ECC4

Delta-Like 3 Plays a Pivotal Role in Gastrointestinal Neuroendocrine Malignancy

Delta-like 3 (DLL3) functions as a Notch signaling ligand that possesses unusual structural characteristics while simultaneously inhibiting Notch signaling. DLL3 maintains a recognized function in lung neuroendocrine carcinoma but its role and expression status in the gastrointestinal tract and GI neuroendocrine carcinoma (GI-NEC) have not yet been fully determined. Matsuo et al.investigates these aspects, aiming to elucidate DLL3's role and expression in the GI tract and GI-NEC. To assess DLL3 mRNA levels, RT-qPCR on GI-NEC cell lines ECC4, ECC10, and ECC12 revealed significant upregulation (ECC4: 2000-fold, ECC10: 1800-fold, ECC12: 3000-fold) of DLL3 mRNA compared to other cancer cell lines (Fig. 1A). The Western blot analysis validated the observed trend at the protein level (Fig. 1B). ECC cells displayed DLL3 mRNA levels that matched those found in SCLC cells. Using double fluorescent immunocytochemistry both DLL3 and CHGA showed cytoplasmic localization (Fig. 2A), while electron microscopy displayed DLL3 inside neurosecretory granules (Fig. 2B and C). To investigate DLL3 functions in GI-NEC cells, they silenced DLL3, resulting in significant growth inhibition (Fig. 3A). The elevation of cleaved PARP levels along with caspase 3 and caspase 9 demonstrated an augmentation of apoptosis as shown in Figure 7B. The Hoechst 33342 stain demonstrated an increase in apoptotic cells within ECC cells treated with siR-DLL3 (Fig. 3B). Treatment with the pan-caspase inhibitor Z-VAD-FMK led to restored cell growth and lower levels of cleaved PARP expression as shown in Figure 3D, E. Additionally, DLL3 silencing hindered spheroid formation in ECC4 cells (Fig. 3F and G).

Fig. 1. The expression levels of delta-like 3 (DLL3) in gastrointestinal-neuroendocrine carcinoma ECC cell lines (Matsuo K, Taniguchi K, et al., 2019).

Fig. 2. The morphological features of delta-like 3 (DLL3) expression in GI-NEC ECC cell lines (Matsuo K, Taniguchi K, et al., 2019).

Fig. 3. The effects of gene silencing of delta-like 3 (DLL3) in ECC cell lines (Matsuo K, Taniguchi K, et al., 2019).

Shared Expression of Mucin12 in Ascaris lumbricoides and the Human Small Intestine

Accounting for the widespread infection caused by A. lumbricoides, the study highlights its ability to thrive in the human small intestine by potentially sharing antigenic features with host proteins. Using molecular techniques and screening, Hayashi et al. identified DNA clones showing antigenic similarities between A. lumbricoides and human mucin12, suggesting an immune evasion strategy. To confirm proteins cross-reacting with human mucin12, western blotting was conducted. As illustrated in Fig. 4, mucin12 protein appeared in the lysates of human gastrointestinal cells CACO-2 and ECC4, showing bands around 45 kDa and 85 kDa when immunostained with polyclonal anti-mucin12 antibody (Fig. 4b). In contrast, A. lumbricoides crude antigen displayed bands of 37 kDa and 100 kDa using the same antibody (Fig. 4b). Moreover, the main bands, reacting with commercial anti-mucin12 antibody, were approximately 90 kDa in both cell lines (Fig. 4c). In A. lumbricoides crude antigen, a 100 kDa band matched the one detected with polyclonal anti-mucin12 antibody (Fig. 4c). These findings reveal different proteins detected in A. lumbricoides and human gastrointestinal cells with a common anti-mucin12 antibody.

Fig. 4. Western blotting for mucin12 protein in A. lumbricoides crude antigen and human gastrointestinal cells (Hayashi I, Kanda S, et al., 2019).