Phenolic component of tobacco smoke as tumor promoter

描述（由申请人提供）：有大量证据表明，烟草烟雾冷凝物的弱酸性酚类组分（TSCPhFr）在多环芳烃（PAH）引发的动物中具有强烈的促肿瘤活性。TSCPhFr中促进肿瘤的酚类组分的身份未知，其促进肿瘤活性的机制也未知。我们观察到非细胞毒性浓度的TSCPhFr减弱（±）-抗-苯并[a]芘-7，8-二醇-9，10-环氧化物[BPDE]-诱导的p53/NF-kB应答并促进JB 6细胞中的锚定非依赖性细胞生长（AICG）。DNA损伤诱导的NF-κ B活化在p53介导的细胞凋亡中起重要作用。我们推测，TSCPhFr的肿瘤促进活性是由于废除p53的积累和其对DNA损伤多环芳烃的细胞凋亡的功能。我们的研究包括（1）使用反相HPLC从TSCPhFr中纯化肿瘤促进组分，（2）使用化学和基因组抑制剂确定BPDE诱导的p53应答和NF κ B活化的抑制与细胞凋亡抑制和AICG活性增加的对应性，（3）如果上述目的2中的发现是肯定的，则确定TSCPHFr对BPDE诱导的p53介导的NFkB活化的作用（RAF/MAPK途径）、凋亡调节蛋白例如cIAP 1、cIAP 2、存活素和bcl-2家族蛋白的表达（Bax和bcl-2）、PARP降解和半胱天冬酶激活，（4）如果上述目的2的结果为阴性，然后在BPDE处理的细胞中确定TSCPhFr对已知参与p53非依赖性凋亡的p73应答的影响，并检查p73应答的抑制与AICG活性和使用基因组抑制剂的凋亡抑制的对应性，以及（5）如果抑制BPDE诱导的p53，而不是凋亡，与AICG活性有对应关系，然后确定TSCPhFr是否可以克服BPDE诱导的细胞周期阻滞，抑制p21 WAF 1（p21）反式激活，上调G1期细胞周期蛋白（D1/D3/E）和相关的cdk活性（cdk 2，cdk 4）。在我们的研究中，我们将使用JB 6 cl 41细胞，一种广泛用于肿瘤促进研究的模型细胞系。该提案的总体目标是确定TSCPhFr中的促肿瘤成分，并了解酚类成分在PAH启动的细胞中增强促肿瘤作用的潜在机制。从这些研究中获得的数据将有助于评估烟草烟雾成分带来的相关健康风险，并将有助于预防癌症的治疗策略。该项目中进行的研究结果将为进一步详细研究TSC酚类组分对PAH引发的肿瘤促进机制开辟途径。识别促肿瘤酚类成分并了解烟草烟雾中酚类成分发挥促肿瘤作用的机制不仅有助于评估烟草烟雾成分带来的健康风险，还有助于开发通过化学手段或遗传操作消除烟草中特定促肿瘤酚类成分的策略。

DESCRIPTION (provided by applicant): There is considerable evidence of strong tumor-promoting activity of weakly acidic phenolic fraction of tobacco smoke condensate (TSCPhFr) in polynuclear aromatic hydrocarbons (PAHs)-initiated animals. The identity of the tumor promoting phenolic component(s) in TSCPhFr is not known nor the mechanism(s) underlying its tumor-promoting activity. We observed TSCPhFr at non-cytotoxic concentrations attenuates ()-anti-benzo[a]pyrene-7,8- diol-9,10-epoxide [BPDE]-induced p53/NF-kB responses and promotes anchorage- independent cell growth (AICG) in JB6 cells. DNA damage-induced NF-kB activation has important role in p53- mediated apoptosis. We hypothesize that the tumor promoting activity of TSCPhFr is due to the abrogation of p53 accumulation and its function toward apoptosis in response to DNA damaging PAHs. Our studies include (1) purification of the tumor promoting component(s) from TSCPhFr using reverse phase HPLC, (2) determine the correspondence of inhibition of BPDE-induced p53 response and NFkB activation with apoptosis inhibition and increased AICG activity using chemical and genomic inhibitors, (3) if the findings in aim 2 above are affirmative then determine the effect of TSCPHFr on BPDE-induced p53-mediated NFkB activation (RAF/MAPK pathway), the expression of apoptosis regulatory proteins e.g. cIAP1, cIAP2, survivin and bcl-2 family proteins (Bax and bcl-2), degradation of PARP and activation of caspases, (4) if the findings in aim 2 above are negative, then in BPDE treated cells determine the effect of TSCPhFr on p73 response which is known to be involved in p53- independent apoptosis, and examine the correspondence of the inhibition of p73 response with AICG activity and inhibition of apoptosis using genomic inhibitor and (5) if inhibition of BPDE-induced p53, but not apoptosis, has correspondence with AICG activity then determine whether TSCPhFr can override BPDE-induced cell cycle arrest, inhibit p21WAF1 (p21) transactivation and up-regulate G1 cyclins (D1/D3/E) and associated cdk activities (cdk2, cdk4). In our studies we will use JB6 cl41 cells, a model cell line extensively used for the study of tumor promotion. The overall objective of the proposal is to identify the tumor promoting component/s in TSCPhFr and to understand the underlying mechanism(s) by which the phenolic component(s) elicits tumor promoting effect in PAH-initiated cells. Data obtained from these studies will help in assessing the associated health risk presented by tobacco smoke constituents and will be useful for therapeutic strategies in the prevention of cancer. The findings from studies undertaken in the project will open avenues for further detailed investigations on the mechanism of PAH-initiated tumor promotion by the phenolic fraction of TSC. Identification of tumor promoting phenolic component and understanding of the mechanism(s) by which phenolic component of tobacco smoke exerts its tumor promoting effect will help not only assessing the health risk presented by tobacco smoke constituents but also developing a strategy to eliminate the presence of particular tumor promoting phenolic component/s from tobacco leaf through chemical means or genetic manipulation.