Epimedokoreanin C, A Prenylated Flavonoid Isolated from Epimedium Koreanum, Induces Non-Apoptotic Cell Death with the Characteristics of Methuosis in Lung Cancer Cells

Background: Methuosis is a novel type of non-apoptotic cell death characterized by accumulation of cytoplasmic vacuoles. Identi�cation of molecules that induce methuosis may provide alternative therapeutics for cancers that are refractory to apoptosis. Epimedokoreanin C (EKC) is a prenylated �avonoid isolated from a Chinese herb Epimedium koreanum. Methods: In this study, MTT assay, real-time cell analysis monitoring of cell growth and wound-healing assay were used for cell viability and migration evaluation. Flow cytometry was performed to assess apoptosis. Immunoblot analysis, immuno�uorescence, pull-down assays and live cell imaging with �uorescent tracers were utilized to evaluate the regulation of signaling pathways. Results: EKC reduced cell viability accompanied by extreme vacuolation in lung cancer NCI-H292 cells. The EKC-induced cell death was clari�ed as non-apoptosis based on the absence of apoptotic changes. The vacuoles stimulated by EKC were supposed to be derived from macropinocytosis based on the engulfment of extracellular �uid tracer, Lucifer Yellow. The vacuoles acquired some characteristics of late endosomes supported that EKC-induced cell death could be described as methuosis. Rac1 and Arf6 were found to be regulated inversely after EKC treatment. Blocking Rac1 activation with the speci�c Rac1 inhibitor EHT 1864 prevented the accumulation of vacuoles induced by EKC markedly, suggested that the regulation of Rac1 and Arf6 was at least partial mechanism involved in EKC induced methuosis. EKC synergized the effects of doxorubicin and etoposide, demonstrating the effectiveness of using EKC to synergize conventional chemotherapy. Conclusions: Collectively, EKC was demonstrated as a methuosis-like cell death inducer in NCI-H292 cells. It has the potential to be used as an attractive prototype for developing drugs that could kill apoptosis-resistant cancer cells.


Background
Despite the fact that remarkable progress has been achieved in the understanding of cancer therapies over the past several decades, cancer remains the second leading cause of death in the world [1].Surgery, radiotherapy and chemotherapy are the most widely used treatments for cancers [2].Chemotherapy is the only option for treatment of metastatic cancers [3].Although many new chemotherapeutic drugs especially molecular-targeted approaches have been developed during the past decades, drug resistance remains a major obstacle in cancer treatments [4].Apoptosis is the most recognized form of programmed cell death.As most conventional chemotherapeutic agents work by triggering apoptotic cell death [5], the discovery of alternative agents targeting nonapoptotic cell death pathway might be used as a new strategy to kill cancer cells that are resistant to apoptosis.
On the basis of distinguished morphological and molecular characteristics, several non-apoptotic forms of cell death have been identi ed.Cytoplasmic vacuoles are morphological features of several nonapoptotic cell deaths including autophagy, necroptosis and paraptosis [6][7][8].Methuosis is a newly de ned form of non-apoptotic cell death characterized by the accumulation of massive vacuoles in cytoplasm.These vacuoles are originated from macropinosomes and endosomes showing impaired recycling and tra cking [9,10].As a novel nonapoptotic mode of cell death, methuosis has attracted particular attention as a target for cancer treatments.Several methuosis inducers have been reported, for example, indole-based chalcones MIPP and MOMIPP triggered cell death by methuosis in glioblastoma [10,11]; an ursolic acid derivative led cell death via methuosis [12]; a 4,6-disubstituted Aza-indole compound 13 induced methuosis and displayed cytotoxicities against a panel of cancer cells [13]; a quinoline derivative vacquinol also caused methuosis in glioblastoma [14] and CX-4945 induced methuosis in cholangiocarcinoma cells [15].These reported results con rmed that the identi cation of methuosis inducers could provide both probes for studying the speci c molecular mechanism of the nonconventional cell death pathway, and promising therapeutic agents for the treatment of cancers.
Isoprenylated avonoids represented a subgroup of natural avonoids possessing at least one prenyl in their structure.Increasing evidences suggested that prenylation was a determinant factor for the enhancement of cytotoxicity of avonoids.This made prenylated avonoids as attractive compounds for obtaining anti-cancer leading structures [16,17].In the course of screening for bioactive anticancer molecules, epimedokoreanin C (EKC), a prenylated avonoid obtained from Epimedium koreanum, caught our attention because of the induction of extreme cytoplasmic vacuolization in NCI-H292 cells.In the present study, we reported that EKC induced cell death with the speci c characteristics of methuosis in NCI-H292 cells.We also discussed the regulation of Rac1, Arf6 and PIKfyve expression.This compound may be considered as a prototype for a class of therapeutic agents that could be used to treat tumors resistant to conventional apoptosis-inducing agents.

Cell culture
Human lung cancer NCI-H292 cells, bronchial epithelial 16HBE cells were purchased from Shanghai Cell Bank (Shanghai, China), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Grand Island, NY, USA) and Dulbecco's Modi ed Eagle Medium (DMEM) respectively.All culture media were supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin.All cells were maintained at 37 °C in a humidi ed incubator containing 5% CO 2 .

Chemicals and reagents
Epimedokoreanin C and its analogues, epicornunins A, B, F and epimedokoreanin B, together with other 22 prenylated avonoids, were puri ed by our laboratory from the leaves of Epimedium koreanum [37].The puri cation procedure and the identi cation of EKC were described (Additional le 1).

Antibodies
Details of the primary antibody and dilutions used for these studies are listed in Table 1.Alexa Fluor 594 and 488 anti-rabbit IgG antibodies were from Proteintech Group (Wuhan, China).GAPDH and β-actin were purchased from Santa Cruz Biotechnology.

MTT assay
Cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight.The cells were then treated with DMSO or EKC for different times, after treatment, 20 µl of 2.0 mg/ml MTT solution was added in each well and incubated at 37 °C for an additional 4 hr.The plates were centrifuged and the medium was removed, then 100 µl of DMSO was added to each well to dissolve the formazan crystals.The optical density value was measured at 570 nm with a microplate reader (Biorad, Model 680, USA).

Wound-healing assay
Cells were seeded in 6-well plates at a density of 5 × 10 5 cells per well and incubated overnight.The wounds were made by straight scratching the cell surface with a 20 ul pipette tip.Cells were washed with PBS gently, and then incubated in medium with 2% FBS with DMSO or EKC.Pictures of the entire scratched areas in each group were acquired at 12 h and 24 h using a phase-contrast microscope (Olympus, IX71, Japan).The wound area was quanti ed using Image J software.The wound healing rate was calculated as follows: (area of original wound-area of wound at different time points)/area of original wound × 100%.

Apoptosis assay
Apoptosis was detected by ow cytometry using an Annexin V-FITC/ PI kit according to the manufacturer's instruction.In brief, cells were seeded in 6-well plates at a density of 4 × 10 5 cells per well, after treatment with EKC for 48 h, cells were harvested, washed, centrifuged and then resuspended in binding buffer containing annexin V-FITC and PI.After staining, cells were analyzed using a FACS Calibur ow cytometer (BD Biosciences, San Jose, CA, USA).Cells treated with 15 µM cisplatin were served as positive control.

Autophagic ux measurement
Autophagic ux was detected by using the mRFP-GFP-LC3 adenovirus.NCI-H292 cells cultured on coverslips were infected with mRFP-GFP-LC3 adenovirus according to the manufacture's protocol.After infection, cells were treated with or without EKC for 2 h or 24 h.Autophagic ux was detected using a urescence microscope system (Olympus, IX71, Japan).

mRNA extraction and real-time quantitative reverse transcription-polymerase chain reaction
Trizol reagent was used to extract the total mRNA of cells.Equal amount of RNA was reverse transcribed to cDNA using the PrimeScript RT reagent Kit (Takara) according to the manufacturer's instruction.Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Premix Ex Taq Kit (Takara) and the condition was an initial cycling for 2 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 15 s at 55 °C and 20 s at 72 °C.The data presented are relative mRNA levels normalized to GAPDH, the value from the untreated group was set as 1.The primer sequences are list in the Table 2. 2.10 Live cell imaging with uorescent tracers NCI-H292 cells were seeded in coverglass bottom dishes (confocal dishes) and allowed to attach overnight.Cells were treated with 15 µM EKC for 12 h and then incubated with MitoTracker Red, LysoTracker Red and ER-Tracker according to manufacturer's protocol respectively.Images were collected by confocal microscope (LSM 700, Zeiss, Germany).For Lucifer Yellow (LY) staining, cells were incubated with EKC 15 µM and Lucifer Yellow (0.5 mg/ml) simultaneously for 24 h, then washed with PBS and photographed with a orescence microscope.For acridine orange staining, cells cultured in 24-wells plate were treated with 15 µM EKC for 12 hr, then were incubated with 10 µM acridine orange for additional 30 min in serum-free medium.Pictures were obtained by a urescence microscope system (Olympus, IX71, Japan).

Immuno uorescence
After NCI-H292 cells were attached on glass cover slips, EKC (15 µM) was added into the medium and treated cells for 12 h.At the end of incubation, cells were xed in methanol, then introduced Rab7, LAMP1 and Rab5 primary antibody (1:100) and incubated at 4 °C overnight.Afterwards, incubated cells with secondary antibody Alexa Fluor 488 or 594-conjugated at room temperature for 1 h.Fluorescent images were taken with a uorescence microscope.

Immunoblot analysis
After treatments, cell lysate was prepared by using a sample buffer (50 mM Tris-HCl [PH 6.8], 2% SDS, 10% glycerol, 100 mM DTT, 0.1% bromophenol blue).Equal amounts of proteins were loaded and electrophoresed on SDS PAGE gel followed by a transfer onto nitrocellulose membrane (Millipore).After blocking, the membrane was incubated with a diluted primary antibody solution at 4 °C overnight, and then the membrane was exposed to a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h.Signals were visualized using the enhanced chemiluminescence (ECL) detection reagents (Millipore, Billerica, MA, USA).GAPDH and β-actin were used as internal controls.
2.13 Cytotoxicity assay using xCELLigence system Cytotoxicity assay was conducted using the xCELLigence system (ACEA Biosciences, San Diego, CA, USA), which allows for dynamic monitoring of cell growth in response to treatment in real-time.In this experiment, NCI-H292 cells were seeded onto E-plates with the number of 8000 per well, after an overnight incubation, cells were exposed to EKC (6.5 µM), doxorubicin (0.3 and 0.5 µM), etoposide (30 and 50 µM) or in combination.The impedances were measured per 30 min for 70 h and expressed as CI value.
2.14 Pull-down assays to measure Rac1 and Arf6 activation Rac1 and Arf6 activation assays were performed using the Cytoskeleton Rac1 and Arf6 activation biochem kits.NCI-H292 cells were seeded in 100 mm dishes and incubated overnight.Then, cells were treated with 12.5 µM EKC or DMSO for 12 h.Cells were lysed and used for pull-down assays according to the manufacturer's instructions.The assays either use PAK-PBD conjugated sepharose beads to selectively bind active Rac1 in whole cell lysates, or GGA3-PBD beads to pull down Arf6.The active Rac1 or Arf6 collected on the beads were subjected to immunoblot analysis.On a separate immunoblot, the total Rac1 or Arf6 was determined.

Statistical analysis
Results are presented as the mean ± standard deviation (SD).To determine the signi cant difference between two groups, one way analysis of variance (ANOVA) test and post hoc multiple comparison Bonferroni test were used.p < 0.05 was considered to be signi cant.

The obtaining of EKC and its analogues
Epimedium koreanum is a good resource of prenylated avonoids.Our phytochemical study on the leaves of this plant led to the isolation of EKC and other 26 prenylated avonoids.The isolation process and structures of the isolated prenylated compounds were shown in Additional le 1 and Additional le 2.
The structure of the compound was determined by analysis of the spectroscopic data (Fig. 1A). 1 H NMR and 13 C NMR data of EKC were listed as below.3.2.EKC induced cell death and cytoplasmic vacuolization in NCI-H292 cells.
During our screening of anticancer candidates from prenylated avonoids, the effects on cell viability of these 27 compounds were determined by MTT assay rst (Additional le 2).EKC inhibited cell growth in a dose-and time-dependent manner in non-small cell lung cancer NCI-H292 cells, the IC 50 value for 48 h was 17.04 µM.Meanwhile, in an immortalized normal human bronchial epithelial cell line 16HBE, the cell viability after 48 h of EKC treatment was almost unaffected (Fig. 1B).Thus, EKC was more toxic to the tested lung cancer cells compared to normal bronchial epithelial cells.In terms of cytotoxity, EKC was not really a promising candidate, but it captured our attention because of its cytoplasmic vacuoles inducing effect.We found that EKC caused a quick and striking accumulation of numerous phase-lucent cytoplasmic vacuoles within 12 h in NCI-H292 cells, these morphological effects of EKC persisted for 48 h and beyond (Fig. 1C).In contrast, 16HBE cells and the other normal human bronchial epithelial cell line, Beas2B were relatively resistant to EKC-induced vacuolization (Fig. 1D).The vacuolization effects in NCI-H292 cells induced by EKC at a dose of 15 µM were demonstrated to be reversible, if cells were treated for 12 h, then placed in EKC-free medium for culturing additional 12 h, the vacuolization was almost completely resolved (Fig. 1E).The effects of EKC on other four cancer cell lines, including A549, Calu-1, HepG2 and PANC-1, were also evaluated.Similar to the results with NCI-H292 cells, EKC induced dramatic cytoplasmic vacuolization in all of the four cell lines (Additional le 3).
Four closely related compounds with > 75% structure similarity to EKC were isolated from E. koreanum together, they showed almost no vacuole-inducing activity or induced a small amount of vacuoles (Additional le 4).This suggested that the effect of EKC was probably due to its interaction with speci c intracellular targets rather than non-speci c effects.

EKC induced cell death was distinct from apoptosis
Apoptosis is the most common form of programmed cell death.To investigate if the cell death induced by EKC was related with apoptosis, annexin V-FITC/PI double staining and DAPI staining were performed.
As shown in Fig. 2A and 2B, neither signi cant increase of the number of apoptotic cell nor obvious morphological changes in the nuclei were observed with EKC treatment, indicating that apoptosis may not be critically involved in EKC-induced cell death.For further determination, the activation of apoptosis markers including caspase-9, caspase-3 and PARP was also examined.As depicted in Fig. 2C, no obvious cleavage of caspase-9, caspase-3 and PARP was observed in 24 h after EKC exposure.Consistent with this assumption, pretreatment with pan-caspase inhibitor Z-VAD(OMe)-FMK (Z-VAD) for 1 h prior to EKC treatment did not block both the loss of cell viability and vacuolization induced by EKC (Fig. 2D and 2E).
These ndings suggested that EKC-induced cell death was non-apoptotic and caspase-independent.

EKC induced methuosis in NCI-H292 cells
Cytoplasmic vacuolation has been observed in several types of non-apoptotic cell deaths.To determine the origin of EKC-induced vacuoles, different subcellular compartments staining dyes including acridine orange (AO), MitoTracker red, LysoTracker red and ERTracker red were used.Live cell imaging showed that the vacuoles could sequester AO to change to orange uorescence (Fig. 3A), which indicated the acidic nature of the vacuoles.None of MitoTracker red, LysoTracker red and ERTracker red was observed to be incorporated into the vacuoles (Fig. 3B-3D), indicating that the vacuoles were not derived from mitochondria, lysosome or endoplasmic reticulum.
The formation of large vacuoles is one of the most important characteristics of macropinocytosis.We next turned our attention to determine whether the vacuoles induced by EKC were correlated with macropinocytosis.One of the typical features of macropinocytosis is the incorporation of extracellularphase uid tracers.Lucifer Yellow (LY) is a well-established uorescent tracer used to de ne micropinocytosis [18].As shown in Fig. 3E, LY incorporated into most of the phase-lucent vacuoles induced by EKC, suggested that EKC-induced vacuoles might be derived from macropinosomes.
Normally, once internalized into cells, macropinosomes pesist for only about 5 to 20 minutes, during which they either recycle back to the cell surface or merge with lysosomes [18,19].While in methuosis, macropinosomes bypass the normal endosomal tra cking pathway, immediately recruit Rab7 and form progressively larger LAMP1-positive structures [20].For further investigation of whether EKC induced methuosis, immuno uorescence assay against two late endosome markers, Rab7 and LAMP1, was performed.As seen in Fig. 3F, Rab7 and LAMP1 were detected on the membrane of EKC-induced vacuoles.Rab5, a marker of early endosome, was also detected, but it was not labeled on the membrane of the vacuoles.The protein levels of Rab7 and LAMP1 were also detected, as shown in Fig. 3G, EKC treatment increased the levels of Rab7 and LAMP1 in dose-and time-dependent manners.Combined with aforementioned LysoTracker staining results, we could conclude that the vacuoles induced by EKC possessed the characteristics of late endosomes, but did not merge with lysosomal compartments.These characteristics were consistant with methuosis, suggesting that EKC was a methuosis inducer.

EKC induced PIKfyve phosphoinositide kinase inhibition and Akt suppression
PIKfyve, a class Ш phosphoinositide (PI) kinase, plays crucial role in the regulation of tra cking events associated with the endocytic pathway [21,22].It has been reported that PIKfyve was one candidate protein target of a methuosis inducer, MOMIPP [23].To explore whether EKC inhibited PIKfyve expression, the protein level of PIKfyve was detected by immunoblot assay.As depicted in Fig. 4A, EKC treatment decreased the protein levels of PIKfyve dose-and time-dependently.Akt suppression was required for the cytotoxicity of PIKfyve inhibitors, the phosphorylation of Akt was shown to be down-regulated concurrently (Fig. 4A).
PIKfyve also plays a crucial role in ensuring phagosomal maturation [24].Under the same conditions in which EKC induced the accumulation of cytoplasmic vacuoles, immunoblotting analysis revealed a dose-as well as time-dependent increase of two biomarkers of autophagosome, LC3-П and SQSTM1, indicating the accumulation of autophagosomes (Fig. 4B).To determine whether the accumulation of autophagosomes resulted from induction of autophagy or from disruption of autophagic ux, cells were transfected with mRFP-GFP-LC3 adenovirus, as shown in Fig. 4C, distinct yellow puncta were observed in EKC treated cells.Therefore, EKC disrupted autophagic ux by preventing autolysosome formation.
Ba lomycin A1 (Baf A1), an H + -vacuolar ATPase inhibitor, has been shown to prevent formation of cytoplasmic vacuoles that are induced by inhibition of PIKfyve PI kinase [25].To determine whether or not Baf A1 also prevented induction of the cytoplasmic vacuoles that were induced by EKC, NCI-H292 cells were treated by Baf A1, EKC, or both Baf A1 and EKC, the results showed that Baf A1 prevented EKCinduced cytoplasmic vacuolation almost completely (Fig. 4D), suggesting that EKC inhibited PIKfyve activity.

EKC affected the activities of Rac1 and Arf6 GTPases
It has been reported that methuosis triggered by over-expression of activated H-Ras required activation of the Rac1 GTPase with a concomitant deactivation of another GTPase, Arf6 [26].In EKC treated cells, we rst detected the total protein level of Rac1 and Arf6, the results revealed that EKC treatment had no signi cant effect on the expression of Rac1, while decreased the total level Arf6 (Fig. 5A).The mRNA levels were examined by qPCR, and the mRNA level of Rac1 was not changed, while the mRNA level of Arf6 decreased (Fig. 5B).Futhermore, the active GTP-bound forms of Rac1 and Arf6 were measured in pull-down assays using fusion proteins that bind speci cally to the activated forms of Rac1 and Arf6.The results showed that the amounts of active Rac1 (Rac1-GTP) increased although the total level of Rac1 did not change in EKC treated cells.In contrast, EKC had the opposite effect on Arf6, both total level and active Arf6 decreased, however Arf6-GTP decreased more signi cant than total Arf6 after EKC treatment, that is the ratio of active/total Arf6 decreased (Fig. 5C).These results indicated that EKC treatment induced activation of the Rac1 GTPase, with a concomitant reduction in the activation state of Arf6 GTPase.For further exploration, NCI-H292 cells were pretreated with a highly speci c Rac inhibitor EHT 1864 for 24 h, then incubated with EKC in the presence of EHT 1864 for additional 24 h, as illustrated in Fig. 5D, EKC induced cell viability decrease was antagonized by EHT 1864; in Fig. 5E, most of the vacuoles were precluded by EHT 1864.These results suggested that the mechanisms of vacuolization induced by EKC is probably similar with that induced by Ras, that is, might be depended on reverse regulation of Rac1 and Arf6.

EKC inhibited NCI-H292 cell migration
It has been demonstrated that Arf6 regulates cancer cell invasion through the activation of MEK/ERK signaling pathway, and downregulation of Arf6 correlate with impaired cell migration [27,28].We evaluated the effect of EKC on the migration of NCI-H292 cells by performing a scratch wound-healing assay.As shown in Fig. 6A, EKC treatment at the dose of 6.5, 12.5 µM could signi cantly reduce cell migration in NCI-H292 cells.We also examined the phosphorylation as well as total ERK levels by immunoblot analysis.EKC treatment decreased the levels of phosphorylation ERK in dose-and timedependent manners, while no obvious change of total ERK was observed (Fig. 6B).

EKC sensitized NCI-H292 cells to doxorubicin and etoposide
To determine the effects of EKC on the sensitivity to conventional chemotherapy agents, NCI-H292 cells were exposed to doxorubicin/etoposide in the presence of 6.5 µΜ of EKC, the viability of cells was monitored using a real time cell analyzer (RTCA).As shown in Fig. 7, both doxorubicin and etoposide displayed a reduction in cell growth, whereas treatment with 6.5 µΜ of EKC alone resulted in slight growth inhibition.However, co-treatment of EKC with doxorubicin/etoposide signi cantly reduced cell growth as judged by decreased cell index (CI) compared with doxorubicin and etoposide treatment alone.

Discussion
Apoptosis is a mechanism of programmed cell death and is essential for cell development and homeostasis.Apoptotic cells undergo characteristic changes in cell morphology, including cell rounding, plasma membrane blebbing and nuclear fragmentation [29,30].In cancer cells, modulation of apoptosis plays a signi cant role in the progression of malignancy.Alongside the discoveries of the mechanisms underlying apoptosis, targeting apoptotic pathways has been approved as a primary therapeutic strategy [31].However, many cancer cells developed resistance to most of the apoptosis-based cancer therapies.
In this sense, targeting non-apoptotic forms of cell death therapeutics may have important implications.
Several forms of non-apoptotic programmed cell death including autophagy, paraptosis, necroptosis, oncosis and ferroptosis has been reported in the past three decades [32].Methuosis is one of the most recent additions to the non-apoptotic cell death forms.As a unique form of non-apoptotic cell death, methuosis was initially de ned in glioblastoma cells after overexpression of Ras [33].In recent years, some methuosis inducers were identi ed and all of these compounds displayed meaningful effects in killing cancer cells [10][11][12][13][14][15].This indicated that small molecules with the capacity to induce methuosis may be of considerable interest as potential therapeutics for cancers that are resistant to apoptosis.
Natural compounds are good resources of methuosis inducers, as most of aforementioned methuosis inducers are natural compounds related.Therefore natural compounds have gained increasing interests for the future discovery of non-apoptotic cell death based anticancer therapeutics [34,35].Epimedium koreanum is a traditional medicinal plant recorded in Chinese Pharmacopoeia.The aerial part of this plant has been used for alleviating a variety of disorders [36].Previous phytochemical investigations revealed that prenylated avonoids are major constituents of E. koreanum [37].During our exploration of cytotoxic prenylated avonoids, we performed a systematic isolation of E. koreanum, as a result, 27 prenylated avonoids were obtained.In the cell viability assay, EKC caught our attention because of the induction of catastrophic vacuolization in NCI-H292 lung cancer cells.The phenotype of EKC-induced cell death was identi ed as methuosis based on the following features: (1) Shortly after being exposed to EKC, extreme cytoplasmic vacuolization was observed (Fig. 1C); (2) The vacuoles were decorated with late endosomal markers Rab7 and LAMP1, but did not sequester with lysosomes (Fig. 3B, 3F); (3) Consistant with a non-apoptotic mechanism, cell death could not be prevented by caspase inhibitor, and nuclear chromatin condensation was not observed (Fig. 2B, 2D).Small G proteins in the Rab and Arf family are present on endosomes and coordinate the tra cking of cargo proteins.It was reported that methuosis induced by overexpression of Ras required the activation of Rac1 and inactivation of Arf6, two GTPases implicated in macropinocytosis and endosome recycling [26].Our ndings indicated that Rac1 was activated while Arf6 was inactivated under EKC treatment, a Rac1 inhibitor EHT 1864 precluded the vacuole-inducing effect of EKC, suggesting that inversed regulation of Rac1 and Arf6 was involved in EKC-induced methuosis (Fig. 5C, 5E).
PIKfyve is a critical regulator that has been implicated in various tra cking events associated with the endocytic pathway [38].PIKfyve is required for the maturation of both endosome and phagosome [24].Our results indicated that EKC treatment decreased the expression level of PIKfyve, also blocked the formation of autophagolysosome (Fig. 4C).Considering the function of PIKfyve, we hypothesized that EKC-induced vacuoles were blocked to merge with lysosomes might be involved with down-regulation of PIKfyve.
Based on our ndings, we suggested a working model of EKC depicted in Fig. 8.For the normal process of macropinocytosis in mammalian cells, macropinosomes are internalized rst, then the macropinosomes rapidly acquire Rab5, EEA1 and sorting nexins, which promote fusion with early endosomes.The latter retain the capacity to recycle to the cell surface, but after several additional minutes they lose Rab5 and acquire Rab7 and LAMP1, taking on the characteristics of late endosomes.Ultimately, the vesicles dissipate as they merge with lysosomes [10].EKC induced vacuoles were suggested to bypass the normal endosomal tra cking pathway and undergo abnormal homotypic fusions to form progressively larger LAMP1-positive structures.The possible reasons were that the decline of Arf6 and PIKfyve blocked vacuoles recycling and maturation to late endosomes.
The key observation that EKC effectively induced methuosis, as well as synergized with doxorubicin or etoposide in NCI-H292 cells, raises the possibility that further development of this compound could lead to useful therapeutic agents for treating cancers that are resistant to drugs that commonly work by inducing apoptosis.

Conclusions
In conclusion, EKC was identi ed as an inducer of methuosis, an unconventional non-apoptotic cell death form.Inverse regulation of Rac1 and Arf6 contributed to EKC induced cytoplasmic vacuolization, this might be related to the mechanism of action of EKC.Combined treatment of EKC with doxorubicine and etoposide enhanced the e cacy of chemotherapeutic drugs dramatically.EKC could be considered as a candidate for the development of methuosis-based therapies against cancers which are resistant to chemo-drugs that commonly work by inducing apoptosis.EKC sensitized NCI-H292 cells to doxorubicin/etoposide treatment.Cells were pretreated with 6.5 μM EKC for 4 h before the addition of DMSO, doxorubicin (0.3 and 0.5 μM) or etoposide (30 and 50 μM).Cell index was measured using a xCELLigence system for 70 h.

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Table 2
Primer sequences used in the RT-PCR