Journal of Photochemistry and Photobiology B: Biology 133 (2014) 1–10

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Autophagy inhibition sensitizes bladder cancer cells to the photodynamic effects of the novel photosensitizer chlorophyllin e4 Du Lihuan a, Jiang Ning b, Wang Guozeng b, Chu Yiwei c, Lin Wei c, Qian Jing c, Zhang Yuanfang d, Zheng Jingcun b,1, Chen Gang a,⇑,1 a

Department of Urology, Jin Shan Hospital, Fudan University, Shanghai, China Department of Urology, Gongli Hospital, Shanghai, China Department of Immunology, Fudan University, Shanghai, China d Department of Urology, Hua Shan Hospital, Fudan University, Shanghai, China b c

a r t i c l e

i n f o

Article history: Received 27 October 2013 Received in revised form 12 February 2014 Accepted 15 February 2014 Available online 27 February 2014 Keywords: Autophagy Photodynamic Chlorophyllin e4 Bladder cancer Apoptosis

a b s t r a c t We previously developed a novel photosensitizer, chlorophyllin e4, and found that chlorophyllin e4 mediated-PDT could kill 5637 and T24 cells by inducing apoptotic cell death. Here, we further investigated the new mechanism of autophagy and determined its relevance to apoptosis in e4-PDT. We demonstrated that chlorophyllin e4 was located in both lysosome and mitochondria, and autophagy also occurred in bladder cancer cells upon e4-PDT. More importantly, autophagy played a pro-survival role, and its inhibition enhanced e4-PDT-associated apoptotic cell death because cells pretreated with the typical autophagy inhibitor either 3-methyladenine or Bafilomycin A1 exhibited much lower cell viability and higher apoptotic cell death. Thus, these data imply that the combination of PDT, when mediated by our new photosensitizer chlorophyllin e4, and an autophagy inhibitor might be a promising approach to the elimination of non-muscle invasive bladder cancer. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Bladder cancer is among the most frequently occurring urological malignancies, with an estimated 386,300 new cases and 150,200 deaths in 2008 worldwide [1]. Most bladder cancers initially present as non-muscle invasive bladder cancers (NMIBC) and thus have a high disease recurrence rate and high potential to progress to advanced stage disease [2]. Furthermore, NMIBC was found to be resistant to current therapies, including postoperational chemo- or immuno-therapies [3]. Therefore, efforts to develop innovative and effective therapeutic strategies for this disease are greatly needed.

Abbreviations: NMIBC, non-muscle invasive bladder cancers; PDT, photodynamic therapy; 5-ALA, 5-Aminolevulinic acid; SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy; 3-MA, 3-methyladenine; SEM, standard errors of the mean; AVO, acidic vesicular organelles; FCM, flow cytometry; TURBt, transurethral bladder tumor resection; PE, phosphatidylethanolamine; Du et al., autophagy inhibition plus photodynamic therapy for bladder cancer. ⇑ Corresponding author. Address: Department of Urology, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, China. Tel.: +86 13918043956. E-mail address: [email protected] (G. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jphotobiol.2014.02.010 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

Photodynamic therapy (PDT) is considered to be very well suited for NMIBC therapy, as the bladder can be accessed by endoscopy and the tumors are often limited to the mucosa or sub-mucosa. PDT relies on three major parameters: oxygen, a photosensitizer, which is considered the most important parameter, and light, to which photosensitized target tissues or cells are subsequently exposed to generate cytotoxic agents that lead to cell death [4]. Recently, our research group developed a novel photosensitizer, chlorophyllin e4, which belongs to the family of chlorophyll derivatives. Additionally, we previously showed that e4-PDT exhibited significant photo-cytotoxicity in 5637 and T24 cells, and that apoptosis might be one of its associated mechanisms [5]. We have determined that our new photosensitizer chlorophyllin e4, the most important factor in PDT, has the following benefits [5]: first, it is a pure compound with a clear and stable chemical structure (Fig. 1), thus allowing all our experiments to be performed under visible light. Additionally, it has a very good optical property with an absorption peak of 665.5 nm, making it optimal for PDT. Second, it is easily dissolved in aqueous solutions. Third, it is obtained from abundant crude materials and therefore has a much lower cost than compounds such as 5-Aminolevulinic acid (5-ALA), which is currently the most commonly used photosensitizer and has a high synthesis cost, and we have also obtained

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2.3. Subcellular location of chlorophyllin e4 Bladder cancer cells were seeded into 35-mm dishes at a density of 1  105 cells per dish. When the cells reached 80% confluence, they were first co-cultured with 2 lg/ml (5637) or 10 lg/ ml (T24) of chlorophyllin e4 for 2 h, and were then incubated with 75 nM (5637) and 50 nM (T24) of LysoTrackerÒ Green Probe (Invitrogen, 1043147, Eugene, Oregon) for 30 min. Lastly, the stained cells were observed by confocal laser scanning microscopy (Leica, TCS-SP8). The excitation wavelength for e4 was 405 nm, according to our previous spectral analysis [5]. The fluorescence excitation and emission wavelengths of the lysosome probe were 504 nm and 511 nm, respectively. 2.4. Photosensitization Fig. 1. Chemical structure of chlorophyllin e4 sodium.

patent protection for the synthesis of chlorophyllin e4(No. CN200510024984.8). Lastly, it is rapidly cleared from the body. Therefore, we think that chlorophyllin e4 might be an effective photosensitizer, especially in developing countries such as China. Autophagy is an essential and evolutionarily conserved lysosome degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates, damaged self-cellular cytosol and organelles [6,7]. Apart from its vital homeostatic role, autophagy was also found to be activated during environmental stress and various disorders such as nutrient starvation, infection, myopathy and cancer [8]. Moreover, autophagy might also play an essential role in PDT [9,10], although this remains in dispute. Autophagy might be involved in the repair of photo-damaged cellular components to promote cell viability, given that the silencing of the autophagy-related gene Atg7 resulted in enhanced photodynamic effects on mouse leukemia L1210 cells [11]. Autophagy was also reported to play a possible pro-death role, as its inhibition makes MCF-7 cells more resistant to PDT [12]. Collectively, the literature suggests that PDT very likely induces autophagy, which might play different roles during the PDT process. However, it is unknown whether chlorophyllin e4-mediated PDT could also induce autophagy in 5637 and T24 cells. Furthermore, the role of autophagy in e4-PDT for bladder cancer cells is unknown. Therefore, in this study, we aimed to investigate the following questions: can e4-PDT modulate autophagy in 5637 and T24 cells? What is the relationship between autophagy and apoptosis in e4-PDT for the two cell lines? Does the induction of autophagy have a pro-survival or a pro-death role in this process?

2. Materials and methods 2.1. Preparation of chlorophyllin e4 The chlorophyllin e4 synthesis procedure was performed according to our specifications, and was conducted as we had described in our earlier work [5].

The photosensitization procedure was also conducted as previously described in our earlier work [5]. Briefly, cells that reached 80% confluence were first co-cultured for 2 h with e4 at a concentration of 2 lg/ml for 5637 and 10 lg/ml for T24 cells. Next, the cells were exposed to a 40 mW/cm2 650 nm laser for 100 s after replacing the e4 solution with fresh complete culture medium (with 10% FBS). 2.5. Western blotting 5637 and T24 cells were seeded into 60-mm dishes at a density of 3  105 cells per dish, cultured overnight and treated with or without e4-PDT. Next, whole cells were harvested at the indicated times (0 h, 1 h, 2 h and 3 h after PDT). After that, they were lysed in a lysis-buffer that contained protease and phosphatase inhibitors. A total of 40 lg of total protein from each cell sample was separated by electrophoresis on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA). The membranes were then blocked in 5% non-fat milk for 1 h at room temperature and incubated overnight with primary antibodies (Beclin 1, Cell Signaling, 3738; p62, Cell Signaling, 8025; LC3, Sigma, L7543; GAPDH, Kangchen, KC-5G5) at 4 °C. The membranes were then incubated with a secondary antibody at room temperature for 1 h. Lastly, immunoreactive bands on the incubated membranes were visualized with an ECL Kit (CW Bio, CW0049A). Cells without e4-PDT were used as blank controls. 2.6. Autophagosomal small molecule probe staining This procedure was conducted as described in the protocol. Bladder cancer cells were seeded into 24-well plates at a density of 5  104 cells per well and cultured overnight. Next, the cells were exposed to e4-mediated photosensitization. After 1 h, the cells were stained with the Cyto-IDÒ Autophagy Detection Reagent (ENZO, ENZ-51031-K200) at 37 °C for 30 min in the dark. The excitation and emission wavelengths for the Cyto-ID fluorescence staining dye are 463 nm and 534 nm. The negative controls were set as described above in western blotting. 2.7. Acridine orange staining

2.2. Tumor cells and culture conditions The human bladder cancer cell lines 5637 and T24 were obtained from the Shanghai Institute of Biological Science, and cultured in RPMI-1640 medium supplemented with 10% FBS plus 1% penicillin–streptomycin (Thermo Scientific, Peking, China) at 37 °C with full humidity and 5% CO2.

Briefly, 5637 and T24 cells were seeded into 24-well plates at a density of 5  104 cells per well and pretreated with e4 for 2 h, after which they were exposed to the laser. Subsequently, at 2 h after PDT, the cells were stained with 1 mmol/L of acridine orange (Sigma, 235474) in PBS with 5% FBS at 37 °C for 15 min. The stained cells, excited at the wavelengths of 488 nm, were then observed under a confocal laser scanning microscopy (Leica, TCS-SP8), with

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the emission wavelengths at 510–536 nm and 650 nm. The negative controls were also determined as described above.

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3. Results 3.1. Chlorophyllin e4 locates in lysosome

2.8. Transmission electron microscopy 5637 and T24 cells were seeded into 6-well plates at a density of 2  105 cells per well, treated with or without e4-PDT and then fixed for transmission electron microscopy(TEM) analysis at 1 h after PDT. Briefly, both cell types were collected after PDT and fixed in 2.5% glutaraldehyde for 2 h, washed three times with 0.1 M phosphate buffer solution, fixed with a 1% OsO4 buffer and washed again with 0.1 M phosphate buffer solution. Next, the samples were dehydrated at 4 °C in the following alcohol series (15 min per step): 50% alcohol, 70% alcohol, 90% alcohol, 90% alcohol plus 90% acetone (1:1) and 100% acetone, respectively. The samples were then embedded in pure acetone with embedding liquid (2:1) overnight, followed by pure embedding liquid for 3 h. Next, the samples were solidified at 37 °C overnight, at 45 °C for 12 h and at 60 °C for 24 h. Lastly, the cell samples were cut into ultrathin sections on an ultra-microtome (KB-I), stained with 3% lead citrate plus uranyl acetate and observed by electron microscope (Philips CM20). The negative control was determined as described above. 2.9. Cell counting Kit-8 assay This procedure was conducted according to the manufacturer’s protocol (Dojindo Laboratories, Kumamoto, Japan, EQ829). Both cell lines pretreated by 5 mmol/L of 3-methyladenine (3-MA) (Sigma, M9281) or 500 nmol/L Bafilomycin A1 (BioVision, 1829-50, 250) for 1 h were divided into four groups: the blank control group, 3-MA group (Bafilomycin A1 group), e4-PDT group, e4-PDT + 3-MA group (e4-PDT + Bafilomycin A1 group). 5637 and T24 cells were seeded into 96-well plates at a density of 1  104 cells per well and were cultured overnight; after photosensitization, they were incubated at 37 °C in 5% CO2 for 12 h. Next, 10 ll of the Cell Counting Kit-8 solution were added into each well, and the cells were incubated for an additional 2 h. Lastly, the absorbance at 450 nm in each well was measured with a micro-plate reader (Infinite M200 pro, TECAN). 2.10. Measurement of cell apoptosis We used an Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen, V13241) to detect cell apoptosis. Both the 5637 and T24 cells were seeded into 60-mm dishes at a density of 3  105 cells per dish and were cultured overnight. The two cell lines were divided into four groups according to the treatments that they received: the blank control group, e4-PDT alone group, e4-PDT + 3-MA group and e4-PDT + Bafilomycin A1group. The cells were then harvested at 12 h after PDT and washed twice with ice-cold PBS. Next, they were re-suspended in 500 ll Binding Buffer and stained with 5 ll AnnexinV-FITC plus 5 ll PI at 37 °C for 10 min in the dark. Finally, CyAn ADP (Beckman Coulter, Beckman) was used immediately to determine the cell fluorescence. 2.11. Statistical analysis The statistical analysis was performed with the SPSS17.0 software package (SPSS, Inc., Chicago, IL). Values were expressed as the means ± standard errors of the mean (SEM). All experiments were repeated at least three times. The significance of the differences between the controls and each experimental group was analyzed with an unpaired Student’s t Test, and p < 0.05 was considered statistically significant.

As shown in Fig. 2, after 2 h incubation with the cancer cells, the e4 granular particles emitted red fluorescence (auto-fluorescence) that served to indicate the particles’ location in the 5637 and T24 cells (Fig. 2b and e). Additionally, the LysoTrackerÒ Green Probe, which specifically stains lysosomes with green fluorescence, was found to be restricted to the cytoplasm of both cells (Fig. 2a and d). Interestingly, the overlay images of e4 and the green probe clearly demonstrated that the signal distributions of the two agents were almost identical (Fig. 2c and f). This finding revealed that chlorophyllin e4 was primarily located in the lysosomes and mitochondria. The latter location was investigated in our previous studies [5].

3.2. Chlorophyllin e4 mediated PDT induces autophagy First, we examined the expression levels of autophagy-related proteins (Beclin1, LC3 and p62) in e4-PDT-treated cells to monitor whether e4-PDT could induce autophagy and, more importantly, when autophagy occurred after PDT. As shown in Fig. 3, the Beclin1 expressions levels in both the 5637 and T24 cells significantly increased in a time-dependent manner, compared to those in the blank control groups. Specifically, there was a notable increase of Beclin1 in the 5637 cells from 1 h to 2 h after e4-PDT, followed by a significant decrease at 3 h after PDT (Fig. 4c). In the T24 cells, evident Beclin1 expression was observed at 1 h and 2 h after e4PDT, with a mild decrease from 3 h after PDT (Fig. 4f). LC3, an ubiquitin-like protein also known as the microtubuleassociated protein-1 light chain 3, is the most widely known autophagy-related protein [13]. Fig. 3 demonstrates that both cells had a time-dependent increase in LC3-II expression or the proportion of LC3-II:LC3-I, compared to the blank controls. We can clearly see an obvious increase in the proportion of LC3-II:LC3-I at 1 h after PDT in 5637 cells, and this proportion became more evident at 2 h after PDT, with a significant decrease at 3 h post-PDT (Fig. 4a). Similar results were observed in T24 cells, as the proportion of LC3-II:LC3-I was very prominent at 1 h and 2 h after PDT, but began to weaken at 3 h after PDT (Fig. 4d). The p62 protein, which serves as a link between LC3 and ubiquitinated substrates [14], is incorporated into the completed autophagosome and degradation in autolysosomes, thus playing as a readout of autophagic degradation. Fig. 3 shows that both 5637 and T24 cells had a time-dependent decrease in p62 expression, compared to the blank controls. There was the lowest level of p62 at 1 h after PDT in 5637 cells, and then followed by an evident increase at 2 h and 3 h after PDT (Fig. 4b). It was similar in T24 cells, as the expression of p62 was observed to reach the minimum at 1 h post-PDT, then began to increase significantly from 2 h after PDT (Fig. 4e). We next assessed autophagy induction using the Cyto-IDÒ Green autophagy detection dye, a new and well-known agent that only weakly stains lysosomes and can act as a selective marker of autophagosomes in live cells as GFP-LC3. It stains autophagosomes with green fluorescence and forms punctate structures, and therefore cells with such structures are counted as autophagy-positive. As shown in Fig. 5, at 1 h after e4-PDT, the treated cells elicited detectable increases in green fluorescence intensity and punctate structure distribution in the perinuclear regions and focally throughout the cytoplasm in the 5637 and T24 cells (Fig. 5a and c), while these findings could not be observed in the control cells (Fig. 5b and d).

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Fig. 2. Intracellular location of chlorophyllin e4 in 5637(a–c) and T24 cells (d–f). The fluorescence micrograph illustrates the locations of LysoTrackerÒ Green probes, which are identical to those of the lysosomes. Arrowheads indicate punctate fluorescence in the lysosome of the cancer cells (a and d), the auto-fluorescence of chlorophyllin e4 (b and e) and overlapped fluorescence of the lysosome tracker and chlorophyllin e4 (c and f) (magnification, 2000). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Autophagy was assessed by the appearance of punctate vesicle structures in both 5637 and T24 cells via Cyto-ID fluorescence staining. Cells with punctate structures, which represent autophagosomes, were counted as autophagy-positive cells. The arrowheads denoted green punctate structures that were distributed in both the perinuclear region and the cytoplasm in 5637 (a) and T24 cells (c). (a) 5637 cells with e4-PDT; (b) 5637 cells without e4-PDT; (c) T24 cells after e4-PDT and (d) T24 cells without e4-PDT (magnification, 400). ⁄ p < 0.05.

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Fig. 4. Autophagy responses were measured by the appearance of acidic vesicular organelles (AVO), using acridine orange staining. The arrowheads indicate punctate red fluorescence in the AVO, distributed in the peri-nuclear regions in 5637 (a) and T24 cells (c), and all the cells exhibit yellowish-green fluorescence in cytoplasm and nucleus. (a) 5637 cells after e4-PDT; (b) 5637 cells without e4-PDT; (c) T24 cells with e4-PDT and (d) T24 cells without e4-PDT. Scale bar, 25 lm.

Autophagy can also be detected by staining cells with an acidotropic dye, acridine orange, that emits a bright red fluorescence at acidic pH levels and forms acidic vesicular organelles when autophagosomes fuse with lysosomes to generate into autolysosomes (late stage autophagy). Furthermore, acridine orange can also permeate the nuclei of live cells, emits yellow fluorescence when it binds to RNA, labels the nuclear DNA, cytoplasm with green fluorescence [15]. In Fig. 6, we can see clearly the cells showed a significant increase in particles emitting bright red fluorescence, which indicates acidic vesicular organelles (AVO), in the cytoplasm of 5637 and T24 cells at 2 h after e4-PDT (Fig. 6a and c), while there were no such structures in the control groups (Fig. 6b and d). All cells exhibited the yellowish-green fluorescence of the acridine orange dye in the cytoplasm and nucleus. Lastly, TEM was used to observe the formation of autophagosomes; this is known as the ‘‘Golden Hallmark’’ of autophagy [16,17]. As shown in Fig. 7, the appearance of double-membrane autophagosomes that contained engulfed bulk cytoplasm and cytoplasmic organelles could be easily observed in both the 5637 and T24 cells at 1 h after e4-PDT (Fig. 7a and c). However, these were not observed in the control groups (Fig. 7b and d). 3.3. Inhibition of autophagy promotes e4-PDT-induced cell death To evaluate whether e4-PDT-induced autophagy contributes to cell survival or death, two commonly used autophagy

inhibitors, 3-MA, which can inhibit class I as well as class III PtdIns 3Ks; and Bafilomycin A1, an inhibitor of vacuolar-type ATPases, which can elevate lysosomal pH, were used to pre-treat both 5637 and T24 cells; the cells were subsequently treated with e4-PDT. The Cell Counting Kit-8 assay was first used to detect the effects of 3-MA and Bafilomycin A1 on cell survival after PDT. The growth inhibition rates of the 5637 and T24 cells were calculated as follows: 100%(OD value of control group-OD value of treatment group)/OD value of control group. Fig. 8 shows the different OD values for each group of 5637 and T24 cells. In both cell lines, a statistically significant difference was observed in the 3-MA/Bafilomycin A1 + e4-PDT group compared to e4-PDT group (p < 0.05), and the three above groups experienced much lower OD values than the blank control groups (p < 0.01). However, both the 3-MA groups and Bafilomycin A1 groups did not differ from the blank control groups in the 5637 cell lines (p = 0.089; 0.909, respectively). A similar result was observed in the T24 cell lines (p = 0.171; 0.273, respectively). Furthermore, in 5637 cells, treatment with 3-MA + e4-PDT and Bafilomycin A1 + e4-PDT resulted in a remarkable 95.58% and 96.62% cell death, while e4-PDT alone induced 85.69% cell death. However, treatment with 3-MA, Bafilomycin A1 alone only induced 3.21%, 2.42% cell death, respectively. The situation was similar in T24 cells. Treatment with 3-MA + e4-PDT, Bafilomycin A1 +

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Fig. 5. Autophagy responses were measured by western-blotting to determine the Beclin1 and p62 protein expression, as well as the LC3 I to LC3 II conversion after e4-PDT.

e4-PDT, e4-PDT, 3-MA and Bafilomycin A1 induced 94.18%, 96.21%, 82.44%, 6.01% and 3.22% cell death, respectively. No cell death was observed in either control groups. To determine the effects of autophagy inhibition on cell apoptosis after e4-PDT, AnnexinV-FITC and PI dual staining was performed. In Fig. 9, flow cytometry (FCM) analysis clearly shows that the proportions of AV-positive cells and AV/PI-positive cells in the 5637 and T24 cells, which is considered an indicator of apoptotic cell death, significantly increased in the e4-PDT + 3-MA and Bafilomycin A1 + e4-PDT groups, compared to the e4-PDT only group (p < 0.05). Specifically, in the 5637 cells, e4-PDT plus 3-MA, BafilomycinA1 + e4-PDT remarkably induced an apoptotic cell rate of 82.77 ± 1.69% and 78.17 ± 2.89%, respectively, while e4-PDT alone only induced an apoptosis rate of 62.14 ± 1.99%; in the T24 cells, the same groups had apoptosis rates of 85.17 ± 1.44%, 88.59 ± 1.17% and 54.09 ± 1.72%, respectively. Additionally, the e4-PDT groups, 3-MA plus e4-PDT groups and Bafilomycin A1 + e4-PDT groups demonstrated much higher apoptosis rates than did the blank control groups (p < 0.05) in both the 5637 and T24 cell lines. The blank groups had apoptosis rates of only 0.37 ± 0.27% and 0.21 ± 0.15% in the 5637 and T24 cells, respectively (Fig. 9).

4. Discussion PDT has been evaluated as a promising treatment for NMIBC, which is resistant to transurethral bladder tumor resection (TURBt), chemo-therapy and immuno-therapy [3]. An ideal photosensitizer, which is the most important parameter for PDT, should have characteristics of biological stability, photochemical efficiency, and selective accumulation in target tissues, compared to the surrounding normal tissues, and more importantly, minimal toxicity [18]. Our new photosensitizer chlorophyllin e4, which is extracted from the traditional Chinese herb Excrementum bombycis, would meet all of these requirements. Therefore it should be an effective photosensitizer for PDT. Regarding the mechanism by which PDT kills cancer cells, until now, three interdependent factors were known: damage to the vasculature, the activation of nonspecific immune responses and direct cytotoxicity [19]. This direct toxicity is due to ROS-induced damage to cytoplasmic proteins and organelles such as the mitochondria: this damage can induce apoptosis, one of the major causes of PDT-induced cell death [20]. Interestingly, recent studies demonstrated that PDT could also simultaneously induce autophagy in cancer cells [10,21], indicating that autophagy might be

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Fig. 6. Densitometric analyses of the three autophagy-related proteins expression relative to GAPDH post-e4-PDT.

Fig. 7. Autophagy was evaluated by transmission electron microscopy. Arrowheads denote the typical appearance of double-membrane autophagosomes that contain engulfed bulk cytoplasm and cytoplasmic organelles in 5637cells (a) and T24 cells (c). (a) 5637 cells after e4-PDT; (b) 5637 cells without e4-PDT; (c) T24 cells with e4-PDT and (d) T24 cells without e4-PDT. Scale bar, 500 nm.

involved in PDT-mediated cell death. We therefore hypothesized that e4-PDT could also induce autophagy in bladder cancer cells, independent of apoptotic cell death [5]. As we know, PDT exerts its cytotoxicity mainly by generating ROS within a very limited range (

Autophagy inhibition sensitizes bladder cancer cells to the photodynamic effects of the novel photosensitizer chlorophyllin e4.

We previously developed a novel photosensitizer, chlorophyllin e4, and found that chlorophyllin e4 mediated-PDT could kill 5637 and T24 cells by induc...
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