Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway
Abstract:BackgroundProhibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored.MethodsFL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB … Show more
“…These results indicate that down regulation of Akt1/2 level in DLBCL cells exposed to FL3 may be mediated at least by reduction of AKT mRNA levels and by a caspase-dependent cleavage of the protein. Finally, as PHBs are known to be substrates of Akt [36, 37], we indirectly analyzed PHB1 phosphorylation and our results strongly suggest decreased PHB phosphorylation in FL3-treated DLBCL cells (Additional file 1: Figure S6). Fig.…”
Section: Resultsmentioning
confidence: 88%
“…In this study we used the flavagline FL3 to gain insight into the cytoplasmic role of PHBs in DLBCL cells. Indeed, by binding to PHB, flavaglines were shown to inhibit PHB-mediated signaling pathways, and this interaction did not target mitochondria [36, 43]. As expected, FL3 triggered apoptosis of DLBCL cell lines and also in vivo in a xenograft murine model.…”
Section: Discussionmentioning
confidence: 91%
“…However, further studies would be needed to precise their relative contribution which could vary according the DLBCL subtype. Interestingly, phosphorylation of PHBs by Akt was recently demonstrated to be required for PHB translocation and localization in the mitochondria, as well as for tumor cell proliferation [36, 37]. It was proposed that, by inhibiting Akt/PHBs interaction, FL3 may decrease the PHB localization within mitochondria leading to mitochondria dysfunction and thus apoptosis.…”
BackgroundDiffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL.MethodsPHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK–MNK–eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect.ResultsAs in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model.ConclusionOur data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.
“…These results indicate that down regulation of Akt1/2 level in DLBCL cells exposed to FL3 may be mediated at least by reduction of AKT mRNA levels and by a caspase-dependent cleavage of the protein. Finally, as PHBs are known to be substrates of Akt [36, 37], we indirectly analyzed PHB1 phosphorylation and our results strongly suggest decreased PHB phosphorylation in FL3-treated DLBCL cells (Additional file 1: Figure S6). Fig.…”
Section: Resultsmentioning
confidence: 88%
“…In this study we used the flavagline FL3 to gain insight into the cytoplasmic role of PHBs in DLBCL cells. Indeed, by binding to PHB, flavaglines were shown to inhibit PHB-mediated signaling pathways, and this interaction did not target mitochondria [36, 43]. As expected, FL3 triggered apoptosis of DLBCL cell lines and also in vivo in a xenograft murine model.…”
Section: Discussionmentioning
confidence: 91%
“…However, further studies would be needed to precise their relative contribution which could vary according the DLBCL subtype. Interestingly, phosphorylation of PHBs by Akt was recently demonstrated to be required for PHB translocation and localization in the mitochondria, as well as for tumor cell proliferation [36, 37]. It was proposed that, by inhibiting Akt/PHBs interaction, FL3 may decrease the PHB localization within mitochondria leading to mitochondria dysfunction and thus apoptosis.…”
BackgroundDiffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL.MethodsPHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK–MNK–eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect.ResultsAs in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model.ConclusionOur data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.
“…Our previous work showed that growth arrest and DNA damage-inducible 45 alpha gene (GADD45a) may play a repressive role in BC cell proliferation by delaying cell cycle progression in the G2/M phase 3. GADD45a was first found and defined by Fornace et al in 1989 when researchers found that some mRNAs were increased after exposure to a variety of exogenous and endogenous stresses associated with growth arrest, including ultraviolet (UV) radiation 4.…”
BackgroundBladder cancer (BC) is one of the most prevalent malignancies of the genitourinary system, yet the underlying mechanism of BC progression still remains unclear. Growth arrest and DNA damage-inducible 45 alpha (GADD45a) is a repressive gene implicated in cell cycle regulation, as well as in human cancers development. However, its role in BC remains to be determined.MethodsFirst, quantitative real-time polymerase chain reaction (PCR) and Western blot assays were used to detect GADD45a expression in BC tissues and adjacent non-tumor tissues, as well as in bladder cancer cell lines, respectively. Then, cell counting kit-8 (CCK-8) assays, colony formation assays, and flow cytometry assays were used to measure the ability of cell growth, proliferation and cell cycle distribution. Lentiviral infection technology was used to increase gene expression, while siRNA interfering technology was used to knockdown gene expression. Finally, nude mice were used to construct tumor-burdened models in vivo by injecting tumor cells subcutaneously.ResultsPCR results showed that the level of GADD45a mRNA and protein levels were lower in BC tissues than in adjacent normal tissues. After increasing GADD45a expression, both the ability of growth and proliferation of BC cells were seriously impaired. Additionally, the upregulation of GADD45a expression resulted in BC cell cycle in G2/M and S phases in a p53-regulated pathway.ConclusionGADD45a-mediated cell cycle inhibition is regulated by p53 in bladder cancer cells.
“…Different mechanisms by which FL3 targets cancer cells were reported in the literature. It was shown that in urothelial carcinoma cells, FL3 can directly binds to Prohibitin 1 (PHB) preventing its phosphorylation by Akt, leading to a decrease of PHB in mitochondria, which causes a mitochondria-related apoptosis and cell cycle inhibition 5 , 6 . PHB is an ubiquitous and evolutionarily conserved protein expressed in different cellular compartments including the nucleus, cytoplasm and mitochondria 7 , it is involved in diverse biological processes such as cell proliferation, resistance to apoptosis, maintenance and integrity of mitochondria 7 , 8 .…”
Glioblastoma (GBM) is one of the most aggressive types of cancer, which begins within the brain. It is the most invasive type of glioma developed from astrocytes. Until today, Temozolomide (TMZ) is the only standard chemotherapy for patients with GBM. Even though chemotherapy extends the survival of patients, there are many undesirable side effects, and most cases show resistance to TMZ. FL3 is a synthetic flavagline which displays potent anticancer activities, and is known to inhibit cell proliferation, by provoking cell cycle arrest, and leads to apoptosis in a lot of cancer cell lines. However, the effect of FL3 in glioblastoma cancer cells has not yet been examined. Hypoxia is a major problem for patients with GBM, resulting in tumor resistance and aggressiveness. In this study, we explore the effect of FL3 in glioblastoma cells under normoxia and hypoxia conditions. Our results clearly indicate that this synthetic flavagline inhibits cell proliferation and induced senescence in glioblastoma cells cultured under both conditions. In addition, FL3 treatment had no effect on human brain astrocytes. These findings support the notion that the FL3 molecule could be used in combination with other chemotherapeutic agents or other therapies in glioblastoma treatments. Flavaglines are natural products isolated from Aglaia genus plants possessing unique anticancer properties. One synthetic flavagline, called FL3, is known for its anticancer effects without being toxic to healthy cells 1,2. Flavaglines were isolated for the first time in 1982 based on their strong anti-leukemic activity 3. Cytotoxic effects of flavaglines has been reported in a lot of cancer cell lines, such as lung, breast, and colon cancer 4 , leading to the inhibition of proliferation and thus inducing cell cycle arrest or apoptosis in tumor cells. Different mechanisms by which FL3 targets cancer cells were reported in the literature. It was shown that in urothelial carcinoma cells, FL3 can directly binds to Prohibitin 1 (PHB) preventing its phosphorylation by Akt, leading to a decrease of PHB in mitochondria, which causes a mitochondria-related apoptosis and cell cycle inhibition 5,6. PHB is an ubiquitous and evolutionarily conserved protein expressed in different cellular compartments including the nucleus, cytoplasm and mitochondria 7 , it is involved in diverse biological processes such as cell proliferation, resistance to apoptosis, maintenance and integrity of mitochondria 7,8. Also, FL3 was shown to selectively kill cancer stem-like cells through the p38 mitogen-activated protein kinase (MAPK)-dependent caspase-3-dependent pro-apoptotic
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
