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Rnd3 regulates lung cancer cell proliferation through notch signaling.

Tang Y, Hu C, Yang H, Cao L, Li Y, Deng P, Huang L - PLoS ONE (2014)

Bottom Line: The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells.Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation.Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Xiangya Hospital, the Central South University, Changsha, Hunan, P.R. China.

ABSTRACT
Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.

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Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.(A) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (B) & (C) A western blot to detect phosphorylated MYPT1, NICD in cells. (D) & (E) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (F) & (G) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *p<0.05 compared to control (group 0); #p<0.05 compared to group 1; $p<0.05 compared to group 3. Data represent means ± S.D.
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pone-0111897-g006: Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.(A) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (B) & (C) A western blot to detect phosphorylated MYPT1, NICD in cells. (D) & (E) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (F) & (G) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *p<0.05 compared to control (group 0); #p<0.05 compared to group 1; $p<0.05 compared to group 3. Data represent means ± S.D.

Mentions: H358 cell line was derived from lung bronchioalveolar carcinoma and H520 cell line was derived from lung squamouse cell carcinoma. However, the major subtype of NSCLC was lung adenocarcinoma. So we confirmed our study in one lung adenocarcinoma cells, A549, as shown in Fig. 6. Rnd3 was down-regulated along with up-regulated Rho Kinase and Notch1 signaling in A549 cells compared to HBEC cells (Fig. 6A and 6B). We quantified the up-regulation fold in change in two signaling. The results were showed in Fig. 6C. Rho Kinase signaling were up-regulated by almost 4 fold evidenced by the pMYPT1 level; the Notch1 signaling were up-regulated by 5 fold evidenced by NICD level. Inhibition of Notch1 signaling by Compound E blocked the proliferation of A549 cells (Fig. 6D and 6E). The cytotoxicity of Compound E in A549 cell was also evaluated by Trypan blue staining. As showed in Fig. S6 (picture 9 to 12), the 5 nM Compound E caused no significant death compared to baseline condition in A549 cells. Mechanistically, Rnd3 regulated the cell proliferation through NICD/Hes1 signaling. Overexpression Rnd3 suppressed the NICD and Hes1 expression in a dosage dependent manner in A549 cells (Fig. 6F and 6G). Our findings in A549 cells were consistent with the results from H358 and H520 cells, suggesting the Rnd3-NICD-Hes1 signaling was a generally molecular mechanism to regulate NSCLC cells growth. Together, higher NICD and Hes1 expression and lower Rnd3 expression were observed in H520, H358 and A549 cells compared to HBEC cells. Reintroduction of Rnd3 inhibited NICD and Hes1 expression, which resulted in decreased proliferation rates of H520 and H358 cells. Blockage of Notch1 signaling inhibited the cell proliferation in H358, H520 and A549 cells. Rnd3 regulated the NICD abundance through prompting its proteasomal degradation in NSCLCs.


Rnd3 regulates lung cancer cell proliferation through notch signaling.

Tang Y, Hu C, Yang H, Cao L, Li Y, Deng P, Huang L - PLoS ONE (2014)

Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.(A) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (B) & (C) A western blot to detect phosphorylated MYPT1, NICD in cells. (D) & (E) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (F) & (G) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *p<0.05 compared to control (group 0); #p<0.05 compared to group 1; $p<0.05 compared to group 3. Data represent means ± S.D.
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Related In: Results  -  Collection

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pone-0111897-g006: Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.(A) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (B) & (C) A western blot to detect phosphorylated MYPT1, NICD in cells. (D) & (E) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (F) & (G) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *p<0.05 compared to control (group 0); #p<0.05 compared to group 1; $p<0.05 compared to group 3. Data represent means ± S.D.
Mentions: H358 cell line was derived from lung bronchioalveolar carcinoma and H520 cell line was derived from lung squamouse cell carcinoma. However, the major subtype of NSCLC was lung adenocarcinoma. So we confirmed our study in one lung adenocarcinoma cells, A549, as shown in Fig. 6. Rnd3 was down-regulated along with up-regulated Rho Kinase and Notch1 signaling in A549 cells compared to HBEC cells (Fig. 6A and 6B). We quantified the up-regulation fold in change in two signaling. The results were showed in Fig. 6C. Rho Kinase signaling were up-regulated by almost 4 fold evidenced by the pMYPT1 level; the Notch1 signaling were up-regulated by 5 fold evidenced by NICD level. Inhibition of Notch1 signaling by Compound E blocked the proliferation of A549 cells (Fig. 6D and 6E). The cytotoxicity of Compound E in A549 cell was also evaluated by Trypan blue staining. As showed in Fig. S6 (picture 9 to 12), the 5 nM Compound E caused no significant death compared to baseline condition in A549 cells. Mechanistically, Rnd3 regulated the cell proliferation through NICD/Hes1 signaling. Overexpression Rnd3 suppressed the NICD and Hes1 expression in a dosage dependent manner in A549 cells (Fig. 6F and 6G). Our findings in A549 cells were consistent with the results from H358 and H520 cells, suggesting the Rnd3-NICD-Hes1 signaling was a generally molecular mechanism to regulate NSCLC cells growth. Together, higher NICD and Hes1 expression and lower Rnd3 expression were observed in H520, H358 and A549 cells compared to HBEC cells. Reintroduction of Rnd3 inhibited NICD and Hes1 expression, which resulted in decreased proliferation rates of H520 and H358 cells. Blockage of Notch1 signaling inhibited the cell proliferation in H358, H520 and A549 cells. Rnd3 regulated the NICD abundance through prompting its proteasomal degradation in NSCLCs.

Bottom Line: The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells.Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation.Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Xiangya Hospital, the Central South University, Changsha, Hunan, P.R. China.

ABSTRACT
Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.

Show MeSH
Related in: MedlinePlus