NOX2-Mediated TFEB Activation and Vacuolization Regulate

Subscriber access provided by AUSTRALIAN NATIONAL UNIV

Article

NOX2-mediated TFEB activation and vacuolization regulate lysosome-associated cell death induced by Gypenoside L, a saponin isolated from Gynostemma pentaphyllum Kai Zheng, Yingchun Jiang, Chenghui Liao, Xiaopeng Hu, Yan Li, Yong Zeng, Jian Zhang, Xuli Wu, Haiqiang Wu, Lizhong Liu, Yifei Wang, and Zhendan He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02296 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

TOC 210x192mm (300 x 300 DPI)

ACS Paragon Plus Environment


NOX2-mediated

TFEB

activation

and

vacuolization

Page 2 of 36

regulate

lysosome-associated cell death induced by Gypenoside L, a saponin isolated from Gynostemma pentaphyllum

Kai Zheng,1,2,†,* Yingchun Jiang,1,† Chenghui Liao,1 Xiaopeng Hu,1 Yan Li,3 Yong Zeng,3 Jian Zhang,1 Xuli Wu,1 Haiqiang Wu,1 Lizhong Liu,1 Yifei Wang,2,* and Zhendan He1,*

1

Department of Pharmacy, School of Medicine; Shenzhen Key Laboratory of Novel

Natural Health Care Products; Innovation Platform for Natural small molecule Drugs; Engineering Laboratory of Shenzhen Natural small molecule Innovative Drugs; Shenzhen University, Shenzhen, China; 2

College of Life Science and Technology, Jinan University, Guangzhou, China;

3

The First Affiliated Hospital of Kunming Medical University, Kunming, China;

†

These authors contributed equally to this work.

*Correspondence: Kai Zheng: School of Medicine, Shenzhen University, Nanhai Ave 3688, Shenzhen 518060, Guangdong, China. Tel/fax: +86 755 86671909; Email: [email protected] Zhendan He: School of Medicine, Shenzhen University, Nanhai Ave 3688, Shenzhen 518060,

Guangdong,

China;

Tel/fax:

+86

755

86671909;

Email:

[email protected]. Yifei Wang: Biomedicine Research and Development Center, Jinan University, No. 601 Huangpu Road West, Guangzhou, 510632, Guangdong, China; Email, [email protected].

Key words: Gypenoside L, TFEB, lysosome biogenesis, NOX2, Vacuolization.


Page 3 of 36


Abstract Downregulation of apoptotic signal pathway and activation of protective autophagy mainly contribute to the chemo-resistance of tumor cells. Therefore, exploring efficient chemotherapeutic agents or isolating novel natural products that can trigger non-apoptotic and non-autophagic cell death such as lysosome-associated death is emergently required. We have recently extracted a saponin, Gypenoside L (Gyp-L), from Gynostemma pentaphyllum and showed that Gyp-L was able to induce non-apoptotic cell death of esophageal cancer cells associated with lysosome swelling. However, contributions of vacuolization and lysosome to cell death remain unclear. Herein, we reveal a critical role for NADPH oxidase NOX2-mediated vacuolization and transcription factor EB (TFEB) activation in lysosome-associated cell death. We found that Gyp-L initially induced the abnormal enlarged and alkalized vacuoles, which were derived from lipid-rafts dependent endocytosis.

Besides,

NOX2

was

activated

to

promote

vacuolization

and

mTORC1-independent TFEB-mediated lysosome biogenesis. Finally, raise lysosome pH could enhance Gyp-L induced cell death. These findings suggest a protective role of NOX2-TFEB-mediated lysosome biogenesis in cancer drug resistance and the tightly interaction between lipid rafts and vacuolization. In addition, Gyp-L can be utilized as an alternative option to overcome drug-resistance though inducing lysosome associated cell death.

1



1 2

Page 4 of 36

Introduction Chemotherapy

frequently

deregulates

apoptotic

signal

pathway,

activates

3

anti-apoptotic system and stimulates protective autophagy, leading to the acquirement of

4

drug-resistance of different cancer cells.1,2 Consequently, exploring novel and efficient

5

chemotherapeutic agent triggering non-apoptotic and non-autophagic cell death, such as

6

lysosome-associated cell death, is emergently required.3 Lysosomes contain hydrolytic

7

enzymes and act as the main digestive organelles to degrade materials within their lumen

8

to maintain cellular homeostasis.4 The lysosome also represents a central hub for

9

signaling transduction, controlling cellular response to different stimulus, regulating cell

10

death and so on.5 Besides, increasing evidences suggest an important role of lysosome in

11

mediating drug resistance or promoting cell death.6

12

The underlying mechanisms of lysosome biogenesis are beginning to be clarified and

13

the transcription factor EB (TFEB) and TFE3 have emerged as master regulators of the

14

expression of many lysosome and autophagy proteins.7-9 TFEB activation is negatively

15

regulated by the mammalian target of rapamycin (mTOR) complex 1, a protein complex

16

regulating cellular response to growth signals and energy levels on the lysosome

17

membrane8,10-12 Small guanosine triphosphatase (GTPases) and Ragulator, a pentameric

18

protein complex that comprises LAMTOR1 (p18), LAMTOR2 (p14), LAMTOR3 (MP1),

19

LAMTOR4 (HBXIP) and LAMTOR5 (C7orf59), are necessary for the translocation and

20

activation of mTOR to the lysosome surface.13,14 When nutrients are sufficient, TFEB is

21

phosphorylated by mTOR at Ser211 or Ser142, and subsequently is released from the

22

lysosome surface, sequestered in the cytosol by chaperones 14-3-3 proteins. On the

23

contrary, starvation inactivates mTORC1 and TFEB dissociates from 14-3-3 complex,

24

migrates to the nucleus, and binds to the promoters of a specific gene network known as

25

coordinated lysosomal expression and regulation (CLEAR). Moreover, the lysosome

26

calcium efflux and the protein kinase C (PKC) pathway have also been reported to

27

regulate TFEB nuclear localization.15,16 Of particular interest, except for regulating

28

lysosome-autophagy homeostasis, TFEB signal is also involved in cell death process

29

triggered by several anticancer agents.17-21

30

Considerable efforts have been devoted to exploring or identifying novel anticancer 2


Page 5 of 36


31

compounds from different natural plants. Gynostemma pentaphyllum, also known as the

32

“ miracle green” and “ southern ginseng”, has been widely used as a traditional tea in Asia.

33

G. pentaphyllum is renowned as having powerful antioxidant and adaptogenic effects and

34

is traditionally used to maintain the normal healthy functions of respiratory, cardiovascular,

35

digestive and liver purported to increase longevity. G. pentaphyllum is also extensively

36

used as a health supplement in beverages, food, cosmetics and wash supplies, such as

37

face washes and bath oils. Bioactive constitutes of G. pentaphyllum include gypenosides,

38

sterols and flavonoids. Gypenosides are the major extracts and have been described to

39

possess different anticancer activities.22-26 However, the specific functional components

40

as well as the detailed molecular mechanisms of gypenoside-induced cell death remain

41

unclarified. Previously we have identified and demonstrated that Gypenosides L (Gyp-L),

42

a saponin isolated from G. pentaphyllum, induces vacuolization and lysosome swelling in

43

esophageal cancer cells.27 Besides, Gyp-L inhibited autophagic flux and induced

44

non-apoptotic cell death through reactive oxygen species (ROS)-mediated Ca2+ signaling.

45

However, contributions of vacuolization and lysosome to cell death remain unclear.

46

In this study, we reveal that the giant vacuoles induced by Gyp-L are abnormally

47

enlarged alkalized endo-lysosomes, likely resulting from the fusion of lipid rafts

48

dependent-endosomes and lysosomes. We further demonstrate that this vacuolization

49

leads to the mTORC1-independent, NADPH oxidase NOX2-dependent nucleus

50

translocation of TFEB and the enhanced lysosome biogenesis, which antagonize

51

Gyp-L-induced lysosome associated cell death. These findings suggest that Gyp-L may

52

represent as a novel therapeutic option for inducing lysosome-associated cell death to

53

overcome drug-resistance in cancer therapy.

54 55

Results

56

Gyp-L induces lysosome biogenesis in esophageal cancer cells

57

Previously we demonstrated that Gyp-L induces non-apoptotic and non-autophagic

58

cell death in esophageal cancer cells, which is associated with lysosome swelling and

59

fusion.27 However, the contribution of lysosome to cell death has not been clearly clarified. 3



60

In this work, we first examined whether Gyp-L triggers lysosome biogenesis, a process

61

requiring integration of the endocytic and biosynthetic pathways. Apparently, Gyp-L

62

treatment significantly induced lysosome biogenesis in a concentration dependent

63

manner as the immunofluorescence of lysosome specific label Lyso-tracker Red has been

64

largely increased (Fig. 1A and Fig. S1A). Flow cytometry assay has also been performed,

65

which showed that Gyp-L increased lysosome generation in both ECA-109 and TE-1 cells

66

in a time dependent manner (Fig. 1B). Besides, confocal microscopy experiment showed

67

an enhanced expression and cluster of LAMP1 (lysosomal associated membrane protein

68

1), a lysosome marker (Fig. 1C). Such dose-dependent enhancement was further

69

confirmed by western blotting (Fig. 1D). In addition, we performed acridine orange assay

70

(AO) to analyze the lysosome production. AO is a fluorescent acid dye that accumulates in

71

acidic spaces and emits red light when excited by blue light. Under normal pH conditions,

72

the dye emits green light. The fluorescence microscopy images of AO staining were

73

shown (Fig. S1B). Obviously, the red fluorescent signal was largely increased in

74

Gyp-L-treated cells. The Red/Green ratio of fluorescent intensity was also calculated by

75

flow cytometry assay, which demonstrated that the increment of the red fluorescence

76

signal was due to an enhanced generation of lysosome (Fig. 1E). Taken together, these

77

results indicated that Gyp-L induces lysosome biogenesis in esophageal cancer cells.

78 79

Gyp-L induces lysosome alkalization

80

Treatment with Gyp-L triggers massive vacuolization, with the formation of

81

cytoplasmic vacuoles possessing various sizes. To assess the possible involvement of

82

lysosome in this vacuolization event, ECA-109 cells were transfected with GFP-LAMP1

83

(green fluorescent protein tagged LAMP 1) or GFP-Vector, treated with Gyp-L for 12 h and

84

stained with Lyso-tracker Red. Obviously, significant GFP signal were seen surrounding

85

these vacuoles in Gyp-L-treated cells (Fig. 2A and Fig. S2), which were consistent with

86

our previous report.27 However, these giant LAMP1-positive vacuoles were not stained by

87

Lyso-tracker Red, suggesting that these Gyp-L-induced giant vacuoles were abnormal

88

lysosome (Fig. 2A). On the contrary, these giant vacuoles were clearly stained brownish

89

red by neutral red, a pH indicator that is red below pH of 6.8, yellow above pH of 8.0, and 4


Page 6 of 36

Page 7 of 36


90

changes from red to yellow in pH between 6.8 and 8.0, while few red staining structures

91

were seen in untreated control cells (Fig. 2B). To further confirm Gyp-L-mediated

92

alterations in the lysosomal pH, we used a ratiometric fluorescence probe, LysoSensor

93

Yellow/Blue DND-160, to selectively label lysosome and to quantitatively measure the

94

inner pH. LysoSensor appears blue in acidic compartments when the pH is neutral or

95

alkaline and green/yellow when the pH is acidic. Obviously, unlike control cells presenting

96

green fluorescence, a blue fluorescence was observed in cells-treated with Gyp-L,

97

indicating an elevated pH (Fig. 2C). Besides, there was a significant difference between

98

control (pH ~4.5) and Gyp-L treatment (pH ~7) when lysosome pH was quantitatively

99

measured by a fluorescence spectrophotometer. These results suggested that the

100

cytoplasmic vacuoles induced by Gyp-L were abnormally enlarged and alkalized

101

lysosome-like structures and the inner pH of the vacuoles is higher than that of functional

102

lysosomes. Finally, we tested the interaction between lysosome biogenesis and

103

vacuolization by using CHX, a protein synthesis inhibitor. Remarkably, CHX inhibited

104

Gyp-L-induced lysosome biogenesis whereas vacuole formation (percentages of cells

105

with vacuoles) was only slightly affected (Fig. 2D). In summary, Gyp-L treatment firstly

106

induced vacuole formation and their subsequently fusion with each other and/ or with

107

lysosome, accompanying alteration of their inner pH and lysosome biogenesis.

108 109

Lipid rafts-mediated endocytosis promotes Gyp-L-induced vacuolization

110

and cell death

111

Next, we speculated that the cytoplasmic vacuoles and cell death provoked by Gyp-L

112

were likely to be related to a particular endocytic pathway. To characterize the association

113

of endocytic vacuoles and Gyp-L-induced vacuoles, FITC-labeled dextran was used to

114

check for the cellular uptake. As expected, FITC-dextran located at Gyp-L-induced

115

vacuoles (Fig. S3A). Flow cytometry assay also suggested an increased uptake of

116

FITC-dextran in the presence of Gyp-L. The fact that the fluorescence signals of

117

FITC-dextran were restricted in the vacuoles also indicated that lysosomal integrity was

118

still intact in cells treated with Gyp-L. Thus we tested which of the known endocytic

119

pathways was essential for the vacuolization by using specific chemical inhibitors. 5



120

Inhibition of clathrin-mediated endocytosis by dynasore (Fig. S3B), or inhibition of

121

macropinocytosis by 5-(N,N-dimethyl) amiloride hydrochloride (DMA) (Fig. S3C) did not

122

affect the vacuolization and cell death induced by Gyp-L. On the other hand,

123

methyl-β-cyclodextrin (MβCD), an inhibitor of lipid rafts-based endocytosis, abolished the

124

vacuolization in Gyp-L-treated cells (Fig. 3A-C). MβCD also inhibited the lysosome

125

biogenesis and alleviated cell death triggered by Gyp-L. Considering that MβCD rapidly

126

extracts cholesterol from cell membranes, these results suggested an indispensable role

127

for lipid rafts in vacuolization and cell death. Besides, we tested the effect of U18666A, a

128

cholesterol transport inhibitor preventing the egress of cholesterol from late endosomes

129

and lysosomes, on Gyp-L-induced cell death and lysosome biogenesis and no significant

130

influence was observed (Fig. 3C and 3D), indicating that Gyp-L required the mobilization

131

of cholesterol. Next, we used fluorescein isothiocyanate (FITC)-conjugated cholera toxin

132

beta subunit (CTxB) to label the lipid raft marker ganglioside GM1 and to examine raft

133

organization in response to Gyp-L treatment. In cells treated with Gyp-L, GM1 was

134

drastically reorganized to form distinct patches at the membrane and the cytoplasm,

135

indicating lipid-cluster formation (Fig. 3E). However, such translocation of GM1 was

136

notably inhibited by MβCD. We further examined the expression of caveolin 1, a protein

137

critical for lipid rafts translocation,28 and found that Gyp-L also up-regulated its expression,

138

suggesting a possible role for caveolin-1 in cellular uptake of Gyp-L (Fig. 3F). In addition,

139

we detected intracellular cholesterol using Filipin III, a fluorescent high-affinity and

140

cholesterol-binding agent, via fluorescence microscopy. In untreated cells, Filipin III was

141

detected as an even distribution throughout the cell membrane (Fig. 3G). However, diffuse

142

and uniform Filipin III staining was observed throughout the cell in the presence of Gyp-L,

143

which was further impaired by MβCD. Finally to explore whether sphingolipids, another

144

important components of lipid rafts, are also crucial for vacuolization and cell death, we

145

added fumonisin B1 (FB1) to Gyp-L-treated cells (Fig. 3H). FB1 blocks both the salvage

146

pathway and the de novo synthesis of sphingolipids. No significantly reduction of FB1 was

147

observed, which further confirmed the essential function of cholesterol mobilization in

148

Gyp-L-induced lysosome biogenesis, vacuolization and cell death. Taken together, these

149

results indicated that lipid rafts-dependent endocytosis was essential for Gyp-L-induced 6


Page 8 of 36

Page 9 of 36


150

cytoplasmic vacuolization, and the vacuoles were abnormally enlarged and alkalized

151

endo/lysosomes, likely formed through the fusion of endosomes and lysosomes.

152 153

Gyp-L does not require cholesterol de novo synthesis

154

To exclude the possibility that MβCD-mediated inhibition of vacuolization and cell

155

death was independent of its destructive effect on lipid rafts, we restored the lipid rafts by

156

cholesterol (CHO) replenishment. As shown in Figure 4A, CHO replenishment recovered

157

the fluorescence intensity of CTxB staining decreased by MβCD, as well as the

158

redistribution of filipin III staining caused by Gyp-L and MβCD (Fig. 4B). Notably, the

159

inhibitory effect of MβCD on Gyp-L-induced vacuolization and lysosome biogenesis were

160

also reversed by adding CHO (Fig. 4C). The same CHO replenishment also maintained

161

the cytotoxicity of Gyp-L (Fig. 4D). Moreover, to test whether de novo synthesis of

162

cholesterol is involved in Gyp-L treatment, we tested the effect of lovastatin (Lov), an

163

inhibitor of HMG-CoA reductase that restricts the conversion and synthesis of cholesterol,

164

on Gyp-L-induced cell death, vacuolization and lysosome biogenesis. As shown in Figure

165

4E and 4F, Lov treatment only

166

difference was found in the mRNA expression levels of other critical enzymes of de novo

167

synthesis of cholesterol under the treatment of Gyp-L (Fig. 4G). These results indicated

168

that only the translocation of cholesterol from the cell membrane to endo-lysosome is

169

critical for Gyp-L.

showed a minor inhibitory effect. Consistently no

170 171

Na+ Ionophore inhibits Gyp-L-induced cell death

172

In seeking to understand the mechanism of lysosome alkalization and cell death, we

173

examined the role of P2X4 on endosome-lysosome fusion and vacuolization. P2X4 is an

174

endo-lysosomal calcium channel that is activated by endo-lysosome lumen alkalization

175

and

176

calcium-dependent calmodulin (CaM) activation.29 However, knockdown of P2X4 by

177

siRNA or inhibiting CaM by chemical inhibitor W7 did not affect Gyp-L-induced

178

vacuolization (data not shown), and cell death (Fig. S4A and S4B), implying that other

179

calcium-independent mechanisms are involved in Gyp-L-mediated endosome-lysosome

promotes

vacuole

enlargement

and

endosome-lysosome

7


fusion

through


180

fusion.

181

Next we considered a possible role of osmotic imbalance in lysosome swelling. The

182

exchange of osmotically active monovalent cations in intracellular compartments, such as

183

Na+, K+, can alter the ionic balance and osmotic properties, resulting in water influx.30 To

184

determine whether redistribution of intracellular ions has a general impact on

185

Gyp-L-induced vacuolization and cell death, we assessed the effects of several

186

ionophores, including monensin (for Na+), nigericin (for K+), valinomycin (for K+),

187

gramicidin A (for monovalent cations), and ionomycin (for Ca2+). The results that only

188

monensin blocked Gyp-L-induced cell death and vacuoles formation indicated Na+

189

imbalance occurring during Gyp-L triggered cholesterol-dependent endocytosis and

190

subsequent endosome/lysosome fusion and cell death (Fig. 5A and 5B). Next to inhibit

191

water influx, aquaporin water channel inhibitor phloretin was used.31 Unexpectedly, low

192

concentration (50 µM) of phloretin did not affect lysosome biogenesis, vacuolization and

193

cell death, whereas high concentration (100 µM) promoted Gyp-L-induced cell death (Fig.

194

S5A-5D). These results suggested that lysosome swelling induced by Gyp-L is not

195

osmotically regulated.

196 197 198

mTOR-independent nuclear translocation of TFEB promotes lysosome

199

biogenesis

200

Those Gyp-L-induced abnormally enlarged and alkalized endo-lysosomes likely

201

represent a type of lysosome stress to the cell, leading to disrupted lysosome balance and

202

homeostasis. Next we investigated the mechanism of lysosome biogenesis and its

203

contribution to cell death. In ECA-109 cells, Gyp-L up-regulated several TFEB/TFE3

204

targeted genes (Fig. 6A). Gyp-L also induced nuclear translocation of EGFP-tagged TFEB,

205

but not TFE3 (Fig. 6B and 6C). In contrast, Torin 1, a mTOR inhibitor, as well as

206

chloroquine (CQ),11 induced nuclear translocation of both EGFP-TFEB and EGFP-TFE3.

207

Substantially, such Gyp-L-mediated TFEB nuclear translocation was strongly suppressed

208

by MβCD, further implying that vacuolization leads to nucleus translocation of TFEB and

209

lysosome biogenesis. Although total cytoplasmic TFEB protein level was not changed, we 8


Page 10 of 36

Page 11 of 36


210

observed an augmented accumulation of nuclear TFEB in response to Gyp-L (Fig. 6D). In

211

addition, knockdown of TFEB by siRNA significantly reduced Gyp-L and Torin 1-triggered

212

lysosome biogenesis (Fig. 6E). These results suggested that TFEB participates in

213

Gyp-L-induced lysosome biogenesis. Considering that active mTOR has been reported to

214

phosphorylate TFEB and inhibit its translocation,10,12 we therefore tested whether Gyp-L

215

activated TFEB and induced lysosome biogenesis through inhibiting mTOR activity.

216

Inhibition or activation of mTOR by specific inhibitors did not increase or decrease

217

Gyp-L-induced lysosome biogenesis respectively (Fig. 6F). More surprisingly, Gyp-L

218

increased the phosphorylation of mTOR and its substrates, such as ribosomal S6 kinase

219

(S6K) and eIF4E-binding protein (4E-BP1), in both ECA-109 and TE-1 cells (Fig. 6G). The

220

activated mTOR in the nucleus was also found to be enhanced by Gyp-L (Fig. 6D). These

221

results indicated that Gyp-L activated mTOR. Therefore, Gyp-L induced TFEB nuclear

222

translocation through other mechanisms, but not mTOR-inactivation.

223

In addition, PKC and lysosomal calcium signals have been described to promote

224

TFEB nuclear translocation.15,16 Therefore several chemical inhibitors were utilized to

225

examine their roles in Gyp-L-induced lysosome biogenesis (Fig. 6H). As shown in Figure

226

6I, all the inhibitors did not significantly change the induction of lysosome biogenesis,

227

implying the involvement of other unclarified mechanisms of TFEB activation. Finally we

228

examined the expression of several regulators that promote the translocation of mTOR to

229

the lysosome surface where it is activated by amino acid stimulation and subsequently

230

inhibits TFEB (Fig. 6J). Though western blot assay, we found that Gyp-L reduces all the

231

protein levels of LAMTOR1, LAMTOR2 and LAMTOR3 in a concentration-dependent

232

manner in both ECA-109 and TE-1 cells. Besides, Gyp-L upregulated the expression of

233

several Rag GTPases which activate mTORC1 in response to amino acids. These results

234

suggested that Gyp-L promotes TFEB nuclear targeting and lysosome biogenesis though

235

a mTOR-independent pathway.

236 237

NOX2 alkalinizes vacuoles and triggers TFEB nuclear translocation

238

Previous several studies have demonstrated that the recruitment of NADPH oxidase,

239

in particular the NOX2 complex, to the early phagosomes controls endosome or lysosome 9



Page 12 of 36

240

alkalization and vacuole enlargement,32-34 therefore we examined whether NOX2 was

241

responsible for Gyp-L-induced lysosome alkalization and subsequent lysosome

242

biogenesis. The NADPH oxidase NOX2 consists of several subunits, including small

243

GTPase, cytosolic proteins (p47phox, p67phox and p40phox) and membrane-associated

244

proteins (gp91phox/Nox2 and p22phox). We first tested the activity of NADPH oxidase

245

and found that Gyp-L enhanced NADPH oxidase activity in a dose-dependent manner

246

(Fig.

247

diphenyleneiodonium chloride (DPI) (Fig. 7A). We then examined the involvement of

248

NOX2 by detecting the superoxide-generating enzyme p47phox, a component of the

249

NADPH oxidase NOX2 complex. In resting cells, p47phox is located in the cytosol and

250

during activation p47phox migrates to the plasma membrane and binds to gp91phox.

251

Microscopy images showed that upon Gyp-L stimulation, p47phox was considerably

252

up-regulated and accumulated at the membrane of plasma and giant vacuoles (Fig. 7B).

253

Besides, pretreatment with apocynin (ACN), a specific NOX2 inhibitor, considerably

254

reduced the NADPH oxidase activity (Fig. 7A). These results clearly suggested the

255

activation of NOX2. Disruption of lipid rafts by MβCD also inhibited the activation of NOX2,

256

which was consistent with a previous report that NOX2 localized to plasma membrane

257

lipid rafts for activation.35 In addition, inhibiting NOX2 activity by ACN and DPI remarkably

258

inhibited lysosome biogenesis and vacuolization (Fig. 7C). Nuclear translocation of

259

EGFP-TFEB caused by Gyp-L was also obviously impaired, whereas no influence of ACN

260

and DPI on Torin1-mediated nuclear targeting of TFEB was observed (Fig. 7D).

261

Furthermore, analyzing the nuclear accumulation of TFEB using western blot also

262

obtained the similar results (Fig. 7E). Because NOX2 activity generates and mediates the

263

sustained production of low levels of superoxide anions in the phagocytic lumen to cause

264

alkalization, we therefore examined the effects of several ROS scavengers on lysosome

265

production. Unexpectedly, reducing ROS production by NAC, TEMPOL and Trolox did not

266

diminish the inducing capacity of Gyp-L (Fig. 6I). Further works are required to determine

267

the mechanism through which NOX2 promotes TFEB nuclear translocation. Taken

268

together, these above results indicated that vacuole formation triggered by lipid

269

rafts-dependent Gyp-L endocytic entrance activates NOX2 to regulate endo/lysosome

7A).

Such

increment

was

reduced

by

a

NADPH

10


oxidase

inhibitor

Page 13 of 36


270

alkalization and TFEB-mediated lysosome biogenesis.

271 272

Raising lysosome pH enhances Gyp-L-mediated cell death

273

We finally analyzed the contribution of NOX2-mediated lysosome increment and their

274

normal function to Gyp-L-induced cell death. Different chemical inhibitors of lysosomal

275

proteases, including Z-FA-FMK (inhibiting cathepsin B), Z-FY-CHO (inhibiting cathepsin L)

276

and Cat-I (inhibiting cathepsin L, L2, S, K, B), were used and their inhibitory effects on the

277

enzymatic activities of cathepsins were firstly confirmed by measuring the activities of

278

cathepsin B and L (Fig. S6). However, pretreatment of these inhibitors did not ameliorate

279

Gyp-L-induced cell death (Fig. 8A), suggesting that Gyp-L-induced cell death did not

280

require the activation of cathepsin. On the contrary, raising lysosome pH by V-ATPase

281

inhibitors (bafilomycin A (Baf) and concanamycin A (CMA)) (Fig. 8B), or by the weak base

282

lysosomotropic amine CQ27 significantly enhanced the cytotoxicity of Gyp-L, which was

283

consistent with the relationship between the Gyp-L-induced abnormal lysosome

284

alkalization and cell death. Together, these results indicated that the maintenance of

285

lysosome acidic or proteolytic environment antagonizes Gyp-L-induced cell death.

286 287

Discussion

288

Screening and identifying anticancer compounds from active natural products are of

289

particular

290

pharmacological properties of G. pentaphyllum, including anti-inflammatory, anti-oxidative

291

and anticancer activities. In this study, we demonstrated that Gyp-L, a gypenoside

292

isolated

293

lysosome-associated cell death in esophageal cancer cells. We first confirmed that

294

vacuole formation in esophageal cancer cells induced by Gyp-L was abnormal enlarged

295

and alkalized lysosome, which originated from lipid-rafts dependent endocytosis. We also

296

deciphered the critical role for NOX2 in vacuolization, lysosome alkalization and

297

mTOR-independent TFEB activation. Accordingly, TFEB nuclear translocation-mediated

298

lysosome biogenesis and the maintenance of lysosome acidic partly antagonized

299

Gyp-L-triggered cell death (Fig. 9).

interest.

from

G.

Increasing

researches

pentaphyllum,

have

induced

demonstrated

lipid-raft

11


and

the

different

NOX2-mediated


300

Lysosome-associated cell death always accompanies vacuolization, which is

301

attributed to the non-canonical cell death including methuosis, paraptosis, oncosis, and

302

necroptosis.36-39 In most cases, the original resource of vacuole remains largely unclear

303

and is identified as the late endosome or lysosome in some studies.40-43 In the present

304

work, we demonstrated that Gyp-L induced the abnormal enlargement and alkalization of

305

lysosomes. Besides, such vacuolization derived from endosome-lysosome fusion but not

306

amphisome (Fig. 2A), the result of lysosome/autophagosome fusion. Indeed, as our

307

previous works showed,27,44 Gyp-L inhibited the fusion between autophagosome and

308

lysosome and had no effect on the endosomal trafficking pathway. These results were

309

consistent with the fact that destruction of endosomes or endo-lysosomes are common

310

reasons for the vacuolization.36 In addition, our results deciphered an important role of

311

lipid-rafts in vacuolization and lysosome associated cell death (Fig. 3). Previously the

312

lipid-rafts have been reported to control endosome dynamics through the MEK–ERK

313

pathway.45 Lipid rafts have also been implied to regulate lysosomal functions through their

314

interaction with several lysosome membrane proteins, such as vacuolar ATPase

315

(V-ATPase), chloride channels CLCN6 and CLCN7.28,46 Here we provided clear evidences

316

that disruption of lipid-rafts impairs vacuolization and lysosome associated cell death,

317

which was consistent with the finding that lysosomes usually contained higher levels of

318

lipid rafts in some lysosomal storage disease.47,48 Besides, we demonstrated an

319

enhanced expression of caveolin 1 by Gyp-L. Indeed, caveolin 1 has been reported to

320

promote the localization of lipid rafts at lysosome membranes and the disassembly of

321

V-ATPase.49 Therefore, lysosomal alkalization may partly be caused by the excessive

322

caveolin 1-mediated V-ATPase dysfunction. Furthermore, we demonstrated that the

323

activity of Ca2+ release-activated intracellular P2X4 channel, is not required for

324

endo-lysosome fusion and vacuolization. It has been suggested that the increment of

325

cytosol Ca2+ is essential for the fusion of intracellular organelles, partly through

326

P2X4-CaM pathway.29,50,51 Besides, we found that Gyp-L triggered the Ca2+ signals to

327

promote cell death.27,44 However, inhibition of Ca2+ or P2X4-CaM pathway did not affect

328

the fusion and vacuolization, implying the involvement of other Ca2+ independent

329

mechanisms. Considering that Gyp-L-induced vacuolization and fusion were inhibited by 12


Page 14 of 36

Page 15 of 36


330

Na+ ionophore (Fig. 5), it is interesting to investigate whether TPC2, another channel

331

localizing at lysosomal membrane to conduct Na+ and Ca2+ release from endo-lysosomes,

332

facilitates vesicle fusion.

333

Except for fusion, lysosome alkalization is currently a matter of considerable interest

334

and the NADPH oxidase NOX2 has been described to control the phagosome-endosome

335

pH.32,33 Herein we also demonstrated that Gyp-L induced the lysosome alkalization

336

through NOX2. Upon Gyp-L treatment, NOX2 was activated and recruited to the surface

337

of lysosomes (Fig. 7A and 7B), possibly mobilized by lipid rafts.35 In contrast, inhibition of

338

NOX2 significantly reduced vacuole formation. More importantly, NOX2 exerted such

339

function not through the production of ROS, as ROS scavenger has no influence on

340

vacuolization. Further works are required to test whether NOX2 controls lysosome pH by

341

modulating luminal redox environment.34 In addition, we found that NOX2 mediated the

342

regulation of lysosome biogenesis via TFEB, which has not been reported in the literature.

343

Recently it was reported that activation of NOX2 impaired both autophagy and lysosome

344

formation through activated PI3K/Akt/mTOR pathway.52 We revealed here that NOX2

345

induced lysosome biogenesis through mTOR-independent TFEB activation, which was

346

not associated with ROS production. The subcellular localization of NOX2 within lipid-rafts

347

and lysosome surface may provide a spatially privileged communication,53 facilitating

348

NOX2-mediated directly or indirectly TFEB activation. Considering that NOX1 has the

349

ability to bind with 14-3-3 proteins,54 it is therefore interesting to postulate that NOX2 may

350

interact with 14-3-3 proteins and compete for TFEB-14-3-3 interaction to release and

351

activate TFEB. Furthermore, the phenomena that TFEB nuclear translocation acted as a

352

consequence of vacuolization suggested that TFEB exerted an attempt to restore the

353

lysosomal imbalance and cell death by up-regulating the expression of several lysosome

354

proteins.55 Consistently, CQ, a lysosomal trophic agent that raises the lysosome pH, has

355

also been reported to induce vacuolization-associated cell death and TFEB translocation

356

in tumor cells.11,56 CQ is part of a large set of compounds leading to phospholipidosis (for

357

instance, accumulation of lipid lamellar bodies in endosomal/lysosomal compartment,

358

possibly to the alkalization), which might explain the fact that CQ significantly enhanced

359

the cytotoxicity of Gyp-L.27 Finally, it is also interesting to investigate the interaction 13



360

between vacuolization and TFEB-mediated lysosome biogenesis,57 as well as the function

361

of NOX2 in other circumstances. Alternatively, NOX2-mediated lysosome biogenesis

362

provides important insights into lysosomal adaptation to signals (e.g. pathogenic microbe

363

infection) other than nutrients.

364

In conclusion, our reports demonstrated that Gyp-L induced lysosome biogenesis,

365

alkalization and lysosome-associated cell death through NOX2-TFEB axis. Our results

366

also indicated that in the case of cells with abnormal endo-lysosome, TFEB is activated

367

and translocated into the nucleus to promote the expression of lysosome related genes

368

and attempt to alleviate the imbalance of lysosome to avoid lysosome-associated cell

369

death. In addition, the identification of drug-like compounds from natural plants that can

370

trigger lysosome-associated cell death, such as Gyp-L, may provide new opportunities to

371

overcome drug resistance in the treatment of cancers.

372 373

Methods and Materials

374

Cell lines, Chemicals and Antibodies

375

ECA-109 cells (TCHu 69) and TE-1 cells (TCHu 89) were purchased from the Cell

376

Bank of China Science Academy (Shanghai, China) and cultured in RPMI 1640 (Gibco).

377

HUVEC cells (CRL-1730, ATCC) were cultured in DMEM (Gibco).

378

CQ (C6628), TUDCA (T0557), 2-APB (D9754), methyl-β-cyclodextrin (C4555),

379

Monensin (m5273), Nigericin (481990), Valinomycin (V0627), CHX (C7698), Gramicidin A

380

(G5002), Ionomycin calcium salt (407953), Cholesterol (C4951), NAC (A9165),

381

concanamycin A (C5275), W7 (A3281), U0126 (U120) and SP600125 (S5567) were

382

purchased from Sigma-Aldrich. BAPTA-AM (B018) was from Dojindo. Z-VAD-FMK

383

(S7023), Dynasore (S8047), Lovastatin (S2061), Phloretin (S2342), Bafilomycin A1

384

(S1413), Rapamycin (S1039), TEMPOL (S2910), 3-MA (S2767), Torin 1 (S2827), FK506

385

(S5003) and Staurosporine (S1421) were purchased from Selleck. Trolox (H828379) was

386

purchased from Macklin. 5-(N,N-dimethyl) amiloride hydrochloride (DMA) (sc-202459),

387

Fumonisin B1 (sc-201395), PI(4,5)P2 (sc-221508), Diphenyleneiodonium chloride (DPI)

388

(sc-202584) and Apocynin (ACN) (sc-203321) were from Santa Cruz. U18666A (662015) 14


Page 16 of 36

Page 17 of 36


389

was from Merck. All the cytotoxicities of chemical inhibitors were tested and used in a

390

concentration without affecting cell viability.

391

Anti-caveolin

1

(3267),

anti-mTOR

392

anti-phospho-4E-BP1

393

anti-phospho-p70-S6K (Thr389)

394

anti-TFEB (37785), anti-p47phox (4301), anti-RagA (4357), anti-RagB (8150), anti-RagC

395

(5466), anti-RagD (4470), anti-LAMTOR1 (8975), anti-LAMTOR2 (8145), anti-LAMTOR3

396

(8168), anti-Histone H3 (4499), anti-GAPDH (5174), anti-mouse IgG (7076) and

397

anti-rabbit IgG (7074) HRP-linked antibodies were purchased from Cell signaling

398

Technology.

(2855),

(2983),

anti-phospho-mTOR

anti-phospho-p70-S6K

(Ser371)

(2974), (9208),

(9234), anti-LAMP1 (9097), anti-TFE3 (14779),

399 400

Detection of cell death

401

MTT (Sigma-Aldrich, M2128) assay was performed to examine the effects of Gyp-L,

402

all the chemical inhibitors and siRNAs on cell viability as previously described.27

403

Esophageal cancer cells were treated with different concentrations of drugs for 24 h. Then

404

10 µl MTT (5 mg/ml) was added and incubated for another 4 h. Finally 100 µl DMSO was

405

added to dissolve the insoluble formazan product before being measured at 490 nm by a

406

multiscanner autoreader (M450, Bio-rad, USA).

407 408

Quantitative Real-Time PCR (RT-PCR)

409

TRIzol reagent (Invitrogen) was used to extract total RNA. Total RNA (100 ng) was

410

reverse transcribed and analyzed by using a Bio-Rad CFX96 real-time PCR system. The

411

primer sequences of genes are available in supplemental table S1. The mRNA expression

412

level of GAPDH was used as control.

413 414

Western blot analysis

415

As described previously,27 total cell lysates were extracted by using RIPA buffer (Beyotime,

416

P0013B) supplemented with 1 mM PMSF. A protein extraction kit was used to

417

disassociate nuclear and cytoplasmic proteins according to manufacturer’s protocol

418

(Beyotime, P0027). The lysates were electrophoresed by SDS-PAGE and immunoblotted 15



Page 18 of 36

419

with primary antibodies. The targeted proteins were visualized with specific detection

420

reagents (Thermo, 34080). Quantification of each protein relate to GAPDH or H3 was

421

measured by densitometric analysis using Image J software.

422 423

Acridine orange (AO) Staining

424

ECA-109 cells pretreated with Gyp-L for 12 h or 24 h were incubated with 5 µg/ml AO

425

(Sigma-Aldrich,

426

phosphate-buffered saline (PBS) buffer, fluorescent photographs were obtained using a

427

fluorescence microscope (Nikon Ti-u). The red or green fluorescence intensity of AO was

428

also calculated by flow cytometry assay (BD FACS Calibur).

A6014)

for

another

0.5

h.

After

three

times

washing

with

429 430

Lysosome biogenesis assay

431

Lysosome biogenesis was evaluated using LysoTracker Red (Beyotime, C1043) staining.

432

The cells pretreated with inhibitors and Gyp-L (80 µg/ml) for indicated times were stained

433

with 50 nM LysoTracker Red for another 0.5 h. After extensive washing, cells were

434

suspended in PBS and images were observed using a fluorescence microscope (Nikon

435

Ti-u). The fluorescence intensity of LysoTracker Red was calculated using software Image

436

J. For flow cytometry assay, cells were transferred into tubes, stained with LysoTracker

437

Red and quantified by flow cytometry using a FACS machine (BD FACS Calibur, USA).

438 439

Dextran uptake Assay

440

At 24 h after Gyp-L treatment, cells were loaded with FITC-labelled 10-kD dextran (1

441

mg/ml) for 3 h and chased for another 2 h. The cells were then fixed immediately, and

442

images were taken using a fluorescence microscopy (Nikon Ti-u) or analyzed by flow

443

cytometry (BD FACS Calibur, USA) .

444 445

RNA interference and transfection

446

ECA-109 or TE-1 cells were transfected with 2 µg siRNA targeting TFEB (sc-38509, Santa

447

Cruz), P2X4 (sc-42569, Santa Cruz) or control nontargeting siRNA, as well as the

448

transfection of 2 µg plasmids GFP-LAMP1 (C10507, Invitrogen), EGFP-TFEB (38119, 16


Page 19 of 36


449

Addgene) or EGFP-TFE3 (38120, Addgene), using Lipofectamine 3000 (Invitrogen,

450

L3000015) according to the manufacturer’s instructions.

451 452

NADPH oxidase activity

453

The lucigenin-based chemiluminescence assay was used to assess the NADPH oxidase

454

activity. Equal amounts of proteins were sequentially added with reaction buffer containing

455

10

456

000000010107824001), incubated at 37 °C for 10 min. Luminescence was measured with

457

a Fluostar Optima microplate reader (Molecular Devices SpectraMax I3, USA). The

458

NADPH oxidase activity was calculated as Relative light unit (RLU)/mg protein.

µM

lucigenin

(Santa

Cruz,

2315-97-1)

and

100

µM

NADPH

(Sigma,

459 460

Cathepsin activity analysis

461

The catalytic activities of cathepsin B (CSTB, Abnova, KA0766) and cathepsin L (CSTL,

462

Abcam, ab65306) were measured according to the instruction. In brief, add 50-200 µg cell

463

lysates (in 50 µl of cell lysis buffer) to a 96-well plate, and add 50 µl reaction buffer to each

464

samples, mix with 2 µl substrate (200 µM final concentration)(Ac-RR-AFC for CSTB and

465

Ac-FR-AFC for CSTL). Then incubate the reaction mixture at 37 °C for 2 h. The samples

466

were read in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission

467

filter.

468 469

Lysosomal pH measurement

470

LysoSensor Yellow/Blue DND-160 (Thermo Fisher, L7545), a ratiometric probe, was used

471

to stain lysosome and measure lysosomal pH. The LysoSensor dye exhibits blue

472

fluorescence in neutral environments and green/yellow fluorescence in acidic conditions.

473

ECA-109 cells were firstly incubated with LysoSensor Yellow/Blue DND-160 (1 µM) for 5

474

min and then washed with PBS. The fluorescence of cells was detected by fluorescence

475

microscopy exciting at 365 nm and light emitting at 450 nm (Blue) or 510 nm (green) in

476

response to excitation at 365 nm. In addition, the lysosomal pH was quantitatively

477

measured by a spectrophotometer.

478 17



479

Immunofluorescence analysis

480

The cells were washed twice with PBS, fixed in 4% paraformaldehyde (Coolaber, SL1830)

481

for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 5-10 min. After blocking in

482

PBS containing 5% bovine serum albumin (Gibco, 1027-106), the cells were probed with

483

anti-LAMP1 antibody and appropriate Alexa Fluor-conjugated secondary antibodies. In

484

addition, cell nuclei was stained with DAPI-PBS (1 mg/ml) for 15 min. Fluorescence

485

images were obtained using a Zeiss LSM510 Meta confocal system (Carl Zeiss

486

Microimaging, USA).

487

For co-localization assay of LAMP1 and LysoTracker Red, ECA-109 cells were firstly

488

transfected with LAMP1-GFP (2 µg) or GFP-Vector for 24 h and were then incubated with

489

Gyp-L for another 24 h. The cells were then stained with LysoTracker Red and viewed by

490

fluorescence microscopy. For neutral red staining, the cells pretreated with Gyp-L were

491

stained by neutral red solution (Beyotime, C0125). After incubation for 10 min, cells were

492

washed and observed under brightfield microscope. For lipid rafts staining, cells were

493

loaded with1 µg/ml FITC-conjugated cholera toxin beta subunit (CtxB) (C1655,

494

Sigma-Aldrich) for 15 min on ice, removed by PBS washing and incubated at 37°C for 30

495

min.

496

For cholesterol staining, Filipin III (Sigma, F4767) was used to label cholesterol. After

497

treatments, the cells were fixed with 4% paraformaldehyde for 10 min, quenched in 50

498

mM NH4Cl for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. Then the cells

499

were blocked in the solution containing 2% BSA (Sigma, A7906), and stained with 100

500

µg/ml of filipin III for 45 min. Fluorescence photographs were analyzed with a microscopy.

501 502

Statistical analysis

503

Data were expressed as mean value ± standard deviation from three independent

504

experiments. The statistical analyses were performed using the Student’s two-tailed t-test,

505

and the statistically significance sets as **, p < 0.01; *, p < 0.05.

506 507 508

Acknowledgments 18


Page 20 of 36

Page 21 of 36


509

This work was funded by the National Natural Science Foundation of China (No.

510

31500285, 31540012, 30570421 and 81603341), the Natural Science Foundation of

511

Guangdong Province (No. 2015A030310529, the Shenzhen strategic emerging industry

512

development

513

KQCX20140522111508785, CXZZ20150601110000604, JCYJ20140414170821276 and

514

JCYJ20150324141711557) and the China Postdoctoral Science Foundation (Grant

515

2015M570726).

project

funding

(ZDSYS201506031617582,

SFG2013-180,

516 517

Supporting Information description

518

The Supplemental information includes six Figures.

519 520

Conflicts of Interest

521

The authors declare no potential conflicts of interest.

522 523

References

524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545

1.

Mohammad, R.M.; Muqbil, I.; Lowe, L.; Yedjou, C.; Hsu, H.Y.; Lin, L.T.; et al. Broad targeting of resistance to apoptosis in cancer. Semin Cancer Biol 2015. 35 Suppl:S78-103.

2.

Yoshida, G.J. Therapeutic strategies of drug repositioning targeting autophagy to induce cancer cell death: from pathophysiology to treatment. J Hematol Oncol 2017. 10(1):67.

3.

Yu, F.; Chen, Z.; Wang, B.; Jin, Z.; Hou, Y.; Ma, S.; Liu, X. The role of lysosome in cell death regulation. Tumour Biol 2016. 37(2):1427-36.

4.

Settembre, C.; Fraldi, A.; Medina, D.L.; Ballabio, A. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat Rev Mol Cell Biol 2013. 14: 283–296.

5.

Xu, H.; Ren, D. Lysosomal physiology. Annu Rev Physiol 2015. 77: 57–80.

6.

Zhitomirsky, B.; Assaraf YG. Lysosomes as mediators of drug resistance in cancer. Drug Resist Updat 2016. 24:23-33.

7.

Martina, J.A.; Diab, H.I.; Lishu, L.; Jeong-A, L.; Patange, S.; Raben, N.; et al. The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Sci Signal 2014. 7: ra9.

8.

Settembre, C.; Di Malta, C.; Polito, V.A.; Garcia Arencibia, M.; Vetrini, F.; Erdin, S.; et al. TFEB links autophagy to lysosomal biogenesis. Science 2011. 332:1429–1433.

9.

Sardiello, M.; Palmieri, M.; di Ronza, A.; Medina, D.L.; Valenza, M.; Gennarino, V.A.; et al. A gene network regulating lysosomal biogenesis and function. Science 2009. 325: 473–477.

10. Settembre, C.; Zoncu, R.; Medina, D.L.; Vetrini, F.; Erdin, S.; Erdin, S.; et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J 2012. 31: 1095–1108. 11. Roczniak-Ferguson, A.; Petit, C.S.; Froehlich, F.; Qian, S.; Ky, J.; Angarola, B.; et al. The 19



546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589

transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci Signal 2012. 5: ra42. 12. Martina, J.A.; Chen, Y.; Gucek, M.; Puertollano, R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 2012. 8: 903–914. 13. Martina, J.A.; Puertollano, R. Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. J Cell Biol 2013. 200: 475–491. 14. Sancak, Y.; Bar-Peled, L.; Zoncu, R.; Markhard, A.L.; Nada, S.; Sabatini, D.M. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 2010. 141(2):290-303. 15. Medina, D.L. Di Paola, S.; Peluso, I.; Armani, A.; De Stefani, D.; Venditti, R.; et al. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nat Cell Biol 2015. 17: 288– 299. 16. Li, Y.; Xu, M.; Ding, X.; Yan, C.; Song, Z.; Chen, L.; et al. Protein kinase C controls lysosome biogenesis independently of mTORC1. Nat Cell Biol 2016. 18(10):1065-77. 17. Giatromanolaki, A.; Sivridis, E.; Kalamida, D.; Koukourakis, M.I. Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis. Clin Breast Cancer 2016. pii: S1526-8209(16)30497-9. 18. Blessing, A.M.; Rajapakshe, K.; Reddy Bollu, L.; Shi, Y.; White, M.A.; Pham, A.H.; et al. Transcriptional regulation of core autophagy and lysosomal genes by the androgen receptor promotes prostate cancer progression. Autophagy 2017. 13(3):506-521. 19. Zhang, J.; Wang, J.; Xu, J.; Lu, Y.; Jiang, J.; Wang, L.; et al. Curcumin targets the TFEB-lysosome pathway for induction of autophagy. Oncotarget 2016. 7(46):75659-75671. 20. Klein, K.; Werner, K.; Teske, C.; Schenk, M.; Giese, T.; Weitz, J.; et al. Role of TFEB-driven autophagy regulation in pancreatic cancer treatment. Int J Oncol 2016. 49(1):164-72. 21. Yonekawa, T.; Gamez, G.; Kim, J.; Tan, A.C.; Thorburn, J.; Gump, J.; et al. RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis. EMBO Rep 2015. 16(6):700-8. 22. Shi, L.; Pi, Y.; Luo, C.; Zhang, C.; Tan, D.; Meng, X. In vitro inhibitory activities of six gypenosides on human liver cancer cell line HepG2 and possible role of HIF-1α pathway in them. Chem Biol Interact. 2015, 238, 48-54. 23. Liu, J.S.; Chiang, T.H.; Wang, J.S.; Lin, L.J.; Chao, W.C.; Inbaraj, B.S.; Lu, J.F.; Chen BH. Induction of p53-independent growth inhibition in lung carcinoma cell A549 by gypenosides. J Cell Mol Med. 2015, 19, 1697-1709. 24. Yan, H.; Wang, X.; Niu, J.; Wang, Y.; Wang, P.; Liu, Q. Anti-cancer effect and the underlying mechanisms of gypenosides on human colorectal cancer SW-480 cells. PLoS One 2014, 9, e95609. 25. Cheng, T.C.; Lu, J.F.; Wang, J.S.; Lin, L.J.; Kuo, H.I.; Chen, B.H. Antiproliferation effect and apoptosis mechanism of prostate cancer cell PC-3 by flavonoids and saponins prepared from Gynostemma pentaphyllum. J Agric Food Chem. 2011, 59, 11319-11329. 26. Xie, Z.H.; Liu, W.; Huang, H.; Slavin, M.; Zhao, Y.; Whent, M.; Blackford, J.; Lutterodt, H.; Zhou, H.P.; Chen, P.; Wang, T.T.Y.; Wang, S.; Yu, L. Chemical Composition of Five Commercial Gynostemma pentaphyllum Samples and Their Radical Scavenging, Antiproliferative, and Anti-inflammatory Properties. J Agric Food Chem. 2010, 58, 11243–11249. 27. Liao, C.; Zheng, K.; Li, Y.; Xu, H.; Kang, Q.; Fan, L.; et al. Gypenoside L inhibits autophagic flux and 20


Page 22 of 36

Page 23 of 36


590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633

induces cell death in human esophageal cancer cells through endoplasm reticulum stress-mediated Ca2+ release. Oncotarget 2016. 7(30):47387-47402. 28. Bosch, M.; Mari, M.; Herms, A.; Fernandez, A.; Fajardo, A.; Kassan, A.; et al. Caveolin-1 deficiency causes cholesterol dependent mitochondrial dysfunction and apoptotic susceptibility. Curr Biol 2011. 21:681-6. 29. Cao, Q.; Zhong, X.Z.; Zou, Y.; Murrell-Lagnado, R.; Zhu, M.X.; Dong, X.P. Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion. J Cell Biol 2015. 209(6):879-94. 30. Florey, O.; Gammoh, N.; Kim, S.E.; Jiang, X.; Overholtzer, M. V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation. Autophagy 2015. 11(1):88-99. 31. Schorn, C.; Frey, B.; Lauber, K.; Janko, C.; Strysio, M.; Keppeler, H.; et al. Sodium overload and water influx activate the NALP3 inflammasome. J Biol Chem 2011. 286:35-41. 32. Savina, A.; Jancic, C.; Hugues, S.; Guermonprez, P.; Vargas, P.; Moura, I.C.; et al. NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 2006. 126(1):205-18. 33. Savina, A.; Peres, A.; Cebrian, I.; Carmo, N.; Moita, C.; Hacohen, N.; et al. The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation selectively in CD8(+) dendritic cells. Immunity 2009. 30(4):544-55. 34. Rybicka, J.M.; Balce, D.R.; Khan, M.F.; Krohn, R.M.; Yates, R.M. NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes. Proc Natl Acad Sci U S A 2010. 107(23):10496-501. 35. Oakley, F.D.; Smith, R.L.; Engelhardt, J.F. Lipid rafts and caveolin-1 coordinate interleukin-1beta (IL-1beta)-dependent activation of NFkappaB by controlling endocytosis of Nox2 and IL-1beta receptor 1 from the plasma membrane. J Biol Chem 2009. 284(48):33255-64. 36. Overmeyer, J.H.; Kaul, A.; Johnson, E.E.; Maltese, W.A. Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis. Mol Cancer Res 2008. 6: 965-977. 37. Weerasinghe, P.; Buja, L.M. Oncosis: an important non-apoptotic mode of cell death. Exp Mol Pathol 2012. 93: 302-308. 38. Overholtzer, M.; Mailleux, A.A.; Mouneimne, G.; Normand, G.; Schnitt, S.J.; King, R.W.; et al. A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell 2007. 131: 966-979. 39. Sperandio, S.; Poksay, K.; de Belle, I.; Lafuente, M.J.; Liu, B.; Nasir, J.; et al. Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix. Cell Death Differ 2004. 11: 1066-1075. 40. Overmeyer, J.H.; Young, A.M.; Bhanot, H.; Maltese, W.A. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells. Mol Cancer 2011. 10: 69. 41. Nara, A.; Aki, T.; Funakoshi, T.; Uemura, K. Methamphetamine induces macropinocytosis in differentiated SH-SY5Y human neuroblastoma cells. Brain Res 2010. 1352:1-10. 42. Papini, E.; de Bernard, M.; Milia, E.; Bugnoli, M.; Zerial, M.; Rappuoli, R.; et al. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc Natl Acad Sci USA 1994. 91: 97209724. 43. Genisset, C.; Puhar, A.; Calore, F.; de Bernard, M.; Dell’Antone, P.; Montecucco, C. The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes. Cell Microbiol 2007. 9: 1481-1490. 21



634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671

44. Zheng, K.; Liao, C.; Li, Y.; Fan, X.; Fan, L.; Xu, H.; et al. Gypenoside L.; Isolated from Gynostemma

672

Figure Legends

673

Fig.1. Lysosome biogenesis induced by Gyp-L: (A) Gyp-L enhances LysoTracker Red

674

staining. ECA-109 cells were treated for 12 h with Gyp-L (80 µg/ml) and stained with

675

LysoTracker Red. Scale Bars: 20 µm. (B) Quantifications of Gyp-L-induced lysosomes

pentaphyllum.; Induces Cytoplasmic Vacuolation Death in Hepatocellular Carcinoma Cells through Reactive-Oxygen-Species-Mediated Unfolded Protein Response. J Agric Food Chem 2016. 64(8):1702-11. 45. Nada, S.; Hondo, A.; Kasai, A.; Koike, M.; Saito, K.; Uchiyama, Y.; et al. The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK-ERK pathway to late endosomes. EMBO J 2009. 28:477-89. 46. Foster, L.J.; De Hoog, C.L.; Mann, M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A 2003. 100:5813-8. 47. Simons, K.; Gruenberg, J. Jamming the endosomal system: lipid rafts and lysosomal storage diseases. Trends Cell Biol 2000. 10:459-62. 48. Hayer, A.; Stoeber, M.; Ritz, D.; Engel, S.; Meyer, H.H.; Helenius, A. Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation. J Cell Biol 2010. 191:615-29. 49. Shi, Y.; Tan, S.H.; Ng, S.; Zhou, J.; Yang, N.D.; Koo, G.B.; et al. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy. Autophagy 2015. 11(5):769-84. 50. Pryor, P.R.; Mullock, B.M.; Bright, N.A.; Gray, S.R.; Luzio, J.P. The role of intraorganellar Ca2+ in late endosome–lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. J Cell Biol 2000. 149:1053–1062. 51. Morgan, A.J.; Platt, F.M.; Lloyd-Evans, E.; Galione, A. Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease. Biochem J 2011. 439:349–374. 52. Pal, R.; Palmieri, M.; Loehr, J.A.; Li, S.; Abo-Zahrah, R.; Monroe, T.O.; et al. Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy. Nat Commun 2014. 5:4425. 53. Whitehead, N.P.; Yeung, E.W.; Froehner, S.C.; Allen, D.G. Skeletal muscle NADPH oxidase is increased and triggers stretch-induced damage in the mdx mouse. PLoS ONE 2010. 5: e15354. 54. Kim, J.S.; Diebold, B.A.; Babior, B.M.; Knaus, U.G.; Bokoch, G.M. Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding. J Biol Chem 2007. 282(48):34787-800. 55. Zhitomirsky, B.; Assaraf, Y.G. Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis. Oncotarget 2017. doi: 10.18632/oncotarget.15155.Epub ahead of print 56. Zhou, T.; Ye, L.; Bai, Y.; Sun, A.; Cox, B.; Liu, D.; et al. Autophagy and Apoptosis in Hepatocellular Carcinoma Induced by EF25-(GSH)2: A Novel Curcumin Analog. PLoS ONE 2014. 9(9): e107876. 57. Lin, J.; Shi, S.S.; Zhang, J.Q.; Zhang, Y.J.; Zhang, L.; Liu, Y.; et al. Giant Cellular Vacuoles Induced by Rare Earth Oxide Nanoparticles are Abnormally Enlarged Endo/Lysosomes and Promote mTOR-Dependent TFEB Nucleus Translocation. Small 2016. 12(41):5759-5768.

22


Page 24 of 36

Page 25 of 36


676

(fold induction of LysoTracker Red staining) were analyzed by flow cytometry and showed

677

in the right panel. n=3 independent experiments. A representative cytofluorimetry graph of

678

ECA-109 cells was shown in left panel. (C,D) Gyp-L enhances the expression of LAMP1

679

in a dose-dependent manner. ECA-109 cells were treated with different concentrations of

680

Gyp-L (0, 20, 40, 60, 80 µg/ml) for 24 h and cell lysates were subjected to western blot.

681

ImageJ densitometric analysis of the LAMP1/GAPDH from LAMP1 immunoblots (mean ±

682

SD of 3 independent experiments). (E) AO staining assay. ECA-109 cells were stained

683

with AO (5 µg/ml) for 30 min and analyzed by flow cytometry. The ratio of red and green

684

was also calculated.

685 686

Fig.2. Gyp-L-induced vacuoles are alkalized lysosomes: (A) The representative

687

fluorescent microscopy image of ECA-109 cells transfected with LAMP-GFP and stained

688

with LysoTracker Red. The cells were transfected with LAMP-GFP for 24 h before the

689

Gyp-L treatment and LysoTracker Red staining. Scale Bars: 10 µm. (B) The

690

representative pictures of ECA-109 cells treated with Gyp-L (80 µg/ml) for 12 h and

691

stained with neutral red. Schematic diagram of pH confirmation of the vacuoles by

692

overlapping the pH range of the dyes is shown in the lower panel. (C) ECA-109 cells were

693

treated with Gyp-L for 12 h, and stained with LysoSensor Yellow/Blue DND-160 (1 µM) for

694

5 min; the fluorescence of live cells were detected by a fluorescence microscopy. The pH

695

was determined and calculated by a spectrophotometer. (D) Effect of CHX on

696

vacuolization and lysosome biogenesis. ECA-109 and TE-1 cells were treated with Gyp-L

697

(80 µg/ml) in the presence or absence of CHX (5 µg/ml) for 12 h to detect the lysosome

698

production, or for 24 h to test the cell viability. * indicates a significant difference from the

699

controls. **, p < 0.01.

700 701

Fig. 3. Lipid rafts is involved in vacuolization and cell death: (A,B) MβCD reduces the

702

vacuoles formation and lysosome production. ECA-109 cells and TE-1 cells were treated

703

with Gyp-L in the presence or absence of MβCD (1 mM). * indicates a significant

704

difference from the controls. **, p < 0.01. Scale Bars: 10 µm. (C,D) Effects of MβCD (1 mM)

705

and U18666A (5 µg/ml) on Gyp-L-induced cell death (C) or AO staining (D). (E) Lipid rafts 23



706

staining. ECA-109 cells incubated with the FITC-conjugated CtxB (30 µg/ml) were fixed

707

and visualized by fluorescent microscopy. (F) Gyp-L up-regulates the expression of

708

Caveolin 1 in a dose-dependent manner. ECA-109 cells were treated with different

709

concentrations of Gyp-L (0, 20, 40, 60, 80 µg/ml ) for 24 h and cell lysates were subjected

710

to western blot for Caveolin 1 and GAPDH. ImageJ densitometric analysis of the

711

Caveolin1/GAPDH from caveolin 1 immunoblots (mean ± SD of 3 independent

712

experiments). (G) Cholesterol indicator filipin III staining of Gyp-L and MβCD-treated cells.

713

(H) Effect of FB 1 (30 µM) on cell viability.

714 715

Fig. 4. Cholesterol replenishment rescues the lowering effect of MβCD. (A) ECA-109 cells

716

were pretreated with MβCD (1 mM) for 1 h, then incubated in the presence or absence of

717

cholesterol (CHO, 30 mg/ml) and then stained with CTxB. Scale Bars: 10 µm. (B)

718

ECA-109 cells were treated with Gyp-L and MβCD in the presence or absence of CHO

719

and then stained with filipin. (C,D) Cholesterol replenishment overcomes the effect of

720

MβCD disruption on vacuolization, lysosome biogenesis and cell death. (E,F) Lovastatin

721

(10 µM) has slight effects on Gyp-L treatment. (G) The mRNA expression levels of several

722

genes involved in cholesterol de novo synthesis. ACC: Acetyl-CoA carboxylase; SREBP:

723

Sterol-regulatory element binding protein; FASN: fatty acid synthase; HMGCR: HMG-CoA

724

reductase.

725 726

Fig. 5. Na+ ionophore inhibits Gyp-L-induced vacuolization and cell death. (A,B) ECA-109

727

cells and TE-1 cells were treated with Gyp-L in the presence or absence of monensin

728

(Mon, 10 µM ), nigericin (Nig, 1.5 µM), valinomycin (Val,10 µM) gramicidin A (Gra, 5 µM)

729

and ionomycin (Ion, 2µM) for 12 h or 24 h.

730 731

Fig.6. Gyp-L induces TFEB nuclear translocation. (A) Gyp-L induces the expression of

732

lysosomal genes. n=3 independent experiments; comparisons were made between

733

DMSO and Gyp-L treatment. (B,C) Images of the subcellular locations of TFEB-EGFP or

734

TFE3-EGFP in ECA-109 cells treated with Gyp-L, Torin1 (1 µM) or CQ (20 µM). Scale

735

Bars: 10 µm. Quantification of nuclear translocation of TFEB or TFE3 are shown in the 24


Page 26 of 36

Page 27 of 36


736

right panel. n = 3 independent experiments. (D) Subcellular fractionations and western

737

blotting show that Gyp-L treatment increases the nuclear abundance of TFEB and

738

p-mTOR. (E) Quantification of lysosomes (fold induction of LysoTracker staining) of

739

ECA-109 cells or TE-1 cells treated with TFEB siRNA and Gyp-L or Torin1. (F) Inhibition of

740

mTOR does not affect lysosome production. The cells were treated with Gyp-L in the

741

presence or absence of 3-MA (10 µM), Rapamycin (1 µM) and U0126 (10 µM) for 12 h. (G)

742

Western blots showing the activation of mTOR signal by Gyp-L treatment in a

743

dose-dependent manner. Different concentrations of Gyp-L (0, 20, 40, 60, 80, 100 µg/ml)

744

were used. (H) Schematic illustration of possible signals regulating TFEB nuclear

745

translocation. (I) Effect of chemical inhibitors on Gyp-L-induced lysosome biogenesis. The

746

cells were treated with Gyp-L and 2-APB (20 µM), TUCDA (40 µM), NAC (5 mM),

747

TEMPOL (2 mM), FK506 (5 µM), PI(4,5)P2 (1 µM), SP600125 (10 µM), BAPTA-AM (10

748

µM) and Staurosporine (100 nM) for 12 h. (J) Western blotting of the expression of several

749

subunits of mTOR complex after Gyp-L treatment.

750 751

Fig. 7. NOX2 promotes TFEB activation. (A) Relative activity of NADPH oxidase.

752

ECA-109 cells were treated with Gyp-L and ACN (10 µM) or DPI (10 µM) for 12 h. (B)

753

Subcellular location of NOX2 subunit p47phox. Scale Bars: 10 µm. (C-E) ACN and DPI

754

reduce lysosome production and vacuolization (C), or the percentage of cells with nuclear

755

TFEB-EGFP (D), or nuclear accumulation of TFEB (E). ImageJ densitometric analysis of

756

the TFEB/GAPDH from three immunoblots (mean ± SD of 3 independent experiments).

757 758

Fig. 8. Raising lysosomal pH enhances cell death. (A) Cell viability is not affected by the

759

inhibition of lysosomal cathepsins by chemical inhibitors Z-FA-FMK (10 µM), Z-FY-CHO

760

(10 µM) and Cat-I (10 µM). (B) Raising lysosomal pH by Baf (75 nM) or CMA (2 nM)

761

enhances cell death.

762 763

Fig. 9. Schematic illustration of the involvement of NOX2-mediated lysosome alkalization

764

and TFEB activation in lysosome-associated cell death in esophageal cancer cells. Gyp-L

765

initially induces the lipid rafts-dependent vacuolization, which is derived from 25



766

endosome-lysosome fusion and alkalized through NOX2 activation, and subsequently

767

causes the NOX2-mediated TFEB activation. Then the nuclear translocation of TFEB

768

induces lysosome biogenesis attempting to alleviate the imbalance of lysosome to avoid

769

lysosome-associated cell death.

770 771 772

26


Page 28 of 36

Page 29 of 36


Figures

Fig.1

27



Fig. 2

28


Page 30 of 36

Page 31 of 36


Fig.3

29



Fig. 4

30


Page 32 of 36

Page 33 of 36


Fig. 5

31



Fig. 6

32


Page 34 of 36

Page 35 of 36


Fig. 7

33



Fig. 8

Fig. 9

34


Page 36 of 36

NOX2-Mediated TFEB Activation and Vacuolization Regulate

Recommend Documents