ABSTRACT AIM: Physcion is a major bioactive ingredient in the traditional Chinese medicine Radix et Rhizoma Rhei, which has an anthraquinone chemical structure and exhibits a variety of

pharmacological activities including laxative, hepatoprotective, anti-inflammatory, anti-microbial and anti-proliferative effects. In this study we investigated the effect of physcion on

human nasopharyngeal carcinoma _in vitro_ and _in vivo_, as well as the mechanisms underlying the anti-tumor action. METHODS: The nasopharyngeal carcinoma cell line CNE2 was treated with

physcion, and cell viability was detected using MTT and colony formation assays. Flow cytometry was used to assess the cell cycle arrest, mitochondrial membrane potential loss, apoptosis,

(10, 20 mg·kg−1·d−1, ip) for 30 d. RESULTS: Treatment with physcion (5, 10, and 20 μmol/L) dose-dependently suppressed the cell viability and colony formation in CNE2 cells. Physcion (10 and

20 μmol/L) dose-dependently blocked cell cycle progression at G1 phase and induced both caspase-dependent apoptosis and autophagy in CNE2 cells. Furthermore, physcion treatment induced

excessive ROS generation in CNE2 cells, and subsequently disrupted the miR-27a/ZBTB10 axis, resulting in repression of the transcription factor Sp1 that was involved in physcion-induced

apoptosis and autophagy. Moreover, physcion-induced autophagy acted as a pro-apoptotic factor, and possibly contributed to physcion-induced apoptosis. In the xenograft murine model,

administration of physcion dose-dependently suppressed the tumor growth without affecting the body weight. Furthermore, the anti-tumor effects of physcion were correlated with downregulation

of Sp1 and suppression of miR-27a in the tumor tissues. CONCLUSION: Physcion induces apoptosis and autophagy in human nasopharyngeal carcinoma by targeting Sp1, which was mediated by

ROS/miR-27a/ZBTB10 signaling. The results suggest that physcion is a promising candidate for the treatment of human nasopharyngeal carcinoma. SIMILAR CONTENT BEING VIEWED BY OTHERS _HEDYOTIS

DIFFUSAE_ HERBA-_ANDROGRAPHIS_ HERBA INHIBITS THE CELLULAR PROLIFERATION OF NASOPHARYNGEAL CARCINOMA AND TRIGGERS DNA DAMAGE THROUGH ACTIVATION OF P53 AND P21 Article 09 November 2021

MIR-122-5P REGULATES ERASTIN-INDUCED FERROPTOSIS VIA CS IN NASOPHARYNGEAL CARCINOMA Article Open access 01 May 2024 INDUCTION OF FERROPTOSIS IN HUMAN NASOPHARYNGEAL CANCER CELLS BY

CUCURBITACIN B: MOLECULAR MECHANISM AND THERAPEUTIC POTENTIAL Article Open access 04 March 2021 INTRODUCTION Nasopharyngeal carcinoma (NPC), a malignancy particularly prevalent in southern

China, Taiwan, Southeast Asia, and North Africa, accounts for the majority of tumors originating in the nasopharynx1. It differs from other types of head and neck cancers because most NPC

patients present with adjacent regional invasion as well as neck lymph node metastasis at the time of diagnosis2. A number of genetic, environmental and microbial factors have been shown to

contribute to the development of NPC3. An increase in serum Epstein-Barr virus (EBV)-related antibodies as well as circulating and intratumoral EBV DNA were associated with NPC, suggesting a

role of EBV in the etiology and development of NPC4. In addition, a variety of chemicals widely distributed in herbal medicines and foods, including phorbol esters and n-butyrate, have been

shown to initiate the EBV lytic cycle and thus play a role in the carcinogenesis of NPC5. A recent study by Fang _et al_ reported that repeated contact with these chemicals results in

recurrent reactivation of EBV and alters cancer hallmark gene expression in NPC cells6. Consumption of food or herbs containing the above chemicals, smoking and consumption of salty food

have also been identified as moderate risk factors for NPC7,8. However, epidemiological studies also found that a number of foods, such as fresh vegetables, fruits, and green tea, were

correlated with a lower risk of NPC9. Further studies have shown that a few naturally occurring chemicals have anti-neoplastic effects against NPC9. However, the underlying mechanisms are

not clear. Transcription factors play crucial role in biological processes and carcinogenesis by regulating gene expression10. Sp1, a member of the zinc-finger Sp family of proteins, was one

of the first transcription factors to be identified in mammalian cells10. Sp1 is expressed ubiquitously in various mammalian cells and is implicated in the transcription of many genes that

contain GC boxes in their promoter11, particularly housekeeping genes and those involved in cell growth and development. Sp1 is overexpressed in a number of human epithelial tumors

originating from different tissues, including breast12, thyroid13, pancreas14, stomach15, and lung16, and plays a role in the regulation of cell growth17, angiogenesis (VEGF and VEGF

receptors)18, and metastasis19,20. Recently, Zhang _et al_ reported that higher levels of Sp1 were correlated with advanced tumor stage in NPC, and knockdown of Sp1 by siRNA inhibited

proliferation and led to cell cycle arrest in NPC cells21, indicating the potential of Sp1 as a therapeutic target in NPC treatment. Physcion has an anthraquinone chemical structure and is

one of the major bioactive ingredients in the traditional Chinese medicine Radix et Rhizoma Rhei22. Physcion has been reported to have a variety of pharmacological properties including

laxative, hepatoprotective, anti-inflammatory and anti-microbial activities22,23,24,25. Recently, physcion has been found to induce apoptosis26,27,28,29, block cell cycle arrest27, and

suppress metastasis30 in a variety of human cancer cells. In the present study, our results revealed that physcion suppressed the growth of NPC cells _in vitro_ and _in vivo_. Moreover, we

provided experimental evidence showing that physcion had anti-proliferative effects by modulating Sp1 via generation of ROS and regulating the miR-27a/ZBTB10 axis. MATERIALS AND METHODS CELL

CULTURE The nasopharyngeal carcinoma cell line CNE2 was obtained from Fuheng Biology Inc (Shanghai, China) and maintained at a density of 1×105 cells in DMEM medium (Gibco BRL Co, Ltd,

Gaithersburg, MD, USA) containing 10% FBS (HyClone, Logan, UT, USA), 100 IU/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich, St Louis, MO, USA). The cells were maintained in a CO2

incubator at 37 °C with 90% humidity and 5% CO2. Three times every week, the cells were passaged via trypsinization. Human dermal fibroblasts and primary skin fibroblasts were maintained in

FibroLife medium (Qingyuanhao Biotech Inc, Beijing, China). Immortalized human melanocyte PIG1 cells were maintained in Medium 254 (Gibco BRL Co, Ltd, Gaithersburg, MD, USA). CELL VIABILITY

ASSAY Cells were plated at a density of 5.0×103 cells/well in 96-well culture plates for 24 h before challenge by different concentrations of physcion for 24 and 48 h. The cell viability was

determined by using a CellTiter96® Aqueous One Cell Proliferation Assay Kit (Promega, Madison, WI, USA) according to the instructions of the manufacturer. The relative cell viability was

determined after normalization to untreated cells. COLONY FORMATION ASSAY CNE2 cells suspended in DMEM agarose medium containing physcion at different concentrations were seeded in each well

of a 6-well plate over a bottom layer of solidified DMEM agarose medium. Cultures were maintained for 14 d without fresh medium feeding at 37 °C in a humidified atmosphere of 95% air and 5%

CO2. Then, cell colonies with over 50 cells were enumerated and stained with crystal violet before being photographed using a digital camera (Nikon DXM1200, Tokyo, Japan). CELL CYCLE

ANALYSIS Cell cycle analysis was conducted following treatment with physcion using a Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China) according to the manufacturer's

instructions. Briefly, cells were collected and fixed with 70% cold ethanol at 4 °C overnight. DNA was stained with 0.05 mg/mL propidium iodide and 2.0 mg/mL RNase for 30 min at room

temperature. Cells were then subjected to FACScan flow cytometry (Beckman Coulter Inc, Fullerton, CA, USA) for cell cycle analysis. The percentage of cells in G1, S, and G2 phases of the

cell cycle was calculated using Cell Lab Quanta SC software (Beckman Coulter Inc, Fullerton, CA, USA). CELL APOPTOSIS ASSAY Cell apoptosis was assessed using a FITC Annexin V Apoptosis

Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, CNE2 cells were treated with various concentrations of physcion for

48 h, collected and washed with ice-cold PBS, and then suspended in 500 μL of annexin V binding buffer. Next, a 100 μL aliquot was taken, 2 μL of annexin V-FITC and 2 μL of PI were added,

and the mixture was incubated for 5 min at room temperature in the dark. After the addition of 400 μL of binding buffer, 1×104 cells were analyzed on a FACScan flow cytometer (Beckman

Coulter Inc, Fullerton, CA, USA) using CellQuest software. Annexin V-FITC positive cells were considered to be undergoing apoptosis, and those negative for FITC were considered to be alive.

TDT-MEDIATED DUTP-BIOTIN NICK-END LABELING (TUNEL) ASSAY TUNEL assays were conducted in CNE2 cells using a TUNEL Kit (Guava Technologies, Hayward, CA, USA) as previously described31.

Apoptosis was determined by flow cytometry, and the data were analyzed using Guava TUNEL Software. APOPTOSIS DETECTION BY MORPHOLOGICAL CHANGES USING HOECHST STAINING Hoechst 33258 staining

was also performed to detect apoptotic cells. Apoptosis was indicated by the presence of condensed or fragmented nuclei that bind Hoechst 33258 with high affinity. CNE2 cells were treated

with various concentrations of physcion for 48 h, washed with PBS, and fixed with pre-cooled methanol at 500 μL/well for 10 min. Then, the cells were stained with 1 μmol/L Hoechst 33258

(Sigma-Aldrich, MO, USA) for 10 min and analyzed on a Leica fluorescence microscope. Two hundred cells in three randomly selected fields were counted and scored for the incidence of

apoptotic chromatin. CASPASE-3, CASPASE-8, AND CASPASE-9 ACTIVITY ASSAYS After the CNE2 cells were treated with various concentrations of physcion for 48 h, the cytosolic proteins were

extracted in hypotonic cell lysis buffer and subjected to a Caspase Activity Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. The activity was determined

by measuring the absorbance at 405 nm using a microplate reader (Tecan Group Ltd, Männedorf, Switzerland). MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) ASSAY The changes in MMP were examined

using the fluorochrome dye JC-1 following a standard protocol. Briefly, CNE2 cells were challenged with the indicated dose of physcion for 48 h before the cells were harvested and incubated

with JC-1. The cells were then gently rinsed with PBS to remove excessive dye before the fluorescence signal was quantitatively analyzed by flow cytometry (Beckman Coulter Inc, Fullerton,

CA, USA). QUANTIFICATION OF AUTOPHAGIC CELLS BY FLOW CYTOMETRY The autophagic cells were detected and quantified by acridine orange (AO) staining as previously described32. Briefly, CNE2

cells were stained with AO (1 μg/mL) for 15 min following treatment with various concentrations of physcion. AO-stained cells were then rinsed with PBS, and the samples were observed under

an inverted microscope. Orange represents the acidic autophagic vacuoles, while bright green represents the cytoplasm and nucleus. Then, the fluorescence signal was detected by a FACScan

flow cytometer (Beckman Coulter Inc, Fullerton, CA, USA). In flow cytometric analysis of the AO-stained cells, the cytoplasm and nucleolus in non-autophagic cells showed green fluorescence

(500–550 nm, FL-1 channel), whereas AVO in autophagic cells (quadrant A1) showed bright red fluorescence (650 nm, FL-3 channel). Because the red fluorescence intensity indicates the number

of AVO in autophagic cells, autophagic cells can be quantified based on the intensity of red fluorescence. DETERMINATION OF MIRNA AND MRNA EXPRESSION For miRNA detection, total miRNA was

extracted using a commercial kit (Applied Biosystems, Carlsbad, CA, USA), and miR-27a was quantified by real-time PCR with a TaqMan Probe (Applied Biosystems, Carlsbad, CA, USA). For mRNA

detection, total RNA was extracted using a commercial kit (RNeasy Mini kit, Qiagen, Dusseldorf, Germany), and the primers for Sp1 and ZBTB10 were synthesized based on published sequence by

Sangon Biotech (Shanghai, China)33. First-strand cDNA was synthesized from 1 μg RNA using a Reverse Transcription System (TaKaRa, Dalian, China) according to the manufacturer's

instructions, and PCR was performed using SYBR Green Master Mix (Solarbio Co, Beijing, China). Gene expression was analyzed using U6 or GAPDH as an internal standard. SP1 OVEREXPRESSION IN

CNE2 CELLS The transfection of the Sp1-overexpressing plasmid was performed as previously described34. Briefly, CNE2 cells were seeded in 96-well culture plates in culture medium and grew to

70% confluence before transfection. The Sp1 overexpression plasmid was constructed by inserting a full-length cDNA fragment obtained with reverse transcription and PCR with a specific

primer and Sp1 primers into the pCMV vector (Beyotime, Shanghai, China). Then, the resulting pCMV-Sp1 vector was cloned into CNE2 cells to induce exogenous Sp1 expression. CNE2 cells

transfected with an empty pCMV vector were used as the controls. Stable Sp1-overexpressing clones were selected 48 h after transfection. KNOCKDOWN OF SP1 USING SIRNA The siRNA oligos for Sp1

gene knockdown were purchased from Cell Signaling (Boston, MA, USA), and a scramble sequence (RiboBio, Guangzhou, China) was used as a control. The CNE2 cells in logarithmic growth phase

were seeded in 6-well plates at a density of 3×105 cells per well, incubated overnight and then transfected with siRNA or scramble siRNA using Lipofectamine 2000 transfection reagent

(Invitrogen, Grand Island, NY, USA) according to the manufacturer's protocol. Transfected cells were incubated for another 48 h, and the knockdown was verified by Western blot analysis.

MEASUREMENT OF REACTIVE OXYGEN SPECIES (ROS) The cellular ROS level was determined using flow cytometry. Briefly, after CNE2 cells were treated with different concentrations of physcion for

48 h, cells were further incubated with 10 mmol/L DCFHDA at 37 °C for 1 h. Subsequently, the cells were harvested, rinsed and resuspended in PBS before the fluorescence (FL1 channel) was

analyzed by flow cytometry (Beckman Coulter Inc, Fullerton, CA, USA) using an excitation wavelength of 480 nm and an emission wavelength of 525 nm. MIR-27A TRANSFECTION The lentiviral

constructs miR-27a mimic and miR-con and a miR-27a inhibitor were obtained from Qiagen (Dusseldorf, Germany). CNE2 cells were seeded in each well of a 96-well plate, incubated overnight, and

then transfected with either the miR-27a mimic, miR-27a inhibitor, or control miR-con according to the manufacturer's instructions (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA).

The transfection efficiency was confirmed by qPCR analysis. WESTERN BLOTS Western blot analysis was performed following standard protocols. Briefly, following incubation with various

concentrations of physcion for 48 h, CNE2 cells were harvested from flasks and lysed with ice-cold lysis buffer (Beyotime, Shanghai, China) for 30 min on ice. Then, the collected proteins

were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, MA, USA). Proteins were probed with specific primary antibodies, and a rabbit polyclonal antibody to β-actin was

used as a gel loading control. Specific primary antibodies against cyclin D1, p21, p27, β-actin, AIF, PARP, caspase-3, COX IV, and LC3B were purchased from Beyotime (Shanghai, China), and

antibodies against cyclin E, DR4, DR5, TRAIL, Fas, Sp1, Endo G, and ZBTB10 were purchased from Abcam (Shanghai, China). After another wash with TBST, secondary antibody (HRP-conjugated

secondary antibody, Boster, Wuhan, China) was added for blot detection. Signals were detected using chemiluminescent substrate (KP, Guildford, UK), and the blot intensity was quantified

using BandScan software (Glyko, Novato, CA, USA). HUMAN NASOPHARYNGEAL CARCINOMA XENOGRAFT MODEL The xenograft experiments were conducted in accordance with CAPN (China Animal Protection

Law), and the protocols were approved by the Medical Ethics Committee of Qingdao University. Tumors were inspected every 3 d. Once tumor volume reached 100 mm3, mice (obtained from the

Medical Animal Center, Qingdao University) were randomly assigned to 3 groups (10 mice each group) as follows: (A) vehicle (0.9% sodium chloride plus 1% DMSO), (B) physcion (20 mg·kg−1·d−1,

dissolved in vehicle), and (C) physcion (10 mg·kg−1·d−1, dissolved in vehicle). Physcion was administered via intraperitoneal (ip) injections. Tumor volumes and body weight were measured

every 3 d. Tumor volume was calculated as volume = (length×width2), where width was the shortest measurement. All mice were euthanized 30 d after the first day of treatment. Tumor histology

examination was performed by staining the formalin-fixed, paraffin-embedded tumor sections with hematoxylin and eosin. The protein levels and miR-27a levels in tumor tissues were determined

by Western blots and RT-PCR analysis, respectively. STATISTICAL ANALYSIS The data are presented as the mean±SD (standard deviation) and represent the results of three separate experiments

conducted in quadruplicate unless otherwise stated. All statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc, La Jolla, CA, USA). Values are presented as

the mean±SD. Statistical comparisons were performed by one-way ANOVA followed by Dunnett's _t_-test. Differences with a _P_ value less than 0.05 were defined as statistically

significant. RESULTS PHYSCION INHIBITS THE PROLIFERATION OF NPC CELLS To explore the anti-proliferative effect of physcion, CNE2 cells were incubated with an increasing concentration of

physcion for 24 and 48 h. At a concentration of 5 μmol/L of physcion, no change in cell viability was observed, regardless of the treatment period (24 and 48 h). For the 10 μmol/L physcion

treatment, a slight reduction in viability was observed at 24 h, and a significant decrease in viability was found at 48 h (Figure 1A). When the concentration increased to 20 μmol/L, the

anti-proliferative effect of physcion increased. Compared to the untreated control cells, the viability of CNE2 cells treated with 20 μmol/L physcion was significantly reduced to 65% and 45%

at 24 and 48 h, respectively. To further demonstrate the effect of physcion on cell growth, a colony formation assay was also performed. The results from colony formation assays showed that

physcion treatment resulted in a reduction in colony number and size (Figure 1B). These results indicate that physcion can reduce the viability of CNE2 cells in a time- and dose-dependent

manner. In contrast, physcion treatment for 48 h did not significantly decrease the viability of control proliferating cells, including human dermal fibroblasts (HDF), immortalized human

melanocyte PIG1 cells and primary human skin fibroblasts (FB) (Figure 1A), indicating that physcion selectively exerts cytotoxic effect in cancerous cells. PHYSCION BLOCKS CELL CYCLE

PROGRESSION To determine the involvement of cell cycle arrest in physcion-mediated inhibition of cell growth, we examined the cell cycle distribution of CNE2 cells following physcion

treatment for 48 h. As shown in Figure 1C, our results indicated that physcion treatment results in the accumulation of cells in the G1 phase with a corresponding decrease of the cell

population in the S phase and G2 phase. Cyclins promotes S phase, while p21 and p27 arrest cells at G1 phase35. Therefore, the level of cyclin D1, cyclin E, p21, and p27 was examined to

determine whether physcion affects the expression of cell cycle-regulated molecules. As shown in Figure 1D, both cyclin D1 and cyclin E levels in CNE2 cells were substantially downregulated

by physcion in a dose-dependent manner. In contrast, the expression levels of p21 and p27 were markedly elevated following physcion treatment. Collectively, these results suggested that

physcion induces G1 arrest by modulating key molecules in cell cycle regulation. PHYSCION INDUCES CASPASE-DEPENDENT APOPTOSIS As shown in Figure 2A, flow cytometric analysis showed that

physcion treatment strongly induced apoptosis in CNE2 cells in a dose-dependent manner. The apoptosis-inducing effect of physcion was also demonstrated by TUNEL assays and Hoechst staining,

as shown in Figure 2B and 2C, respectively, which also demonstrated an increase in apoptotic cell death in a dose-dependent manner. Because apoptosis in cells can occur via caspase-dependent

and caspase-independent pathways, which are mediated by different factors, we investigated the effect of physcion on the expression or activity of key mediators in both apoptosis pathways.

As shown in Figure 2D, the release of AIF and Endo G, two crucial mediators in caspase-independent apoptosis pathway, from the mitochondria to the cytosol was not markedly altered by

physcion, regardless of the concentration, ruling out the involvement of caspase-independent pathways in physcion-induced apoptosis. In contrast, the activity of caspase-3, which is an

indicator of the caspase-dependent apoptotic pathway, was significantly increased by physcion treatment (Figure 2E). We also performed Western blot assays to confirm the activation of

caspase-3. As shown in Figure 2F, physcion significantly activated caspase-3 and promoted the cleavage of PARP, confirming that physcion induces apoptosis in CNE2 cells in a

caspase-dependent manner. Caspase-dependent apoptosis can be triggered by extrinsic stimuli through cell surface death receptors or intrinsic stimuli through mitochondrial signaling. The

mitochondria-mediated intrinsic apoptosis pathway is characterized by loss of mitochondrial membrane potential (MMP), release of cytochrome _c_ from the mitochondria to the cytosol and

activation of caspase-9. As shown in Figure 3A, 3B, and 3C, physcion treatment resulted in a significant decrease in MMP and the mitochondrial level of cytochrome _c_ as well as an increase

in the cytosolic level of cytochrome _c_ and caspase-9 activity. However, we examined the effect of physcion on the expression of major factors in the extrinsic apoptosis pathway, including

DR4, DR5, TRAIL and Fas, and the activity of caspase-8. Western blot analysis showed that physcion caused a significant increase in TRAIL, Fas and DR5 expression but only a slight elevation

in DR4 levels (not significant) (Figure 3D). In addition, significant activation of caspase-8 by physcion was also observed (Figure 3B). Taken together, our results shown that physcion

induced apoptosis in CNE2 cells via both extrinsic and intrinsic pathways. PHYSCION INDUCES AUTOPHAGY IN CNE2 CELLS Next, we investigated whether physcion induced autophagy in CNE2 cells.

Autophagy is characterized by the formation of the acidic vesicular organelles, which was confirmed by AO staining (Figure 4A). Based on the flow cytometric analysis, approximately 6% and

13% autophagic populations were produced by 10 and 20 μmol/L physcion, respectively (Figure 4B). In addition, we conducted Western blot analyses to examine the level of LC3 protein, a

prominent autophagy marker. As shown in Figure 4C, physcion at both 10 and 20 μmol/L significantly increased the level of LC3B-II, indicating that physcion induced autophagy in CNE2 cells.

For further confirmation of autophagy induction by physcion, the autophagic flux was also monitored. It is well established that P62 is efficiently degraded by autophagy, and autophagic flux

induction correlates with decreased P62 level36. In our study, we also found that physcion treatment decreased the level of P62 in a dose-dependent manner (Figure 4C). Increased conversion

of LC3B-II in combination with decreased P62 level suggested that physcion treatment resulted in autophagic activation. SUPPRESSION OF SP1 IS INVOLVED IN PHYSCION-INDUCED APOPTOSIS AND

AUTOPHAGY The role of Sp1 in apoptosis has been well documented in a number of cancer cells, including nasopharyngeal carcinoma37,38. Interestingly, a recent study by Yang _et al_ found that

downregulated expression of Sp1 was associated with induction of autophagy in the human myeloid leukemia cell line K-56239. Therefore, we investigated whether physcion induced apoptosis and

autophagy in CNE2 cells by modulating Sp1. Our results revealed that physcion repressed both the mRNA and protein expression of Sp1 in a dose-dependent manner (Figure 5A and 5B). To verify

the role of Sp1 in apoptosis and autophagy regulation in CNE2 cells, Sp1 was knocked down using siRNA targeting Sp1 (Supplementary Figure 1). As shown in Figure 5C and 5D, knockdown of Sp1

in CNE2 cells resulted in an increase in both the apoptotic and autophagic cell populations, suggesting that downregulation of Sp1 in cells promote both apoptosis and autophagy. Then, a

Sp1-overexpressing plasmid was utilized to assess the involvement of Sp1 in the apoptosis- and autophagy-inducing effect of physcion (Supplementary Figure 2). Our results showed that Sp1

overexpression in CNE2 cells significantly abrogated the physcion-induced apoptosis and autophagy, indicating that physcion promoted apoptosis and autophagy, at least partially, by

modulating Sp1. PHYSCION MODULATES SP1 BY INDUCING ROS GENERATION AND THUS REGULATING THE MIR-27A/ZBTB10 AXIS A number of studies have revealed that Sp1 expression can be suppressed in tumor

cells by excessive ROS40. Therefore, we examined whether ROS induction was involved in the physcion-induced suppression of Sp1 expression in CNE2 cells. As shown in Figure 6A, physcion

treatment led to a significant increase in intracellular ROS levels. The physcion-mediated repression of Sp1 expression was further confirmed using the ROS activator CoCl2 and the inhibitor

NAC. As shown in Figure 6B and 6C, CoCl2, which served as a positive control, resulted in significant repression in Sp1 at both the mRNA and protein levels. In contrast, a combination of

physcion and NAC almost completely abolished the physcion-induced repression of Sp1 expression. Our findings suggested that physcion modulated Sp1 expression by generating ROS. A number of

studies have shown that downregulation of miR-27a and subsequent induction of the miR-regulated “Sp repressor” ZBTB10P mediate ROS-dependent downregulation of Sp1 in tumor cells40.

Therefore, we examined the effects of physcion on the miR-27a/ZBTB10 axis to elucidate the mechanism by which physcion modulates the expression of Sp1. As shown in Figure 6C and 6D, physcion

treatment substantially increased the expression of ZBTB10 and significantly decreased the cellular level of miR-27a in a dose-dependent manner. To confirm the role of miR-27a/ZBTB10

signaling in regulation of Sp1 expression, a miR-27a inhibitor and mimic were utilized (Supplementary Figure 3). As shown in Figure 6E, the miR-27a inhibitor significantly increased the

expression of ZBTB10 and thus reduced the expression of Sp1, indicating the role of miR-27a/ZBTB10 signaling in the regulation of Sp1 expression. Additionally, regulation of Sp1 by physcion

through miR-27a/ZBTB10 signaling was also confirmed using miR-27a mimics that decreased ZBTB10 expression, which abrogated the effect of physcion on Sp1 (Figure 6E). Moreover, the link

between ROS generation and miR-27a/ZBTB10 was also demonstrated in this study (Figure 6F and 6G). Collectively, our results indicated that physcion induces excessive ROS generation and

disrupts the miR-27a/ZBTB10 axis, resulting in repression of Sp1. PHYSCION-INDUCED ROS GENERATION AND REGULATION OF MIR-27A/ZBTB10 ARE ALSO INVOLVED IN APOPTOSIS AND AUTOPHAGY Based on the

results described above, we performed experiments to investigate the involvement of the regulatory factors of Sp1 in physcion-induced apoptosis and autophagy. Corresponding to the modulatory

effect of these factors on Sp1, the physcion-mediated induction of apoptosis and autophagy was significantly abolished by transfection with miR-27a mimics and NAC (Figure 7A and 7B). Our

results also showed that the miR-27a inhibitor and CoCl2 induced significant apoptosis and autophagy in CNE2 cells, indicating the role of miR-27a and ROS in the regulation of apoptosis and

autophagy. CROSSTALK BETWEEN PHYSCION-INDUCED APOPTOSIS AND AUTOPHAGY In the following experiments, an inhibitor of autophagy as well as apoptosis was used to investigate the role of

autophagy in the anti-tumor effects of physcion. As shown in Figure 8A, inhibition of autophagy with 3-MA (autophagy inhibitor) significantly attenuated the physcion-induced decrease in cell

viability (3-MA itself has no marked effect on cell viability), indicating that physcion-induced autophagy contributed to the anti-proliferative effects and served as a form of cell death.

Mounting evidence has indicated that agents that can trigger ROS generation mediate crosstalk between autophagy and apoptosis41,42. In this study, our results showed that Z-VAD-FMK (caspase

inhibitor) itself slightly increased autophagic cell death (not significant), whereas a combination of physcion and Z-VAD-FMK significantly increased the proportion of autophagic cells

relative to physcion as single agent, as demonstrated by the flow cytometric results and the change in LC3B-II level, indicating that apoptotic cell death was partially transformed into

autophagic cell death in the case of apoptosis inhibition. Then, the effect of autophagy inhibition on physcion-induced apoptosis was explored. As demonstrated by the flow cytometric results

and the change in caspase-3 activity, autophagy inhibition significantly enhanced physcion-induced apoptosis in CNE2 cells, suggesting that autophagy acts as a pro-apoptotic factor here.

PHYSCION SUPPRESSES TUMOR GROWTH _IN VIVO_ IN A XENOGRAFT MURINE MODEL A xenograft murine model was used to evaluate the _in vivo_ anti-tumor effects of physcion in nasopharyngeal carcinoma.

As shown in Figure 9A, physcion significantly suppressed tumor growth in a dose-dependent manner without markedly affecting the body weight of the animals. Histological examination of the

tumor tissues also revealed a larger number of dead tumor cells in the physcion-treated group relative to the controls (Figure 9B). Consistent with the tumor growth results, Western blot

assays showed that physcion treatment induced apoptotic and autophagic cell death _in vivo_, as demonstrated by the elevated level of cleaved caspase-3 and LC3B-II, respectively (Figure 9C).

Moreover, our results also showed that inhibition of tumor growth by physcion correlated with decreased expression of Sp1 and miR-27a in tissues, indicating that physcion inhibits tumor

growth by inducing apoptosis and autophagy via modulation of Sp1 and miR-27a signaling. DISCUSSION As early as 2005, the anti-tumor effect of physcion was noted in colon cancer and leukemia

cells43. However, the anti-tumor mechanisms of physcion were recently reported in 2014 by two research groups that found that physcion blocked cell cycle progression and induced apoptosis in

human breast cancer cells and cervical carcinoma cells, respectively27,28. Recently, studies by Han's group have demonstrated that physcion not only acts as an apoptosis-inducing agent

but also potentially suppress the metastasis of colorectal cancer29,30,43. Moreover, the modulatory effect of physcion on the metabolic function of tumor cells was also reported in a recent

study, which showed that physcion suppressed cancer cell proliferation and tumor growth in nude mouse xenografts by inhibiting 6-phosphogluconate dehydrogenase44. In the present study, we

provide the first evidence for the autophagy-inducing ability of physcion. The results presented here clearly demonstrated that physcion is effective at inducing apoptotic and autophagic

cell death in the nasopharyngeal carcinoma cell line CNE2. Moreover, our findings also suggest that physcion induces apoptosis and autophagy in nasopharyngeal carcinoma by targeting Sp1,

which was mediated by ROS/miR-27a/ZBTB10 signaling. Apoptosis, defined as type-I programmed cell death (PCD), is considered a major route by which chemotherapeutic agents eradicate cancer

cells30. Two distinct pathways, caspase-independent and caspase-dependent pathways, have been proposed to account for the apoptotic cell death. AIF promotes chromatin condensation and

fragmentation of the nucleus after translocation from the intermembrane space of the mitochondria to the nucleus, mediating caspase-independent apoptosis45. In addition, Endo G, a

pro-apoptotic protein located in the intermembrane space of the mitochondria, can also mediate caspase-independent apoptosis by directly digesting nuclear DNA following entrance into the

nucleus46. Therefore, AIF and Endo G are considered markers of the caspase-independent apoptotic pathway. In the current study, our results showed that physcion did not cause a detectable

release of AIF or Endo G from the mitochondria to the cytosol, ruling out the involvement of the caspase-independent pathway in physcion-induced apoptosis in CNE2 cells. By examining the

activation of caspase-3 and PARP, we confirmed that physcion induced apoptosis in a caspase-dependent manner. Caspase-dependent apoptosis can occur via the intrinsic mitochondrial pathway

and the extrinsic death receptor pathway47. Consistent with previous _in vitro_ studies in breast cancer cells27, our results showed that physcion induced apoptosis in CNE2 cells via both

the intrinsic and extrinsic pathways. However, an early study by Wijesekara _et al_28 and a recent study by Chen _et al_29 showed that physcion induced apoptosis in cervical carcinoma and

colorectal cancer cells, respectively, only through the intrinsic mitochondrial pathway. Taken together, these findings suggest that the mechanism by which physcion induces apoptosis might

be cell specific and related to activation of distinct signaling elicited by physcion. Autophagy in normal cells is a fundamental process for maintaining homeostasis and housekeeping

functions through lysosomal degradation of cytoplasmic organelles or cytosolic components. Autophagy comprises a series of sequential phases, including initiation, elongation, autophagosome

maturation and autophagosome-lysosome fusion, and is initiated by sequestering cytosolic proteins or organelles into autophagosomes that then fuse with lysosomes to form autolysosomes for

the degradation of the sequestered contents by lysosomal hydrolases48. In the context of cancer cells, autophagy has been documented in response to various metabolic stress and anticancer

agents, but the role of autophagy in cancer remains controversial49. It has been found that autophagy can serve as a survival pathway in cancer cells50. In contrast, autophagic cell death,

defined as Type II PCD, which is presumed to result from excess autophagy, acts as a route for cancer cell elimination51. Here, we found that the physcion-induced reduction in cell viability

was partially abolished by an autophagy inhibitor, 3-MA, indicating that physcion-induced autophagy acts as contributing factor to cell death. Mounting evidence has shown that autophagy and

apoptosis are important cellular processes that crosstalk with each other42. For example, autophagy can promote apoptosis by degrading inhibitor of apoptosis proteins (IAPs) or

Fas-associated phosphates 1 (Fap 1)52. However, the cross inhibition between autophagy and apoptosis has also been noted53. In an early report in a nasopharyngeal carcinoma cell model, Chow

_et al_ found that wogonin-induced autophagy interfered with the process of apoptosis54. However, our results showed that inhibition of apoptosis enhanced physcion-induced autophagy while

inhibition of autophagy impaired physcion-induced apoptosis, suggesting that physcion-induced autophagy acts as pro-apoptotic factor, and CNE2 cells tend to die via autophagy following

apoptosis inhibition in response to physcion challenge. These mixed results suggest that the complex interplay between autophagy and apoptosis might occur in a stimulus-specific manner even

in the same tumor cells, which needs to be clarified with further studies. The role of Sp1 in cancer has been extensively explored16. Sp1 is overexpressed in a number of human malignancies

and can promote cancer development by modulating the expression of pro-oncogenic genes important for cell growth (cyclin D1, EGFR, and c-Met)17, survival (bcl-2 and survivin)17, angiogenesis

(VEGF and VEGF receptors)18, and metastasis19,20. However, Sp1 has also been shown to act as a tumor suppressor. High levels of Sp1 in highly invasive lung adenocarcinoma cells were

associated with increased E-cadherin expression and metastasis inhibition55. In addition, Chuang _et al_ found that Sp1 cooperates with p53 to induce apoptosis in a variety of cancer cells,

including lung adenocarcinoma, cervical adenocarcinoma, breast carcinoma, colon carcinoma and glioma cells56. Recently, Zhang _et al_ reported that aberrant overexpression of Sp1 was

observed in advanced tumor stages in NPC, and knockdown of Sp1 by siRNA inhibited proliferation and led to cell cycle arrest in NPC cells21, suggesting the potential of Sp1 as a therapeutic

target in NPC treatment. Consistent with Zhang's report, our results from both _in vitro_ and _in vivo_ experiments also showed that the anti-tumor effect of physcion is associated with

downregulation of Sp1, supporting the role of Sp1 as an oncogene in nasopharyngeal carcinoma. MicroRNAs (miRNAs), a group of endogenous small non-coding RNAs (20–24 nucleotides), modulate

the biological function of tumor cells through negative regulation of the stability or translational efficiency of their target mRNAs57. Accumulating evidence has demonstrated that miRNAs

play crucial roles in the regulation of apoptotic and autophagic pathways58, including miR-27a. The involvement of miR-27a in apoptosis has been reported in a variety of human

malignancies59,60. In the context of nasopharyngeal carcinoma, upregulated miR-27a was found in patients and correlated with advanced TNM stage61. Consistent with these findings, we found

that delayed tumor growth by physcion was associated with downregulation of miR-27a. In addition, the present study provided the first experimental evidence for the involvement of miR-27a in

autophagic cell death of tumor cells. Collectively, these results highlighted that potential of miR-27a as a therapeutic target for nasopharyngeal carcinoma. In conclusion, our study is the

first report demonstrating that physcion has anti-cancer effects in nasopharyngeal carcinoma _in vitro_ and _in vivo_ by blocking cell cycle progression as well as inducing apoptosis and

autophagy. Moreover, our results showed that physcion promotes both apoptotic and autophagic cell death by modulating Sp1 via generation of ROS and subsequently inhibiting miR-27a signaling.

Considering the crucial role of mitochondria in ROS generation and the lipophilic chemical structure of physcion, which allows diffusion of physcion across the mitochondrial membrane, it is

highly possible that physcion predominantly targets the mitochondria to activate the anti-tumor cascade. However, further studies need to be performed to assess this hypothesis. Moreover,

the compelling results of this report warrant further study to evaluate the therapeutic value of physcion as a potential anti-neoplastic agent. AUTHOR CONTRIBUTION Ming-jie PANG and Zhun

YANG initiated the project and participated in the design of the study; Zhun YANG, Xing-lin ZHANG, Zhao-fang LIU, Jun FAN, and Hong-ying ZHANG contributed to the study design and performed

the experiments; Ming-jie PANG and Zhun YANG wrote the paper. All authors contributed to the data analysis. CHANGE HISTORY * _ 03 DECEMBER 2024 This article has been retracted. Please see

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carcinoma with deep sequencing. _J Exp Clin Cancer Res_ 2016; 35: 17. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This study is supported by

the Fund project of the Health Department of Shandong province, Study on Gene Therapy for Head and Neck Cancer Patients after Surgery (No 2005-jz006). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Otolaryngology, Qingdao Municipal Hospital, 266011, Qingdao, China Ming-jie Pang, Zhun Yang, Zhao-fang Liu & Jun Fan * Department of Oncology, Qingdao

Municipal Hospital, 266011, Qingdao, China Xing-lin Zhang * Department of Dermatology, Qingdao Municipal Hospital, 266011, Qingdao, China Hong-ying Zhang Authors * Ming-jie Pang View author

publications You can also search for this author inPubMed Google Scholar * Zhun Yang View author publications You can also search for this author inPubMed Google Scholar * Xing-lin Zhang

View author publications You can also search for this author inPubMed Google Scholar * Zhao-fang Liu View author publications You can also search for this author inPubMed Google Scholar *

Jun Fan View author publications You can also search for this author inPubMed Google Scholar * Hong-ying Zhang View author publications You can also search for this author inPubMed Google

Scholar CORRESPONDING AUTHOR Correspondence to Zhun Yang. ADDITIONAL INFORMATION Supplementary information is available at Acta Pharmacologica Sinica's website. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY FIGURE S1 Knockdown of Sp1 with siRNA in CNE2 cells. **P<0.01 vs. control. (JPG 160 kb) SUPPLEMENTARY FIGURE S2 Overexpression of Sp1 in CNE2 cells. **P<0.01

vs. control. (JPG 153 kb) SUPPLEMENTARY FIGURE S3 Effect of miR-27 mimic and inhibitor on miR-27 a expression. **P<0.01 vs. control. (JPG 135 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR

FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT SLIDE FOR FIG. 5 POWERPOINT SLIDE FOR FIG. 6 POWERPOINT SLIDE FOR FIG. 7 POWERPOINT

SLIDE FOR FIG. 8 POWERPOINT SLIDE FOR FIG. 9 POWERPOINT SLIDE FOR FIG. 10 ABOUT THIS ARTICLE CITE THIS ARTICLE Pang, Mj., Yang, Z., Zhang, Xl. _et al._ RETRACTED ARTICLE: Physcion, a

naturally occurring anthraquinone derivative, induces apoptosis and autophagy in human nasopharyngeal carcinoma. _Acta Pharmacol Sin_ 37, 1623–1640 (2016).

https://doi.org/10.1038/aps.2016.98 Download citation * Received: 11 April 2016 * Accepted: 24 July 2016 * Published: 03 October 2016 * Issue Date: December 2016 * DOI:

https://doi.org/10.1038/aps.2016.98 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently

available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * physcion * human nasopharyngeal carcinoma * CNE2 cells *

apoptosis * autophagy * Sp1 * miR-27a