Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple?negative?breast?cancer cells

Article abstract of DOI:10.1186/s13045-020-01016-8

Background: TNBC is the most aggressive breast cancer with higher recurrence and mortality rate than other types of breast cancer. There is an urgent need for identification of therapeutic agents with unique mode of action for overcoming current challenge

Full text of DOI:10.1186/s13045-020-01016-8

et al. J Hematol Oncol
(2021) 14:19
Ding
https://doi.org/10.1186/s13045-020-01016-8
Open Access
RESEARCH
Identifcation of a small molecule
as inducer of ferroptosis and apoptosis
through ubiquitination of GPX4
in triple negative breast cancer cells
Yahui Ding1, Xiaoping Chen1, Can Liu1, Weizhi Ge1, Qin Wang1, Xin Hao1, Mengmeng Wang2, Yue Chen1*
and Quan Zhang1*
Abstract
Background: TNBC is the most aggressive breast cancer with higher recurrence and mortality rate than other types
of breast cancer. There is an urgent need for identifcation of therapeutic agents with unique mode of action for over-
coming current challenges in TNBC treatment.
Methods: Diferent inhibitors were used to study the cell death manner of DMOCPTL. RNA silencing was used to
evaluate the functions of GPX4 in ferroptosis and apoptosis of TNBC cells and functions of EGR1 in apoptosis. Immu-
nohistochemical assay of tissue microarray were used for investigating correlation of GPX4 and EGR1 with TNBC.
Computer-aided docking and small molecule probe were used for study the binding of DMOCPTL with GPX4.
Results: DMOCPTL, a derivative of natural product parthenolide, exhibited about 15-fold improvement comparing
to that of the parent compound PTL for TNBC cells. The cell death manner assay showed that the anti-TNBC efect of
DMOCPTL mainly by inducing ferroptosis and apoptosis through ubiquitination of GPX4. The probe of DMOCPTL
assay indicated that DMOCPTL induced GPX4 ubiquitination by directly binding to GPX4 protein. To the best of our
knowledge, this is the frst report of inducing ferroptosis through ubiquitination of GPX4. Moreover, the mechanism
of GPX4 regulation of apoptosis is still obscure. Here, we frstly reveal that GPX4 regulated mitochondria-mediated
apoptosis through regulation of EGR1 in TNBC cells. Compound 13, the prodrug of DMOCPTL, efectively inhibited
the growth of breast tumor and prolonged the lifespan of mice in vivo, and no obvious toxicity was observed.
Conclusions: These fndings frstly revealed novel manner to induce ferroptosis through ubiquitination of GPX4 and
provided mechanism for GPX4 inducing mitochondria-mediated apoptosis through up-regulation of EGR1 in TNBC
cells. Moreover, compound 13 deserves further studies as a lead compound with novel mode of action for ultimate
discovery of efective anti-TNBC drug.
Keywords: GPX4, Ubiquitination, EGR1, Ferroptosis, Triple negative breast cancer
Introduction
*Correspondence: yuechen@nankai.edu.cn; zhangquan@nankai.edu.cn;
Ferroptosis is a new form of programmed cell death
zhangquan612@163.com
characterized by the accumulation of iron-dependent
lipid peroxides, and is morphologically, biochemically,
and genetically distinct from apoptosis, necrosis, and
autophagy [1]. In recent years, more and more research
1 State Key Laboratory of Medicinal Chemical Biology, College
of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,
Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353,
People’s Republic of China
Full list of author information is available at the end of the article
© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Ding et al. J Hematol Oncol
(2021) 14:19
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revealed that ferroptosis was implicated in various TNBC cells [23]. Terefore, inducing ferroptosis is a new
human diseases, including Alzheimer’s, Parkinson’s and efective strategy for treatment of TNBC.
Huntington’s diseases, stroke, periventricular leukoma-
Sesquiterpene lactones (SLs) have attracted extensive
lacia (PVL), intracerebral hemorrhage, kidney degen- attention because of their potent bioactivity, such as
eration [2, 3]. Moreover, emerging evidence indicated anti-cancer, anti-infammatory and anti-malaria [24, 25].
that ferroptosis may also have a tumor-suppressor Parthenolide (1, PTL, Fig. 1), a prominent germacrane-
function for cancer therapy. It was reported that many type SL, showed promising anti-cancer property. Never-
cancer cells, such as liver cancer, difuse large B cell theless, PTL has some disadvantages, such as poor oral
lymphomas (DLBCL), renal cell carcinoma, gastric can- bioavailability, unstable under chemical and physiological
cer, rhabdomyosarcoma, ovarian cancer cells, are sus- conditions and poor water solubility [26, 27]. DMAPT, a
ceptible to ferroptosis [4, 5]. Small molecule ferroptosis derivative of PTL, efectively increased the water solu-
inducers had a strong inhibition of tumor growth and bility and oral bioavailability, which has advanced into a
enhanced the sensitivity of chemotherapeutic drugs phase I clinical trial for treatment of acute myeloid leu-
[6]. Combination of chemotherapeutic drugs such as kemia (AML) [25]. Recently, we developed the other PTL
tmozolomide, cisplatin, doxorubicin with ferroptosis derivative, ACT001, to be in clinical trial in Australia and
inducer erastin resulted in a remarkable synergistic China for treatment of glioblastoma [28].
efect on tumor treatment [3]. Terefore, induction of
ferroptosis has become a novel potential therapeutic induced ferroptosis [29], we rationalized that an alterna-
strategy for cancer therapy [7–12].
tive way to lead to cancer cell death by inducing ferrop-
As previous studies suggested that GPX4 inhibition
Breast cancer is the most commonly being diagnosed tosis would be through ubiquitination of GPX4 to reduce
cancer and the leading cause of cancer death for women. abundance of GPX4. In this study, we revealed a PTL
Worldwide, there are about 2.1 million newly diagnosed derivative (2, DMOCPTL, Fig. 2) as an inducer of ferrop-
female breast cancer cases, and the incidence and mor- tosis and apoptosis through ubiquitination of GPX4 and
tality of female breast cancer is 24.2% and 15%, respec- a potential therapeutic agent for TNBC. To the best our
tively, in 2018 [13]. Breast cancer accounts for 30% all knowledge, herein, this is the frst report of inducing fer-
new cancer diagnoses in women in USA [14]. Although roptosis through ubiquitination of GPX4.
5-year relative survival rate for breast cancer is 90% in
USA [14], the prognosis is still far from satisfactory, espe- Materials and methods
cially for triple-negative breast cancer (TNBC). TNBC Chemistry
is the most aggressive breast cancer with higher recur- Synthesis of compound 4. To a solution of 3 (500 mg,
rence and mortality rate than other types of breast cancer 2.74 mmol) and TBSCl (455 mg, 3.0 mmol) in CH2Cl2
[15]. TNBC represents 15–20% of breast carcinomas and was added imidazole (204 mg, 3.0 mmol) at 0 °C. Te
is characterized by lack of expression of estrogen recep- mixture was stirred overnight, quenched with NH4Cl,
tors (ER), progesterone receptors (PR) and human epi- and extracted with CH2Cl2. Te CH2Cl2 layer was dried
dermal growth factor receptor 2 (HER-2) [16, 17]. TNBC over anhydrous Na2SO4 and concentrated under reduced
patients cannot beneft from targeted therapy, such as pressure to provide 4 (731 mg, 2.47 mmol, yield 90%).
endocrine or anti-HER2 therapy and chemotherapy is
Synthesis of compound 5. To a solution of tri-
still the only validated therapy option for treatment of methyl phosphonoacetate (1.48 mL, 9.17 mmol) in
TNBC in clinical practice [18]. Terefore, there is an anhydrous THF (20 mL) was added LiHMDS (1.0 M,
urgent need for identifcation of therapeutic agents with 9.17 mL, 9.17 mmol) at − 20 °C. After 1.5 h, the mix-
unique mode of action for overcoming current challenges ture was cooled to − 78 °C. Compound 4 (1.36 g,
in TNBC treatment.
4.59 mmol) in THF was added to the mixture. Te
It was reported that ferroptosis inducer erastin could reaction mixture was quenched with saturated NaCl
selectively target MDA-MB-231 cells and efciently and extracted with ethylacetate. Te organic layer was
induce ferroptosis in TNBC cells [19]. Sulfasalazine
could induce ferroptosis in breast cancer cells with low
ER expression [20]. Combination of siramesine and lapa-
tinib induced ferroptosis in MDA-MB-231 and SKBR3
cells [21]. Kufe et al., identifed that targeting MUC1-C/
xCT signaling pathway induced ferroptosis of TNBC cells
[22]. Lee and Tseng et al., reported that cystine starvation
could trigger ferroptosis through CHAC1 degradation of
Fig. 1 Structures of parthenolide (1), DMAPT, and ACT001
glutathione via GCN2-eIF2α-ATF4 pathway in human

Ding et al. J Hematol Oncol
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Fig. 2 The antiproliferative activity of DMOCPTL in TNBC cells. a The chemical structure of DMOCPTL. b The efect of DMOCPTL on the viability of
MDA-MB-231 and SUM159 cells after being treated for 72 h. c The representative pictures and colony number of MDA-MB-231 and 4T1 cells after
treatment of DMOCPTL at concentrations of 0.05 μM, 0.1 μM and 0.25 μM, respectively. d The efect of DMOCPTL and PTL at a concentration of
0.5 μM on colony formation in MDA-MB-231 cells. Analysis results represented mean SD, *P<0.05, **P<0.01, ***P<0.001
dried over anhydrous Na2SO4 and concentrated under
Synthesis of compound 8a. A mixture of 6a (110 mg,
reduced pressure to give a crude oil. Te resulting oil 0.4 mmol), LiOH•H2O (336 mg, 8 mmol, 20 eq) and
was dissolved in THF, which was added TBAF in THF THF–H2O (1:1) (4 mL) was stirred at 40 °C for 12 h.
(5 mL, 1 N). After 12 h, the reaction was quenched Te pH of the mixture adjusted to 2–3 with 2 N HCl.
with NH4Cl, extracted with ethylacetate. Te organic Te resulting mixture was extracted with ethylacetate.
layer was dried over anhydrous Na2SO4 and concen- Te organic layer was dried over anhydrous Na2SO4 and
trated under reduced pressure to give an oil, which concentrated under reduced pressure to give a crude
was purifed with silica gel column chromatography to oil. Te mixture of the crude oil, 7 (211 mg, 0.8 mmol,
1
provide a white solid 5 (915 mg, yield 84%). H NMR 2 eq), EDCI (155 mg, 0.8 mmol, 2 eq), DMAP (1.2 mg,
(400 MHz, MeOD-d4) δ 8.19 (d, J = 16.2 Hz, 1H), 6.79 0.01 mmol), and Et3N (110.9 μL, 0.8 mmol, 2 eq) in
(d, J = 16.2 Hz, 1H), 6.20 (s, 2H), 3.95 (s, 6H), 3.86 (s, 1 mL CH2Cl2 was stirred at room temperature for 12 h
3H). 13C NMR (100 MHz, MeOD-d4) δ 171.3, 162.4, and quenched with sat. NaHCO3. Te resulting mixture
162.3, 137.4, 115.7, 104.9, 92.5, 55.9, 51.7. HRMS (ESI) was extracted with CH2Cl2. Te CH2Cl2 layer was dried
cald for C12H15O5 [M + H]+ 239.0914, found 239.0910.
over anhydrous Na2SO4 to give a crude oil, which was
Synthesis of compounds 6a-6e.
A mixture of purifed by silica gel column chromatography to pro-
5 (100 mg, 0.42 mmol, 1 eq), K2CO3 (232.1 mg, vide a white solid 8a (109 mg, 0.22 mmol, 54% for two
1.68 mmol, 4 eq) and diferent alkynyl bromide steps). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J=16.2 Hz,
(1.47 mmol, 3.5 eq) in 4 mL anhydrous DMF was heated 1H), 6.71 (d, J=16.2 Hz, 1H), 6.20 (d, J=5.2 Hz, 3H),
to 40 °C for 4 h. Te reaction was quenched with NaCl 5.74 (t, J=8.1 Hz, 1H), 5.53 (d, J=3.1 Hz, 1H), 4.73 (q,
and extracted with ethylacetate. Te organic layer was J=4.9 Hz, 3H), 4.60 (d, J=12.4 Hz, 1H), 3.95–3.75 (m,
dried over anhydrous Na2SO4 to give a crude oil, which 7H), 3.20–3.04 (m, 1H), 2.94 (d, J=9.4 Hz, 1H), 2.58 (t,
was purifed with silica gel column chromatography to J=2.3 Hz, 1H), 2.54–2.13 (m, 6H), 1.65 (dd, J=19.5,
provide white solid 6a-6e in yields of 67%–88%.
6.7 Hz, 1H), 1.60–1.54 (m, 3H), 1.14 (t, J=12.6 Hz, 1H).

Ding et al. J Hematol Oncol
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13C NMR (100 MHz, CDCl3) δ 169.7, 168.8, 161.4, 161.0,
138.8, 136.4, 135.6, 131.0, 120.6, 116.9, 106.4, 100.1, 91.4,
81.2, 78.0, 76.3, 67.1, 63.5, 60.1, 56.0, 55.9, 42.9, 36.8, 26.3,
25.3, 24.0. HRMS (ESI) cald for C29H32NaO8 [M+Na]+
531.1989, found 531.1992.
4.00 (t, J=6.1 Hz, 2H), 3.87 (d, J=13.5 Hz, 6H), 3.12 (t,
J=9.3 Hz, 1H), 2.94 (d, J=9.6 Hz, 1H), 2.50–2.12 (m,
8H), 1.96 (s, 1H), 1.89–1.75 (m, 2H), 1.64 (m, 6H), 1.55
(s, 3H), 1.14 (t, J=12.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 169.7, 168.9, 162.7, 161.5, 138.9, 136.7, 135.7,
130.9, 120.5, 116.2, 105.6, 91.0, 84.4, 81.2, 68.6, 68.0, 67.0,
63.5, 60.1, 55.9, 42.9, 36.8, 28.8, 28.3, 26.3, 25.3, 25.3, 24.0,
18.5, 18.2. HRMS (ESI) cald for C33H40NaO8 [M+Na]+
587.2615, found 587.2618.
8b (white solid, 30%) was synthesized following the
1
similar procedure for 8a. H NMR (400 MHz, CDCl3) δ
8.11 (d, J=16.2 Hz, 1H), 6.70 (d, J=16.2 Hz, 1H), 6.20
(d, J=3.2 Hz, 1H), 6.12 (s, 2H), 5.74 (t, J=8.1 Hz, 1H),
5.53 (d, J=2.8 Hz, 1H), 4.73 (d, J=12.5 Hz, 1H), 4.60 (d,
J=12.3 Hz, 1H), 4.13 (t, J=7.0 Hz, 2H), 3.93–3.77 (m,
7H), 3.13 (t, J=9.6 Hz, 1H), 2.94 (d, J=9.4 Hz, 1H), 2.77–
2.63 (m, 2H), 2.55–2.13 (m, 6H), 2.07 (s, 1H), 1.70–1.63
(m, 1H), 1.25 (s, 3H), 1.14 (t, J=12.5 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 169.7, 168.8, 162.0, 161.5, 138.9,
136.5, 135.7, 131.1, 131.0, 129.0, 120.5, 116.6, 91.1, 81.3,
70.3, 67.1, 66.2, 63.5, 60.1, 55.9, 43.0, 36.8, 26.4, 25.3, 24.1,
19.7, 18.2. HRMS (ESI) cald for C30H34NaO8 [M+Na]+
545.2146, found 545.2148.
Synthesis of 10a
A mixture of azide (80 mg, 0.097 mmol) and 8a (60 mg,
0.118 mmol), CuSO4 (0.014 mmol), sodium ascorbate
(0.014 mmol), tert-butanol (1 mL) and water (0.5 mL)
was stirred overnight at room temperature. Ten, 3 mL
water was added and the reaction mixture was extracted
(3×10 mL) with EtOAc. Te combined organic layers
were washed with saturated brine, dried over anhydrous
Na2SO4, and concentrated to give crude product, which
was purifed on a silica gel column to give compound
10a.
8c (white solid, 44%) was synthesized following the
1
similar procedure for 8a. H NMR (400 MHz, CDCl3) δ
1H NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 8.06 (d,
J=16.2 Hz, 1H), 7.77 (s, 2H), 7.69 (s, 1H), 7.51 (s, 1H),
7.26 (d, J=9.3 Hz, 2H), 7.05 (dd, J=9.6, 2.4 Hz, 2H),
6.97–6.91 (m, 2H), 6.66 (d, J=16.2 Hz, 1H), 6.33 (s,
2H), 5.70 (t, J=8.3 Hz, 1H), 5.59 (d, J=3.2 Hz, 1H),
5.24 (s, 2H), 4.73–4.61 (m, 2H), 4.62–4.57 (m, 2H),
3.85 (s, 6H), 3.67 (m, 10H), 3.62–3.56 (m, 4H), 3.54 (d,
J=10.5 Hz, 7H), 3.46 (t, J=5.5 Hz, 4H), 3.34 (s, 7H), 2.92
(d, J=9.5 Hz, 1H), 2.53 (s, 2H), 2.41 (d, J=6.9 Hz, 3H),
2.37–2.25 (m, 2H), 2.23–2.08 (m, 3H), 1.55 (s, 4H), 1.29–
1.28 (m, 15H).
8.09 (d, J=16.2 Hz, 1H), 6.68 (d, J=16.2 Hz, 1H), 6.18
(s, 1H), 6.10 (s, 2H), 5.72 (t, J=8.0 Hz, 1H), 5.52 (s, 1H),
4.71 (d, J=12.4 Hz, 1H), 4.59 (d, J=12.4 Hz, 1H), 4.10 (t,
J=5.9 Hz, 2H), 3.85 (s, 7H), 3.10 (t, J=9.4 Hz, 1H), 2.92
(d, J=9.4 Hz, 1H), 2.53–2.10 (m, 8H), 2.01 (dd, J=12.0,
5.4 Hz, 3H), 1.68–1.60 (m, 1H), 1.54 (s, 3H), 1.12 (t,
J=12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 169.6,
168.8, 162.5, 161.5, 138.8, 136.5, 135.6, 130.8, 120.4,
116.3, 105.7, 91.0, 83.3, 81.2, 69.2, 67.0, 66.3, 63.4, 60.1,
55.8, 42.9, 36.8, 28.1, 26.3, 25.2, 24.0, 18.1, 15.2. HRMS
(ESI) cald for C31H36NaO8 [M+Na]+ 559.2302, found
559.2305.
8d (white solid, 64%) was synthesized following the
Synthesis of compound 12 in two methods
1
similar procedure for 8a. H NMR (400 MHz, CDCl3)
Method A: A solution of 7 (1.1 g, 4.16 mmol) in dimeth-
ylamine (20.8 mL, 41.6 mmol, 2 N in THF) was stirred
for 1 h at 0 °C. Te solvent was removed under reduced
pressure. Te residue was purifed by silica gel column
chromatography (CH2Cl2: MeOH=50:1) to aford the
desired product 11 (1.2 g, 93%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ 5.58 (t, J=7.9 Hz, 1H), 4.10 (dd,
J=30.7, 13.1 Hz, 2H), 3.85 (t, J=9.2 Hz, 1H), 3.38 (s, 1H),
2.81 (d, J=9.4 Hz, 1H), 2.73 (dd, J=12.9, 5.1 Hz, 1H),
2.61 (dd, J=12.9, 5.4 Hz, 1H), 2.51–2.38 (m, 4H), 2.30–
2.18 (m, 8H), 2.17–2.06 (m, 2H), 1.67–1.55 (m, 1H), 1.52
(s, 3H), 1.12–1.02 (m, 1H). 13C NMR (100 MHz, CDCl3)
δ 177.0, 141.2, 127.5, 81.7, 66.3, 64.2, 60.0, 57.6, 45.8,
44.3, 42.1, 37.2, 27.7, 26.0, 23.9, 18.1. HRMS (ESI) calcd
for C17H28N2O4 [M+H]+ 310.2013, found 310.2015.
To a solution of 2,6-dimethoxylcinnamic acid (712 mg,
3.42 mmol), compound 11 (705 mg, 2.28 mmol), EDCI
(655.6 mg, 3.42 mmol) and DMAP (27.8 mg, 0.228 mmol)
in 25 mL CH2Cl2 was added TEA (0.45 mL, 3.42 mmol)
δ 8.10 (d, J=16.2 Hz, 1H), 6.68 (d, J=16.2 Hz, 1H),
6.19 (d, J=3.4 Hz, 1H), 6.09 (s, 2H), 5.73 (t, J=8.1 Hz,
1H), 5.52 (d, J=3.0 Hz, 1H), 4.72 (d, J=12.4 Hz, 1H),
4.59 (d, J=12.4 Hz, 1H), 4.02 (t, J=6.2 Hz, 2H), 3.86 (d,
J=13.4 Hz, 7H), 3.18–3.04 (m, 1H), 2.93 (d, J=9.4 Hz,
1H), 2.55–2.12 (m, 8H), 1.97 (t, J=2.3 Hz, 1H), 1.96–1.88
(m, 2H), 1.72 (dt, J=14.7, 7.4 Hz, 2H), 1.64 (dd, J=18.3,
6.8 Hz, 1H), 1.55 (s, 3H), 1.13 (t, J=12.7 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ 169.7, 168.8, 162.6, 161.5,
138.9, 136.6, 135.7, 130.9, 120.5, 116.3, 105.6, 91.0, 84.0,
81.2, 68.9, 67.6, 67.0, 63.5, 60.1, 55.9, 42.9, 36.8, 28.3, 26.3,
25.3, 25.1, 24.0, 18.3, 18.2. HRMS (ESI) cald C32H38NaO8
[M+Na]+ for 573.2459, found 573.2462.
8e (white solid, 42%) was synthesized following the
1
similar procedure for 8a. H NMR (400 MHz, CDCl3) δ
8.11 (d, J=16.1 Hz, 1H), 6.69 (d, J=16.2 Hz, 1H), 6.20
(s, 1H), 6.09 (s, 2H), 5.74 (t, J=7.8 Hz, 1H), 5.53 (s,
1H), 4.72 (d, J=12.4 Hz, 1H), 4.60 (d, J=12.5 Hz, 1H),

Ding et al. J Hematol Oncol
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at 0 °C. Te mixture was stirred for 8 h at room tempera- aford the desired product 12 (985 mg, 96%) as a white
ture. Te reaction was quenched with saturated aque- solid.
ous NaHCO3 and extracted with CH2Cl2 (3×75 mL).
Synthesis of compound 13. To a solution of com-
Te combined organic layers were washed with satu- pound 12 (1.15 g, 2.3 mmol) in methanol (23 mL), fuma-
rated brine, dried over Na2SO4, and concentrated to give ric acid (267 mg, 2.3 mmol) was added. Te mixture
an oily crude product, which was purifed on a silica gel was stirred for 6.5 h and concentrated under vacuum to
column [DCM:MeOH=50:1] to yield compound 12 aford compound 13 as a white amorphous solid (1.3 g,
1
1
(987.2 mg, 86%) as a white solid. H NMR (400 MHz, yield 92%). H NMR (400 MHz, DMSO-d6) δ 12.95 (s,
CDCl3) δ 8.16 (d, J=16.3 Hz, 1H), 7.28 (d, J=8.7 Hz, 2H), 8.00 (d, J=16.3 Hz, 1H), 7.37 (t, J=8.4 Hz, 1H),
1H), 6.89 (d, J=16.3 Hz, 1H), 6.56 (d, J=8.4 Hz, 2H), 6.78 (d, J=16.3 Hz, 1H), 6.72 (d, J=8.4 Hz, 2H), 6.61 (s,
5.67 (t, J=7.8 Hz, 1H), 4.84 (d, J=12.8 Hz, 1H), 4.66 (d, 2H), 5.59 (t, J=7.6 Hz, 1H), 4.79 (d, J=12.7 Hz, 1H), 4.57
J=12.8 Hz, 1H), 3.97–3.80 (m, 7H), 2.84 (d, J=9.3 Hz, (d, J=12.6 Hz, 1H), 4.03 (t, J=9.5 Hz, 1H), 3.86 (s, 6H),
1H), 2.73 (d, J=4.3 Hz, 1H), 2.65 (d, J=5.9 Hz, 1H), 2.77–2.54 (m, 4H), 2.39–2.23 (m, 4H), 2.18 (s, 6H), 2.10
2.58–2.26 (m, 6H), 2.23 (s, 6H), 2.15 (d, J=12.5 Hz, 2H), (dd, J=24.6, 10.8 Hz, 3H), 1.65 (t, J=11.4 Hz, 1H), 1.48
1.58 (d, J=20.6 Hz, 4H), 1.10 (t, J=12.8 Hz, 1H). 13C (s, 3H), 0.94 (t, J=12.3 Hz, 1H). 13C NMR (100 MHz,
NMR (100 MHz, CDCl3) δ 177.2, 168.3, 160.2, 136.3, DMSO-d6) δ 177.1, 167.2, 166.2, 159.6, 135.8, 135.2,
136.1, 131.6, 128.5, 120.2, 112.2, 103.8, 81.3, 66.2, 64.0, 134.2, 132.2, 128.1, 119.4, 110.7, 104.1, 80.4, 66.0, 63.2,
60.0, 58.5, 55.9, 45.9, 44.7, 43.3, 37.1, 27.2, 25.0, 23.9, 18.1. 59.8, 57.9, 56.0, 45.2, 43.3, 42.6, 36.6, 25.6, 24.2, 23.1, 17.5.
HRMS (ESI) calcd for C28H38NO7 [M+H]+ 500.2643, HRMS (ESI) calcd for C28H38NO7 [M+H]+ 500.2643,
found 500.2644.
found 500.2645.
Method B. Synthesis of compound 2. To a mix-
ture of 7 (53 mg, 0.2 mmol), EDCI (115 mg, 0.6 mmol), Cell culture
DMAP (1.2 mg, 0.01 mmol) and 2,6-dimethoxylcinnamic Human triple negative breast cancer cell lines MDA-
acid (0.3 mmol, 1.5 eq) in CH2Cl2 2 mL was added tri- MB-231, SUM159, BT574, and mouse breast cancer
ethylamine (83.4 μL, 0.6 mmol). After 12 h, saturated cell 4T1 were purchased from ATCC and were cultured
NaHCO3 was added. Te resulting mixture was extracted in 1640 medium supplement with 10% FBS under a 5%
with CH2Cl2. Te CH2Cl2 layer was dried over anhy- CO2 humidifed atmosphere at 37 °C. Hs578T and MDA-
drous Na2SO4, and concentrated under reduced pres- MB-468 breast cancer cells were cultured in DMEM
sure to give crude product, which was purifed by silica medium supplement with 10% FBS under a 5% CO2
gel column chromatography to aford compound 2 (yield humidifed atmosphere at 37 °C.
1
83%). H NMR (400 MHz, CDCl3) δ 8.16 (d, J=16.3 Hz,
1H, H-18), 7.30–7.22 (m, 1H, overlap with CHCl3, MTT assay
H-4′), 6.83 (d, J=16.3 Hz, 1H, H-17), 6.54 (d, J=8.4 Hz, Human triple negative breast cancer cells were seeded
2H, H-3′, H-5′), 6.19 (d, J=3.5 Hz, 1H, H-13), 5.73 into 96 well plate at the density of 4000 cells/200µL/well.
(t, J=8.1 Hz, 1H, H-1), 5.53 (d, J=3.2 Hz, 1H, H-13), After the adherent cell growth, DMOCPTL was added
4.74 (d, J=12.4 Hz, 1H, H-14), 4.60 (d, J=12.4 Hz, 1H, with a series concentration for 72 h. Ten, 20 µL thiazolyl
H-14), 3.96–3.75 (m, 7H, H-6, H-19, H-18), 3.14–3.02 blue tetrazolium bromide (MTT, 5 mg/mL) was added
(m, 1H, H-7), 2.91 (d, J=9.4 Hz, 1H, H-5), 2.49–2.11 (m, and incubated for additional 4–6 h. Te supernatant was
6H, H-2, H-3, H-8, H-9), 1.71–1.60 (m, 1H, H-8), 1.54 discarded and the precipitate was dissolved with DMSO.
(s, 3H, H-15), 1.12 (t, J=12.6 Hz, 1H, H-3). 13C NMR Ten, the absorbance at 570 nm was measured.
(100 MHz, CDCl3) δ 169.6(C-12), 168.4(C-16), 160.2(C-
2′, C-6′), 138.8(C-11), 136.5(C-18), 135.5(C-10), 131.7(C- Cell colony formation assay
4′), 130.9(C-1), 120.5(C-13), 119.5(C-17), 111.9(C-1′), Breast cancer cells MDA-MB-231 and 4T1 were seeded
103.7(C-3′, 5′), 81.2(C-6), 67.1(C-14), 63.4(C-5), 60.1(C- into 6 well plate with a concentration of 500 cells/1 mL/
4), 55.9(C-19, C-20), 42.8(C-7), 36.7(C-3), 26.2(C-8), well. After 8–12 h, compounds at diferent concentration
25.1(C-9), 24.0(C-2), 18.1(C-15). HRMS (ESI) cald for were added and incubated for 10 days. Ten, the cells
C26H34NO7 [M+NH4]+ 472.2330, found 472.2329.
were fxed with 4% polyoxymethylene at room tempera-
A solution of compound 2 (930 mg, 2.05 mmol) and ture for 15 min. After being washed with PBS, the cells
dimethylamine (10.5 mL, 21.0 mmol, 2 N in THF) at 0 °C were stained with crystal violet solution at room temper-
was stirred 1 h at 0 °C. Te solvent was removed under ature for 15 min. Te excess crystal violet was removed
reduced pressure. Te residue was purifed by silica gel and washed with water for 3 times. Te number of colo-
column chromatography (CH2Cl2: MeOH=50:1) to nies was counted.

Ding et al. J Hematol Oncol
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Cell death manner assay
48 h, the efcacy of siRNA was confrmed by western
To investigate the mechanism of DMOCPTL in breast
cancer proliferation inhibition, the manners of cells
death were analyzed. Te cell apoptosis inhibitor
Z-VAD-FMK, cell autophagy inhibitor 3-methylad-
enine, cell necrosis inhibitor necrostatin-1, and fer-
roptosis inhibitors N-Acetyl-L-cysteine, deferoxamine
and Ferrostatin-1 were purchased and stored at –20 °C.
MDA-MB-231 cells were seeded with a density of at
5000 cells/200µL/well into 96 well plate. After 8–12 h,
DMOCPTL was added at diferent concentrations. Fol-
lowing that 10 µM Z-VAD-FMK, 5 mM 3-methylade-
nine, 10 µM necrostatin-1, 3 mM N-acetyl-L-cysteine,
200 µM deferoxamine and 10 µM Ferrostatin-1 were
added and co-incubated with DMOCPTL, respec-
tively, for 72 h. Ten, 20 µL thiazolyl blue tetrazolium
bromide (MTT, 5 mg/mL) was added and incubated
at 37 °C for additional 4 h. Ten, supernatant was dis-
carded and 200 μL DMSO was added to dissolve the
precipitate. After 15 min, the absorbance was measured
at 570 nm. Te percentage of inhibition was calculated.
blot assay.
Immunofuorescence assay
MDA-MB-231 cells were cultured on gelatin-coated glass
coverslips for 24 h and treated with DMOCPTL for 48 h.
Ten, the cells were fxed in 4% paraformaldehyde for
20 min and permeabilized with Triton X-100 for 15 min.
After blocked with horse serum for 30 min, the cells
were incubated with GPX4 antibody over night at 4 °C.
After the incubation, the cells were washed fve times
with PBST and incubated with the anti-rabbit IgG-FITC
secondary antibody for 1 h at room temperature. Ten,
the cell nucleus was stained with DAPI and analyzed
using fuorescence microscopy. As to fow cytometry
assay, MDA-MB-231 cells with DMOCPTL treatment
for 48 h were collected, fxed with 4% paraformaldehyde
for 20 min, permeabilized with Triton X-100 for 15 min,
incubated with GPX4 antibody overnight, then incubated
with corresponding FITC-conjunct second antibody. Flu-
orescence intensity of GPX4 expression was analyzed by
fow cytometry.
Cell apoptosis assay
Western blot assay
MDA-MB-231 and SUM159 cells were collected and
washed with cold PBS. Ten, the cells were suspended
with 100 μL of binding bufer; 5 μL of Annexin V-APC
and 5 μL PI were added and incubated for 15 min in
dark at room temperature. Ten, the cells were ana-
lyzed by fow cytometry within 1 h.
MDA-MB-231 and SUM159 cells were collected and
lysed with RIPA bufer for 30 min on ice. Ten, the pro-
tein (50 μg) from each sample were separated by 12%
tris-acrylamide gel electrophoresis and transferred onto
PVDF membrane. After blocking with 5% skim milk for
1 h at room temperature, Primary antibodies against
GPX-4, EGR1, Bax, Bcl-2, Bcl-xl, cytochrome C, caspase
3, caspase 9 and PARP was used and incubated at 4 °C
overnight on rotary shaker. After washing with PBST for
5 times, the membrane was probed with goat anti-rabbit
IgG highly cross-adsorbed secondary antibody (1:10,000)
for 2 h at room temperature. Ten, the membrane was
washed for 5 times and developed with ECL reagent.
Flow cytometry with reactive O2 species assay
MDA-MB-231 and SUM159 cells in logarithmic growth
period were digested and divided into control group,
DMOCPTL-treated group. Te cells were washed with
PBS for 3 times and DMOCPTL was added. Ten,
10 µM DCF-DA or BODIPY 581/591 C11 were added
and co-incubated with DMOCPTL in an incubator at
37 °C. Te cells were collected by centrifugation and
analyzed by fow cytometry within 1 h.
Probe pull‑down assay
Te pull-down experiments were performed using the
probe of DMOCPTL. In vitro pull-down assay, MDA-
MB-231 and SUM159 cells were collected and lysed,
respectively, in RIPA bufer supplemented with protease
Fe2+ intensity assay
MDA-MB-231 was collected and washed with cold PBS inhibitors. Ten, probe 10 was added and incubated for
bufer. Ten, cells were incubated with PGSK at 10 µM 2 h at room temperature with diferent concentrations.
at 37 °C for 30 min in dark. Ten, cells were collected, Ten, excessive pre-cooled methanol was added and
washed and analyzed by fow cytometry within 1 h.
incubated at−80 °C for 30 min to precipitate the protein.
After centrifuged at 14,000×g for 15 min, the precipi-
tated proteins were dissolved and incubated with strepta-
vidin conjunct agarose A+G beads at 4 °C overnight.
Ten, the streptavidin beads were washed three times
with PBS bufer, and the bead-bound proteins were col-
lected and detected by western blot experiments.
RNA interference
Breast cancer cell at about 40–50% confuence was
transfected with siRNA using lipofectamine™ 2000
according to the manufacturer’s instructions. After

Ding et al. J Hematol Oncol
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Page 7 of 21
In vitro ubiquitination assay
As to in situ pull-down assay, MDA-MB-231 and
SUM159 cells were seeded into 6 well plate. After 12 h,
probe 8a was added at diferent concentrations and incu-
bated for additional 6 h. Ten, the cells were collected
and lysed, respectively, in RIPA bufer supplemented with
protease inhibitors. After the centrifugation, the super-
natant was collected and a mix of TBTA (0.1 mM), TCEP
(1 mM), biotin-N3 (100 µM) and CuSO4 (1 mM) was
added to conjunct biotin on probe 8a. Ten, excessive
pre-cooled methanol was added and incubated at−80 °C
for 30 min to precipitate the protein. After centrifuged
at 14,000×g for 15 min, the precipitated proteins were
dissolved and incubated with streptavidin conjunct aga-
rose A+G beads at 4 °C overnight. Ten, the streptavi-
din beads were washed three times with PBS bufer, and
the bead-bound proteins were collected and detected by
western blot experiments or sliver staining.
MDA-MB-231 cells in logarithmic growth period were
plated into 6-well plate with the density at 1×105 cells/
mL/well. Ten, DMOCPTL at the concentration of
0.05 µM, 0.1 µM, 0.2 µM, 0.5 µM and 1 µM were added
and incubated for 24 h. Ten, 10 µM MG-132 was added
to inhibit the protein degradation for 4 h. After that
the cells were collected by centrifugation and washed
with PBS, subsequently suspended with RIPA bufer for
30 min on ice. After centrifugation, the GPX4 antibody
was added with 1:100 dilution for 2 h on ice, then 10
µL agarose G was added and co-incubated at 4 °C over-
night on rotary shaker. Te samples were collected and
washed with PBS for 3 times, then the SDS-PAGE pro-
tein electrophoresis was performed and the protein was
transferred onto PVDF membrane. Following that the
membrane was blocked in 5% skim milk for 1 h at room
temperature, and subsequently was incubated with ubiq-
uitin antibody (P4D1) at 4 °C overnight on rotary shaker.
Co‑localization assay
MDA-MB-231 cells were cultured on gelatin-coated glass
coverslips for 24 h and treated with probe 10a for 6 h at
2 µM. Te cells were fxed in 4% paraformaldehyde for
20 min and permeabilized with Triton X-100 for 15 min.
After being blocked with horse serum for 30 min, the
cells were incubated with anti-GPX4 antibody (rabbit)
and anti-Ubiquitin (mouse) over night at 4 °C. After the
incubation, the cells were washed fve times with PBST
and incubated with the anti-rabbit IgG-FITC secondary
antibody and anti-mouse IgG-AF647 secondary antibody
for 2 h at room temperature. After being washed for 3
times, the cell nucleus was stained with DAPI and ana-
lyzed using fuorescence microscopy.
Immuno‑coprecipitation assay
MDA-MB-231 and SUM159 cells were collected and
lysed with RIPA bufer for 30 min on ice. Te indicated
protein in the cell lysates was immunoprecipitated using
ubiquitin antibody. After incubated with ubiquitin anti-
body at 4 °C for 6 h, 10 µL agarose A+G was added and
co-incubated at 4 °C overnight on rotary shaker. Te
samples were washed with PBS and visualized using an
ECL detection system.
Pharmacodynamics
Compound 13 was injected into three male SD rats by
intravenous administration at a dose of 1 mg/kg. Ten,
the blood samples from jugular sinus of rats were col-
lected at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h,
4 h, 6 h and 8 h after administration. After being cen-
trifugated at 12000 rpm for 1 min, the supernatant was
collected, respectively. Ten, equal amount of acetoni-
trile was added into the sample, which was vortexed for
2 min, centrifugated at 12000 rpm for 5 min, and then the
supernatant was collected and analyzed by LC/MS. Te
pharmacodynamic of 13 on SD rats was calculated by
DAS 3.3.
Bioinformatics analysis
Te relationship between GPX4 and gene functional
states was analyzed by CancerSEA (http://biocc.hrbmu
.edu.cn/CancerSEA/). Te expression level of EGR1 in
normal breast tissue and breast cancer tissues with difer-
ent stages was analyzed in TCGA by UALCAN analysis
(http://ualcan.path.uab.edu). Te immunohistochemical
staining intensity of EGR1 in normal breast tissue and
breast cancer tissues with diferent stages were analyzed
by Human Protein Atlas database (http://www.proteinatl
as.org/).
Acute toxicity assay in Bar b/c mice
To verify the toxicity of 13, Bar b/c mice were adminis-
trated by intravenous administration at a dose of 50 mg/
kg or oral administration with 13 at a dose of 500 mg/kg
or vehicle control. During the experiment, their behav-
ior was observed, and the body weight was recorded
every day. Te level of GPT, GOT and Cr in serum was
detected by ELISA assay and the major organs of mice
including liver, spleen, lung, kidney, heart, and brain were
Docking simulations
Molecular Docking simulations were performed with the
software of AutoDock. Te crystal structure of the pub-
lished GPX4 (PDB code: 5H5S) was retrieved from the
RCSB Protein Data Bank. Te solvent molecules within
the protein structure were removed in the docking calcu-
lations, and the best ligand pose was chosen according to
the dock score.

Ding et al. J Hematol Oncol
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Page 8 of 21
weighted, fxed with 4% paraformaldehyde and sectioned
to 4 µm slides. After dewaxing and hydration, HE stain-
ing was performed. Te histology and morphology were
observed under microscope.
was 44 1, 36.7 2.5, 33.3 1.5 and 0 with the treatment
of DMOCPTL at 0, 0.05, 0.1 and 0.25 µM, respectively.
Moreover, we compared the efcacy of DMOCPTL and
PTL on inhibiting colony formation of MDA-MB-231
cells. As shown in Fig. 2d, the number of colonies was
82.7 5.5, 71.5 1.5 and 0 after treatment with vehicle,
PTL or DMOCPTL at the same concentration of 0.5 µM.
Tese results indicated that DMOCPTL could signif-
cantly inhibit colony formation of TNBC cells. Moreover,
the inhibitory efect of DMOCPTL was evidently higher
than that of PTL.
In vivo anti‑tumor activity assay
Te experimental procedures of the animal experiments
were permitted by the Animal Care and Use Committee
at Nankai University. 4T1 triple negative breast cancer
cells (1×105) were injected on the Bar b/c mouse’s breast
fat pad. Ten, compound 13 was administrated through
intravenous injection (7.5 mg/kg) according to body
weight. Body weights and tumor size were measured
every other day. Te tumor growth inhibition was calcu-
lated and the tumor weight was recorded when the mice
were sacrifced. Te tumor was collected and crushed
with cell lysate bufer. Ten, the expression of GPX4 and
EGR1 were analyzed in vehicle and 13-treated group by
western blot assay. Furthermore, to analysis the efect
of 13 on the overall survival compound 13 was admin-
istrated orally (50 mg/kg) according to body weight on
tumor animal model. After administration for 6 times
every other day the overall survival and body weight were
recorded and analyzed.
DMOCPTL induced apoptosis and ferroptosis of TNBC cells
To reveal the mechanism of DMOCPTL, the death man-
ners were analyzed. In this experiment, the apoptosis
inhibitor Z-VAD-FMK, cell autophagy inhibitor 3-meth-
yladenine, cell necrosis inhibitor necrostatin-1, and fer-
roptosis inhibitors N-acetyl-L-cysteine, deferoxamine
and Ferrostatin-1 were used. As shown in Fig. 3, there
was no signifcant change in the percentage of cell viabil-
ity after the combination of DMOCPTL and cell necrosis
inhibitor necrostatin-1, which indicated that DMOCPTL
could not induce cell necrosis (Fig. 3a). Meanwhile, the
percentage of cell viability was not afected clearly after
the combination of DMOCPTL and cell autophagy
inhibitor 3-methyladenine, which suggested that
DMOCPTL could not induce cell autophagy (Fig. 3b).
We then investigated whether the efect of DMOCPTL
was achieved by inducing cell apoptosis. Te apoptosis
inhibitor Z-VAD-FMK was combined with DMOCPTL
for treatment of MDA-MB-231 cells for 48 h. Te efect
of DMOCPTL was inhibited by cell apoptosis inhibitor
Z-VAD-FMK, which prompted us to infer that the efect
of DMOCPTL may be through inducing cell apoptosis
(Fig. 3c). As cell apoptosis inhibitor could not completely
inhibit the efect of DMOCPTL, we further analyzed
Immunohistochemical assay
Te tumors isolated from the mice were fxed with 4%
paraformaldehyde and sectioned to 4 µm slides. As
to IHC assay, after dewaxing, hydration and antigen
retrieval, the slides were incubated with primary antibod-
ies at 4 °C overnight. After washed with PBST for 3 times,
the slides were incubated with biotinylated secondary
antibody for 1 h. Ten, the slides were developed by DAB.
Results
DMOCPTL inhibited the antiproliferation of TNBC cells
Sesquiterpene lactone PTL showed moderate activity whether DMOCPTL could induce ferroptosis. Te efect
against breast cancer [30]. To improve the anticancer of DMOCPTL was inhibited by cell ferroptosis inhibitors
efect of parthenolide, we designed and synthesized a N-Acetyl-L-cysteine, deferoxamine and Ferrostatin-1
series of parthenolide derivatives. Ultimately, we identi- which demonstrated that the efect of DMOCPTL was
fed that DMOCPTL exhibited the most potent anti- achieved partly by inducing cell ferroptosis (Fig. 3d–f).
TNBC activity [31]. As shown in Fig. 2b, DMOCPTL Tese results suggested that the efect of DMOCPTL
signifcantly inhibited proliferation of MDA-MB-231 may be achieved by inducing apoptosis and ferroptosis of
and SUM159 cells in a dose dependent manner. To fur- TNBC cells.
ther investigate the inhibitory efect of DMOCPTL on
In order to investigate the efect of DMOCPTL on
proliferation of TNBC cells, the colony formation of cell apoptosis, the cell apoptosis assay was performed
MDA-MB-231 and 4T1 cells assay was performed. After by Annexin V/PI staining. Te percentage of apop-
treatment of DMOCPTL for 10 days, the number of colo- tosis cells was the sum of early apoptosis (Annexin
nies was counted. Te number of colonies was 82.7 5.5, V+/PI-) and late apoptosis (Annexin V+/PI+). As
68 1, 60 3 and 0 with treatment of DMOCPTL at dif- shown in Fig. 4b, the percentage of cell apoptosis was
ferent concentrations of 0, 0.05, 0.1 and 0.25 µM, respec- 3.60 1.53, 4.27 1.22, 12.60 1.95 and 75.60 1.30
tively, in human breast cancer MDA-MB-231 cells. As to after the treatment of DMOCPTL at the concentration
mouse breast cancer 4T1 cells, the number of colonies of 0, 0.2 µM, 0.5 µM and 1 µM, respectively.Furthermore,

Ding et al. J Hematol Oncol
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Fig. 3 DMOCPTL induced apoptosis and ferroptosis of TNBC cells. a The percentage of cell viability after the combination of DMOCPTL (0.5 μM)
with necrosis inhibitor necrostatin-1 (10 μM) for 72 h. b The percentage of cell viability after the combination of DMOCPTL (0.5 μM) with
3-methyladenine (5 mM) for 72 h. c The percentage of cell viability after the combination of DMOCPTL (0.5 μM) with Z-VAD-FMK (10 μM) for 72 h. d
The percentage of cell viability after the combination of DMOCPTL (0.5 μM) with ferroptosis inhibitors N-Acetyl-L-cysteine (5 mM), e deferoxamine
(200 μM), and f Ferrostatin-1 (10 μM). Analysis results represented mean SD, **P<0.01, ***P<0.001
DMOCPTL reduced GPX4 protein level by inducing GPX4
ubiquitination
the mechanism of DMOCPTL inducing apoptosis was
studied by western blot assay. Te relative levels of anti-
apoptotic proteins Bcl-2 and Bcl-xl were decreased with
a dose-dependent manner, while the relative level of
apoptotic protein Bax was dramatically increased with
a dose-dependent manner. Bax, Bcl-2 and Bcl-xl were
important mitochondrial proteins which regulated the
release of cytochrome C. Moreover, DMOCPTL could
lead to the release of the cytochrome C and the cleavage
of caspase 9, caspase 3 and PARP, which induced apop-
tosis of MDA-MB-231 cells. Tese results suggested that
DMOCPTL could induce apoptosis of MDA-MB-231
cells by mitochondria pathway (Fig. 4c, d).
It is well known that GPX4 was a signifcant negative reg-
ulator of ferroptosis [32, 33]. Western blot results showed
that the protein level of GPX4 was highly expressed
in MDA-MB-231, SUM159 and MDA-MB-468 cells
(Fig. 5a). DMOCPTL could reduce GPX4 protein level
in a dose-dependent manner in both MDA-MB-231 and
SUM159 cells (Fig. 5b, c). Moreover, the fow cytometry
assay and immunofuorescence assay also suggested that
DMOCPTL down-regulated the level of GPX4 with a
dose-dependent manner (Fig. 5d, e).
Ubiquitination is an enzymatic process that involves
the binding of a ubiquitin protein to a substrate protein.
Te most common result of ubiquitination is the degra-
dation of the protein via the proteasome. We rationalized
that an alternative way to lead to cell death by inducing
ferroptosis would be through inducing ubiquitination of
GPX4. We treated MDA-MB-231 cells with proteasome
inhibitor MG132 and the result showed that GPX4 was
accumulated with the treatment of MG132. Tis result
suggested that GPX4 was modifed by ubiquitination and
the degradation of GPX4 in MDA-MB-231 occurs via the
ubiquitin–proteasome system (Fig. 5f). Co-immunopre-
cipitation analysis with anti-Ubiquitin antibody also indi-
cated that GPX4 was ubiquitinated in MDA-MB-231 and
We then investigated the mechanism of DMOCPTL
inducing cell ferroptosis. Te accumulation of intra-
cellular ROS was increased after the treatment of
DMOCPTL in a time- and dose-dependent manner
(Fig. 4e–h). Te lipid ROS, a key characteristic of ferrop-
tosis, was increased synchronously after the treatment
of DMOCPTL in a time- and dose-dependent man-
ner (Fig. 4i, j). Moreover, we detected the Fe2+ intensity,
the results showed that Fe2+ intensity was signifcantly
increased (PGSK intensity decreased) after the treatment
of DMOCPTL for 48 h (Fig. 4k, l). Tese above investiga-
tions suggested that DMOCPTL could induce apoptosis
and ferroptosis of TNBC cells.

Ding et al. J Hematol Oncol
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Page 10 of 21
Fig. 4 DMOCPTL induced apoptosis and ferroptosis of TNBC cells. a The representative images of cell apoptosis in MDA-MB-231 cells for 48 h
at diferent concentrations of 0.2 μM, 0.5 μM and 1 μM, respectively. b The statistical results of cell apoptosis assays. c Western blot analysis of
caspase 9, cleaved caspase 9, caspase 3, cleaved caspase 3, PARP, and cleaved PARP after treatment of DMOCPTL for 48 h in MDA-MB-231 cells
at diferent concentrations of 0.05 μM, 0.1 μM and 0.2 μM, respectively. d Western blot analysis of apoptosis related proteins of mitochondrial
pathway after the treatment of DMOCPTL for 48 h in MDA-MB-231 at diferent concentrations of 0.05 μM, 0.1 μM and 0.2 μM, respectively. e The
representative images and statistical results of ROS after the treatment of DMOCPTL with a dose-dependent manner for 2 h in MDA-MB-231 cells.
f The representative images and statistical results of ROS after the treatment of DMOCPTL with a dose-dependent manner for 2 h in SUM159
cells. g The representative images and statistical results of ROS after the treatment of DMOCPTL with a time-dependent manner in MDA-MB-231
cells. h The representative images and statistical results of ROS after the treatment of DMOCPTL with a time-dependent manner in SUM159 cells.
i The statistical results of lipid ROS after the treatment of DMOCPTL with a dose-dependent manner for 2 h in MDA-MB-231 and SUM159 cells. j
The statistical results of lipid ROS after the treatment of DMOCPTL with a time-dependent manner for 2 h in MDA-MB-231 and SUM159 cells. k
The representative images and statistical results of intracellular Fe2+ by PGSK assay after the treatment of DMOCPTL for 48 h in MDA-MB-231 cell.
l The fuorescence intensity of PGSK after the treatment of DMOCPTL for 48 h in MDA-MB-231 cells by immunofuorescence assay. Analysis results
represented mean SD, *P<0.05, **P<0.01, ***P<0.001
SUM159 cells (Fig. 5g). Terefore, the ubiquitination of confocal microscopy results showed that DMOCPTL
GPX4 after the treatment of DMOCPTL was analyzed. colocalized with GPX4 and ubiquitin (Fig. 6k). To further
Te result indicated that DMOCPTL could increase the reveal the E3 enzymes involved in GPX4 ubiquitination
ubiquitination of GPX4 with a dose-dependent man- induced by DMOCPTL, in situ pull down assay with
ner (Fig. 5h). Tese data demonstrated that DMOCPTL probe 8a was performed (Fig. 6l).
could reduce GPX4 level by inducing ubiquitination of
GPX4.
GPX4 was overexpressed in breast cancer
GPX4 expression in breast cancer was still little investi-
gated. To analyze GPX4 expression in breast cancer, we
investigated the related functional states of GPX4 in Can-
DMOCPTL induced ubiquitination by directly binding
to GPX4
To further get understanding of the mechanism of cerSEA (http://biocc.hrbmu.edu.cn/CancerSEA/). GPX4
DMOCPTL reducing level of GPX4, interaction of expression distribution with t-SNE showed that breast
DMOCPTL with GPX4 was studied. DMOCPTL was cancer cells with high GPX4 expression tended to clus-
docked with the GPX4 (PDB code: 5H5S). As shown the ter together in two patients (Fig. 7b, d), which suggests
optimal pose of DMOCPTL oriented the styryl ring into that GPX4 may promote the malignant progression of
the hydrophobic pocket, forming strong hydrophobic breast cancer. Furthermore, apoptosis, hypoxia, invasion,
interactions with Trp163 and Pro182. Meanwhile, the cell cycle and DNA damage were signifcantly related
oxygen atom of methoxyl group formed hydrogen bond to GPX4 expression in breast cancer cells (Fig. 7a, c, e,
with the side chain of Lys 162 (3.41 Å) and Ile156 (3.17 Å) f, g, h, i). Te IHC assay showed that GPX4 protein was
(Fig. 6a). DMOCPTL was tightly bound into the active signifcantly increased in breast cancer tissue compared
site of GPX4, and the target binding site is consistent with normal breast tissue and related with breast can-
with Sakamoto’s result [34]. Te docking of DMOCPTL cer stages, which indicated the prominent role of GPX4
with GPX4 protein suggested that DMOCPTL may in breast cancer (Fig. 7j, k). Moreover, the level of GPX4
directly interact with GPX4. To further analyze the inter- was higher in TNBC than non-TNBC in clinical sam-
action of DMOCPTL with GPX4, we synthesized a series ples, which suggested that GPX4 was very signifcant for
of DMOCPTL probes (Fig. 6d, e). Te anti-TNBC activ- TNBC (Fig. 7l).
ity showed that probe 8a showed a comparable IC50 value
with that of DMOCPTL (Fig. 6b, c). Ten, the in vitro GPX4 knockdown induced ferroptosis and apoptosis
pull-down experiment showed that DMOCPTL could of TNBC cells
bind with GPX4 directly in MDA-MB-231 and SUM159 To further investigated the role of GPX4 in TNBC cells,
cells (Fig. 6f, g). To better imitate the intracellular envi- GPX4 protein was knocked down by siRNA. Te number
ronment and membrane permeability of DMOCPTL, of colonies was clearly decreased after GPX4 knockdown
in situ pull-down assay was performed. Te results also in MDA-MB-231 and SUM159 cells (Fig. 8a). Moreover,
indicated that DMOCPTL could bind GPX4 directly knockdown of GPX4 by siRNA increased the accumula-
in MDA-MB-231 and SUM159 cells (Fig. 6h, i). To fur- tion of lipid ROS (Fig. 8b, c), the intensity of intracellu-
ther visualize the interaction of GPX4 and DMOCPTL, lar Fe2+ (Fig. 8d, e), and intracellular ROS (Fig. 8f).Tese
we synthesized a fuorescent probe 10a (Fig. 6j). Te

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Ding et al. J Hematol Oncol
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Fig. 5 DMOCPTL regulated GPX4 protein level by inducing GPX4 ubiquitination. a Western blot analyzed GPX4 expression in fve TNBC cell lines.
b Western blot analyzed GPX4 expression in MDA-MB-231 cells and SUM159 cells c after the treatment of DMOCPTL for 24 or 48 h at diferent
concentrations. d MDA-MB-231 cells with DMOCPTL treatment for 48 h were collected and incubated with GPX4 antibody and corresponding
FITC-conjunct second antibody. The fuorescence intensity of GPX4 expression was analyzed by fow cytometry. e The fuorescence intensity of
GPX4 after the treatment of DMOCPTL for 48 h in MDA-MB-231 cells by immunofuorescence assay. f The protein level GPX4 after treatment of
proteasome inhibitor MG132 at 3 h and 6 h. g MDA-MB-231 and SUM159 cell lysates were incubated with IgG or anti-Ub antibody at 4 °C for 6 h,
then agarose A+G was added, co-incubated at 4 °C overnight on rotary shaker. The samples were washed with PBS and analyzed by western blot
assay. h DMOCPTL was added into MDA-MB-231 cells and incubated for 24 h. The proteins were collected and incubated GPX4 antibody for 2 h
on ice, then agarose G was added and co-incubated at 4 °C overnight. The ubiquitination level of GPX4 was analyzed by western blot assay with
anti-Ub antibody. Analysis results represented mean SD, *P<0.05, **P<0.01
GPX4 induced apoptosis through up‑regulation of EGR1
in TNBC cells
results demonstrated that GPX4 regulated ferroptosis in
TNBC cells.
Early growth response-1 (EGR1) is an early response gene
that is involved in growth, diferentiation, apoptosis, neur-
ite outgrowth, and wound healing [35]. As reported, EGR1
was closely related to cell apoptosis and ROS production.
Moreover, ROS production induced EGR1 expression to
induce cell apoptosis. GPX4 was a key regulator of fer-
roptosis by induced ROS especially lipid ROS production.
Accordingly, we rationally speculated that GPX4 would
induce EGR1 expression and cell apoptosis. We investi-
gated the gene transcription level in breast cancer cells
using the UALCAN database (http://ualcan.path.uab.edu/
index.html). EGR1 was signifcantly decreased in breast
cancer tissues compared to normal breast tissues (Fig. 9a),
and EGR1 gene expression was negative correlation with
breast cancer stages (Fig. 9b). Moreover, the IHC assay
showed that the EGR1 protein expression in breast cancer
tissue was clearly decreased compared with that in nor-
mal breast tissue (Fig. 9c). To verify the efect of EGR1 in
It was reported that knockdown of GPX4 induced
apoptosis of glioma cells [32]. Accordingly, we specu-
lated that GPX4 knockdown might induce apoptosis of
breast cancer cells. To verify our speculation, we evalu-
ated apoptosis related proteins by western blot assay.
Te results showed that the anti-apoptotic proteins
Bcl-2 and Bcl-xl of GPX4-siRNA group were decreased,
while the apoptotic protein Bax and Bim were dra-
matically increased compared with the control-siRNA
group (Fig. 8g). Furthermore, the percentage of apop-
tosis induced by CCCP (an apoptosis inducer) was sig-
nifcantly increased after GPX4 knockdown (Fig. 8h, i).
Tese results showed that GPX4 regulated ferroptosis
and apoptosis in TNBC cells.

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Fig. 6 DMOCPTL regulated ubiquitination by directly binding to GPX4. a Docking of DMOCPTL into the active site of GPX4 (PDB code: 5H5S).
The protein structure is shown as a cartoon diagram with selected residues labeled (left). Surface representation of the protein residues (Middle).
2D representation of binding interaction of DMOCPTL and GPX4 (right). b The IC50 values of DMOCPTL probes in MDA-MB-231 cells after the
treatment for 72 h by MTT assay. c The efect of DMOCPTL and probe 8a on the viability of MDA-MB-231 cells after treatment for 72 h. d The
scheme for synthesis of probes 8a–8e. e The scheme for synthesis of probe 10. f The workfow of probe 10 for identifying DMOCPTL-interacting
proteins by in vitro pull-down assay in MDA-MB-231 and SUM159 cells. g MDA-MB-231 and SUM159 cell lysates were incubated with probe 10 for
2 h at room temperature. After incubated with streptavidin conjunct agarose A+G beads, the bead-bound proteins were collected and detected
by western blot experiments. h The workfow of probe 8a for identifying DMOCPTL-interacting proteins by in situ pull-down assay in MDA-MB-231
and SUM159 cells. i MDA-MB-231 and SUM159 cells after the treatment of probe 8a for 6 h were collected and performed click reaction to
conjugate biotin with probe 8a. After incubated with streptavidin conjunct agarose A+G beads, the bead-bound proteins were collected and
detected by western blot experiments. j The scheme for synthesis of probe 10a. k The co-localization of GPX4 (green), Ubiquitin (purple), probe 10a
(red); the nucleus DAPI (blue) in MDA-MB-231 cells by immunofuorescence assay. l MDA-MB-231 cells after the treatment of probe 8a for 6 h were
collected and performed click reaction to conjugate biotin with probe 8a. After incubated with streptavidin conjunct agarose A+G beads, the
bead-bound proteins were collected and analyzed by sliver staining

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Fig. 7 GPX4 was overexpressed in breast cancer. a The Functional state analysis of GPX4 in breast cancer cells in patient 1. b The expression
distribution of GPX4 in breast cancer cells in CancerSEA database in patient 1. Every point represents a single cell. c The correlation of GPX4 with
cell apoptosis was analyzed in patient 1. d The expression distribution of GPX4 in breast cancer cells in CancerSEA database in patient 2. Every
point represents a single cell. e The Functional state analysis of GPX4 in breast cancer cells in patient 2. The correlation of GPX4 with hypoxia (f),
invasion (g), cell cycle (h) and DNA damage (I) were investigated in breast cancer cells in CancerSEA database in patient 2. Every point represents
a single cell. j IHC analysis of GPX4 expression in breast cancer tissue with diferent stages and normal breast tissue. k The statistical results of GPX4
expression in breast cancer tissue with diferent stages and normal breast tissue. l The IHC results of GPX4 expression in TNBC and non-TNBC breast
cancer tissues. Analysis results represented mean SD, *P<0.05, **P<0.01, ***P<0.001
(See fgure on next page.)
Fig. 8 GPX4 knockdown induced cell ferroptosis and apoptosis of breast cancer cells. a The representative pictures and statistical results of
colony formation in MDA-MB-231 and SUM159 cells after GPX4 knockdown. b MDA-MB-231 cells were transfected with GPX4 siRNA for 48 h, and
then cells were collected and incubated with BODIPY for 1 h to detect the lipid ROS level by fow cytometry. The representative images (left) and
statistical results (right) of lipid ROS after GPX4 knockdown. c The representative images (left) and statistical results (right) of lipid ROS after GPX4
knockdown in SUM159 cells by fow cytometry assay. d MDA-MB-231 cells were transfected with GPX4 siRNA for 48 h, and then cells were collected
and incubated with PGSK for 1 h to detect intracellular Fe2+ level by fow cytometry. The representative images (left) and statistical results (right)
of intracellular Fe2+ after GPX4knock down in MDA-MB-231 cells by PGSK assay. e The representative images (left) and statistical results (right)
of intracellular Fe2+ after GPX4 knock down in SUM159 cells by fow cytometry assay. f MDA-MB-231 cells were transfected with GPX4 siRNA for
48 h, and then cells were collected and incubated with DCF-DA for 1 h to detect ROS level by fow cytometry. The representative images (left)
and statistical results (right) of ROS after GPX4 knockdown. g Western blot analysis of apoptosis related proteins of mitochondrial pathway after
GPX4 knockdown. h The representative images of cell apoptosis in MDA-MB-231 and SUM159 cells after GPX4 knockdown with the treatment of
CCCP. i The statistical results of cell apoptosis in MDA-MB-231 and SUM159 cells after GPX4 knockdown with the treatment of CCCP. Analysis results
represented mean SD, *P<0.05, **P<0.01, ***P<0.001
mitochondria-mediated apoptosis of TNBC cells, west- the relative level of apoptotic protein Bax was dramati-
ern blot assay was performed. Te relative levels of anti- cally decreased after EGR1 knockdown (Fig. 9d). Further-
apoptotic proteins Bcl-2 and Bcl-xL were increased, while more, EGR1 protein was signifcantly increased after GPX4

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Fig. 9 GPX4 regulated apoptosis through upregulation of EGR1 in TNBC cells. a The expression analysis of EGR1 in normal individuals and breast
cancer tissues by UALCAN. b The expression analysis of EGR1 in breast cancer tissue with diferent stages and normal breast tissue by UALCAN. c
The IHC analysis of EGR1 expression in breast cancer tissue and normal breast tissue in the human protein atlas datasets. d Western blot analysis of
apoptosis related proteins of mitochondrial pathway after EGR1 knockdown. e Western blot analysis of EGR1 expression after GPX4 knockdown in
MDA-MB-231 cells. f Western blot analysis of EGR1 expression after GPX4 knockdown in SUM159 cells. g Western blot analysis of EGR1 expression
after DMOCPTL treatment for 24 h or 48 h in MDA-MB-231 cells (left) and SUM159 cells (right). h The representative images of cell apoptosis in
MDA-MB-231 cells after EGR1 knockdown with the treatment of CCCP or DMOCPTL (left). The statistical results of cell apoptosis in MDA-MB-231 cells
after EGR1 knockdown with the treatment of CCCP or DMOCPTL (right). Analysis results represented mean SD, *P<0.05, **P<0.01, ***P<0.001
DMOCPTL could inhibit growth of TNBC and prolong
knockdown (Fig. 9e, f). DMOCPTL clearly increased
survival life of mice in vivo and had no obvious toxicity
Taking account of signifcant anti-TNBC activity of
DMOCPTL in vitro and its novel mechanism induc-
ing mitochondria-mediated apoptosis and ferroptosis
through ubiquitination of GPX4, we planned to evaluate
its potency to inhibit TNBC in vivo and investigate its
efect on GPX4 and apoptosis related proteins in vivo.
However, DMOCPTL showed low solubility in water.
In order to improve the water solubility of DMOCPTL,
we designed to convert DMOCPTL into a prodrug 13.
Michael-type addition of 7 with dimethylamine gave
EGR1 expression both in MDA-MB-231 and SUM159
cells with a dose dependent manner (Fig. 9g). Moreover,
the percentage of apoptosis induced by CCCP (an apopto-
sis inducer) and DMOCPTL were signifcantly decreased
after EGR1 knockdown (Fig. 9h), which indicated that
EGR1 inhibited cell apoptosis and the apoptosis inducing
efect of DMOCPTL was rescued by EGR1 knockdown.

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Fig. 10 The prodrug of DMOCPTL. a Schematic diagram for synthesis of compound 13, a prodrug of DMOCPTL. b Concentration–time curve of
compound 13 and DMOCPTL in PBS (pH=7.4)
Fig. 11 The toxicity study of compound 13 by oral administration at a dose of 500 mg/kg. a The level of GPT, GOT and Cr in serum after oral
administration of 13 for 72 h in Bar b/c mice. b The organ coefcient of liver, spleen, lung, brain, heart and kidney were calculated. The organ
coefcient was calculated by organ weight divided by body weight. c The histomorphology of liver, spleen, brain, kidney, lung, spleen and heart
were analyzed by HE staining
alcohol 11, subsequent EDCI/DMAP–mediated cou-
To explore the safety of 13, compound 13 was admin-
pling with 2,6-dimethoxycinnamic acid produced ester istrated orally at a dose of 500 mg/kg. Te level of GPT,
12. Alternatively, direct Michael addition of 2 selec- GOT and Cr in serum was detected, and no obvious
tively occurred on γ-butyrolactone moiety produced change was observed comparing with that of vehicle con-
12 without infuence of the cinnamyl moiety. Finally, trol group (Fig. 11a). Te weight and histomorphology
12 and equivalent fumaric acid in methanol gave its of the major organs including liver, spleen, lung, kidney,
salt 13 (Fig. 10a). Compound 13 could gradually release heart, and brain in 13-group showed no obvious change
DMOCPTL in PBS solution (pH=7.4) (Fig. 10b).
compared with vehicle control group (Fig. 11b, c).

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Fig. 12 DMOCPTL could inhibit TNBC and prolong survival life of mice in vivo and had no obvious toxicity. a Compound 13 was injected to
SD rats by intravenous administration at a dose of 1 mg/kg. Then, the blood sampling from jugular sinus of rats were collected at 2 min, 5mins,
15mins, 30mins, 1 h, 2 h, 3 h, 4 h, 6 h and 8 h after administration. The samples were treated and analyzed by LC/MS. The pharmacodynamics
of 13 on SD rats was calculated by DAS 3.3. b The body weight of mice after intravenous administration of 13 at a dose of 50 mg/kg compared
with vehicle group. c The body weight of mice after treatment of 13 at a dose of 7.5 mg/kg compared with vehicle group. d The statistical results
of tumor volume after treatment of 13 at a dose of 7.5 mg/kg. e The picture of tumors after administration of 13 compared with vehicle group.
f The weight of tumors after the treatment of compound 13 compared with vehicle group. g The levels of GPX4 and EGR1 protein expression
in tumors by western blot assay. h The statistical results of GPX4 and EGR1 protein expression in tumors. i The IHC assay of GPX4, EGR1, and
mitochondria-mediated apoptosis related proteins Bax and Bcl-2 in tumors. PBS instead of primary antibody was used as negative control. Breast
cancer marker MUC16 was used as positive control. j The statistical results of GPX4, Bax (k), EGR1 (l) and Bcl-2 (m) by IHC assay after 13 treatment.
n The body weight of mice after oral administration of 13 at a dose of 500 mg/kg. o The body weight of mice during treatment of 13 at a dose
of 50 mg/kg every other day compared with vehicle group. p The statistical results of survival life after treatment of 13 at a dose of 50 mg/kg and
clinically used drug ADR as a positive control by intraperitoneal administration at a dose of 2 mg/kg every 2 days. Analysis results represented
mean SD, *P<0.05, **P<0.01
Te pharmacodynamics of 13 was also investigated, observed after treatment of 13 (Fig. 12b). To determine
and the results are shown in Fig. 12a. To further iden- the efect of compound 13 in vivo, orthotopic breast can-
tify the safety of 13, acute toxicity assay was performed. cer mouse model was used. Te 4T1 cells were implanted
Bar b/c mice were treated with 13 at a dose of 50 mg/ into the mammary fat pads of Bar b/c mice. After treat-
kg by intravenous injection or vehicle control for 7 days. ment of 13, the tumor sizes of mice and their body
No obvious toxic reaction and loss of body weight was weights were monitored and recorded every 2 days. Te

Ding et al. J Hematol Oncol
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results showed that compound 13 signifcantly inhib- Erastin and RSL5, the other directly inhibits GPX4 activ-
ited the tumor growth in vivo with no apparent loss of ity without decreasing GSH, including RSL3 [2]. In con-
body weight compared with control group at the dose of trast to these mechanisms, it was reported that FINO2
7.5 mg/kg (Figs. 12c–e). Te tumor weight was also sig- could initiate ferroptosis through GPX4 inactivation
nifcantly reduced after treatment of compound 13 com- and iron oxidation [37] and FIN56 induced ferroptosis
pared with control group (Fig. 12f). Te protein level of by degrading GPX4 with requirement of the enzymatic
GPX4 in tumor was decreased signifcantly in compound activity of acetyl CoA carboxylase [38].
13-treated group compared with vehicle group (Fig. 12g,
Here, we identify a derivative of PTL, DMOCPTL, as a
h). Te relative level of EGR1 was dramatically increased potential anti-TNBC agent, which can induce ferroptosis
in compound 13-treated group compared with vehicle and apoptosis through ubiquitination of GPX4. Tis is the
group (Fig. 12g, h). Te tumors were harvested for IHC frst report of inducing ferroptosis through ubiquitina-
staining. Te IHC implied that 13 treatment markedly tion of GPX4 by binding to GPX4 directly. DMOCPTL
down-regulated the expression levels of GPX4 and Bcl-2, could signifcantly inhibit the proliferation of TNBC cells
and up-regulated the expression levels of EGR1 and Bax and the inhibitory activity was evidently potent than its
(Fig. 12i–m).
mother compound PTL. GPX4 played prominent role in
Furthermore, we planned to study the efcacy of 13 by breast cancer and was signifcantly increased in breast
oral administration. Prior to the study of the anti-breast cancer tissue compared with normal breast tissue and
cancer efcacy in vivo, an acute toxicity study of 13 by related with breast stages. Moreover, the expression of
oral administration was conducted in Bar b/c mice. A GPX4 in TNBC was higher than non-TNBC, which indi-
single oral administration of 13 at a dose of 500 mg/kg cated the signifcance of GPX4 in TNBC cells. Te mech-
was administered to the mice. Tese mice were observed anism study suggested that DMOCPTL bound to GPX4
for 14 days. During the study period, all mice remained leading to ubiquitination of GPX4, which induced ferrop-
alive, and no signifcant side efect was observed. Te tosis and EGR1-mediated apoptosis of TNBC cells. Previ-
body weight of Bar b/c mice was not changed obviously ous study had indicated that the loss of GPX4 expression
(Fig. 12n). Tese results demonstrated that oral adminis- could induce apoptosis [33]. Moreover, Lu et al.’s study
tration of 13 was well-tolerated in Bar b/c mice at a dose demonstrated that GPX4 knockdown could induce apop-
of 500 mg/kg in course of treatment and during the post- tosis of glioma cells [32]. However, the mechanism for
treatment period. After the oral administration of 13 GPX4 regulation of apoptosis is still obscure. Here, we
with 50 mg/kg for 6 times every other day, no apparent frstly reveal that GPX4 regulated apoptosis through up-
change of the body weight was observed in comparison regulation of EGR1 in TNBC cells (Figs. 8 and 9).
with vehicle group (Fig. 12o). Te overall survival assay
Acute toxicity study suggested that compound 13, the
showed that compound 13 could signifcantly prolong prodrug of DMOCPTL, showed no obvious toxicity by
the life span compared with control group. Tese results intravenous administration and oral administration at
indicated that compound 13 could prolong survival life a dose of 50 mg/kg and 500 mg/kg, respectively. Fur-
of mice in vivo and had no obvious toxicity (Fig. 12p).
thermore, in vivo experiment indicated that compound
Te above results indicated that compound 13 was ef- 13 efectively inhibited the growth of breast tumor and
cacious in inhibiting the growth of breast tumor and pro- prolonged the life span of mice in vivo, and no obvious
longing of survival span of mice in vivo and no obvious toxicity was observed during the experiment. Te level
toxicity was observed.
of GPX4 was reduced, and EGR1 was increased in tumor
for 13-treated group compared with vehicle group.
Discussion
TNBC is the most malignant breast cancer among vari-
ous breast cancer subtypes. Its prognosis is far from
satisfactory. TNBC remains a very challenging disease.
Tere is an urgent and unmet need to discover efective
drugs with novel mechanism for treatment of TNBC [36].
Induction of ferroptosis is a new efective strategy for
treatment of TNBC [19–23]. GPX4 is an essential regula-
tor of ferroptotic cell death [29]. Small molecule ferrop-
tosis inducers could be summarized into two classes: one
directly acts on mitochondrial VDAC2/3, inhibits cystine
uptake by system Xc− and ultimately results in lipid ROS
accumulation in an NADH-dependent manner, including
Conclusions
In summary, these fndings indicated that DMOCPTL
could induce ferroptosis and EGR1-mediated apoptosis
of TNBC cells through ubiquitination of GPX4 (Fig. 13).
Tis is the frst report of inducing ferroptosis through
ubiquitination of GPX4.Moreover, our results link GPX4
to EGR1 leading to apoptosis of TNBC cells. On basis
of these investigations, we propose that compound 13
deserves further studies as a promising lead compound
for discovery of novel anti-TNBC drug.

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Fig. 13 Schematic diagram of our proposed model of mechanism for how small molecule DMOCPTL induced ferroptosis and apoptosis of TNBC
cells
Availability of data and materials
Supplementary Information
All data generated or analyzed during this study are included either in this
The online version contains supplementary material available at https://doi.
article or in Additional fle 1.
org/10.1186/s13045-020-01016-8.
Ethics approval and consent to participate
Not applicable.
Additional fle 1. NMR copies of compounds.
Consent for publication
This manuscript has not been previously published and is not under consid-
eration for publication elsewhere.
Abbreviations
PVL: Periventricular leukomalacia; DLBCL: Difuse large B cell lymphomas;
TNBC: Triple-negative breast cancer; ER: Estrogen receptors; PR: Proges-
terone receptors; HER-2: Human epidermal growth factor receptor 2; PTL:
Parthenolide; AML: Acute myeloid leukemia; MTT: Thiazolyl blue tetrazolium
bromide.
Competing interests
The authors declare no competing interests.
Author details
1 State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy
and Tianjin Key Laboratory of Molecular Drug Research, Nankai University,
Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic
of China. 2 Accendatech Company, Ltd., Tianjin 300384, People’s Republic
of China.
Acknowledgements
Not applicable.
Authors’ contributions
Q.Z. and Y.C. designed research; Y.D., X.C., W.G., X.H., and M.W. performed
research; Q.Z., Y.D., X.C., C.L., W.G., Q.W. and X.H. analyzed data; Q.Z. and
Y.D. wrote and revised the paper. All authors read and approved the fnal
manuscript.
Received: 24 July 2020 Accepted: 2 December 2020
Funding
This study was supported by the National Natural Science Foundation of
China (NO. NO. 81872764 to Q.Z.; NO. 81903456 to Y.D.), The National Science
Fund for Distinguished Young Scholars (NO. 81625021) to Y.C., Guang-
dong Joint Fund of the National Natural Science Foundation of China (NO.
U1801288).
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