Role of p38MAPK in apoptosis and autophagy responses to photodynamic therapy with Chlorin e6

Photodiagnosis and Photodynamic Therapy (2015) 12, 84—91

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt

Role of p38MAPK in apoptosis and autophagy responses to photodynamic therapy with Chlorin e6 Qin Xue a,b, Xiaobing Wang a,∗, Pan Wang a, Kun Zhang a, Quanhong Liu a,∗ a

Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, Shaanxi, China b Department of Urology, Xijing Hospital, Fourth Military, Medical University, Xi’an, China Available online 17 December 2014

KEYWORDS PDT; Ce6; p38MAPK; Autophagy; Apoptosis

Summary Background: Photodynamic therapy (PDT) has been undergoing clinical evaluation for the treatment of colorectal cancer. But the molecular mechanism of photodynamic injury in human colorectal cancer cells still remains unclear. Methods: Chlorin e6 (Ce6) was used to photosensitize SW620 cells. The inhibitory effect of PDT was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide tetrazolium) assay and colony forming assay. Apoptosis was determined by nuclear DAPI (4 -6diamidino-2-phenylindole) staining and Annexin V-PE/7-AAD assay. Monodansylcadaverine (MDC) staining was used to evaluate the abundance of autophagic vacuoles in PDT treated cells. The apoptosis and autophagy associated proteins were analyzed by western blotting. Moreover, we applied siRNA p38MAPK and p38MAPK inhibitor SB203580 to dissect its effect on cellular response to PDT in SW620 cells. Results: Ce6 mediated PDT (Ce6-PDT) induced apparent autophagy and apoptosis with dependent on ROS (reactive oxygen species) generation. When p38MAPK was inhibited by siRNA or inhibitor SB203580, a marked enhancement of apoptosis and autophagy in SW620 cells was detected after PDT. Moreover, autophagy inhibitor 3-methyladenine/Bafilomycin A1 greatly aggravated PDT induced photodamage in SW620 cells. Conclusion: Ce6-PDT induced ROS production to activate p38MAPK probably to prevent SW620 cells from photodamage. Inhibition of p38MAPK activation accelerated cell apoptosis, meanwhile enhanced autophagy may act as a cytoprotective process in SW620 cells. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding authors at: Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, Shaanxi, China. Tel.: +86 29 853 10275. E-mail addresses: [email protected] (X. Wang), [email protected] (Q. Liu).

http://dx.doi.org/10.1016/j.pdpdt.2014.12.001 1572-1000/© 2014 Elsevier B.V. All rights reserved.

Role of p38MAPK in apoptosis and autophagy

Introduction Colorectal cancer is the second most fatal cancer in the world [1]. Surgery remains the most effective curative treatment for colorectal cancer, but the risk of recurrence is high. It is usually treated with chemotherapy or radiation. However, chemotherapy sensitivity is poor, especially for the advanced metastatic colorectal cancer, it also brings patients intolerable side effects [2,3]. Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of different types of cancer. PDT consists of mainly three components, which are light, oxygen, and a photosensitizer. Once the sensitizer is irradiated with light of a specific wavelength, it absorbs photons of the wavelength and transfers the excitation energy to molecular oxygen which is in turn metamorphosed to its diamagnetic form to generate singlet oxygen [4] or other reactive oxygen species (ROS) such as superoxide anion, hydroxyl radicals, and hydrogen peroxide [5]. Of the ROS, singlet oxygen plays a central role for cytotoxicity in PDT [6], or even the amount of singlet oxygen would determine the PDT efficacy. The molecular mechanisms of PDT induced antitumor activities are complicated. The mitogen-activated protein kinase (MAPK) family represents an important group of signaling proteins that can regulate many fundamental cellular processes. The MAPKs, which mainly consist of the extracellular signal-regulated kinase (ERK1/2), c-Jun Nterminal kinases (JNK) and p38MAPK, have been shown to regulate a wide variety of cellular activities, including cell survival and death [7]. In general, ERK regulates cellular proliferation, cell differentiation, cell cycle and cell survival [8]. JNK activation is usually associated with apoptosis induction [9]. Whereas, p38MAPK relates to pro-apoptosis or anti-apoptosis depend on the specific stimulus and cell type [10]. Recent study also indicates that the p38MAPK expression level in colorectal cancer tissues was significantly higher than that in peripheral normal tissues [11]. H-ALA or ALA mediated PDT in H460 cells were closely related to the activation of p38MAPK and JNK signaling pathway [12]. Blocking p38MAPK signaling pathways increases the propensity of the cells to undergo PDT-induced apoptosis [13]. It also reported the protection mechanism of HeLa cells against HypoxiaPDT induced apoptosis was found to be a rapid activation of p38MAPK [14]. In contrast, the enhanced phosphorylation of p38MAPK in response to Pc 4-PDT suggests a possible role in promoting apoptosis within the colon tumor xenografts [15]. The step in PDT-induced apoptosis is promoted by phosphop38MAPK in LY-R cells [16]. Thus it can be seen that p38MAPK may play distinct or even opposite role under different PDT conditions, such as the applied sensitizers, cell models, light exposure parameters, and so on. Pharmacological blockage of the p38MAPK pathway in colorectal cancer cells could also cause autophagic cell death [17]. Autophagy is active in colorectal cancer. However, the role of autophagy in colorectal cancer is paradoxical. Studies indicate that activation of autophagy can suppress the tumor function, but it also contributes to colorectal tumorigenesis. The evidence suggests autophagy has an important role in colorectal tumorigenesis [18], and by regulating autophagy activity, it may bring some positive influence in colorectal cancer treatment.

85 This study is to investigate the role of p38MAPK in the process of Ce6-PDT induced autophagy and apoptosis in human colorectal cancer SW620 cells. Furthermore, we explored the possibility to improve PDT efficacy by manipulating p38MAPK activity.

Materials and methods Chemicals and reagents Chlorin e6 (Ce6) was purchased from Sigma Chemical Company (St Louis, MO, USA) and the purity was greater than 95%. Ce6 was dissolved in sterilized PBS (0.1 M, pH 7.4) at a stock concentration of 2.5 mg/ml at −20 ◦ C. Bafilomycin A1 (BafA1) was obtained from Millipore (Boston, MA, USA), Dimethyl sulfoxide (DMSO), 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI), 3-methyladenine (3-MA), Histidine (His), Monodansylcadaverine (MDC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma Chemical Company (St. Louis, MO, USA). Inhibitor of p38MAPK (SB203580 (SB)) was from Cell Signaling Technology (Beverly, USA). Antibodies used in Western blotting: Rabbit anti-human antibodies to microtubule-associated protein 1 light chain 3 (LC3), Caspase-3, ␤-actin was purchased from Cell Signaling Technology (Beverly, USA).

Cell culture The human colorectal cancer SW620 cells were obtained from the cell bank of the Chinese Academy of Science, Shanghai, China. The cell line was maintained in L-15 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (100 U/ml penicillin and 100 ␮g/ml streptomycin) and 1% glutamine in 100 cm2 tissue culture flasks under a humidified 5% CO2 and 95% air atmosphere at 37 ◦ C.

Photodynamic treatment procedure SW620 cells were cultured in 24 well plates to 80% of confluence, and subjected to photodynamic treatment. Before laser light exposure, cells were incubated with different concentration of Ce6 in serum-free L-15 medium for 4 h, allowing sufficient time for cells uptake the sensitizer rapidly to achieve a maximum level. Then the medium was replaced with L-15 medium without Ce6 and cells were irradiated with light per well. The semiconductor laser (excitation wavelength: 650 nm) was used as a source for evoking photodynamic effect. Irradiance was measured by the radiometer system (Institute of Photonics & Photontechnology, Department of Physics, Northwest University). The light doses of 3, 6, 12 J/cm2 were applied in this study. After irradiation, the cells were incubated in a 5% CO2 incubator at 37 ◦ C before further investigation.

Cell viability detection The MTT assay was used to monitor PDT-induced cell damage in the presence and absence of Ce6 on SW620 cells.

86 Cells with different treatments (100 ␮l, 4 × 104 cells) in 96well flat-bottomed tissue culture plates were irradiated with different light dose (3—12 J/cm2 ) after incubated with various concentrations of Ce6 (0.5, 1.0, 2.0 ␮g/ml). And cell viability was determined at 24 h (up) and 48 h (down) postPDT by adding 10 ␮l MTT solution (5 mg/ml in PBS) to each well and the mixture was incubated for 4 h at 37 ◦ C in a CO2 incubator. The formazan crystals were dissolved in DMSO and the absorbance at 570 nm was recorded using a microplate reader (Bio-Tek ELX800, USA) against the reference value at 630 nm. The results were shown as percentage of control.

DAPI staining for apoptotic morphology observation DAPI is a fluorescence probe, which binds to natural double-stranded DNAs and shows the change of the nuclei morphology. DAPI staining was performed to detect changes of nuclei morphology of SW620 cells after PDT treatment. The cells were irradiated with 3 J/cm2 light dose in the presence of 0.5 ␮g/ml Ce6. At 24 h after PDT treatment, cells were stained with DAPI (4 ␮g/ml) at room temperature for 30 min. Then, the stained cells were washed three times with PBS and observed using a fluorescence microscopy with standard excitation filters (Nikon E-600, Japan).

MDC staining assay for autophagy detection MDC is a marker for lysosomal activity and fused autolysosomes. In this study, MDC staining was used to confirm the abundance of autophagic vacuoles in PDT treated cells. Briefly, at different times post PDT (0.5 ␮g/ml Ce6, 3 J/cm2 laser light), cells were washed with PBS and stained with MDC at a final concentration of 20 mM for 20 min at 37 ◦ C. After labeling, cells were washed with PBS, and images were obtained with a fluorescence microscope (Nikon E600). Excitation wave-length was 360—380 nm, and emission wavelength was 520—560 nm.

Western blot analysis SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel) and immunoblotting were performed according to standard procedures. Briefly, after PDT treatment, cells were harvested and collected by centrifugation, then lysed with RIPA buffer (containing 50 mM Tris—HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF,1 ␮M leupeptin and 0.01 ␮M aprotinin) on ice. The protein content of the lysate was measured using the BCA protein assay reagent. Similar amount of protein was analyzed on SDS-PAGE and transferred onto polyvinylidene fluoride membranes (0.22 ␮m, Millipore). Membranes were then incubated at room temperature for 1 h in blocking buffer (5% low-fat milk powder in tris-buffered saline-tween 20 (0.05%) (TBST)). The membranes were then incubated overnight at 4 ◦ C with primary antibodies. The bound primary antibodies were then tagged with IRDye 680 conjugated IgG (Li-cor, Biosciences, Lincoln, NE) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE) and autographs

Q. Xue et al. were quantified using Bio-Rad Quantity One software (BioRad). ␤-actin was used to ensure equal loading.

Annexin V-PE/7-AAD staining assay Apoptotic cells were measured with the Guava Nexin Assay (Millipore, USA) according to the manufacturer’s protocol. Cells in each group were digested and 100 ␮l sample (4 × 104 cells) was added with 100 ␮l Annexin V-PE/7-ADD binding buffer and then incubated for 20 min at room temperature in the dark. The Annexin-V (+) was determined by flow cytometry and analysis was performed using FCS Express V3 software. About 3000 cells were counted in each group.

Colony forming assay SW620 cells were plated at 4 × 105 cells/ml onto 24-well microtiter plates. After 24 h of culture, to allow cells to attach, 0.5 ␮g/ml Ce6 was added with or without the applied inhibitors (1 mM 3-MA, 40 ␮M BafA1). After Ce6-loading, cells were irradiated with 3 J/cm2 light dose. Post PDT treatment, cells were harvested by trypsinization and diluted in gradient ratio by L-15 with 10% FBS, and seeded into 24well plates at a density of 200 cells/well and cultured for 14 days. Colonies were fixed with formaldehyde at 4 ◦ C for 15 min after washed by PBS, and then stained with Giemsa for 30 min. Colonies that contained 50 or more cells were counted under light microscope. For each experimental condition, colonies from four different wells were counted. The proliferative ability of SW620 cells was calculated as the ratio of each experimental condition to the control one.

siRNA transfection siRNA against p38MAPK and control siRNA were obtained from Sangon Biotech Co. (Shanghai, China). Transfection was performed using TurboFect Transfection Reagent (Thermo, 00110997) according to the manufacturer’s protocol.

Statistical analysis All values were expressed as means ± SD of three independent experiments. Differences among different groups were assessed with one-way ANOVA. Statistical significance was established at a value of p < 0.05.

Results Ce6-PDT induced apoptosis and autophagy in SW620 cells Cell viability of SW620 cells was evaluated by MTT assay at 24 h and 48 h after different treatments. The result clearly demonstrated that cell viability after Ce6-PDT was reduced in a drug and light-dose dependent manner, while Ce6 alone and light alone showed no cellular toxicity (Fig. 1A). To determine whether Ce6-PDT (0.5 ␮g/ml Ce6 + 3 J/cm2 laser light) induced cell apoptosis, cells were detected by

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Fig. 1 Ce6-PDT induced apoptosis and autophagy in SW620 cells. (A) Cell viability was determined at 24 h (up) and 48 h (down) post Ce6-PDT by MTT. (B) Annexin V-PE/7-ADD staining and flow cytometry of apoptosis populations after PDT. Data are means ± SD of three independent experiments and *p < 0.05 versus control treatment. (C) Staining of apoptotic nuclei by DAPI. (D) Caspase-3 activation and (E) LC3 conversion in Ce6-PDT treated SW620 cells were determined by western blot. Significant difference between Control and a treatment was indicated by *p < 0.05, **p < 0.01 versus control. (F) Staining of AVOs by MDC. (G) SW620 cells were pretreated with or without BafA1 for 1 h followed by PDT, LC3II were detected. The ratios of LC3II/I, Cleaved caspase-3/␤-actin were statistically analyzed. Data are presented as mean ± SD of three independent experiments. # p < 0.05, ## p < 0.01 between groups.

Annexin-V staining with flow cytometry. At 12 h after PDT treatment, compared with control, the PDT group triggered cellular apoptotic response with the Annexin-V positive cells increased to 15.75% (Fig. 1B). To further confirm the apoptosis of PDT, morphological changes and the downstream apoptotic biomarkers were examined. The classic apoptotic features were observed at 24 h post Ce6-PDT treatment with DAPI staining. As shown in Fig. 1C, the control cells displayed a weak fluorescence and had no visible nuclei changes. While in the PDT group, typical apoptosis characteristics such as nuclear condensation with more enhancing DAPI staining and altered nuclei morphology were observed. In addition, apoptotic executioner caspase-3 was detected from 0.25 h to 24 h post Ce6-PDT treatment (Fig. 1D). The result showed that the caspase-3 cleavage was evidently increased at early 0.5 h post Ce6-PDT treatment then gradually enhanced with the incubation time, reached a peak value after 12 h and slightly decreased at 24 h after treatment, suggesting that caspase-3 was rapidly activated to execute apoptosis induction by Ce6-PDT. Besides, we also observed obvious autophagic response of SW620 cells to

Ce6-PDT treatment. The autophagy marker protein LC3, was converted from its I type to II at 0.5 h post Ce6-PDT, then the conversion rate gradually enhanced and displayed very remarkable at 4 h and 8 h after Ce6-PDT treatment (Fig. 1E). MDC staining confirmed cells in control group exhibited weak and diffused MDC staining throughout the cytoplasm and very little punctate staining was observed. While in Ce6-PDT treated cells, the MDC staining was visibly enhanced and its distribution pattern was changed from diffusion to punctuated accumulation, and the phenomenon was more evident as the incubation time increased until 8 h post PDT (Fig. 1F). However MDC staining is only an assay for acidic regions and would not be wholly reliable. The LC3-1 lipidation assay is specific for autophagy, but in order to determine the effect of PDT on the autophagic flux, it is necessary to determine LC3-II formation with and without the blocking of autophagosome digestion. As described recently by Klionsky et al. [19], an increased LC3I to LC3II conversion seen on western blots in response to a given cellular stress could reflect stimulation of autophagy (i.e., increased ON-rate) or the inhibition of the degradation/processing

88 of autophagosomes (i.e., decreased OFF-rate). Hence, in PDT-treated cells it is highly recommended to carry out a thorough evaluation of ‘‘autophagic flux,’’ i.e., the complete degradation of autophagosomes and their cargo, to distinguish between stimulation of the ON-rate or decreased OFF-rate. BafA1, an inhibitor of the vacuolar H+ -ATPase, inhibits lysosome-autophagosome fusion and/or the degradation of autolysosomal LC3II [20]. Hence, BafA1 is often used to assess autophagic flux. Comparing of SW620 subjected to Ce6-PDT, in the absence or presence of BafA1, revealed an effectiveness in enhancing the accumulation of LC3II (Fig. 1G). From these experiments it is clear that initial photosensitization by Ce6 stimulates the ON-rate of autophagy, thus indicating that in this PDT paradigm lysosomes can complete autophagosome processing.

Ce6-PDT induced autophagy and apoptosis were correlated with the accumulation of ROS in SW620 cells ROS, a group of highly reactive molecules, especially singlet oxygen, have been shown to play a key role in PDT induced cell death. By pretreated with singlet oxygen scavenger His [21] before PDT, the results showed that His (10 mM) significantly prevented cytotoxicity of SW620 cells caused by Ce6-PDT (Fig. 2A). In addition, His inhibited Ce6-PDT induced autophagy as demonstrated by inhibition of the formation of MDC punctate staining (Fig. 2B) and the conversion of LC3 (Fig. 2D). We also found that large condensed chromatin and nuclear damage caused by Ce6-PDT were decreased (Fig. 2C) with His pretreatment, and the cleavage of caspase-3 and PARP were also reduced (Fig. 2D). The results suggest ROS, dominated by singlet oxygen participated in regulating autophagy and apoptosis induction by Ce6-PDT treatment.

Effects of p38MAPK on Ce6-PDT induced autophagy and apoptosis in SW620 cells SB203580, a specific inhibitor of p38MAPK, has been widely used to assess the role of p38MAPK in a wide array of biological systems [22]. As shown in Fig. 3A and B, cells pretreated with 10 ␮M SB203580 resulted in significant enhancement of Ce6-PDT induced photodamage in SW620 cells. And, the formation of acidic vesicular organelles and the conversion of LC3 were also visibly increased with PDT in the presence of 10 ␮M SB203580 (Fig. 3C and D). To determine whether this inhibitory effect of p38MAPK on the proliferative activity of SW620 cells depended on the induction of apoptotic cell death, the apoptosis detection assay was performed on Ce6-PDT treated SW620 cells, using Annexin V-PE/7-ADD staining. The percentage of Annexin V (+) apoptotic cells increased from 15.75% to 24.55% in PDT groups compared with pretreatment with SB203580 groups (Fig. 3E). Similarly, compared with PDT group, more enhanced caspase-3 activation and PARP cleavage during apoptosis (Fig. 3D) were observed when cells were pretreated with 10 ␮M SB203580. Accordingly, when p38MAPK was knockdown, the colony forming ability was significant inhibited after PDT treatment, approximately 50% of cells forming colonies at day 14

Q. Xue et al. of culture, but many small colonies containing fewer than 50 cells were observed, suggesting that cells were in the quiescent state, while co-treatment with 3-MA or BafA1 again strongly potentiated the inhibitory effect of colony-forming ability (Fig. 3F). These results suggest that autophagy may play a pro-survival role in the process of Ce6-PDT in SW620 cells with knockdown of p38MAPK.

Discussion PDT employs a sensitizer and visible light to produce singlet oxygen and other reactive oxygen species, which lead to subcellular damage at sites where the photosensitizer accumulates [23]. Although PDT is a promising treatment for solid tumors, the precise molecular mechanisms of PDTinduced tumor destruction are not fully understood. PDT has been shown to trigger cellular apoptotic and autophagic responses and cause direct tumor cell killing or indirect vascular destruction, thus eliminating tumor or inhibiting tumor growth [24]. In this work, we used one of the frequently employed photosensitizers, Ce6 [25], to study the effects of cell death exposed to PDT on human colorectal cancer SW620 cells. It shows that the cytotoxicity of PDT could not only be totally explained by the induction of apoptosis, autophagy also occurred in response to Ce6-PDT in human colorectal cancer SW620 cells. Autophagy is a relatively newly described cellular response to various cancer therapies [26]. The issue of whether autophagy plays a pro-survival or pro-death role after toxicant exposure is hotly contested. In the presence of light, activated photosensitizer can generate reactive oxygen species which are toxic to tumors. ROS, a group of highly reactive molecules, especially singlet oxygen have been shown to play a key role in PDT induced cell death [27]. In our study, singlet oxygen scavenger His obviously decreased autophagic response, cell apoptosis and cell viability loss induced by PDT (Fig. 2), suggesting singlet oxygen is the key component participated in the Ce6-PDT photochemical process. ROS has been reported to activate p38MAPK pathway [28—30]. p38MAPK activation may function as a downstream signaling of ROS and would be a core component in Ce6PDT induced apoptosis and autophagy signaling pathway. There are differing reports concerning the role of p38MAPK activation in apoptosis, cell growth, and survival. For example, p38␣ deficiency caused increased proliferation and decreased apoptosis, showing that p38MAPK promotes apoptosis in tumor cells [31]. In contrast, p38MAPK activation was involved in the H2 O2 — induced cell proliferation in Chinese hamster lung fibroblast cells [32], and in protecting cells from Hypecirin-induced phototoxicity [33]. p38MAPK has been reported to be highly activated in colorectal cancer. It is interesting to know whether p38MAPK promoted proliferation or death in SW620 cells by Ce6-PDT treatment. In our study, we demonstrated that pharmacological inhibition of p38MAPK caused increased PDT cytotoxic. By using SB203580, it was clearly seen that more enhanced cell apoptosis was observed after PDT, implying that p38MAPK might have the function of anti-apoptosis in SW620 cells after Ce6-PDT treatment. In this study, when SW620 cells were pre-treated with SB203580, a p38␣/␤ specific inhibitor,

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Fig. 2 Ce6-PDT induced apoptosis, autophagy and growth inhibition in SW620 cells were correlated with an increase of cellular ROS. SW620 cells were pretreated with or without 10 mM His before PDT. (A) Cell viability was determined at 24 h up and 48 h down post-PDT by MTT assay. Data are means ± SD of three independent experiments. *p < 0.05 versus PDT treatment. (B) Autophagic cells were detected with MDC staining. (C) Apoptotic nuclei were observed with DAPI staining. (D) Western blot analysis of Caspase-3, LC3I, LC3II and Cleaved PARP expression after Ce6-PDT treatment. The ratios of LC3II/I, Cleaved caspase-3/␤-actin and Cleaved PARP/␤-actin were statistically analyzed. *p < 0.1 and **p < 0.01 versus Control; # p < 0.05 between groups.

it promoted autophagic response to Ce6-PDT treatment. These findings suggest that p38MAPK could mediate ROSinduced apoptosis as well as autophagy in Ce6-PDT treated SW620 cells. Autophagy induction after mitochondrial damage has recently been found to be accompanied by the production of ROS. ROS in the induction of cell death are closely related with MAPK pathways. In our subsequent studies, when combined with siRNA p38MAPK in SW620 cells, autophagy inhibitor 3-MA/BafA1 further aggravated Ce6-PDT induced cell apoptosis (Fig. 3F). The results implied that autophagy was a cytoprotective process in SW620 cells with knockdown p38MAPK after Ce6-PDT treatment. This may be based on the ability of autophagy to recycle photo-damaged mitochondria before cytochrome C can be released [34]. It is evident that the apoptosis and autophagy pathways share several key signaling components and several opportunities for crosstalk exist [35—37]. Of note, the results show that Ce6-PDT induced necrotic cell death (indicated by Annexin V (+) and 7-ADD (+) in Fig. 1B) also occurred in SW620 cells, suggesting that autophagy might be also involved in the process of necrotic cell death in our study. The interplay between necrotic cell

death and autophagy is very complex. Generally considered, autophagy is an important cell survival mechanism, especially in cells under stress conditions. In present study, inhibition of autophagy activation by 3-MA/BafA1 promoted necrotic cell death, indirectly demonstrating the prosurvival function of autophagy via suppression of necrosis. In addition, PARP-mediated cell death with the involvement of autophagy may be one of the forms of programmed necrosis [38]. Several studies have demonstrated that DNA damage induced by ROS, DNA damage agents, or ionizing radiation led to the following sequential events, including PAPR activation, ATP depletion, AMPK activation, mTOR suppression and induction of autophagy [39]. In our study, we confirmed that Ce6-PDT enhanced the level of PARP cleavage via enhancement ROS generation, as shown in Fig. 2C. Further investigations are required to get full understanding of the contributions of autophagy for determining the outcome of combined autophagy inhibition and apoptosis induction. This study suggests that autophagy could represent a promising field in cancer treatment and p38MAPK may be a potential therapeutic target to enhance the efficacy on clinical evaluation for the treatment of colorectal cancer.

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Fig. 3 SB203580 improved Ce6-PDT induced apoptosis and autophagy. (A) The cell viability was determined at 24 h and 48 h postPDT by MTT assay. Data are means ± SD of three independent experiments and *p < 0.05 and **p < 0.01 versus PDT treatment. (B) Cell proliferative inhibition with or without SB203580. Data are means ± SD of three independent experiments and *p < 0.05, **p < 0.01 versus PDT treatment, ## p < 0.01 between groups. (C) Staining of AVOs by MDC. (D) Western blot analysis of Caspase-3, LC3I, LC3II and Cleaved PARP expression after Ce6-PDT treatment. The ratios of LC3II/I, Cleaved caspase-3/␤-actin and Cleaved PARP/␤-actin were statistically analyzed. *p < 0.05 and **p < 0.01 versus Control; # p < 0.05 between groups. (E) Annexin V-PE/7-ADD staining and flow cytometry of apoptosis populations. Data are means ± SD of three independent experiments and *p < 0.05 and **p < 0.01 versus Control; # p < 0.05 between groups. (F) Cell proliferative inhibition. Data are means ± SD of three independent experiments and *p < 0.05 and **p < 0.01 versus PDT treatment, # p < 0.05 between groups.

Thus, regular autophagy by inhibition of p38MAPK to affect apoptosis may be a useful strategy for colorectal cancer PDT treatment.

Acknowledgement This work was supported by National Natural Science Foundation of China (No. 81472846).

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