Cell Death by Gallotannin Is Associated with Inhibition of the JAK/STAT Pathway in Human Colon Cancer Cells

Abstract

Background: Gallotannin (GT) is a polyphenol that possesses interesting anticancer properties. However, the mechanisms underlying its antitumor effects have not been well defined.

Objective: This study was designed to clarify the mechanisms underlying GT antitumor effects in colon cancer cell lines.

Methods: Three isogenic HCT116 cell lines (p53^+/+, p53^-/-, and p21^-/-) were treated with GT for different time points then Western blot, flow cytometry, and senescence analysis were performed to examine the effect of GT on Mitogen-activated protein kinase (MAPK) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) effectors, STAT3 downstream apoptotic targets, Sub-G1 phase, and programmed cell death induction. Transfection using Invitrogen Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, Massachusetts) were used to identify the role of p53 and p21 in the p53^-/- and p21^-/- cell lines.

Results: Both low and high GT concentrations caused MAPKs activation marked by upregulation of extracellular signal-regulated kinase (p-ERK). The preincubation with the antioxidant Tiron (Sigma-Aldrich, St Louis, Missouri) showed that GT's antitumor effects were not mediated by reactive oxygen species. We then examined the effect of GT on the JAK/STAT pathway, which is known to be activated in colorectal cancer. GT totally inhibited the JAK/STAT pathway effectors JAK2, STAT1, and STAT3 and their downstream apoptotic regulators B-cell lymphoma-extra large (Bcl-x_L) and c-Myc in all 3 cell lines. HCT116 cancer cells exhibited differential sensitivity to GT with p21^-/- cells being the most sensitive and p53^+/+ cells that express p21 protein being the least sensitive. In p53^+/+ cells, GT induced senescence, whereas in p53^-/- and p21^-/- cells, GT induced apoptosis in a caspase independent manner marked by Poly(ADP-Ribose) Polymerase (PARP) cleavage, Bcl-2 downregulation, and upregulation of the Bcl-2 associated X (Bax) to B-cell lymphoma 2 (Bcl-2) ratio. In addition, the sub-G1 phase exceeded 50% in p21^-/- cells.

Conclusions: Considered together, our results indicate that GT is potent inhibitor of the JAK/STAT pathway in colon cancer irrespective of the p53 and p21 status, which provides insights into its mechanism of anticancer activities and future potential for clinical translation. (Curr Ther Res Clin Exp. 2020; 81:XXX-XXX).

Keywords: Apoptosis; JAK/STAT; caspase; colon cancer; gallotannin; senescence.

Graphical abstract

Fig. 1

(A) Treatment of HCT116 (p53^+/+, p53^−/−, and p21^−/−) cells with gallotannin (GT) did not significantly modulate the levels of MAPK proteins except for a slight increase in the levels of p-ERK (Tyr 204). In HCT116 (p53^−/−) cells p-JNK increased 15 hours posttreatment. The cells were treated at 50% confluency with 40 µg/mL GT for 6, 15, 24, 48, and 72 hours. Whole-cell lysates were then immunoblotted with p-ERK (Tyr 204), p-JNK (Thr 183, Tyr 185), and p-p38 (Tyr 182) antibodies. The membranes were also probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody to ensure equal loading. (B) Treatment of HCT116 (p53^+/+, p53^−/−, and p21^−/−) cells with GT increased the levels of p-ERK (Tyr 204) proteins. The cells were treated at 50% confluency with 20 µg/mL GT for 6, 15, 24, 48, and 72 hours. Whole-cell lysates were then immunoblotted with p-ERK (Tyr 204), p-JNK (Thr 183, Tyr 185), and p-p38 (Tyr 182) antibodies. The membranes were also probed with GAPDH antibody to ensure equal loading.

Fig. 2

(A) Gallotannin (GT) treatment reduces the proliferation of HCT116 (p53^+/+, p53^−/−, and p21^−/−) cells and Tiron addition does not counteract this antiproliferative activity. Cells were treated at 60% confluency with either 1 or 2 mM Tiron for 1.5 hours followed by addition of GT (20, 40, or 60 µg/mL) for 48 hours. Cell proliferation was determined by the Cell Titer96 nonradioactive cell proliferation assay as described in the Materials and Methods section. Each value is the mean (SD) of 3 separate experiments each done in triplicates. *P < 0.05 and **P < 0.01 defined the statistical significance from control using 1-way ANOVA test. (B) Treatment of HCT-116 (p53^+/+, p53^−/−, and p21^−/−) cells with GT showed a decrease in the protein expression levels of STAT3, STAT1, p-STAT1 (Tyr 701), and p-STAT3 (Tyr 705) JAK2 as well as p-JAK2 (Tyr1007/1008). The cells were treated at 50% confluency with 40 µg/mL GT for 6, 15, 24, 48, and 72 hours. The membranes were also probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody to ensure equal loading. Whole-cell lysates were immunoblotted with STAT1 and STAT3, p-STAT1 (Tyr 701), p-STAT3 (Tyr 705), JAK2, and p-JAK2 (Tyr1007/1008) antibodies.

Fig. 3

Treatment of HCT116 (p53^+/+, p53^−/−, and p21^−/−) cells with gallotannin (GT) (A) downregulated the 2 anti-apoptotic proteins, Bcl-x_L and c-Myc; (B) did not modulate the Bax/Bcl-2 ratio in HCT116 (p53^+/+) cell line that is increased in HCT116 (p53^−/−) and (p21^−/−) cell line; and (C) induced PARP cleavage. The cells were treated at 50% confluency with 40 µg/mL GT for 6, 15, 24, 48, and 72 hours. Whole-cell lysates were then immunoblotted with specific antibody. The membranes were also probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody to ensure equal loading.

Fig. 4

(A) Treatment of HCT116 (p21^−/−) cells with gallotannin (GT) induced cell death as evidenced by the accumulation of cells in the subG0/G1 phase. Cells were treated at 50% confluency with GT (20, 40, and 60 µg/mL) for 48 hours. Cells were then harvested and DNA stained with propidium iodide (PI) for flow cytometric analysis of DNA content with FACScan flow cytometry. The percentage of cells in each of the cell cycle phases was calculated using Cell Quest. TUNEL assay (Roche Diagnostics, Basel, Switzerland) indicates that GT induced apoptosis in HCT-116 (p21^−/−) cells at 40 and 60 mg/mL 48 hours posttreatment. Cells were treated at 50% confluency with either <0.1% ethanol (control) or GT (40 or 60 mg/mL) for 48 hours. Cells were then harvested and DNA fragmentation was evaluated by TUNEL assay and reading by flow cytometry as described in the Materials and Methods section. The percentage of apoptotic cells under each treatment was obtained with Cell Quest. Each value is the mean of 2 separate experiments performed in duplicates. *P < 0.05 and ^⁎⁎P < 0.01 defined the statistical significance from control using 1-way ANOVA test. (B) GT treatment inhibits the proliferation of HCT116 (p53^−/−) and HCT116 (p21^−/−) cells, whereas concomitant treatment with the different caspase inhibitors had no effect on the antiproliferative effects of GT. Cells were treated at 50% confluency with 20 µM caspase inhibitors followed by addition of GT (0, 40, or 60 µg/mL) for 48 hours. Cell proliferation was determined by the Cell Titer 96 nonradioactive cell proliferation assay as described in the Materials and Methods section. Each value is the mean (SD) of 3 separate experiments each done in triplicates. *P < 0.05 and ^⁎⁎P < 0.01 defined the statistical significance from control using 1-way ANOVA test. (C) Treatment of HCT116 (p53^−/−) cells and HCT116 (p21^−/−) cells induced slight reduction in the levels of pro-caspase-3 and pro-caspase-9 protein levels; however, it failed to trigger caspase cleavage. The cleaved caspase product expressed by Molt-4 cells subjected to irradiation is used as positive control for pro-caspase-3 cleavage. The cells were treated at 50% confluency with 40 µg/mL GT for 6, 15, 24, 48, and 72 hours. Whole-cell lysates were then immunoblotted with caspase-3 and caspase-9 antibodies. The membranes were also probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody to ensure equal loading.

Fig. 5

(A) Treatment of HCT116 (p53^+/+) with gallotannin (GT) decreased p-Rb (Ser 780) expression and induced increase in Rb expression. Treatment of HCT116 (p53^−/− and p21^−/−) cells decreased the levels of p-Rb (Ser 780) but did not induce Rb expression. The cells were treated at 50% confluency with 40 µg/mL GT for 6, 15, 24, 48, and 72 hours. Whole-cell lysates were then immunoblotted with p-Rb (Ser 780) and Rb antibodies. The membranes were also probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody to ensure equal loading. (B) GT-induced senescence only in HCT116 (p53^+/+) cells but failed to induce it in HCT116 (p53^−/−) cells transfected with pCMV-p53 plasmid or pCMV-p53mt135 and in HCT116 (p53^−/−) cells. Cells were treated at 50% confluency with GT (20 and 40 µg/mL) for 72 hour. Senescence induction was assayed by the Senescence β-galactosidase Staining kit as described in the Materials and Methods section. Five random images were then taken for each sample and the percentage of stained cells was calculated in each image. The percentage of senescent cells in each sample represents the average of the percentages of stained cells in the 5 regions. ^⁎⁎P < 0.01 defined the statistical significance from control using 1-way ANOVA test. (C, D) Treatment of HCT116 (p53^−/−) cells, transfected with p53 plasmid (pCMV-p53) or plasmid harboring p53 mutant p53m plasmid (pCMV-p53mt135) with 40 µg/mL GT induced significant p53 expression in cells transfected with pCMV-p53 plasmid but failed to express p21 proteins. In cells transfected with pCMV-p53mt135, p53 was slightly induced but not p21. Treatment of both types of transfected cells with 20 µg/mL GT failed to induce both p53 and p21 proteins. The cells were treated at 50% confluency with 20 or 40 µg/mL GT for 6, 15, 24, 48, or 72 hours. Whole-cell lysates were then immunoblotted with p53 and p21 antibodies. The membranes were also probed with GAPDH antibody to ensure equal loading.