Receptor-interacting protein 3 (RIP3) is a serine/threonine protein kinase, which has extensive substrates including its cognate kinase RIP1 and multiple metabolic enzymes involving oxidative phosphorylation1, 2. RIP3 has been shown to be essential for development, immunity and some physiological or pathophysiological responses to exogenous and endogenous stimuli3, 4, 5. In 2009, three groups independently reported that RIP3 acted as a molecular switch between apoptosis and necrosis (also called as necroptosis) 6, 7, 8. Specifically, RIP3 could turn tumor necrosis factor (TNF)-induced cell death from apoptosis to necrosis6. Most of small-molecule anticancer drugs elicit their anticancer effects via apoptotic induction9. However, it is unclear whether RIP3 affects the cellular sensitivity to small-molecule anticancer drugs.
We treated A cells (NIH 3T3 cells, murine fibroblasts, RIP3−/−; typically undergoing apoptosis in response to TNF stimulation) and N cells (NIH 3T3 cells, murine fibroblasts, RIP3+/+; undergoing necroptosis in response to TNF stimulation)6 with small-molecule anticancer drugs of different mechanisms of action. Detection by sulforhodamine B assays showed that A cells and N cells displayed differential sensitivity only to the examined topoisomerase I (Top1) inhibitors camptothecins. RIP3-deficient A cells revealed 32.6- and 40.2-fold higher sensitivity than RIP3-proficient N cells to SN38 and chimmitecan10, respectively (Figure 1A). Such differential sensitivity was reflected as higher apoptosis rates (Figure 1B) and more rapid G2/M arrest (Figure 1C) in A cells than in N cells treated with chimmitecan. Consistently, exposure to chimmitecan caused faster reduction of the target protein Top1 in A cells than in N cells (Figure 1D); and the treatment with chimmitecan or SN38 drove more γ-H2AX formation (a molecular marker for DNA double-strand breaks; Figure 1E) and produced more foci of phosphorylated ataxia telangiectasia mutated kinase (p-ATM) at Ser 1981 (sensing DNA double-strand breaks; Figures 1F and 1G) in A cells than in N cells. These results collectively indicate that the differential sensitivity of the RIP3-deficient A cells and the RIP3-proficient N cells to the examined Top1 inhibitors in all the tested events from inducing DNA double-strand breaks through sensing the damage signals to eliciting the final biological effects including cell cycle arrest, apoptosis and proliferation/growth inhibition.
Both A cells and N cells were kindly gifted from Prof Jiahuai HAN, who used them to successfully demonstrate the role of RIP3 in regulating TNF-inducing cell death6. The differential sensitivity of the RIP3-deficient and proficient murine fibroblasts to the Top1 inhibitors suggests a role of RIP3 in determining the cellular sensitivity to those agents. In this case, however, RIP3 does not seem to directly affect the choice of cell death between apoptosis and necrosis, because no significant difference of the cellular sensitivity to the other examined anticancer drugs except camptothecins was detectable (data not shown) and because RIP3-based differential responses of A cells and N cells to the Top1 inhibitors took place actually at the levels of the target protein Top1 and DNA double-strand breaks, not only at the level of cell death. In contrast, our data seem to imply that RIP3 is involved in the DNA damage induced by the Top1 inhibitors and/or subsequent repair. As a clinically important class of anticancer drugs, Top1 inhibitors are extensively used to treat various solid tumors such as colon and lung cancers. Occurrence of drug resistance to those inhibitors is a serious obstacle to successful therapy in the clinic11. The differential sensitivity of A cells and N cells to SN38 and chimmitecan also suggests that RIP3 could contribute to cellular resistance to Top1 inhibitors. Actually, we detected and found that RIP3 was expressed at high levels in cancer cells originated from different human tissues including blood (K562), liver (SMMC-7402 and SMMC-7721), colon (HCT116), stomach (MKN45), lung (A549), breast (MDA-MB-468, MDA-MB-231, T47D, and BT549), cervix (HeLa) and bone (Rh30) (data not shown). Inhibition of RIP3 might be an alternative approach to circumventing drug resistance. However, the exact molecular mechanisms remain to be further clarified.
Taken together, our results demonstrate for the first time the differential sensitivity of the RIP3-deficient A cells and the RIP3-proficient N cells to Top1 inhibitors, suggesting a potential new role of RIP3 in Top1 inhibitor-induced DNA damage/repair and cellular resistance to Top1 inhibitors, probably independently of its regulation in the choice of cell death modes.
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We thank Prof Jia-huai HAN (Xiamen University, China) for his kind gifts of RIP3-deficient A cells and RIP3-proficient N cells. This work was supported by grants from the National Natural Science Foundation of China (No 81025020 and No 81021062), the National Basic Research Program of China (No 2012CB932502), the National Science & Technology Major Project of China (No 2012ZX09301-001-002) and the State Key Laboratory of Drug Research of China (No SIMM1105KF-02).
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