LCLC-103H

Canonical BMP Signaling Pathway Is Active in LCLC‑103H Lung Cancer Cells

Bone morphogenetic protein (BMP) signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. BMP-4 treatment of LCLC-103H lung cancer cells triggered the activation of canonical SMAD-dependent BMP signaling. This was confirmed by the detection of a strong increase in the phosphorylated form of SMAD1, 5, and 9 (pSMAD1/5/9) as early as 30 min after the treatment (Fig. 1a, b). Using Western blot analysis, the phosphorylation events were detected with 50 ng/ml and 100 ng/ml, but not with 2 ng/ml BMP-4 concentration. The increase in the amount of pSMAD1/5/9 stayed significant even 24 hours after the treatment, compared to the vehicle control cells. The activation of SMAD-dependent signaling was successfully blocked by the BMP receptor type I antagonist, LDN-214117 when used alone or as a pre-treatment before BMP-4 stimulation.

The ability of BMP-4 to activate and that of LDN214117 to block SMAD-dependent BMP signaling was also observed on ID1, a BMP target gene. A steady increase of ID1 protein starting from 30 min up to 3 and 24 h after stimulation of the investigated lung cancer cell line with 50 and 100 ng/ml BMP-4 (Fig. 1a, c). The effect was completely diminished 3 h after LDN-214117 incubation of LCLC-103H cells. A 30-min pre-treatment with LDN-214117 before BMP-4 incubation was not as efficient in blocking ID1 production, suggesting a time-dependent effect of LDN-214117 activity on gene regulation level and/or a BMP signaling pathway other than SMAD-dependent that is responsible for ID1 targeting.

Fig. 1 BMP-4 activates SMAD-dependent BMP signaling pathway in LCLC-103H cells via phosphorylation of SMAD1/5/9. (Mihajlović J, et al., 2019)

Ionomycin Induces Apoptosis in LCLC-103H Cells

Ubiquitous calpains (μ- and m-calpain) have been repeatedly implicated in apoptosis. In the course of calpain localization studies, LCLC-103H cells overexpressing the catalytic subunit of μ-calpain rapidly underwent cell death after treatment with ionomycin. To monitor this process in cells not overexpressing calpain, LCLC-103H cells were labeled transiently with GFP or stably with a histone-ECFP chimera. The first signs of ionomycin-induced cell death were detected 3 h after the addition of the ionophore. Typical hallmarks of apoptosis, including cell blebbing and fragmentation, were prevented for 24 h by preincubation with the selective calpain inhibitor AC27P (Fig. 2A and B). Untreated cells overexpressing GFP (Fig. 2C) or the chimeric-ECFP protein were not impaired in their vitality, ruling out that ectopic expression of either protein is cytotoxic for this cell line.

The addition of 2 μm ionomycin to LCLC-103H cells caused an instantaneous increase in intracellular Ca²⁺ concentration from 50 to 180 nm (Fig. 2D). Remarkably, calcium concentrations were raised transiently to 0.8-1.5 μm. Concomitantly, calpains were activated, as reflected by almost a double increase of Suc-LLVY-amc cleavage inhibited by AC27P (Fig. 2E). In addition, nuclear condensation was detected in the histone-ECFP-labeled cells 3 h after addition of ionomycin (Fig. 3A), and it was inhibited by preincubation with AC27P (Fig. 3C). DNA and protein analysis in ionomycin-treated cultures revealed DNA fragmentation (Fig. 3D) and PARP cleavage to an 85-kDa fragment typical of caspase-mediated apoptosis (Fig. 3F). The same observations were made in etoposide-treated LCLC-103H cells (Fig. 3B) and COS 7 cells after ionomycin addition (Fig. 3E), suggesting an apoptotic process, although its onset was delayed in comparison to ionomycin-treated LCLC-103H cells. These findings demonstrate that ionomycin induces apoptosis in these cells in a calpain-dependent manner.

Fig. 2 Induction of cell death and calpain activation in LCLC-103H cells by ionomycin. (Gil-Parrado S, et al., 2002)

Fig. 3 Hallmarks of apoptosis in ionomycin-treated LCLC-103H cells. (Gil-Parrado S, et al., 2002)