Figure 1.
CAPE-containing propolis augments the apoptotic effect of butyrate on CC cells.
(A). Representative western blot analysis of HCT-R cells exposed to mock (M), 5 mM butyrate (B), 1.2 µg/ml CAPE, butyrate and CAPE (BC), 100 µg/ml propolis (P), or butyrate and propolis (BP) for 19 h. (B, C) HCT-R (B) and HCT-116 (C) cells were exposed to mock (M), 5 mM butyrate (B), 1.2 µg/ml CAPE (C), butyrate and CAPE (BC), 100 µg/ml propolis (P), or butyrate and propolis (BP) for 50 h. Propolis preparation contains 0.3 µg CAPE per 100 µg powder. A minimum three independent experiments were carried out with triplicate samples each, values are mean ± SD.
Figure 2.
Role of JNK signaling in the apoptotic response of HCT-R cells to butyrate and propolis.
(A). A representative western blot analysis of HCT-116 and HCT-R cells exposed to mock (M), or 5 mM butyrate and 100 µg/ml propolis (BP) for 19 h. (B) A representative immunoblot analysis of HCT-R cells exposed to mock (M), or 5 mM butyrate and 100 µg/ml propolis (BP), 20 µM SP600125 (S) or SP600125 and butyrate/propolis (BPS) for 19 h. (C) Suppression of JNK signaling augments apoptosis of HCT-R cells exposed to butyrate and propolis. HCT-R cells were exposed for 50 h to mock (M), 20 µM SP600125 (S), the combination of 5 mM butyrate and 100 µg/ml propolis (BP), or SP600125, butyrate, and propolis (BPS). Apoptosis was measured by flow cytometry with PE Annexin V Apoptosis Detection Kit I (BD Biosciences, #559763). A minimum three independent experiments were carried out with triplicate samples each, values are mean ± SD. (D) A representative western blot analysis of HCT-R cells treated as in (B), and developed with antibodies to the phosphorylated forms of cJUN and STAT3.
Figure 3.
Role of cJUN in the apoptosis of butyrate/propolis-treated HCT-R cells.
(A) Silencing of cJUN expression via nucleofection of cJUN siRNA suppresses total and phosphorylated (p) cJUN levels in HCT-R cells, as ascertained by western blot analyses. (B) A representative apoptotic analysis of HCT-R cells that were nucleofected with control or cJUN siRNA. Cells were exposed to mock or butyrate/propolis treatment for 24 h, and analyzed at 48 h post-nucleofection. Each treatment was in triplicate, values are mean ± SD. (C) Detection of TAM67-GFP in HCT-116 (H) or HCT-R (R) cells transfected stably with control (c) or TAM67 (t) expression vector. (D) AP1 luciferase transcription assays of control (C) and TAM67 (T) cells exposed for 19 h to mock (m) or 5 mM butyrate and 100 µg/ml propolis (bp) treatment. (E) Apoptotic assays of control and TAM67-expressing HCT-116 cells exposed for 24 h to mock or 5 mM butyrate and 100 µg/ml propolis were carried out as described in Methods. Three independent experiments were carried out with triplicate samples each, values are mean ± SD. (F) Expression of TAM67-GFP, endogenous cJUN, and beta-catenin in nuclear lysates of HCT-116 cells transfected with GFP (control) or TAM67-GFP vector. Cells were exposed to butyrate/propolis as in Fig. 1A for 19 h. TAM67 and endogenous cJUN were visualized with an antibody to the DNA-binding domain of c-JUN (sc-44, Santa Cruz Biotechnology). (G) Immunoprecipitations with nuclear lysates described in (F) with a control (IgG) antibody or a cJUN antibody that recognizes the amino-terminal domain of the endogenous cJUN protein (sc-45, Santa Cruz Biotechnology). Beta-catenin that co-immunoprecipitates with endogenous cJUN was labeled with an antibody from Santa Cruz Biotechnology (sc-53483). (H,I) Luciferase transcription assays of control (C) and TAM67 (T) cells exposed for 19 h to mock (m) or 5 mM butyrate and 100 µg/ml propolis (bp) were carried out as described in Methods. Three independent experiments were carried out with duplicate samples each, values are mean ± SD.
Figure 4.
Role of JAK/STAT signaling in the apoptosis of butyrate/propolis-treated HCT-R cells.
(A). A representative western blot analysis of HCT-R cells exposed to mock (M), 5 mM butyrate and 100 µg/ml propolis treatment (BP), 5 mM butyrate and 1.2 µg/ml CAPE (BC) for 19 h. (B). A representative immunoblot of HCT-R cells exposed for 19 h to mock (M), 1 µM JAK inhibitor pyridone 6 (Ji), 5 mM butyrate and 100 µg/ml propolis treatment (BP), the combination of JAK inhibitor, butyrate, and propolis (Ji/BP), 5 mM butyrate and 1.2 µg/ml CAPE (BC), and the combination of JAK inhibitor and butyrate/CAPE (Ji/BC). (C and D) Apoptotic analyses of HCT-R cells exposed for 35 hours to (M), 5 mM butyrate and 100 µg/ml propolis treatment (BP), 1 µM JAK inhibitor (Ji), 5 mM butyrate and 1.2 µg/ml CAPE (BC), JAK inhibitor and butyrate/propolis (Ji/BP), or JAK inhibitor and butyrate/CAPE (Ji/BC). A minimum of three independent experiments were carried out with triplicate samples each, values are mean ± SD.
Figure 5.
Compensatory survival signaling in apoptotic CC cell populations.
Butyrate hyperinduces WNT/beta-catenin activity in CC cells with mutations in the pathway by increasing the formation of beta-catenin/TCF4 (BCT) complexes [1], [2]. High levels of BCT complexes induce directly pro-apoptotic genes such as BAX and BOK [47]. However, due to signaling heterogeneity, only a fraction of all cells hyperactivates the pathway and undergo apoptosis within 24 hours of exposure to butyrate [1]. Similar to cells undergoing compensatory proliferation [13], [15], apoptotic CC cell populations increase the expression of TGFbeta, WNT5A, and WNT11 [3], [16], and these molecules activate pro-survival pathways, such as JNK and AKT signaling [3], (Figs. 1,2). JNKs activate cJUN and JAK/STAT signaling [22], [23]. Inhibition of JNK augments apoptosis induced by butyrate/propolis treatment (Fig. 2C), as does the inhibition of its downstream target, the JAK/STAT pathway (Fig. 4). The role of pcJUN in the apoptotic response of CC cells to butyrate/propolis exposure is complex: at relatively lower levels, cJUN may contribute to proliferation; whereas, when hyperinduced together with beta-catenin/TCF complexes it may induce apoptosis. Asterisks mark the pathways inhibited by propolis.