Study On The Effect Of P38MAPK On Ce6-mediated Photodynamic Therapy To Induce Apoptosis And Autophagy In Human Colorectal Cancer Cells SW620

Background and purpose:Colorectal cancer is the third most fatal cancer in the world,the rate of morbidity and mortality has been increased in our country of these years.The chemotherapy sensitivity of colorectal cancer is poor,especially for the advanced metastatic colorectal cancer.Therefore it is important to find a new method with little adverse reactions and highly specific for treatment of colorectal cancer.In recent years,photodynamic therapy(PDT)has become a promising novel treatment for cancer.It has many advantages,like effective,safe,low side effects and cost,etc.Chlorin e6(Ce6)is a second-generation photosensitizer,which is a derivative of natural chlorophyll a by partial saturation of one of the four pyrrole rings.It has been shown to have better selectivity and prolonged retention time in tumor cells and clear faster from the organism and ow toxicity.However,the mechanism of PDT killing cancer cells remains unknown.Studies have indicated that PDT can induce cell autophagy or apoptosis through activating MAPK pathways.p38MAPK pathway is an important member of the mitogen-activated protein kinase(MAPK)family,which involves cell survival,differentiation and apoptosis.The aim of this study is to investigate roles of p38MAPK in the process of Ce6-PDT induced autophagy and apoptosis in human colorectal cancer SW620 cells(represent a final stage of colorectal cancer development),and to further explore the enhanced PDT efficacy in p38MAPK-knockdown SW620 cells by manipulating autophagy activity.Methods and Results:Part 1 Ce6-PDT induced apoptosis and autophagy in SW620 cells The aim of study is to indicate cell viability after Ce6-PDT,typical features of apoptosis and autophagy by morphological observation,biochemical analysis and protein detection.Cell viability was determined post Ce6-PDT by MTT.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.To determine whether Ce6-PDT(0.84[μM Ce6 + 3 J/cm2 laser light)induced cell autophagy,cells were observed by fluorescence microscope stained with MDC.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;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 8 h after Ce6-PDT treatment;Hence,BafAl is often used to assess autophagic flux.Comparing of SW620 subjected to Ce6-PDT,in the absence or presence of BafAl,revealed an effective in enhancing the accumulation of LC3 Ⅱ.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%;Typical apoptosis characteristics such as nuclear condensation with more enhancing DAPI staining and altered nuclei morphology were observed;In addition,apoptotic executioner Caspase-3 were detected from 0.25 h to 24 h post Ce6-PDT treatment,which 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 Caspase-3 was rapidly activated to execute apoptosis induction by Ce6-PDT.Part 2 Ce6-PDT induced autophagy and apoptosis were correlated with the accumulation of ROS in SW620 cells The data shows that the percentage of cells with DCF fluorescence increased significantly after PDT,suggesting amount of intraocular ROS was formed.By pretreated with ROS general scavenger NAC(5 mM)and singlet oxygen scavenger His(10 mM)before PDT,The results showed that NAC or His both greatly eliminated accumulation of intracellular ROS and inhibited Ce6-PDT induced autophagy as demonstrated by inhibition of the formation of MDC punctate staining and the conversion of LC3.We also found that large condensed chromatin and nuclear damage caused by Ce6-PDT were decreased with NAC or His pretreatment,and the cleavage of Caspase-3 and PARP were also reduced.The results suggest ROS,dominated by singlet oxygen participated in regulating autophagy and apoptosis induction by Ce6-PDT treatment.In addition,NAC and His both significantly prevented cytotoxicity of SW620 cells caused by Ce6-PDT.Part 3 Activation of p38MAPK To assess whether Ce6-PDT could activated MAPKs,the states of phosphorylation of p38MAPK,JNK and ERK were investigated by Western blotting.As shown,activation of p38MAPK was shown immediately after PDT and reached maximum after 1 h incubation,then slightly decreased.The time for phospho-JNK to increase was similar to phospho-p38MAPK,but the expression level was relatively lower.Compared with control,no significant increase in phosphorylation of ERK1/2 was observed.We also observed that ROS scavenger NAC reduced the activation of p38MAPK apparently.However,we inhibited the activation of p38MAPK by SB203580,the percentage of cells with higher DCF fluorescence wasn’t significantly changed compared with PDT alone.The data demonstrated Ce6-PDT induced p38MAPK activation depending on ROS production in SW620 cells.Part 4 Effects of inhibition of p38MAPK on Ce6-PDT induced autophagy and apoptosis in SW620 cells To further confirm the role of p38MAPK in Ce6-PDT induced apoptosis and autophagy in SW620 cells,we applied the siRNA knockdown p38MAPK/SB203580 to dissect its effect on cellular apoptotic and autophagic response to Ce6-PDT treatment.Subsequent analysis shows both Ce6-PDT induced apoptosis and autophagy was enhanced by inhibition of p38MAPK.In autophagy detection,the formation of acidic vesicular organelles by MDC stating and the conversion of LC3 induced by Ce6-PDT were significantly increased.In apoptosis evaluation,more casepase-3 activation and Annexin V(+)apoptotic cells were detected.Moreover,the percentage of Annexin V(+)cells was further increased when pretreatment with 3-MA and BafAl respectively.Accordingly,when p38MAPK was inhibited,the colony forming ability was significant inhibited after PDT treatment,approximately 50%of cells forming colonies at day 14 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.These results suggest that autophagy may play a pro-survival role in the process of Ce6-PDT in SW620 cells with knockdown of p38MAPK.Conclusion:Taken together,this study suggests that Ce6-PDT induced activation of p38MAPK involving ROS production,instinctively prevented auophagy and apoptosis.Auophagy probably to protect SW620 cells from photodamage,suggesting p38MAPK may be a potential therapeutic target to enhance the efficacy on clinical evaluation for treatment of colorectal cancer.