scholarly journals Parthenolide induces autophagy and apoptosis of breast cancer cells associated with the PI3K/AKT/mTOR pathway

2021 ◽  
Author(s):  
Yanyan Han ◽  
Jinfeng Yang ◽  
Yan Sun ◽  
Shujun Fan ◽  
Ying Lu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Underlying Mechanism ◽  
Mtor Pathway ◽  
Dapi Staining ◽  
Anticancer Effects ◽  
Phosphoinositide 3 Kinase

Abstract Background: Breast cancer is an aggressive malignancy that is unresponsive to conventional therapies. Parthenolide has been demonstrated to have anticancer effects against various types of cancer, including breast cancer. The aim of the present study was to investigate the effect and underlying mechanism of parthenolide in human breast cancer. Methods: Autophagy was measured through immunofluorescence and western blotting. DAPI staining and flow cytometry analysis were used to measure apoptosis. Western blot analysis was used to investigate the mechanism of autophagy induced by parthenolide on the expression levels of phosphoinositide 3‑kinase (PI3K), AKT, phosphorylated (p‑) AKT, mammalian target of rapamycin (mTOR), ATG13 and ATG14. Furthermore, apoptosis was confirmed via western blot analysis. Conclusion: Parthenolide inhibits breast cancer cell proliferation and induces autophagy and apoptosis, and suggested that the PI3K/AKT/mTOR pathway serves an important role in autophagy and apoptosis.

Apoptosis induction mediated through PI3-kinase/AKT/mTOR pathway using anti-ROR1 monoclonal antibody in chronic lymphocytic leukemia cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7087-7087
Author(s):  
Amir Hossein Daneshmanesh ◽  
Mohammad Hojat Farsangi ◽  
Ali Moshfegh ◽  
Salam Khan ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Signaling Molecules ◽  
Mtor Pathway ◽  
Lymphocytic Leukemia ◽  
Type I ◽  
Aberrant Expression ◽  
Downstream Signaling

7087 Background: The PI3K/AKT/mTOR is a central pathway activated in many types of cancer. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase regulating cell growth, proliferation and survival. In CLL cells PI3K pathway is constitutively activated leading to AKT activation with subsequent phosphorylation of other downstream signaling molecules. ROR1 is a type I transmembrane RTK, overexpressed and constitutively phosphorylated in CLL. A unique anti-ROR1 mAb directed against CRD region of ROR1 was capable of inducing direct apoptosis as well as dephosphorylating the ROR1 molecule. Here, we investigated the apoptotic effect of the anti-ROR1 mAb and effects on the PI3K/AKT/mTOR pathway using primary CLL cells. Methods: Apoptosis was detected by the MTT assay and Annexin V/PI methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the ROR1 mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-24 h. Results: ROR1 detection on surface of the CLL cells was 80-85% and apoptotic frequency 45-50%. Western blot analysis showed decreased levels of p-AKT, p85 isoform of p-PI3K and p-mTOR in treated compared to untreated samples. No changes in the phosphorylation levels of ERK and PKC proteins were seen. Conclusions: Incubation of CLL cells with the anti-ROR1 mAb induced apoptosis of CLL cells. Apoptosis was preceded by dephosphorylation of PI3K, AKT and mTOR proteins indicating deactivation of these proteins by the ROR1 mAb. In untreated CLL cells no effect was noted. Furthermore no dephosphorylation of PKC or ERK was seen. We suggest that activation of mTOR might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. Further studies are warranted to understand better the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs.

TIPE1 suppresses the invasion and migration of breast cancer cells and inhibits epithelial-to-mesenchymal transition primarily via the ERK signaling pathway

2019 ◽  
Vol 51 (10) ◽  
pp. 1008-1015 ◽  
Author(s):  
Shusheng Qiu ◽  
Wei Hu ◽  
Qiuhong Ma ◽  
Yi Zhao ◽  
Liang Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
And Migration

Abstract Tumor necrosis factor α-induced protein 8-like-1 (TIPE1) functions as an activator or a repressor in a tumor cell type-specific manner. However, the role of TIPE1 in breast cancer, especially regarding metastasis, is unknown. In this study, we aimed to investigate the TIPE1 expression in breast cancer tissues, the biological functions, and the underlying mechanisms of TIPE1 regarding the metastatic properties of breast cancer cells. The results of immunohistochemical staining and western blot analysis indicated that TIPE1 expression was associated with tumor size and lymph node metastasis, and the expression of TIPE1 was downregulated in the tissues of patients with lymph node metastasis. Transwell and wound healing assay results showed that TIPE1 inhibited the invasive and migratory capacities of breast cancer cells. Moreover, the epithelial-mesenchymal transition (EMT) was suppressed in TIPE1-overexpressing cells, as demonstrated by western blot analysis. In addition, western blot analysis also showed that TIPE1 reduced the expression levels of MMP2 and MMP9 and decreased the phosphorylation level of ERK. These results suggested that TIPE1 might suppress the invasion and migration of breast cancer cells and inhibit EMT primarily via the ERK signaling pathway. Our findings revealed the anti-tumor metastasis role of TIPE1 in breast cancer and TIPE1 might be a new candidate prognostic indicator and a potential molecular target for the treatment of breast cancer.

Relationship between BRCA1 promoter methylation and sensitivity of breast cancer cell lines to cisplatin and paclitaxel

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21083-21083 ◽  
Author(s):  
C. Collins ◽  
D. Huo ◽  
J. Xu ◽  
W. K. Bleibel ◽  
M. E. Dolan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cell Lines ◽  
Breast Cancer Cell ◽  
Promoter Methylation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cell Lines ◽  
Brca1 Promoter ◽  
Brca1 Promoter Methylation

21083 Background: While BRCA1 germline mutations are uncommon, and contribute to fewer than 5% of breast cancer cases, epigenetic alterations in BRCA1 occur more frequently. BRCA1 promoter methylation has been detected in 10–30% of breast tumors. Given the role of BRCA1 in DNA repair and cell cycle regulation, we hypothesize that cells with decreased expression of BRCA1 secondary to promoter methylation will be sensitive to DNA damaging agents and resistant to microtubule inhibitors, as has previously been shown for cells deficient in BRCA1 secondary to mutation. Methods: BRCA1 methylation was determined using methylation specific PCR (MSP) as previously described (Wei et al, Cancer Research 2005). The relative sensitivities of BRCA1 methylated, mutated and competent cells to cisplatin and paclitaxel were determined in five representative breast cancer cell lines using the AlamarBlue cytotoxicity assay. Exponentially growing cells were treated with increasing concentrations of cisplatin and paclitaxel for 96 hours. IC50 values and 95% confidence intervals (CI) were calculated from sigmoidal dose response curves fitted with SAS 9.1 Proc NLIN. Western blot analysis for BRCA1 was performed on each cell line. Results: Conclusions: Only one of the two BRCA1 methylated cell lines studied (UACC3199) was sensitive to cisplatin and resistant to paclitaxel, as hypothesized. While both cell lines are methylated, western blot analysis revealed that both express BRCA1, but to a lesser degree than unmethylated cells. BRCA1 methylation, as assessed by non-quantitative MSP, does not correlate with sensitivity to cisplatin and resistance to paclitaxel. Quantification of BRCA1 promoter methylation may better predict chemosensitivity. Identification of the degree of BRCA1 methylation which does correlates with sensitivity to cisplatin and resistance to paclitaxel could improve treatment selection for patients with breast cancer. This work was supported by the US Army Grant W81XWH-04–1-0545. [Table: see text] No significant financial relationships to disclose.

scholarly journals Suppression of CCT3 inhibits the proliferation and migration in breast cancer cells

2020 ◽  
Author(s):  
Gang Xu ◽  
Shanshan Bu ◽  
Xiushen Wang ◽  
He Zhang ◽  
Hong Ge
Keyword(s):  
Breast Cancer ◽  
Signal Transduction ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Signal Transduction Proteins ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background CCT3 is a subunit of chaperonin-containing TCP-1 (CCT), which folds many proteins involved in cancer development and plays an important role in many cancers. However, the role of CCT3 in breast cancer is still unclear. Methods CCT3 expression was knocked down by transfecting breast cancer cells with lentiviral shRNA. The proliferation of breast cancer cells (HCC1937 and MDA-MB-231) was detected by Celigo image cytometry and MTT assay, the migration of the cells was measured by Transwell analysis, cell cycle distribution and apoptosis was detected by flow cytometry, and changes in signal transduction proteins were detected by western blot analysis. Results The expression of CCT3 was significantly suppressed by transduction with lentiviral shRNA; CCT3 knockdown significantly reduced the proliferation and metastasis ability of breast cancer cells (HCC 1937 and MDA-MB-231), increased the proportion of cells in S phase, and decreased the proportion of cells in G1 phase compared to those in shControl cells. There was no significant change in the number of cells in the G2/M phase. Apoptosis analysis showed that knockdown of CCT3 induced apoptosis in breast cancer cells. Western blot analysis showed that the expression of many signal transduction proteins was changed after suppression of CCT3. Conclusion CCT3 is closely related to the proliferation and migration of breast cancer and may be a novel therapeutic target.

Expression and function of the four and a half LIM-only protein 2 (FHL2) in breast cancer

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10109-10109
Author(s):  
B. T. Martin ◽  
K. Kleiber ◽  
M. Kaufmann ◽  
K. Strebhardt
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Reporter Gene ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Soft Agar ◽  
Normal Breast ◽  
Breast Tissues

10109 Background: FHL2 belongs to the expanding family of proteins that possess highly conserved double Zn-finger motifs, CX2CX16–23HX2CX2CX2CX16–21CX2 (C/H/D), and are involved in protein-protein interactions and transcriptional regulation. FHL2 was primarily described as “down regulated in rhabdomyosarcama cells LIM domain protein” (DRAL). Further studies also indicated a connection of FHL2 with the tumor biology. Its expression has been shown to be increased in tumors of epithelial and melanoma cell origin. A recent publication describes the interaction of FHL2 with the breast cancer susceptibility gene 1 (BRCA1) but to date little is known about the function of FHL2 in breast cancer. Methods: Tumor samples of 61 breast cancer patients and 8 normal breast tissues were lysed and subjected to Western blot analysis using a monoclonal FHL2-specific antibody; Tissue specimens of normal breast and breast cancer were histologically examined with the monoclonal antibody used for the Western blot analysis; Luciferase assays were carried out in MCF-7 breast cancer cells, using AP1-dependent reporter plasmids. Anchorage-independent growth was assayed by colony formation in soft agar. Results: FHL2 is overexpressed in almost all human breast cancer tissues tested but not in normal breast tissues and only low levels of FHL2 expression were present in four premalignant ductal carcinoma in situ (DCIS). Co-immunoprecipitation assays and GST-pull down experiments revealed that FHL2 interacts with the proto-oncogene c-jun in breast cancer cells. Reporter gene assays demonstrated that FHL2 represses c-jun-mediated activation of an idealized AP-1-regulated reporter gene in a dose-dependent manner. This repression was also seen on the naturally occurring AP-1-dependent IL2 promoter. The repression domain was identified within the N-terminal half of the FHL2 molecule. Finally, we show that stable expression of FHL2 leads to anchorage-independent growth of breast cancer cells in soft agar. Conclusions: Taken together, our data suggest that by modulating the activity of transcription factors such as c-jun, the expression of FHL2 in breast cancer might contribute to the malignant potential of breast tumors. No significant financial relationships to disclose.

scholarly journals Autophagy and Mitophagy Promotion in a Rat Model of Endometriosis

2021 ◽  
Vol 22 (10) ◽  
pp. 5074
Author(s):  
Rosalba Siracusa ◽  
Ramona D’Amico ◽  
Daniela Impellizzeri ◽  
Marika Cordaro ◽  
Alessio Filippo Peritore ◽  
...  
Keyword(s):  
Western Blot ◽  
Rat Model ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Immunohistochemical Analysis ◽  
Reproductive Age ◽  
Serine Threonine Kinase ◽  
Autophagy Pathway ◽  
The Impact

Endometriosis is a gynecological condition affecting patients in reproductive age. The aim of this paper was to assess the effects of the autophagy and mitophagy induction in a rat model of endometriosis. Endometriosis was induced by the injection of uterine fragments, and rapamycin (0. 5 mg/kg) was administered once per week. One week from the induction, rats were sacrificed, and laparotomy was performed to collect the endometriotic implants and to further process them for molecular analysis. Western blot analysis was conducted on explanted lesions to evaluate the autophagy pathway during the pathology. Elevated phospho-serine/threonine kinase (p-AKT) and mammalian target of rapamycin (mTOR) expressions were detected in vehicle-treated rats, while Beclin and microtubule-associated protein 1A/1B-light chain 3 II (LC3II) expressions were low. Additionally, samples collected from vehicle groups indicated low Bnip3, Ambra1, and Parkin expressions, demonstrating impaired autophagy and mitophagy. Rapamycin administration reduced p-AKT and mTOR expressions and increased Beclin and LC3II, Bnip3, Ambra1, and Parkin expressions, activating both mechanisms. We also evaluated the impact of the impaired autophagy and mitophagy pathways on apoptosis and angiogenesis. Rapamycin was administered by activating autophagy and mitophagy, which increased apoptosis (assessed by Western blot analysis of Bcl-2, Bax, and Cleaved-caspase 3) and reduced angiogenesis (assessed by immunohistochemical analysis of vascular endothelial grow factor (VEGF) and CD34) in the lesions. All of these mechanisms activated by the induction of the autophagy and mitophagy pathways led to the reduction in the lesions’ volume, area and diameter.

scholarly journals Differential Role of Rapamycin in Epidermis-Induced IL-15-IGF-1 Secretion via Activation of Akt/mTORC2

2017 ◽  
Vol 42 (5) ◽  
pp. 1755-1768 ◽  
Author(s):  
Yang Bai ◽  
Rui Xu ◽  
Xueyuan Zhang ◽  
Xiaorong Zhang ◽  
Xiaohong Hu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Low Dose ◽  
Underlying Mechanism ◽  
High Dose ◽  
Mtorc1 Pathway ◽  
Hematoxylin And Eosin

Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism. Methods: C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis. Results: This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway. Conclusion: This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent.

scholarly journals Critical Roles of the PI3K-Akt-mTOR Signaling Pathway in Apoptosis and Autophagy of Astrocytes Induced by Methamphetamine

2019 ◽  
Vol 17 (1) ◽  
pp. 96-104 ◽  
Author(s):  
Han-Qing Liu ◽  
Ya-Wen An ◽  
A-Zhen Hu ◽  
Ming-Hua Li ◽  
Jue-Lian Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Mtor Signaling Pathway ◽  
Induced Apoptosis

AbstractThis study aimed to reveal potential roles of the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in apoptosis and autophagy of astrocytes induced by methamphetamine (METH). A Cell Counting Kit-8 (CCK-8) was used to determine the reduction in proliferation of U-118 MG cells induced by METH. Hoechst 33258 and flow cytometry were used to observe the astrocytes. Western blot analysis was performed to evaluate protein expression and phosphorylation levels. METH inhibited the proliferation of U-118 MG cells and induced apoptosis and autophagy. Western blot analysis showed that the ratio of LC3-II/I was increased, whereas the expression of Bcl-2 was decreased. The phosphorylation cascade of kinases in the PI3K-Akt-mTOR signaling pathway was significantly inhibited by METH exposure, as were proteins downstream of mTORC1, such as p70s6k, rps6, 4EBP1 and eIF4E. METH inhibited proliferation of U-118 MG cells and induced apoptosis and autophagy. The PI3K-Akt-mTOR signaling pathway likely plays a critical role in these effects.

scholarly journals UNBS5162 inhibits colon cancer growth via suppression of PI3K/Akt signaling pathway

2018 ◽  
Vol 34 ◽  
pp. 99-104 ◽  
Author(s):  
Fan Zhang ◽  
Hui-zeng Lv ◽  
Ji-ming Liu ◽  
Xiao-yong Ye ◽  
Cun-chuan Wang
Keyword(s):  
Colon Cancer ◽  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Underlying Mechanism ◽  
Akt Signaling ◽  
Expression Level ◽  
Akt Signaling Pathway ◽  
Hct116 Cells

Colon cancer is a common cause of cancer-related death worldwide. However, the underlying mechanism of tumor progression of colon cancer remains far from being elucidated. In the present study, we report the role of UNBS5162 in colon cancer. UNBS5162 is a naphthalimide that can intercalate into DNA and suppress the expression level of CXCL chemokines. Here, we investigated its effect on cell proliferation, mobility and apoptosis in HCT116 cells, and explored the underlying mechanism. A CCK8 assay revealed that UNBS5162 can block the proliferation of colon cancer cells. Base on a Transwell assay, we showed that cell migration and invasion ability of HCT116 cells are inhibited by UNBS5162. In addition, Annexin V-FITC/PI assay and Western blot analysis were performed to detect whether UNBS5162 could induce cell apoptosis. The results indicated that UNBS5162 increases the number of apoptotic cells remarkably. Furthermore, Western blot analysis demonstrated that UNBS5162 down-regulates the expression level of Bcl2, and up-regulates that of Bax as well as the level of activated Caspase-3. Moreover, we examined the impact of UNBS5162 on PI3K/Akt signaling pathway. UNBS5162 substantially inhibited the phosphorylation of Akt and its downstream effector mTOR, and reduced the expression of p-70. Taken together, these results suggest that UNBS5162 should be considered as a potent therapeutic anticancer agent that targets the PI3K/AKT signaling pathway.

The expression profiles of apoptosis-related genes induced usnic acid in SK-BR-3 breast cancer cell

2020 ◽  
Vol 39 (11) ◽  
pp. 1497-1506
Author(s):  
RŞ Özben ◽  
D Cansaran-Duman
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Western Blot ◽  
Differential Expression ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Usnic Acid ◽  
Apoptosis Pathway

This study aims to determine whether usnic acid (UA) could induce the expression of apoptosis-related genes in apoptosis pathway. The current study has enabled us to better understand the target of UA in the treatment of breast cancer. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Based on the previous study and the results of this study, UA had the most antiproliferative effect on SK-BR-3 breast cancer cell line. We examined differential expression of 88 apoptosis-related genes by quantitative real-time polymerase chain reaction using the apoptosis primary library panel in SK-BR-3 breast cancer cell. We observed a difference in the significant differential expression of 74 apoptosis-related genes in breast cancer after SK-BR-3 cells applied to UA (7.21 µM) for 48 h. The expression level of 56 of these 74 differentiated apoptosis-related genes increased ( p < 0.05), but the expression level of the other 18 related genes decreased ( p < 0.05). In order to evaluate the mechanism of apoptosis of UA, Western blot analysis was performed with Bcl-2, Bax, Caspase-3, and Caspase-9 antibodies. According to the Western blot analysis, we obtained similar results with gene-expression data. These results suggest that UA showed a cytotoxic effect in SK-BR-3 cells through activation of the mitochondrial apoptotic pathway. The obtained results from gene expression revealed that the effect of UA on apoptosis pathway is critical for clinical research.

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