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Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

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Knockdown of Scribble promotes cisplatin-related drug resistance.A, apoptotic cells were quantified in Scribble knockdown (KD) cells in the CRL-1848 cell line following cisplatin treatment (15 μg/ml, 24 h). Apoptosis was measured using an annexin V apoptosis detection kit and expressed as the percentage of total cells that were in late apoptosis or were both annexin V-PE and PI positive. Left side was the Scribble KD efficiency examined by Western blot. B, cell cycle distributions was measured using flow-cytometric analysis in Scribble KD cells in CRL-1848 cell line when compared with control cells under cisplatin treatment. C, apoptosis was measured using annexin V detection kit in Scribble overexpression (O.E.) cells in the HTB-177 cell line, left side was the Scribble overexpression efficiency examined by Western blot. D, cell cycle distributions were measured using flow-cytometric analysis in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment. E, the CRL-1848 cell line Scribble KD or control cells were treated with different concentrations of cisplatin for 24 h. Cell viability was determined using an MTT assay; cell viability was expressed as the percent of total cell number. F, immunoblots of untreated and 24-h cisplatin-treated cells (15 μg/ml, 24 h) in the CRL-1848 cell line control and Scribble KD cells. Primary antibodies were specific for Scribble, cleaved PARP, cleaved caspase 3 or GAPDH (loading control). G, immunofluorescence of Scribble KD or control cells following 24 h of cisplatin exposure in the CRL-1848 cell line. Left column (blue), DAPI (nuclei); middle column (red), cleaved caspase 3; right column, merged images.
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Figure 1: Knockdown of Scribble promotes cisplatin-related drug resistance.A, apoptotic cells were quantified in Scribble knockdown (KD) cells in the CRL-1848 cell line following cisplatin treatment (15 μg/ml, 24 h). Apoptosis was measured using an annexin V apoptosis detection kit and expressed as the percentage of total cells that were in late apoptosis or were both annexin V-PE and PI positive. Left side was the Scribble KD efficiency examined by Western blot. B, cell cycle distributions was measured using flow-cytometric analysis in Scribble KD cells in CRL-1848 cell line when compared with control cells under cisplatin treatment. C, apoptosis was measured using annexin V detection kit in Scribble overexpression (O.E.) cells in the HTB-177 cell line, left side was the Scribble overexpression efficiency examined by Western blot. D, cell cycle distributions were measured using flow-cytometric analysis in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment. E, the CRL-1848 cell line Scribble KD or control cells were treated with different concentrations of cisplatin for 24 h. Cell viability was determined using an MTT assay; cell viability was expressed as the percent of total cell number. F, immunoblots of untreated and 24-h cisplatin-treated cells (15 μg/ml, 24 h) in the CRL-1848 cell line control and Scribble KD cells. Primary antibodies were specific for Scribble, cleaved PARP, cleaved caspase 3 or GAPDH (loading control). G, immunofluorescence of Scribble KD or control cells following 24 h of cisplatin exposure in the CRL-1848 cell line. Left column (blue), DAPI (nuclei); middle column (red), cleaved caspase 3; right column, merged images.

Mentions: To test the effects of the polarity gene Scribble on the cellular process of apoptosis, we generated stable Scribble KD gene expression in different cancer cell lines. We observed that there was no significant difference in the percentage of apoptosis between Scribble KD cells and control cells by general culture conditions. The platinum chemotherapeutic agent cisplatin is the first-line for lung cancer patients (24). Surprisingly, following cisplatin treatment, Scribble KD lines exhibited significant attenuation in the percentage of apoptotic cells compared with control cells (Fig. 1A). Meanwhile, flow-cytometric analysis documented that G1 phase, S phase, and G2/M phase remained almost the same in control cells, Scribble KD cells, and Scribble overexpression cells. Consistent with previous data in mammary gland cells (9), it indicated that Scribble low expression had no significant effect on the regulation of cell cycle and proliferative activity (Fig. 1B). Moreover, cells engineered to overexpress Scribble exhibited significantly increased levels of apoptosis upon cisplatin treatment (Fig. 1C). Meanwhile, there is no significant difference in cell cycle distributions in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment (Fig. 1D). To determine whether Scribble KD cells developed resistance to cisplatin, cells were treated with different concentrations of cisplatin for 24 h. A cell viability assay revealed that the percentage of surviving cells was enhanced by Scribble KD (Fig. 1E). The 50% inhibition concentration (IC50) for cisplatin in control and Scribble KD cells was 16.28 ± 0.06 and 26.33 ± 6.08 μg/ml, respectively. These data suggested that loss of Scribble can enhance cellular drug resistance to cisplatin.


Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Knockdown of Scribble promotes cisplatin-related drug resistance.A, apoptotic cells were quantified in Scribble knockdown (KD) cells in the CRL-1848 cell line following cisplatin treatment (15 μg/ml, 24 h). Apoptosis was measured using an annexin V apoptosis detection kit and expressed as the percentage of total cells that were in late apoptosis or were both annexin V-PE and PI positive. Left side was the Scribble KD efficiency examined by Western blot. B, cell cycle distributions was measured using flow-cytometric analysis in Scribble KD cells in CRL-1848 cell line when compared with control cells under cisplatin treatment. C, apoptosis was measured using annexin V detection kit in Scribble overexpression (O.E.) cells in the HTB-177 cell line, left side was the Scribble overexpression efficiency examined by Western blot. D, cell cycle distributions were measured using flow-cytometric analysis in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment. E, the CRL-1848 cell line Scribble KD or control cells were treated with different concentrations of cisplatin for 24 h. Cell viability was determined using an MTT assay; cell viability was expressed as the percent of total cell number. F, immunoblots of untreated and 24-h cisplatin-treated cells (15 μg/ml, 24 h) in the CRL-1848 cell line control and Scribble KD cells. Primary antibodies were specific for Scribble, cleaved PARP, cleaved caspase 3 or GAPDH (loading control). G, immunofluorescence of Scribble KD or control cells following 24 h of cisplatin exposure in the CRL-1848 cell line. Left column (blue), DAPI (nuclei); middle column (red), cleaved caspase 3; right column, merged images.
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Figure 1: Knockdown of Scribble promotes cisplatin-related drug resistance.A, apoptotic cells were quantified in Scribble knockdown (KD) cells in the CRL-1848 cell line following cisplatin treatment (15 μg/ml, 24 h). Apoptosis was measured using an annexin V apoptosis detection kit and expressed as the percentage of total cells that were in late apoptosis or were both annexin V-PE and PI positive. Left side was the Scribble KD efficiency examined by Western blot. B, cell cycle distributions was measured using flow-cytometric analysis in Scribble KD cells in CRL-1848 cell line when compared with control cells under cisplatin treatment. C, apoptosis was measured using annexin V detection kit in Scribble overexpression (O.E.) cells in the HTB-177 cell line, left side was the Scribble overexpression efficiency examined by Western blot. D, cell cycle distributions were measured using flow-cytometric analysis in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment. E, the CRL-1848 cell line Scribble KD or control cells were treated with different concentrations of cisplatin for 24 h. Cell viability was determined using an MTT assay; cell viability was expressed as the percent of total cell number. F, immunoblots of untreated and 24-h cisplatin-treated cells (15 μg/ml, 24 h) in the CRL-1848 cell line control and Scribble KD cells. Primary antibodies were specific for Scribble, cleaved PARP, cleaved caspase 3 or GAPDH (loading control). G, immunofluorescence of Scribble KD or control cells following 24 h of cisplatin exposure in the CRL-1848 cell line. Left column (blue), DAPI (nuclei); middle column (red), cleaved caspase 3; right column, merged images.
Mentions: To test the effects of the polarity gene Scribble on the cellular process of apoptosis, we generated stable Scribble KD gene expression in different cancer cell lines. We observed that there was no significant difference in the percentage of apoptosis between Scribble KD cells and control cells by general culture conditions. The platinum chemotherapeutic agent cisplatin is the first-line for lung cancer patients (24). Surprisingly, following cisplatin treatment, Scribble KD lines exhibited significant attenuation in the percentage of apoptotic cells compared with control cells (Fig. 1A). Meanwhile, flow-cytometric analysis documented that G1 phase, S phase, and G2/M phase remained almost the same in control cells, Scribble KD cells, and Scribble overexpression cells. Consistent with previous data in mammary gland cells (9), it indicated that Scribble low expression had no significant effect on the regulation of cell cycle and proliferative activity (Fig. 1B). Moreover, cells engineered to overexpress Scribble exhibited significantly increased levels of apoptosis upon cisplatin treatment (Fig. 1C). Meanwhile, there is no significant difference in cell cycle distributions in Scribble overexpression HTB-177 cells when compared with control cells under cisplatin treatment (Fig. 1D). To determine whether Scribble KD cells developed resistance to cisplatin, cells were treated with different concentrations of cisplatin for 24 h. A cell viability assay revealed that the percentage of surviving cells was enhanced by Scribble KD (Fig. 1E). The 50% inhibition concentration (IC50) for cisplatin in control and Scribble KD cells was 16.28 ± 0.06 and 26.33 ± 6.08 μg/ml, respectively. These data suggested that loss of Scribble can enhance cellular drug resistance to cisplatin.

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

Show MeSH
Related in: MedlinePlus