Acetylshikonin induces apoptosis through the endoplasmic reticulum stress‐activated PERK/eIF2α/CHOP axis in oesophageal squamous cell carcinoma

Abstract Acetylshikonin (AS) is an active component of Lithospermum erythrorhizon Sieb. et Zucc that exhibits activity against various cancers; however, the underlying mechanisms of AS against oesophageal squamous carcinoma (ESCC) need to be elusive. The research explores the anti‐cancer role and potential mechanism of AS on ESCC in vitro and in vivo, providing evidences for AS treatment against ESCC. In this study, we firstly demonstrated that AS treatment effectively inhibits cell viability and proliferation of ESCC cells. In addition, AS significantly induces G1/S phage arrest and promotes apoptosis in ESCC cell lines. Further studies reveal that AS induces ER stress, as observed by dose‐ and time‐dependently increased expression of BIP, PDI, PERK, phosphorylation of eIF2α, CHOP and splicing of XBP1. CHOP knockdown or PERK inhibition markedly rescue cell apoptosis induced by AS. Moreover, AS treatment significantly inhibits ESCC xenograft growth in nude mice. Elevated expression of BIP and CHOP is also observed in xenograft tumours. Taken together, AS inhibits proliferation and induces apoptosis through ER stress‐activated PERK/eIF2α/CHOP pathway in ESCC, which indicates AS represents a promising candidate for ESCC treatment.

Endoscopic resection combined with radiotherapy and chemotherapy represent the standard treatment for ESCC 4,5 ; however, outcomes remain unsatisfactory because of the high recurrence rate and the development of drug and radiation resistance. 6The 5-year survival rate of patients with ESCC is less than 30%. 7Therefore, it is of importance to explore novel chemotherapeutic agents to treat ESCC.
The endoplasmic reticulum (ER) as a specialized organelle involves in protein synthesis, folding and transportation through dynamic structural changes 8 ; thus, the maintenance and homeostasis of the ER environment are necessary.Various endogenous or exogenous stimuli may damage the morphology and function of the ER (ER stress), which promotes the misfolding of proteins and subsequently primes the cell to respond and restore function by activation of unfolded protein response (UPR). 9The activated UPR helps protein fold properly and removes misfolded proteins to maintain ER homeostasis. 10However, sustained ER stress may cause the cell to switch from a pro-survival response to a pro-death mode, which results in apoptotic or autophagic cell death. 11There are three signalling pathways related to the UPR, protein kinase RNA-dependent-like ER kinase (PERK), inositol-requiring protein-1 (IRE1) and activating transcription factor 6 (ATF6).Sustained ER stress induces death signals through each of these ways. 12,13Thus, identifying small molecules that trigger apoptosis mediated by ER stress represents a novel strategy for cancer therapy.
Acetylshikonin (AS) is a fat-soluble naphthoquinone pigment that exhibits broad bioactivity, such as anti-tumour, anti-inflammation and antibacterial activity. 149][20][21][22][23] But the anti-cancer effects and mechanisms of AS in ESCC have not been defined.It was reported that AS induces apoptosis via ER stress in human hepatocellular carcinoma cells. 24 demonstrated that AS treatment triggered sustained ER stress and activated the PERK/eIF 2α pathway, resulting in up-regulation of CHOP.Both inhibition of PERK and siRNA-mediated knockdown of CHOP rescued cells from AS-induced apoptosis.Our findings indicate that AS is an attractive candidate for further development as ESCC therapy.

| Compounds and reagents
The extraction, separation and purification of AS have been described previously. 25AS was prepared to 10 mM with DMSO and

| Cell lines and culture
Six cell lines of ESCC were purchased from the National Collection of authenticated cell cultures.Cell culture has been described previously. 26In brief, KYSE450 and TE10 cells were cultured in MEM and DMEM, respectively, with 10% FBS and 1% antibiotic mix.KYSE180, KYSE510, EC109 and EC9706 cells were maintained in RPMI-1640 media supplied with 10% FBS and 1% antibiotic mix.

| Cell viability assay
The KYSE450 cells were grown in 96-well plates at 4 × 10 3 cells/well, while the other cell lines were seeded at 5 × 10 3 cells/well.After adhesion, all cells were treated with DMSO or AS for 24 h and then subjected to CCK-8 assay. 26IC 50 values were determined by GraphPad Prism 8.0.The results were from three independent experiments.

| Clonogenicity assay
The clonogenicity assay was described previously with minor modifications. 27Briefly, the KYSE180 cells (1 × 10 3 cells per well) were incubated with AS for 2 weeks.Then the cold methanol was added to fix the cells.After washing with PBS, the colonies were visualized and counted after staining with 10% Giemsa dye.

| Cell apoptosis analysis
KYSE180 and KYSE450 cells were cultured in 6-well plate overnight and then incubated with AS for 24 h.The cells were collected to investigate apoptosis by flow cytometer (BD FACS Canto II) using the apoptosis assay kit.For combination assays, after transfecting with siRNAs for 24 h or pretreating with PERK inhibitor for 2 h, KYSE180 and KYSE450 were treated with AS for 24 h.

| Cell cycle assay
The KYSE450 cells were seeded and treated as indicated.The cells were collected and suspended with cold ethanol for fixation.After stained with propidium iodide (PI), the distribution of cell cycle was analysed by flow cytometer.

| Western blot assay
The treated cells were collected, and total proteins were prepared by SDS buffer containing protease inhibitors.After centrifugation, the supernatants were collected and quantified by the BCA assay.
The 20 μg protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes.After blocking, the membranes were incubated with primary antibody and secondary antibody, respectively.
Finally, immunoreactive bands were shown after detecting by the ECL reagent.GAPDH served as control for normalizing the amount of protein.

| XBP1 splicing analysis
After AS treatment, total RNA was harvested and reversely transcribed for XBP1 splicing analysis.Specific primers for XBP1 were used to amplify XBP1 transcripts by RT-PCR.The products were digested with Pst I as reported previously. 21After digestion, the fragments were applied to the 2% agarose gel to identify the spliced and unspliced products of XBP1.

| SiRNA Transfection
The sequences of CHOP siRNA were described previously. 31A scramble siRNA was used as control.The KYSE180 and KYSE450 cells were incubated with 10 nm of scramble siRNA or siRNA duplex (OBiO Technology) using INTERFERin (Polyplus) according to the protocol.The transfected cells were exposed to AS for 24 h.Tumour size was determined with Vernier callipers.The formula was used to calculate tumour volume is that V = (L × W 2 )/2, where V means tumour volume, L represents length and W is width.The control group mice were treated with a vehicle solution (0.3% 2-hydroxypropyl-beta cyclodextrin).The low-dose group and high-dose group mice were intraperitoneally injected with 10 mg/kg AS and 20 mg/kg AS, respectively.All mice were treated with AS every 2 days.The body weight and tumour volumes were monitored every 2 days.After 3 weeks, all mice were sacrificed.The tumours were collected for weighing and immunohistochemistry analysis.

| Immunohistochemistry
The fixed and paraffin-embedded samples were sectioned into 5 μm slices.The specimens were dewaxed in xylene and then rehydrated by gradually decreasing concentrations of ethanol (100%-75%).After washed, the slides were heated in 0.01 M citrate buffer (pH 6.0) for antigen retrieval.After spontaneous cooling, the samples were rinsed with PBS (pH 7.4) thrice on a shaking table, treated with 3% H 2 O 2 in the dark at regular temperature for 25 min and subsequently rinsed with PBS (pH 7.4) thrice.The samples were blocked with 3% BSA for 30 min and incubated with primary antibodies overnight at 4°C in a humidified chamber.Ki67 (1:500), CHOP (1:1000) and BIP (1:800) antibodies were used.
After washing, the samples are incubated with secondary antibody labelled with HRP and DAB, the slides were counterstained with haematoxylin and images were captured.The tumour specimens were also analysed by H&E staining.

| Data statistical analysis
The results were analysed by GraphPad Prism 8.00 and displayed as the mean ± SD.Two-tailed Student's t-test was used to determine the significance between two groups.The p < 0.05 was set as statistical significance.

| AS inhibited the viability, proliferation and cell cycle progression in ESCC cells
To determine the anticancer effect of AS (Figure 1A) on ESCC cells, the KYSE450, KYSE180, KYSE510, TE10, EC109 and EC9706 cells were treated with increasing dosages of AS for 24 h.Cell viability was assessed by the CCK8 assay.As shown in Figure 1B, the viability of all cell lines was significantly decreased following AS treatment.The IC 50 values varied from 1.69 to 13.25 μΜ (Table 1).Among which, KYSE180 and KYSE450 were more sensitive to AS as evidenced by lower IC 50 values.We also found that AS dose-dependently inhibited the clonogenicity of KYSE180 cells (Figure 1C).The KYSE180 cells could hardly develop a colony when the AS concentration was greater than 1.2 μΜ (Figure 1D).
To confirm whether the AS-induced proliferation inhibition due to cell cycle arrest, the KYSE450 cells were exposed to AS and then the proportion of cell phases was analysed by flow cytometer.The results indicated that AS significantly blocked cell cycle at G1/S phase as evidenced by increasing proportion of G1/S phase (Figure 1E).
Interestingly, low dosages (<2 μΜ) of AS significantly exhibited S phase arrest.When AS concentration was more than 2 μΜ, the cell cycle was blocked at G1 phase in KYSE450 cells (Figure 1F).
Taken together, AS suppressed the viability and proliferation in ESCC cells and blocked cell cycle progression.AS may be a potent cytotoxic agent for ESCC treatment.

| AS-induced apoptosis in KYSE180 and KYSE450 cells
To determine whether AS-triggered apoptosis in ESCC cells, the KYSE180 and KYSE450 cells were incubated with AS for 24 h.The results demonstrated that AS-induced morphological characteristics of apoptosis in the two cell lines in a dose-dependent pattern (Figure 2A).We further examined the apoptotic rate by flow cytometry.AS sharply triggered apoptosis in KYSE180 and KYSE450 cells due to the increased proportion of FITC-positive cells (Figure 2B).The apoptotic rate reached 28.6% when treated with 6 μM AS in KYSE180 cells (Figure 2C).For KYSE450 cells, the apoptotic rate was 22.8% following 3 μM AS treatment (Figure 2D).
We further investigated the changes of apoptosis-related proteins by WB.We found the levels of cleaved PARP and caspase 3 were no difference when the AS was below 2 μM.However, cleaved PARP and caspase 3 were significantly increased when the AS was more than 4 μM.In addition, the levels of cleaved PARP and caspase 3 were dramatically increased when AS exposure was more than 12 h (Figure 2E,F).However, Bax levels were not increased by AS.These data indicated that AS dose-and time-dependently triggered apoptosis by a caspase-dependent manner in KYSE180 and KYSE450 cells.

| AS-triggered apoptosis by activating the ER stress pathway
It was reported that natural naphthoquinones triggered apoptosis through activation of the ER stress pathway in numerous cancers. 32,33To determine whether ER stress was induced by AS treatment, we examined several well-known proteins associated with ER stress following AS treatment.BIP, PDI and Ero1-Lα were time-dependently up-regulated with AS treatment in the two ESCC cell lines (Figure 3A).In addition, eIF 2α phosphorylation increased markedly at 6 h and maintained an elevated level up to 24 h; however, calnexin was significantly decreased.We also observed that CHOP, an ER stress-associated pro-apoptotic protein, exhibited the similar observation to eIF 2α phosphorylation.
5][36][37][38] To determine whether AS-triggered apoptosis in ESCC cells through the up-regulation of CHOP, we knocked down CHOP with siRNA and measured apoptosis in KYSE180 and KYSE450 cells.We found that AS-triggered apoptosis was markedly reduced in the absence of CHOP as evidenced by a reduction of FITC-positive cells (Figure 4A,C).The apoptotic rate decreased from 38.8% to 5.8% following treatment with 6 μM AS in KYSE180 cells (Figure 4B).

| AS delayed ESCC growth in nude mice
Since AS markedly suppressed the viability and proliferation of ESCC cells, we determined its potential to inhibit ESCC tumour growth.We successfully implanted KYSE180 cells into nude mice to establish a tumour xenograft model.After treatment of AS, we found AS dose-dependently inhibited the growth of KYSE180 xenografts as evidenced by the smaller tumour volume (Figure 6A,C).
Analysis of tumours indicated that AS dose-dependently decreased tumour weight and volume compared with the control (Figure 6D); however, the body weights decreased after 3 weeks (Figure 6B).
Further immunohistochemistry analysis confirmed that AS inhibited ESCC growth as proved by a dramatical reduction in Ki67-positive cells (Figure 6E).Furthermore, expression level of BIP and CHOP also increased in xenograft tumours, which indicated AS-induced ER stress in vivo (Figure 6E).

| DISCUSS ION
Natural small molecules derived from traditional Chinese medicine are valuable resources for the development of anti-tumour drugs because of their novel structure and diverse bioactivites.Lithospermum erythrorhizon, also called 'Zicao' is an herbal plant that has been used traditionally to treat inflammation, infection, burns and carbuncles in China. 42Recently, many studies have revealed that Zicao possesses multiple chemical ingredients and exhibits wound healing, anti-cancer and anti-inflammatory activities.3][44] AS is a derivative of shikonin, and its pharmacological effects, particularly its anti-cancer effect, has attracted significant attention 45,46 ; however, it is unclear whether AS exhibits a growth-inhibitory effect on ESCC.In the present study, The ER is the most important organelle that participate in the synthesis and folding of protein, stress-sensing and calcium storage. 47Cellular stress, including oxidative injury, hypoxia, calcium depletion and viral infection, impairs ER homeostasis, leading to the accumulation of misfolded and unfolded proteins, which activates URP and evokes ER stress. 48,49The UPR works through three signal sensors, PERK, ATF6 and IRE1α.They are normally inactive and interact with the chaperone GRP78/BIP. 50These molecules are activated by dissociation from BIP when ER stress occurs. 47tivated PERK selectively phosphorylates and inactivates eIF 2α , which causes global translation inhibition and improves the translation of ATF4 and CHOP. 34The active IRE1α splices an intron into XBP1 mRNA, converting it from an unspliced form to a spliced form.
The spliced XBP1 is translated into the XBP1 protein, which regulates the expression of many UPR-responsive genes. 51Generally, ER stress plays a cytoprotective role, but severe or sustained ER stress induces apoptosis through various pathways.Here, we found that AS-triggered sustained ER stress and activated PERK and IRE1α, resulting in eIF 2α phosphorylation and XBP1 splicing.In the absence of eIF 2α activity, the ATF4 expression was markedly up-regulated, which sequentially enhanced CHOP expression and induced apoptosis.CHOP knockdown markedly reduced apoptosis induced by AS.Therefore, AS may trigger apoptosis through the ER stress-activated PERK-eIF 2α /CHOP axis; however, AS also activates IRE1α and ATF3.3][54][55][56] We could not exclude whether IRE1α and ATF3 participate in apoptosis triggered by AS in ESCC cells which need further exploration.Furthermore, we also demonstrated that AS treatment significantly reduced tumour growth in nude mice and triggered ER stress.
In conclusion, our research indicated that AS inhibited ESCC cell proliferation and triggered apoptosis.AS effectively induced sustained ER stress and promoted apoptosis via the PERK/eIF 2α / CHOP cascade.We proved that AS might be a promising candidate for ESCC treatment.
Animal studies were approved by the animal ethics committee of Shandong First Medical University.Thirty athymic BALB/c nude mice were housed in SPF level of animal house with the 12-h light and 12-h dark cycle.100 μl suspension containing 5 × 10 6 cells of KYSE180 was implanted subcutaneously into the right flank of a mouse.All mice were randomly classified into three groups, the control group, low-dose group and high-dose group, until the tumour volume reached approximately 100 mm 3 .

F I G U R E 1 25 F
Acetylshikonin (AS) inhibited viability and proliferation of ESCC cells.(A) The chemical structure of AS. (B) AS decreased ESCC cells viability in a dose-dependent manner.The cell viability of six ESCC cell lines was estimated by CCK8 assay after treating with AS for 24 h.(C) AS inhibited clonogenicity of KYSE180 cells in a dose-dependent manner.(D) The numbers of colonies were shown in the statistical chart.*p < 0.05, **p < 0.01.Data were presented as mean ± SD of three independent experiments.(E) AS-induced cell cycle arrest in KYSE450 cells.(F) After AS treatment, KYSE450 cells exhibited S phase arrest at low concentrations and G1 phase arrest at medium to high concentrations.TA B L E 1 IC50 of acetylshikonin in six cell lines of ESCC.I G U R E 2 Acetylshikonin (AS) induced apoptosis in KYSE180 and KYSE450 cells.(A) The morphology of KYSE180 and KYSE450 cells changed with AS treatment.The magnification was 200× and the scale bar represented 50 μm.(B) AS dramatically increased Annexin V-positive cells in KYSE180 and KYSE450 cells.The cells were treated with indicated AS for 24 h, stained with Annexin V/PI and analysed by flow cytometry.The percentages of Annexin V-positive cells were shown.(C, D) The quantitative data of panel.The data were displayed as the mean ± SD (n = 3).*p < 0.05, **p < 0.01.(E, F) KYSE180 and KYSE450 cells were treated with different concentrations of AS for 24 h or treated with 4 μM of AS for different periods.The expression levels of apoptosis-related proteins were detected by WB.GAPDH was used as an internal control.
ing of the X-box-binding protein 1 (XBP1) in AS-treated cells.It was shown that the spliced XBP1 mRNA was dose-dependently up-regulated in responding to AS administration, which indicated that F I G U R E 3 Acetylshikonin (AS) induced apoptosis through activating the ER stress pathway.(A) KYSE180 and KYSE450 cells were treated with different concentrations of AS for 24 h or with the 4 μM of AS for different periods, and WB was performed to detect the expression levels of proteins related to ER stress pathway.GAPDH-normalized quantitative data are shown below the panel.(B) AS increased the shear of IRE1 to XBP1.(S: sheared XBP1 fragments; U: unsheared XBP1 fragments).(C) AS significantly increased the mRNA levels of ER stressrelated gene in a dose-dependent manner.F I G U R E 4 Acetylshikonin (AS) induced apoptosis through up-regulation of CHOP in KYSE180 and KYSE450 cells.(A) Knockdown of CHOP dramatically blocked AS-induced apoptosis in KYSE180 and KYSE450 cells, as tested by Annexin V and PI staining and analysed by flow cytometry.The percentages of Annexin V-positive cells were shown.(B) The quantitative data of panel.The data were displayed as the mean ± SD (n = 3).****p < 0.0001.(C) The efficacy of CHOP siRNA by real-time PCR.The data were analysed by graphpad prism 8 and displayed as the mean ± SD (n = 3).**p < 0.01, ****p < 0.0001.the IRE1/XBP1 axis was activated in KYSE180 and KYSE450 cells (Figure 3B).Next, we analysed gene expression levels by qRT-PCR in AS-treated cells.The results indicated that AS markedly induced BIP, CHOP, activating transcription factor 3 (ATF3) and activating transcription factor 4 (ATF4) at the transcriptional level.Meanwhile, the activating transcription factor 6 (ATF6) mRNA levels were only slightly up-regulated with AS administration (Figure 3C).Taken together, AS-induced sustained ER stress and activated PERK/eIF 2α and IRE1/XBP1 signalling.

F I G U R E 5
servations indicated that CHOP played critical role in AS-induced apoptosis.Activation of PERK/eIF 2α cascade is associated with CHOP expression in response to ER stress. 39-41To characterize the function of the PERK/eIF 2α axis in AS-triggered CHOP up-regulation, we pretreated the KYSE180 and KYSE450 cell lines with GSK2606414, a PERK inhibitor for 2 h to inhibit PERK activity, and assessed CHOP expression and apoptosis induced by AS treatment.The results demonstrated that GSK2606414 alone scarcely affected cell viability, whereas AS-induced CHOP was markedly decreased by GSK2606414 (Figure 5A,D).Consistently, cell viability was significantly restored and apoptosis decreased after GSK2606414 Acetylshikonin (AS) induced apoptosis through activated PERK/eIF 2α / CHOP axis in KYSE180 and KYSE450 cells.(A) The down-regulation of CHOP in the presence of GSK2606414 (PERKi), as tested by WB.GAPDH-normalized quantitative data are shown.(B) Inhibition of PERK dramatically blocked AS-induced apoptosis in KYSE180 and KYSE450 cells, as tested by Annexin V and PI staining and analysed by flow cytometry.The percentages of Annexin V-positive cells were shown.(C) The quantitative data of panel.The data were displayed as the mean ± SD (n = 3).****p < 0.0001.(D) The cell viability inhibited by AS was significantly restored in the presence of GSK2606414 (PERKi).treatment (Figure 5B-D).These data indicated that the AS-activated PERK/eIF 2α pathway up-regulated CHOP expression, leading to apoptosis in KYSE180 and KYSE450 cells.
we found that AS exerted anticancer activity in ESCC cells as proved by lower IC 50 values.AS not only effectively decreased cell viability and proliferation, but also inhibited cell cycle progression in ESCC cell lines.Furthermore, AS-induced apoptosis in ESCC cell lines.