Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells

AIM: To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells.METHODS: 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry.Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine.RESULTS: We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58±5.03 (control 201±27.2, P＜0.05, n = 4) in SMMC 7721-k3 HCC, and to 254±25.04 (control 302±30.1, P＜0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2±3.3 in SMMC 7721-k3 HCC (control 27±13.1), and to 24.3±3.2 in melanoma cells (control 67.5±10.1, P＜0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited.Furthermore, 3 μmol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11±0.94%) more significantly than all-trans retinoic acid (P＜0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27kip1, and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells.CONCLUSION: 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27kip1 expression might be associated with 4-HPR-induced apoptosis.

4-Hydroxy phenyl Retinamide Preferentially Targets FLT3 Mutated Acute Myeloid Leukemia via ROS Induction and NF-κB Inhibition

FMS-like tyrosine kinase 3(FLT3)mutation is strongly associated with poor prognosis in acute myeloid leukemia(AML).Though many FLT3 inhibitors have been developed for clinical application with 34%-56%complete remission rate,patients would develop resistance sooner or later after initial response to tyrosine kinase inhibitors(TKIs),such as gilteritinib.And increasing studies have shown that several resistance related mutations of FLT3 emerged during the AML progression.Thus,further investigation is warranted for these FLT3mu,AML patients to achieve a better treatment outcome.4-Hydroxyphenyl retinamide(4-HPR)has been investigated extensively in animal models and clinical trials as an anticancer/chemopreventive agent and is currently used for protection against cancer development/recurrence,with minimal side effects.In this study,we performed gene-set enrichment analysis and found that down-regulated genes induced by 4-HPR were associated with FLT3-ITD gene sets.CD34+ AML stem/progenitor cells separated from 32 AML samples were treated with 4-HPR.Correlation analysis showed that AML cells with FLT3-ITD genetic alteration were more sensitive to 4-HPR treatment than those without FLT3-ITD.Next,we treated 22 primary AML cells with 4-HPR and found that 4-HPR was more toxic to AML cells with FLT3-ITD.These results indicated that 4-HPR was preferentially cytotoxic to all FLT3-ITD AML cells irrespective of stem/progenitor cells or blast cells.4-HPR-induced reactive oxygen species(ROS)production and NF-kB inhibition might be the reason of 4-HPR selectivity on FLT3 mutated AML cells.

Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in Ectopic and Orthotopic Neuroblastoma Xenografts

Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective. Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malignant neuroblastoma resists traditional treatments and further studies are needed for effective therapeutic interventions. We evaluated synergistic efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells in culture and activation of multiple pathways for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice. Combination of 4-HPR and GST synergistically reduced cell viability, caused subG1 accumulation, increased caspase-3 activity for apoptosis in vitro and reduced tumor growth in vivo. Western blotting indicated that combination therapy down regulated Id2 to induce differentiation, increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 leading to an increase in Bax:Bcl-2 ratio, increased mitochondrial Bax level, caused mitochondrial release of Smac/Diablo, down regulation of the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3, and activation of calpain and caspase-3 in SH-SY5Y xenografts. Accumulation of apoptosis-inducing-factor (AIF) in cytosol and increase in caspase-4 activation suggested involvement of mitochondrial pathway and endoplasmic reticulum (ER) stress, respectively, for apoptosis in SH-SY5Y xenografts. In situ immunofluorescent labelings of SH-SY5Y and SK-N-BE2 xenograft sections showed overexpression of calpain, caspase-12, and caspase-3, and AIF, suggesting induction of mitochondrial caspase-dependent and caspase-independent pathways for apoptosis. Collectively, synergistic effects of 4-HPR and GST induced mitochondrial pathways and also ER stress for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice.

Role of dihydroceramides in the progression of acute-on-chronic liver failure in rats

Background:Previously,dihydroceramide(d18:0/24:0)(dhCer(d18:0/24:0))was reported to be a potential biomarker for acute-onchronic liver failure(ACLF)prognosis.In this study,we further explored the role of dhCer(d18:0/24:0)in the progression of ACLF to validate the biomarker using ACLF rat model.Methods:ACLF rats were sacrificed at 4 and 8 h post-D-galactosamine(D-gal)/lipopolysaccharide(LPS)administration to investigate the liver biochemical markers,prothrombin time and liver histopathology.Change in dhCer and other sphingolipids levels were investigated by high-performance liquid chromatography coupled to tandem mass spectrometry(HPLC-MS/MS).Rats were treated with N-(4-hydroxyphenyl)retinamide(4-HPR)to examine the mortality rate and its role in improving ACLF.Results:LPS/D-gal administration resulted in significant elevation in alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels.Prothrombin time was prolonged and histopathological examination showed abnormality.HPLC-MS/MS results showed total dhCer levels in ACLF group(64.10±8.90 pmol/100 mL,64.22±6.78 pmol/100 mL for 4 and 8 h,respectively)were decreased significantly compared with control group(121.61±23.09 pmol/100 mL)(P<0.05).In particular,dhCer(d18:0/24:0),dhCer(d18:0/20:0),and dhCer(d18:0/22:0)levels were decreased.Treatment with 4-HPR significantly increased the levels of dhCers,including dhCer(d18:0/24:0)compared with ACLF group,for the level of dhCer(d18:0/24:0)in 4-HPR group was 20.10±8.60 pmol/100 mL and the level of dhCer(d18:0/24:0)in ACLF group was 9.74±2.99 pmol/100 mL(P<0.05).This was associated with reduced mortality rate and prolonged survival time.The ALT and AST in 4-HPR group were significantly decreased compared with ACLF group.The prothrombin time of 4-HPR group(41.49 s)was significantly lower than the prothrombin time of ACLF group(57.96 s)(P<0.05).4-HPR also decreased plasma ammonia levels slightly,as the plasma ammonia levels in 4-HPR group and ACLF group were 207.37±60.43,209.15±60.43 mmol/L,respectively.Further,4-HPR treatment improved histopathological parameters.Conclusions:DhCer,especially dhCer(d18:0/24:0),is involved in the progression of ACLF.Increasing the levels of dhCer can reduce the mortality rate of ACLF rats and alleviate liver injury.