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Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma

  • Authors:
    • Wei Mao
    • Jian Ding
    • Yu Li
    • Ruofei Huang
    • Baoxin Wang
  • View Affiliations

  • Published online on: June 16, 2022
  • Article Number: 521
  • Copyright: © Mao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Head and neck squamous cell carcinoma (HNSCC) is a worldwide public health problem; its incidence is increasing and it is now the sixth most common cancer type worldwide. As indicated by existing studies, ferroptosis contributes to HNSCC progression and Tanshinone IIA (TanIIA) may exert therapeutic effects via affecting ferroptosis. However, the underlying mechanisms have remained to be clarified. Therefore, the main aim of the present study was to screen and investigate the key genes in regulating ferroptosis of the human hypopharynx squamous carcinoma cell line FaDu and further elucidate the mechanism of action of TanIIA. A list of ferroptosis‑related genes was obtained from the FerrDb database. RNA‑sequencing expression (level 3) profiles and corresponding clinical information (cases, n=502; normal controls, n=44) were downloaded from The Cancer Genome Atlas dataset for HNSCC ( The limma package in R software was used to study the differentially expressed mRNAs. Adjusted P<0.05 and Log2(fold change) >1 or <‑1 were defined as the threshold for the differential expression of mRNAs. The ClusterProfiler package (version 3.18.0) in R was employed to analyze the Gene Ontology functional terms associated with potential targets and perform a Kyoto Encyclopedia of Genes and Genomes pathway analysis. The R package ggplot2 was used to draw the boxplot and the pheatmap package was used to draw the heatmap. The DEG‑related protein‑protein interaction network was built with the Search Tool for the Retrieval of Interacting Genes and proteins database and then the visualization was performed using Cytoscape. Ferritin heavy chain 1 (FTH1), transferrin (TF) and TF receptor were screened out using a Wayne diagram, which was drawn by the Venn Diagram package in R. Kaplan‑Meier survival analysis and the log‑rank test were used to compare differences in survival between the groups. The receiver operating characteristic (v 0.4) (ROC) curve analysis was used to compare the predictive accuracy of mRNAs. FTH1 was screened out and the expression results were verified using The Human Protein Atlas data. Immunohistochemistry and immunofluorescence were used to localize FTH1 expression in FaDu cells. Furthermore, Cell Counting Kit‑8 and Transwell assays were used to detect the cell survival and invasion ability, respectively. Furthermore, western blot analysis was performed to analyze protein expression. The results of the present study indicated that three validated ferroptosis marker genes were differentially expressed in HNSCC, among which FTH1 was significantly associated with poorer survival. TanIIA was demonstrated to significantly affect FaDu cell survival and invasiveness and markedly attenuate FTH1 expression. To conclude, the ferroptosis gene FTH1 is highly expressed in HNSCC and TanIIA significantly inhibited HNSCC, partially by suppressing FTH1.


Head and neck squamous cell carcinoma (HNSCC) is one of the most lethal malignancies with >800,000 new-onset cases reported per year. Mortality rates are increased with the stage of the disease and rates >50% have been identified in critically ill patients (1). HNSCC originates from epithelial cells of the upper respiratory tract and esophageal mucosa; of note, HNSCC exhibits high heterogeneity due to the complex anatomy and genetic variations in that area. Over the past few years, research on the correlation between ferroptosis and tumors has become a major research hotspot. Several studies confirmed that the expression levels of ferroptosis proteins exhibited a wide variation in HNSCC, thereby significantly regulating cell proliferation (2) and epithelial-mesenchymal transformation (3) and even affecting cisplatin resistance (4). However, systematic studies focusing on ferroptosis and HNSCC have been continuously lacking thus far and research on novel precision therapy targets remains in its infancy.

Over the past decade, research on the significance of ferroptosis in cancer has gained momentum. Ferroptosis is another type of programmed necrotic cell death involving iron-dependent lipid peroxidation and oxidative stress is thought to be a principal cause of ferroptosis (5). Due to cancer cells' high iron levels and their increased sensitivity to ferroptosis induction, ferroptosis has been proposed to be promising for cancer therapeutics. A growing number of studies indicated that for numerous types of solid tumor, positive treatment effects were achieved by targeting ferroptosis-related genes.

Besides ferroptosis agonists and inhibitors, multiple drugs and Traditional Chinese Medicine (TCM) monomers were confirmed to effectively affect the level of ferroptosis and exert antitumor effects. Kong et al (6) proved that baicalin exerted its anticancer activity via ferritin heavy chain 1 (FTH1) in both 5637 and KU19-19 cells. Zhu et al (7) proposed that artemisinin and its derivatives may be used in the future as cancer therapies with broader applications due to their induction of ferroptosis. Tanshinone IIA (TanIIA) has long been confirmed to have a pivotal role in HNSCC treatment. Studies in other organs indicate that TanIIA exerts a significant impact on ferroptosis. For example, it has been proved that Tan IIA induced ferroptosis in BGC-823 and NCI-H87 gastric cancer cells via upregulating p53 expression and downregulating xCT expression in gastric cancer (8). However, the association between TanIIA and ferroptosis in HNSCC has remained elusive.

TanIIA, a pharmacologically active component isolated from the rhizome of the Chinese herb Salvia miltiorrhiza Bunge (Danshen), has been reported to possess pleiotropic effects, such as anti-inflammatory effects, antioxidant and anti-atherogenic effects. Recent studies have suggested that TanIIA exhibits highly potent antitumor activity by inducing apoptosis and triggering autophagy in tumor cells (9). A previous study by our group confirmed TanIIA has a significant effect to promote FaDu cell apoptosis by causing cell cycle arrest at the S phase, which resulted in observably higher apoptotic cell fractions and downregulation of survivin protein expression (10). In addition, TanIIA was confirmed to exert its protective effect on endothelial cells by inhibiting ferroptosis via activation of NRF2(11). However, the exact target and mechanisms by which TanIIA regulates ferroptosis remain to be elucidated.

In the present study, data acquired from FerrDb were mined to comprehensively analyze the correlations between ferroptosis marker genes and HNSCC. Cell survival, invasion and expression status were evaluated after treatment with TanIIA, with the goal of uncovering the function of TanIIA.

Materials and methods

Data sources

The RNA-sequencing expression (level 3) profiles and corresponding clinical information for HNSCC were downloaded from The Cancer Genome Atlas (TCGA) dataset (, including 502 tumor samples and 44 normal controls. Ferroptosis-related genes were acquired from the FerrDb database [Zhou and Bao (12), 2020] which includes 125 ferroptosis marker genes, of which 9 genes have been verified.

Differentially expressed gene (DEG) analysis

The limma package in R (version 3.4.2; was used to analyze the differentially expressed mRNAs among samples. ‘Adjusted P<0.05 and Log2 (Fold Change) >1 or Log2(Fold Change) <-1’ were defined as the threshold for the differential expression of mRNAs. Spearman's correlation analysis was used to describe the correlation between the 9 marker genes. P<0.05 was considered to indicate statistical significance (Table SI). The DEG-related protein-protein interaction (PPI) network was established using the Search Tool for the Retrieval of Interacting Genes and proteins (STRING) database ( (13) (Table SII), followed by visualization using Cytoscape software (version 3.8.2; (14).

To further confirm the underlying function of potential targets, the data were analyzed by functional enrichment. To better understand the role of these mRNAs in carcinogenesis, the ClusterProfiler package (version: 3.18.0) in R was employed to analyze the Gene Ontology function of potential targets and the Kyoto Encyclopedia of Genes and Genomes pathway. The R software ggplot2 package was used to draw the boxplot, while the R software pheatmap package was used to draw the heatmap.

Validated ferroptosis marker genes and HNSCC correlation analysis

The multi-gene correlation pheatmap was displayed by the R software package. Spearman's correlation analysis was used to determine the correlation between the 9 validated ferroptosis markers. A Venn diagram analysis was carried out between genes encoding ferroptosis markers and validated markers that are associated with DEGs using the Interacti-Venn website (; Table SIII). By the above means, FTH1, transferrin (TF) and TF receptor (TFRC) were screened out. The Human Protein Atlas ( was used to validate FTH1 expression in the alimentary system (which includes the larynx and hypopharynx).

The expression distributions of FTH1, TFRC and TF genes in tumor tissues and normal tissues were downloaded from the TCGA dataset. The current-release (V8) GTEx datasets were obtained from the GTEx data portal ( website. Data were analyzed using R software v4.0.3 (R Foundation for Statistical Computing). P<0.05 was considered to indicate statistical significance.

Analyses of prognosis

Correlations between the expression levels of FTH1, TFRC and TF genes and HNSCC overall survival were separately analyzed through the Kaplan-Meier method and the log-rank test. Time-receiver operating characteristic (ROC; v 0.4) (15) analysis was used to compare the predictive accuracy of mRNA. All the analytic methods and R packages were implemented by R (foundation for statistical computing 2020) version 4.0.3. Furthermore, images of protein immunohistochemistry (IHC) stains for FTH1, TFRC and TF in tumor tissues were obtained from the Human Protein Atlas (16).

Cell acquisition

FaDu and human hypopharyngeal cell lines were purchased from Procell Life Science & Technology Co., Ltd. The obtained cells were cultured and passaged as primary cells and then cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% double antibody (penicillin-streptomycin mixture) (MilliporeSigma) in an incubator with 5% CO2 at 37˚C. The cells were subcultured to the third passage.

IHC method

FTH1 expression was visualized using the IHC method. FaDu cells were seeded on to cover slips at 5x104/ml density and then fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 15 min at 37˚C, permeabilized using 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 20 min blocked using 5% bovine serum albumin (MilliporeSigma) for 1 h at 37˚C Heat-mediated antigen retrieval was performed with Tris/EDTA buffer (Gibco; Thermo Fisher Scientific, Inc.). A total of 100 µl reaction enhancer was added to the samples and incubated for 20 min at 37˚C before IHC staining (cat. no. PV-6000; Zhongshanjinqiao). The samples were then incubated with HRP-labeled secondary antibody (cat. no. A18781; 1:1,000 dilution; Thermo Fisher Scientific, Inc.) for 30 min at 37˚C, after which the nuclei were counterstained with diaminobenzidine (1:1,000; Beyotime Institute of Biotechnology, Inc.) for 5-8 min. Following washing with tap water, samples were stained with hematoxylin (Beyotime Institute of Biotechnology, Inc.) for 20 sec, then dehydrated with different concentrations of ethanol and clarified using dimethyl benzene (MilliporeSigma) solution. Images were captured with an inverted phase-contrast microscope (CH30; Olympus Corp.). A total of 5 images were acquired from different fields of view of each slide and the staining of yellow particles was regarded as positive expression.

Immunofluorescence (IF) method

IF assays were conducted to determine the cellular location of FTH1 protein expression. The pre-treatment procedure was the same as that used for IHC. IF was performed with FTH1 (5 µg/ml; cat. no. ab65080; Abcam) antibody for 12 h at 4˚C in the dark, followed by incubation with DyLight 488 conjugated, goat anti-human IgG (cat. no. ab96907; 1:1,000; Abcam.). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Beijing Solarbio Science & Technology, Co., Ltd.). A total of five fluorescence images were captured using a fluorescence microscope (Leica DMi8; Leica Microsystems GmbH) with different excitation wavelengths for the same field (dylight488 maximum emission is 518 nm; DAPI maximum emission is 454 nm).

Cell survival

A Cell Counting Kit-8 assay (CCK-8; MedChemExpress) (17) was adopted to detect cell survival. TanIIA (cat. no. HY-N0135; MedChemExpress) was dissolved with DMSO using ultrasonication, followed by treatment for 24, 48 and 72 h at individual concentrations of 0.25, 0.5 or 1.0 mg/l as the treatment group. Cells were grown in 96-well plates and cell survival was measured after 24 h following the manufacturer's protocol. CCK-8 stain and serum-free medium were mixed at a volume ratio of 1:10 and incubated in 5% CO2 at 37˚C for 1 h. The absorbance at 450 nm wavelength (optical density value) was then measured with a microplate reader (Multiskan-MK3; Thermo Fisher Scientific, Inc.).

Tumor invasive ability

The tumor cell invasion capacity was analyzed using a Transwell chamber (8.0 µm pore size; Corning, Inc.). BD Matrigel (cat. no. 354248; BD Biosciences), which had been frozen in a -80˚C freezer, was kept at 4 degrees overnight for 24 h, after which 300 µl serum-free medium was added to 60 µl Matrigel® to prepare a mixed solution and the upper chambers were coated with it in 24-well plates. FaDu cells (1x105) were then seeded into five 96-well plates (1x103 cells/well), with five parallel wells for each cell group. Furthermore, 500 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS was added to the lower chamber. The cells were then incubated for 24 h at 37˚C in a 5% CO2 incubator. Transwell chambers were removed and cells that had transgressed through the membrane to the lower side were washed 2 times with PBS and fixed with 5% glutaraldehyde (Beijing Solarbio Science & Technology, Co., Ltd.) at 4˚C. The attached cells were stained with crystal violet (0.5% crystal violet in 20% methanol) for 5-10 min and then washed two times with PBS. Images were acquired using an Axiovert 40 CFL inverted microscope (Zeiss AG) and invaded cells were counted and recorded. Five fields per slide were used in the analysis and each experiment was performed in triplicate.

Western-blot analysis

FaDu and normal mucosa cells were seeded in 6-well plates at 5x104/ml density (8 ml) and incubated overnight in a 5% CO2 incubator at 37˚C. Cells were first starved in serum-free medium for 48 h, then treated with complete medium with 10% FBS for 12 h and treated with TanIIA at 0.5 mg/l for another 12 h. Cells cultured in a drug-free medium were used as the control group. Total proteins were extracted using One Step Animal Tissue/Cell Active Protein Extraction buffer (RIPA; Thermo Fisher Scientific, Inc.) Protein concentration was determined via BCA assay (cat. no. 23227; Thermo Fisher Scientific, Inc.). Equal amounts of total protein (40 µg) were loaded in each protein lane. Samples were separated by 10% SDS-PAGE (Beijing Solarbio Science & Technology, Co., Ltd.) using a Bio-Rad Electrophoresis System (Bio-Rad Laboratories, Inc.). The proteins were transferred to a nitrocellulose membrane (cat. no. IFPL00010; Merck KGaA) for gel electrophoresis separation, followed by blocking with 5% skimmed milk (cat. no. 232100; BD Difco) for 1 h. Tris-buffered saline with Tween-20 (Beijing Solarbio Science & Technology, Co., Ltd.) was used to wash the membranes, and subsequently, they were incubated at 4˚C overnight with the following primary antibodies: FTH1 (1 µg/ml; 21 kDa; cat. no. ab65080; Abcam); GAPDH (1:500 dilution; 36 kDa; cat. no. ab8245; Abcam). Subsequently, membranes were incubated with a 1:10,000 dilution of HRP-labeled secondary antibody (cat. no. 7076s; Cell Signaling Technology, Inc.) at room temperature for 1 h. GAPDH was used as a normalization control. The membrane was visualized using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.) and analysis was performed using Image Lab software (v4.0; Bio-Rad Laboratories, Inc.).

Statistical analysis

Statistical analyses were performed using SPSS version 21.0 (IBM Corporation) and GraphPad Prism 8 (GraphPad Software, Inc.). Each experiment was performed in triplicate and the mean ± SD. The normality of distribution of data was assessed using Shapiro-Wilk test. Data comparisons between two groups were performed using an unpaired t-test. Comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey's multiple-comparisons test. P<0.05 was considered to indicate a statistically significant difference.


Correlation between HNSCC and ferroptosis marker genes

The gene expression data and clinical characteristics of 502 HNSCC and 44 normal tissue samples from the TCGA database were included in the present study (Table SIV). The limma package in the R software was used to study the differentially expressed mRNAs. A total of 1,457 genes were significantly upregulated and 715 downregulated (Fig. 1). Enrichment analysis indicated that the DEGs were enriched in ‘cell cycle’. Next, 125 ferroptosis genes were downloaded from the FerrDb database, among which nine were validated ferroptosis-related markers (PTGS2, CHAC1, FTH1, SLC40A1, TF, TFRC, GPX4, HSPB1 and NFE2L2). Pearson correlation analysis was conducted to assess the correlations of expressions between each pair of genes of the above 9 ferroptosis marker genes (Fig. 2A) and the PPI network of these genes was further mapped (Fig. 2B). The results indicated that the expression of most of the gene pairs exhibited a statistical correlation (Table SI). The gene annotations and scores are presented in Table SII.

FTH1, TFRC and TF expression in HNSCC

Venn diagram analysis was used and FTH1, TFRC and TF were screened out as common genes expressed in HNSCC and validated ferroptosis markers (Fig. 2C). Subsequently, the expression distributions of these genes in tumor tissues and normal tissues were analyzed and the results indicated that all the three genes were significantly differentially expressed in HNSCC (FTH1, P=5.5x10-10; TFRC, P=1.3x10-13; and TF, P=2.1x10-17; Fig. 2E-G).

FTH1 is an independent prognostic factor in HNSCC

The associations between ferroptosis marker-related DEGs and survival were then analyzed. The log-rank test was used to compare differences in survival between groups with high and low expression of the genes FTH1 [hazard ratio (HR): 1.562, 95%CI: 1.191-2.051, P=0.00129], TFRC (HR: 1.228, 95%CI: 0.94-1.606, P=0.132) and TF (HR: 0.854, 95%CI: 0.653-1.115, P=0.246; Fig. 3A). Time-receiver ROC analysis was used to compare the predictive accuracy for FTH1 [3-year area under the ROC curve (AUC)=0.585, 95%CI: 0.531-0.639], TFRC (3-year AUC=0.525, 95%CI: 0.470-0.480) and TF (3-year AUC=0.514, 95%CI: 0.460-0.569; Fig. 3B).

Based on the formal results, FTH1, TFRC and TF expression was next validated using Oncomine and the Human Protein Atlas database (Fig. 3C). The brown color indicates positive IHC staining. FTH1 expression was then observed in the digestive system heatmap, indicating that it was mainly expressed in squamous epithelial cells and fibroblasts (Fig. 2D). The above results indicated that FTH1 is an independent prognostic factor for HNSCC.

FTH1 protein expression

IHC observation and an IF assay were further performed to determine the localization and FTH1 expression in HNSCC cells. It is generally considered that FTH1 expression is localized to the cytoplasm and membrane (18). The IHC staining result was fully consistent with this (Fig. 4A). An IF assay was then performed for further confirmation and the results complied with IHC, i.e., fluorescence signals were present in the cytoplasm, while no expression was observed in the nucleus (Fig. 4B).

TanIIA downregulates FTH1 expression in FaDu cells

FTH1 expression in FaDu cells was measured by western blot analysis (Fig. 4C). Normal mucosa cells were used as the control group. FTH1 in FaDu cells was significantly overexpressed compared with the control group (P<0.05). Different concentrations (0.25, 0.5 and 1 mg/l) of TanIIA were adopted for FaDu cell treatment. The data obtained confirmed that TanIIA at 0.5 mg/l effectively downregulated FTH1 expression in FaDu cells. Furthermore, it is noteworthy that the P-value in the normal mucosa group indicated no significance compared with that of the normal mucosa + TanIIA group. Whether such a result indicated selectivity of TanIIA should be further investigated in depth.

TanIIA effectively reduces FaDu cell survival and invasion

A CCK-8 assay was adopted for detection of cell survival (Fig. 5A-C). TanIIA treatment was performed at concentrations of 0.25, 0.5 and 1.0 mg/l, and the survival rate was measured at 24, 48 and 72 h, respectively, by determining the mean value. The proportion of live cells in the treatment group (48 h: 70.3%; 72 h: 52.29%) decreased significantly (P<0.05) as compared with the control group (48 h: 67.83%; 72 h: 54.35%). The effect of TanIIA exhibited a clear dose-effect relationship (Fig. 5D). A Transwell assay was then performed to observe the invasion of FaDu cells (Fig. 5E). As indicated from the results, FaDu cells exhibited a stronger migration and invasion ability than normal hypopharyngeal cells and treatment with TanIIA significantly attenuated the invasion ability of FaDu cells (P<0.05; Fig. 5F). Specifically, under treatment with TanIIA (0.5 mg/l) (Fig. 5G), although the invasion ability of HNSCC (mean=146) remained higher than that of the control (mean=119), the difference was not statistically significant (P=0.0557), thereby demonstrating that TanIIA treatment effectively inhibited the invasion ability of FaDu cells.


The global annual occurrence of head and neck cancers exceeds 0.5 million, out of which 90% are squamous cell carcinoma, HNSCC (19). The development of HNSCC refers to a multi-step process involving multiple signal transduction pathways and complex cross-talk among the pathways. The rapid advancement of microarray technology has significantly boosted gene expression profiling, gene sequencing and convenient diagnosis of diseases due to its high-throughput characteristics and rapid detection ability. Numerous studies have confirmed that ferroptosis has complex roles in tumor progression (20) and regulate the tumor microenvironment (21).

Increasing research studies have confirmed that ferroptosis levels are closely related to HNSCC initiation, progression, recurrence, metastasis and immune escape. Ferroptosis (22) has been referred to as the ‘new land of cell death’. Distinct from apoptosis, necrosis and autophagy, it refers to an iron-dependent, novel form of programmed cell death with diverse and complex targets involved (e.g., PTGS2, NOX1, FTH1, COX2, GPX4 and ACSl4). A tissue microarray study indicated that patients with high expression of GPX4 had poor survival and the expression of GPX4 has been negatively associated with the immunogenic cell death-related protein calreticulin in an HNSCC tissue array (23). An increasing number of ferroptosis-related targets have been identified as independent prognostic markers for HNSCC (24).

As a widely used TCM compound, TanIIA has already been investigated as a primary or adjuvant therapy with beneficial effects on HNSCC patient survival (10). The latest studies have confirmed that TanIIA increased the level of lipid peroxides and decreased glutathione levels in gastric cancer cells, both of which are markers of ferroptosis (25). Guan et al (8) proved that TanIIA caused decreased intracellular glutathione levels and cysteine levels and increased intracellular reactive oxygen species (ROS) levels. Furthermore, p53 knockdown attenuated TanIIA-induced lipid peroxidation and ferroptosis in a BGC-823 xenograft model. The above studies revealed that TanIIA has significant impacts on cancer cells, partially by inducing ferroptosis. Therefore, it is reasonable to assume that TanIIA may regulate HNSCC progression through ferroptosis-related marker genes.

In the present study, by listing all DEGs in HNSCC and validated marker genes for ferroptosis, three possible targets were screened out: FTH1, TFRC and TF. In a TGCA prognostic analysis, FTH1 was indicated to be an independent prognostic factor of HNSCC.

Iron is an essential element required by cells and has been described as a key player in ferroptosis (26). Ferritin is a protein complex that stores iron in a bioavailable, nontoxic form (27). FTH1 is the main subunit for ferritin's function, which covers a ferroxidase active center responsible for regulating the oxidation and integration of ferric ions. Upstream of the transcription start site of ferritin heavy chain contained an antioxidant response element, which could protect cells from oxidative damage, thereby avoiding apoptosis by responding to oxidative reactions (28). This feature makes FTH1 closely related to numerous biological processes such as oxidative stress (29), cell differentiation (30) and neuronal functions (31). Previous work has shown that FTH1 expression is increased in several human cancers including squamous cell carcinoma, the study by Yang et al (32) indicated that long non-protein-coding RNA FTH1 pseudogene 3 was significantly up-regulated in Esophageal squamous cell carcinoma tissues and cells and was critical to the course of tumor formation. Also, the FTH1 expression level is associated with the survival rate of oral squamous cell carcinoma (33). Silencing of FTH1 or reducing its expression has been proved can significantly inhibit carcinogenesis, mainly by promoting cell death (34), inhibiting metastasis (35), and attenuating tumor cell invasion (36). Here we proved that FTH1 is a validated ferroptosis marker gene that significantly impacts HNSCC prognosis, which is mainly expressed in squamous epithelial cells and fibroblasts in the human digestive system.

TanIIA is a pharmacologically active lipophilic component of Salvia miltiorrhiza extract, which shares a history of high repute in TCM (37). TanIIA has demonstrated potent tumor inhibitory effects against various types of tumor cell (38); the underlying mechanism involves regulation of the cell cycle (39), suppresses cell proliferation and induces apoptosis (40), inhibits tumor invasion and metastasis, inhibits angiogenesis (41) and reverses tumor multi-drug resistance (42). Xu et al (43) demonstrated that TanIIA enhances the antitumor activity of chemotherapeutic drugs and increases the sensitivity of FaDu cells to radiotherapy. Ding et al (44) examined the underlying mechanisms and indicated that TanIIA exerted a strong radiosensitizing effect due to enhanced ROS generation and autophagy. Here, CCK-8 and Transwell assays were used to determine the effects of TanIIA on the cell survival and invasion ability in an attempt to examine its pharmacological effects, indicating that at certain concentrations, TanIIA significantly promoted FaDu cell death and attenuated its invasion ability with a dose-effect relationship.

It is generally accepted that FaDu cells exhibit the biological characteristics of high proliferation and invasion (45), which makes treatment of HNSCC relatively difficult. In patients with early-stage HNSCC, surgery and radiotherapy act as the mainstays of treatment, whereas 70-80% of patients are already locally advanced or advanced at the initial presentation. For these patients, the 5-year survival rate was only 40% or even less (46). As reported by a previous study, the median survival time of patients was only 10 months once HNSCC had recurred or metastasized (47). Thus, adjuvant therapies are critical to HNSCC treatment. To date, cisplatin remains the treatment of choice for HNSCC, whereas its development of resistance and toxicity cannot be ignored. Given all the above, finding an effective drug with fewer and less severe side effects is a promising solution. The present study proved that a certain concentration of TanIIA (≥0.5 mg/l) was able to significantly attenuate the protein expression of the prognostic factor FTH1.

In summary, the present study highlights a novel perspective in the clinical treatment of HNSCC and provides a theoretical basis for developing TanIIA for use in co-treatment for HNSCC. By identifying the relationship between ferroptosis markers and HNSCC, a ferroptosis coregulated gene was screened out, which was able to significantly affect HNSCC prognosis. The present study was the first, to the best of our knowledge, to indicate that TanIIA treatment drastically inhibited FTH1 expression in FaDu cells, significantly affected cell survival and suppressed the invasive capacity of FaDu cells. However, the present study had several limitations. FTH1 serves as a vital regulator of ferroptosis, which is generally considered a suppressor of ferroptosis (48), its specific gene function still requires further study using the knockdown or knockout method. The STRING data of the present study indicated that FTH1 is associated with ferroptosis markers including GPX4, SLC40A1, TFRC and NFE2L2, so the downstream signaling pathways are worth further investigating. In addition, as TanIIA is a natural antineoplastic drug, it is also important to perform clinical research on whether dose escalation increases with long-term use and if this would bring side effects.

Supplementary Material

Correlation analysis between verified targets of ferroptosis.
Protein-protein interactions of ferroptosis markers.
Venn diagrams of HNSCC and ferroptosis markers.
Clinical characteristics of samples from The Cancer Genome Atlas.


The authors would like to thank Dr Pin Dong (Otolaryngology-Head and Neck Surgery, Shanghai General Hospital, Shanghai, China) for reviewing this article for technical accuracy and for providing valuable suggestions.


Funding: This study was supported by The National Natural Science Foundation of China (grant no. 81800893).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

WM and BW designed the experiments and drafted the manuscript. WM and JD performed the experiments and analyzed and interpreted the data. YL helped with data collection and analysis. RH coordinated the research and participated in the experimental design. All authors were involved in critically revising the manuscript, and have read and approved the final manuscript. WM and BW confirm the authenticity of all the raw data.

Ethics approval and consent to participate

This work was approved by the Ethics Committee of Shanghai General Hospital (Shanghai, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



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Mao W, Ding J, Li Y, Huang R and Wang B: Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma. Exp Ther Med 24: 521, 2022
Mao, W., Ding, J., Li, Y., Huang, R., & Wang, B. (2022). Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma. Experimental and Therapeutic Medicine, 24, 521.
Mao, W., Ding, J., Li, Y., Huang, R., Wang, B."Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma". Experimental and Therapeutic Medicine 24.2 (2022): 521.
Mao, W., Ding, J., Li, Y., Huang, R., Wang, B."Inhibition of cell survival and invasion by Tanshinone IIA via FTH1: A key therapeutic target and biomarker in head and neck squamous cell carcinoma". Experimental and Therapeutic Medicine 24, no. 2 (2022): 521.