Aspirin enhances the sensitivity of colon cancer cells to cisplatin by abrogating the binding of NF-κB to the COX-2 promoter

Abstract

Cisplatin is one of the most potent chemotherapeutic agents for the treatment of colon cancer. Nevertheless, the unavoidability of the notable toxicity and the development of the acquired resistance severely restricted its clinical application. Aspirin and some other non-steroidal anti-inflammatory drugs have been used to prevent colon tumorigenesis as chemopreventive agents. Here, we explored the possibility of aspirin as an adjuvant drug to boost the anti-cancer effect of cisplatin for colon cancer. We found that aspirin significantly enhanced the cisplatin-mediated inhibitions of cell proliferation, migration and invasion and the induction of apoptosis in colon cancer cells. The combined treatment of aspirin and cisplatin suppressed the expression of the anti-apoptotic protein Bcl-2 and the EMT-related proteins, up-regulated the levels of the cleaved PARP and Bax, and blocked the PI3K/AKT and RAF-MEK-ERK signaling pathway. In addition, we demonstrated that the enhanced effect of aspirin on the cisplatin-induced inhibition of tumor cell growth was also mediated through the suppression of the binding activity of NF-κB to the COX-2 promoter. The combination of aspirin and cisplatin effectively attenuated the translocation of NF-κB p65/p50 from the cytoplasm to the nucleus, and abrogated the binding of NF-κB p65/p50 to the COX-2 promoter, thereby down-regulating COX-2 expression and PGE₂ synthesis. Moreover, the in vivo study also verified the enhanced anti-tumor activity of such combined therapy in colon cancer by targeting the NF-κB/COX-2 signaling. Our results provided new insights into understanding the molecular mechanisms of aspirin in sensitizing cisplatin-mediated chemotherapeutic effect in colon cancer and indicated a great potential of this combined therapy for cancer treatment.

Keywords: COX-2; NF-κB; aspirin; cisplatin; colon cancer.

Figure 1

Aspirin synergizes the inhibiting effect of Cisplatin on cell viability in colon cancer cells. (A) Human colon cancer cells SW620, LoVo, RKO and DLD-1 were treated with different concentrations of Aspirin for 48 h, and the cell viability was tested by MTT assay (n=6). (B) Human colon cancer cells were treated with indicated dose of Cisplatin alone or Cisplatin combined with 2 mM Aspirin for 48 h, and the cell viability was tested by MTT assay (n=6). (C) IC50 values of Cisplatin were calculated by software CVXPT32 in colon cancer cells upon treatment with Cisplatin alone or Cisplatin combined with 2 mM Aspirin. Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Aspirin synergizes the inhibiting effect of Cisplatin on cell proliferation in colon cancer cells. (A) Human colon cancer cells RKO and LoVo were treated with Cisplatin alone or Aspirin alone or their combination at indicated dose for 48 h. The representative images of cell morphology were taken by inverted microscope, Scale bars, 200 μm. (B) The colony formation assay of human colon cancer cells RKO and LoVo treated with Cisplatin alone or Aspirin alone or their combination at indicated dose was taken to detect the capacity of cell proliferation (n=4). (C) The expression levels of main PI3K/AKT signaling pathway-related proteins p110-α, p110-β, p110-γ, p-p85, p-PDK1, Akt, p-Akt(S473) and Pten in human colon cancer cell RKO and LoVo treated with Cisplatin alone (15 μM) or Aspirin alone (10 mM) or their combination for 48 h were detected by Western blot assay (n=3). (D) Human colon cancer cells RKO and LoVo were treated with Cisplatin alone (15 μM) or Aspirin alone (10 mM) or their combination for 48 h, and the expression levels of main RAF-MEK-ERK signaling pathway-related proteins p-c-Raf, p-mek1/2, Erk1/2 and p-Erk1/2 were detected by western blot assay (n=3). (E) Human colon cancer cells RKO and LoVo were pretreated with PI3K inhibitor LY294002 (50 μM) or MEK inhibitor U0126 (10 μM) for 8 h, and then incubated with combination of Aspirin and Cisplatin. After 48 h, cell viability was determined by MTT analysis (n=6). (F) Human colon cancer cells RKO and LoVo were transfected with specified siRNAs for 24 hours, and then incubated with the combination of Aspirin and Cisplatin. After 48 h, cell viability was determined by MTT analysis (n=6). All the data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Aspirin increased the inhibiting effect of Cisplatin on cell migration and invasion in colon cancer cells. (A) A wound-healing assay of human colon cancer cells RKO and LoVo treated with Cisplatin alone or Aspirin alone or their combination at indicated dose for 56 h was performed to detect the capacity of cell migration. Scale bars, 500 μm. The cell migration rate was calculated through the quantification of migration distance (n=4). (B) The capacity of cell invasion of human colon cancer cells RKO and LoVo treated with Cisplatin alone or Aspirin alone or their combination at indicated dose for 96 h was analyzed by a transwell assay. The invaded cells were stained by crystal violet and the representative images were taken by inverted microscope (n=4). Scale bars, 200 μm. (C) The expression levels of main EMT- related proteins N-cadherin, β-catenin, Vimentin, MMP2, MMP9, Snail and Slug in human colon cancer cell RKO treated with Cisplatin alone (15 μM) or Aspirin alone (10 mM) or their combination for 48 h were detected by western blot assay (n=3). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Aspirin enhanced the Cisplatin-induced apoptosis in colon cancer cells. (A) A FACS analysis was used to detect cell apoptosis in human colon cancer cells RKO and LoVo treated with Cisplatin alone or Aspirin alone or their combination at indicated dose for 48 h. The percentage of apoptotic cells was further calculated (n=4). (B) Human colon cancer cells RKO and LoVo treated with cisplatin alone or aspirin alone or their combination at indicated dose for 48 h were stained with acridine orange and ethidium bromide to observe cell apoptosis. The representative images were taken by inverted fluorescence microscope. Scale bars, 200 μm (n=3). (C) The expression levels of main apoptosis-related proteins PARP, cleaved PARP, Bcl-2 and Bax in human colon cancer cell RKO treated with Cisplatin alone (15 μM) or Aspirin alone (10 mM) or their combination for 48h were detected by western blot assay (n=3). (D) Immunofluorescence assay of human colon cancer cell LoVo treated with Cisplatin alone (2.5 μM) or Aspirin alone (4 mM) or their combination for 48 h was implemented to monitor cytochrome-c release from intermembrance space of mitochondria into cytoplasm. The representative images were taken by laser scanning confocal microscope. Scale bars, 25 μm (n=3). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Cotreatment with Aspirin and Cisplatin suppressed NF-κB/COX-2 signaling pathway in colon cancer cells. (A) The expression level of COX-2 in the whole cell lysate of human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin(10mM /5mM) for 48 hours was analyzed by Western blot (n=3). (B) Human colon cancer cells RKO and LoVo were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h. The amount of PGE₂ in cell culture media was detected by Prostaglandin E₂ High Sensitivity in vitro competitive ELISA Kit (n=4). (C) Human colon cancer cells RKO and LoVo were incubated with combination of Aspirin and Cisplatin at indicated dose for 48 h after pretreatment with COX-2-selective inhibitor celecoxib (CB) at indicated dose for 8 h, and then cell viability was determined by MTT analysis (n=6). (D) Human colon cancer cells RKO and LoVo were treated with Cisplatin alone (15μM /5μM) or Aspirin alone (10mM /5mM) or their combination for 48 h. The expression levels of p65, p50, COX-2 and c-myc in nucleus, p65, p50, p-p65, IKKα, IKKβ, p-IKKα/β, p-IκBα and IκBα in cytoplasm were respectively detected by western blot assay (n=3). (E) Immunofluorescence assay of human colon cancer cell RKO treated with Cisplatin alone (15μM) or Aspirin alone (10mM) or their combination for 48 h was implemented to observe the subcellular localization of p65 and p50. The representative images were taken by laser scanning confocal microscope. Scale bars, 25 μm (n=3). (F) The streptavidin-biotin pulldown assay was performed to test the binding of p65 and p50 at COX-2 promoter region in human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h (n=3). (G) Human colon cancer cells RKO and LoVo were transfected with COX-2 promoter (-892/+9 fragments) driven-luciferase plasmids and pRL-TK Renilla luciferase construct (Promega). After 24 hours, the cells were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 hours. Then luciferase activities were measured according to the Dual-Luciferase Assay System protocol (Promega) (n=4). (H) Human colon cancer cells RKO and LoVo were pretreated with NF-κB activator LPS or NF-κB inhibitor QNZ at indicated dose for 8 h, and then incubated with combination of Aspirin and Cisplatin at indicated dose. After 48 h, cell viability was determined by MTT analysis (n=6). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Aspirin synergizes the inhibiting effect of Cisplatin on tumor growth in a xenograft mouse model of human colon cancer cells. Human colon cancer LoVo cells (5×10⁶, 5×10⁵ in 100 ul PBS) were injected subcutaneously into the left and right flank of each athymic nude mice respectively. The four randomly assigned groups (n=6 for each group) were used: (1) non-drug therapy as negative control; (2) the treatment with Cisplatin (3 mg/kg) through intraperitoneal injection every three days; (3) a daily treatment of Aspirin(100 mg/kg) through intragastric administration; (4) the combination therapy of Cisplatin and Aspirin. (A) The representative images of the measurement of tumor diameters. (B) Dynamic development of tumor volume during the therapy. (C) Tumor weight of nude mice from each group at the moment when mice were sacrificed. (D) Images of xenograft tumor harvested after therapy. (E) The expression levels of β-catenin, N-Cadherin, Bcl-2, p-Akt(S473), p-p65, p-Erk1/2, COX-2, p65 and p50 in tumor tissue lysates were detected by western blot assay (n=6). (F) HE staining and immunohistochemical staining assay to show tissue morphological variations and the expressions of N-Cadherin, p-Akt(S473), p-p65, p-Erk1/2, COX-2, p65 and p50 in tissue sections. The representative images were taken by upright microscope. Scale bars, 100 μm (n=6). (G) Serum creatinine (Cr) and Blood Urea Nitrogen (BUN) levels of mice in each group were measured by the detection kit (n=6). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.

Figure 7

The schematic of the molecular mechanisms involved in the synergistic anti-tumor effect of Aspirin and Cisplatin in colon cancer. Aspirin in combination with Cisplatin enhanced the inhibition of cell proliferation, migration and invasion by reducing COX-2 mediated prostaglandin E2 synthesis and attenuating downstream PI3K/AKT, RAF-MEK-ERK and NF-κB/COX-2 signaling activities. Meanwhile, combination with Aspirin and Cisplatin could induce more apoptosis through triggering cytochrome-c release.