PLCD1 Suppressed Cellular Proliferation, Invasion, and Migration via Inhibition of Wnt/ß-Catenin Signaling Pathway in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Phospholipase C delta 1 (PLCD1) has been found to be abnormally expressed in various cancers. However, the potential roles of PLCD1 in esophageal squamous cell carcinoma (ESCC) are still unknown. METHODS: Western blot and qPCR were used to explore PLCD1 expression in various ESCC cells. MTT, colony formation assays, wound-healing assay, and transwell cell invasion assay were used to examine the cell viability in vitro. Western blot, qPCR, and luciferase assays were used to investigate the effects of PLCD1 on Wnt/ß-catenin signaling pathway. The xenograft models in nude mice were established to explore the roles of PLCD1 in vivo. RESULTS: We found that the expression of PLCD1 in ESCC cells was significantly downregulated than that in normal esophageal epithelial cells. In addition, upregulation of PLCD1 decreased the capacity of TE-1 and EC18 cells in proliferation, invasion, and migration. Then, the expression of ß-catenin/p-ß-catenin, C-myc, cyclin D1, MMP9, and MMP7 was investigated. PLCD1 activity was found to be negatively associated with the expression of ß-catenin, C-myc, cyclin D1, MMP9, and MMP7. Finally, the activity of PLCD1 in inhibiting ESCC proliferation in vivo was validated. CONCLUSION: The inhibitory effects of PLCD1 on the proliferation, invasion, and migration of TE-1 and EC18 cells might be associated with inhibition of Wnt/ß-catenin signaling pathway. PLCD1 played a key role in inhibiting ESCC carcinogenesis and progression in patients with ESCC.

Enhanced transient receptor potential channel-mediated Ca2+ influx in the cells with phospholipase C-δ1 overexpression: its possible role in coronary artery spasm.

We reported that coronary spasm was induced in the transgenic mice with the increased phospholipase C (PLC)-δ1 activity. We investigated the effect of enhanced PLC-δ1 on Ca2+ influx and its underlying mechanisms. We used human embryonic kidney (HEK)-293 and coronary arteries smooth muscle cells (CASMC). Intracellular free Ca2+ concentration ([Ca2+ ]i ; nm) was measured by fura-2, and Ca2+ influx was evaluated by the increase in [Ca2+ ]i after addition of extracellular Ca2+ . Acetylcholine (ACh) was used to induce Ca2+ influx. ACh-induced peak Ca2+ influx was 19 ± 3 in control HEK-293 cells and 71 ± 8 in the cells with PLC-δ1 overexpression (P < 0.05 between two groups). Nifedipine partially suppressed this Ca2+ influx, whereas either 2-APB or knockdown of classical transient receptor potential channel 6 (TRPC6) blocked this Ca2+ influx. In the human CASMC, ACh-induced peak Ca2+ influx was 29 ± 6 in the control and was increased to 45 ± 16 by PLC-δ1 overexpression (P < 0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or knockdown of TRPC6 blocked it. ACh-induced Ca2+ influx was enhanced by PLC-δ1 overexpression, and was blocked partially by nifedipine and completely by 2-APB. TRPC-mediated Ca2+ influx may be related to the enhanced Ca2+ influx in PLC-δ1 overexpression.

Coronary vasospasm induced in transgenic mouse with increased phospholipase C-δ1 activity.

BACKGROUND: We reported that phospholipase C (PLC)-δ1 activity was enhanced 3-fold in patients with coronary spastic angina. We detected variant PLC-δ1 with replacement of arginine 257 by histidine (R257H) showing increased enzymatic activity. We tested the hypothesis that increased PLC-δ1 activity causes enhanced coronary vasomotility. METHODS AND RESULTS: We generated transgenic (TG) mice with human R257H variant PLC-δ1 in vascular smooth muscle cells. PLC enzymatic activity in the coronary artery was increased by 2.57 and 1.89 times, respectively, in homozygous and heterozygous TG compared with wild-type (WT) mice. ST elevation after ergometrine occurred in 17 of 18 homozygous TG, 6 of 20 heterozygous TG, and 3 of 22 WT mice (P<0.01, homozygous TG versus WT; P<0.05, homozygous TG versus heterozygous TG; P=NS, heterozygous TG versus WT). ST elevation was associated with bradyarrhythmias in homozygous TG mice. Focal coronary artery narrowing was documented with the microvascular filling technique in 3 of 5 homozygous TG mice after ergometrine but not in any of 7 WT mice (P<0.05). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in homozygous TG mice (P<0.01) but not in heterozygous TG or WT mice. Coronary perfusion pressure increase after prostaglandin F2α was similar among homozygous TG, heterozygous TG, and WT mice. Cultured rat aortic smooth muscle cells transfected with variant PLC-δ1 showed a higher PLC activity than those with WT PLC-δ1 (P<0.05) and furthermore showed greater intracellular Ca2+ response to acetylcholine in variant than in WT PLC-δ1 (P<0.05). CONCLUSIONS: Increased PLC-δ1 activity enhances coronary vasomotility such as that seen in patients with coronary spastic angina.

Phospholipase C-delta1 expression is linked to proliferation, DNA synthesis, and cyclin E levels.

We previously reported that phospholipase C-delta1 (PLC-delta1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate ( Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-delta1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-delta1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-delta1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-delta1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-delta1 and nuclear phospholipid metabolism in regulating cell cycle progression.

Characterization of a novel tumor-suppressor gene PLC delta 1 at 3p22 in esophageal squamous cell carcinoma.

Deletion of 3p is one of the most frequent chromosomal alterations in many solid tumors, including esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor-suppressor genes at 3p. Recently, our loss of heterozygosity study revealed that 3p22 was frequently deleted in ESCC and a candidate tumor-suppressor gene (TSG), phospholipase C-delta 1 (PLC delta 1), was identified within the 3p22 region. In this study, absent expression of PLC delta 1 was detected in 26 of 50 (52%) primary ESCCs and 4 of 9 (44.4%) ESCC cell lines, which was significantly associated with DNA copy number loss and promoter hypermethylation (P < 0.05). Functional studies showed that PLC delta 1 was able to suppress both in vitro and in vivo tumorigenic ability of ESCC cells, including foci formation, colony formation in soft agar, and tumor formation in nude mice. The tumor-suppressive mechanism of PLC delta 1 was associated with its role in the cell cycle arrest at the G(1)-S checkpoint by up-regulation of p21 and down-regulation of phosphorylated Akt (Ser(473)). In addition, down-regulation of PLC delta 1 protein was significantly correlated with ESCC metastasis (P = 0.014), which was associated with its function in increasing cell adhesion and inhibiting cell mobility. Taken together, our results suggest that PLC delta 1 plays an important suppressive role in the development and progression of ESCC.