ANXA11 regulates the tumorigenesis, lymph node metastasis and 5-fluorouracil sensitivity of murine hepatocarcinoma Hca-P cells by targeting c-Jun

Abstract

Annexin A11 (Anxa11) is associated with various cancers. Using a pair of syngeneic murine hepatocarcinoma cells, Hca-P with ~25% and Hca-F with ~75% lymph node metastatic (LNM) potentials, we demonstrated Anxa11 involvement in hepatocarcinoma lymphatic metastasis. Here, ANXA11 acted as a suppressor for the tumorigenicity, LNM and 5-FU resistance of Hca-P via c-Jun. We constructed monoclonal Hca-P cell line with stable ANXA11 knockdown. Although Bax and Bcl-2 levels increased in shRNA-Anxa11-transfected Hca-P, ANXA11 downregulation showed no clear effect on Hca-P apoptosis. ANXA11 downregulation promoted in vitro migration and invasion capacities of Hca-P. In situ adhesion potential of Hca-P cells toward LN was significantly enhanced following ANXA11 downregulation. Consistently, ANXA11 downregulation promoted the in vivo tumor growth and LNM capacities of Hca-P cells. ANXA11 knockdown enhanced the chemoresistance of Hca-P cells specifically toward 5-FU instead of cisplatin. Its downregulation increased c-Jun (pSer73) and decreased c-Jun (pSer243) levels in Hca-P. c-Jun (pSer243) downregulation seemed to be only correlated with ANXA11 knockdown without the connection to 5-FU treatment. Interestingly, compared with scramble-Hca-P cells, the levels of c-Jun and c-Jun (pSer73) in shRNA-Anxa11-Hca-P cells were upregulated in the presences of 0.1 and 1.0 mg/L 5-FU. The levels changes from c-Jun and c-Jun (pSer73) in Hca-P cells showed a more obvious tendency with the combination of ANXA11 knockdown and 5-FU treatment. ANXA11 level regulates LNM and 5-FU resistance of Hca-P via c-Jun pathway. It might play an important role in hepatocarcinoma cell malignancy and be a therapeutic target for hepatocarcinoma.

Keywords: Anxa11; c-Jun; chemoresistance; hepatocarcinoma; lymphatic metastasis.

Figure 1. Anxa11 knockdown by RNAi

A. Relative Anxa11 mRNA levels in Hca-P, shAnxa11- Hca-P and scramble-Hca-P cells were determined by qRT-PCR using GAPDH as internal reference. B. WB assay of ANXA11 levels in Hca-P, shAnxa11-Hca-P and scramble-Hca-P cells. GAPDH was the internal reference. Triplicate independent measurements were performed for WB assays. No statistical significances for the differences between Hca-P and scramble-Hca-P cells at both mRNA and protein levels for Anxa11. ** Refers to the difference is of statistical significance (P < 0.01).

Figure 2. Anxa11 knockdown on Hca-P apoptosis

A. Flow cytometry showed Anxa11 level had no clear effect on Hca-P apoptosis (P>0.05). B. WB assay of the levels of Bax, Bcl-2, PARP and cleaved PARP in shAnxa11-Hca-P and scramble-Hca-P cells. Anxa11 knockdown shows no clear effect on the levels of PARP and cleaved PARP. Bax (** P <0.01) and Bcl-2 (* P<0.05) were up-regulated with statistical significances in shAnxa11-Hca-P compared with scramble-Hca-P cells. Anxa11downregulation exhibited no influence on Bax/Bcl-2 ratio (P>0.05). Three independent measurements were performed for each protein molecule.

Figure 3. Influence of Anxa11 downregulation on in vitro migration, invasion and in situ LN adhesion potentials of Hca-P cells

A. and B. Anxa11 downregulation significantly enhanced the migration ability A1. and invasion capacity A2. of Hca-P cells, **P<0.01. C. and D. Anxa11 downregulation significantly promoted in situ inguinal and axillary LNs adhesion capacities of Hca-P cells, **P<0.01. Three independent measurements were performed for migration, invasion and LN adhesion assays.

Figure 4. Anxa11 knockdown on tumorigenicity and LNM capacity for Hca-P cells

A. shAnxa11-Hca-P-transplaneted mice showed increased tumor growth speed than scramble-Hca-P-transplanted ones. Results were represented as the mean tumor volume ± SD of each group. The tumor volume differences were with P<0.05 on the 11^th and 15^th days, and with P<0.01 on the 18^th and 21^st days after cell inoculation. B. Images of the primary tumors formed on the footpads of mice on the 21^st day after cell inoculations. C. Photos of inguinal and axillary LNs from shAnxa11-Hca-P-tanspanted mice and scramble-Hca-P-transplanted mice taken at the magnifications of 200× (left) and 400× (right).

Figure 5. Anxa11 knockdown influence on Hca-P cell drug sensitivity to 5-FU and cisplatin

Dose-response curves for shAnxa11-Hca-P and scramble-Hca-P cells to 5-FU A. and cisplatin B. treatments. Cell viability was determined by CCK-8 assay. The viability differences between the two cell lines at 0.1, 1.0 and 10 mg/L 5-FU administrations were of statistical significances (P<0.01). C. Triplicate WB assays of the levels of cleaved PARP with the treatments of different concentrations of 5-FU in shAnxa11-Hca-P and scramble-Hca -P cells. D. Hoechst 33258 staining assay for shAnxa11-Hca-P and scramble-Hca-P cells with the treatments of different concentrations of 5-FU.

Figure 6. ANXA11 is linked to c-Jun pathway on Hca-P sensitivity to 5-FU

A. WB images of c-Jun, c-Jun (pSer73) and c-Jun (pSer243) in shAnxa11-Hca-P and scramble-Hca-P in 0, 0.01, 0.1 and 1.0 mg/L 5-FU. B. Quantified relative levels of c-Jun, c-Jun (pSer73) and c-Jun (pSer243) in shAnxa11-Hca-P vs scramble-Hca-P cells. Triplicate independent experiemnts were performed for each assay. * and ** refer to the differences are in statistical significances with P<0.05 and 0.01, respectively.