The Anticancer Effect Of Extract Of Medicinal Mushroom Sanghuangprous Vaninii Against Human Cervical Cancer Cell Via Endoplasmic Reticulum Stress-mitochondrial Apoptotic Pathway

The genus Sanghuangprous,comprising 14 species,is a group of fungi called“Sanghuang”.Among them,9 species were found in China and medicinally used for resolving the symptoms of gynecological tumors including vaginal bleeding,leucorrhea,abdominal pain and abdominal mass.Sanghuangprous vaninii is a perennial lignicolous mushroom with the largest market share compared with other Sanghuangprous species in traditional Chinese medicine.As a traditional medicine mushroom,“Sanghuang”is firstly recorded as“Sang’er”in“Shen Nong Materia Medica”.The term of“Sanghuang”firstly appeared in“Yao Xing Lun”in Tang dynasty.Previous studies have revealed that polysaccharides,flavonoids,terpenoids,steroids and styrylpyranones are the main constituents of Sanghuangprous vaninii.Nowadays,there are promising pharmacological studies on its anti-tumor activity,antioxidant effect and anti-inflammatory action etc.This mushroom is potential for gynecological cancers therapy.However,there is a lack of scientific evidence on its anti-tumor activity against human cervical cancer,the most common gynecological cancer.Objective:To identify the anti-tumor potential of the extract of Sanghuangprous vaninii（ESV）on human cervical cancer Si Ha cells,explore detailed mechanisms of ESV inducing cancer cell death and to provide scientific basis for the further clinical application of Sanghuangprous vaninii.Methods:The main active ingredients including polysaccharides,flavonoids,polyphenols,and triterpenoids were detected by UV spectrophotometry.The spectrophotometric assay via anthrone-sulfuric acid,folin-phend,sodium nitrite-aluminum nitrate,vanillin-glacial acetic acid methods respectively was applied to determine the content of these ingredients.The antiproliferation activities were evaluated by CCK-8 assay on six human cancer cell lines,human large cell lung cancer cell（H460）,human cervical cancer cell（Si Ha）,human breast cancer cell（MDA-MB-231）,human hepatoma cell（SMMC-7721）,human hepatoma cell（BEL-7402）and human neuroblastoma cell（SH-SY5Y）.Transmission electron microscope was applied to determined the cellular ultrastructure changes.The cell cycle distribution,quantification of apoptotic cells,mitochondrial transmembrane potential,ROS level,and cytosolic calcium level were determined by flow cytometer.Changes in cell cycle distribution of Si Ha cells were observed by PI staining.Quantitative measurement of apoptotic cells was conducted by dual staining with Annexin V-FITC/PI.Alterations of mitochondrial membrane potential were determined by staining with a JC-1 probe.Determination of intracellular ROS level was obtained by staining with DCFH-DA.The cytosolic Ca²⁺level was determined by staining with Fluo-3 AM.Western blot analysis was used to explore the alterations in the expression levels of endoplasmic reticulum stress-marked and apoptosis-related proteins.The in-vivo anti-tumor effect was identified by mouse xenograft model.Results:Facts have proved that triterpenoids,polyphenols and flavonoids from S.vaninii contributed significantly to the anti-tumor capacity.ESV significantly inhibited the proliferation of Si Ha cells in vivo and vitro.Blocking cell cycle and causing cell apoptosis contributed to cell death induced by ESV.ESV triggered cell cycle arrest at G0/G1 phase.ESV was considerably effective in apoptotic induction of Si Ha cells,especially in early apoptotic induction.Mechanistically,ESV induced endoplasmic reticulum stress evidenced by the elevated expressions of GRP78 and CHOP,which accompanied by the release of Ca²⁺.The Ca²⁺overload and oxidative stress facilitated the collapse of mitochondrial membrane potential and subsequently activated caspase-3and-9,which eventually lead to cell apoptosis.Conclusion:Our results revealed that Sanghuangprous vaninii was effective against human cervical cancer Si Ha cells in vitro and vivo.S.vaninii blocked cell cycle and induced cell apoptoisis,in which the ER stress-mitochondrial pathway might be involved.