| Background and purposeWith the worsening human living environment, dietary structure diversification, lifestyle differences, incidence and mortality of cancer presented the rising tendency year by year. Traditional chemoradiotherapy and surgery have many shortcomings such as poor effect, low sensitivity and malignant matastasis, etc. Therefore it is urgent to find a new therapy with little adverse reactions and highly specific for cancer treatment. In recent years, photodynamic therapy (PDT) has become a promising novel treatment strategy for cancer. It has many advantages, like safe, effective, low side effects, strong specificity and cost, etc. Photosensitizers are critical factor of PDT. One of the earliest clinical applied photosensitizers is Photofrin, which has been approved by FDA for treating skin cancer, breast cancer, esophageal cancer, bladder cancer and head and neck cancer in the worldwide. However, its undefined active components, strong skin photosensitization and high cost have limited the clinical application in our country. Sinoporphyrin sodium (DVDMS) was recently purified, identified and modified from Photofrin and the purity was as high as 98.7%. Early results suggested that DVDMS has many advantages in terms of water solubility, skin phototoxicity, production of singlet oxygen, tumor growth inhibition and selectivity to tumor tissue and so on. In addition, DVDMS was granted intellectual property rights in China.Previous studies have reported DVDMS-PDT can inhibit malignant metastasis and induce cell apoptosis. However, there have been few reports on the role of F-actin filaments in tumor cell migration. And, when the novel photosensitizer DVDMS mediated photodynamic therapy induced cell apoptosis and autophagy, the expression changes of upstream MAPK family and heme oxygenase 1(HO-1) need to be further explored.The aim of this study is to investigate the role of F-actin filaments in cell migration inhition induced by DVDMS-PDT. Whereafter, we futher explored the relative molecular mechanism in process of DVDMS-PDT trigged cell apoptosis and autophagy and expression changes of MAPK family and heme oxygensase 1.Methods and ResultsPart 1 Cell proliferation, apoptosis and migration effect post DVDMS-PDT was investigated in human breast cancer MDA-MB-231 cellsMTT assay and Viacount assay displayed that cell viability after DVDMS-PDT was significantly decreased in a drug and light-dose dependent manner, while DVDMS alone showed no obvious cytotoxicity. Annexin-V staining indicated that DVDMS-PDT triggered cell early apoptotic response and the percentage of apoptosis gradually increased with the incubation time prolonged. Besides, scratch assay demonstrated DVDMS-PDT effectively inhibited migration of MDA-MB-231 cells.Part 2 Collapse of F-actin filaments triggered by DVDMS-PDT was associated with the accumulation of ROS in MDA-MB-231 cellsAccording to flow cytometry analyses, cells subjected to DVDMS-PDT significantly enhanced ROS level, which indicated that the phototoxic effect was mainly medicated by ROS. Given the key role of ROS in the cytotoxicity caused by PDT and the short existence time and diffusion distance of radical products derived from the photosensitizer during PDT process, we further detected the DVDMS distribution. The results showed that DVDMS mainly located in the mitochondria of MDA-MB-231 cells. On the basis of this finding, we speculated that mitochondria may be the main damaging targets. Besides, fluorescence microscope detected that DVDMS-PDT caused remarkable collapse of F-actin filaments. In order to detect the role of ROS in collapse of F-actin filaments, we pretreated cells with NAC (ROS scavenger). The results demonstrated that the disrupted F-actin filament bundles appeared again in the cytoplasm, which indicated that F-actin filaments disruption was possibly caused by ROS production.Part 3 The influence of F-actin filaments disruption on cell migration and apoptosisTo further evaluate the effect of F-actin filaments on cell migration and apoptosis, jasplakinolide (F-actin filaments stabilizer) was employed in this study. Thick actin bundles and a patchy appeared in the cytoplasm when cells were pretreated with jasplakinolide. Transwell assay was performed to assess the change of cell migration under the condition that jasplakinolide can stabilize the depolymerized filament. The results showed that jasplakinolide significantly promoted cell migration, which indicated that F-actin filaments disruption was the main factor in cell migration inhibition. However, Annexin V-PE staining indicate cell apoptosis ratio appear to be not obviously changed with or without jasplakinolide.Part 4 DVDMS-PDT induced apoptosis and autophagy in human esophageal cancer Eca-109 cellsThe aim of this study is to indicate typical of apoptosis and autophagy by flow cytometry analysis, morphological observation and protein detection. MTT assay displayed that Eca-109 cell viability after DVDMS-PDT was significantly decreased in a drug and light-dose dependent manner, while DVDMS alone showed no obvious cytotoxicity. Cell apoptosis post DVDMS-PDT was determined by Annexin V staining. The results clearly demonstrated that DVDMS-PDT induced Eca-109 cells apoptosis. In addition, apoptotic executioner Caspase-3 were detected from 0 h and 24 h post DVDMS-PDT, which showed that the Caspase-3 cleavage was evidently increased at 3 h post DVDMS-PDT treatment and 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 activated to execute apoptosis induction by DVDMS-PDT. To determine whether DVDMS-PDT induced cell autophagy, cells were observed by fluorescence microscope stained with MDC. The results showed that MDC staining was visbly enhanced and its distribution pattern was changed from diffusion to punctuated accumulation. The autophagy marker protein LC3, was converted from I type to II type at 3 h post DVDMS-PDT, then the conversion rate gradually increased and displayed very remarkable at 24 h after DVDMS-PDT. In order to further analyze the role of autophagy in cell death induced DVDMS-PDT, we pretreated cells with autophagy inhibitor LY294002. In the presence of LY294002, cleaved Caspase-3 decreaed detected by western blotting.Part 5 MAPK pathway activation and heme oxygenase-1(HO-1) rapid expression were correlated with the accumulation of ROS in Eca-109 cellsTo assess whether DVDMS-PDT could activate MAPK, the states of phosphorylation of p38MAPK, JNK and ERK were investigated by western blotting. Activations of p38MAPK and JNK were detected after DVDMS-PDT treatment and reached maximum after 3 h incubation, then gradually decreased. In addition, the phosphorylation of ERK 1/2 has not been detected. Moreover, the expression of heme oxygenase 1(HO-1) rapidly enhanced at 0 h after DVDMS-PDT treatment. In order to detect the role of ROS in MAPK pathway activation and heme oxygenase-1(HO-1) rapid expression, we pretreated Eca-109 cells with ROS scavenger NAC. The results showed that NAC reduced the activation of p38MAPK and the up-regulation expression of HO-1. The data demonstrated that DVDMS-PDT induced p38MAPK activation and rapid expression of HO-1 depending on ROS production in Eca-109 cells.ConclusionIn summary, this study demonstrated that DVDMS-PDT triggered cell apoptosis and collapse of F-actin filaments through the induction of ROS in MDA-MB-231 cells. F-actin filaments contributed to cell migration but produced no obvious effect on cell apoptosis. Moreover, DVDMS-PDT induced Eca-109 cells apoptosis and autophagy. Furthermore, our thorough investigation suggested that the activation of p38MAPK and rapid expression of HO-1 was associated with ROS production. |
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