An NAD(P)H oxidase regulates growth and transcription in melanoma cells
An NAD(P)H oxidase regulates growth and transcription in melanoma cells
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DOI：
10.1152/ajpcell.00496.2001
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发表时间：
2002-06-01
影响因子：
5.5
通讯作者：
Hoidal, JR
中科院分区：
文献类型：
--
作者：
Brar, SS;Kennedy, TP;Hoidal, JR
恶性黑色素瘤细胞自发产生活性氧簇(ROS)，促进转录因子核因子-kappaB(NF-kappaB)的结构性激活。虽然抗氧化剂和NAD(P)H氧化酶的抑制剂显著降低了构成核因子-kappaB的活性并抑制了细胞的增殖(11)，但负责黑色素瘤细胞中ROS产生的酶的性质尚未确定。为了解决这个问题，我们现在已经确定了黑色素瘤细胞中ROS产生的来源。我们报道，ROS是由分离的、无胞浆的黑色素瘤质膜产生的，并被NAD(P)H氧化酶抑制剂抑制。RT-PCR和测序证实黑色素瘤细胞吞噬细胞NAD(P)H氧化酶的p22(Phox)、gp91(Phox)和p67(Phox)组分以及gp91(Phox)同系物NOX4；免疫分析法检测到细胞膜上p22(Phox)和gp91(Phox)的蛋白产物。正常人表皮黑素细胞仅表达p22(Phox)和NOX4。NAD(P)H氧化酶抑制剂和p22(Phox)和NOX4的反义寡核苷酸而不是正义寡核苷酸可以抑制黑色素瘤的增殖。此外，黄素蛋白抑制剂二苯基碘抑制核蛋白与核因子-kappaB和cAMP反应元件共识寡核苷酸的结构性DNA结合，而不影响DNA与激活蛋白-1或OCT-1的结合活性。这表明，由NAD(P)H氧化酶以自分泌方式产生的ROS可能在恶性黑色素瘤的生长信号中发挥作用。
Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappaB activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD( P) H oxidase inhibitors. The p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22(phox) and NOX4. Melanoma proliferation was reduced by NAD( P) H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappaB and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD( P) H oxidase may play a role in signaling malignant melanoma growth.