The prognostic evaluation of advanced CRC patients places increased importance on longitudinal peripheral blood immune status. This study aimed to identify prognosis associated longitudinal immune markers and construct dynamic prognostic models for advanced CRC patients with first-line chemotherapy. Metastatic CRC patients treated with standard first-line palliative chemotherapy were retrospectively collected at Shanghai General Hospital from May 2013 to May 2020. Lymphocyte subsets, inflammatory indices, and tumor markers in peripheral blood were repeatedly assessed before each chemotherapy cycle. Joint models were used to identify significant longitudinal prognostic markers. A dynamic prognostic model was established using random forests for time-dependent predictors, and internally validated using tenfold cross-validation. Increased levels of CRP, CEA, CA199, and IL-6, as well as the CD4 + CD29 + cell proportion and the CD4 + CD45RO + /CD4 + ratio were identified as significant risk factors for overall survival (OS) in metastatic CRC patients. Conversely, the increased levels of CD3-CD19 + cell proportion and the CD4 + CD45RA + /CD4 + ratio were identified as favorable factors for OS. A dynamic prognostic model demonstrated good discriminative ability, with AUC values of 0.827, 0.787, 0.726, and 0.693 for 2-, 3-, 4-, and 5-year predictions, respectively. A high ratio of CD4 + CD45RA + /CD4 + before the third to fourth chemotherapy cycle was associated with significantly better OS. Normal CRP and IL-6 levels in the early phase of first-line chemotherapy indicated a good prognosis. This study highlights the prognostic significance of measuring longitudinal immune status in advanced CRC patients and develops an internally validated dynamic prediction model. External validation is needed before clinical implementation.

BACKGROUND AND OBJECTIVE:All-trans retinoic acid (ATRA), an effective differentiation inducer, has been applied clinically to treat acute promyelocytic leukemia (APL). Unfortunately, it is not as potent in other kinds of acute myeloid leukemia (AML). Ethacrynic acid (EA), a classical powerful diuretic, can increase reactive oxygen species (ROS) contents, which can assist ATRA in inducing differentiation in AML cells. Here, we investigated the effect of EA combined with ATRA (EA+RA) on some AML cells except APL.METHODS:Apoptosis and differentiation were determined by morphology, cell viability, Annexin-V assay and CD11c expression. Western blot analysis and the detection of ROS and mitochondrial transmembrane potentials (MMP) were used to investigate the mechanisms.RESULTS:AML cells exhibited differentiation and/or apoptosis after EA+RA treatment. EA+RA increased the intracellular ROS contents. EA+RA-induced apoptosis was accompanied by MMP attenuation and caspase-3/7 activation. EA+RA-induced differentiation was along with MEK/ERK and Akt activation and increased expression of PU.1, CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPε. N-acetyl-L-cysteine (NAC), an antioxidant, thoroughly reduced EA+RA-increased ROS, and also inhibited MMP attenuation, the activation of caspase- 3/7, MEK/ERK and Akt pathways, the elevation of PU.1 and C/EBPs, and apoptosis and differentiation. However, MEK or PI3K specific inhibitors only suppressed EA+RA-triggered differentiation and the elevation of PU.1 and C/EBPs, but not ROS levels.CONCLUSION:EA+RA induced cell apoptosis through ROS dependent MMP attenuation and caspase 3/7 activation while inducing differentiation by ROS-MEK/ERK-PU.1/C/EBPs and ROS-Akt-PU.1/C/EBPs pathways. In summary, it may provide innovative ATRA-based combination therapy strategies for AML patients via ROS.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid–liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild‐type (WT) NBs, PML A216V derived from arsenic‐resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled‐coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS‐crippled and uncrippled NBs showed aggregation‐ and ring‐like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS‐driven NB formation was the prerequisite for partner recruitment, post‐translational modifications (PTMs), and PML‐driven cellular regulations, such as ROS stress control, mitochondria production, and PML‐p53‐mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.

BACKGROUND AND OBJECTIVE:All-trans retinoic acid (ATRA) is only effective in acute promyelocytic leukemia (APL), but not in other subtype of acute myeloid leukemia (AML). Salinomycin targets tumor cells rather than non-tumorigenic cells, and WNT/β-catenin pathway inhibition is one of the mechanisms of its anti-tumor activity. There is a crosstalk between RA and WNT/β-catenin pathway. Here, we investigate the effect of the combination of salinomycin and ATRA (S+RA) in non-APL AML cells.METHODS:Apoptosis was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11c expression and morphology. To explore the underlying mechanisms, Western blot analysis and mitochondrial transmembrane potentials (ΔΨm) were used.RESULTS & DISCUSSION:S+RA induced differentiation and apoptosis in AML cell lines and AML primary cells. S+RA inhibited the β-catenin signal pathway as determined by the decreased protein levels of β-catenin, the low-density lipoprotein receptor-related proteins 6 (LRP6), and its downstream proteins such as survivin, c-Myc, caspase-3/7, cdc25A and cyclinD1 and reduced phosphorylation level of GSK3β S9. S+RA also increased the protein levels of CCAAT/enhancer-binding proteins (C/EBPs) and PU.1 and collapsed Δψm. The above molecular and cellular changes induced by S+RA were inhibited by β-catenin specific activator and promoted by β-catenin specific inhibitor.CONCLUSION:S+RA induced differentiation by β-catenin-inhibition-mediated up-regulation of C/EBPs and PU.1 and suppression of c-Myc. S+RA triggered apoptosis through β-catenin-inhibition-regulated ΔΨm collapse and caspase-3/7 activation. Taken together, our findings may provide novel therapeutic strategies for AML patients by targeting the WNT/β-catenin pathway.

目的:研究低剂量索拉非尼与全反式维A酸(all-trans retinoic acid,ATRA)联合,对野生型Fms样酪氨酸激酶3(Fms like tyrosine kinase3,FLT3)的急性髓系白血病(acute myeloid leukemia,AML)细胞的效应及机制.方法:以野生型FLT3的AML细胞系HL-60、U937以及ATRA耐药的HL-60细胞系——HL-60Res为体外模型,以细胞表面分化抗原CD11b和形态学观察评估细胞分化;应用蛋白质印迹法研究Raf蛋白激酶、促分裂原活化的蛋白激酶(mitogenactivated protein kinase,MEK)和胞外信号调节激酶(extracellular signal-regulated kinase,ERK)的活化状态、PU.1、C/增强子结合蛋白(enhancer-binding protein,EBP)β和C/EBPε的蛋白含量.结果:低剂量(0.1～0.5μmol/L)索拉非尼促进ATRA诱导HL-60、U937和HL-60Res 3株细胞系分化.两药联合活化Raf、MEK和ERK,并上调PU.1、C/EBPβ和C/EBPε的蛋白含量,MEK的特异性抑制剂曲美替尼可抑制两药诱导的细胞分化、MEK/ERK活化以及PU.1、C/EBPβ和C/EBPε的蛋白含量上调.结论:索拉非尼与ATRA联合,通过Raf/MEK/ERK通路上调PU.1、C/EBPβ和C/EBPε的蛋白含量,诱导野生型FLT3的AML细胞分化.

Abstract Background Midostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations. Methods Cell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells. Results In this study, 0.1 − 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 − 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation. Conclusions Midostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

Systemic chemotherapy combining biological targeted therapies is the standard therapy for patients with metastatic colorectal cancer (mCRC), but effective markers are needed to identify clinical responders. Circulating tumour cells (CTCs) have been associated with prognosis in patients with mCRC. This study aimed to explore the relationship between CTC number and the clinical response of patients with advanced CRC.

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib-ATRA (tra-ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra-ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

BackgroundAll-trans retinoic acid (ATRA) is considered to be the sole clinically useful differentiating agent in the treatment of acute myeloid leukemia (AML). However, it has been effective only in acute promyelocytic leukemia (APL) but not other subtypes of AML. Therefore, finding strategies to sensitize cells to ATRA may develop ATRA-based therapy in the treatment of non-APL AML patients.MethodsCell proliferation was assessed by cell growth. Cell death was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11b expression and morphology. To explore the underlying mechanisms, we studied the role of PKCβ, MEK, ERK, AKT, PU.1, C/EBPβ and C/EBPε by Western-blotting analysis.ResultsIn this study, a clinically achievable concentration of enzastaurin enhanced ATRA-induced differentiation of AML cell lines, HL-60 and U937 as well as non-APL AML primary cells, while it also restored ATRA sensitivity in ATRA-resistant cell line, HL-60Res. Mechanistically, in all these cell lines, enzastaurin-ATRA (enz-ATRA) enhanced the protein levels of PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε. The activity of protein kinase C β (PKCβ) was suppressed by enz-ATRA treatment in HL-60 and HL-60Res cells. However, another PKCβ-selective inhibitor mimicked the cellular and molecular effects of enzastaurin only in HL-60 cells. Only in U937 cells, enz-ATRA activated MEK and ERK, and a MEK specific inhibitor suppressed enz-ATRA-triggered differentiation and reduced the protein levels of PU.1, C/EBPβ and C/EBPε. Enz-ATRA activated Akt in HL-60 and HL-60Res cells. However, an Akt inhibitor blocked enz-ATRA-triggered differentiation and restored the protein levels of PU.1, C/EBPβ and C/EBPε only in HL-60Res cells. Therefore, PKCβ inhibition, MEK/ERK and Akt activation are involved in enz-ATRA-induced differentiation in HL-60, U937 and HL-60Res cells, respectively by modulation of the protein levels of C/EBPβ, C/EBPε and PU.1.ConclusionsEnzastaurin, at the clinically achievable concentration, enhances ATRA-induced differentiation of AML cells by PKCβ inhibition, MEK/ERK and Akt activation. This study may provide a potential therapeutic strategy for AML patients.

ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1–2 μm in diameter. The mechanism of PML nuclear body biogenesis remains elusive. Here, PML RBCC is successfully purified. The gel filtration and ultracentrifugation analysis suggest a previously unrecognized sequential oligomerization mechanism via PML monomer, dimer, tetramer and N-mer. Consistently, PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces. Structure-based perturbations in these B1 interfaces not only impair oligomerization in vitro but also abolish PML sumoylation and nuclear body biogenesis in HeLa Pml -/- cell. More importantly, as demonstrated by in vivo study using transgenic mice, PML-RARα (PR) F158E precludes leukemogenesis. In addition, single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. Altogether, these results not only define a previously unrecognized B1-box oligomerization in PML, but also highlight oligomerization as an important factor in carcinogenesis.

All-trans retinoic acid (ATRA) resistance continues to be a critical problem in acute promyelocytic leukemia (APL)-relapsed patients. In this study, a clinically achievable concentration of enzastaurin synergized with ATRA to induce differentiation and apoptosis in ATRA-resistant APL cell lines, NB4-R1 and NB4-R2. Mechanistically, although enzastaurin is a protein kinase Cβ (PKCβ) inhibitor, PKCβ may not be required since the activity of PKCβ was not suppressed by enzastaurin-ATRA (enz-ATRA) co-treatment, and another PKCβ-selective inhibitor did not mimic the effects of enzastaurin. An MEK inhibitor but not a RAF-1 inhibitor suppressed enz-ATRA treatment-triggered differentiation, activation of MEK/ERK and up-regulation of CCAAT/enhancer binding protein β (C/EBPβ) and/or PU.1. Therefore, RAF-1-independent MEK/ERK signaling was required for enz-ATRA treatment-induced differentiation via modulation of the protein levels of C/EBPβ and/or PU.1. Enz-ATRA treatment collapsed mitochondrial transmembrane potential without the activation of caspase-3, -6 and -7. Moreover, caspase-3/7- and caspase-6-specific inhibitors had no inhibitory effect on enz-ATRA treatment-triggered apoptosis. Therefore, enz-ATRA treatment-induced apoptosis was mitochondria-dependent but caspase-independent. Enz-ATRA treatment degraded PML-RARα, which may be involved in enz-ATRA treatment-induced dual effects and may also be beneficial for APL eradication. These findings may provide a potential therapy for ATRA-resistant APL patients.

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein β and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein β and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein β and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.

Fibronectin 1 (FN1) is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense, metastasis, and implicated in various biochemical processes. However, its effects on the development and progression of human cancer, especially colorectal cancer (CRC), are unclear. To evaluate the relationship between the expression of FN1 and the histopathologic parameters of patients with CRC or the proliferation, migration, and invasion of colorectal cancer cell lines, we screened FN1 as a new candidate gene which promotes development of CRC, in an independent dataset (The Human Protein Atlas website). Here, we reported that FN1 was elevated in CRC tissues compared with normal colon tissues. Further, FN1 expression level was correlated with age, lymph vascular invasion, and survival rate. Knockdown of FN1 in two CRC cell lines, LOVO, and SW1116, significantly inhibited cell proliferation, migration and invasion, and induced cell apoptosis. Western blot analysis showed that down-regulation of FN1 significantly decreased the expression of Bcl-2, MMP-9, Twist, and increased the expression of Bax, Caspase-3, and E-cadherin in LOVO and SW1116 cells. Then, we found that the protein ITGA5 was identified as a binding partner of FN1 and ITGA5 overexpression reversed FN1-induced tumorigenesis of CRC in vitro. Taken together, FN1 suppressed apoptosis and promoted viability, invasion, and migration in CRC through interacting with ITGA5. FN1 may be a prognostic factor and potential target for CRC treatment.

Purpose The relationship between the pharmacokinetics of irinotecan and outcomes of advanced colorectal cancer is unclear, and few studies have examined individualized irinotecan-based chemotherapy depending on plasma 7-ethyl-10-hydroxy camptothecin (SN-38) levels and dihydropyrimidine dehydrogenase (DPD) activity, particularly for the UGT1A1*6 or UGT1A1*28 heterozygous type. Methods This study retrospectively explored the relationship among plasma SN-38 level 1.5 hours after critical enzyme for irinotecan (CPT-11) administration (CSN-38 1.5h), plasma SN-38 level 49 hours after CPT-11 administration (CSN-38 49h), DPD activity, and clinical outcomes for the UGT1A1*6 and UGT1A1*28 heterozygous types. Results CSN-38 1.5h and CSN-38 49h of the UGT1A1*6 or UGT1A1*28 heterozygous type were close to those of UGT1A1*6 and UGT1A1*28 wild-types; some of those with relatively high CSN-38 1.5h levels obtained better median progression-free survival (mPFS), whereas others with higher CSN-38 49h concentrations showed a relatively high incidence of adverse reactions possibly because of the decreased activity of DPD. Conclusion Increasing the dosage of CPT-11 according to CSN-38 1.5h may improve the efficacy in patients with lower CSN-38 1.5h levels. For cases with comparably low DPD activity, advisable primary and subsequent dose adjustment of 5-fluorouracil based on plasma 5-fluorouracil levels may be a practical strategy for reducing the occurrence of adverse reactions for personalized treatment of the UGT1A1*6 or UGT1A1*28 heterozygous type.

Objective To investigate the role of phosphoribosyl pyrophosphate synthetase 2 (PRPS2) in lung cancer cell proliferation, tumor growth in vitro, and tumorigenic ability in vivo.Methods Lung cancer A549 cells cultured in vitro were divided into 3 groups: the control group (scramble control shRNA lentivirus infecting cells for 12 h), Lenti-sh1 group (lentivirus Lenti-sh1 infecting cells for 12 h) and Lenti-sh2 group (lentivirus Lenti-sh2 infecting cells for 12 h).These results indicated that the mRNA and protein expression levels of PRPS2 were effectively reduced in the control group as compared with that of the other two groups (both P<0.05).These results indicated that the expression levels of PRPS2 were reduced by PRPS2 shRNA lentivirus in A549 lung cancer cells.Colony formation assay was used to detect the clone number of three groups.The cell proliferation rate was evaluated by a CCK8 assay and cell cycle was detected by flow cytometry.RT-PCR and Western blotting were used to detect the mRNA and protein expression levels of APC, β-catenin, Gsk3β in A549 cells of WNT signaling pathway.Tumors were generated by subcutaneously injecting lung cancer A549 cells from the control, Lenti-sh1 and Lenti-sh2 groups into the right forelimb axillaries of Balb/c nude mice (each group contained 6 mice).The mice were sacrificed at the 6th week to evaluate the weight and volume of tumors.Results Compared with the control group, the number of cell colonies in the Lenti-sh1and Lenti-sh2 groups were reduced (P<0.05).The absorbance value of the Lenti-sh1and Lenti-sh2 groups at 72 and 96h was lower than that of the control group (P<0.05).A significantly higher percentage of cells accumulated in the G0/G1 phase of the cell cycle in the Lenti-sh1and Lenti-sh2 groups as compared with that of the control group.However, the percentage of cells in the S and G2/M phase was lower than that of the control group (all P<0.05).The mRNA and protein levels of APC and β-catenin in the Lenti-sh1and Lenti-sh2 groups were lower than that of the control group but the mRNA and protein levels of Gsk-3β were higher than that of the control group (all P<0.05).The tumor weight and volumes of the the Lenti-sh1and Lenti-sh2 groups decreased significantly as compared with those of the control group (both P<0.05).Conclusion Silencing PRPS2 gene may suppress lung cancer growth, proliferation in vitro, and tumorigenic ability in vivo by regulating the WNT signaling pathway.

All-trans retinoic acid (ATRA) resistance has been a critical problem in acute promyelocytic leukemia (APL) relapsed patients. In this study, dasatinib synergized with ATRA to trigger differentiation in ATRA-resistant APL cell lines. The combined treatment activated RAF-1, MEK and ERK as well as enhanced ATRA-promoted up-regulation of the protein level of PU.1, C/EBPβ and C/EBPε. U0126 (MEK specific inhibitor) and sorafenib tosylate (RAF-1 specific inhibitor) suppressed the combined treatment-induced differentiation, ERK phosphorylation and the up-regulation of C/EBPs and PU.1. Sorafenib tosylate also attenuated the MEK activity. However, the combined treatment did not enhance Ras activity and Ras inhibitor neither blocked MEK activation nor inhibited differentiation. Therefore, the combined treatment induced differentiation via Ras independent RAF-1/MEK/ERK. Earlier than RAF-1 activation, dasatinib suppressed Lyn activity, the predominant activated Src family kinase (SFK) and dephosphorylated RAF-1 at S259. Furthermore, SFK inhibitor, PP2 did suppress Lyn activity and mimicked the effect of dasatinib on ATRA-induced differentiation as well as decreased phosphorylation of RAF-1 at S259. Thus, it was suggested that Lyn inhibition might activate RAF-1 by the dephosphorylation of RAF at S259 and lead to differentiation. In conclusion, the combination of dasatinib and ATRA could overcome ATRA resistance through Lyn inhibition-mediated activation of RAF-1/MEK/ERK.

MicroRNAs (miRNAs) are short, non-coding and endogenous RNAs that played as important roles in the proliferation and metastasis of tumors. In this study, we determined the role of miR-18a in the regulation of HCC cell motility. We showed that miR-18a expression was upregulated in human HCC tissues and cell lines. Moreover, Elevated expression of miR-18a promoted the HCC cell proliferation and migration. KLF4 was identified as a direct target of miR-18a in HCC cells. Furthermore, overexpression of KLF4 attenuated the effects of miR-18a on the regulation of HCC cell motility. The expression of KLF4 was negatively associated with the expression of miR-18a expression in HCC tissues. We also showed that the cell cycle inhibitor p21 was aberrantly downregulated in HCC cells, whereas this inhibition was reversed by miR-18a inhibitor. These data indicated that miR-18a may play a positive role in hepatocellular carcinoma by promoting the proliferation and migration of HCC cells through targeting KLF4 as well as downstream p21.

ABSTRACT It remains uncertain whether there is an correlation between clinical pharmacokinetic parameters and outcomes for metastatic colorectal cancer especially with UGT1A1 *28 and *6 wild type (*1/*1-*1/*1) for serious events associated with Irinotecan are largely excluded. This study retrospectively analyzed the relationship between Irinotecan metabolite levels and outcomes of UGT1A1 *1/*1-*1/*1 genotype arrangement. Blood samples (n = 244) were collected for analysis of plasma DPD activity (before first chemotherapy) and SN-38 levels (1.5 and 49 hour after CPT-11 administration). Clinical variables such as toxicity and outcomes were then assessed. Of the *1/*1 -*1/*1 genotype combination, the median progression free survival of the CSN-38 1.5 h > 50.24 ng/ml subset was remarkably longer than that of the CSN-38 1.5 h ≤ 50.24 ng/ml subset. However, there were no differences between the CSN-38 49 h > 15.25 ng/ml subgroup and the ≤ 15.25 ng/ml group. It was lower DPD activity that responsible for the relatively higher incidence of bone marrow hypocellular, diarrhea, and oral mucositis in the CSN-38 1.5 h > 50.24 ng/ml and CSN-38 49 h > 15.25 ng/ml subsets. Therefore, plasma SN-38 levels is related to outcomes for UGT1A1 *1/*1-*1/*1 genotype, to improve efficacy, patients with CSN-38 1.5 h lower than 50.24 ng/ml, CPT-11 dosage could be added in next chemmotherapy on SN-38 plasma level monitoring. Additionally, in patients with DPD activity below 3.18 before treatment, appropriate reduction of 5-FU dose could be considered to minimize the incidence of adverse events.

Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia (AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinase C (PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA‑induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen‑activated protein kinase kinase (MEK) and extracellular signal‑regulated kinase (ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer‑binding protein β (C/EBPβ) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine‑enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA‑induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

目的:探讨全反式维A酸(all-trans retinoic acid,ATRA)诱导U937细胞分化的可能机制.方法:以急性单核细胞性白血病细胞株U937细胞为体外模型,以细胞表面分化抗原CD11b、四唑氮蓝(nitro-blue tetrazolium,NBT)还原实验和形态学观察,评估细胞分化.应用蛋白印迹法,检测ATRA对C/EBPβ、C/EBPe和PU.1蛋白表达水平的影响.结果:MEK特异性抑制剂U0126预处理不影响ATRA诱导U937细胞分化.蛋白印迹检测显示,ATRA可提高PU.1、C/EBPβ和C/EBPε蛋白水平.结论:ATRA诱导U937细胞分化可能与PU.1、C/EBPβ、C/EBPe的蛋白表达调控相关,而并不涉及MEK-ERK信号转导途径.