low-， medium-， high-dose groups and the 5-FU group compared to the model group （all P<0.05）. The ATL-Ⅱ medium-， high-dose groups and the 5-FU group showed statistically significant differences compared to the low-dose group （all P<0.05）. The 5-FU group was significantly different from the ATL-Ⅱ medium- and high-dose groups （both P<0.05）. Statistically significant differences in p-MEK/MEK expression levels were found among the ATL-Ⅱ low-， medium-， high-dose groups and the 5-FU group compared to the model group （all P<0.05）. The 5-FU group was significantly different from the low-dose group （P<0.05）. Conclusions Atractylenolide Ⅱ inhibits the activity of the ERK/MAPK signaling pathway， reduces the expression of PD-L1， enhances the infiltration of CD8⁺ T cells and NK cells， and promotes tumor cell apoptosis， thereby it can exert an anti-cancer effect on colon cancer.

Key words: Colonic neoplasms, Atractylenolide Ⅱ, Neoplasm invasiveness, Apoptosis, B7-H1 antigen