5.2
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
View/Download PDF

Translate this page into:

Original article
2021
:14;
202106
doi:
10.1016/j.arabjc.2021.103177

Anticancer effects of Cryptotanshinone against lung cancer cells through ferroptosis

, , , , ,
a Department of Thoracic Surgery, Tianjin First Central Hospital, School of Medicine, Nankai University, Tianjin 300192, China
b College of Pharmacy, Nankai University, Tianjin 300350, China
c Department of Medical Oncology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China

⁎Corresponding authors at: No 24 Fukang Road, Nankai District, Tianjin 300192, China (X. Li), NO.9, Beiguan Street, Tongzhou District, Beijing, Beijing 101149, China (L. Ma). xiaopingli_tjmu@tom.com (Xiaoping Li), lima.oncology@yahoo.com (Li Ma)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Lung cancer is considered as one of the most commonly diagnosed malignancies disturbing public health with high rate of mortality worldwide. Cryptotanshinone (CTN), a natural chinese quinoid diterpene isolated from the roots of a herb Salvia miltiorrhiza, has been shown to have anti-tumor properties by inducing apoptosis. CTN inhibits lung cancer invasion, lung tumorigenesis and proliferation of cancer cells. Here, we report a novel mode of death other than apoptosis induced by CTN. We showed that CNT induces caspase-dependent death as well as non-apoptotic ROS-mediated death. Cell survival assay showed the short-term effects of CTN on cell survival of cancer cells. To better understand the CTN-induced mode of death, lung cancer cells titrated with CTN and caspase activity, ROS generation and lipid peroxidation was measured. The EC50 for ROS generation and lipid peroxidation was massively lower than that of caspase activation. CTN-treated cells showed higher iron load and reduced GPx4 activity. In addition, Cytoglobin was upregulated and ferroportin was downregulated in response to CTN. We also showed that CTN induced the iron-dependent lipid peroxidation by blocking the transferrin receptors using anti-TfR antibody. Caspase-3 inhibitor, QVD-OPh, failed to prevent CTN-induced death in lung cancer cell lines. All our data suggests that CTN induces primarily ferroptosis in lung cancer cells although caspase-dependent cell death also explains ̴20% of the death.

Keywords

Ferroptosis
Caspase-dependent death
Lung cancer
Cryptotanshinone
ROS generation
1

1 Introduction

Lung cancer is considered as one of the major health issues and is among the top 20 causes of cancer-associated mortalities worldwide, occurring in both genders and at different ages (Siegel et al., 2016). Lung cancers are divided into two major types based on the differences in clinical behavior and purposes of treatment: Small Cell Lung Cancer (SCLC) responsible for approximately 15% and Non-Small Cell Lung Cancer (NSCLC) accounts for 85% of all lung cancer cases (Sakashita et al., 2014). The majority of patients do not respond well to the current treatments such as chemotherapy and/or radiotherapy and the 5-year survival rate is < 15% (Youlden et al., 2008). Therefore, discovering novel treatments for lung cancer using effective molecules is of great importance.

Traditional Chinese medicines (TCMs), made from natural compounds, have attracted increasing attention due to less sides effects than the chemical compounds for the treatment of cancer (Efferth et al., 2007). These herbal compounds are widely distributed, cheaper and more easily available to patients which make them potential therapeutic candidates.

Salvia miltiorrhiza Bunge also known as Danshen is a well-known Chinese herb that is widely used for the treatment of cardiovascular, cerebrovascular and inflammatory diseases (Gao et al., 2014; Woo et al., 2013). Active components of S. miltiorrhiza mainly include hydrophilic phenolic acids such as salvianic acid A, caffeic acid, and rosmarinic acid and lipophilic tanshinones (Fang et al., 2008). The former components have direct radical scavenging and antioxidant activities, while the latter components show anti-inflammatory, antibacterial, and antitumor activities. In vitro studies in PC–3 cells showed that acetonitrile extract of S. miltiorrhiza induced cell cycle arrest and apoptosis by generating the intracellular ROS. In vivo studies showed that oral administration of the Salvia acetonitrile extract decreased the prevalence and growth of PC–3 tumor xenografts in nude mice. S. miltiorrhiza significantly induced cell cycle arrest at G1/S phase by upregulation p21 and downregulating cyclin-dependent kinase 2 (CDK2), CDK4 and cyclin D1 proteins. (Lee et al., 2017).

The protective role of tanshinone have been reported in gastric adenocarcinoma, prostate cancer, breast cancer, colorectal cancer, and lung cancer (Liu et al., 2019; Sung et al., 2015). It can remove blood stasis in cancer patients and thus it is of most potent anticancer herbs. In the in vitro model of the breast cancer, Salvia miltiorrhiza Bunge extract downregulated MMP-9 and thus reduced the metastasis and invasion of MCF-7 cancer cells (Kim et al., 2017). The dihydrotanshinone has been reported to inhibit the proliferation of glioma cells and induce the mitochondrial-mediated apoptosis (Cao et al., 2017). In leukemia cells, 15,16-dihydrotanshinone I promoted the phosphorylation of JNK and upregulated Fas L which in turn can upregulate the Bad and Bax proteins and leads to activation of caspase 3/8/9 (Liu et al., 2015).

Among the major tanshinones isolated to date, cryptotanshinone, tanshinone I, tanshinone IIA, and dihydrotanshinone, tanshinone IIA shows multiple anti-cancer activities by inducing apoptosis and inhibiting the angiogenesis and metastasis (Kim et al., 2011). Tanshinones also enhance the cytotoxic effects of anti-cancer agents such as TNF-α, 5-fluorouracil, and γ-irradiation by sensitizing the cells to chemotherapy and radiotherapy as well as producing ROS and induce ROS-mediated ER stress (Su, 2012; Ye et al., 2012; Lee et al., 2009; Park et al., 2012). Cryptotanshinone (CTN), the major component of Danshen, enhances ROS generation in human hepatoma and breast cancer cell line models and induces ER stress-mediated apoptosis (Zhang et al., 2018). Anticancer properties of CTN have been reported in several cancer models such as prostate cancer, leukemia, gliomas, hepatic carcinomas, pancreatic, colorectal, melanoma and lung cancer. In lung cancer, CTN prevents cell growth by inhibiting IGF-1R/PI3K/Akt signaling, leading to inhibition of cell proliferation and migration (Tan et al., 2011). Many of the TCMs have been shown to inhibit cancer cell proliferation, migration and invasion and enhance the programmed cell death especially apoptosis (Cain et al., 2000).

Given the inhibitory effects of CTN on tumorigenesis and cell death induction, the present study aimed to further investigate the mode of death induced by CPT on human lung carcinoma A549 and NCI-H520 cells, and better understand the underlying mechanisms by which it plays the protective effects. We, here, report for the first time the induction of ferroptosis in CTN-treated lung cancer cells as the main mode of death.

2

2 Material and methods

2.1

2.1 Cell culture

The A549, NCI-H520 non-small cell lung cancer cell line, and BAES-2B were purchased from American Type Culture Collection (ATCC). The cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, China) medium supplemented with 4 mM L-glutamine (GIBCO–BRL, Basel, Switzerland) and 10% fetal bovine serum (FBS), 100 Units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, China). The Fe(NO3)3 and ferric ammonium citrate (FAC), were used as additives to the supplemented media in select experiments. The cells were incubated at 37 °C, 5% CO2 and 95% air. Prior to each experiment 3.0 × 105 cells were seeded in a 6-well plate.

2.2

2.2 Lipid peroxidation (LPO) assay

The medium was aspirated from the cell monolayer and treated with CTN or TfR antibody (BD Biosciences). After 24 h incubation, the cells were collected and resuspended in 1 mL of PBS, and were washed three times with PBS pH 7.4. The LPO assay was performed by LPO (MDA) Assay Kit according to the manufacture protocol (Sigma-Aldrich, Germany).

2.3

2.3 4-HNE assay

The level of 4-HNE was measured according to manufacturere’s instruction (Abcam). Breifly, 50 µL of samples were added to wells of 4-HNE conjugate coated plate and incubate for 10 mins. Then 50 µL of the diluted anti-4-HNE antibody was added and incubate for 1 h. After washing, 100 µL of diluted Secondary Antibody-HRP Conjugate per well was added and incubate for 1 h. Then 100 µL of warm Substrate Solution was added and incubated for 20 mins, followed by addition of 100 µL Stop Solution. The plate was read at 450 nm.

2.4

2.4 RTqPCR

The level of mRNA was examined using Luna® Cell Ready One-Step RT-qPCR Kit (NEB) according to the manufacturer’s protocol.

2.5

2.5 Caspase-3 like activity

Caspase-3 like activity was measured using DEVD-AMC as described (Han et al., 2020). Briefly, 3.0 × 105 cells were harvested and resuspended in extraction buffer containing 50 mM 4-1- piperazineethanesulfonic acid (HEPES) pH 7.4, 10 mM KCl, 5 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N0,N0- tetraacetic acid, 2 mM MgCl2,1mM DTT plus cytochalasin B 10 µg.mL-1, PMSF 100 µM, and protease inhibitor cocktail. Cells were lysed by freezing–thawing in liquid nitrogen and vigorous mixing. The lysate centrifuged for 1 h at 100,000 g. Protein concentration was determined by Bradford assay. 20 µL of extract was mixed with 180 µL of assay buffer containing HEPES 20 mM, 10 µM DEVD-AMC, and 2 mM DTT. Fluorescence intensity was normalized to the protein concentration.

2.6

2.6 Iron load measurement

Iron assay kit (Sigma) was used to measure the level of iron. Briefly, 50 μL samples was made up to 100 μL per well with Assay Buffer in 96-well format. 5 μL of iron assay buffer was added to each sample. Iron Reducer was then added to reduce Fe3 + to Fe2 + and the mixture was incubated for 30 min at 25 °C. Iron Probe was then added to wells containing standard and test samples. The absorbance of the plate was recorded at 593 nm after 60 min incubation at 25 °C.

2.7

2.7 ROS detection assay

Abcam's Cellular Reactive Oxygen Species Detection Assay Kit (Cambridge, UK) was used according to manufacturer to quantify the ROS production.

2.8

2.8 Cell death assay

Propidium iodide (1 µg mL−1) and Hoechst 33,342 (1 µg mL−1) was used to visualize dead cells. The PI positive cells were dead. Cell death analyses were completed using Operetta high-content imaging system and the percentage of cell death was calculated as the number of PI positive cells/number of Hoechst positive cells) × 100.

2.9

2.9 Statistical analysis

Grouped analysis was performed using Two-way ANOVA to calculate the p-value.

3

3 Results

3.1

3.1 Cryptotanshinone induces cell death in a dose-dependent manner

Cell death was first studies by PI and Hoecsht 33,342 staining over a range of concentration to identify the IC50 concentrations for both A549 adenocarcinoma and NCI-H520 squamous lung cancer cell lines. BAES-2B cells were used as the normal human bronchial epithelium control (Jin et al., 2020). The cells treated with a range of CTN (1, 3, 10, 30, 60, 100, 300 and 500 µM), cell death assay was performed after 24 h incubation and IC50 was calculated. The IC50 was 57.95 µM and 67.65 µM for A549 and NCI-H520, respectively (Fig. 1A). The IC50 values were similar to those reported before (Chen et al., 2012). CTN did not show significant effect on normal BAES-2B cells except for high concentration (300 and 500 µM) suggesting the small adverse effects on normal cells.

CTN induces caspase-dependent and ROS mediated death. (A) A549 and NCI-H520 cells were treated for 24 h with a range of CTN concentrations and cell death was calculated by dual PI/Hoecsh 33,342 staining. (B) Caspase-3 like activity was measured using DEVD-AFC substrate (* p-value > 0.0001). (C) ROS generation was measured in the presence and absence of CTN (* p-value = 0.0001 and ** p-value = 0.0019). All experiments were performed in triplicate. The error bars are mean ± SD.
Fig. 1 CTN induces caspase-dependent and ROS mediated death. (A) A549 and NCI-H520 cells were treated for 24 h with a range of CTN concentrations and cell death was calculated by dual PI/Hoecsh 33,342 staining. (B) Caspase-3 like activity was measured using DEVD-AFC substrate (* p-value > 0.0001). (C) ROS generation was measured in the presence and absence of CTN (* p-value = 0.0001 and ** p-value = 0.0019). All experiments were performed in triplicate. The error bars are mean ± SD.

3.2

3.2 Cryptotanshinone induces ROS generation and caspase activity in lung cancer cell lines

CTN has been reported to induce caspase-independent death in human rhabdomyosarcoma as well as breast cancer and prostate cancer cell lines (Zhang et al., 2019). To see if CTN induces similar mode of death, the cells were treated with IC50 concentrations and caspase-3 like activity was measured using fluorescent substrate. Surprisingly, CTN increased the caspase-3 like activity in both lung cancer cell lines, by 2.2 and 2 folds for A549 and NCI-H520 respectively (Fig. 1B). BAES-2B control cells also showed slight increase in caspase-3 like activity when treated with CTN. This data suggests that CTN can trigger different mode of death in lung cancer cell lines. We also measured the ROS generation in the cells treated with CTN. ROS generation was increased 1.9 and 1.6 folds in A549 and NCI-H520 respectively (Fig. 1C) suggesting that CTN induces a type of death that is mediated by caspase-3 and ROS.

3.3

3.3 CTN induces caspase-3 activity and ROS generation in a dose-dependent manner that are different to cell death

We showed that CTN induces caspase-3 dependent death in lung cancer lines. To get a better understanding of whether this CTN-induced caspase activity is the cause of death we titrated a range of CTN concentrations and measured caspase-3 like activity. As shown in Fig. 2A, the concentrations lower than 30 µM did not induce caspase activity in any of the cell lines tested. The BAES-2B cells showed a linear correlation between caspase-3 activity and CTN concentration and the maximum activity is ̴50% of those measured in cancerous cell lines. The EC50 was then calculated for A549 and NCI-H529 which was 102.3 and 81.45 respectively. The EC50 values were higher than the IC50s suggesting that caspase-3 activity is probably not the main cause of death induced by CTN. As CTN was shown to induce the generation of ROS, then the cells were exposed to different concentrations of CTN and ROS generation was measured (Fig. 2B). The EC50 of ROS generation by CTN for A549 and NCI-H529 was 30.48 and 53.09 respectively. The EC50 values were much lower than IC50 of CTN-induced death, suggesting that ROS generation is the main cause of death.

Titration of CTN in caspase-3 like and ROS generation assay. (A) Caspase-3 like activity was measured 24 h after treatment with a range of CTN concentrations and EC50 was calculated. (B) ROS generation was measured 24 h after treatment with a range of CTN concentrations and EC50 was calculated. All experiments were performed in triplicate. The error bars are mean ± SD.
Fig. 2 Titration of CTN in caspase-3 like and ROS generation assay. (A) Caspase-3 like activity was measured 24 h after treatment with a range of CTN concentrations and EC50 was calculated. (B) ROS generation was measured 24 h after treatment with a range of CTN concentrations and EC50 was calculated. All experiments were performed in triplicate. The error bars are mean ± SD.

3.4

3.4 CTN induces the lipid peroxidation

Aiming to identify the mode of death induced by CTN and knowing that ROS generation plays a key role, we then tested the ability of CTN to induce lipid peroxidation by measuring the production of malondialdehyde (MDA). The normal BAES-2B cells showed very low lipid peroxidation in the nM range (300 nM) and CTN induced the production of MDA up to ̴1 µM (Fig. 3A). However, the level of MDA was higher in cancerous cell lines even in the absence of CTN. The NCI-H520 showed higher MDA than A549 (1.97 versus 1.46 µM). Treatment with CTN significantly increased the lipid peroxidation to 2.47 and 3 µM in A549 and NCI-H520 cells, respectively. This shows that the ROS generation induced by CTN can also induce lipid peroxidation in lung cancer cell lines. Then we titrated CTN and measured the MDA production to obtain the EC50. EC50 values were 34.87 and 48.25 for A549 and NCI-H520 cells, respectively (Fig. 3B). The EC50 values for lipid peroxidation was very close to that of ROS generation. 4-hydroxynonenal (4-HNE), a lipid peroxidation marker was also measured (Ye et al., 2021). CTN increased the level of 4-HNE by 2.92 fold in A549 and 2.96 fold in NCI-H520 cells (Fig. 3C), consistent with MDA level shown before.

CTN-induced lipid peroxidation. (A) Cancer cells were treated with 40 µM CTN and MDA was measured (* p-value = 0.0111 and ** p-value = 0.0013). (B) Cancer cells were titrated over a range of CTN concentrations and EC50 was calculated. (C) Cancer cells were treated with 40 µM CTN and 4-HNE was measured (* p-value > 0.0001). All experiments were performed in triplicate. The error bars are mean ± SD.
Fig. 3 CTN-induced lipid peroxidation. (A) Cancer cells were treated with 40 µM CTN and MDA was measured (* p-value = 0.0111 and ** p-value = 0.0013). (B) Cancer cells were titrated over a range of CTN concentrations and EC50 was calculated. (C) Cancer cells were treated with 40 µM CTN and 4-HNE was measured (* p-value > 0.0001). All experiments were performed in triplicate. The error bars are mean ± SD.

3.5

3.5 CTN alters ferroptotic markers in lung cancer cells

To further investigate that CTN induces ferroptosis, we looked at the ferroptosis-related markers. We showed that iron load increased in A549 and NCI-H520 cells compared to BEAS-2B cells, likely due to higher level of ROS in cancerous cells. However, CTN increased iron load by 2.66 and 2 folds in A549 and NCI-H520 cells respectively (Fig. 4A). GPx4 activity was also measured as a key antioxidant defense system. The GPx4 activity was reduced in response to CTN treatment in both A549 and NCI-H520 although the inhibitory effect on A549 cells was stronger (Fig. 4B). Cytoglobin has been reported as a ferroptosis inducing protein in cancer cells (Conrad et al., 2018). RT-PCR analysis showed the upregulation of Cytoglobin in both lung cancer cells (Fig. 4C). The iron flow related protein, ferroportin, was also downregulated in response to CTN in A549 and NCI-H520 (Fig. 4D). All this data suggested that CTN alters the key components of ferrotosis in lung cancer cells.

CTN altered the key components of ferroptosis machinery. (A) Fe2+ levels was measured in lung cancer cells to show the iron load in response to CTN (* p-value < 0.0001). (B) GPx4 activity was measured in A549 and NCI-H520 cells (* p-value > 0.0001). (C) and (D) the mRNA level of Cytoglobin (* p-value = 0.0129 and ** p-value = 0.0378) and ferroportin (* p-value = 0.0001 and ** p-value = 0.0003) was analyzed by RT-PCR in CTN-treated lung cancer cells.
Fig. 4 CTN altered the key components of ferroptosis machinery. (A) Fe2+ levels was measured in lung cancer cells to show the iron load in response to CTN (* p-value < 0.0001). (B) GPx4 activity was measured in A549 and NCI-H520 cells (* p-value > 0.0001). (C) and (D) the mRNA level of Cytoglobin (* p-value = 0.0129 and ** p-value = 0.0378) and ferroportin (* p-value = 0.0001 and ** p-value = 0.0003) was analyzed by RT-PCR in CTN-treated lung cancer cells.

3.6

3.6 CTN-induced lipid peroxidation is iron dependent

As lipid peroxidation is a characteristics of ferroptosis, we tested if it is iron-dependent as in ferroptotic cells (Hao et al., 2016). Therefore, the MDA production was measured in the CTN treated cells as well as in the cells in which transferrin receptors were blocked using anti-TfR antibody. MDA production was massively reduced in the cells with blocked transferrin receptors (Fig. 5). In A549 cell, co-treatment with CTN and anti-TfR antibody reduced MDA to the background level. However, this reduction was even more in the NCI-H520 cells and MDA was 49% lower than that of untreated cells. This suggest that lipid peroxidation is more dependent on transferrin receptors in NCI-H520 cells than A549 cells.

CTN induced lipid peroxidation is associated with ferroptotic features. Cancer cells were treated with Anti-TfR antibody and exposed to CTN and the production of MDA was measured (* p-value = 0.0006 and ** p-value > 0.0001). All experiments were performed in triplicate. The error bars are mean ± SD.
Fig. 5 CTN induced lipid peroxidation is associated with ferroptotic features. Cancer cells were treated with Anti-TfR antibody and exposed to CTN and the production of MDA was measured (* p-value = 0.0006 and ** p-value > 0.0001). All experiments were performed in triplicate. The error bars are mean ± SD.

3.7

3.7 CTN induces apoptosis to the lower level than ferroptosis

To further investigate the mode of death induced by CTN, caspase-3 inhibitor was used to block the caspase-dependent death and see how cells respond to CTN. CTN and 100 µM QVD-OPh did not change the level of death in normal BAES-2B cells (Fig. 6A). CTN treatment decreased the level of viability by 50% and 40% in A549 and NCI-H520 cells, respectively. However. Caspase-3 inhibition led to 32% and 25% death A549 and NCI-H520 cells, respectively. This suggests that only 19% and 13% of cell death induced by CTN is through induction of caspase-dependent apoptosis.

Caspase-3 inhibition failed to prevent CTN-induced death in lung cancer cells. (A) Caspase-3 cells treated with QVD-OPh and cell death was measured in the presence and absence of CTN. All experiments were performed in triplicate. The error bars are mean ± SD. (B) Schematic representation of the proposed model by which CTN induces death in lung cancer cells.
Fig. 6 Caspase-3 inhibition failed to prevent CTN-induced death in lung cancer cells. (A) Caspase-3 cells treated with QVD-OPh and cell death was measured in the presence and absence of CTN. All experiments were performed in triplicate. The error bars are mean ± SD. (B) Schematic representation of the proposed model by which CTN induces death in lung cancer cells.

4

4 Discussion

CTN has been shown to play apoptotic roles by inducing the generation of ROS although some reports showed caspase-independent death. Autophagy has been shown to be the mode of death in lung cancer (Zhang et al., 2019; Li et al., 2015). Here, we further investigated the cell death induced by CTN. We showed that CTN kills the A549 and NCI-H520 cells in a dose-dependent manner with the IC50 of 57.95 µM and 67.65 µM respectively. Treating the cells with the IC50 concentrations induced caspase-3 activity as well as ROS generation (Fig. 1). Caspase-dependent death was in contrast with previous reports on human rhabdomyosarcoma, breast cancer and prostate cancer (Zhang et al., 2019). This finding suggests that CTN is a potent molecule to induce different modes of death depending on the physiological condition, cell content and maybe the stages of the cancers. If this is true then CTN is able to target different intracellular components which yet to be identified.

If the caspase-dependent cell death (apoptosis) was the actual cause of death we expected to see EC50 similar to IC50. Surprisingly the EC50 values was greater than the IC50 (102.3 versus 57.95 µM for A549 and 81.45 versus 67.65 µM for NCI-H520). The higher EC50 for caspase-3 like activity suggests that CTN kills the cells primarily by a different signaling rather than apoptosis (Fig. 2). As CTN induces the generation of ROS, we titrated CTN to see how it correlates to the mode of death. The EC50 of CTN-mediated ROS generation was lower than the IC50 values (30.48 versus 57.95 µM and 53.09 versus 81.45 for A549 and NCI-H529 respectively). This suggests that ROS generation is the main cause of death (Fig. 2). CTN was shown before to inhibit the activation of Signal transducer and activator of transcription 3 (Stat3) pathways in colorectal cancer cell. Stat3 modulates the expression of genes that mediate as inflammation and cell growth and induces multiple cancer types (Lo et al., 2017). In addition, CTN was also shown to suppress Stat3 signaling and improved cardiac function by inhibition of fibrosis (Gao et al., 2018). However, the positive regulatory role of Stat3 in mediating ferroptosis in human pancreatic ductal adenocarcinoma was reported before (Yan et al., 2020). As CTN targeted Stat3 in various cancer types, it remained to be studied if Stat3 plays an important role in lung cancer. Moreover, further studies are required to determine whether CTN targets Stat3 to modulate apoptosis or ferroptosis in lung cancer cells.

ROS generation takes place in a variety of physiological conditions and is associated with a range of cell death modalities such as autophagy, necroptosis and ferroptosis (Filomeni et al., 2015; Tian et al., 2020; Su et al., 2019; Park et al., 2019). To further investigate how CTN-induced ROS generation, we measured the lipid peroxidation by measuring the MDA production, a characteristic of ferroptotic death. Ferroptosis, a form of regulated non-apoptotic cell death is associated with iron-dependent lipid peroxidation, during which oxygen interacts to lipid and produces lipid hydroperoxides (Hao et al., 2016). MDA was produced with the EC50 of 34.87 and 48.25 for A549 and NCI-H520, respectively. Accordingly, the level of 4-HNE, the marker of lipid peroxidation, was also increased in response to CTN (Fig. 3). This is consistent with the EC50 calculated in ROS generation assay as the values are very close. Ferroptosis is an iron-dependent process, thus to ensure that this ROS and MDA production was iron-dependent, we blocked the transferrin receptors, the importers of iron to intracellular space by anti-TfR antibody and measure MDA. As shown in Fig. 5, blocking the transferrin receptor reduced the MDA in CTN treated cells. GPx4 is the primary defense system against oxidative stress, which is inactivated or downregulated in ferroptotic cells (Ricci et al., 2003). Therefore, it was likely that CTN targets GPx4 and reduces the anti-oxidative stress. Measuring the GPx4 activity confirmed that CTN reduced the activity. To further investigate CTN-induced ferroptosis, we showed that iron load increased in the CTN-treated cells. We also showed the alteration of key component of ferroptosis machinery by analyzing the mRNA level of Cytoglobin and ferroportin. This data was consistent with the recently published data showing the role of Cytoglobin in sensisting the colon cancer cells to ferroptosis (Conrad et al., 2018). These findings showed that CTN can induce ferroptotic death in addition to caspase-dependent death in lung cancer cell lines.

To identify the main cause of death in lung cancer cell lines, the caspase-3 activity was blocked by pharmacological inhibitor, QVD-OPh, and cell viability was tested. Inhibition of caspase-3 activity was responsible for only 19% and 13% for A549 and NCI-H520 cells, respectively (Fig. 6B). This data is consistent with the higher EC50 of CTN for caspase-3 like activity than that of ROS and MDA production.

We here propose a model based on which CTN induces death in cancer cells. In this model CTN primarily can block the activity of GPx4. However, it is not known if this inhibition is due to the direct interaction between CTN and GPx4 or is a result of indirect effect and contribution of upstream proteins. This GPx4 inactivation induces ROS generation, lipid peroxidation and leads to ferroptotic death. On the other hand, CTN activates caspase-3 and enhances apoptosis but to a lower level. Further studies are required to discover the cross-talk between caspase-3 and ROS. It has been shown before that caspase-3 activity can disrupt the mitochondrial membrane and induces mitochondrial-mediated ROS generation (38). In this case, apoptosis induction takes place first to amplify the signal for ferroptosis. Furthermore, ROS can promote the caspase activity. In this scenario, ferroptosis signaling takes place first, and then the signal is amplified with the contribution of caspase-3 and a feedback loop on mitochondria.

Acknowledgement

This study was supported by Tianjin Natural Science Foundation Project (No. 20 JCYBJC 00600)

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. , , , , , , . Apaf-1 Oligomerizes into Biologically Active ∼700-kDa and Inactive ∼1.4-MDa Apoptosome Complexes. J Bio Chem.. 2000 Mar 3;275(9):6067-6070.
    [Google Scholar]
  2. , , , . Salvia miltiorrhiza extract dihydrotanshinone induces apoptosis and inhibits proliferation of glioma cells. Bosn J Basic Med Sci 2017 May 8
    [Google Scholar]
  3. , , , , , , . Cryptotanshinone activates p38/JNK and inhibits Erk1/2 leading to caspase-independent cell death in tumor cells. Cancer Prev Res (Phila Pa).. 2012 May;5(5):778-787.
    [Google Scholar]
  4. , , , , , , . Regulation of lipid peroxidation and ferroptosis in diverse species. Genes Dev.. 2018 May 1;32(9–10):602-619.
    [Google Scholar]
  5. , , , , . From traditional Chinese medicine to rational cancer therapy. Trends Mol Med.. 2007 Aug;13(8):353-361.
    [Google Scholar]
  6. , , , , , , . Tanshinone IIA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit. J Ethnopharmacol.. 2008 Jan;115(2):217-222.
    [Google Scholar]
  7. , , , . Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ.. 2015 Mar;22(3):377-388.
    [Google Scholar]
  8. , , , , , , . Ferroptosis is a lysosomal cell death process. Biochem Biophys Res Commun.. 2018 Sep 10;503(3):1550-1556.
    [Google Scholar]
  9. , , , , , , . Salvia miltiorrhiza (Danshen) inhibits L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes. J Ethnopharmacol. 2014 158 Pt A:397–403
    [Google Scholar]
  10. , , , , . Human lung epithelial BEAS-2B cells exhibit characteristics of mesenchymal stem cells. PLoS One.. 2020 Jan 3;15(1):e0227174
    [Google Scholar]
  11. , , , , , , . Cryptotanshinone Induces Pro-death Autophagy through JNK Signaling Mediated by Reactive Oxygen Species Generation in Lung Cancer Cells. Anticancer Agents Med Chem.. 2016;16(5):593-600.
    [Google Scholar]
  12. , , , , . Cryptotanshinone attenuates the stemness of non-small cell lung cancer cells via promoting TAZ translocation from nuclear to cytoplasm. Chin Med.. 2020 Jun;30(15):66.
    [Google Scholar]
  13. , , , , , , . Cryptotanshinone enhances TNF-α-induced apoptosis in chronic myeloid leukemia KBM-5 cells. Apoptosis.. 2011 Jul;16(7):696-707.
    [Google Scholar]
  14. , , , , , , . Salvia miltiorrhiza extract inhibits TPA-induced MMP-9 expression and invasion through the MAPK/AP-1 signaling pathway in human breast cancer MCF-7 cells. Oncol Lett.. 2017 Sep;14(3):3594-3600.
    [Google Scholar]
  15. , , , , , . Cryptotanshinone, a Stat3 inhibitor, suppresses colorectal cancer proliferation and growth in vitro. Mol Cell Biochem.. 2015 Aug;406(1–2):63-73.
    [Google Scholar]
  16. , , , , , , . Cryptotanshinone Inhibits STAT3 Signaling to Alleviate Cardiac Fibrosis in Type 1-like Diabetic Rats. Phytother Res.. 2017 Apr;31(4):638-646.
    [Google Scholar]
  17. , , , . Acetonitrile extract of Salvia miltiorrhiza Radix exhibits growth-inhibitory effects on prostate cancer cells through the induction of cell cycle arrest and apoptosis. Oncol Lett.. 2017 May;13(5):2921-2928.
    [Google Scholar]
  18. , , , . Reactive oxygen species-mediated kinase activation by dihydrotanshinone in tanshinones-induced apoptosis in HepG2 cells. Cancer Lett.. 2009 Nov 18;285(1):46-57.
    [Google Scholar]
  19. Liu J-J, Wu H-H, Chen T-H, Leung W, Liang Y-C. 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies. Int J Mol Sci. 2015 Aug 17;16(8):19387–19400. 14. Zhang Y, Jiang P, Ye M, Kim S-H, Jiang C, Lü J. Tanshinones: sources, pharmacokinetics and anti-cancer activities. Int J Mol Sci. 2012 Oct 22;13(10):13621–13666.
  20. , , , , , , . Therapeutic effects of ten commonly used chinese herbs and their bioactive compounds on cancers. Evid Based Complement Alternat Med.. 2019 Sep;15(2019):6057837.
    [Google Scholar]
  21. , , , , , , . Cryptotanshinone induces ER stress-mediated apoptosis in HepG2 and MCF7 cells. Apoptosis.. 2012 Mar;17(3):248-257.
    [Google Scholar]
  22. , , , , , , . Quantitative proteomic analyses reveal that GPX4 downregulation during myocardial infarction contributes to ferroptosis in cardiomyocytes. Cell Death Dis.. 2019 Nov 4;10(11):835.
    [Google Scholar]
  23. , , , . Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol.. 2003 Jan 6;160(1):65-75.
    [Google Scholar]
  24. , . Tanshinone IIA potentiates the efficacy of 5-FU in Colo205 colon cancer cells in vivo through downregulation of P-gp and LC3-II. Exp Ther Med.. 2012 Mar;3(3):555-559.
    [Google Scholar]
  25. , , , , , , . Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev.. 2019 Oct;13(2019):5080843.
    [Google Scholar]
  26. , , , , , , . Cytotoxic effects of solvent-extracted active components of Salvia miltiorrhiza Bunge on human cancer cell lines. Exp Ther Med.. 2015 Apr;9(4):1421-1428.
    [Google Scholar]
  27. , , , . Genes and Pathology of Non-Small Cell Lung Carcinoma. Semin Oncol.. 2014 Feb;41(1):28-39.
    [Google Scholar]
  28. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016 Feb;66(1):7–30.
  29. , , , , , , . Anti-cancer natural products isolated from chinese medicinal herbs. Chin Med.. 2011 Jul 22;6(1):27.
    [Google Scholar]
  30. , , , , , , . ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death. Oxid Med Cell Longev.. 2020 Oct;16(2020):1295382.
    [Google Scholar]
  31. , , , , , , . Cardiovascular Protective Effects of Adjunctive Alternative Medicine (Salvia miltiorrhiza and Pueraria lobata) in High-Risk Hypertension. Evid Based Complement Alternat Med.. 2013 Mar;7(2013):132912
    [Google Scholar]
  32. , , , . The International Epidemiology of Lung Cancer: geographical distribution and secular trends. J Thorac Oncol.. 2008 Aug;3(8):819-831.
    [Google Scholar]
  33. , , , , , , . Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells. J Cell Mol Med.. 2021 Feb;21
    [Google Scholar]
  34. , , , , , . Combination treatment with dihydrotanshinone I and irradiation enhances apoptotic effects in human cervical cancer by HPV E6 down-regulation and caspases activation. Mol Cell Biochem.. 2012 Apr;363(1–2):191-202.
    [Google Scholar]
  35. , , , . Multiple cell death modalities and their key features (Review) Wrld Acd Sci. 2020 Mar:11.
    [Google Scholar]
  36. , , , , , , . Cryptotanshinone inhibits cellular proliferation of human lung cancer cells through downregulation ofIGF-1R/PI3K/Akt signaling pathway. Oncol Rep.. 2018 Nov;40(5):2926-2934.
    [Google Scholar]
  37. , , , . BAP1 suppresses tumor development by inducing ferroptosis upon SLC7A11 repression. Mol Cell Oncol.. 2019;6(1):1536845.
    [Google Scholar]

Fulltext Views
1,105

PDF downloads
1,028
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles: