Cell culture RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen (Shanghai, China); antibodies for MMP-1, MMP-2, MMP-9 and MMP-10 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); antibodies for phospho-ERK1/2, phospho-Akt (Ser473), phospho-JNK, phospho-p38, phospho-p65 (NF-κB), phospho-PI3K (55/85), ERK1/2, Akt, JNK, p38 and p65NF-κB were purchased from Cell Signaling Technology (Beverly, MA, USA); antibody for β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA); inhibitors of U0126, LY294002 and SB239063 were purchased from Beyotime Institute of Biotechnology (Shanghai, China); Hexamethonium (Hex) bromide was from Sigma-Aldrich (Shanghai, China). Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase and goat anti-rat IgG antibody conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology, Inc.

The LOVO and SW620 human CRC cells were obtained from the American Type Culture Collection (ATCC; USA). The cells were grown in RPMI-1640 medium containing 10% of FBS, glutamine and antibiotics (penicillin/streptomycin), and were maintained in a humidified incubator at 37°C with a supply of 5% CO₂/95% air atmosphere.

Cell invasion assays were carried out using modified Boyden chambers consisting of Transwell (8-µm pore size; Corning Costar Corp., Cambridge, MA, USA) membrane filter inserts into 24-well tissue culture plates. In brief, the upper surfaces of the membranes were coated with 50 µl Matrigel (BD Biosciences, San Jose, CA, USA) 37°C for 6 h. SW620 or LOVO cells (1.0×10⁵) in 200 µl serum-free RPMI-1640 were added to each Transwell chamber supplied with or without nicotine or/and inhibitors as indicated. Cell culture media with 20% of FBS were added in the lower chamber. Cells were allowed to invade toward the underside of the membrane under cell culture condition for 48 h before the non-invading cells were removed by wiping the upper side of the membrane with a cotton swab and the invaded cells were fixed with ice-cold methanol, then the inserts were stained with 0.1% crystal violet in 20% ethanol for 30 min. The cells that invaded to the underside of the membrane were quantitated by cell counting under the light microscopy in five predetermined fields at a magnification of ×100.

The gelatin zymography assay was used to determine the activities of MMP-2 and MMP-9. Cell culture supernatants were collected and concentrated in Amicon Ultra-4 Centrifugal Filter Devices before loading with SDS sample buffer and electrophorese on a 10% SDS polyacrylamide gel polymerized with 5 mg/ml gelatin. After electrophoresis, the gels were renatured by soaking for 30 min at room temperature in 2.5% Triton X-100. To visualize the bands, the gels were incubated in a developing buffer [50 mM Tris-HCl buffer (pH 7.4), 10 mM CaCl₂] overnight at 37°C before they were stained with 0.5% Coomassie brilliant blue R-250 dissolved in 10% acetic acid and 30% carbinol, and were destained in the washing solution without dye. Gelatinolytic bands were observed as clear zones against the blue background.

Total cellular protein was prepared by lysing the cells with 1xRIPA buffer (CST, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined by the Bradford protein assay. A total of 40 µg protein lysates from each sample were separated in 10% SDS-PAGE, and were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated overnight on ice with primary antibodies against MMP-1, MMP-2, MMP-9, MMP-10, phospho-ERK1/2, phospho-Akt, phospho-JNK, phospho-p38, phospho-p65 (NF-κB), phosph-PI3K (55/85), ERK1/2, Akt, JNK, p38, p65 (NF-κB) and β-actin (all with 1:1,000 dilution). After washing, the membranes were probed with a horseradish peroxidase-conjugated goat anti-rabbit or anti-rat secondary antibody followed detection of signals with the FluorChem E system (Protein Simple, Santa Clara, CA, USA).

All assays were performed in triplicate, and experiments were repeated at least three times. Data are presented as the means ± SEM. Significant differences between two groups were determined by Student's t-test with significance set at p<0.05.

Firstly, we investigated whether nicotine affects the invasion of CRC cells in a Transwell assay and BD Matrigel™ was used to imitate the ECM. LOVO and SW620 colon cancer cells were grown in the upper chamber in serum-free media without or with the supplement of nicotine in a final concentration of 0.1, 1 and 10 µM, respectively (11–14). Results showed that both starved LOVO and SW620 cells were able to invade through the Matrigel with the attracting of serum rich media in the lower chamber. Certain numbers of invaded cells stained in blue were counted after 48 h (Fig. 1, controls). The presence of nicotine increased the number of the invaded cells in a dose-dependent manner, and for both of the LOVO and SW620 cells (Fig. 1).

Next, we detected the activities and expressions of metastasis-related MMPs for LOVO and SW620 human CRC cells without or with the presence of nicotine. A gelatin zymography assay was applied to detect the activity for secreted MMP-9 and -2. In brief, LOVO and SW620 human CRC cells were grown in serum-free medium containing various concentrations of nicotine as indicated above, 24 h before the cell culture supernatants were collected and concentrated for gelatin zymography assay. As indicated in Fig. 2A, there were gelatinolytic bands for MMP-9 and MMP-2, respectively. The nicotine treatments deepen the bands for both MMPs, and in both cell lines.

In a similar experimental setting, western blot assays were applied to detect the expression of a panel of MMPs including MMP-1, -2, -9 and -10 in LOVO cells. As shown in Fig. 2B, the nicotine treatments increased the expression of MMP-1, -2 and -9, but had no effect on MMP-10.

We investigated the activation of relevant intracellular MAPK (JNK, p38, ERK), NF-κB and PI3K/Akt signaling pathways caused by nicotine stimulation. To this end, serum-starved LOVO cells were stimulated without or with 10 µM nicotine for 15, 30, 60 and 120 min, respectively, and then were subjected to western blot assays examining the phosphorylation of JNK, p38, ERK, NF-κB (p65), Akt and PI3K (p55/p85). As shown in Fig. 3, with total amount of protein was the same, the presence of nicotine brought up the phosphorylation levels of p38, ERK, Akt and PI3Kp55, but did not change that of JNK and NF-κB (p65).

Having shown that nicotine stimulates the activation of MAPK/ERK, MAPK/p38 and PI3K (p55)/Akt signaling pathways, we then tried to dissect their relationship to the observed bioactivities for nicotine in regards of stimulating metastasis related MMPs expression in colon cancer cells. To this end, a panel of pharmaceutical inhibitors was applied before the cells were exposed to nicotine stimulation. The activities and expressions of MMPs were assayed by both gelatin zymography and western blotting. In addition, we also included Hex, a pharmaceutical inhibitor for nAChR in the experimental setting to illustrate the role for receptor binding of nicotine.

As detailed in Fig. 4A, western blot results revealed that the pre-incubation of cells with the p38 MAPK inhibitor SB239063 and nAChR antagonist Hex, but not selective MAPK/ERK inhibitor U0126 and the PI3Ks inhibitor LY294002 attenuated the stimulative effect of nicotine for MMP-1, -2 and -9 expression levels. Similar results are also shown in Fig. 4B, as the gelatin zymography assay result revealed that the SB239063 and Hex attenuated the stimulative effect of nicotine on the activation of MMP-2 and -9.

We further examined whether p38 MAPK also was affected by stimulative effect of nicotine on LOVO cell invasion in vitro. In this regards, we performed the Transwell chamber invasion assay as described above with an additional step of pre-incubation of the cells with SB239063. Results showed that compared to nicotine-treated cells, the application of SB239063 indeed decreased the number of invaded cells (p<0.05), almost down to control levels, i.e. without nicotine treatment (Fig. 5).

Finally, we addressed the question whether activation of p38 MAPK in LOVO cells is indeed dependent on the activation of nAChRs complex. To this end, LOVO cells were pre-treated with SB239063 or Hex for 1 h, respectively, before cells were exposed to nicotine stimulation for 24 h and subsequently subjected to western blotting. As shown in Fig. 6, the application of Hex abolished the enhanced p38 phosphorylation caused by nicotine stimulation, to an extent similar to SB239063, the known p38 MAPK inhibitor treatment.