Clinical efficacy of all-trans retinoic acid for treating adult T cell leukemia

Purpose We previously reported that all-trans retinoic acid (ATRA) inhibited growth in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and in fresh cells from patients with adult T cell leukemia (ATL). Here, we confirmed the clinical effects of ATRA in 20 patients with ATL. Materials and methods The 20 patients (n = 20) with a median age of 56 (range 35–73) years who were diagnosed with ATL received ATRA orally. Results The efficacy of treatment was as follows: no complete response (CR), a partial response (PR) in 40% of the patients, no change (NC) in 45% of the patients, and a progressive disease (PD) in 15% of the patients. In seven acute-type ATL patients, a PR was achieved in two (28.5%), NC was observed in two (28.5%), and a PD was observed in three (42.8%). In three lymphoma-type ATL patients, a PR (100%) was achieved. Among four chronic-type ATL patients, a PR was achieved in one (25%) and NC was observed in the remaining three (75%). In six smoldering-type ATL patients, a PR was achieved in two (33.3%) and NC was observed in four (66.6%). The major side effects were headache (n = 5), transient liver dysfunction (n = 2), hyperlipidemia (n = 2), and anorexia (n = 1). Conclusion These results indicated that ATRA might be a useful agent for the safe treatment of ATL.     

In vitro down-regulation of bcl-2 expression by all-trans retinoic acid in AML blasts

 Using flow cytometry, we have investigated the effects of 0.5 μM all-trans–retinoic acid (ATRA) on bcl-2 expression in the blast cells of 25 acute myeloblastic leukemia (AML) patients and the HL-60 cell line after incubation for 6 days. We observed a significant decrease of bcl-2 expression after treatment with ATRA in 12 of 25 AML samples and the HL-60 cells. The mean fluorescence intensity (MFI) ratio for the bcl-2 levels of the ATRA responders (n=12) was reduced to 7.9±4.8 following incubation with ATRA compared with 10.9±6.5 (mean±SD) for control samples incubated without ATRA (p=0.011). There was no significant difference between the baseline bcl-2 MFI ratio in the ATRA responders (11.14±7, n=12) and the non responders (14.18±11.3, n=13;p=0.432). The down-regulation of bcl-2 expression by ATRA was not significantly associated with CD34-negative or -positive AML. There was no correlation between AML subtypes and regulation of bcl-2 expression by ATRA. Complete remission and overall survival were not significantly improved in bcl-2 down-regulated cases. Our data confirm that ATRA can down-regulate the bcl-2 expression in AML blasts. Because many chemotherapeutic agents also operate through the activation of programmed cell death and bcl-2 levels are positively associated with resistance to apoptosis, ATRA can be used in combination chemotherapy to increase the chemosensitivity of some patients with AML.     

Fatal thromboembolism in acute promyelocytic leukaemia treated with a combination of all-trans retinoic acid and aprotonin

We describe a case of acute promyelocytic leukaemia where the combined use of all-trans retinoic acid and an antifibrinolytic (aprotinin) may have contributed to worsening of coagulopathy.     

全反式维甲酸诱导胰腺癌细胞凋亡机制研究

目的 维甲酸类药物调节肿瘤细胞生长、分化和凋亡是其防治肿瘤的基础。本研究观察全反式维甲酸(ATRA)诱导体外胰腺癌细胞凋亡的可能机制。方法 胰腺癌细胞Patu8988与ATRA共同孵育。通过DNA断裂分析、TUNEL标记证实凋亡存在，并用流式细胞仪检测凋亡细胞比例。用RT-PCR和Western blot方法检测凋亡诱导过程中p53、bcl-2和bax基因的水平及其表达变化。结果 ATRA处理组细胞凋亡比例明显增加。TUNEL标记和DNA断裂分析发现典型凋亡特征。ATRA处理组bax和phospho-p53(Ser 46)表达上调，但bcl-2表达下调。结论 ATRA能诱导胰腺癌细胞凋亡，其分子机制可能与bcl-2/bax和p53的表达相关。     

Down-regulation of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34 antigen expression

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 μM ) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14–0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 ± 6.9 following incubation with ATRA compared to 47.6 ± 8.2 (mean ± SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 ± 5.7, n = 8) and the non-responders (41.3 ± 3.9, n = 17) (mean ± SEM) (P = 0.28). The down- regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients’ cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P<0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.     

A unique case of acute promyelocytic leukemia showing monocytic differentiation after ATRA (all-trans retinoic acid) therapy

Acute promyelocytic leukemia (PML) is characterized by a reciprocal translocation between chromosomes 15 and 17 resulting in a chimeric PML and retinoic acid receptor alpha (RARA) oncogene. The resultant fusion protein (PML/RARA) is thought to block differentiation of bone marrow cells arrested at the promyelocytic stage. In vitro and in vivo studies have shown that the large majority of APL cells undergo granulocytic maturation after ATRA therapy. We report a unique case of a PML/RARA positive APL patient exhibiting extensive monocytic differentiation after ATRA therapy as documented by morphology, flow cytometry, and FISH studies. We discuss potential dual capability for granulocytic/monocytic differentiation of PML/RARA positive APL cells and implications of monocytic differentiation in the management of APL patients treated with ATRA.     

Interleukin 10 abolishes the growth inhibitory effects of all-trans retinoic acid on human myeloma cells

Recently, it was disclosed that all-trans retinoic acid (ATRA) inhibits myeloma cell growth by downregulating the interleukin 6 (IL-6)/IL-6 receptor (IL-6R) auto/paracrine loop, and upregulating p21/Cip1 cyclin-dependent kinase inhibitor (CDK-I), thereby inducing apoptosis with a decrease in Bcl-2 protein expression. To elucidate and generalize the effects of ATRA on the proliferation and cellular biology of myeloma cells, 12 human myeloma cell lines established in our laboratory were utilized. Two out of the 12 lines showed enhanced growth on supplementation of ATRA and were characterized by IL-10 production, downregulation of membrane Fas and reduced upregulation of p21/Cip1 CDK-I message. These characteristics may prove important for the clinical use of ATRA and should be considered before starting ATRA therapy for myeloma.     

急性早幼粒细胞白血病经全反式维甲酸缓解后维持治疗的临床探讨

目的：探索急性早幼粒细胞白血病（APL）经全反式维甲酸（ATRA）治疗达完全缓解（CR）后理想的维持治疗方案，提高APL的长期无病生存时间.方法：将我院21例初治的APL患者均经ATRA治疗达CR后，维持治疗方法为三氧化二砷（As2O3）、ATRA及6-巯基嘌呤（6-MP）加甲氨喋呤（MTX）序贯交替治疗3年;观察治疗的效果及不良反应，监测微小残留病.结果：21例患者对上述治疗均有较好的顺从性，有11例患者维持治疗超过2年，无复发病例;PML/RARα融合基因初诊时均为阳性，12例持续转阴，其中维持治疗超过2年的11例患者均转阴;治疗相关的不良反应少见且较轻.结论：用上述维持治疗经ATRA达CR的APL初步取得了令人满意的效果，其不良反应较传统的联合化疗轻，作者认为As2O3、ATRA与6-MP加MTX序贯交替治疗可能是APL理想的维持治疗方法。     

维甲酸对甲状腺癌细胞钠/碘转运体基因的调节作用

以RTPCR法检测维甲酸对甲状腺癌细胞钠/碘转运体(NIS)基因表达的影响,发现在5～50μmol/L的剂量区间,维甲酸能促进NIS基因表达,提示维甲酸在甲状腺癌及其转移灶的治疗中可能起到重要作用。     

甲胎蛋白激活PI3K/AKT信号促使肝癌Bel 7402细胞抗全反式维甲酸诱导凋亡

目的 研究甲胎蛋白(AFP)对肝癌细胞内PI3K/AKT信号传递的影响,以及其在癌细胞耐受全反式维甲酸(ATRA)中的作用. 方法 四甲基偶氮唑盐(MTT)法检测ATRA对人肝癌Bel 7402细胞增殖的影响;显微照相观察细胞形态学的改变;流式细胞分析细胞凋亡;激光共聚焦显微镜观察AFP与PTEN的共定位;免疫共沉淀(Co-IP)技术研究AFP与PTEN相互作用;Westemblot法分析磷酸化的蛋白激酶B(pAKT)和Src表达,构建干扰AFP表达的载体(AFP-siRNA)并转染Bel 7402细胞;用PI3K特异性阻断剂Ly294002处理细胞,分析Ly294002的作用效果.通过t检验分析组间的统计学差异. 结果 MTT分析显示,人肝癌Bel 7402耐受ATRA的细胞毒性;共聚焦显微镜观察显示AFP与PTEN共定位于细胞质;Co-IP技术研究发现AFP能与PTEN结合;MTT分析发现,Bel 7402细胞转染AFP-siRNA载体后24 h后,细胞生长显著受抑制,与对照组比较,差异有统计学意义(P＜0.05),而且AFP-siRNA载体和ATRA共同处理组,细胞生长受到抑制更为显著,与对照组、单独处理组比较,差异均有统计学意义(P＜ 0.01);干扰AFP表达能显著增加Bel 7402细胞对ATRA敏感性,并能抑制pAKT和Src的表达;Ly294002能抑制AFP促进Bel 7402细胞表达pAKT和Src的作用.结论 肝癌细胞内表达的AFP能与PTEN结合并抑制PTEN对AKT的去磷酸化作用,肝癌细胞内高表达的AFP能激活PI3K/AKT信息通路对抗ATRA诱导的凋亡.     

Simultaneous induction of matrix metalloproteinase-9 and interleukin 8 by all-trans retinoic acid in human PL-21 and NB4 myeloid leukaemia cells

All-trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase-9 (MMP-9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL-21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP-9 were much higher in NB4 cells than in PL-21 cells. Stimulation with ATRA significantly increased MMP-9 levels approximately three- to fivefold in both PL-21 and NB4-conditioned media. MMP-9 mRNA levels increased in ATRA-treated cells and was almost in parallel with the increase in MMP-9 activity, suggesting that ATRA induced MMP-9 by activating its gene expression. ATRA can induce interleukin 8 (IL-8) in APL cells. IL-8, chemokine for neutrophils and a potent inducer of MMP-9, was also induced by ATRA in PL-21 cells. However, recombinant IL-8 did not induce MMP-9 expression. In addition, a neutralizing antibody against IL-8 did not inhibit ATRA-induced MMP-9 expression in either cell type. These observations suggest that ATRA can induce both MMP-9 and IL-8, but IL-8 is not involved in ATRA-induced MMP-9 expression. As MMP-9 can truncate and activate IL-8, simultaneous induction of MMP-9 and IL-8 by ATRA could activate leucocytes excessively, causing the hyper-inflammatory events in retinoic acid syndrome.     

维甲酸诱导分化治疗甲状腺癌的临床研究

目的通过全反式维甲酸(ATRA)诱导分化治疗失分化甲状腺癌,并对其诱导分化的指标及疗效进行评价。方法15例失分化甲状腺癌,其中滤泡状癌5例,乳头状癌8例,滤泡乳头混合性癌2例。口服ATRA1~1.5mg·kg-1·d-1,持续治疗30d或60d,分别测定ATRA诱导分化治疗前后转移灶的摄碘变化、转移灶的大小及血清甲状腺球蛋白(Tg)水平,并进行比较。结果15例患者经ATRA诱导分化治疗,7例病灶的131I摄取增高;7例转移灶缩小;12例测定Tg的患者中,同时具有Tg降低、病灶摄碘增高和病灶缩小或无变化的4例,占33%。结论ATRA对失分化甲状腺癌有一定疗效,并值得进一步研究。     

Inhibition of growth and induction of apoptosis by all-trans retinoic acid in lymphoid cell lines transfected with the PML/RARα fusion gene

The interaction of an exogenous PML/RARα fusion gene associated with acute promyelocytic leukaemia, with all- trans retinoic acid (ATRA) was examined in two lymphoid cell lines. L1210 and MOLT-4 cells were transfected with PML/RARα cDNA in the expression vector pGD and stable transformants (L1210 PML/RAR α and MOLT-4 PML/RAR α) were selected with G418. ATRA inhibited the growth of these stable transformants, as assessed by [³H]thymidine incorporation, in a dose-dependent manner, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also induced apoptosis, as assessed by fragmentation of genomic DNA, in L1210 PML/RAR α and MOLT-4 PML/RAR α cells but not in control cells. The exogenous PML/RARα fusion gene therefore probably mediates the effects of ATRA on cell growth and apoptosis in these cell lines.     

全反式维甲酸对各种人甲状腺肿瘤细胞株增殖、摄碘及甲状腺特异基因表达的影响

对4种甲状腺癌细胞株予全反式维甲酸处理,四甲基偶氮唑盐法检测细胞增殖,同位素法测定摄碘功能,半定量RT-PCR检测甲状腺特异基因及维甲酸受体表达.结果 显示全反式维甲酸可抑制FTC-133细胞增殖,促进其摄碘及甲状腺特异基因的表达,但对C643、HTH74及XTC.UC1细胞无影响,提示不同甲状腺肿瘤细胞对全反式维甲酸的反应性不相同.     

Appearance of del(11q) in two patients with acute promyelocytic leukaemia treated with all-trans retinoic acid and combination chemotherapy

Two acute promyelocytic leukaemia patients, treated with all-trans retinoic acid and combination chemotherapy, acquired a deletion of 11q within 12 months of diagnosis. One patient died in relapse, with both t(15;17) and del(11q) cell lines co-existing. Patient 2 remains in remission with del(11q) in 70% metaphases, despite normal marrow morphology. No deletion of the MLL gene was identified in the latter patient. The early appearance of a del(11q) is unusual, particularly without morphological evidence of myelodysplasia. We hypothesize that the del(11q) was therapy-induced but the absence of other genetic lesions has resulted in no accompanying morphological changes.     

全反式维甲酸对亚砷酸钠致人肝细胞损伤保护性作用的研究

目的 观察全反式维甲酸(ATRA)对亚砷酸钠致人肝细胞系(HHL)-5细胞损伤的保护性作用,探讨可能机制.方法 采用细胞培养方法,体外培养HHL-5细胞48 h后进行实验,实验分为4组:正常组、ATRA组、亚砷酸钠组、ATRA+亚砷酸钠组.用细胞增殖实验(WST)观察HHL-5细胞的活力;生物化学方法测定各组HHL-5细胞内超氧化物歧化酶(SOD)、谷胱甘肤过氧化物酶(GSH-Px)的活力及丙二醛(MDA)的含量和细胞培养液中谷草转氨酶(AST)的活力;透射电镜观察各组细胞超微结构的变化.结果 亚砷酸钠组HHL-5细胞活力(0.57±0.02)与正常组(0.70±0.01)比较,差异有统计学意义(P＜ 0.05);SOD、GSH-Px、MDA、AST [(153.84±2.35 )U/mg Prot、(0.08±0.02)U/mg Prot、(4.15±0.50)nmol/mg Prot、(265.43±4.62)×103 U/L]与正常组[(237.41±18.30) U/mg Prot、(0.93±0.02)U/mg Prot、(2.26±0.40)nmol/mg Prot、(177±9.85)×103 U/L]比较,差异有统计学意义(P均.＜0.05).ATRA+亚砷酸钠组HHL-5细胞活力(0.65±0.04)与亚砷酸钠组比较,差异有统计学意义(P＜0.05);SOD、GSH-Px、MDA、AST[(286.85±3.39)U/mg Prot、(0.56±0.09)U/mg Prot、(3.36±0.37)nmol/mg Prot、(220.02±1.07)×103 U/L]与亚砷酸钠组比较,差异有统计学意义(P均＜ 0.05).电镜结果显示,亚砷酸钠组同正常组及ATRA组比较,细胞表面微绒毛减少,双层核膜结构不清,胞质内可见空泡样变,肝糖原凝集;ATRA+亚砷酸钠组上述损伤程度减轻.结论 ATRA通过提高HHL-5细胞内抗氧化酶的活力,清除或者减少氧自由基对细胞的损伤,从而发挥保护作用.     

维甲酸诱导失分化型甲状腺癌的观察

应用全反式维甲酸(ATRA)诱导失分化型甲状腺癌(dDTC)患者,5例患者(5/9,55.6%)ATRA诱导有效;5例患者(5/8,62.5%)131I治疗获益.3例患者(3/12,25.O%)因神经系统的不良反应,不能耐受而放弃ATRA诱导.ATRA是诱导dDTC分化的有效方法,可以提高dDTC病灶的摄碘能力,但使用中应关注ATRA的不良反应.