Dioscin induces ferroptosis and synergistic cytotoxicity with
chemotherapeutics in melanoma cells
Yijie Xie*
, Guangxiong Chen
Department of Dermatology, The Affiliated People’s Hospital of Ningbo University, 315100, Ningbo, Zhejiang, China
article info
Article history:
Received 31 March 2021
Accepted 8 April 2021
Available online 18 April 2021
Keywords:
Dioscin
Melanoma
Ferropotosis
ROS
MDA
Iron
abstract
In this study, we evaluated the anti-tumor effects of dioscin, a steroidal saponin, on melanoma cells.
Dioscin significantly inhibited cell viability and induced cell death of melanoma cells in a time- and dose￾dependent manner. Furthermore, dioscin increased the concentration of intracellular ferrous irons, MDA
and ROS. This effect could be inhibited by L-g-glutamyl-p-nitroanilide (GPNA), compound 968 and fer￾roptosis inhibitor ferrostatin-1 (Fer-1). Furthermore, dioscin induced ferroptosis by affecting the
expression of transferrin and ferroportin which are regulators of intracellular levels of iron. Finally,
dioscin in combination with various chemotherapeutic agents showed synergistic effects against mela￾noma cells. Our data suggested that dioscin exerted anti-tumor effects in melanoma cells by inducing
ferroptosis. Dioscin alone or with other agents might be applied as a promising strategy to treat
melanoma.
© 2021 Elsevier Inc. All rights reserved.
1. Introduction
Melanoma is an aggressive skin cancer with a constantly
growing incidence worldwide [1]. Although great progress has
been made in the treatment of melanoma, the prognosis of mela￾noma is still dismal and the 10-year survival rate of melanoma
patients diagnosed at advanced stage is less than 10% [2]. Promising
treatment strategies have been developed such as molecular tar￾geting agents and immune checkpoint inhibitors. However, only a
small fraction of melanoma patients has a durable response and
acquired resistance hampered the application of both therapeutics
[3,4]. Hence, it’s necessary to identify novel effective therapeutic
agents for melanoma.
Amounting evidence suggested that traditional Chinese medi￾cine (TCM) herbs possess potential antitumor effects against
various cancers when used alone or combined with other chemo￾therapeutic agents [5]. TCM has also been widely accepted as a
complementary and alternative therapy because of its relatively
safety [6]. Dioscin is a steroidal saponin that can be pruified from
the roots of Dioscorea plants [7]. Dioscin has various biological
activities such as anti-inflammatory, anti-viral, anti-fungal, anti￾allergic and anti-oxidant [7]. Dioscin was also found to inhibit the
tumorigenesis of various cancers such as prostate cancer, lung
cancer, endometrial carcinoma, colorectal cancer and cervical
cancer [8e12]. However, the mechanisms underlying the anti￾cancer effects were still not fully understood.
Ferroptosis is a relatively new identified form of regulated cell
death that featured by requirement of free ferrous iron [13]. The
process of ferroptosis is accompanied with the generation of lipid
peroxidation products and reactive oxygen species (ROS) that
derived from iron metabolism. Targeting ferroptosis might be a
promising strategy for the treatment of various cancers.
In the present study, we investigated the anti-melanoma effects
of dioscin. The results suggested that dioscin inhibited cell prolif￾eration and triggered ferroptosis in melanoma cells. Our data pro￾vided insights into the anti-tumor functions of dioscin which could
be applied in the treatment of melanoma.
2. Materials and methods
2.1. Cell culture and chemical reagents
Human melanoma cell lines (A375, G361 and WM115) and hu￾man immortal keratinocyte line (HaCaT) were obtained from the
ATCC (American Type Culture Collection, USA). All cells were
maintained in RPMI1640 medium (Gibco, USA) supplemented with
10% FBS (fetal bovine serum, Sigma-Aldrich, USA) and cultured in a
humidified incubator at 37
C with 5% CO2. Dioscin (Selleck, USA)
* Corresponding author.
E-mail address: [email protected] (Y. Xie).
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Biochemical and Biophysical Research Communications 557 (2021) 213e220
was dissolved in dimethyl sulfoxide (DMSO) to give a stock con￾centration of 10 mM and stored at 20
C. Rapamycin, cisplatin,
dacarbazine and vemurafenib were obtained from Selleck (USA). All
other routine chemicals were obtained from Sigma-Aldrich (USA).
2.2. Cell viability assay
MTT assay was performed to evaluate the cytotoxic effect. Cells
were seeded into 96-well paltes at the density of 5  103 cells/well.
After treated with various doses of dioscin (0, 2, 4, 8, 12 mM) for
various time (24, 48, 72 h) at 37
C. 20 ml MTT solution (Sigma￾Aldrich, USA) was added into each well and incubated for 4 h at
37
C, and the absorbance was measured at 450 nm and the data
was read by the ELX-800 spectrometer reader (Bio-Tek In￾struments, USA).
2.3. Cell death assay
As described earlier, cell death was measured using a Cell Death
ELISA Detection Kit (Roche Diagnostics, Switzerland) according to
the manufacturer’s guide [14].
2.4. ROS detection
The levels of ROS were measured using the peroxide-sensitive
fluorescent probe DCF-DA (Sigma-Aldrich, USA) according to the
manufacturer’s protocol. After treated with various doses of dioscin
for 48 h, cells were incubated with 20 mg/ml DCF-DA for 0.5 h at
37
C. Then cells were collected and analysed by a flow cytometry
(FACS Calibur, Becton-Dickinson, USA).
2.5. Measurement of MDA and Fe2þ levels
The intracellular MDA and Fe2þ levels were measured using the
Lipid peroxidative Assay kit (Abcam, UK) and Iron Assay Kit
(Abcam, UK) according to the manufacturer’s guide.
2.6. Cell transfection
For the transfection of siRNA, the scramble negative control (si￾NC) and siRNA targeting transferrin (si-transferrin) were designed
and synthesized by GenePharma Ltd (China). For the over￾expression of ferropotin, the negative control (empty vector
pcDNA3.1) and ferropotin overexpression vector (pcDNA.ferropo￾tin) were designed and synthesized by RioBio Ltd (China). The
siRNA or plasmids were introduced into cells at the concentrations
of 20 nM and 10 ng/well, respectively. Transfections were con￾ducted using the Lipofectamine 3000 (Life Technologies, USA) ac￾cording to the manufacturer’s guide.
2.7. Combination index (CI) analysis
The synergistic effect between dioscin and various chemother￾apeutic agents were calculated according to the previous study [15].
2.8. Western blotting assay
Total protein was extracted from the cells using the RIPA lysis
buffer. 20 mg of protein were resolved using the 12% SDS-PAGE and
transferred to PVDF membrane (Millipore, USA). The PVDF mem￾brane was blocked with 5% skimmed milk at room temperature for
1 h. Then the PVDF membrane was incubated with primary anti￾bodies against Ferritin, Transferrin receptor, Transferrin, Ferro￾portin and GAPDH at 4
C overnight. Membrane was next incubated
with HRP-conjugated secondary antibody at room temperature for
1 h. All antibodies were obtained from Abcam. The western blotting
results were visualized using the enhanced chemiluminescence
(ECL) kit (Sigma-Aldrich, USA) according to the manufacturer’s
guide.
2.9. Statistical analysis
All data are presented as mean ± SD. An independent samples
Student’s t-test was used to compare the difference of two groups.
One-way analysis of variance (ANOVA) followed by the Tukey’s test
was used for multigroup comparisons. A P-value less than 0.05
(P < 0.05) was considered for significant differences. All statistical
analysis was performed using the Prism 9.0 software (GraphPad
Software Inc, USA).
3. Results
3.1. Dioscin inhibits cell viability and induces cell death in
melanoma cells
To evaluate the anti-tumor effects of dioscin on melanoma cells,
MTT assay was firstly applied to examine changes in cellular
viability of normal immortal human keratinocyte line (HaCaT) and
human melanoma cell lines (A375, G361 and WM115). As shown in
Fig. 1A, the cell viability of A375, G361 and WM115 was significantly
inhibited by dioscin in dose- and time- dependent manner while
the cell viability of HaCaT was lightly affected. Next, apoptotic ELISA
assay was applied to measure the cellular death. Similar to the MTT
assay results, dioscin induced little death in HaCaT cell while
induced noticeable death in a dose- and time- dependent manner
in melanoma cells (Fig. 1B). Thus, these data indicated that dioscin
repressed cell viability and induced cell death of melanoma cells
while had little effects on human keratinocyte.
3.2. Dioscin induces lipid peroxidation and iron accumulation in
melanoma cells
Next, we investigated whether ferroptosis was involved in the
cell death induced by dioscin in melanoma cells. It was recognized
that lipid peroxidation and iron accumulation served as two
essential signalling events in the process of ferroptosis [16]. Since
malondialdehyde (MDA) is one of the most important end￾products of lipid peroxidation, we firstly examined the accumula￾tion of MDA in melanoma cells. As shown in Fig. 2A, treatment of
dioscin lead to the upregulation of MDA in both A375 and
WM115 cells. Meanwhile, incubation with dioscin also caused
elevated levels of Fe2þ in both A375 and WM115 cells (Fig. 2B).
Amounting evidence suggested that glutaminolysis plays a critical
role in ferroptosis. L-g-glutamyl-p-nitroanilide (GPNA) and com￾pound 968 are two pharmacological inhibitors that can block the
process of glutaminolysis [17]. Ferrostatin-1 (Fer-1) is a synthetic
compound that acts as a hydroperoxyl radical scavenger to inhibit
ferroptosis [18]. In order to examine whether dioscin induces fer￾roptosis via regulating glutaminolysis, we treated cells with dioscin
in the presence of GPNA/968/Fer-1 or not. As shown in Fig. 2C and
D, MDA and Fe2þ accumulation was significantly inhibited by
GPNA, Compound 968 and Fer-1. Meanwhile, repression of cellular
viability caused by dioscin could be reversed by treatment of GPNA/
968/Fer-1 in melanoma cells (Fig. 2E). Furthermore, cell death
induced by dioscin also be abrogated by the presence of GPNA/968/
Fer-1 in melanoma cells. These results suggested that dioscin trig￾gered ferroptosis via Gln and its metabolic process-glutaminolysis.
Y. Xie and G. Chen Biochemical and Biophysical Research Communications 557 (2021)
3.3. Dioscin regulates ROS accumulation and expression of iron
regulatory proteins in melanoma cells
It is well known that the cytotoxicity of ferroptosis is relied on
the generation of ROS. We then measure the level of ROS over a 24 h
time course following treatment with various doses of dioscin. As
shown in Fig. 3A, treatment of dioscin lead to the generation of ROS
in a dose-dependent manner which could be inhibited by Fer-1.
According to previous studies that transferrin and ferroportin
cooperatively regulate cellular iron levels via transporting iron into
or out of cells [19]. Thus, we examined whether dioscin could affect
the expression of iron regulatory proteins. As shown in Fig. 3B,
treatment of dioscin lead to the upregulation of transfeerin and
downregulation of ferroportin. At the same time, the expression of
ferritin and transferrin receptor was little affected (Fig. 3B). In order
to explore the role of transferrin, transferrin was significantly
downregulated after transfection with siRNA (Fig. 3C). MTT assay
showed that the inhibitory effects of dioscin on viability of mela￾noma cells was significantly attenuated after silencing of trans￾ferrin (Fig. 3D). Meanwhile, cell death triggered by dioscin was also
Fig. 1. Dioscin inhibited the viability and induced cell death of melanoma cells.
A, HaCaT, A375, G361 and WM115 cells were treated with various doses of dioscin (0 mM, 2 mM, 4 mM, 8 mM, 12 mM) for different time (24 h, 48 h, 72 h), then cell viability was assayed
by the MTT assay. B, HaCaT, A375, G361 and WM115 cells were treated with various doses of dioscin (0 mM, 2 mM, 4 mM, 8 mM, 12 mM) for different time (24 h, 48 h, 72 h), then cell
death was assayed. Data was presented as mean ± SD; *P < 0.05; **P < 0.01.
Y. Xie and G. Chen Biochemical and Biophysical Research Communications 557 (2021)
Fig. 2. Dioscin induced lipid peroxidation and Fe2þ accumulation in melanoma cells.
A, A375 and WM115 cells were treated with various doses of dioscin (8 mM, 12 mM) for different time (12 h, 24 h), the lipid accumulation was measured by MDA assay. B, A375 and
WM115 cells were treated with various doses of dioscin (8 mM, 12 mM) for different time (12 h, 24 h), the Fe2þ levels were assayed. C, A375 and WM115 cells were treated with
dioscin (12 mM) in combination with GPNA (5 mM) or compound 968 (20 mM) or Fer-1 (1 mM) for 24 h, then MDA levels were assayed. D, A375 and WM115 cells were treated with
dioscin (12 mM) in combination with GPNA (5 mM) or compound 968 (20 mM) or Fer-1 (1 mM) for 24 h, then Fe2þ levels were assayed. E, A375 and WM115 cells were treated with
dioscin (12 mM) in combination with GPNA (5 mM) or compound 968 (20 mM) or Fer-1 (1 mM) for 24 h, then cell viabilities were assayed. F, A375 and WM115 cells were treated with
dioscin (12 mM) in combination with GPNA (5 mM) or compound 968 (20 mM) or Fer-1 (1 mM) for 24 h, then cell death was assayed. Data was presented as mean ± SD; *P < 0.05;
**P < 0.01.
Y. Xie and G. Chen Biochemical and Biophysical Research Communications 557 (2021) 213e220
greatly abrogated by downregulation of transferrin (Fig. 3E). Then
the role of ferroportin was also studied. As shown in Fig. 3F, fer￾roportin was successfully upregulated after transfection with a
vector expressing ferroportin. Forced expression of ferroportin
attenuated the inhibitory effects of dioscin on viability of mela￾noma cells (Fig. 3G). In addition, overexpression of ferroportin also
decreased dioscin-induced cell death in melanoma cells (Fig. 3H).
Taken together, these data confirmed that dioscin treatment led
to the ferroptosis in melanoma cells.
3.4. Dioscin synergizes with chemotherapeutics to induce cell death
in melanoma cells
Finally, we tested the effects of dioscin alone and in combination
with various anti-tumor agents. Dioscin showed synergistic effects
with various chemotherapeutic agents (rapamycin, cisplatin, dacr￾bazine, vemurafenib) to induce cell death in melanoma cells
(Fig. 4A, B, C, D). Calculation of CI (combination index) suggested
that there is a strong synergistic effect between dioscin and various
chemotherapeutic agents (Table 1). Hence, dioscin may be applied
alone or in combination with other agents to fight against
melanoma.
4. Discussion
In the current study, we found that dioscin, a natural steroid
saponin, exerts markedly anti-tumor effects against melanoma
cells. Mechanism investigations revealed that dioscin treatment led
Fig. 3. Dioscin regulates ROS accumulation and expression of iron regulatory proteins in A375 and WM115 cells.
A, A375 and WM115 cells were treated with various doses of dioscin (0 mM, 4 mM, 8 mM, 12 mM) or dioscin (12 mM) in combination with Fer-1 (1 mM) for 24 h, then the levels of ROS
were assayed. B, A375 and WM115 cells were treated with various doses of dioscin (0 mM, 4 mM, 8 mM, 12 mM) for 24 h, then the expression of indicated proteins were measured by
western blotting. C, A375 and WM115 cells were transfected with si-NC or si-transferrin for 24 h, then the expression of transferrin was measured by western blotting. D, A375 and
WM115 cells were transfected with si-NC or si-transferrin for 24 h, then cells were treated with or without dioscin (12 mM) for another 24 h, cell viability was assayed. E, A375 and
WM115 cells were transfected with si-NC or si-transferrin for 24 h, then cells were treated with or without dioscin (12 mM) for another 24 h, cell death was assayed. F, A375 and
WM115 cells were transfected with empty vector (pcDNA3.1) or pcDNA.ferroportin for 24 h, then the expression of ferroportin was measured. G, A375 and WM115 cells were
transfected with vector or pcDNA.ferroportin for 24 h, then cells were treated with or without dioscin (12 mM) for another 24 h, cell viability was assayed. H, A375 and WM115 cells
were transfected with empty vector or pcDNA.ferroportin for 24 h, then cells were treated with or without dioscin (12 mM) for another 24 h, cell death was assayed. Data was
presented as mean ± SD; **P < 0.01.
Y. Xie and G. Chen Biochemical and Biophysical Research Communications 557 (2021) 213e220
to the upregulation of MDA, intracellular ferrous iron and ROS.
Ferroptosis inhibitors such as GPNA, Compound 968 and Fer-1
significantly attenuated the anti-tumor effects of dioscin. More￾over, we also confirmed that upregulation of transferrin and
downregulation of ferroportin played an essential role in the anti￾melanoma effects of dioscin. All these data suggested that dioscin
exerts anti-melanoma effects via induction of ferroptosis.
In recent years, great attentions have been paid on the naturally
derived anti-tumor agents due to their safety and encouraging anti￾tumor effects. Dioscin is a naturally occurring glucoside saponin
that can be isolated from the roots of wild yam (Dioscorea villosa)
[20]. Although dioscin has been reported to inhibit the tumori￾genesis of various cancers including the melanoma, the underlying
mechanisms of anti-tumor effects of dioscin are still elusive. In line
with previous investigations, our findings also confirmed that
dioscin decreased viability and induced cell death of melanoma
cells [21]. To the best of our knowledge, our study revealed the
ferroptosis-inducing ability of dioscin for the first time. Ferroptosis
is a newly established form of cell death which is genetically,
morphologically, and biochemically distinct from other forms of
apoptotic and non-apoptotic cell death [22]. Ferroptosis is featured
by accumulation of intracellular ferrous iron and ROS [22]. Tumor
cells, including the melanoma cells, are highly relied on iron for
their dysregulated proliferation because iron is necessary for syn￾thesis of DNA. Hence, targeting ferroptosis is a promising strategy
to treat human cancer. In the current study, we observed that
dioscin treatment led to the upregulation of intracellular ferrous
iron, MDA and ROS in melanoma cells. Our findings are in line with
previous studies. For instance, dioscin has been found to trigger
accumulation of ROS in lung squamous cell carcinoma, cervical
carcinoma cells and liver carcinoma cells [9,23,24]. Noteworthy,
dioscin has been reported to reduce MDA and ROS level in rat in￾testinal crypt cells IEC-6 [25]. Dioscin has also been found to
abrogate the doxorubicin-induced upregulation of ROS and MDA in
mouse liver cells [26]. This controversial role of dioscin in the
regulation of ROS might be caused by different cell types and/or
microenvironment, thereby more investigations are required.
Fig. 4. Dioscin acted synergistically with various chemotherapeutics to induce cell death of melanoma cells.
A, A375 and WM115 cells were treated with dioscin (12 mM) or Rapamycin (100 ng/ml) alone or in combination for 24 h, then cell death was measured. B, A375 and WM115 cells
were treated with dioscin (12 mM) or cisplatin (1 mM) alone or in combination for 24 h, then cell death was measured. C, A375 and WM115 cells were treated with dioscin (12 mM) or
dacarbazine (50 mg/ml) alone or in combination for 24 h, then cell death was measured. D, A375 and WM115 cells were treated with dioscin (12 mM) or vemurafenib (0.5 mM) alone
or in combination for 24 h, then cell death was measured. Data was presented as mean ± SD; **P < 0.01.
Table 1
Combination Index (CI) values for melanoma cells treated with dioscin and various chemotherapeutic agents.
Agents CI value
A375 WM115
Dioscin (12 mM) Rapamycin (100 ng/ml) 0.567 0.522
Dioscin (12 mM) Cisplatin (1 mM) 0.573 0.413
Dioscin (12 mM) Dacarbazine (50 mg/ml) 0.508 0.533
Dioscin (12 mM) Vemurafenib (0.5 mM) 0.431 0.594
CI < 1 indicate synergistic effect; CI ¼ 1 indicate additive effect; CI > 1 indicate antagonistic effect.
Y. Xie and G. Chen Biochemical and Biophysical Research Communications 557 (2021) 213e220
It was well-documented that metabolism process is dramati￾cally changed known as the Warburg effect [27]. One feature of
Warburg effect is cellular addiction to glutamine. Tumor cells will
normally switch from oxidative metabolism to a highly glycolytic
metabolic status [28]. Thus, targeting glutamine metabolism may
serve as a promising strategy to fight against tumors. Given that
glutamine metabolism was also essential for ferroptosis, tumor
cells might particularly vulnerable to ferroptosis. In the current
study, anti-tumor effects induced by dioscin were markedly
attenuated by GPNA and compound 968, both of which are in￾hibitors of glutamine metabolism. Moreover, the blockage effect of
GPNA/968 on dioscin is similar to the ferroptosis inhibitor
ferrostatin-1. These results confirmed that dioscin triggered cell
death relied on the initiation of ferroptosis.
Initiation of ferroptosis is relied on the accumulation of intra￾cellular ferrous iron which is regulated by the balance between iron
uptake, storage and export. This dynamic process is regulated by
various proteins such as transferrin, ferroportin and ferritin [29]. In
this study, we showed that dioscin treatment led to the upregula￾tion of transferrin and downregulation of ferroportin. Following
investigations showed that silencing of transferrin or over￾expression of ferroportin could abrogated the anti-melanoma ef￾fects of dioscin. These results confirmed that dioscin exerts anti￾tumor effects relied on the regulation of iron transport and initia￾tion of ferroptosis in melanoma cells.
Rational combination of various agents is a promising strategy
for the treatment of cancer. To this end, previous studies reported
that dioscin in combination with other agents showed synergistic
effects against cancers. For instance, dioscin in combination with
epirubicin synergistically inhibited the progression of lung cancer
[30]. It was also reported that dioscin with HSV-tk-mediated sui￾cide gene therapy showed synergistic effects against melanoma
[31]. We found that dioscin had strong synergistic effects with
various clinically approved chemotherapeutics such as rapamycin,
cisplatin, dararbazine and vemurafenib. Considering that
dacarbazine-based combination chemotherapy is frequently used
for the treatment of melanoma, our findings provided new insight
into the clinical application of dioscin in the treatment of
melanoma.
There are some limitations of our study. Firstly, due to the
limited experimental conditions, we were unable to observe the
morphological changes of cells after treatment of dioscin. Secondly,
the effects of dioscin on xenograft mice were not investigated in our
study. Thirdly, it was not examined whether there’s any other sig￾nalling pathways were involved in the ferroptosis induced by
dioscin.
In summary, we study demonstrated that dioscin exerts anti￾tumor effect in melanoma cells via inducing ferroptosis which
was confirmed by generation of ROS and upregulation of intracel￾lular iron via regulating transferrin and ferroportin. Our data
revealed that dioscin can be applied for the treatment of melanoma
alone or in combination with other agents. This study also high￾lights targeting ferroptosis as a promising strategy for the treat￾ment of melanoma.
Declaration of competing interest
None.
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