Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway
Authors: Liqiang Qian
Guoliang Wu
Zheng Wang
Genhai Shen
Yan Liu
Zhimin Li
Tingting Bi
Quangen Gao
Journal: Cytotechnology (2016)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Apoptosis; EGFR; Gallbladder carcinoma; Invasion; Lupeol
Affiliations: Department of General Surgery, Wujiang No.1 People's Hospital, Suzhou, 215200, China., Graduate School, Xuzhou Medical College, Xuzhou, 221004, China., Department of General Surgery, Wujiang No.1 People's Hospital, Suzhou, 215200, China. .

Abstract:  The cytostatic drug from fruits and other plant derived products have acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Lupeol, a dietary triterpene, present in many fruits and medicinal plants, has been shown to possess many pharmacological properties including anti-cancer effect in both in vitro and in vivo assay systems. However, the cancer proliferative and invasive inhibitory effects and molecular mechanisms on gallbladder carcinoma GBC-SD cells have not been studied. In the present study, GBC-SD cells were treated by lupeol and subjected to methyl thiazolyl tetrazolium analysis, Hoechst 33342 staining, annexin V/propidium iodide double-staining, transwell chamber assay and Western blot analysis. In addition, GBC-SD xenograft tumors were established in male nude BALB/c mice, and lupeol was intravenously administered to evaluate the anti-cancer capacity in vivo. Our results showed that lupeol inhibited the proliferation, migration, invasion and induced apoptosis of GBC-SD cells in a dose-dependent manner in vitro. Furthermore, the expression of p-EGFR, p-AKT and MMP-9 levels were significantly down-regulated. These protein interactions may play a pivotal role in the regulation of apoptosis and invasion. More importantly, our in vivo studies showed that administration of lupeol decreased tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the down-regulation of p-EGFR and MMP-9 in tumor tissues following lupeol treatment, consistent with the in vitro results. Taken together, our findings indicated that lupeol can induce apoptotic cell death and inhibit the migration as well as invasion of GBC-SD cells. The mechanism may be associated with the suppression of EGFR/MMP-9 signaling. These results might offer a therapeutic potential advantage for human gallbladder carcinoma chemoprevention or chemotherapy. 

Retracted: No

PubPeer comments:
 Figure 5 appears to have two sets of images which overlap. *** ![file] , 
 *[left]* Fig. 8.  Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification x400). bu2013d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification x400). *[right]* Fig. 8 from [Liu et al (2015)] .  Lupeol downregulates p-AKT, p-ERK, and PCNA in PCNA-1 xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification, u00d7400). bu2013d The expression of p-AKT, p-ERK, and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification, u00d7400). Unexpected overlaps between different conditions and IHC stains. ![] , 
 *[left]* Fig. 7.  Lupeol inhibits the growth of GBC-SD cells in vivo. a GBC-SD cells were subcutaneously injected into mouse right axillary fossa to establish xenograft models and all mice were divided into four groups (n = 6 per group): control (sterile physiological saline), 5-Fu (20 mg kg), and lupeol (30 and 60 mg kg) treatment groups. Lupeol significantly reduced the tumor weight in vivo. *[right]* Fig. 7 from [Liu et al (2015)] .  Lupeol inhibits the growth of PCNA-1 cells in vivo. The PCNA-1 cells were subcutaneously injected into mouse right axillary fossa to establish xenograft models, and mice were treated with either lupeol (30 and 60 mg kg) or control (positive: 20 mg kg 5-Fu; negative: sterile physiological saline). Lupeol significantly reduced the tumor growth in vivo. Four representatives of eight tumors in each group are shown . ![file] Control or 5-FU? , 
 Thanks so much for pointing out these errors for us. After checking the data, we are so sorry not to notice the duplicated images. During the imageu2019s preparation, multiple photographs of random fields in two directions per sample were taken, and then cut to the same size in the Photoshop. We think that confusion appeared when selecting representative pictures of Figure 5. Also, since the experiments and immunohistochemistry related to these articles were basically carried out at the same time, there was a possibility of partial overlap in the preservation of images and the selection of representative images, and it was easy to make a mistake. we will contact the editor as soon as possible to replace these images. We apologize for these mistakes to the journal editors, reviewers and readers for any inconvenience, Thanks again for your comments. Best Wishes. , 
 Top to bottom: Fig 3a from [Liu et al (2016a)] .  Combination treatment with OMT and 5-Fu-induced cell apoptosis in HCC cells. a The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . At right, panels rotated through 90 and 270 deg. Fig. 3a from [Liu et al (2015)] .  Hoechst staining of PCNA-1 cells treated with 15, 30, and 60 u03bcM lupeol for 36 h. a The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . Fig 3a from [Liu et al (2016b)] .  Oxymatrine treatment induced apoptosis in human hepatoma cells. A, The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . Fig 3 from [Liu et al (2016c)] .  Apoptotic nuclear morphology changes induced by lupeol (15, 30, and 60 lM) treatment for 36 h, were observed by Hoechst 33342 staining in GBC-SD cell lines . Surprisingly many overlaps, given the differences in cell-lines and conditions. ![file] , 
 #2 *[left]* Fig. 8.  Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification x400). bu2013d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification x400). *[right]* Fig. 8 from [Liu et al (2015)] .  Lupeol downregulates p-AKT, p-ERK, and PCNA in PCNA-1 xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification, u00d7400). bu2013d The expression of p-AKT, p-ERK, and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification, u00d7400). ![] In addition, Fig 6d from [Liu et al (2016)] .  Representative images of hematoxylin and eosin (H E) staining of tumor sections . PCNA row. ![file] Unexpected overlaps between different conditions and IHC stains. I have added some rectangles for the PCNA rows. , 
 Retracted [3 October 2022] . --------------------- The Editors-in-Chief have retracted this article at the request of the first author. Concerns were raised regarding a number of figures in this article, specifically. - The invasion panel in Fig. 5a contains overlapping images. The first author has stated that some of the data could not be recovered. The Editors-in-Chief therefore no longer have confidence in the reliability of the data reported in the article. ,