p38γ Activation and BGP (Biliary Glycoprotein) Induction in Primates at Risk for Inflammatory Bowel Disease and Colorectal Cancer-A Comparative Study with Humans

Abstract

Colorectal cancer (CRC) is a common cause of cancer-related deaths largely due to CRC liver metastasis (CRLM). Identification of targetable mechanisms continues and includes investigations into the role of inflammatory pathways. Of interest, MAPK is aberrantly expressed in CRC patients, yet the activation status is not defined. The present study assessed p38γ activation in CRC patients, cancer cells, and tissues of cotton top tamarin (CTT) and common marmoset (CM). The primate world is an overlooked resource as colitis-CRC-prone CTT are usually inure to liver metastasis while CM develop colitis but not CRC. The results demonstrate that p38γ protein and phosphorylation levels are significantly increased in CRC patients compared to normal subjects and CTT. Furthermore, p38γ phosphorylation is significantly elevated in human CRC cells and hepatoblastoma cells but not in CM colon. Additionally, carcinoembryonic antigen (CEA) and biliary glycoprotein (BGP) are induced in the CRC patients that showed p38γ phosphorylation. Inhibition of p38 MAPK in CRC cells showed a significant decline in cell growth with no effect on apoptosis or BGP level. Overall, p38γ is activated in CRC tumorigenesis and likely involves CEA antigens during CRLM in humans but not in the CTT or CM, that rarely develop CRLM.

Keywords: BGP; CEA; CRC; blood group antibodies; p38γ; p87; pp38γ.

Figure 1

Increased protein expression and activation of p38γ in human cancerous colonic tissue compared to normal subjects. (a) Whole tissue extracts from colon cancer patients and normal subjects were prepared and 25 µg of protein subjected to SDS-gel electrophoresis and Western blot analysis using antibodies against p38γ and pp38γ. Equal loading was confirmed using an antibody against total p38 antibody. Representative blotting is shown for four normal (N) and four colon cancer (CRC) subjects. (b) Densitometric values expressed as fold increase of ratio of phosphorylated p38γ/total p38. (c) Densitometric values expressed as fold increase of the ratio of p38γ/total p38. The data was analyzed using the paired, two-tailed Student’s t-test, and the results were expressed as fold change ± SEM of 10 colon cancer patients and 10 normal subjects * p-value < 0.001.

Figure 2

Presence of p38γ and pp38γ in preclinical and human tissue. (a) Whole tissue extracts from normal subject, CTT liver, and two derived from human cancerous colorectal tissue were prepared and subjected to SDS-PAGE and Western blot analysis using specific antibodies to pp38γ. Equal loading was confirmed using antibodies against p38γ. Normal subject (lane 1), CRC patients (lane 2, lane 3), and CTT liver (lane 4). (b) Densitometric values (means ± SEM) expressed as ratio of pp38γ/p38γ. * p value < 0.001. (c) Whole tissue extracts prepared from common marmoset (CM-C. jacchus) colon tissues (lanes 1–6) not at risk representing a negative control, in contrast CM small intestinal ileum are potentially at risk and a positive control (doxorubicin treated breast cancer cell line) were all similarly subjected to SDS-PAGE and Western blot analysis using specific antibodies to p38 and p38γ. None of the CM tissues have p38γ but all express total p38 (α and β) (n = 6). (d) Whole tissue extracts prepared from different organs of common marmoset, liver (lanes 1 and 2), gall bladder (lane 3), colon (lane 4), and ileum (lane 5) were subjected to SDS-PAGE and Western blot analysis using specific antibody to p38γ (n = 2).

Figure 3

Increased protein expression of carcinoembryonic antigen (CEA) and biliary glycoprotein (BGP) from cancerous and corresponding normal tissues from patients with CRC were compared to normal colon from patients without colon cancer. (a) Whole tissue extracts from colon cancer (CRC) subjects and normal (N) subjects (included in this figure are four samples shown in Figure 1a with an additional sample making a total of five) were subjected to Western blot analysis using specific antibodies to CEA and BGP. Equal loading was confirmed with β-actin. A representative blot is shown for five normal subjects and five CRC subjects. (b) Densitometric values are expressed as fold increase of the ratio of CEA/β-actin (mean± SEM) from 10 CRC and 10 normal patients, * p-value < 0.007. (c) Densitometric values are expressed as a fold increase of the ratio of BGP/β-actin (mean± SEM) from 10 CRC and 10 normal patients, * p-value < 0.005. As shown in (a) the CRC patients exhibited increased protein expression of BGP and CEA compared to normal subjects.

Figure 4

Increased phosphorylation of p38γ and protein expression of CEA in CRC cell lines. (a) Western blot using specific antibodies to p38γ and pp38γ in cell extracts from IEC-6 rat intestinal epithelial cells (lane 1), HCT 116 human CRC cells (lane 2), HT-29 human CRC cells (lane 3), Caco-2 human CRC cells (lane 4), SW620 human CRC cell line (lane 5), and HepG2 human hepatoblastoma carcinoma cell line (lane 6). (b) Densitometric values expressed as fold increase of the ratio of pp38γ/p38γ mean ± SEM (n = 4) * p-value < 0.05. The above results show the increased phosphorylation in CRC cell lines and HepG2 cells compared to normal IEC-6 cells. (c) Whole cell extracts from different cell lines were subjected to Western blotting with specific antibodies to CEA. (d) Densitometric values showing the fold increase of the ratio of CEA/β-actin mean ± SEM (n = 4) * p-value < 0.05. As shown in (c) the CEA levels are uniformly increased in colon cancer cell lines and HepG2 cell line.

Figure 5

Cell growth inhibition of human CRC HT-29 and HCT 116 cell lines by losmapimod and doramapimod. HCT-116 and HT-29 CRC cells were treated with DMSO (untreated) or with different doses of losmapimod (LOS) and doramapimod (DORA) for 24, 48, and 72 h. Determination of viable/live cells was carried out by MTT assay as described in Section 2. (a,b) show the dose dependent inhibition of cell growth at 48 and 72 h by losmapimod and doramapimod in HCT-116 cells. Similarly, (c,d) show the dose dependent inhibition in HT-29 cells. The results are shown as mean ± SEM (n = 6); * p-value < 0.005. The dose is on the x-axis, and the viability of cells on y-axis.

Figure 6

Inhibition of p38 MAPKs did not alter the caspase 8 or caspase 3 activity in CRC cells. HCT-116 and HT-29 CRC cell lines were treated with different doses of losmapimod or doramapimod or DMSO (control) for 48 h. Whole cell extracts were prepared and subjected to SDS-PAGE. Western blotting was performed using specific antibodies to caspase 8 and caspase 3. Equal loading was confirmed with β-actin. As shown above, neither caspase 3 or caspase 8 levels were altered by losmapimod or doramapimod.

Figure 7

Lack of inhibition of biliary glycoprotein (BGP) protein levels by losmapimod or doramapimod by in colon cancer cells. (a,b) HCT-116 and (c,d) HT-29 CRC cells were treated with different doses of losmapimod or doramapimod or DMSO (control) for 48 h. Whole cell extracts were prepared and SDS-PAGE was performed followed by Western blotting with specific BGP antibodies. Equal loading was confirmed with β-actin. Panels (b,d) depict the resultant densitometric values showing fold change of the ratio of BGP/β-actin in HCT-116 and HT-29 cells.

Figure 8

Expression of tumor metastasis associated antigens in tamarin and marmoset colonic tissue. Tissue extracts were obtained from the Callitrichidae family of nonhuman primates; cancerous (CTT n = 5) and corresponding normal mucosa (CTT n = 5) and (CM n = 5). The expression of tumor metastasis associated antigens were determined by ELISA. Antibody and antigen group designations: BGP-Biliary glycoprotein (CEACAM1), CEA Superfamily 53.5, CEA family 46.1, T84.66,FH6 Sial LeX fucoganglioside 6B, FH2 sialylated Lewis X, KH1 Extended Lewis Y blood group, Sialyl Tn α-sialyl Tn, CaCo3/61 fucosylated aminoproteoglycan, Adnab-9 Paneth cell glycoprotein p87. Positivity status was determined by an OD-background ≥0.05 as previously described [9]. Significant differences comparisons were made to BGP by nonparametric tests and the * indicates a p value < 0.002. Only CTT extracts were available for most of the blood group antigen testing. Additional comparisons to human can be found as described [34]

Figure 9

The correlation between BGP and Adnab-9 epitope expression in colonic tissues of CTT and CM. The scatter gram plot shows a statistically significant correlation between BGP antibody binding and Adnab-9 antibody binding by ELISA as previously published in [34].

Figure 10

The correlation between Adnab-9 antibody binding and T84.66 antibody binding to CTT cancerous and normal tissue extracts. The scatter gram plot shows a statistically significant correlation between Adnab-9 and CEA. There was no correlation between Adnab-9 and anti-CEA T84.66 binding in CM tissues.

Figure 11

Normal CTT tissue correlation of BGP versus CEA in ng/mL shows an inverse relationship. The scatter gram plot shows a statistically significant correlation between BGP and CEA concentration. There was a similar correlation between BGP and CEA concentration when expressing the results in ng/mg protein in these normal-appearing mucosal sample CTT extracts. These extracts shown here are identical to the normal CTT shown in Figure 8, Figure 9 and Figure 10. There was no BGP versus CEA concentration correlation seen in the cancer tissue extracts. The CEA concentrations were obtained using the TOSOH Medics kit [25].