Overexpression of centrosomal protein Nlp confers breast carcinoma resistance to paclitaxel
Authors: Binghe Xu
Weihong Zhao
Qimin Zhan
Yongmei Song
Journal: Cancer Biology & Therapy (2012)
Discipline: CLINICAL MEDICINE
Keywords: Nlp; centrosome maturation; spindle formation; tumorigenesis; breast cancer
Affiliations: Department of Medical Oncology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Abstract:  Nlp (ninein-like protein), an important molecule involved in centrosome maturation and spindle formation, plays an important role in tumorigenesis and its abnormal expression was recently observed in human breast and lung cancers. In this study, the correlation between overexpression of Nlp and paclitaxel chemosensitivity was investigated to explore the mechanisms of resistance to paclitaxel and to understand the effect of Nlp upon apoptosis induced by chemotherapeutic agents. Nlp expression vector was stably transfected into breast cancer MCF-7 cells. With Nlp overexpression, the survival rates, cell cycle distributions and apoptosis were analyzed in transfected MCF-7 cells by MTT test and FCM approach. The immunofluorescent assay was employed to detect the changes of microtubule after paclitaxel treatment. Immunoblotting analysis was used to examine expression of centrosomal proteins and apoptosis associated proteins. Subsequently, Nlp expression was retrospectively examined with 55 breast cancer samples derived from paclitaxel treated patients. Interestingly, the survival rates of MCF-7 cells with Nlp overexpressing were higher than that of control after paclitaxel treatment. Nlp overexpression promoted G2-M arrest and attenuated apoptosis induced by paclitaxel, which was coupled with elevated Bcl-2 protein. Nlp expression significantly lessened the microtubule polymerization and bundling elicited by paclitaxel attributing to alteration on the structure or dynamics of β-tubulin but not on its expression. The breast cancer patients with high expression of Nlp were likely resistant to the treatment of paclitaxel, as the response rate in Nlp negative patients was 62.5%, whereas was 58.3 and 15.8% in Nlp (+) and Nlp (++) patients respectively (p = 0.015). Nlp expression was positive correlated with those of Plk1 and PCNA. These findings provide insights into more rational chemotherapeutic regimens in clinical practice, and more effective approaches might be developed through targeting Nlp to increase chemotherapeutic sensitivity. 

Retracted: No

PubPeer comments:
 Cell image in Figure1B reappeared in Figure3. Could authors give an explanation? ![file] , 
 We thank Takifugu Stictonotusu2019s comment about this work. Figure 1B showed the localization of Nlp in centrosome by transfecting MCF-7 breast cancer cell line with pEGFP-C3 (vector control) and pEGFP-Nlp (Nlp expression vector) respectively. Figure 3 showed the phenomena of microtubules polymerizing into bundles in the vector control cells (transfection with pEGFP-C3) and the Nlp transfected clone cells (transfection with pEGFP-Nlp) treated by paclitaxel at the different concentrations (0nmol L, 10nmol L, and 20nmol L). tBriefly, pEGFP-C3 uff08empty plasmiduff09 was transfected to MCF-7 cells to establish empty vector cell line as control (MCF-7-GFP), and pEGFP-Nlp (containing full length of Nlp cDNA) was transfected to MCF-7 to establish Nlp overexpression stable cell line (MCF7-GFP-Nlp). The stable transfectants were obtained by selection in the medium containing 400 ug ml of G418. After we obtained stable cell lines, we examined the localization of Nlp in centrosome of MCF-7 cells (Figure 1), and also treated cells with different doses of paclitaxel (0nmol L, 10nmol L, and 20nmol L) followed by examining microtubules polymerizing into bundles (Figure 3). In this situation, the vector control cells in figure 1B were same as the vector control cells treated with 0nmol L (means no paclitaxel treatment). Therefore, we used the images of the vector control cells from the Figure 1B as the vector control cells treated by paclitaxel at 0nmol L (no treatment) in Figure 3. , 
 We thank Takifugu Stictonotusu2019s comment about this work. Figure 1B showed the localization of Nlp in centrosome by transfecting MCF-7 breast cancer cell line with pEGFP-C3 (vector control) and pEGFP-Nlp (Nlp expression vector) respectively. Figure 3 showed the phenomena of microtubules polymerizing into bundles in the vector control cells (transfection with pEGFP-C3) and the Nlp transfected clone cells (transfection with pEGFP-Nlp) treated by paclitaxel at the different concentrations (0nmol L, 10nmol L, and 20nmol L). tBriefly, pEGFP-C3 uff08empty plasmiduff09 was transfected to MCF-7 cells to establish empty vector cell line as control (MCF-7-GFP), and pEGFP-Nlp (containing full length of Nlp cDNA) was transfected to MCF-7 to establish Nlp overexpression stable cell line (MCF7-GFP-Nlp). The stable transfectants were obtained by selection in the medium containing 400 ug ml of G418. After we obtained stable cell lines, we examined the localization of Nlp in centrosome of MCF-7 cells (Figure 1), and also treated cells with different doses of paclitaxel (0nmol L, 10nmol L, and 20nmol L) followed by examining microtubules polymerizing into bundles (Figure 3). In this situation, vector control cells in figure 1B should be same as the vector control cells treated with 0nmol L (means no paclitaxel treatment). Therefore, we used the images of the vector control cells from the Figure 1B as the vector control cells treated by paclitaxel at 0nmol L (no treatment) in Figure 3. , 
 #2 #2 We thank Takifugu Stictonotusu2019s comment about this work. Figure 1B showed the localization of Nlp in centrosome by transfecting MCF-7 breast cancer cell line with pEGFP-C3 (vector control) and pEGFP-Nlp (Nlp expression vector) respectively. Figure 3 showed the phenomena of microtubules polymerizing into bundles in the vector control cells (transfection with pEGFP-C3) and the Nlp transfected clone cells (transfection with pEGFP-Nlp) treated by paclitaxel at the different concentrations (0nmol L, 10nmol L, and 20nmol L). tBriefly, pEGFP-C3 uff08empty plasmiduff09 was transfected to MCF-7 cells to establish empty vector cell line as control (MCF-7-GFP), and pEGFP-Nlp (containing full length of Nlp cDNA) was transfected to MCF-7 to establish Nlp overexpression stable cell line (MCF7-GFP-Nlp). The stable transfectants were obtained by selection in the medium containing 400 ug ml of G418. After we obtained stable cell lines, we examined the localization of Nlp in centrosome of MCF-7 cells (Figure 1), and also treated cells with different doses of paclitaxel (0nmol L, 10nmol L, and 20nmol L) followed by examining microtubules polymerizing into bundles (Figure 3). In this situation, vector control cells in figure 1B should be same as the vector control cells treated with 0nmol L (means no paclitaxel treatment). Therefore, we used the images of the vector control cells from the Figure 1B as the vector control cells treated by paclitaxel at 0nmol L (no treatment) in Figure 3. , 
 #4 Out of curiosity, why is the feature circled in yellow in Figure 3 below absent in Figure 1B? Were these images edited to appear differently? *** ![file] ,