1,25-二羟维生素D3治疗恶性肿瘤临床前研究进展

大量体内外实验证实1,25-二羟维生素D3[1,25(OH)2D3]具有抗肿瘤活性.1,25(OH)2D3抗肿瘤机制尚未完全明确,但认为与其能抑制肿瘤增殖、诱导细胞凋亡、促进细胞分化、降低肿瘤侵袭性及抑制肿瘤血管形成有关.国内外众多学者以1,25(OH)2D3为基础,联合细胞毒药物、放疗、地塞米松等制剂干预肿瘤,发现能增强1,25(OH)2D3抗肿瘤活性,有望成为未来的抗癌剂.     

全反式维甲酸诱导大肠癌细胞分化的实验研究

作者采用全反式维甲酸对大肠癌细胞株ＬｏＶｏ进行体外实验，结果细胞增殖受抑制，呈时间剂量依赖关系，细胞周期改变，被阻滞于Ｇｏ／Ｇ１期，Ｓ期比例下降，分化标志酶ＡＬＰ比活性增高，双层软琼脂糖培养细胞集落形成能力降低，提示全反式维甲酸具有诱导ＬｏＶｏ细胞分化作用，实验结果有重要的临床指导意义。     

全反式维甲酸诱导分化舌癌细胞株的动物实验研究

目的：研究全反式维甲酸（ＶＴＲＡ）对舌癌细胞株的体内诱导分化作用。方法：人舌癌细胞株分别经下列方法处理后接种于；裸鼠皮下，（１）未经药物处理，（２）未经药物处理的舌癌细胞接种后立即行ＡＴＲＡ灌胃，（３）经１０μｍｏｌ／Ｌ ＡＴＲＡ处理３天或６天舌癌细胞，观察肿瘤的生长状态，倍增时间和抑瘤率，３ 一处死动物取出瘤块行病理学检查。结果：经ＡＴＲＡ处理后，肿瘤生长速率减慢，倍增时间延长和抑瘤率增高，光镜     

血清1,25-二羟维生素D3水平与肺癌临床病理特征及预后的关系

目的探讨血清1,25-二羟维生素D3水平与肺癌临床病理特征及预后的关系。方法将103例肺癌患者纳入肺癌组,另将同期体检的62例健康体检者作为对照组,采集2组晨起空腹外周静脉血,应用酶联免疫吸附法(ELISA)检测血清1,25-二羟维生素D3水平,比较2组血清1,25-二羟维生素D3水平,并分析肺癌患者血清1,25-二羟维生素D3水平与临床病理特征及预后的关系。结果ELISA检测结果显示,肺癌组的血清1,25-二羟维生素D3水平[(29.24±5.37)μg/l]明显低于对照组[(40.16±6.26)μg/l],2组相比差异具有统计学意义(P<0.05)。不同性别、年龄、肿瘤直径、病理分型的肺癌患者血清1,25-二羟维生素D3水平相比,差异无统计学意义(P>0.05);但不同TNM分期、组织分化程度、有无淋巴结转移及远端转移的肺癌患者血清1,25-二羟维生素D3水平相比,差异有统计学意义(P>0.05)。1,25-二羟维生素D3缺乏组(≤20μg/l)2年生存率为30.00%(18/60),正常组(>20μg/l)患者的2年生存率为63.83%(30/47),缺乏组患者2年生存率明显低于正常组患者(P<0.05)。结论血清1,25-二羟维生素D3水平降低可能与肺癌的发生及恶性程度有关,提高1,25-二羟维生素D3水平可能有助于改善肺癌患者的预后。     

1,25二羟基维生素D3对体外培养的HL-60细胞的作用

[目的]研究1,25二羟基维生素D3[1,25(OH)2D3]对人HL-60细胞株生长、分化、凋亡及对维生素D受体(VDR)蛋白表达的影响.[方法]在体外用不同浓度的1,25(OH)2D3作用于对数生长期的HL-60细胞.甲基噻唑基四唑(MTT)比色法分析细胞增殖抑制作用,硝基四氮唑蓝(NBT)还原实验法分析HL-60细胞的分化指标,末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记染色法(TUNEL)检测细胞晚期凋亡的变化,免疫细胞化学法检测VDR蛋白表达.[结果]不同浓度的1,25(OH)2D3作用后均可抑制HL-60细胞的生长,呈剂量和时间依赖性;可诱导HL-60细胞向成熟粒细胞分化并促其凋亡.1,25(OH)2D3作用前后HL-60细胞均表达VDR蛋白,药物作用后VDR蛋白表达上调.[结论]1,25(OH)2D3在一定浓度范围内可抑制HL-60细胞增殖,诱导细胞分化及凋亡,使VDR蛋白表达上调.     

1,25-二羟基维生素D3增强卡铂对人肺癌A549细胞的杀伤效果

目的 观察活性维生素D[1,25-dihydroxyvitamin D3,1,25(OH)2D3]和卡铂(carboplatin,CBP)两种药物单独及联合作用对人肺癌A549细胞的抗肿瘤效应,并探讨其可能的作用机制.方法 本研究分1,25(OH)2D3组,CBP组以及联合作用组.用CCK-8法测定细胞抑制率,使用激光扫描共聚焦显微镜进行维生素D受体(vitamin D receptor,VDR)的鉴定,用流式细胞仪进行细胞周期、细胞活性氧(reactive oxidative species,ROS)及线粒体膜电位(mitochondrial membrane potential,MMP)的分析.结果 1,25(OH)2D3和CBP单独使用均可以抑制A549细胞的生长,且两者联合作用时具有协同作用,1,25(OH)2D3能显著提高CBP对A549细胞的抑制率.通过免疫荧光测得A549细胞中有较高维生素D受体的表达.细胞周期分析显示,经1,25(OH)2 D3和CBP处理后的A549细胞,细胞周期发生变化,G0/G1期细胞数增多,S期和G2/M期细胞相应减少.两者联合作用后,以上趋势更加明显.与正常对照组相比较,各处理组均可使细胞ROS的释放增加(P＜0.05),MMP下降(P＜0.05),其中两种药物联合作用时细胞ROS的产生最多,MMP下降最为显著.结论 1,25(OH)2D3可以抑制A549细胞的增殖,并且与CBP联合作用时能显著增强其对肺癌细胞的杀伤能力.1,25(OH)2D3与CBP的协同作用与阻滞细胞周期的有序进行有关,并通过提高ROS和降低MMP抑制肺癌细胞的增殖.     

全反式维甲酸诱导再分化在16例甲状腺癌治疗中的应用

目的：探讨全反式维甲酸（ATRA）在分化型甲状腺癌（DTC）放射性碘治疗过程中的临床应用。方法：16例分化型甲状腺癌转移患者,131I治疗中转移灶不摄取或轻度摄取131I,服ATRA2个月后再行131I治疗,7天后SPECT显像,对转移灶部位进行感兴趣区（ROI）计数,并和ATRA治疗前SPECT显像进行比较,评价ATRA治疗前后131I摄取变化情况。结果：16例患者服用ATRA后,其中7例131I摄取增加,治疗有效率43.7%。结论：ATRA治疗能促进部分失分化DTC细胞的再分化。     

全反式维甲酸联合细胞因子对NB_4细胞增殖分化作用的体外研究

目的 :探讨新的药物协同 ATRA诱导早幼粒白血病细胞分化作用 ,降低临床 ATRA剂量 ,提高ATRA疗效。方法 :NB4细胞株为实验模型 ,采用细胞生物学研究方法 ,体外研究 IFNγ、TNF和 G- CSF各结合ATRA对 NB4细胞增殖和分化的作用。结果 :在诱导分化和抑制白血病克隆增殖中 ,IFNγ和 TNF具协同作用 ,以IFNγ作用最明显 ,在 ATRA浓度为 10 - 8mol/ L 时 ,比较各细胞因子联合 ATRA对 NB4细胞作用后 NBT的变化 ,发现联合 IFNγ组 NBT仍达 92 % ,高于其它各组 (P<0 .0 1) ,细胞周期变化 :联合 IFNγ作用后使 G0 / G1 期积聚最明显 ,由对照的 5 0 .5 %增至 86 .2 % ,半固体克隆增殖抑制实验显示 ,ATRA与 IFNγ联合用药组具明显抑制 NB4细胞克隆增殖活性作用。结论 :细胞因子 IFNγ与 ATRA联合具较强协同诱导 NB4细胞分化和抑制克隆增殖作用 ,这对临床联合 IFNγ诱导早幼粒白血病分化治疗提高 ATRA疗效有一定价值。     

全反式维甲酸联合细胞因了对NB4细胞增殖分化作用的体外研究

目的：探讨新的药物协同ＡＴＲＡ诱导早幼粒白血病细胞分化作用,降低临床ＡＴＲＡ剂量提高ＡＴＲＡ疗效。方法：ＮＢ４细胞株为实验模型,采用细胞生物学研究方法,体外研究ＩＦＮγ、ＴＮＦ和Ｇ－ＣＳＦ各结合ＡＴＲＡ对ＮＢ４分化的作用。结果：在诱导分化和抑制白血病克隆增中,ＩＦＮγ和ＴＮＦ具协同作用,以ＩＦＮγ作用最明显,在ＡＴＲＡ浓度为１０＾－８ｍｏｌ／Ｌ时,比较各细胞因子联合ＡＴＡ对ＮＢ４细胞作用后ＮＢＴ的     

全反式维甲酸诱导再分化在16例甲状腺癌治疗中的应用

目的:探讨全反式维甲酸(ATRA)在分化型甲状腺癌(DTC)放射性碘治疗过程中的临床应用.方法:16例分化型甲状腺癌转移患者,131I治疗中转移灶不摄取或轻度摄取131I,服ATRA 2个月后再行131I治疗,7天后SPECT显像,对转移灶部位进行感兴趣区(ROI)计数,并和ATRA治疗前SPECT显像进行比较,评价ATRA治疗前后131I摄取变化情况.结果:16例患者服用ATRA后,其中7例131I摄取增加,治疗有效率43.7%.结论:ATRA治疗能促进部分失分化DTC细胞的再分化.     

全反式维甲酸对神经母细胞瘤细胞系体外生长的影响

目的　体外观察全反式维甲酸 (alltransretinoicacid ,ATRA)对神经母细胞瘤细胞系 (SK N SH)细胞生长的影响。方法　SK N SH细胞持续培养在含ATRA的 96孔板中 ,采用MTT法检测ATRA的体外抗增殖效果 ,用相差倒置显微镜及透射电镜观察细胞形态变化。结果　 0 .1～ 1μmol/L浓度的ATRA对SK N SH细胞可产生明显的抗增殖作用 ;其抗增殖作用呈时间和剂量依赖关系。 1μmol/LATRA作用 12天 ,细胞生长抑制率达 77.0 3%。ATRA能诱导SK N SH细胞分化成熟 ,先是细胞出现神经树 (轴 )突样胞突 ,培养 10天后多个细胞形成类似神经节样形态结构 ,“神经节”之间形成类神经纤维 ;电镜观察细胞核浆比例下降 ,无特征性细胞凋亡形态变化。结论　ATRA通过诱导分化作用 ,而明显抑制神经母细胞瘤细胞增殖。     

Differential and antagonistic effects of 9-cis-retinoic acid and vitamin D analogues on pancreatic cancer cells in vitro

Retinoids and vitamin D are known to exert important anti-tumour effects in a variety of cell types. In this study the effects of 9-cis-retinoic acid (9cRA) the vitamin D analogues EB1089 and CB1093 on three pancreatic adenocarcinoma cell lines were investigated. All compounds caused inhibition of in vitro growth but the vitamin D analogues were generally the more potent growth inhibitors. They were also more effective on their own than in combination with 9cRA. Growth arrest correlated with an increased proportion of cells in the G0/G1 phase. Apoptosis was induced in the three cell lines by 9cRA, whereas neither EB1089 nor CB1093 had this effect. Furthermore, addition of EB1089 or CB1093 together with 9cRA resulted in significantly reduced apoptosis. Our results show that retinoic acids as well as vitamin D analogues have inhibitory effects on pancreatic tumour cells but different and antagonistic mechanisms seem to be employed.     

Synergistic effect of retinoic acid and 1,25-dihydroxyvitamin D3 on the differentiation of the human monocytic cell line U937

The human-derived leukemia cell lines HL-60 and U937 are known to differentiate into more mature phagocytic cells in the presence of retinoic acid or 1,25-dihydroxyvitamin D₃. We studied the effects of combinations of these two agents on cell growth and differentiation. These treatments were found to increase inhibition of cell proliferation. A dramatic enhancement of functional properties was observed in U937, but not HL-60 cells exposed to combinations of the two inducers. We investigated the conditions required to obtain the highest synergistic effects on the differentiation of U937 cells. These effects were found to be highly dose-dependent. We found that synergism required the simultaneous presence of both inducers and did not occur upon sequential exposure to each agent used separately.     

Inhibition of lung carcinogenesis by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in the A/J mouse model: evidence of retinoid mitigation of vitamin D toxicity

9-cis-Retinoic acid (9cRA) and 1alpha,25-dihydroxyvitamin D3 (1,25D) show promise as potential chemopreventive agents. We examined 9cRA and 1,25D, alone and in combination, for their potential to inhibit carcinogen (NNK)-induced lung carcinogenesis in A/J mice. A/J mice (n=14/group) were treated with 9cRA (7.5, 15, or 30 mg/kg diet), 1,25D (2.5 or 5.0 microg/kg diet), or a combination of 9cRA (15 mg/kg diet) plus 1,25D (2.5 microg/kg diet) for 3 weeks before and 17 weeks after carcinogen injection. Lung tumor incidence, tumor multiplicity, plasma 1,25D levels and kidney expression of vitamin D 24-hydroxylase (CYP24) were determined. Compared to carcinogen-injected controls, mice receiving 9cRA supplementation had significantly lower tumor multiplicity at all doses (decreased 68-85%), with body weight loss at the higher doses of 9cRA. Mice receiving 1,25D supplementation had significantly lower tumor incidence (decreased 36 and 82%) and tumor multiplicity (decreased 85 and 98%), but experienced significant body weight loss, kidney calcium deposition, elevated kidney CYP24 expression and decreased fasting plasma 1,25D levels. Although, there was no apparent influence on chemopreventive efficacy, addition of 9cRA to 1,25D treatment effectively prevented the weight loss and kidney calcification associated with 1,25D treatment alone. These data demonstrate that 9cRA and 1,25D, alone or combined, can inhibit lung tumor promotion in the A/J mouse model. Combining 1,25D with 9cRA has the potential to mitigate the toxicity of 1,25D, while preserving the significant effect of 1,25D treatment against lung carcinogenesis. The underlying mechanism behind this effect does not appear to be related to retinoid modulation of vitamin D catabolism.     

1,25D3 Enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

BACKGROUND:

1,25 dihydroxyvitamin D₃ (1,25D₃) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin is a current standard chemotherapy regimen for bladder cancer. The authors investigated whether 1,25D₃ could enhance the antitumor activity of gemcitabine and cisplatin in bladder cancer model systems.

METHODS:

Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D₃ followed by gemcitabine and cisplatin. Apoptosis was assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate‐based caspase activity assay. The cytotoxic effects were examined by using 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model.

RESULTS:

1,25D₃ pretreatment enhanced gemcitabine and cisplatin‐induced apoptosis and the activities of caspases 8, 9, and 3 in T24 and UMUC3 cells. 1,25D₃ synergistically reduced gemcitabine and cisplatin‐suppressed surviving fraction in T24 cells. 1,25D₃, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by gemcitabine and cisplatin or 1,25D₃ and gemcitabine and cisplatin. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D₃, gemcitabine and cisplatin, or 1,25D₃ and gemcitabine and cisplatin. 1,25D₃ and gemcitabine and cisplatin combination enhanced tumor regression compared with 1,25D₃ or gemcitabine and cisplatin alone.

CONCLUSIONS:

1,25D₃ potentiates gemcitabine and cisplatin‐mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis. Cancer 2010; 116:3294‐303. © 2010 American Cancer Society.     

Role of 24-hydroxylase in vitamin D3 growth response of OVCAR-3 ovarian cancer cells

Vitamin D and its analogues are potent regulators of cell growth and differentiation both in vivo and in vitro. We studied the effects of 25-hydroxyvitamin D(3) [25(OH)D(3)], 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and vitamin D analogue, EB 1089, on the growth of a human ovarian cancer cell line, OVCAR-3. We also studied the expression of vitamin D metabolising enzymes 24-hydroxylase (24OHase) and 1alpha-hydroxylase (1alphaOHase). Our results showed that high concentrations (10 and 100 nM) of 1,25(OH)(2)D(3) inhibited a cell proliferation, whereas low concentration (0.1 nM) stimulated growth of the OVCAR-3 cells. In the concentration range of 10-500 nM a prohormone, 25(OH)D(3), stimulated growth. An amount of 1 nM EB 1089 and 100 nM 1,25(OH)(2)D(3) inhibited growth with an equal magnitude. The expression of 24OHase was strongly induced by 1,25(OH)(2)D(3) and EB 1089 in OVCAR-3 cells, and analysis of vitamin D metabolites showed the functionality of 24OHase. An inhibition of 24OHase activity with a novel 24OHase inhibitor enhanced growth-inhibiting effects of 1,25(OH)(2)D(3) and suppressed the growth stimulation of 100 nM 25(OH)D(3). We also report the expression of a vitamin D activating enzyme, 1alphaOHase, in 7 ovarian cancer cell lines. The production of 1,25(OH)(2)D(3) in OVCAR-3 cells was low, possibly due to an extensive activity of 24OHase or a low 1alphaOHase activity. These results suggest that in ovarian cancer cells vitamin D metabolizing enzymes might play a key role in modulating the growth response to vitamin D. The possible mitogenic effects of vitamin D should be considered when evaluating treatment of ovarian cancer with vitamin D.     

Comparative effects of 1,25(OH)2D3 and EB1089 on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells

1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active metabolite of vitamin D, inhibits breast cancer cell growth both in vivo and in vitro. In addition to its anti-proliferative effects, 1,25(OH)₂D₃ induces morphological and biochemical markers of apoptosis in MCF-7 cells. In the studies reported here, we compared the effects of 1,25(OH)₂D₃ and EB1089, a low calcemic vitamin D analog, on cell cycle kinetics and apoptosis in MCF-7 cells. Both vitamin D compounds reduced viable MCF-7 cell number in a time and dose dependent manner, with EB1089 approximately 50 fold more potent than 1,25(OH)₂D₃. Flow cytometric analysis indicated that both agents induced cell cycle arrest in G₀/G₁ which was associated with accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein. MCF-7 cells treated with either 1,25(OH)₂D₃ or EB1089 for 48 h exhibited characteristics of apoptosis, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and DNA fragmentation. Cells treated with either agent exhibited up regulation of proteins associated with mammary gland regression (clusterin and cathepsin B) and down regulation of the anti-apoptotic protein bcl-2. These studies demonstrate that, despite its lower calcemic activity in vivo, the vitamin D analog EB1089 induces effects that are indistinguishable from those of 1,25(OH)₂D₃ on cell number, cell cycle and indices of apoptosis in MCF-7 cells in vitro. In addition, since both agents rapidly down regulate estrogen receptor, disruption of estrogen dependent signalling may play a role in the induction of apoptosis by vitamin D compounds in MCF-7 cells.     

Loss of growth inhibitory effects of retinoic acid in human breast cancer cells following long-term exposure to retinoic acid

Although retinoids are known to be inhibitory to breast cancer cell growth, a key remaining question is whether they would remain effective if administered long-term. We describe here the long-term effects of all- trans retinoic acid on two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1. Although both cell lines were growth inhibited by retinoic acid in the short-term in either the absence or the presence of oestradiol, prolonged culture with 1 microM all- trans retinoic acid resulted in the cells acquiring resistance to the growth inhibitory effects of retinoic acid. Time courses showed that oestrogen deprivation of the cell lines resulted in upregulation of the basal non-oestrogen stimulated growth rate such that cells learned to grow at the same rate without as with oestradiol, but the cells remained growth inhibited by retinoic acid throughout. Addition of 1 microM all- trans retinoic acid to steroid deprivation conditions resulted in reproducible loss of growth response to both retinoic acid and oestradiol, although the time courses were separable in that loss of growth response to retinoic acid preceded that of oestradiol. Loss of growth response to retinoic acid did not involve loss of receptors, ER as measured by steroid binding assay or RARalpha as measured by Northern blotting. Function of the receptors was retained in terms of the ability of both oestradiol and retinoic acid to upregulate pS2 gene expression, but there was reduced ability to upregulate transiently transfected ERE- and RRE-linked reporter genes. Despite the accepted role of IGFBP3 in retinoic acid-mediated growth inhibition, progression to retinoic acid resistance occurred irrespective of level of IGFBP3, which remained high in the resistant MCF7 cells. Measurement of AP1 activity showed that the two cell lines had markedly different basal AP1 activities, but that progression to resistance was accompanied in both cases by a lost ability of retinoic acid to reduce AP1 activity. These results warn of potential resistance which could arise on long-term treatment with retinoic acid in a clinical situation and echo the problems of progression to endocrine resistance. It seems that whatever the constraints imposed on growth, these cells have a remarkable ability to escape from growth inhibition. However, the ability of retinoic acid to delay progression to oestrogen resistance is encouraging for endocrine therapy, and the concentration-dependence of retinoic acid resistance suggests that progression is not absolute but could be manipulated by dose.     

Cooperation between BRCA1 and vitamin D is critical for histone acetylation of the p21waf1 promoter and for growth inhibition of breast cancer cells and cancer stem-like cells

Carriers of germline mutations in the BRCA1 gene have a significant increased lifetime risk for being diagnosed with breast cancer. The incomplete penetrance of BRCA1 suggests that environmental and/or genetic factors modify the risk and incidence among mutation carriers. Nutrition and particular micronutrients play a central role in modifying the phenotypic expression of a given genotype by regulating chromatin structure and gene expression. The active form of vitamin D, 1α,25-dihydroxyvitamin D₃, is a potent inhibitor of breast cancer growth. Here we report that two non-calcemic analogues of 1α,25-dihydroxyvitamin D₃, seocalcitol (EB1089) and QW-1624F2-2, collaborate with BRCA1 in mediating growth inhibition of breast cancer cells and breast cancer stem-like cells. EB1089 induces a G1/S phase growth arrest that coincides with induction of p21waf1 expression only in BRCA1-expressing cells. A complete knockdown of BRCA1 or p21waf1 renders the cells unresponsive to EB1089. Furthermore, we show that in the presence of ligand, BRCA1 associates with vitamin D receptor (VDR) and the complex co-occupies vitamin D responsive elements (VDRE) at the CDKN1A (p21waf1) promoter and enhances acetylation of histone H3 and H4 at these sites. Thus, BRCA1 expression is critical for mediating the biological impact of vitamin D₃ in breast tumor cells.