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[Autophagy 5:4, 451-460; 16 May 2009]; ©2009 Landes Bioscience

Cathepsins have long been considered as housekeeping mole-cules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

Introduction

Lysosomes, which mediate the turnover of normal and abnormal macromolecules and organelles, are involved in endocytosis, exocytosis, inactivation of pathogens and antigen processing and presentation.1 A previous study2 and recent works have described a pathway of apoptotic cell death involving lysosomal disruption with release of proteolytic enzymes into the cytosol.3,4 Lysosomal proteases, also called cathepsins, represent the largest group of prote-olytic enzymes in the lysosomes. Among the human cathepsins, cat L, B and D are ubiquitously expressed and are the most abundant in the lysosomes. They participate in certain programmed cell death pathways from different stimulations, including p53-induced apop-tosis, oxidative stress and growth factor deprivation.5-7

Resveratrol (3,4'-trihydroxystilbene, RSV), a phytoalexin present in significant concentrations in red wine, grapes and nuts, has been demonstrated to exert a variety of biological actions in humans. Several studies demonstrated that RSV is an effective antioxidant8 with antineoplastic potentials for cell cycle arrest and apoptosis.9-11 Furthermore, RSV was recently shown to induce cell death through a mechanism involving autophagy in ovarian and colon cancer cell lines.12,13

Autophagy is a lysosomal degradation pathway for cytoplasmic material, which is activated during stress conditions, such as amino acid starvation or viral infection.14 Mammalian cells use autophagy during short periods of starvation to degrade nonessential cellular components in order to liberate nutrients for vital biosynthetic reactions. Autophagy also plays a role in development, growth regula-tion, cancer and longevity.14 The proteolytic activities of lysosomal proteases, such as cat L, B, D, are involved in the execution of autophagy.15,16

Although RSV exerts antiproliferative and proapoptotic activities in various cancer cell types, the underlying mechanisms and types of proteases involved in autophagy remain unclear. In addition, RSV’s effect on uterine cervical cancer cells is not yet known. Squamous cell carcinoma antigen (SCCA) has been used as a serologic tumor marker in uterine cervix cancer.17 One of the isoform genes, SCCA 1 has been shown to neutralize the cysteine proteinases, cat L, K and S,18 and attenuates apoptosis induced by anticancer drugs, TNF-a or human interleukin-2-activated natural killer (NK) cells.19

*Correspondence to: Ai-Li Shiau; Department of Microbiology and Immunology; National Cheng Kung University Medical College; 1 Dashiue Road; Tainan 701 Taiwan; Tel.: 886.6.2353535x5629; Fax: 886.6.2363715; Email: alshiau@mail.ncku.edu.tw/ Cheng-Yang Chou; Department of Obstetrics and Gynecology; National Cheng Kung University Hospital; 138 Sheng-Li Road; Tainan 704 Taiwan; Tel.: 886.6.2766685; Fax: 886.6.2766185; Email: chougyn@mail.ncku.edu.tw

Submitted: 08/08/08; Revised: 12/18/08; Accepted: 12/19/08

Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/7666

Research Paper

Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cellsKeng-Fu Hsu,1,2 Chao-Liang Wu,1,3 Soon-Cen Huang,4 Ching-Ming Wu,5 Jenn-Ren Hsiao,6 Yi-Te Yo,7 Yu-Hung Chen,3 Ai-Li Shiau1,7,* and Cheng-Yang Chou2,*

1Institute of Clinical Medicine; Departments of 2Obstetrics and Gynecology, 3Biochemistry and Molecular Biology, 5Cell Biology and Anatomy, 6Otolaryngology, and 7Microbiology and Immunology; National Cheng Kung University Medical College; Tainan, Taiwan; 4Department of Obstetrics and Gynecology; Chi Mei Medical Center; Tainan, Taiwan

Key words: cathepsin L, resveratrol, squamous cell carcinoma antigen 1, SCCA 1, cervical cancer

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Cathepsin L in autophagy, apoptosis in cervical cancer

The purpose of this study was to study the effect of RSV on human cervical cancer cells and to better under-stand RSV’s underlying mechanisms. Here we show that RSV induced cell growth arrest and death in uterine cervical cancer cells. Chronic treatment of RSV caused apoptotic cell death through autophagy, in which cat L played an important role in this death pathway. Moreover, SCCA 1 plays an antiapoptotic role through inhibition of cat L activity in cervical cancer cells.

Results

RSV inhibits cell growth and induces apoptotic cell death in cervical cancer cells. Treatment of four cervical cancer cell lines, HeLa, Cx, SiHa and SKGIIIb, with RSV for 48 h at different concentrations resulted in cell growth inhibition (Fig. 1A). In direct contrast, treatment of primary cultures of normal cervical epithelial cells with RSV at 100 and 200 μM showed little effect in cell growth (data not shown). RSV treat-ment with 100 μM for up to 96 h significantly reduced the number of viable cervical cancer cells (Fig. 1B). In HeLa and Cx cancer cells, the release of cytochrome c (Fig.1C) and the activation of caspase 3 (Fig. 1D) increased 24 h after treatment with RSV. Furthermore, flow cytometry analysis showed RSV induced apoptotic cell death along the exposure time (Fig. 1E). The percentage of apoptotic cells was ~26% in HeLa cells and ~31% in Cx cancer cells after RSV treatment for 72 h compared with 2.5% in HeLa cells and 3% in Cx cancer cells at 0 h (Fig. 1F).

RSV induces autophagic response in cervical cancer cells. HeLa cells were treated with 100 μM of RSV and observed for the forma-tion of autophagosomes at 6, 12, 24, 48 and 72 h intervals. To prevent autophagy induced by nutrient consumption, we replaced the culture medium and RSV every 24 h. RSV-induced autophago-some formation in HeLa cells was noted as early as 6 h after exposure. Peak autophagosome formation occurred at 24 h, and then was

gradually decreased (Fig. 2A). To further monitor the induction of autophagy response, we employed Cx transfected cells that transiently express the chimeric fluorescent protein GFP-LC3. Upon induction of autophagy, LC3 is modified to a membrane-bound form LC3-II (a LC3-phospholipid conjugate) that translocates and aggregates onto the membrane of autophagic vacuoles.23 As shown in Figure 2B, the number of GFP-LC3 positive dots gradually increased at 6 h after exposure to RSV, with peak autophagosome formation at the 24 h, and then decreased. Since the amount of LC3-II is a good early indicator for the formation of autophagosomes, we measured LC3-II protein in RSV-treated HeLa and Cx cells in the presence of protease inhibitors, E64 (10 μg/ml) and PSTA (10 μg/ml), to prevent LC3 protein degradation.31 Following RSV treatment, Figure 2C shows that the increased expression of LC3-II within a time period as early as 6 h. To further verify RSV-induced autophagy, we observed numerous autophagic vacuoles with double-membrane structure in HeLa cells by transmission electron microscopy when cell were treated with RSV (100 μM) at 24 h (Fig. 2D). Correspondingly, control cells exhibited few autophagic features.

Figure 1. Resveratrol (RSV)-induced growth inhibition, cyto-chrome c translocation, caspase-3 (casp-3) activation and apoptotic cell death in cervical cancer cells. (A) dose-depen-dent killing of cervical cancer cells with RSV. Cell viability after 48 h was determined by WST-1 assay. (B) Actual cell number of cervical cancer cells untreated (control) or treated with 100 μM of RSV. Data represent cell counts on days 1, 2, 3 and 4 after exposure to RSV. Triplicate trypan blue viability assays were performed for each cell line; bars, ±SD. (C) cytochrome c translocation in RSV treatment. Cytosolic fractions were obtained from HeLa and Cx cells and treated with 100 uM of RSV for indicated time period. Western blot analysis was done using antibodies against cytochrome c and control marker, calnexin. (D) Western blot analysis of cleaved caspase-3 at 0, 24, 48, 72 h of 100 uM RSV treatment. Cell lysate from HeLa cells treated with 50 uM cisplatin for 24 h was used as a positive control. (E) RSV induced cervical cancer cell apop-tosis. HeLa and Cx cells were incubated with 100 μM RSV for 0, 24, 48, 72 h and double labeled with annexin V-FITC and propidium iodide (PI). In the cytogram from three experiments, apoptotic cells (positive for annexin V) are distributed in the lower and upper right panels (values in percent are given). (F) Histograms of apoptotic cells in HeLa and Cx cultures treated with RSV in the indicated time period. Apoptotic cells were increased with time period. Results shown are the means of three independent experiments; bars, ±SD.

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of cat B and D remained unchanged (data not shown). In addition, cytosolic cat L activity in the cell lysate from RSV-treated cells increased gradually reaching levels up to 132% and 145% over that of the control samples (Fig. 3E).

Cat L mediates RSV-induced apop-totic cell death in cervical cancer cells. Flow cytometry analyses with annexin V-FITC and PI (Fig. 4A) or PI only (Fig. 4B) revealed the percentage of apoptotic cells (in lower and upper right of the cyto-fluorogram). In RSV-treated HeLa cells this was ~26%, and did not change after the addition of PSTA, a cat D inhibitor (Fig. 4A). However, in the presence of E64 (an inhibitor specific for cat B and L) and cat L inhibitor I, the percentages of apoptotic cells decreased to ~15% and ~18%, respectively. Similarly, Figure 4B shows that cell death induced by RSV was reduced from ~23% to 8–9% when E64 or cat L inhibitor I was added. With SCCA 1 being an endogenous cat L inhibitor in human cervical cells, we used siRNA to knockdown cat L or SCC A1 expression to further confirm their involvement in RSV-induced cytotoxicity. The western blot analysis demonstrated that transfection with cat L siRNA (50 nM) or SCC A1 (25 nM) siRNA reduced the expression of cat L or SCCA 1 by ~95% (Fig. 4C). When Cx cells trans-fected with cat L siRNA or SCCA 1 siRNA were treated with 100 μM of RSV, silencing of cat L by siRNA significantly reduced RSV-induced cell apoptosis while SCCA 1 siRNA enhanced the apoptosis.

This phenomenon was detected at 48 h, and increased further at 72 h. Analysis of our observations showed that cat L siRNA partially reduced RSV-induced cell apoptosis from 33.3% to 11.2%, whereas silencing of SCCA 1 enhanced the apoptosis to 45.7% (Fig. 4D and E). As a result, the data suggest that cat L is an essential mediator of RSV-induced apoptosis in cervical cancer cells.

Inhibition of autophagy results in reduced LMP and decreased apoptosis in cervical cancer cells. To confirm that excessive autophagy led to induce LMP and thereby resulted in apoptosis in cervical cancer cells treated with RSV, we used amino acid, ASP and autophagy inhibitors WT and 3-MA to block the autophagy process.34-36 Extra supplementation of certain amino acids has been shown to reduce the process of autophagy. Although 3-MA effec-tively inhibited RSV-induced autophagy responses at appropriate concentrations (5–10 mM), 3-MA also exerted cytotoxic effect on cervical cancer cells after prolonged treatment (data not shown) as previously reported in other tumor cells.12,13 As a result, 3-MA was not used in our further experiments. The immunoblotting for LC3-II expression showed that the addition of autophagy inhibitors

RSV increases LMP resulting in cat L cytosolic translocation and increase of cytosol cat L activity. Lysosomal enzymes, such as cat L, D and B, are involved in the activation of apoptotic cell death.3,7,28,32 The key factor in determining the type of cell death (necrosis vs. apoptosis) mediated by lysosomal enzymes seems to be directly related to the magnitude of lysosomal permeabilization and the amount of proteolytic enzymes released into the cytosol.33 As shown in Figure 3A, relocation of AO from the lysosomes to the cytosol results in decreased red granular fluorescence. Cells with decreased red granular fluorescence are also revealed as ‘pale’ cells in the histogram. An increase in the percentage of ‘pale’ cells was observed in cervical cancer cells after RSV treatment (Fig. 3B). LysoTracker Red imaging in HeLa cells further indicated the presence of red fluorescence in perinucleus (lysosomes) at 0 h and then the dispersion of fluorescence at 72 h (Fig. 3C). To investigate which lysosomal enzyme translocated to cytosol after RSV treat-ment, cytosolic fractions from Cx and HeLa cells were obtained for the detection of cat L, B and D expression. As shown in Figure 3D, expression of cytosolic cath L increased over time, whereas the levels

Figure 2. RSV induced autophagic response in cervical cancer cells. (A) Visualization of activation of autophagy with MDC in HeLa cells. HeLa cells were treated with RSV in 100 μM for 0–72 h, then incu-bated with MDC, and analyzed immediately by fluorescence microscopy. The formation of autophagic vacuoles was suggested by interspersed MDC labeling in the cytoplasm. Microphotographs (x400) were shown at RSV treatment in 24 h as representative. Quantitative results of spot (MDC staining) per cell were from three independent experiments; bars, ±SD. (B) LC3 accumulation observable in Cx cells. Cx cells were first transfected with LC3-GFP, incubated for 24 h and then RSV-treated for 0–72 h, analyzed by a fluorescence microscope followed by quantitation of green spots per cell with LC3 accumulation in cytoplasmic vacuoles. Representative images are shown at 0 h or RSV treated at 24 h. (C) Immunoblotting for LC3-I and LC3-II using lysates from HeLa and Cx cells treated with RSV (100 μM) for 0 to 48 h. The blots were stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. Similar results were observed in at least two independent experiments. (D) Ultrastructural observation of cells treated with RSV. HeLa cells were incubated with RSV (100 μM) for 24 h and processed for electron microscopic examination. The right side panel shows the magnified image of the area indicated by the box in the left side panel. Note the abundance of double-membrane autophagic vacuoles in RSV-treated cells.

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Figure 3. RSV promotes lysosomal mem-brane permeability (LMP) resulting in translocation to the cytosol and ensuing activation of cat L in HeLa and Cx cells. The occurrence of LMP was investigated by using two lysosomotropic fluorescence probes, acridine orange (AO) (A and B) and LysoTracker Red (C). (A) HeLa cells treated with or without ciprofloxacin (CPX), stained with AO in 5 μg/ml for 30 min, were analyzed by flow cytometry using the FL3 channel. HeLa cells treated with CPX in 300 ug/ml for 3 h were used as a positive control. Relocation of AO from the lysosomes to the cytosol results in green fluorescence (white arrow in inset photomi-crographs at fluorescent microscopy, x100) and shown as ‘pale’ cell in histogram. Note the cells with decreased red fluores-cence (‘pale’ cells) increases up to 58.98% when treated with CPX. (B) HeLa and Cx cells treated with 100 μM RSV within the indicated time period and then incubated with AO in 5 μg/ml for 30 min were analyzed by flow cytometry using the FL3 channel. An increase percentage of ‘pale’ cells were observed with RSV treatment. (C) HeLa cells treated with RSV 100 uM for 72 h, stained with lysosomotropic fluores-cence probes LysoTracker Red for 30 min and observed in a confocal microscope. Representative cells are shown. Note the red fluorescence accumulated in perinu-clear granula (lysosomes) at 0 h while dispersed in cytosol at 72 h. (D) Cathepsin L was released from lysosomes to cytosol when treated with RSV. Cx and HeLa cells were treated with RSV in 100 μM in the indicated time period. For the analysis of the translocation of cat L, cytosolic extracts were recovered and was analyzed by western blot analysis. β-actin serves as a loading control. A representative blot of four independent experiments is shown. Blots were scanned and protein expression was quantified by densitometric analysis. The ratio of cat L’s expression increased over the control sample is shown. (E) Increased cytosolic cathepsin L activities in HeLa and Cx cells after RSV treatment. Following treatment with RSV 100 μM, cytosolic fractions of cells were prepared, and cathepsin L activities were assayed by use of the InnoZymeTM Cathepsin L Fluorogenic Activity Kit. Six independent experiments were performed at each of the different time intervals.

WT or ASP decreased LC3-II expression between 6–24 h, indicating the inhibition of autophagic response (Fig. 5A). To monitor the LMP change under such conditions, HeLa cells and Cx Cells were treated with RSV 100 μM, stained with AO, and analyzed with flow cytometry. As shown in Figure 5B, WT significantly reduced LMP from 23.73% to 13.73% in HeLa cells and 33.97% to 16.79% in Cx cells at 72 h. Furthermore, ASP also significantly reduced LMP to 13.93% in HeLa cells and 15.95% in Cx cells. As for apoptotic

change, the flow cytometry analysis showed that WT and ASP significantly reduced apoptotic cell death in these two cells (Fig. 5C). Histograms of apoptotic cells induced by RSV in the absence or pres-ence of WT or ASP for 48 and 72 h are shown in Figure 5D.

RSV reacts with iron extracellularly but neither iron deletion induces cervical cancer cell apoptosis nor supplement of iron abolish cell death induced by RSV. Iron overload as well as iron depletion has been reported to induce lysosomal destabilization and apoptotic

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significant shift to high binding energy, an oxidation state, compared with pure FeSO4 or FeSO4-DFO. The binding energies are at 709.7, 711.2, 712.3 and 715.1 eV, respectively (insert). The former three peaks is the 2p3/2 envelope. The binding energy at 715.1 eV is referred to Fe3+ 2p3/2 satellite peak. It represented that the oxidation process occurred in FeSO4-RSV. From the XPS analysis, there is a strong interaction between Fe2+ and RSV. Further testing was also done to determine whether iron chelating agent DFO could induce cervical cancer cell apoptosis. HeLa and Cx cancer cells were incu-bated with 100 μM or 1 mM DFO for 0, 24, 48, 72 h and double labeled with annexin V-FITC and PI. However, we could not observe cytotoxic effect event treated for 72 h (Fig. 6B). Further, we test whether the iron supplement could abolish cytotoxic effect of RSV in cervical cancer cells. FeSO4 in 50 μM or 100 μM were added to HeLa and Cx cancer cells one hour before the addition of RSV (100 μM). Cells were treated with RSV in the absence or presence of FeSO4 for 0, 24, 48, 72 h and double labeled with annexin V-FITC and PI. No decreased percentage of apoptotic cell death was noted after FeSO4 supple-ment for 72 h (Fig. 6C).

Discussion

The antiproliferative and apoptosis-inducing capabilities as well as inhibition of carcinogenesis of RSV in animal models have been demonstrated with various mechanisms.9-13 In this study, we demonstrate that RSV induces autophagy, inhibits proliferation and induces apoptotic death in cervical cancer cells. These effects are mediated by RSV in which RSV has been shown to destabilize lysosomes which cause lysosomal leakage, increased cytosol translocation, and enzymatic activity of cat L. The increased cat L activity in cytosol induces cytochrome c release from mitochondria, and subsequently results in apoptotic cell death. More importantly, blocking autophagy by ASP or WT will eliminate LMP and retard apoptotic cell death. In addition, SCCA 1, an endogenous cat L inhibitor in cervical cells and a widely used tumor marker in the clinic,17 can counteract the effect of resveratrol. On the basis on our findings, we proposed a working model as illustrated in Figure 7.

In cervical cancer cells, autophagy responses increased significantly after 6 h of treatment with RSV. In contrast, the autophagy response time was as early as 1 h in colon cancer cell.13 Autophagy

responses gradually decreased after reaching its peak at 24 h. The reasons why the autophagy responses decreased after chronic RSV treatment (>24 h) are not yet clear. It is possible that when excessive autophagy cannot overcome the cell-death stimulation, apoptotic cell death does occur and autophagy response decreases. The other possible explanation is the limitation of the assay used which detected

cell death.37 To test the possible role of iron in RSV-induced cyto-toxicity, we first examine the binding ability of RSV with FeSO4 by XPS analysis. In this experiment, we have analyzed Fe 2P3/2 (2p3/2 is the characteristic peak in iron XPS analysis) binding energy of FeSO4, FeSO4-DFO (as positive control) and FeSO4-RSV. As shown in Figure 6A, we observe that the binding energy of FeSO4-RSV has a

Figure 4. Cat L mediates RSV-induced apoptotic cell death in cervical cancer cells. (A) HeLa and Cx cells were incubated with 100 μM RSV in the absence or presence of cathepsin D inhibitor, PSTA; cathepsin L inhibitor, E64 or cathepsin L inhibitor I for 72 h (A) or 48 h (B). At the end of treatments, cells were double labeled with annexin V-FITC and propidium iodide (PI) (A) or PI only (B) for cell viability, and analyzed by flow cytometry. (A) RSV-induced apop-tosis was partially prevented by E64, cat L inhibitor I but not by PSTA. Similar results were observed in at least two independent experiments. (B) Representative flow histograms depicted PI-positive fraction in Cx cells treated with 100 μM RSV in the absence or presence of 10 μM E64, 100 μM PSTA or 10 μM cat L inhibitor I for 48 h. The values shown in each histogram are the percentages of cells in different conditions. Cx cells were plated, transfected with cat L-siRNA in 50 nM or SCCA 1-siRNA in 25 nM and incubated for 48 h. Cells were then treated with 100 μM RSV at the indicated time. Cell lysates were collected and analyzed by western blot analysis (C), and double labeled with annexin V-FITC and propidium iodide (D). In the cytogram, apoptotic cells (positive for annexin V) were distributed in the lower and upper right panels (values in percent are given). (E) Histograms of apoptotic cell death in Cx cells induced by RSV 100 μM in the absence or presence of cat L siRNA, SCCA 1 siRNA for 24, 48 or 72 h. n = 3. *p < 0.01.

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and prognosis in uterine cervix, lung and esophageal cancers.17,43,44 Molecular cloning of the cDNA shows that SCCA belongs to the superfamily of serine proteinase inhibitors (serpins) and contains two tandemly arrayed genes, SCCA 1 and SCCA 2.45 Since cancer cells express significantly higher SCCA 2 and SCCA 1 levels than their normal counterparts,46 it was reasonable to use SCCA for cancer target therapy.22 Regarding the function of SCCA 1 in cervical cancer cells, we found that knocking down SCCA 1 to enhance cat L activity resulted in increased apoptotic cell death induced by RSV. Therefore, SCCA 1 may play a key role in resisting cell death. It may provide a new therapeutic viewpoint in treating SCC from

only the decrease of “induction of autophagy,” rather than recognizing the entire “autophagy program.”38

Although autophagy may initially represent a compensatory response, a housekeeping mechanism, it can eventually contribute a pathologic role i.e., neurodegeneration,39 or apoptosis when overactivation is demon-strated as in the present study. We show here that autophagy preceded apoptosis in cervical cancer cells after RSV treatment. Lysosomes have been reported to initiate the pathway to cell death.3 Active participation of lysosomal proteases in cell death induced by different stimuli have been observed previ-ously.4-7 An essential event in the pathway is the lysosomal membrane permeabilization and the release of lysosomal proteases to the cytosol. The released cathepsins may trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues, and induce cytochrome c release.40 Cat L, B and D were the most abundant lysosomal proteases, and Cat D has been reported to be involved with RSV-induced cell death in colon cancer lines.41 However, only one colon cancer cell line, LDL1, was shown to increase autophagy activity under RSV treatment.13 In ovarian cancer cell lines, RSV could induce autophagocytosis and cell death.12 Meanwhile, the details of the mechanism of how ovarian cancer cells encounter cell death under RSV treatment are not yet fully understood. Our data indicate that cat L plays the most signifi-cant role involved in this process in cervical cancer cells. This observation is unique and different from that reported in hepatocyte and colon cancer cells.7,41

Iron depletion has been reported to induce lysosomal destabilization and apoptotic cells death.37 RSV is a biphenol compound, known to be a potent iron chelator. In our experi-mental condition, treatment with iron chelator DFO up to 72 h does not induce apoptosis in cervical cancer cells. However, since DFO does not pass through cell membranes but is taken up by endocytosis, we cannot exclude the possibility that iron deple-tion may induce apoptosis with extended period of DFO exposure. RSV-induced apoptosis was reported in depending on the class III phosphoinositide 3-phosphate kinase activity and on the formation of autophagolysosomes.13 In the meantime, reactive oxygen species (ROS) were reported as essential for autophagy.42 They may both involved in the mechanisms of RSV-induced autophagy or apoptosis pathway.

Squamous cell carcinoma antigen (SCCA) was first identified by Kato and Torigoe in 1977. It has become a useful tumor marker clin-ically that has been shown to be associated with tumor progression

Figure 5. Inhibition of autophagy resulted in reduced LMP and decreased apoptotic cell death in HeLa and Cx cells. (A) Wortmannin (WT) and asparagine (ASP) inhibited early autophagy activities in RSV-treated cervical cancer cells. Immunoblotting for LC3-II using lysates from HeLa cells treated with RSV (100 uM) in the presence or absence of autophagy inhibitors WT (100 nM), ASP (10 mM) for 0–72 hrs. The blots were stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. Similar results were observed in three independent experiments. (B) WT and ASP reduced RSV induced-LMP in HeLa and Cx cells. For monitoring the LMP change under interference of autophagy inhibition, AO uptake assay was used at end of the RSV treatment. After treatment with RSV 100 μM for 72 h with/without WT (100 nM), ASP (10 mM), cells were stained with AO and analyzed for flow cytometry. (C) WT and ASP significantly reduced apoptotic cell deaths in cervical cancer HeLa and Cx cells. Cells were incubated with 100 μM RSV in the absence or presence of autophagy inhibitors WT or ASP for 72 h. At the end of treatments, cells were double labeled with annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry for cell viability. In (D), histograms show apoptotic cell death induced by RSV 100 μM in the absence or presence of WT, ASP for 48 and 72 h. Data are mean ± SD in three independent experiments. *p < 0.05.

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different parts of the body organs. This may be especially effective when combined with the blocking of the SCCA 1 function and when an apoptosis-inducing agent is used (i.e., RSV, antitumor drugs) to enhance the autophagy-cathepsin-apoptosis pathway.

The relative high concentrations (100 μM) of RSV in our study and others13,41 to elicit cytotoxic effect in vitro raises the question of whether such concentrations can be achieved in vivo in the target organ. Pharmacokinetic studies in animals and humans indicate that RSV is rapidly metabolized in the body, a factor that limits the availability of the drug at target organs remote from the site of absorption.47 In contrast, topic administration and tissue-specific deconjugation of RSV metabolites could contribute to local rise of RV concentrations. However, whether RSV will produce similar effect in vivo remains to be investigated.

In conclusion, we have shown that the persistence of autophagy in chronic treatment of RSV results in cytotoxic effect in cervical cancer cells. Our results also demonstrate that cat L actively involves in the anticarcinogenic effects of RSV in cervical cancer cells and SCCA 1 may offer resistance to RSV-induced apoptotic cell death by inhibiting cat L activity.

Figure 6. RSV reacts with iron extracellularly but its iron-binding capacity is unrelated to cell death induced by RSV in cervical cancer cells. (A) The bind-ing ability of RSV with FeSO4 was examined by XPS analysis. Fe 2p3/2 (2p3/2 is the characteristic peak in iron XPS analysis) binding energy of FeSO4, FeSO4-DFO (as positive control) and FeSO4-RSV were examined. The binding energies are at 709.7, 711.2, 712.3 and 715.1 eV, respectively (insert). The former three peaks is the 2p3/2 envelope. The binding energy at 715.1 eV is referred to Fe3+ 2p3/2 satellite peak. (B) HeLa and Cx cancer cells were incubated with 100 μM or 1 mM DFO for 0, 24, 48, 72 h and double labeled with annexin V-FITC and PI. In (C), FeSO4 in 50 μM or 100 μM were pre-added to HeLa and Cx cancer cells one hour before the addition of RSV (100 μM). Cells were treated with RSV in the absence or presence of FeSO4 for 0, 24, 48, 72 h and double labeled with annexin V-FITC and PI.

Figure 7. Schematic representation of the proposed model for cat L mediat-ing resveratrol-induced autophagy and apoptosis in cervical cancer cells.

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experiment, RSV-treated cells were concomitantly treated with the following cathepsin inhibitors: PSTA (100 μM), E-64 (50 μM), cat L inhibitor I (10 μM) or autophagy inhibitors: WT (100 μM), ASP (10 mM). In iron depletion experiments, iron chelating agent, DFO (100 μM, 1 mM) was added to the culture medium or FeSO4 (50 μM, 100 μM) was added in RSV-treated cells. Inhibitors were added to the culture medium at the 1 h (PSTA) or 3 h (E-64, cat L inhibitor I, WT, ASP) interval before the addition of RSV. The culture medium was then replaced with a fresh medium every 24 h.

Electron microscopy. HeLa cells treated with or without RSV for 24 h were washed in 0.1 M phosphate buffer (pH 7.2) and prefixed in the same buffer with 2% formaldehyde, freshly prepared form formaldehyde and 1.25% glutaldehyde in situ at 4°C for 10 min. The cells were then collected and centrifuged at 1,500 g for 3 min. Pallets were then postfixed in 1% osmium tetroxide (OsO4) at room temperature for 1 h, and dehydrated in increasing concentrations of ethanol and pure propylene oxide (PO). The samples were then embedded in an Epon at room temperature and polymerized in an oven at 55°C for 24 h. Ultra-thin sections of the samples (80 nm) were then cut from the blocks and then contrasted with lead citrate and uranyl acetate. The samples were viewed with a JEOL 1200 transmission microscope at 80 kV (JEOL, Tokyo, Japan).

Visualization and quantitative analysis of intracellular autophagic vacuoles. We used MDC staining and aggregation of GFP-LC3 to quantify the autophagic vacuoles.22,23 To view the MDC-labeled vacuoles, cervical cancer cells grown on coverslips were treated with RSV (100 μM) at different time intervals. At end of the culture, the cells were incubated with 0.05 mM MDC at 37°C for 10 min, washed four times with PBS, and immediately analyzed by fluorescence microscopy (Olympus BX51, Tokyo, Japan). Images of the MDC-labeled vacuoles were obtained with a CCD camera. To transiently transfect the GFP-LC3 into the cells, cervical cancer cells at 70% confluence were transfected with pGFP-LC3 using lipofectamine 2000 according to the manufacturer’s instructions. After 24 h, cells were treated with RSV at different time points intervals and analyzed by fluorescence microscopy. The presence of autophagic vacuoles was suggested by the interspersed punctate MDC labeling in the cytoplasm. The number of MDC-positive or GFP-LC3-positive dots at each time point was then counted. At least 50 cells were counted on each microscopic field at 200x magnifica-tion. A total of five different fields were investigated.

Western blot analysis. Equal amounts of protein from the lysates of cervical cancer cells following various treatments were resolved in SDS-PAGE and transferred to the HybondTM-P PVDF transfer membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). The membrane was blocked in blocking buffer (5% non-fat milk powder, 1x TBS, 0.1% Tween-20) for 1 h followed by overnight incubation with primary antibody at 4°C. The membrane was then sequentially incubated for 1 h at room temperature with HRP-conjugated secondary antibody. Antibody binding was detected by enhanced chemiluminescence with the use of the Luminol Reagent (Santa Cruz) or Immobilon Western chemilumi-nescent HRP substrate (Millipore, Billerica, Massachusetts) followed by exposure to X-ray films or BioSpectrum® Imaging System (UVP, Upland, California). Densitometry analysis was performed using VisionWorks LS analysis software (UVP, Upland, California).

Materials and Methods

Cell culture and reagents. Cervical cancer cell lines SiHa and HeLa were purchased from ATCC (Rockville, Maryland, USA). SKGIIIb was obtained from the JCRB Cell Bank (Tokyo, Japan). Cx, a cervical cancer cell line, was established in this laboratory from a cervical cancer patient.20 Normal cervical epithelial cells were prepared as previously described.21 All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Inc., Carlsbad, California) in a humidified atmosphere of 5% CO2. RSV was purchased from Sigma Chemical Co. (St. Louis, Missouri) and dissolved in DMSO as a stock solution of 100 mM and stored at -20°C until use. Iron chelating agent, deferoxamine mesylate (Desferal,® DFO) was purchased from Novartis Pharma Stein AG (Stein, Switzerland). Pepstatin A (PSTA), an aspartyl protease (cat D) inhibitor, L-asparagine (ASP), trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine proteases (cat B, L) inhibitor, ciprofloxacin (CPX), acridine orange (AO), propidium iodide (PI), wortmannin(WT), monodansylcadaverine (MDC), 3-methylad-enine (3-MA), FeSO4 and anti-β-actin antibody (clone AC-15; 1:25000) were also obtained from Sigma Chemical Co.

Antibodies against cytochrome c (1:500), cat L (1:1000), cat B (1:1000), cat D (1:500) and calnexin (1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Anti-cleaved caspase-3 (Asp175) antibody (1:3000) was obtained from Cell Signaling Technology (Beverly, Massachusetts). SCC A1 antibody (1:2000) was purchased from Abnova Co., (Taipei, Taiwan). FITC-conjugated annexin V and 10x binding buffer were purchased from BD Biosciences (San Diego, California). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG (1:20000) were obtained from Jackson Immunoresearch Laboratories (West Grove, Pennsylvania). The following reagents were also obtained commercially: LysoTracker Red (Molecular Probes, Eugene, Oregon), anti-microtubule-associated protein 1 light chain 3 antibody (LC3; 1:1000, MBL, Nagoya, Japan), cat L inhibitor I (Calbiochem Inc., La Jolla, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, California), and Colorimetric WST-1 assay Kit (TaKaRa, Shiga, Japan).

Cell viability assay. Uterine cervical cancer cells, HeLa, Cx, SiHa and SKGIIIb, grown in 96-well plates overnight were treated with 100 μM RSV for 48 h. Cell viability was determined with colorimetric WST-1 assay as described previously.22 To accurately determine the viable cell numbers, the cells were cultured with 100 μM RSV for 0 to 96 h and then harvested. The number of viable cells was then quantified by trypan blue exclusion.

Cell death measurement. Phosphatidylserine externalization, a marker of early apoptotic events, was detected by binding FITC-conjugated annexin V, whereas counterstaining with PI allowed the detection of cells with permeable cell membrane. Cervical cancer cells were treated with RSV at different time intervals and resus-pended in a 1x binding buffer at a concentration of 106 cells/ml. The cervical cancer cells were then stained with FITC-conjugated annexin V and PI (0.5 μg/ml) for 20 min in the dark. The cells were immediately analyzed by flow cytometry with the FACScan flow cytometer (BD Biosciences) and by the use of CellQuest software (Verity Software House, Topsham, Maine). In different sets of the

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electrophoresis, and analyzed by western blotting using cytochrome c or cat L antibody.

Determination of cytosolic cat L activity. Cytosolic cat L activity was determined by InnoZymeTM Cathepsin L Fluorogenic Activity Kit (Calbiochem, La Jolla, California) according the manufacturer’s protocol with some modifications. Briefly, 50 μL of cytosolic frac-tion proteins was added up to the final reactive to 200 μL in a 96-well plate. Cat L activity was measured in the presence of the CA 074 inhibitor alone, the CA 074 Inhibitor (in 1:200 dilution) and the irreversible cathepsin L inhibitor (Z-FY(t-Bu)-DMK). Cleavage of the fluorogenic peptide substrate, Z-Phe-Arg-7-amido-4-methylcoumarin (AMC) by cat L releases AMC and the fluorescence is measured at 360 nm excitation and 460 nm emission with a fluorescence spectrophotometer (Fluoroskan Ascent FL, Thermo Labsystems, Franklin, Massachusetts). The relative fluorescent units (RFU) were normalized with protein concentrations first, and RFU of cytosolic cat L was determined by subtracting the RFU measured in the presence of CA 074 inhibitor and cat L inhibitor from the RFU measured in the presence of CA 074 inhibitor alone.

Measurements of iron and RSV interaction by X-ray photoelec-tron spectroscopy (XPS). For determining iron binding capacity of RSV, XPS measurements were carried out using a ULVAC-PHI AES-650 spectrometer (ULVAC-PHI Inc., Chigasaki, Japan) as previously reported.30 All RSV, DFO, FeSO4 samples examined were prepared and introduced into the spectrometer under inert gas (N2) to limit the chance that the samples would react with the air or other contaminates. The conditions used for all of the survey scans were as follows: energy range = 1100–0 eV, pass energy = 150 eV, step size = 1 eV, sweep time = 120 s and x-ray spot size = 400 x 400 μm. For the high-resolution spectra an energy range of 40–20 eV was used, depending on the peak being examined, with a pass energy of 10 eV and a step size of 0.1 eV.

Statistical analysis. Results were expressed as the mean ± SD. Statistically significant differences among groups were evaluated with the use of ANOVA. The Newman-Keuls test was used for post-ANOVA individual comparison of the means. p < 0.05 was considered significant.

Acknowledgements

We thank Drs. Noboru Mizushima and Tamotsu Yoshimori (National Institute for Basic Biology, Okazaki, Japan) for kindly providing pGFP-LC3 plasmid. This work was supported by grants from the National Science Council (NSC 95-2314-B-006-124 and NSC 96-2314-B-006-041 to K.F.H.), Center of Excellence for Clinical Trial and Research in Oncology Specialty, Ministry of Health, and Center for Cancer Medicine, National Cheng Kung University Program for Promoting University Academic Excellence to C.Y.C.

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Design of siRNA and transfection. Post-transcriptional silencing of cat L and SCCA 1 expression was achieved by using small inter-ference RNA (siRNA) technology. The human cat L and SCCA 1 siRNA was designed from the human cat L cDNA sequence AAG TGG AAG GCG ATG CAC AAC25 and SCCA 1 cDNA sequence GGC CTT TGT GGA GGT TACA, respectively. Both siRNAs were synthesized by Ambion Inc., (Austin, Texas). Nonspecific control siRNA duplexes (StealthTM RNAi Negative Control Duplex, Invitrogen, Carlsbad, California) were also used in parallel. On the day before transfection, 3 x 105 cells were seeded in 6-well plates and grown in 2.5 ml of DMEM supplemented with 10% fetal bovine serum. The siRNA or control duplexes were transfected into cells with Lipofectamine 2000 and incubated for 48 h. The cells were collected and protein lysates were used to detect cat L and SCCA 1 expression by western blot analysis as described above or further treated with RSV (100 μM) at the indicated time intervals.

Lysosomal stability assay. To determine lysosomal membrane permeability (LMP), HeLa and Cx cells treated with RSV (100 μM) were evaluated with the AO uptake and LysoTracker Red staining assays as previously described.6 AO is a metachromatic fluorophore and a lysosomotropic base. AO retains its charged form by trapping protons inside the acidic vacuolar compartment, preferentially in secondary lysosomes. This results in red fluorescence after excitation with blue light. Increased LMP of initially AO-loaded lysosomes may be monitored as an increase in cytoplasmic diffuse green under fluorescent microscopy.26 Cells were treated with RSV in 0–72 h and exposed to 5 μg/ml of AO for an additional 15 min at the end of RSV treatment. Red lysosomal fluorescence of 10,000 cells per sample was determined using the FL3 (red fluorescence) channel by FACScan flow cytometer (BD Biosciences) and the CellQuest software (Verity Software House, Topsham, Maine) was used for data acquisition and analyses. In some experiments, cells were pretreated with ASP (10 mM) or WT (100 nM) for 3 h, and then cultured with RSV in the presence of ASP or WT. Cells with reduced AO-accumulating lyso-somes were termed ‘pale’ cells as previously reported.6 The quinolone antibiotic ciprofloxacin (CPX), known to accumulate in lysosomes to induce LMP, was used as positive control.27

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