| Colorectal cancer(CRC)is one of the most common malignant tumors in the digestive tract,with increasing incidence and mortality year by year.CRC has the characteristics of high invasion and metastasis,which is an important cause of its deterioration.Due to the high heterogeneity,hidden onset,and rapid development of CRC,many CRC patients have been diagnosed with invasion and metastasis,which often require surgical treatment combined with radiotherapy and chemotherapy.With the emergence of patients with poor postoperative recovery,adverse drug reactions,and drug resistance,there is an urgent need to develop effective,novel drugs with fewer side effects to improve patient’s quality of life and adapt to clinical needs.Epithelial-mesenchymal transformation(EMT)is a key event in the early stage of CRC invasion and metastasis,in which the PI3K/AKT signaling pathway plays an important role and is considered to be the key point of CRC targeted therapy.Compared with anthracyclines,which belong to the same family of class Ⅱ aromatic polyketone,angucyclines showed higher antitumor activity due to their complex and varied structure.In the previous work,we extracted and isolated four angucyclines from marine Streptomyces sp.OC1610.4,which are as follows:Moromycin B(Mor B),Saquayamycin B1(Saq B1),Saquayamycin B(Saq B),and Landomycin N(Lan N).The cytotoxic effects of four kinds of angucyclines on several common tumor cell lines and normal cell lines were detected by MTT assay.The results of structure-activity relationship showed that the angular tetracyclic core of angucyclines is a nonplanar structure,and the introduction of more hydroxyl groups in the A-ring may enhance the antitumor activity of the compound.It is worth noting that the introduction of larger groups on the A-ring may cause steric hindrance,which reduces the activity of the compound.MTT results further showed that among the four compounds,Saq B1 was the most sensitive to CRC cells SW480 and SW620,and had weak damage to normal liver cells.Therefore,Saq B1 was selected in this study to focus on the anti-CRC effect and preliminary mechanism.First,we verified the effect of Saq B1 on the expression of related proteins in the PI3K/AKT pathway by Western blot,cellular immunofluorescence assay,and molecular docking assay.Western blot results showed that the expression levels of PI3K and p-AKT in SW480 and SW620 cells were significantly decreased after the treatment of Saq B1,and similar results were obtained in cell immunofluorescence staining experiments.Furthermore,the computer molecular docking software was used to predict the specific site of binding of compound Saq B1 to PI3K protein,indicating that there is a strong molecular force between Saq B1 and PI3K,which assisted to verify the results of molecular biology experiments.Secondly,after DAPI and JC-1 staining of SW480 cells,it was found that cells treated with Saq B1 showed karyopyknosis,fragmentation of nucleoli,formation of apoptotic bodies,and decreased mitochondrial membrane potential.Flow cytometry showed that the number of apoptotic cells increased with the increasing concentration of Saq B1.We further detected the expression of apoptosis-related proteins by Western blot,and the results showed that the expression of Bax/Bcl-2 in SW480 and SW620 cells was increased after Saq B1 treatment.These results suggest that the PI3K/AKT signaling pathway may be involved in the role of Saq B1 in inducing apoptosis of CRC cells.Further,to determine whether Saq B1 has the effect of inhibiting CRC cell invasion and metastasis,the cell cloning formation assay showed that with the increase of Saq B1 concentration,the size and number of cell clones decreased,and the proliferation and migration ability of SW480 cells gradually decreased.The wound-healing migration assay showed that Saq B1 gradually reduced the wound-healing rate of cells and significantly inhibited the migration of SW480 cells.In the Transwell assay,treatment with Saq B1 showed dose-dependent inhibition of cell migration and invasion in SW480 cells.After treating SW480 and SW620 cells with Saq B1,the expression changes of EMT-related proteins and genes were detected by Western blot and RT-qPCR.The results showed that decreased p-AKT protein expression resulted in decreased NF-κB protein expression,which further inhibited Snail and Slug protein transcription and affected their expression.We found that the expression of mesenchymal proteins N-Cadherin and Vimentin decreased with the increase of Saq B1 concentration.In contrast,the expression of the epithelial adhesion protein E-Cadherin was increased.Similar results were also obtained in the RT-qPCR experiment,where E-Cadherin gene levels were up-regulated,while N-Cadherin and Vimentin gene levels were down-regulated,further verifying our experimental results.In this study,we analyzed the structure-activity relationship of four angucyclines,providing ideas for the further study of angucyclines.The results exhibited that Saq B1 showed great potential cytotoxic activity on CRC cells compared to other tested compounds.We found for the first time that Saq B1 inhibited the proliferative,invasive,and metastatic ability of SW480 and SW620 cells in vitro by regulating the PI3K/AKT signaling pathway and promoting their apoptosis. |
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