Retinoic acid enhances TRAIL-induced apoptosis in cancer cells by upregulating TRAIL receptor 1 expression.
ABSTRACT: Many human cancer cells are sensitive to killing by the proapoptotic ligand TNF-related apoptosis-inducing ligand (TRAIL), which is under study for cancer treatment in clinical trials. The TRAIL receptor (TRAIL-R1; also known as death receptor 4) is a transmembrane receptor that mediates TRAIL-induced apoptosis in cancer cells. In this study, we show that retinoids sensitize cancer cells to TRAIL-induced apoptosis by upregulating expression of TRAIL-R1. All-trans retinoic acid (ATRA) upregulated TRAIL-R1 expression in human cancer cells at the transcriptional level. The ability of ATRA to activate TRAIL-R1 expression was inhibited by retinoic acid receptor (RAR) antagonists or siRNAs, but augmented by several RAR agonists. In analyzing how ATRA induces RAR-dependent transcriptional upregulation of TRAIL-R1, we identified 2 putative retinoic acid response elements termed Pal-17 (a palindrome separated by 17 bases) and DR-11 (a direct repeat separated by 11 bases) in the 5'-flanking region of TRAIL-R1 gene. Deletion of DR-11, but not Pal-17, abrogated the ability of ATRA to stimulate TRAIL-R1 promoter activity. Consistent with this observation, RAR binding to DR-11, but not to Pal-17, was detected by chromatin immunoprecipitation assay in ATRA-treated cells, arguing that DR-11 was responsible for ATRA-mediated activation of the TRAIL-R1 gene. ATRA augmented TRAIL-induced apoptosis of cancer cells, and this activity was attenuated by a blockade to upregulation of TRAIL-R1 expression. Taken together, our findings establish that ATRA accentuates TRAIL-induced apoptosis, reveal a novel mechanism by which retinoids modulate apoptosis, and suggest a novel strategy to augment the anti-cancer activity of TRAIL.
Project description:All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RAR?-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RAR? activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RAR? in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.
Project description:The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia-retinoic acid receptor, alpha fusion protein (PML-RAR?). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates > 80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RAR?). Whereas combination therapy with As2O3 has increased survival further, patients that experience relapse and are refractory to atRA and/or As2O3 is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-xL) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RAR? agonist), and SR11253 (RAR? antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-xL/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment.
Project description:The major oncogenic driver of acute promyelocytic leukemia (APL) is the fusion protein PML-RAR? originated from the chromosomal translocation t(15;17). All-trans retinoic acid (ATRA) and arsenic trioxide cure most patients by directly targeting PML-RAR?. However, major issues including the resistance of ATRA and arsenic therapy still remain in APL clinical management. Here we showed that compound Z-10, a nitro-ligand of retinoid X receptor ? (RXR?), strongly promoted the cAMP-independent apoptosis of both ATRA- sensitive and resistant NB4 cells via the induction of caspase-mediated PML-RAR? degradation. RXR? was vital for the stability of both PML-RAR? and RAR? likely through the interactions. The binding of Z-10 to RXR? dramatically inhibited the interaction of RXR? with PML-RAR? but not with RAR?, leading to Z-10's selective induction of PML-RAR? but not RAR? degradation. Z-36 and Z-38, two derivatives of Z-10, had improved potency of inducing PML-RAR? reduction and NB4 cell apoptosis. Hence, RXR? ligand Z-10 and its derivatives could target both ATRA- sensitive and resistant APL cells through their distinct acting mechanism, and are potential drug leads for APL treatment.
Project description:All-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) administration leads to granulocytic maturation and/or apoptosis of acute promyelocytic leukemia (APL) cells mainly by targeting promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR?). Yet, ~10-15% of APL patients are not cured by ATRA- and ATO-based therapies, and a potential failure of ATRA and ATO in completely reversing PML/RAR?-driven oncogenic alterations has not been comprehensively examined. Here we characterized the in vivo primary responses of dysregulated genes in APL cells treated with ATRA and ATO using a GFP-labeled APL model. Although induced granulocytic differentiation of APL cells was evident after ATRA or ATO administration, the expression of the majority of dysregulated genes in the c-Kit+ APL progenitors was not consistently corrected. Irf8, whose expression increased along with spontaneous differentiation of the APL progenitors in vivo, represented such a PML/RAR?-dysregulated gene that was refractory to ATRA/ATO signaling. Interestingly, Irf8 induction, but not its knockdown, decreased APL leukemogenic potential through driving monocytic maturation. Thus, we reveal that certain PML/RAR?-dysregulated genes that are refractory to ATRA/ATO signaling are potentially crucial regulators of the immature status and leukemogenic potential of APL cells, which can be exploited for the development of new therapeutic strategies for ATRA/ATO-resistant APL cases.
Project description:Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-? fusion (PML-RAR?) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RAR? and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RAR?.Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis.A PML-RAR?- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)? and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBP? and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBP? are newly identified molecular markers for the efficacy of HDACi against APL cells.Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.
Project description:All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells.The effects of ATRA (concentrations from 10-9 to 10-5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10-9 to 10-5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RAR?-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RAR?.RAR? mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RAR? mRNA levels were dose dependent (at concentrations of 10-9 to 10-5 mol/l) with a maximum effect observed at 10-6 mol/l. There were no significant changes in the mRNA levels of RAR? and RAR?. Western blot assay revealed that RAR? protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10-6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RAR? and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RAR? antagonist LE135.ATRA induced upregulation of RAR? in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2.
Project description:With the purpose of creating a multifunctional drug for gastric cancer treatment, a novel all-trans-retinoic acid (ATRA) conjugate with podophyllotoxin (PPT) was designed and synthesized, and its in vitro antiproliferative activity was evaluated against human gastric cancer cell lines using CCK-8 assay. The conjugate, P-A, exhibited significant anticancer activity against MKN-45 and BGC-823 cells with IC50 values of 0.419 ± 0.032 and 0.202 ± 0.055 ?M, respectively. Moreover, P-A efficiently triggered cell cycle arrest and induced apoptosis in MKN-45 and BGC-823 cells due to modulation of cell cycle arrest- (CDK1, CDK2, CyclinA and CyclinB1) and apoptosis- (cleaved caspase-3, -8 and -9) related proteins, respectively. Further mechanism studies revealed that P-A could increase the expression levels of RAR? and RAR?, and decrease the level of RAR? in MKN-45 and BGC-823 cells. Finally, P-A inhibited the ERK1/2 and AKT signaling in the above two cancer cell lines. More importantly, the underlying mechanisms of P-A were similar to those of precursor PPT but different with the other precursor ATRA. Together, the conjugate P-A was a promising candidate for the potential treatment of human gastric cancer.
Project description:The transcription factor Krüppel-like factor 4 (KLF4) plays a critical role in vascular smooth muscle cell (VSMC) differentiation induced by all-trans-retinoic acid (ATRA). Although it has been demonstrated that ATRA stimulation augments both KLF4 protein and mRNA levels in VSMCs, the molecular mechanisms by which ATRA regulates Klf4 transcription are unknown. In this study, we examined the roles of ATRA-selective nuclear retinoic acid receptors (RARs) in the transcriptional regulation of Klf4. The introduction of small interfering RNA and an RAR antagonist demonstrated that RAR?, but not RAR? or RAR?, mediated ATRA-induced Klf4 expression. A luciferase assay for the Klf4 promoter showed that three GC boxes in the proximal Klf4 promoter were indispensible for ATRA-induced Klf4 transcription and that RAR? enhanced Klf4 promoter activity in a GC box-dependent manner. Furthermore, chromatin immunoprecipitation and oligonucleotide pulldown assays demonstrated that the transcription factors KLF4, Sp1, and YB1 directly bound to the GC boxes of the proximal Klf4 promoter. Upon RAR? agonist stimulation, RAR? was recruited to the Klf4 promoter through its interaction with KLF4, Sp1, and YB1 to form a transcriptional activation complex on the three GC boxes of the Klf4 promoter. These results suggest that RAR? serves as an essential co-activator for ATRA signaling and that the recruitment of RAR? to the KLF4-Sp1-YB1 complex, which leads to Klf4 expression in VSMCs, is independent of a retinoic acid response element.
Project description:We recently demonstrated that retinoic acid receptor-? (RAR?) is overexpressed and acts as a tumor promoter in hepatocellular carcinoma (HCC). The oncogenic activity of RAR? is mainly attributed to its physiological interaction with p85? regulatory subunit of PI3K leading to constitutive activation of AKT. Here we report RAR? as a negative regulator of p53 signaling and thus extend the oncogenic potential of RAR? to a new role in controlling the balance between AKT and p53. A natural flavonoid acacetin is then identified to be capable of modulating RAR?-dependent AKT-p53 network. It specifically binds to RAR? and inhibits all-trans retinoic acid (atRA) stimulation of RAR? transactivation. However, the anticancer action of acacetin is independent on its modulation of RAR?-driven transcriptional activity. Acacetin induces cancer cell apoptosis through antagonizing the non-genomic effect of RAR? on AKT and p53. When bound to RAR?, acacetin prevents RAR? from its activation of AKT followed by recovery of the normal p53 signaling. Given the implication of AKT-p53 dysregulation in most HCC, targeting the non-genomic signaling of RAR? that switches AKT-p53 from a pro-survival to a pro-apoptotic program in cancer cells should be a promising strategy for developing novel anti-HCC drugs.
Project description:The refractoriness of acute promyelocytic leukemia (APL) with t(11;17)(q23;q21) to all-trans retinoic acid (ATRA)-based therapy concerns clinicians and intrigues basic researchers. By using a murine leukemic model carrying both promyelocytic leukemia zinc finger/retinoic acid receptor-? (PLZF/RAR?) and RAR?/PLZF fusion genes, we discovered that 8-chlorophenylthio adenosine-3', 5'-cyclic monophosphate (8-CPT-cAMP) enhances cellular differentiation and improves gene trans-activation by ATRA in leukemic blasts. Mechanistically, in combination with ATRA, 8-CPT-cAMP activates PKA, causing phosphorylation of PLZF/RAR? at Ser765 and resulting in increased dissociation of the silencing mediator for retinoic acid and thyroid hormone receptors/nuclear receptor corepressor from PLZF/RAR?. This process results in changes of local chromatin and transcriptional reactivation of the retinoic acid pathway in leukemic cells. Meanwhile, 8-CPT-cAMP also potentiated ATRA-induced degradation of PLZF/RAR? through its Ser765 phosphorylation. In vivo treatment of the t(11;17) APL mouse model demonstrated that 8-CPT-cAMP could significantly improve the therapeutic effect of ATRA by targeting a leukemia-initiating cell activity. This combined therapy, which induces enhanced differentiation and oncoprotein degradation, may benefit t(11;17) APL patients.