Effects Of All-trans Retinoic Acid Research Articles

Retinoic acid reduces kidney injury by regulating oxidative stress, NRF-2, and apoptosis in hyperoxic mice.

Nuclear factor-erythroid-2-related factor-2 (NRF-2) is a cellular resistance protein to oxidants. We investigated the effect of exogenous all-trans retinoic acid (ATRA) on the antioxidant system and NRF-2 in mice kidneys under hyperoxia-induced oxidative stress. Mice were divided into four groups. Daily, two groups were given either peanut-oil/dimethyl sulfoxide (PoDMSO) mixture or 50 mg/kg ATRA. Oxidative stress was induced by hyperoxia in the remaining groups. They were treated with PoDMSO or ATRA as described above, following hyperoxia (100% oxygen) for 72 h. NRF-2 and active-caspase-3 levels, lipid peroxidation (LPO), activities of antioxidant enzymes, xanthine oxidase (XO), paraoxonase1 (PON1), lactate dehydrogenase (LDH), tissue factor (TF), and prolidase were assayed in kidneys. Hyperoxia causes kidney damage induced by oxidative stress and apoptosis. Increased LPO, LDH, TF, and XO activities and decreased PON1 and prolidase activities contributed to kidney damage in hyperoxic mice. After hyperoxia, increases in the activities of antioxidant enzymes and NRF-2 level could not prevent this damage. ATRA attenuated damage via its oxidative stress-lowering effect. The decreased LDH and TF activities increased PON1 and prolidase activities, and normalized antioxidant statuses are indicators of the positive effects of ATRA. We recommend that ATRA can be used as a renoprotective agent against oxidative stress induced-kidney damage.

Inhibitory effect of all-trans retinoic acid on ferroptosis in BeWo cells mediated by the upregulation of heme Oxygenase-1

IntroductionThis study aimed to explore the association between ferroptosis, a newly identified type of cell death, and the role of retinoic acid in developing pregnancy complications. Therefore, the effects of all-trans retinoic acid (ATRA) on ferroptosis susceptibility in BeWo cells were assessed to understand abnormal placental development. MethodsBeWo cells were used as surrogates for cytotrophoblasts. The effect of ATRA on ferroptosis sensitivity was assessed on BeWo cells pretreated with ATRA or dimethyl sulfoxide (DMSO; control), following which the LDH-releasing assay was performed. The effects of ATRA pretreatment on the antioxidant defense system (including glutathione [GSH], mitochondrial membrane potential, and heme oxygenase-1 [HMOX1]) in BeWo cells were assessed using assay kits, RT-qPCR, and HMOX1 immunostaining. To evaluate the effect of ATRA on BeWo cells, HMOX1 was silenced in BeWo cells using shRNA. ResultsATRA pretreatment increased ferroptosis resistance in BeWo cells. Although with pretreatment, qPCR indicated upregulation of HMOX1, no significant change was observed in the GSH levels or mitochondrial membrane potential. This was corroborated by intensified immunostaining for heme oxygenase-1 protein (HO-1). Notably, the protective effect of ATRA against ferroptosis was negated when HO-1 was inhibited. Although HMOX1-silenced BeWo cells exhibited heightened ferroptosis sensitivity compared with controls, ATRA pretreatment counteracted ferroptosis in these cells. DiscussionATRA pretreatment promotes BeWo cell viability by suppressing ferroptosis and upregulating HMOX1 and this can be used as a potential therapeutic strategy for addressing placental complications associated with ferroptosis.

ATRA regulates myoblast differentiation and fusion through the RARα/Pitx2 signaling pathway, causing abnormal development of PFMs in ARM fetal rats.

Anorectal malformation (ARM) is the most common congenital digestive tract anomaly in newborns, and children with ARM often have varying degrees of underdevelopment of the pelvic floor muscles (PFMs). To explore the effects of RARα and Pitx2 on the development of rat PFMs, we constructed a rat ARM animal model using all-trans retinoic acid (ATRA), and verified the expression of RARα and Pitx2 in the PFMs of fetal rats. Additionally, we used rat myoblasts (L6 cells) to investigate the regulatory roles of RARα and Pitx2 in skeletal muscle myoblast differentiation and their interactions. The results indicated a significant decrease in the expression of RARα and Pitx2 in the PFMs of fetal rats with ARM. ATRA can also decrease the expression of RARα and Pitx2 in the L6 cells, while affecting the differentiation and fusion of L6 cells. Knocking down RARα in L6 cells reduced the expression of Pitx2, MYOD1, MYMK, and decreased myogenic activity in L6 cells. When RARα is activated, the decreased expression of Pitx2, MYOD1, and MYMK and myogenic differentiation can be restored to different extents. At the same time, increasing or inhibiting the expression of Pitx2 can counteract the effects of knocking down RARα and activating RARα respectively. These results indicate that Pitx2 may be downstream of the transcription factor RARα, mediating the effects of ATRA on the development of fetal rat PFMs.

Combined treatment of All-trans retinoic acid with Tamoxifen suppresses ovarian cancer.

Ovarian cancer is a malignant tumor of the female reproductive system, and its mortality rate is as high as 70%. Estrogen receptor α (ERα)-positive ovarian cancer accounted for most of all ovarian cancer patients. ERα can promote the growth and proliferation of tumors. The combined effect of All-trans retinoic acid (ATRA) and tamoxifen was obtained by the combination screening of tamoxifen and compound library by MTS. In addition, colony formation assay, flow cytometry analysis, immunofluorescence staining, quantitative real-time polymerase chain reaction (PCR), western blot, and tumor xenotransplantation models were used to further evaluate the efficacy of tamoxifen and ATRA in vitro and in vivo for ER-α-positive ovarian cancer. In our study, we found that All-trans retinoic acid (ATRA) can cooperate with tamoxifen to cause cell cycle arrest and apoptosis and inhibit ERα-positive ovarian cancer in vivo and in vitro. Further exploration of the mechanism found that ATRA can Inhibit genes related to the ERα signaling pathway, enhance the sensitivity of ERα-positive ovarian cancer cells to tamoxifen, and ascertain the effectiveness of tamoxifen and ATRA as treatments for ovarian cancer with an ERα-positive status. Combination of ATRA and tamoxifen is a new way for the treatment of ERα-positive ovarian cancer.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

Differentiation Syndrome Presenting as Acute Kidney Injury in a Patient of Acute Promyelocytic Leukemia on All-trans Retinoic Acid and Arsenic Trioxide Regimen: A Case Report

Introduction: Acute promyelocytic leukemia (APL), classified as Subtype M3 of acute myeloid leukemia, is known for its relatively favorable prognosis, primarily due to the high effectiveness of all-trans retinoic acid (ATRA) in treating the maturation block. However, it is essential to be aware of potential complications such as ATRA syndrome, which can affect 5%–25% of patients and manifest with symptoms such as fever, interstitial pulmonary infiltration, pleural/pericardial effusion, and acute renal failure. Methods: We present the case of a 47-year-old female with a history of hypertension, who experienced rash, fever, and reduced urine output during the induction phase with ATRA. Her serum creatinine levels exhibited a rapid increase from 2.63 mg/dL on October 07, 2022, to 7 mg/dL on October 18, 2022, despite having normal-sized kidneys and normal corticomedullary differentiation. A renal biopsy conducted on October 20, 2022, revealed features consistent with active tubulointerstitial nephritis, with direct immunofluorescence showing no significant glomerular immune deposits. A total of 17 hemodialysis sessions were performed. Results: A subsequent bone marrow examination conducted on October 26, 2022, demonstrated a hypercellular aspirate with normal morphology. However, due to the persistence of renal failure, a repeat biopsy was performed, revealing severe acute tubular injury. Pulse steroids were initiated, and ATRA was discontinued, resulting in an improvement in renal function. Conclusion: This case highlights an instance of acute, early, and severe differentiation syndrome (DS) occurring after induction therapy in a patient with APL. The DS in this case was particularly severe, leading to renal failure necessitating hemodialysis. The prompt initiation of pulse steroids and discontinuation of ATRA proved to be effective in managing this complication. Clinicians should remain vigilant for DS and its potential complications, including acute kidney injury and acute tubulointerstitial nephritis, when treating patients with APL.

All-trans retinoic acid exhibits anti-proliferative and differentiating activity in Merkel cell carcinoma cells via retinoid pathway modulation.

The limited therapies available for treating Merkel cell carcinoma (MCC), a highly aggressive skin neoplasm, still pose clinical challenges, and novel treatments are required. Targeting retinoid signalling with retinoids, such as all-trans retinoic acid (ATRA), is a promising and clinically useful antitumor approach. ATRA drives tumour cell differentiation by modulating retinoid signalling, leading to anti-proliferative and pro-apoptotic effects. Although retinoid signalling is dysregulated in MCC, ATRA activity in this tumour is unknown. This study aimed to evaluate the impact of ATRA on the pathological phenotype of MCC cells. The effect of ATRA was tested in various Merkel cell polyomavirus-positive and polyomavirus-negative MCC cell lines in terms of cell proliferation, viability, migration and clonogenic abilities. In addition, cell cycle, apoptosis/cell death and the retinoid gene signature were evaluated upon ATRA treatments. ATRA efficiently impaired MCC cell proliferation and viability in MCC cells. A strong effect in reducing cell migration and clonogenicity was determined in ATRA-treated cells. Moreover, ATRA resulted as strongly effective in arresting cell cycle and inducing apoptosis/cell death in all tested MCC cells. Enrichment analyses indicated that ATRA was effective in modulating the retinoid gene signature in MCC cells to promote cell differentiation pathways, which led to anti-proliferative and pro-apoptotic/cell death effects. These results underline the potential of retinoid-based therapy for MCC management and might open the way to novel experimental approaches with other retinoids and/or combinatorial treatments.

All-trans retinoic acid alleviates transmissible gastroenteritis virus-induced intestinal inflammation and barrier dysfunction in weaned piglets

BackgroundTransmissible gastroenteritis virus (TGEV) is one of the main pathogens causing severe diarrhea of piglets. The pathogenesis of TGEV is closely related to intestinal inflammation. All-trans retinoic acid (ATRA) is the main active metabolite of vitamin A, which has immunomodulatory and anti-inflammatory properties. However, it is unclear whether ATRA can alleviate TGEV-induced intestinal inflammation and barrier dysfunction in piglets. This study aimed to investigate the effects of ATRA on growth performance, diarrhea, intestinal inflammation and intestinal barrier integrity of TGEV-challenged piglets.MethodsIn a 19-d study, 32 weaned piglets were randomly divided into 4 treatments: Control group (basal diet), TGEV group (basal diet + TGEV challenge), TGEV + ATRA5 group (basal diet + 5 mg/d ATRA + TGEV challenge) and TGEV + ATRA15 group (basal diet + 15 mg/d ATRA + TGEV challenge). On d 14, piglets were orally administered TGEV or the sterile medium.ResultsFeeding piglets with 5 and 15 mg/d ATRA alleviated the growth inhibition and diarrhea induced by TGEV (P < 0.05). Feeding piglets with 5 and 15 mg/d ATRA also inhibited the increase of serum diamine oxidase (DAO) activity and the decrease of occludin and claudin-1 protein levels in jejunal mucosa induced by TGEV, and maintained intestinal barrier integrity (P < 0.05). Meanwhile, 5 mg/d ATRA feeding increased the sucrase activity and the expressions of nutrient transporter related genes (GLUT2 and SLC7A1) in jejunal mucosa of TGEV-challenged piglets (P < 0.05). Furthermore, 5 mg/d ATRA feeding attenuated TGEV-induced intestinal inflammatory response by inhibiting the release of interleukin (IL)-1β, IL-8 and tumor necrosis factor-α (TNF-α), and promoting the secretion of IL-10 and secretory immunoglobulin A (sIgA) (P < 0.05). Feeding 5 mg/d ATRA also down-regulated the expressions of Toll-like receptors and RIG-I like receptors signaling pathway related genes (TLR3, TLR4, RIG-I, MyD88, TRIF and MAVS) and the phosphorylation level of nuclear factor-κB-p65 (NF-κB p65), and up-regulated the inhibitor kappa B alpha (IκBα) protein level in jejunal mucosa of TGEV-challenged piglets (P < 0.05).ConclusionsATRA alleviated TGEV-induced intestinal barrier damage by inhibiting inflammatory response, thus improving the growth performance and inhibiting diarrhea of piglets. The mechanism was associated with the inhibition of NF-κB signaling pathway mediated by TLR3, TLR4 and RIG-I.Graphical

All-trans Retinoic Acid Increased Transglutaminase 2 Expressions in BV-2 Cells and Cultured Astrocytes.

Activation of microglia and astrocytes has been observed in Alzheimer's disease (AD). Transglutaminase 2 (TG2) is reported to be activated in AD and involved in cell proliferation, differentiation, and inflammation. Moreover, amyloid β (Aβ) aggregation is detected as a characteristic pathology in the AD brain, and is known to be a substrate of TG2. All-trans retinoic acid (ATRA) can modify cell proliferation and differentiation, and is reported to have therapeutic effects on AD pathology. We aimed to assess the effects of ATRA in microglia and astrocytes on TG2 expression and glial functions. After treatment with ATRA, TG2 expression and TG activity were assayed in both murine microglia BV-2 cells and cultured rat brain astrocytes. Endocytosis activity in BV-2 cells and Aβ aggregation by astrocytes conditioned medium were also assessed. In both BV-2 cells and cultured astrocytes, ATRA increased TG2 expression and TG activity. The increase was blocked by AGN194310, an RA receptor antagonist. ATRA enhanced the endocytosis activity in BV-2 cells, and the addition of AGN194310 reversed it. The addition of cystamine, a competitive TG inhibitor, also reduced ATRA-enhanced endocytosis activity. On the other hand, Aβ aggregation was potentiated by ATRA-treated astrocytes conditioned medium compared to control astrocytes conditioned medium. These results suggest that ATRA increased TG2 expression and TG activity via RA receptor in microglia and astrocytes. ATRA-enhanced TGs might be involved in phagocytosis and Aβ aggregation. Adequate control of TGs expression and function in microglia and astrocytes can be an important factor in AD pathology.

POU5F1 promotes the proliferation, migration, and invasion of gastric cancer cells by reducing the ubiquitination level of TRAF6

POU5F1 plays an important role in maintaining the cancer stem cell (CSC) -like properties of gastric cancer (GC) cells. The impact of POU5F1 on the proliferation and metastasis of GC was examined, along with the potential of ATRA as a specific therapeutic agent for GC. The dysregulation of POU5F1 expression in GC tissues was analyzed using public databases and bioinformatics techniques, and the disparity in POU5F1 expression between normal gastric tissues and GC tissues was further assessed through western blot, RT-qPCR, and immunohistochemistry. The present study aimed to investigate the impact of POU5F1 on the proliferation, migration, and invasion of GC cells through both in vivo and in vitro experiments. Additionally, the effects of ATRA on the proliferation, migration, and invasion of GC cells were examined using in vivo and in vitro approaches. Our findings revealed a significant upregulation of POU5F1 in GC tissues, which was found to be associated with a poorer prognosis in patients with GC. Moreover, POU5F1 was observed to enhance the proliferation, migration, and invasion of GC cells in vitro, as well as promote subcutaneous tumor growth and lung metastasis of GC cells in vivo. The overexpression of POU5F1 mechanistically triggers the process of Epithelial-mesenchymal transition (EMT) by down-regulating E-Cadherin and up-regulating N-Cadherin and VIM. POU5F1 hinders the ubiquitination of TRAF6 through negative regulation of TRIM59, thereby facilitating the activation of the NF-κB pathway. Furthermore, the administration of ATRA effectively impedes the proliferation, migration, and invasion of GC cells by suppressing the expression of POU5F1. The upregulation of POU5F1 elicits EMT, fosters the initiation of the NF-κB signaling pathway in GC cells, and stimulates the proliferation, invasion, and metastasis of GC cells. All-trans retinoic acid (ATRA) can impede these POU5F1-induced effects, thereby potentially serving as an adjunctive therapeutic approach for GC.

Decider-2: Prospective Randomized Multicenter Phase III Trial of Decitabine and Venetoclax Administered in Combination with All- Trans Retinoic Acid (ATRA) or Placebo in Patients with Acute Myeloid Leukemia Who Are Ineligible for Induction Chemotherapy

Background and Significance DNA-hypomethylating agent (HMA) based therapy is the treatment backbone for older, medically non-fit AML/MDS pts, and studies have been performed with multiple combination partners over the last years (reviewed by Stomper et al., Leukemia 2021). Recently, the combination of azacitidine or decitabine (DEC) with venetoclax (VEN) has become the novel standard for many of these pts. Based on preclinical evidence of priming of AML cells to all- trans retinoic acid (ATRA) by DEC, we have conducted a randomized phase II trial in 200 elderly and medically non-fit AML pts on the effect of ATRA as add-on to DEC (DECIDER-1, Lübbert et al., J. Clin. Oncol 2020). Median overall survival (OS) was 8.2 months with ATRA v 5.1 months without ATRA (hazard ratio, 0.65; 95% CI, 0.48 to 0.89; P = .006). Notably, ATRA did not add toxicity, the combination was active also in AML with adverse genetics, and it appeared to prolong time to secondary treatment resistance. A randomized open-label phase II trial (DEC -/+ ATRA) is also being conducted in higher-risk MDS pts (Zhou et al., ASH 2021, 539). Investigating a triple combination of HMA+VEN+ATRA is a logical next step. We could demonstrate cooperativity between these 3 drugs in AML cell lines irrespective of TP53 mutation status on cell growth inhibition and apoptosis (Bresser et al., ASH 2022, 11537). Study Design and Methods This academic study is a randomized, prospective, double-blind, placebo-controlled, parallel group, multicenter trial. It is planned to randomize 256 pts in a ratio of 1:1 to the treatment arms at ~35 sites in Germany. The primary objective is to compare the efficacy of ATRA v placebo as add-on to the standard treatment DEC+VEN with respect to OS. Secondary objectives are to compare ATRA v placebo as add-on to DAC+VEN with respect to objective best response (i.e. CR/CRi/MLFS/PR), CR with negative measurable residual disease (MRD), probability of survival with objective best response, health-related quality of life and safety. Sample size calculation is based on the primary endpoint OS. The study should have a power of 80% to demonstrate superiority of ATRA vs. placebo with respect to OS at one-sided level alpha of 2.5% under the assumption of a hazard ratio of 0.67. This requires the observation of 191 deaths. With a recruitment period of 3 years and an additional one year follow-up period, it is necessary to include 230 pts. To account for the possibility of up to 10% of the pts with incomplete follow-up, 230/0.9 = 256 pts will be randomized in total (ratio 1:1). Interim safety analyses will be performed after the 2nd cycle of the 10th randomized pt, and after the 3rd cycle of the 50th, the 100th and the 150 th randomized pt, and monitored by an Independent Data Monitoring Committee. Key inclusion criteria are: age ≥18 years; previously untreated AML (WHO 2016); ineligibility for standard induction chemotherapy; ECOG ≤ 2; WBC &amp;lt;25×109/L (prior cytoreduction allowed). Key exclusion criterion is: acute promyelocytic leukemia. Treatment All pts will be treated with DAC+VEN as standard treatment and will additionally either receive ATRA or placebo (Fig. 1). Study treatment is continued until relapse/progression, hematopoietic cell transplantation, death or unacceptable toxicity (whichever occurs first). Correlative investigations include serial MRD measurement, NGS profiling and apoptosis studies. Registration EUDRACT 2020-005495-36; DRKS 00023646 (P001516). Funding Funded by the German Research Foundation (DFG, project no. 367848349/LU 429/13-1); ATRA is provided free of charge by Cheplapharm (Germany). Figure 1 Overview of the study treatment

ATRA Can Correct Impaired Proplatelet Formation By Regulating HIF-1α/SPHK2/S1P Axis in ITP

Introduction Under normal physiological conditions, the bone marrow (BM) niche is hypoxic, and sustained and consistent oxygen gradients result in stable physiological hypoxia. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. Whether hypoxia gradients regulate megakaryocyte (MK) differentiation and maturation and the possible mechanisms of these actions remain unknown. Our previous data showed that impaired proplatelet formation (PPF) contributed to the development of thrombocytopenia in ITP. Recent studies suggest that the S1P signaling pathway in MKs plays a critical role in PPF ( Blood, 2013; J EXP MED, 2012). To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/SPHK2/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies ( Blood, 2021; Lancet Haematology, 2017; Lancet Haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Methods Thirty patients with newly diagnosed ITP and 30 healthy donors were included in our study. The 30 ITP patients received ATRA at a dose of 10 mg orally twice daily for 12 weeks. Bone marrow samples were taken before the first medication (Day 0) and after 12 weeks of ATRA therapy (Day 85). We employed an imaging cytometry platform to perform a comprehensive quantitative analysis of MK distribution and hypoxic status in the bone marrow microenvironment of ITP. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Results We analyzed the hypoxic status of MKs in the endosteal niche and perivascular niche. In healthy controls, MKs in the perivascular niche exhibited a hypoxic profile, defined by robust HIF-1α expression and strong retention of pimonidazole. MKs in the endosteal region were less hypoxic. These results reflect a spatial hypoxia gradient in the bone marrow microenvironment. In contrast, we found that, irrespective of their location, ITP MKs displayed less hypoxia with no defined spatial gradients. ITP-MKs in the perivascular niche displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF). Metabolomics data revealed a decreased S1P level in ITP patients. Downregulated sphingosine kinase 2 (SPHK2) in MKs suppressed S1P production. S1P was essential for cytoskeletal reorganization and PPF regulation. In addition, we demonstrated that hypoxia inducible factor-1α (HIF-1α) mediated SPHK2 activation and S1P production. These data suggested that a disrupted hypoxia gradient contributed to impaired PPF via the HIF-1α/SPHK2/S1P axis. We then investigated the effect of ATRA on PPF. The ITP patients received ATRA at a dose of 10 mg orally twice daily for 12 weeks. For patients who received ATRA, S1P levels, HIF-1α and SPHK2 expression levels were increased compared with the pretreatment levels. And ATRA corrected impaired PPF and restored cytoskeletal reorganization by upregulating HIF-1α. Conclusions A disrupted hypoxia gradient contributed to impaired PPF in the ITP bone marrow environment via the HIF-1α/SPHK2/S1P axis. The ATRA-induced correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

Metabolic reprogramming involves in transition of activated/resting CD4+ memory T cells and prognosis of gastric cancer.

Little is known on how metabolic reprogramming potentially prompts transition of activated and resting CD4+ memory T cells infiltration in tumor microenvironment of gastric cancer (GC). The study aimed to evaluate their interactions and develop a risk model for predicting prognosis in GC. Expression profiles were obtained from TCGA and GEO databases. An immunotherapeutic IMvigor210 cohort was also enrolled. CIBERSORT algorithm was used to evaluate the infiltration of immune cells. The ssGSEA method was performed to assess levels of 114 metabolism pathways. Prognosis and correlation analysis were conducted to identify metabolism pathways and genes correlated with activated CD4+ memory T cells ratio (AR) and prognosis. An AR-related metabolism gene (ARMG) risk model was constructed and validated in different cohorts. Flow cytometry was applied to validate the effect of all-trans retinoic acid (ATRA) on CD4+ memory T cells. Since significantly inverse prognostic value and negative correlation of resting and activated CD4+ memory T cells, high AR level was associated with favorable overall survival (OS) in GC. Meanwhile, 15 metabolism pathways including retinoic acid metabolism pathway were significantly correlated with AR and prognosis. The ARMG risk model could classify GC patients with different outcomes, treatment responses, genomic and immune landscape. The prognostic value of the model was also confirmed in the additional validation, immunotherapy and pan-cancer cohorts. Functional analyses revealed that the ARMG model was positively correlated with pro-tumorigenic pathways. In vitro experiments showed that ATRA could inhibit levels of activated CD4+ memory T cells and AR. Our study showed that metabolic reprogramming including retinoic acid metabolism could contribute to transition of activated and resting CD4+ memory T cells, and affect prognosis of GC patients. The ARMG risk model could serve as a new tool for GC patients by accurately predicting prognosis and response to treatment.

Anti-tumor activity of all-trans retinoic acid in gastric-cancer: gene-networks and molecular mechanisms

BackgroundGastric-cancer is a heterogeneous type of neoplastic disease and it lacks appropriate therapeutic options. There is an urgent need for the development of innovative pharmacological strategies, particularly in consideration of the potential stratified/personalized treatment of this tumor. All-Trans Retinoic-acid (ATRA) is one of the active metabolites of vitamin-A. This natural compound is the first example of clinically approved cyto-differentiating agent, being used in the treatment of acute promyelocytic leukemia. ATRA may have significant therapeutic potential also in the context of solid tumors, including gastric-cancer. The present study provides pre-clinical evidence supporting the use of ATRA in the treatment of gastric-cancer using high-throughput approaches.MethodsWe evaluated the anti-proliferative action of ATRA in 27 gastric-cancer cell-lines and tissue-slice cultures from 13 gastric-cancer patients. We performed RNA-sequencing studies in 13 cell-lines exposed to ATRA. We used these and the gastric-cancer RNA-sequencing data of the TCGA/CCLE datasets to conduct multiple computational analyses.ResultsProfiling of our large panel of gastric-cancer cell-lines for their quantitative response to the anti-proliferative effects of ATRA indicate that approximately half of the cell-lines are characterized by sensitivity to the retinoid. The constitutive transcriptomic profiles of these cell-lines permitted the construction of a model consisting of 42 genes, whose expression correlates with ATRA-sensitivity. The model predicts that 45% of the TCGA gastric-cancers are sensitive to ATRA. RNA-sequencing studies performed in retinoid-treated gastric-cancer cell-lines provide insights into the gene-networks underlying ATRA anti-tumor activity. In addition, our data demonstrate that ATRA exerts significant immune-modulatory effects, which seem to be largely controlled by IRF1 up-regulation. Finally, we provide evidence of a feed-back loop between IRF1 and DHRS3, another gene which is up-regulated by ATRA.ConclusionsATRA is endowed with significant therapeutic potential in the stratified/personalized treatment gastric-cancer. Our data represent the fundaments for the design of clinical trials focusing on the use of ATRA in the personalized treatment of this heterogeneous tumor. Our gene-expression model will permit the development of a predictive tool for the selection of ATRA-sensitive gastric-cancer patients. The immune-regulatory responses activated by ATRA suggest that the retinoid and immune-checkpoint inhibitors constitute rational combinations for the management of gastric-cancer.

An Efficient Suppression of EGFR and B-Raf mRNA Overexpression in the Lung of Benzo[a]pyrene-induced mice by Cationic Lipo-ATRA Nanoformulation.

The molecular drug all-trans retinoic acid (ATRA) acts on cancer cells via different molecular pathways, but its poor bioavailability in cancer cells limits its potency. This study was, therefore, carried out to analyse the oncogene expressions in the lung tissue of benzo[a]pyrene (B[a]P)-induced mice and compare between free ATRA and cationic liposome nanoformulation (lipo- ATRA) treatments. This study was designed to analyse the changes in the expression levels of epidermal growth factor receptor (EGFR) and B-Raf in the lung tissues of B[a]P-induced mice during the cancer development stage itself and to find the suppressive effect of free ATRA and lipo-ATRA. Lung cancer was induced in mice by oral ingestion of 50mg/kg body weight B[a]P weekly twice for four consecutive weeks. Then, the mice were treated with free and lipo-ATRA (0.60mg/kg) for 30 days via i.v injection. The EGFR and B-Raf gene expressions were analyzed in lung cells by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR). The RT-PCR gene band density and the relative quantity (RQ) values from qPCR revealed both EGFR and B-Raf genes to be significantly overexpressed in B[a]P control mice while having very low or no expression in normal mice. This indicates that they function as oncogenes in B[a]P-induced lung carcinogenesis. The lipo-ATRA treatment has shown a highly significant increase in RQ values for both EGFR and BRaf when compared to the free ATRA treatment. The study results have revealed the cationic lipo-ATRA treatment to have enhanced the bioavailability of ATRA in lung tissue due to its significant suppression action on EGFR-mediated oncogenes' expressions. Furthermore, the EGFR and BRaf could be the molecular targets of ATRA action in lung carcinogenesis.

Targeting the retinoic acid signaling pathway as a modern precision therapy against cancers.

Retinoic acid (RA) is a vital metabolite derived from vitamin A. RA plays a prominent role during development, which helps in embryological advancement and cellular differentiation. Mechanistically, RA binds to its definite nuclear receptors including the retinoic acid receptor and retinoid X receptor, thus triggering gene transcription and further consequences in gene regulation. This functional heterodimer activation later results in gene activation/inactivation. Several reports have been published related to the detailed embryonic and developmental role of retinoic acids and as an anti-cancer drug for specific cancers, including acute promyelocytic leukemia, breast cancer, and prostate cancer. Nonetheless, the other side of all-trans retinoic acid (ATRA) has not been explored widely yet. In this review, we focused on the role of the RA pathway and its downstream gene activation in relation to cancer progression. Furthermore, we explored the ways of targeting the retinoic acid pathway by focusing on the dual role of aldehyde dehydrogenase (ALDH) family enzymes. Combination strategies by combining RA targets with ALDH-specific targets make the tumor cells sensitive to the treatment and improve the progression-free survival of the patients. In addition to the genomic effects of ATRA, we also highlighted the role of ATRA in non-canonical mechanisms as an immune checkpoint inhibitor, thus targeting the immune oncological perspective of cancer treatments in the current era. The role of ATRA in activating independent mechanisms is also explained in this review. This review also highlights the current clinical trials of ATRA in combination with other chemotherapeutic drugs and explains the future directional insights related to ATRA usage.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1

BackgroundSorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms.MethodsCCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues.ResultsWe observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models.ConclusionsOur findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients.2_mPhFeBDQdvWNSurq11yxVideo

All-trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells.

To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells. After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR. Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P<0.001) and promote the expression of MMP-2 and TGF-β2 mRNA (P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group (P=0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated (P<0.001). ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.