Panoply™ Human ALK Knockdown Stable Cell Line

Midkine is overexpressed in hepatocellular carcinoma (HCC) and plays a role in tumor progression. Here, researchers found that cultured HCC cells exhibited enhanced resistance to anophthalmia (apoptosis) in vitro, which was positively correlated with midkine expression levels. Midkine treatment significantly inhibited anophthalmia in a dose-dependent manner. Furthermore, in vitro and in vivo experiments showed that knockdown of midkine significantly reduced the sensitivity of HCC cells to anophthalmia, decreased cell survival, and significantly reduced tumor incidence. HCC patients with high midkine expression had higher circulating tumor cell (CTC) counts, fewer apoptotic CTCs, significantly increased recurrence rates, and shorter recurrence-free survival. To understand the molecular mechanism of midkine in HCC progression, researchers conducted in vitro and in vivo studies. The results indicate that midkine plays an important role in enhancing the resistance of HCC cells to anophthalmia, thereby promoting subsequent metastasis. Activation of PI3K/Akt/NF-κB/TrkB signaling by midkine-activated anaplastic lymphomakinase (ALK) is responsible for anoikis resistance.

Here, researchers investigated ALK expression in HCC cells. Western blot analysis showed that 5 out of 7 HCC cell lines exhibited elevated ALK expression (Figure 1A). They knocked down endogenous midkine or ALK expression in PLC/PRF/5 cells, and the knockdown effect was confirmed by Western blot (Figure 1B). Compared with control cells, the expression of Bcl-2 and TrkB was downregulated in midkine-knockdown or ALK-knockdown cells, while the expression of cleaved caspase-3 and Bax was upregulated (Figure 1B), but the midkine-knockdown cells showed significantly enhanced sensitivity to anoikis (Figure 1C). Correspondingly, the enzyme activity of caspase-3 was significantly increased in both midkine-knockdown and ALK-knockdown cells (Figure 1D). Surprisingly, ALK knockdown cells partially regained sensitivity to anodic apoptosis (Figure 1C), and their ability to anchor-independent growth in soft agar (Figure 1E) and invade through Matrigel^TM after 16 hours of incubation in poly-HEMA-coated Transwell chambers was also partially inhibited (Figure 1F). Hep3B cells showed similar results. Furthermore, compared to control cells, ALK knockdown cells responded less strongly to the addition of midkine to promote anchor-independent growth, invasion, and anoikis (Figure 1E and F).

Figure 1. Midkine induces anoikis resistance, growth and invasion of HCC cells through ALK activation. (Sun B, et al., 2017)