The Study Of Dehydrorabelomycin Induces Autophagic Cell Death In The Human Breast Cancer Cells

The occurrence of cancer is one of the main factors threatening human life and living quality.Breast cancer among women is the most common cancer.Basing on the nearest statistics of the International Agency for Research on Cancer(IARC)of the World Health Organization(WHO),the number of new cases of breast cancer has been the first in the world,and the mortality rate is still increasing.Currently,there are chemotherapy,surgical excision and radiotherapy for breast cancer,among which chemotherapy is the most common,effective and highly accepted treatment in clinical practice.However,because most of the common anti-breast cancer drugs are broadspectrum drugs,which have high lethality to normal cells and are easy to produce drugdependent drugs for long-term use,the development of breast cancer treatment drugs is still a research hotspot.There are many ways to develop antitumor drugs.One of the main ways is to modify the structure of the discovered antitumor drugs to obtain new drugs with stronger antitumor effects.Dehydrorabelomycin is of high research value as an intermediate in the biosynthesis of atypical horacanthracene polyketones.However,the antitumor mechanism and related targets of dehydrorabelomycin have not been reported.In this study,we conducted autophagy induction and apoptosis induction of MCF-7 cells by dehydrorabelomycin,and futher discussed the mechanism of antiMCF-7 cells by dehydrorabelomycin.Firstly,the in vitro inhibiting effect of dehydrorabelomycin on the proliferation of several tumor cell lines and the cytotoxicity of normal cell lines were tested by MTT assay.The results displayed that dehydrorabelomycin had an evident inhibiting effect on the proliferation of several breast cancer cell lines and liver cancer cell lines.It has the most obvious inhibiting effect on the proliferation of breast cancer cells MCF-7,and has little toxicity to human normal breast cells MCF-10A and human renal epithelial cells 293T cells.Therefore,we selected MCF-7 cells for subsequent studies on the mechanism of anti-tumor action.The results of JC-1 probe displayed that the mitochondrial membrane potential of the cells treated with dehydrorabelomycin was decreased,suggesting the early apoptosis of the cells.The apoptosis of dehydrorabelomycin treated cells was analyzed by flow cytometry.It was found that the percentage of apoptotic cells increased with the increase of the concentration of dehydrorabelomycin.After DAPI staining,the cells treated by dehydrorabelomycin were observed under an inverted fluorescence microscope.Apoptotic bodies were observed in cells treated by Dehydrorabelomycin,and apoptotic bodies increased with the increase of concentration.Protein western blots displayed that dehydrorabelomycin induced G2/M cycle arrest,Bax、Cleaved caspase-3 expression and Bcl-2 expression were up-regulated,and apoptosis was confirmed by dehydrorabelomycin.Further studies displayed that dehydrorabelomycin promoted autophagy in breast cancer cells.After transfection with mCherry-GFP-LC3 adenovirus,the red fluorescence was decreased,the green fluorescence was increased and the autophagosome was increased,suggesting that autophagy was induced by dehydrorabelomycin.Western Blot results displayed that Beclin 1,LC3B protein expression increased,and P62 protein expression decreased,which suggested that MCF-7 autophagy was induced by dehydrorabelomycin.Finally,Western Blot analysis displayed that the expression of autophagy marker protein induced by dehydrorabelomycin was earlier than that of apoptosis-related protein.Autophagy and apoptosis were significantly reduced when chloroquine,an autophagy inhibitor,was administered in combination with dehydrorabelomycin,which confirmed that dehydrorabelomycin induced autophagy apoptosis.Finally,by Western Blot assay,we found that the expressions of PI3K,P-AKT and mTOR decreased in MCF-7 cells induced by dehydrorabelomycin.It is suggested that autophagy may be induced by inhibition of PI3K/AKT/mTOR pathway by dehydrorabelomycin.The binding energy of dehydrorabelomycin and PI3K was-9.26 Kcal/mol by molecular docking experiments,suggesting a suitable binding site for dehydrorabelomycin and PI3K.In conclusion,the regulation of PI3K/AKT/mTOR signaling pathway by dehydrorabelomycin induced autophagy of human breast cancer cells MCF-7 and then promoted apoptosis,which provided a new idea for the treatment of breast cancer.