Abstract!

Natural compounds from medicinal plants areimportant resources for drug development. Activecompounds targeting apoptosis and autophagyare candidates for anti‑cancer drugs. In this study,we collected Garcinia species from China and ex-tracted them into water or ethanol fractions.Then, we performed a functional screen in searchof novel apoptosis and autophagy regulators. Wefirst characterized the anti‑proliferation activityof the crude extracts on multiple cell lines. HeLacells expressing GFP‑LC3 were used to examinethe effects of the crude extracts on autophagy.Their activities were confirmed by Western blotsof A549 and HeLa cells. By using bioassay guidedfractionation, we found that two caged prenyl-xanthones from Garcinia bracteata, neobractatinand isobractatin, can significantly induce apopto-sis and inhibit autophagy. Our results suggest thatdifferent Garcinia species displayed various de-grees of toxicity on different cancer cell lines. Fur-thermore, the use of a high content screening as-say to screen natural products was an essentialmethod to identify novel autophagy regulators.

Abbreviations!

ATGs: autophagy‑related proteinsBCA: bicinchoninic acidBSA: bovine serum albuminECL: enhanced chemiluminescenceFRET: fluorescent resonance energy transferGFP‑LC3: green fluorescent protein‑fused LC3HCQ: hydroxychloroquineHCS: high content screeningHRP: horseradish peroxidaseHTS: high‑throughput screeningLC3/MAP1LC3B:

microtubule‑associated protein 1 lightchain 3

PARP: poly(ADP‑ribose)‑polymerasePCD: programmed cell deathPI: propidium iodidePPAPs: polycyclic polyprenylated acylphloro-

glucinolsppm: parts per millionSQSTM1: poly‑ubiquitin binding protein p62 or

sequestome 1TBS/T: Tris‑buffered saline/Tween20TCM: traditional Chinese medicineTMS: tetramethylsilane

Supporting information available online athttp://www.thieme‑connect.de/products

Identification and Characterization of AnticancerCompounds Targeting Apoptosis and Autophagyfrom Chinese Native Garcinia Species

Authors Danqing Xu1,2*, Yuanzhi Lao1,2*, Naihan Xu3, Hui Hu1,2, Wenwei Fu1,2, Hongsheng Tan1,2, Yunzhi Gu1,2, Zhijun Song4,Peng Cao5, Hongxi Xu1,2

Affiliations The affiliations are listed at the end of the article

Key wordsl" Garcinial" Clusiaceael" autophagyl" apoptosisl" natural compoundl" caged prenylxanthones

received July 10, 2014revised August 25, 2014accepted October 21, 2014

BibliographyDOI http://dx.doi.org/10.1055/s-0034-1383356Published onlinePlanta Med © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Hongxi XuSchool of PharmacyShanghai University ofTraditional Chinese MedicineCai Lun Road 1200Shanghai 201203ChinaPhone: + 862151323089xuhongxi88@gmail.com

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Introduction!

Dysregulated cell death is a common feature ofcancer, and the modulation of this cellular re-sponse plays an important role in cancer therapy.Apoptosis, autophagy, and necroptosis are impor-tant cell death forms, and resistance to cell deathis considered to be one of the hallmarks of cancer

* Authors contributed equally to this work.

[1]. Apoptosis, also called type I PCD, is mainlycontrolled by the integrity of the outer mem-branes of mitochondria in the cell [2]. The pro‑and anti‑apoptosis Bcl‑2 family proteins are thekey regulators for activating apoptotic pathways.When the anti‑apoptotic proteins are inhibited,the pro‑apoptotic Bax and Bak disrupt the integ-rity of the outer mitochondrial membrane, whichcauses the release of pro‑apoptotic signaling pro-teins (e.g., cytochrome c, Smac/DIABLO). Then,they initiate a cascade of proteolysis involving

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caspases responsible for the execution phase of apoptosis [3]. Au-tophagy (type II PCD) is an evolutionarily conserved membraneprocess that results in the transporting of cellular contents to ly-sosomes for degradation [4]. During tumor development and incancer therapy, autophagy plays paradoxical roles in promotingcell survival and cell death [5]. Autophagy functions as a tumorsuppression mechanism by removing damaged organelles andproteins and preventing genomic instability that drives tumori-genesis. On the contrary, autophagy has been demonstrated topromote the survival of tumor cells under nutrient or chemicalstress [6,7]. Therefore, therapeutic modulation of autophagymay serve as an important and challenging endeavor in cancertreatment [8,9]. The autophagy process involves initiation, elon-gation, closure, maturation, and degradation, which are con-trolled by highly conserved ATGs [10]. LC3/MAP1LC3B, a homo-logue of yeast protein ATG8, serves as a marker protein for auto-phagosomes [11].The complex signaling pathways that control cell death havebeen proven to be targets for anti‑cancer drug discovery [12].Cell‑based tests that quantify cell death are broadly used for drugscreens. However, it is necessary to apply further assays to iden-tify the biochemical cascades and the molecular targets of the ac-tive compounds. During the past decade, many conventional HTScell death detection methods targeting apoptosis and autophagyhave been developed. Caspase activation is a universal marker ofmitochondria‑dependent apoptosis and can be quantified by im-munoblotting methods using antibodies against active forms ofthese enzymes. Moreover, the FRET biosensor containing a cas-pase cleavage site can be applied to HTS for apoptotic inducers[13,14]. GFP‑LC3 protein can be applied to screen autophagy reg-ulators in live cells. The number of GFP‑LC3 puncta is very lowunder normal conditions but rapidly increases when autophagyis activated by rapamycin or stress [15]. However, the increase ofGFP‑LC3 level is not necessarily dependent on autophagy induc-tion. It may be the result of lysosome defects and associated withthe inhibition of autophagy. To confirm the function of chemicalsas either inducers or inhibitors of autophagy, more assay criteria,such as the monitoring of autophagic flux is required [16,17].SQSTM1 is selectively incorporated into autophagosomesthrough direct binding to LC3 and is efficiently degraded by au-tophagy. Thus, the total cellular expression levels of p62 correlatewith autophagic activity [18]. The novel autophagy regulators arenot only lead compounds for drug development but also provideresearchers a useful tool to investigate the complex autophagysignaling pathways [19].Compounds from natural plants are important resources fordrugs against a wide variety of diseases, including cancer. ManyTCMs containing active compounds exhibit antitumor effectsand have been used for various types of cancer treatment. Garci-nia species (Clusiaceae) have been studied for more than 70years, and many bioactive compounds, including xanthones,caged xanthones, PPAPs, and benzophenones, have been identi-fied with anticancer potentials [20]. Gambogic acid, a caged xan-thone from G. hanburyi Hook. f., has been tested in vitro and invivo as a novel anticancer agent that inhibits cell proliferation,angiogenesis, and metastasis [21–23]. Interestingly, many cagedGarcinia xanthones have been identified in the past several dec-ades, and most of them display high cytotoxic efficacy againstvarious cancer cells [24]. These studies suggest that the plant me-tabolites from Garcinia species have unique chemical structuresand potent bioactivities and can be a promising pharmacologicaltarget for drug design and development. Previously, we applied

Xu D et al. Identification and Characterization… Planta Med

bioassay‑guided fractionation by using a caspase‑3 FRET sensorto identify many novel compounds targeting apoptosis in Chinanative Garcinia species [25–29]. Furthermore, the mechanismsof action of some compounds involve the induction of apoptosis,inhibition of autophagic flux, cell cycle arrest, and modulation ofoncogenes, etc. [30–33]. Recently, we constructed HeLa cells sta-bly expressing GFP‑LC3 combined with HCS to identify autopha-gy regulators frommicroRNAs and natural compounds from Gar-cinia species [34,35]. Our studies demonstrated that Garciniaspecies contain many bioactive compounds affecting apoptosisand autophagy pathways. To obtain a comprehensive under-standing of the efficacy of different bioactive compounds, it isnecessary to perform autophagy HCS from crude extracts ofplants to look for novel autophagic regulators. In this study, wecollected different parts (leaves, fruits, bulks, and bulks of roots)from China native Garcinia species, used 95% EtOH and water toobtain crude extracts, respectively, applied a cell viability assayusingmultiple cancer cell lines and selected themost potent frac-tions to perform bioassay‑guided fractionation on GFP‑LC3 HCS.Our results suggested that two active compounds, neobractatinand isobractatin, from G. bracteata C.Y. Wu ex Y.H. Li have strongautophagy inhibition effects.

Results!

Fourteen herbs from Garcinia species were collected and theirdifferent parts, including leaves, twigs, seeds, pericarps, barks,root barks, or fruits, were extracted by water or ethanol to makedifferent types of plant extracts. Three cancer cell lines, includingthe human prostatic cancer cell line PC‑3, human lung adenocar-cinoma epithelial cell line A549, and human colon carcinoma cellline HT29, were used to determine the anti‑proliferation activ-ities of the crude extracts. As shown inl" Table 1, ethanol extractsare more active than water extracts against these cancer celllines. The most potent fractions were highlighted in l" Table 1.We found that the ethanol extracts from G. xanthochymus Hook.f. ex T. Anders., G. bracteata C.Y. Wu ex Y.H. Li, G. lancilimba C.Y.Wu ex Y.H. Li, G. oliganthaMerr., G. esculenta Y.H. Li, G. peduncu-lata Roxb., and G. yunnanensis Hu exhibited high growth inhib-itory activity against all three cancer cell lines. We further testedthe anti‑proliferation activity of the 11 selected Garcinia speciesextracts on 9 other cancer cell lines, and those fractions with IC50values less than 10 µg/mL were highlighted. As shown in l" Table2, they displayed different potencies against various cancer celllines. After the assays, four Garcinia species extracts were se-lected for further analysis, including No. 6 (ethanol extract fromleaves of G. bracteata), No. 18 (ethanol extract from leaves of G.oligantha), No. 60 (ethanol extract from root barks of G. peduncu-lata), and No. 72 (ethanol extract from root barks of G. esculenta).The overall average IC50 values were approximately 20 µg/mL,30 µg/mL, and 10 µg/mL corresponding to colon cancer, breastcancer, and leukemia cell lines, respectively (l" Fig. 1). Our prolif-eration assay suggested that most components with anti‑cancerpotential were extracted by the ethanol solvent. In addition, dif-ferent plants or different parts from the same plants containedvarious components that displayed a distinct effect on multiplecancer cell lines.To further confirm the cytotoxicity of the four chosen crude ex-tracts, A549 cells were used to analyze their effects on cell cycledistribution and cell death by flow cytometry analysis. As shownin l" Fig. 2A, four crude extracts caused Sub‑G1 fraction accumu-

Table 1 Cytotoxicity of seventy six extracts from Garcinia species on three tumor cell lines. Cells were cultured and seeded in 96‑well plates, and extracts weretreated for 72 h. Etoposide was used as positive control. Cell proliferation was detected using a CCK‑8 kit.

No. Name Parts Extract IC50 (µg/ml)

PC‑3 HT‑29 A549

1 G. xanthochymus leaves water > 100 > 100 > 100

2 leaves EtOH 49.47 61.26 38.85

3 twigs water > 100 > 100 > 100

4 twigs EtOH 92.28 75.20 68.64

67 seeds water > 100 > 100 > 100

68 seeds EtOH 35.08 73.08 67.99

69 pericarps water > 100 > 100 > 100

70 pericarps EtOH > 100 > 100 > 100

5 G. bracteata leaves water > 100 > 100 > 100

6 leaves EtOH 22.17 29.84 28.80

7 twigs water > 100 > 100 > 100

8 twigs EtOH > 100 > 100 > 100

9 G. multiflora leaves water > 100 > 100 > 100

10 leaves EtOH > 100 > 100 > 100

11 twigs water > 100 > 100 > 100

12 twigs EtOH > 100 > 100 97.65

13 G. lancilimba leaves water > 100 > 100 > 100

14 leaves EtOH > 100 > 100 > 100

15 twigs water > 100 > 100 > 100

16 twigs EtOH > 100 > 100 > 100

55 barks water > 100 > 100 > 100

56 barks EtOH 31.13 31.08 16.63

17 G. oligantha leaves water 86.90 82.58 41.06

18 leaves EtOH  8.48 12.68  9.95

19 twigs water > 100 > 100 > 100

20 twigs EtOH 11.10 15.35  9.97

21 G. oblongifolia leaves water > 100 > 100 > 100

22 leaves EtOH > 100 > 100 > 100

23 twigs water > 100 > 100 > 100

24 twigs EtOH > 100 > 100 > 100

57 barks water > 100 > 100 72.01

58 barks EtOH 92.69 93.25 57.04

25 G. cowa leaves water > 100 > 100 > 100

26 leaves EtOH > 100 > 100 > 100

27 twigs water > 100 > 100 > 100

28 twigs EtOH 95.14 98.39 60.67

29 G. xipshuanbannaensis leaves water > 100 > 100 > 100

30 leaves EtOH > 100 > 100 > 100

31 twigs water > 100 > 100 > 100

32 twigs EtOH > 100 > 100 > 100

33 G. esculenta leaves water > 100 > 100 > 100

34 leaves EtOH 74.35 82.98 61.20

35 twigs water > 100 > 100 > 100

36 twigs EtOH > 100 > 100 99.28

71 root barks water > 100 > 100 > 100

72 root barks EtOH 62.70 41.34 19.16

73 barks water > 100 > 100 > 100

74 barks EtOH 53.63 47.79 20.64

75 fruits water > 100 > 100 > 100

76 fruits EtOH > 100 > 100 > 100

37 G. nujiangensis leaves water > 100 > 100 > 100

38 leaves EtOH > 100 > 100 > 100

39 twigs water > 100 > 100 > 100

40 twigs EtOH 93.50 > 100 67.34

63 root barks water > 100 > 100 > 100

64 root barks EtOH > 100 > 100 > 100

65 barks water > 100 > 100 > 100

66 barks EtOH > 100 > 100 > 100

41 G. paucinervis leaves water > 100 > 100 > 100

42 leaves EtOH > 100 > 100 > 100

43 twigs water > 100 > 100 > 100

44 twigs EtOH > 100 > 100 > 100continued

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Table 1 Continued

No. Name Parts Extract IC50 (µg/ml)

PC-3 HT-29 A549

45 G. pedunculata leaves water > 100 > 100 > 100

46 leaves EtOH > 100 > 100 > 100

47 twigs water > 100 > 100 > 100

48 twigs EtOH > 100 > 100 > 100

59 root barks water > 100 > 100 > 100

60 root barks EtOH 28.37 18.52 26.69

61 barks water > 100 98.63 41.72

62 barks EtOH >100 99.27 51.17

49 G. yunnanensis twigs water > 100 > 100 > 100

50 twigs EtOH 35.05 40.61 34.54

51 fruits water > 100 > 100 > 100

52 fruits EtOH > 100 97.98 84.58

53 G. mangostana pericarps water > 100 > 100 > 100

54 pericarps EtOH > 100 74.52 62.33

Etoposide 13.04 21.36 2.22

Fig. 1 Cytotoxicity effects of EtOH extracts fromfour Garcinia species on eleven tumor cell lines. Cellswere cultured and seeded in 96‑well plates, and ex-tracts were treated for 72 h. Etoposide was used aspositive control. Cell proliferation was detected us-ing a CCK‑8 kit.

Table 2 Cytotoxicity of eleven extracts from Garcinia species on eleven tumor cell lines. Cells were cultured and seeded in 96‑well plates, and extracts were treat-ed for 72 h. Etoposide was used as positive control. Cell proliferation was detected using a CCK‑8 kit.

IC50 (µg/mL)

No. Prostate

cancer

Colon cancer Lung

cancer

Breast cancer Cervical

cancer

Leukemia

PC‑3 HT‑29 Colo

205

HCT‑15 A549 MCF7 MDA-

tfMB-

231

HeLa RPM-

I‑8226

K562 Molt‑4

2 49.47 61.26 42.46 31.89 38.85 78.84 66.21 28.35 21.57 25.62 11.13

6 22.17 29.84 23.07 26.87 28.80 34.73 19.36 25.13  6.20 12.84  4.76

18  8.48 12.68  9.12 12.05  9.95 18.99 14.71  9.29  4.25  6.34  3.36

20 11.10 15.35 13.95 18.24  9.97 28.46 16.37 13.93  4.34 11.17  4.19

50 35.05 40.61 43.0 37.7 34.54 81.94 80.09 31.13 18.3 25.92  8.57

56 31.13 31.08 16.9 30.12 16.63 32.96 36.42 18.11 14.0 19.0  5.17

60 28.37 18.52 33.71 31.44 26.69 39.91 42.49 19.24 13.82 18.7 15.75

62 > 100 99.27 38.2 33.52 51.17 > 100 > 100 19.46 17.25 58.8 25.16

68 35.08 73.08 37.27 37.76 67.99 > 100 65.7 35.49 32.91 25.09 34.36

72 62.70 41.34 18.74 23.93 19.16 87.50 56.42 10.14 16.44 19.27  9.90

74 53.63 47.79 22.32 30.28 20.64 90.14 63.89 11.10 11.74 23.13  9.20

ETO 13.04 21.36 25.01 26.67 2.22 3.07 6.11 1.02 3.39 2.18 2.05

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Fig. 2 Herbal extracts‑induced apoptosis and cellcycle arrest. A549 cells were treated with 4 Garciniaextracts with indicated concentrations. After 24 h oftreatment, the cells were harvested, fixed in 70%EtOH, and stained with PI. The cell cycle and celldeath were detected by FACS. A Sub‑G1 fraction indifferent concentration treatment. B Cell cycle dis-tribution in different concentration treatment.C Western blot showed the apoptosis‑related pro-teins upon the 4 Garcinia extracts treatment for48 h.

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lation in a dosage‑dependent manner which reflects the numberof dying cells. The effects on G0/G1, S, and G2/M phases were alsoanalyzed. Tests indicated that 50 µg/mL crude extract from theleaves of G. bracteata caused G2/M arrest. Notably, 16.7 µg/mLextract from G. oligantha induced G0/G1 arrest; however, a high-er concentration (50 µg/mL) of extract activated S and G2/M ar-rest. The effects of these fractions on apoptosis were examinedby Western blotting. As shown in l" Fig. 2C, A549 cells were sen-sitive to extracts from G. bracteata, G. oligantha, and G. esculentaas the cleavage of PARP and caspase‑3 was detected in high con-centration treatment. In addition, the crude extract from G. pe-dunculata was able to activate apoptosis in HeLa cells at a 50 µg/mL concentration.Autophagy plays an important role in tumorigenesis and chemo-therapy. We recently reported that compounds from Garciniaspecies might regulate autophagic flux and be beneficial foranti‑cancer efficacy [35]. Here, we used HeLa‑GFP‑LC3 cells andthe HCS platform to investigate whether the crude extracts canregulate autophagy in cancer cells. As shown in l" Fig. 3A, theGFP‑LC3 signal was distributed in the cytosol without significantpuncta formation in the control and low concentration treatmentgroups. In high concentration extract‑treated cells, the GFP‑LC3displayed puncta accumulation, which suggested that the au-tophagy pathway was influenced. During autophagy, the amountof LC3B‑II positively correlates with the number of autophago-somes. Therefore, the conversion from endogenous LC3B–I toLC3B‑II can be used tomonitor the autophagic activity. We exam-ined the effect of the four crude extracts on LC3B conversion inboth A549 and HeLa cells. All tested crude extracts could increasethe LC3B‑II conversion in these two cancer cell lines (l" Fig. 3B).Both induction and suppression of autolysosomal maturation re-sulted in increased numbers of autophagosomes. To distinguish

whether autophagosome accumulation is due to autophagy in-duction or inhibition, we performed an autophagic flux assay.p62 serves as a link between LC3 and ubiquitinated substrates.Inhibition of autophagy correlates with increased levels of p62in mammals and vice versa. We then examined the total cellularamount of p62 that was delivered to the lysosomes for degrada-tion. Immunoblot analysis revealed remarkable changes of p62,which were detected at 48 h after 50 µg/mL drug treatments(l" Fig. 3B). A significant increase of p62 was observed in G. brac-teata‑ and G. oligantha‑treated cells, which suggests that the au-tophagy procedure was suppressed. On the contrary, extractsfrom G. esculenta could induce autophagy, which was reflectedby a reduction of p62.Taken together, our results indicate that different extracts mightcontain distinct active compounds targeting different signalingpathways, such as the cell cycle, cell death, and autophagy. There-fore, the use of phytochemistry to identify single compoundsfrom the crude extracts with bioassay‑guided fractionation isnecessary, and it is helpful to elucidate the mechanism of actionof active compounds.Using these bioactivity screen platforms, we isolated two cagedprenylxanthones, neobractatin and isobractatin, from the etha-nol fraction of the leaves of G. bracteata (l" Fig. 4A). It has beenreported that isobractatin demonstrated cytotoxicity against KBcells (nasopharyngeal carcinoma), A549 cells (lung adenocarci-noma), MCF7 cells (breast cancer), and PC3 cells (prostate cancer)[36,37]. However, the bioactivity of neobractatin has not beenpreviously studied. We investigated the effects of these two com-pounds on cell proliferation and cell death. In CCK8 cell prolifer-ation assays, neobractatin and isobractatin displayed strong inhi-bition of both A549 and HeLa cells, with IC50 values of approxi-mately 2 µM. To examine whether these two compounds could

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Fig. 3 Herbal extracts‑regulated autophagy. HeLacells stably expressing GFP‑LC3 were seeded in 96-well plates and treated with extracts for 48 h. Thenucleus was stained with Hoechst. The images wereacquired with an Opera (GFP ex and em) using a40 x‑H2O objective. A Upper panel: Image fromDMSO treated cells as control. Lower panel: Cellswere treated with indicated extracts, and imageswere acquired after 48 h treatment. B Western blotshowed the autophagy‑related proteins upon the 4Garcinia extracts treatment for 48 h. The quantitiesof p62 and LC3B‑II were quantified in the histo-grams. The intensity was measured by ImageJ soft-ware. (Color figure available online only.)

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induce cell death, we performed flow cytometry analysis toquantify the sub‑G1 population. As shown in l" Fig. 5A, 4 µM ofneobractatin and isobractatin treatment significantly increasedthe sub‑G1 fraction in A549 cells. In addition, we used Westernblotting to check the apoptotic‑related proteins, such as cas-pase‑3 and PARP, upon treatment with these compounds. In bothA549 cells and HeLa cells, 48 h of treatment with neobractatin

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and isobractatin caused a decrease of pro‑caspase 3 and cleavageof PARP, which suggests the activation of apoptosis. Therefore,our data indicate that these two caged prenylxanthones couldsuppress cancer cell growth by inducing apoptosis.In l" Fig. 3B, we show that the crude extract of G. bracteata (6#)inhibited autophagic flux in cancer cells. It was of interest to ex-plore whether neobractatin and isobractatin were the effective

Fig. 5 Neobractatin and isobractatin‑induced ap-optosis in cancer cells. A549 cells were treated withneobractatin and isobractatin. After 24 h of treat-ment, the cells were harvested, fixed in 70% EtOHand stained with PI. The cell cycle and cell deathwere detected by FACS. A Cell cycle attribution ofA549 cells under different concentration treat-ments. Lower and left panel show the Sub‑G1 fac-tion data. B Western blot showed the apoptosis re-lated proteins upon neobractatin and isobractatintreatment for 48 h. (Color figure available onlineonly.)

Fig. 4 Two compounds purified from Garciniabracteata induced cell death in cancer cells.A Chemical structures of isobractatin and neo-bractain. B Cell proliferation curve under com-pounds treatment measured by CCK‑8 kit. IC50 wascalculated by Prism software.

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compounds producing the autophagic flux inhibition effect. Wethen performed Western blotting to examine the effects of thetwo compounds on the autophagic proteins LC3B and p62.l" Fig. 6A indicates that both neobractatin and isobractatin couldincrease LC3B–I to LC3B‑II conversion in A549 and HeLa cells in adosage‑dependent manner. In addition, the accumulation of p62was observed in high‑concentration treatment samples, suggest-ing that both compounds were able to inhibit autophagic flux in

cancer cells. We also used the HCS platform to measure the influ-ence of these compounds on GFP‑LC3 puncta formation. Asshown in l" Fig. 6B, 2 µM neobractatin and isobractatin treat-ment for 48 h induced significant puncta accumulation. To quan-tify the puncta number, we used Hoechst to stain the nucleus andsoftware to identify the cytosolic fraction in each cell (for details,see Materials and Methods). The GFP‑LC3 puncta in single cellscould be automatically counted by the software. The statistical

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Fig. 6 Neobractatin and isobractatin-regulatedautophagy in cancer cells. A A549 (left) and HeLa(right) cells were treated with compounds at differ-ent concentrations. After 48 h of treatment, thecells were harvested, and the indicated proteinswere tested by Western blot. The quantities of p62and LC3B-II were quantified in histograms. The in-tensity was measured by ImageJ software. B HeLacells stably expressing GFP‑LC3 were seeded in a 96-well dish and treated with compounds for 48 h.20 µM HCQ was used as positive control. The im-ages were acquired by an Opera (GFP ex and em)with a 40 x-H2O objective. C The number ofGFP‑LC3 puncta in each cell was calculated by Co-lumbus software. 20 µM HCQ was used as positivecontrol. (Color figure available online only.)

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analysis confirmed that neobractatin and isobractatin causedpuncta formation (l" Fig. 6C). Our results suggest that both neo-bractatin and isobractatin were able to regulate autophagic sig-naling pathways, and they are most likely autophagic flux inhib-itors.In summary, we collected Chinese native Garcinia species andobtained ethanol and water fractions for each sample. We firstused PC‑3, HT‑29, and A549 cancer cell lines to test the cytotox-icity of these crude extracts. Second, we chose the fractions withhigh activity and confirmed their anti‑proliferation ability usingmore cancer cell lines, including those for colon, lung, breast, andcervical cancer, as well as leukemia. Our results suggest that mostactive compounds remained in the ethanol fractions. Later, weselected four ethanol fractions from different plants to investi-gate their effects on cell death and autophagy. Finally, we isolatedtwo caged prenylxanthones, neobractatin and isobractatin, andprovided evidence that they could activate apoptosis and sup-press autophagic flux.

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Discussion!

Natural compounds have served as a major source of drugs, andmore than 50% of pharmaceuticals are derived from naturalproducts. Bioassay‑guided fractionation proved to be an efficientapproach to identify potent components from effective decoc-tions or plants. Our results indicate that these fractions containedactive components inhibiting the proliferation of multiple cancercell lines. Interestingly, these fractions displayed differential ac-tivities for different cell lines. For instance, the extract from G. es-culenta exhibited strong inhibition of lung cancer (A549) and cer-vical cancer (HeLa) cells but only minor toxicity to breast cancercells (MCF and MDA‑MB‑231). These findings suggest that thecomponents in this fraction could activate different signalingpathways. To further investigate the function of these fractions,we chose ethanol extracts from four Garcinia species to analyzetheir effects on cell cycle distribution, apoptosis‑related proteins,and autophagy pathways in the following study. As we expected,all of the samples had the potential to affect the cell cycle, apo-ptosis, and autophagy. By analyzing the protein level of p62, anautophagic flux marker, we found that some fractions containedautophagic flux inhibitors (fraction 6# and 18#), whereas others

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contained an autophagy inducer (fraction 72#). As autophagycontributes to tumorigenesis and cancer cell sensitivity understress, the discovery of novel autophagy regulators might be ben-eficial to anti‑cancer drug discovery. Through further isolationand chemical structure identification, we obtained two cagedprenylxanthones, neobractatin and isobractatin, and character-ized their bioactivities in regards to apoptosis and autophagy.Our data clearly indicate that these two compounds could induceapoptosis and inhibit autophagic flux at a low dosage comparedwith the crude extracts. Taken together, our study findings sug-gest that using cancer cells stably expressing GFP‑LC3 to screenautophagy modulators was an effective way to search for novelcompounds targeting autophagy. Therefore, it would be interest-ing to continue to study other fractions, such as extracts from G.oligantha (18#) and G. esculenta (72#), to identify componentsthat affect autophagy signaling pathways.Cell‑based HCS, which analyzes biological events at subcellularresolution, is widely used in pharmacological research [38]. Pre-viously, we used GFP‑LC3‑expressing HeLa cells to screen ourown pure compounds library from Garcinia species and identi-fied that oblongifolin C was an effective autophagic flux inhibitor[35]. In this study, we used the same cell line to screen our crudeextracts library. The screening setup was optimized in a singlecompound treatment, and the increase of GFP‑LC3 puncta wasquantified automatically. However, the HCS system had difficul-ties in processing the images under the crude extracts treatment.As shown in l" Fig. 3B, the cellular morphology and size changeddramatically upon treatment. Therefore, it was difficult to differ-entiate the nuclear and cytoplasmic regions and to calculate thecell size. This might be due to the complexity of the crude ex-tracts since their active components could have activated multi-ple cellular signaling pathways. To develop a suitable screen plat-form for crude extracts, researchers may consider combiningimaging‑based screenings with Western blots of autophagy-re-lated proteins, such as LC3 and p62 [16]. Alternatively, a novelHTS method based on ATG protein (e.g., ATG4B) activity can beconsidered for application [39].

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Materials and Methods!

Drugs and general procedureThe positive control compound etoposide (E1383, purity ≥98%)and HCQ (H0195, purity ≥98%) were bought from Sigma‑Aldrich.The powder was dissolved in DMSO to make a 10mM solutionstored at − 20°C before usage. All NMR spectra were recorded ona Bruker AV‑400 spectrometer at 400MHz for 1H NMR, HSQC,and HMBC, and at 100MHz for 13C NMR in DMSO‑d6. Chemicalshifts are reported in ppm relative to TMS.

Plant materialThe 38 samples of 14 Garcinia species were collected fromYunnan, Guangxi, Hainan, and Guangdong provinces in China(l" Table 1). The plant materials were authenticated by ProfessorZhao Yiming, Guangxi Medicinal Garden, and Prof. Wang Hong,Xishuangbanna Tropical Botanical Garden, Chinese Academy ofSciences. All voucher specimens (Herbarium No. GAR-01 to GAR-038) were deposited in the Innovative Research Laboratory ofTCM, Shanghai University of TCM.

Preparation of crude extractsEach dried plant sample was ground into a fine powder using apulverizer. A sample of 10.0 g of fine powder was placed in a250‑mL round‑bottom flask in a water bath and extracted for30min twice under reflux with 10 volumes of 95% ethanol or dis-tilled water and then filtered. The filtrate was evaporated to dry-ness under vacuum. These extracts were dissolved in DMSO andfurther diluted with cell culture medium. The final DMSO con-centration was below 1% of total volume of the medium in alltreatments and controls.

Extraction and isolationThe air‑dried trunks of G. bracteata (4.0 kg) was pulverized andextracted with 95% (v/v) ethanol (3 × 8 L) at room temperature,filtered and concentrated to give a crude extract (754.8 g). TheEtOAc‑soluble fraction (262.2 g) was subjected to Si gel columnchromatography (Φ10 × 75 cm, 3.0 kg) with a gradient of petro-leum ether‑acetone as the eluent, and ten fractions (A–J) werecollected. Fraction B (24.8 g) and fraction D (14.5 g) were furtherseparated on Si gel, Sephadex LH‑20, and RP‑C18 Si gel columns togive pure compounds 1–2 (Supporting Information).Compound 1 (isobractatin) and compound 2 (neobractatin) wereidentified based on MS and NMR spectroscopy analysis and bycomparison of their spectroscopic data with published values[36,40]. The purity of these two compounds was greater than98%.

Cell lines and cell cultureAll cell lines from ATCC and the cell bank of the Shanghai Insti-tutes of Biochemistry and Cell Biology, Chinese Academy of Sci-ences, were maintained at 37°C with a 5% CO2 humidified atmo-sphere in growthmedium as recommended by the providers andsubjected to in vitro assays between passages 8 ~ 15.

Cell proliferation assayCell proliferation assays were performed as previously described[41]. Briefly, each cell line was seeded in a 96‑well tissue cultureplate (Corning) at a predetermined density in 180 µL of completemedium, attached overnight and treated using natural productsor compounds for another 72 h. Then, the mediumwas discardedand replaced with 10% CCK‑8 (Dojindo) in complete medium,and the plates were incubated for another 2 h. The OD450 wasmeasured with SpectraMAX 190 spectrophotometer (MDS). Abackground absorbance of the ODblank was subtracted from allwells. The inhibition rate (IR) was determined with following for-mula:

IR (%) = (ODDMSO −ODcompound)/ODDMSO × 100%.

The CCK‑8 assay as above was used for determination of cell via-bility.

Propidium iodide staining for flow cytometryA549 cells were collected and washed with PBS twice and thenfixed with 75% alcohol overnight. After being washed with PBS,RNase (10 µg/mL) was added and incubated for 15min at 37°Cto eliminate the interference of RNA. Cells were then treatedwithPI (Sigma) for another 30min. Cells were washed, and the DNAcontents were detected by FACSCalibur [42].

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Western blottingThe cell lysate was prepared in RIPA buffer and quantified by theBCA method (Pierce). Thirty micrograms of protein per samplewas loaded onto a 4 ~ 12% NuPAGE® Novex SDS gel (Invitrogen).The protein was transferred by an iBlot® dry blotting device (In-vitrogen) onto nitrocellulose membranes. After blocking nonspe-cific binding with TBS/T (0.1%)containing 5% non‑fat milk for 1 hat room temperature, the membrane was incubated in LC3B (sig-ma, L7543), SQSTM1/p62 (MBL, PM045), PARP (Cell SignalingTechnology, CST #9542P), or caspase‑3 (Cell Signaling Technol-ogy, CST #9662P) (1 :1000 in TBS/T containing 3% BSA and gentlyshaken at 4°C overnight. The membrane was washed with TBS/Tthree times to remove the unbound antibody and then incubatedwith the secondary antibody (HRP‑conjugated goat anti‑mouseIgG or goat anti‑rabbit IgG, 1 :5000; KangChen Biotech) for 1 h atroom temperature. Protein bands were visualized with an ECL kit(Pierce).

Green fluorescent protein‑fused LC3 translocation andquantitative analysisHeLa cells stably expressing GFP‑LC3 were generated as previ-ously described [34]. Briefly, HeLa cells were transfected withpEGFP‑LC3 plasmid using lipofectamine 2000 (Invitrogen,11668–019). One day after transfection, the cells were treatedwith 800 µg/mL G418 for 7 days. The surviving cells were contin-ually cultured with 800 µg/mL G418 and named HeLa‑GFP‑LC3cells.The HeLa‑GFP‑LC3 cells were seeded in a 96‑well plate (clear bot-tom, black; PerkinElmer) overnight. Cells were then treated withdifferent concentrations of natural products or compounds intriplicate. After 48 h, the cells were fixed with 4% paraformalde-hyde and washed 3 times with PBS. Image acquisition was per-formed using an Opera High Content Screening System (Perkin-Elmer) using a 40 x‑H2O objective. Datawere analyzed by Colum-bus 2.3, which is software created by Perkin‑Elmer. To quantifythe GFP‑LC3 spots, the following procedures were performed: 1.using the Hoechst channel to define the nuclei region (method A;common threshold 0.45 with area>100 µM2); 2. using the GFPchannel to define the cellular cytoplasm region (method A; indi-vidual threshold 0.15); and 3. spot calculation in each cellʼs exclu-sive nuclear regions (method C; radius ≤ 5.0 µM; contrast > 0.13;spot to region intensity > 1.0; distance ≥ 2.3 px; peak radius1.0 px).

Supporting informationDetails about the isolation and identification of isobractatin andneobractatin, 13C NMR and 1H NMR spectra of isobractatin as wellas 13C NMR, DEPT135, 1H NMR, HSQC, and HSBC spectra of neo-bractatin are available as Supporting Information.

Acknowledgements!

This work was supported by the National Natural Science Foun-dation of China (No. 81303188, 81273403, and 81173485).

Conflict of Interest!

The authors declare no conflict of interest.

Xu D et al. Identification and Characterization… Planta Med

Affiliations1 School of Pharmacy, Shanghai University of Traditional Chinese Medicine,Shanghai, P.R. China

2 Engineering Research Center of Shanghai Colleges for TCM New DrugDiscovery, Shanghai, P.R. China

3 Key Lab in Healthy Science and Technology, Division of Life Science, GraduateSchool at Shenzhen, Tsinghua University, Shenzhen, P.R. China

4 Guangxi Botanic Garden of Medicinal Plants, Nanning, Guangxi, P.R. China5 Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute ofTraditional Chinese Medicine, Nanjing, Jiangsu, P.R. China

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