Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with limited treatment options. Cisplatin (DDP) is used as a mainstay of chemotherapeutic agents in combination with other drugs or radiotherapy for PDAC therapy. However, DDP exhibits severe side-effects that can lead to discontinuation of therapy, and the acquired drug resistance of tumor cells presents serious clinical obstacles. Therefore, it is imperative to develop a more effective and less toxic therapeutic strategy. We and others have previously discovered that dihydroartemisinin (DHA) represents a safe and promising therapeutic agent to preferentially induce cancer cell ferroptosis. In the present study, we find that DHA could intensively strengthen the cytotoxicity of DDP and significantly reduce its effective concentrations both in vitro and in vivo. Combination of DHA and DDP synergistically inhibits the proliferation and induces DNA damage of PDAC cells. Mechanically, the combinative treatment impairs mitochondrial homeostasis, characterized by destroyed mitochondrial morphology, decreased respiratory capacity, reduced ATP production, and accumulated mitochondria-derived ROS. Further studies show that ferroptosis contributes to the cytotoxic effects in PDAC cells under the challenge of DHA and DDP, together with catastrophic accumulation of free iron and unrestricted lipid peroxidation. Moreover, pharmacologic depleting of the free iron reservoir or reconstituted expression of FTH contributes to the tolerance of DHA/DDP-induced ferroptosis, while iron addition accelerates the ferroptotic cell death. In summary, these results provide experimental evidence that DHA acts synergistically with DDP and renders PDAC cells vulnerable to ferroptosis, which may act as a promising therapeutic strategy.
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Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer which is an extremely lethal cancer with poor prognosis and high recurrence rate. PDAC often harbors the universal mutations in the proto-oncogene K-RAS (>90% prevalence in pancreatic cancer), which persistently accelerates and activates various oncogenic events (e.g., uncontrolled proliferation, sustained angiogenesis, metastasis, or invasion), thus leading to metabolic reprogramming and resistance to cell death [1, 2]. Most patients with pancreatic cancer were diagnosed at a late stage even with distant metastasis and died within several months. Although the diagnosis and treatment of pancreatic cancer have achieved great progress, the outcomes of patients are still not satisfactory, especially in those patients with K-Ras oncogenic mutant [3]. The survival benefits of standard chemotherapies are still limited with a median survival of fewer than 6 months [4], the 5-year survival rate for pancreatic cancer patients remains less than 10% [5]. Therefore, it is imperative to develop more effective and less toxic therapies that sensitize cancer cells to chemotherapy agents. Ferroptosis, a new mode of regulated cell death (RCD), is more prone to occur in Ras mutant cancer cells, which might open up a new strategy to solve this problem [6].
Cisplatin (DDP), an effective platinum-based chemotherapeutic agent, has been used to treat various types of solid tumors, including lung, breast, esophageal, ovarian, and pancreatic cancers [7, 8]. The inhibition of proliferation through DNA damage in rapidly dividing cells is the main anticancer mechanism of DDP. Other mechanisms of DDP-induced cytotoxicity are involved in impairing glycolysis, mitochondrial dysfunction, and accumulation of reactive oxygen species (ROS) [9]. However, DDP exhibits severe side-effects that can lead to discontinuation of therapy and acquired drug resistance, which may contribute to the treatment failure in pancreatic cancer [10]. Currently, the depletion of glutathione caused by DDP and the inactivation of glutathione peroxidase were found to play a vital role in its underlying mechanism [11, 39], and NSCLC [40]) could be more suitable to therapies of inducing ferroptosis, and more sensitive to agents that promote ferroptosis. Additionally, subsequent studies have identified that mutant RAS signaling enriches the cellular iron reservoir via transcriptional regulation of iron metabolism genes [30]. In the present study, we have provided evidence to support a means to induce ferroptosis in PDAC via modulation of iron metabolism. First, DDP combined with DHA significantly accelerated the accumulation of labile free iron reflected by the decreased fluorescence of RPA, and increased expression of TFR, which subsequently results in lipid peroxidation. Second, DFO mitigated DHA/DDP-induced ferroptosis in a concentration-dependent manner, while Fe2+ addition accelerated DHA/DDP-induced ferroptotic cell death which was able to be blocked by DFO treatment. Third, reconstituted expression of FTH is able to abolish synergistic cytotoxicity of DHA/DDP through chelation intracellular transitional iron pool. Lastly, pharmacological treatment of iron chelator DFO could weaken the effect of DHA/DDP on ferroptosis in vivo. Our data show that the combination of DHA with DDP is not only tolerable but could also be beneficial through inducing ferroptosis.
DDP is still used as a mainstay of chemotherapeutic agent in combination with other drugs or radiotherapy for pancreatic cancer therapy [41]. However, DDP is commonly associated with acquired drug resistance and high toxicity in clinical settings. Previous research regards that DDP leads to DNA injury and ultimately induces apoptosis [42]. Recent studies have discovered that DDP could induce GSH depletion and GPX4 inactivation, which emerges as an inducer of ferroptosis [12, 43]. Similarly, DDP-resistant or platinum-tolerant cancer cells were also shown to exhibit increased vulnerability to ferroptosis [44]. In our present work, we observed that DDP acts synergistically with DHA to induce oxidative stress which originated from impaired mitochondrial homeostasis. Mechanically, DHA acts synergistically with DDP to suppress mitochondrial oxidative phosphorylation process through inhibiting basal respiratory, maximal respiratory, non-mitochondrial oxygen consumption, thus leading to the reduction of ATP production and enhancement of mitochondrial ROS generation. Iron depletion rescued the mitochondrial respiration and ATP production under the challenge of DHA/DDP, which indicated that free iron accumulation originating from DHA/DDP treatment acts upstream of mitochondrial dysfunction. In addition, we found that co-treatment of DHA with DDP synergistically decreased the expression of GPX4. This may be another mechanism involved in the activation of ferroptotic cell death.
Furthermore, several completed clinical trials (NCT00764036, NCT02353026) and ongoing clinical trials (NCT02633098 and NCT03093129) have shown the efficiency and great tolerance of artemisinins in patients with solid tumors. In addition to inducing apoptosis, both DHA and DDP can trigger ferroptosis in cancer cells and exhibit the synergistic effect through increasing the intracellular free iron. Therefore, our study provides evidence that DHA may be a promising adjuvant to improve the cisplatin-based treatment of patients with pancreatic cancer. We also provide a framework for further understanding and targeting of ferroptosis in cancer therapy.
Conclusion
This study presents strong evidence that DHA could intensively strengthen the cytotoxicity effect of DDP and significantly reduce its effective concentrations both in vitro and in vivo. In particular, ferroptosis contributes to the main cytotoxic effects in PDAC cells under the challenge of DHA and DDP. Our results provide experimental evidence that DHA acts synergistically with DDP and renders PDAC cells vulnerable to ferroptosis, which may act as a promising therapeutic strategy.
Data availability
All data generated during this study are included either in this article or in the supplementary information files.
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Funding
This research was supported by National Science and Technology Major Project for New Drug (No. 2017ZX301033), Zhejiang Public Welfare Technology Application Research Project (Grant Nos.LGF19H080006, LGF21H010008, LGF20H080005), Medical and Health Science and Technology Project of Zhejiang Province (Nos. 2019RC014, 2019RC115, 2021KY842, 2021KY483, 2021KY077). Outstanding Youth Foundation of Zhejiang Provincial People’s Hospital (No. ZRY2020B001).
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J.D., X.T., and Y.W. conceived and designed the experiments. X.W., Y.L., X.R., Q.J., C.Y., and Yi Zhou performed the experiments. W.H., C.Z., and L.W. analyzed the data. H.L. and L.F. contributed with material and data sharing. J.D. and X.W. wrote the paper. Yo. Zhou, X.T., and Y.W. revised and finalized the manuscript. All authors read and approved the final manuscript.
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Animal experiments were performed in strict adherence with the relevant guidelines and regulations of the Animal Care and Use Committee of the Zhejiang Provincial People’s Hospital and approved by the animal ethics committee of the Zhejiang Provincial People’s Hospital.
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Du, J., Wang, X., Li, Y. et al. DHA exhibits synergistic therapeutic efficacy with cisplatin to induce ferroptosis in pancreatic ductal adenocarcinoma via modulation of iron metabolism. Cell Death Dis 12, 705 (2021). https://doi.org/10.1038/s41419-021-03996-y
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DOI: https://doi.org/10.1038/s41419-021-03996-y
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