The role of autophagy in the treatment of colon cancer by chlorin e6 photodynamic therapy combined with oxaliplatin.

BACKGROUND: Photodynamic therapy is a tumour treatment method. Its mechanism mainly induces apoptosis, autophagy, and other ways to cause cell death. Therefore, this study aims to evaluate the therapeutic effect of chlorine e6 photodynamic therapy (Ce6-PDT) combined with oxaliplatin (L-OHP) in colon cancer and to investigate the role of autophagy in L-OHP treatment and Ce6-PDT combined with L-OHP in colon cancer. METHODS: CCK-8 assay, Scratch wound healing assay, and Western Blot (WB) were used to identify drug-resistant colon cancer cell line SW620/L-OHP. Annexin V/FITC assay, laser confocal double immunofluorescence staining method and WB were employed to investigate the apoptosis and autophagy changes in Ce6-PDT combined with L-OHP. RESULTS: Drug resistance cells SW620/L-OHP were developed under the continuous multi-generation of L-OHP treatment, and the expression of ATP-binding cassette subfamily B member 1 (ABCB1) and ATG5 proteins were increased. The results of immunofluorescence showed that LC3B accumulated in SW620 cells and SW620/L-OHP cells under the treatment of L-OHP. The WB results indicated that LC3B and ATG5 protein expression was increasing in SW620 cells and SW620/L-OHP cells. Inhibition of L-OHP-induced autophagy reduces SW620 cells and SW620/L-OHP cells' viability while increasing apoptosis and the Pro Caspase-3 protein expression. The combination of Ce6-PDT and L-OHP decreased the cell viability, the cell migration ability, the Bcl-2 protein expression, and increased the apoptosis rate, Pro Caspase-3 protein expression in SW620 cells. CONCLUSIONS: L-OHP can cause SW620 cells drug resistance. Autophagy plays a protective role in the L-OHP treatment of SW620 cells and SW620/L-OHP cells, and inhibition of autophagy can increase the efficacy of L-OHP. Ce6-PDT combined with L-OHP can further improve the tumor's therapeutic effect, and autophagy inhibition can improve the efficacy of combined therapy.

Inhibition of autophagy enhances apoptosis induced by Ce6-photodynamic therapy in human colon cancer cells.

OBJECTIVE: To evaluate the therapeutic effect of Chlorin e6 photodynamic therapy (Ce6-PDT) in human colorectal cancer cells and investigate the role of autophagy in Ce6-PDT. METHODS: SW480 cells underwent Ce6-PDT with and without pretreatment with the autophagy inhibitor 3-methyladenine (3MA). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated using an Annexin V assay, using a rhodamine 123 (RH123) assay to evaluate mitochondrial membrane potential (MMP), and by measuring Caspase-3 and Bcl-2 protein expression using western blotting. Autophagy was evaluated by directly visualizing acridine orange-stained acidic vesicular organelles (AVOs) using fluorescent microscopy and by measuring LC3Ⅰ/Ⅱand Atg5 expression using western blotting. RESULTS: Ce6-PDT decreased SW480 viability in a dose-dependent manner. Ce6-PDT induced apoptosis in SW480 cells via the mitochondrial apoptosis pathway as indicated by decreased mitochondrial membrane potential, increased Annexin V staining, and increased Caspase-3 expression. Ce6-PDT was also shown to induce autophagy as demonstrated by increased acridine-orange stained AVOs as well as increased expression of the autophagy-associated proteins Atg5. Inhibition of autophagy with 3MA potentiated SW480 cell response to Ce6-PDT and increased the rate of apoptosis in the treated cells. CONCLUSIONS: Ce6-PDT induces autophagy and apoptosis of SW480 cells in a dose-dependent manner. Inhibition of autophagy increases the apoptosis induced by Ce6-PDT. Modulation of autophagy may be a potential therapeutic target for colon cancer cells treated with Ce6-PDT.

Effects of jasplakinolide on cytotoxicity, cytoskeleton and apoptosis in two different colon cancer cell lines treated with m-THPC-PDT.

Colorectal cancer (CRC) is a common malignant tumor, and metastasis is one of the most important challenges in the treatment of CRC. Photodynamic therapy (PDT) is a novel and non-invasive treatment that influence cytoskeleton and to reduce cancer metastases. In addition, cytoskeleton is related to cancer metastases. Two isogenic colorectal cancer cell lines SW480 and SW620 were used in the present study, we found that m-THPC mediated PDT changed the cytotoxicity, apoptosis and cytoskeleton in both cell lines. Interestingly, the expression of intermediate filaments protein cytokeratin18 were different in the two cell lines. In order to further confirm the relationship between cytoskeleton and cell migration, we combined with microfilament stabilizer jasplakinolide (JASP) to observe the effects of microfilaments on cell migration, cytotoxicity and apoptosis. Taken together, these findings suggest that m-THPC-PDT could induce cytoplasmic cytoskeleton destruction in both types of cells, especially on microfilaments and microtubules. Moreover, in SW480 cells, microtubules may participate in the apoptosis process induced by m-THPC-PDT, while microfilaments may participate in the process of m-THPC-PDT inhibiting cell migration. But in SW620 cells, only microfilaments may be involved in m-THPC-PDT induced apoptosis and inhibition of cell migration.

Photodynamic effect of chlorin e6 on cytoskeleton protein of human colon cancer SW480 cells.

BACKGROUND: Photodynamic therapy (PDT) is based on photochemical and photobiological reactions mediated by photosensitizers to achieve a killing effect on diseased cells. It is used in the treatment of malignant tumors, precancerous lesions and infections. OBJECTIVE: In order to provide theoretical data for further study of the mechanism of PDT for colorectal cancer, SW480 cells were treated with Ce6-PDT and effect of photodynamic therapy (Ce6-PDT) on cytoskeleton and E-cadherin protein were observed. METHODS: The survival of SW480 cells was detected by MTT assay. The morphological changes of SW480 cells after Ce6-PDT were observed by scanning electron microscope (ESM). The migration ability was determined by wound healing assay. The distribution of F-actin in the cytoplasm was observed with confocal laser scanning microscope. Western blot analysis was used to detect the expression of cytoskeleton proteins in SW480 cells after Ce6-PDT. RESULTS: Compared with the control group, there was significant difference in cell viability of cells treated with Ce6-PDT (F = 78753.78, P < 0.05). The pseudopodia almost disappeared and cellular atrophy was clearly visible in the cells of Ce6-PDT group. The migration ability of cells treated with Ce6-PDT for 48 h was significantly lower than the control group (F = 11.794, P<0.001). The result of Western blot analysis showed that the expression of F-actin, α-tubulin, ß-tubulin and Vimentin in the cells treated with Ce6-PDT were significantly higher than that in the control group (F = 22.251,8.109, 5.840, 4.685 and 18.754, P < 0.05). The expression of E-cadherin in cells of Ce6-PDT group was significantly higher than that in control group (F = 30.882, P < 0.001). Perhaps Ce6-PDT inhibits the proliferation and migration of colon cancer SW480 cells by enhancing the expression of E-cadherin, causing the disappearance of cell pseudopodia and the destruction of cytoskeleton. CONCLUSIONS: The destruction of cytoskeleton might be one of the reasons for the inhibition of cell proliferation and migration by Ce6-PDT.

[Results of EGFR Mutations Detected in Pleural Effusion and Its 
Clinical Significance in 132 Patients with Advanced Non-small Cell Lung Cancer: 
A Retrospective Study in A Single Center].

BACKGROUND: The lack of pathological quality control standard in detecting epidermal growth factor receptor (EGFR) gene mutation in malignant pleural effusion leads to confusion in the interpretation of detection results and the clinical use of EGFR-tyrosine kinase inhibitor (TKI). Therefore, it is very important to propose quality control standards and guide the detection of EGFR mutation in pleural effusion. The aim of this study is to retrospectively analyze the results of EGFR gene mutation in pleural effusion sediment section according to strict pathological quality control standards, and the therapeutic effect of EGFR-TKIs guided by this detection results. METHODS: From January 2012 to June 2018, the clinical data of patients with pleural effusion collected from Department of Pathology of Peking Union Medical College Hospital were analyzed retrospectively. Among them, 132 patients with relatively complete clinical data and with EGFR gene mutation detection of paraffin-embedded pleural effusion sediment section according to the established quality control standard were included. According to the results of EGFR gene mutation, it was divided into positive group and negative group, and the efficacy of EGFR-TKIs in different groups was compared. RESULTS: After the centrifugation of pleural effusion, the sediment was embedded in paraffin, sectioned, and observed under the microscope after HE staining. If the number of tumor cells ≥100, it met the pathological quality control standard, and it could be used for subsequent EGFR gene mutation detection. EGFR gene mutations were detected in 72 (54.5%) of 132 patients. EGFR-TKIs were used in 69 of 72 mutation positive patients. Of 60 EGFR mutation negative patients, only 15 used EGFR-TKIs. In EGFR mutation positive group, the disease control rate (DCR) was 95.8%, and the median progression-free survival (PFS) was 11 months. In EGFR mutation negative group, the DCR was 0%, and the median PFS was 1 month. The DCR and PFS were significantly different between the two groups (P<0.05). CONCLUSIONS: According to the pathological quality control standards, the embedded section of pleural fluid sediment can be used to detect EGFR gene mutation, and the results can be used to guide the clinical use of EGFR-TKIs.

Enhanced cytotoxicity and apoptosis through inhibiting autophagy in metastatic potential colon cancer SW620 cells treated with Chlorin e6 photodynamic therapy.

Photodynamic therapy (PDT) is a novel and non-invasive treatment that induces apoptosis and autophagy. Autophagy could play a pro-survival role, thus inhibiting autophagic activity might be a promising method to enhance the effectiveness of PDT for tumors. In the present study, photosensitizer Chlorin e6 (Ce6) was found to mainly locate in endoplasmic reticulum, and to a lesser extent in mitochondria and lysosome. Chlorin e6 photodynamic therapy (Ce6-PDT) could kill human colon cancer SW620 cells by inducing apoptotic cell death, and autophagy also induced by Ce6-PDT in colon cancer cells. More importantly, autophagy played a pro-survival role. Its inhibition enhanced Ce6-PDT-associated apoptotic cell death because cells pretreated with the typical autophagy inhibitor 3-methyladenine exhibited higher cytotoxicity and apoptotic cell death.