烟酰胺腺嘌呤二核苷酸(NAD+)不仅对能量代谢至关重要，而且对信号通路也至关重要。聚(ADP核糖)聚合酶(PARP)的激活是NAD+耗竭的主要原因，该激活是对DNA损伤作出的反应。我们以前已经证明，二甲双胍可以引起MCF7乳腺癌细胞中的caspase依赖性细胞死亡和PARP依赖性细胞死亡，但是在高葡萄糖培养基中不会引起MDA-MB-231(231)乳腺癌细胞的死亡。我们假设通过PARP的激活耗竭MCF7细胞中的NAD+有助于二甲双胍引起的细胞死亡。烟酰胺磷酸核糖转移酶(NAMPT)是将烟酰胺(维生素B3)转化为NAD+的关键限速步骤，对于再生正常细胞过程中的NAD+至关重要。证据表明，NAMPT的过表达与肿瘤发生有关。我们假设NAMPT的表达可能决定了癌细胞对二甲双胍的敏感程度。 在本研究中，我们发现二甲双胍随着时间的推移显著降低NAD+水平，并且这可以通过PARP抑制剂延迟。MCF7细胞中预处理NAD+也可以预防细胞死亡和线粒体增大，并且可以保护线粒体免受二甲双胍引起的膜电位丧失的影响。这导致MCF7细胞对二甲双胍的细胞毒性表现出类似于231细胞的耐受性。通过研究这两种乳腺癌细胞系中NAD+调控的差异，我们证明NAMPT在231细胞中的表达水平高于MCF7细胞。当在231细胞中遗传抑制NAMPT时，它们对二甲双胍诱导的细胞死亡变得更加敏感。相反，过表达NAMPT在HEK-293(293)细胞中会使细胞对二甲双胍的生长抑制效果表现出更高的耐受性。添加NAMPT激活剂也降低了MCF7细胞对二甲双胍的敏感性，而NAMPT激活剂P7C3可以保护细胞免受二甲双胍引起的细胞毒性的影响。细胞内NAD+的耗竭是癌细胞对二甲双胍细胞毒性效应敏感性的关键因素。NAMPT在维持足够的NAD+水平中发挥关键作用，表达高水平NAMPT的细胞对于二甲双胍引起的细胞杀伤具有抵抗性。版权所有 © 2023 Zhuang，Haugrud，Schaefer，Messerli和Miskimins。

Nicotinamide adenine dinucleotide (NAD+) is vital for not only energy metabolism but also signaling pathways. A major source of NAD+ depletion is the activation of poly (ADP-ribose) polymerase (PARP) in response to DNA damage. We have previously demonstrated that metformin can cause both caspase-dependent cell death and PARP-dependent cell death in the MCF7 breast cancer cells but not in the MDA-MB-231 (231) breast cancer cells while in high-glucose media. We hypothesize that depletion of NAD+ in MCF7 cells via activation of PARP contributes to the cell death caused by metformin. Nicotinamide phosphoribosyltransferase (NAMPT), a key rate-limiting step in converting nicotinamide (vitamin B3) into NAD+, is essential for regenerating NAD+ for normal cellular processes. Evidence shows that overexpression of NAMPT is associated with tumorigenesis. We hypothesize that NAMPT expression may determine the extent to which cancer cells are sensitive to metformin.In this study, we found that metformin significantly decreases NAD+ levels over time, and that this could be delayed by PARP inhibitors. Pretreatment with NAD+ in MCF7 cells also prevents cell death and the enlargement of mitochondria and protects mitochondria from losing membrane potential caused by metformin. This leads to MCF7 cell resistance to metformin cytotoxicity in a manner similar to 231 cells. By studying the differences in NAD+ regulation in these two breast cancer cell lines, we demonstrate that NAMPT is expressed at higher levels in 231 cells than in MCF7 cells. When NAMPT is genetically repressed in 231 cells, they become much more sensitive to metformin-induced cell death. Conversely, overexpressing NAMPT in HEK-293 (293) cells causes the cells to be more resistant to metformin's growth inhibitory effects. The addition of a NAMPT activator also decreased the sensitivity of MCF7 cells to metformin, while the NAMPT activator, P7C3, protects against metformin-induced cytotoxicity.Depletion of cellular NAD+ is a key aspect of sensitivity of cancer cells to the cytotoxic effects of metformin. NAMPT plays a key role in maintaining sufficient levels of NAD+, and cells that express elevated levels of NAMPT are resistant to killing by metformin.Copyright © 2023 Zhuang, Haugrud, Schaefer, Messerli and Miskimins.