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, 475 (7355), 231-4

Selective Killing of Cancer Cells by a Small Molecule Targeting the Stress Response to ROS


Selective Killing of Cancer Cells by a Small Molecule Targeting the Stress Response to ROS

Lakshmi Raj et al. Nature.

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Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.


Figure 1
Figure 1. Selective killing effect of PL in cancer cells by a small molecule
a, Structure of PL. b, PL treatment induces cell death in cancer cells, but does not induce cell death in normal cells. Human normal cells, including aortic endothelial cells (PAE), breast epithelial cells (76N), keratinocytes (HKC), and skin fibroblasts (HDF), as well as two immortalized breast epithelial cell lines (184B5 and MCF10A), were grown in 12 or 24 well plates and treated with PL at 1–15 μM for 24 h. Cytotoxicity was measured by trypan blue exclusion staining (average of three independent experiments). A variety of human cancer cell lines were treated with PL or DMSO (control) for 24 h. PL was HPLC-purified (~99% purity) prior to the treatment. c, Selective cell death by PL in oncogenically transformed human BJ skin fibroblasts (the left panel) and MCF10A cell lines (right panel). A representative graph for cell viability is shown (mean ± SD of three independent experiments; *p<0.0001). d, The effects of PL on p53 and its target PUMA, and pro-survival proteins were measured by western blot analyses in several cancer cells.
Figure 2
Figure 2. In vivo anti-tumor effect of PL
a, Mammary tumor growth inhibition by PL treatment in MMTV-PyVT transgenic tumor mice. MMTV-PyVT mice spontaneously developed breast adenocarcinoma by ~8 weeks of age. When tumor sizes grew to ~5–6 mm in diameter, the PL (total 2.4 mg/kg), paclitaxel (10 mg/kg) or DMSO (5% V/V) was administered intraperitoneally (i.p.) daily for 13 days (12 mice per group). Mice were then sacrificed and mammary tumors excised and processed for histological examination. b, The size of the grossly dissected tumors was measured. c, Histological morphology of hematoxylin-eosin-stained mammary tissue sections of MMTV-PyVT tumor mice treated with PL or DMSO after 13 days. d, After 13 days of PL treatment (*10 days treatment of paclitaxel was performed due to high toxicity in animals). Values in quantitative bar graphs are mean ± SD of three independent experiments.
Figure 3
Figure 3. PL enhances ROS accumulation in cancer cells by targeting the stress response to ROS
a, PL-mediated modulation of GSH and GSSG. GSH levels were determined after EJ cells were treated with PL or pretreated with NAC for 1 h, followed by PL treatment for 1 h or 3 h. GSSG levels were also determined after EJ cells and 76N cells were treated with PL for 3 hr. b, PL-induced ROS elevation and reversion by NAC. EJ cells were treated with PL (10 μM), paclitaxel (25 nM), or DMSO (basal) for 1 h and 3 h, and also pretreated with 3 mM NAC for 1 h, followed by 10 μM PL for 3 h. c, Reversion of PL-induced ROS accumulation by catalase. EJ or U2OS cells were pretreated with catalase (CAT, 2,000U/ml) for 2 hr, followed by 10 μM PL for 3 h. Comparisons of ROS induced by PL and CAT+PL were also shown by quantitative bar graph. d, PL-induced cell death can be rescued by NAC. EJ cells were treated with PL for 24 h, or treated with 3 mM NAC for 1 h and followed by the treatment of PL or paclitaxel for 24 h. Cell viability was measured by trypan blue exclusion staining assay. Values in quantitative bar graphs are mean ± SD of three independent experiments.
Figure 4
Figure 4. PL does not increase ROS and the ROS-induced DNA-damage response in normal and immortalized non-transformed cells
a, PL does not increase ROS in normal cells (16N). ROS were measured by flow cytometry and shown by quantitative bar graph. b, Selective induction of ROS by PL in oncogenically transformed BJ human fibroblasts (BJ-ELR) but not in non-transformed BJ fibroblasts (BJ-hTERT and BJ-ST). The ROS levels of BJ cells were measured after treating with PL (5 μM) for 8 h. Values in quantitative bar graphs are mean ± SD of three independent experiments. c, The effect of PL on stress-response targets was determined by western blot analysis of p53, p21 and γ-H2AX in PL in normal cells. Etoposide or DMSO as solvent control for 12 and 24 h. β-actin expression used as a loading control.

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