Anticancer Activity Of PMFs Extracts And Main Monomers Form ’Dahongpao’ Tangerine On Prostate Cancer Cells

Polymethoxylated flavones（PMFs）are a class of highly methoxylated flavanoids abundant in citrus fruits,with a series of biological activities.They exhibit anti-oxidation,anti-inflammatory and strong anti-tumor potency.At present,traditional chemotherapy drugs generally have side effects of high toxicity and high drug resistance in chemotherapy of prostate cancer.Therefore,it is necessary to find natural occurring anti-tumor compounds with both good efficacy and few by-effects.‘Dahongpao’tangerine（Citrus tangerina Tanaka）was selected as the experiment material.Ultra-performance liquid chromatography（UPLC）was applied to qualitatively analyze the nine main PMFs in the ethanol extracts of tangerine peels after enrichment and purification.At the same time,human prostate cancer cell lines PC3 and DU145were used to study the effect of the ethanol extracts of tangerine and four main PMFs（tangeretin,5-demethylnobiletin,sinensetin,nobiletin）on prostate cancer cells.Two kinds of monomers with the better anti-tumor effect were selected,including tangeretin and 5-demethylnobiletin.DAPI nucleic acid staining,RT-qPCR,Western blotting,Wound healing assay were used to investigate the effects of 5-demethylnobiletinand on apoptosis,proliferation and migration of prostate cancer cells.Further more,the effect of co-chemotherapy of tangeretin with mitoxantrone（MTX）on prostate cancer cells was also investigated.The main findings are as follows:（1）UPLC test results showed that there were nine kinds of PMFs in the ethanol extracts of tangerine peel after enrichment and purification.And from the highest to the lowest contents were nobiletin 210.87mg/g,tangeretin 55.66mg/g,sinensetin 28.11mg/g,5-demethylnobiletin 24.66mg/g,5,6,7,4’-Tetrathoxyflavone 12.14mg/g,isosinensetin12.9 mg/g,5,7,3’,4’-Tetrathoxyflavone 1.83mg/g,3,5,6,7,8,3’,4’-Hetamethoxyflavone7.68 mg/g,5,7,4’-Trimethoxyflavone 0.64 mg/g.（2）In vitro anti-tumor activity on prostate cancer cells:Tangerine extracts significantly inhibited PC3 and DU145 cells survival in a dose-dependent manner.The IC₅₀ of the extracts to PC3 cells was 7.05μg/mL,and the IC₅₀ to DU145 cells was 26.44μg/mL.Although the four main PMFs all can inhibite cell survival of PC3 and DU145cells,tangeretin and 5DN were proved to exhibit stronger effect to the two cell lines.The IC₅₀ of tangeretin on PC3 cells was 16.47μmol/L,and that of DU145 cells was23.13μmol/L.The IC₅₀ of 5DN was 49.33μmol/L for PC3 cells and 54.14μmol/L for DU145 cells.While the other two PMFs,sinensetin and nobiletin,were not achieve the half suppression of cell viability in PC3 and DU145 cells.（3）The effect of 5-demethylnobiletin（5DN）on prostate cancer cells:5DN can induce the apoptosis of prostate cancer cells and inhibit its proliferation and migration.After 5DN treatment,the transcriptional levels of Bcl-2,AKT1 and AKT2 genes,were significantly decreased,and the transcriptional levels of caspase-3 and Bax genes were significantly increased in two cell lines.In DU145,the transcription level of PTEN mRNA was significantly increased,and the expression level of caspase-9 protein was significantly increased.At the same time,5DN can inhibit the transcription level of c-Myc mRNA,effectively inhibits the proliferation and migration of PC3 cells.（4）The effect of co-chemotherapy of tangeretin with mitoxantrone（MTX）on prostate cancer cells:Both tangeretin and mitoxantrone can inhibit the activity of prostate cancer cell lines PC3 and DU145 in a dose-dependent manner.Co-treatment of tangeretin with mitoxantrone enhance cytotoxic effect of mitoxantrone in prostate cancer cells,by inhibiting colony-forming ability and enhancing cell apoptosis.Among the pathways evaluation,the combination chemotherapy markedly up-regulated caspase-3 and BAX expressions and inhibited the expressions of Blc-2,Akt1 and Akt2in both cell lines.And significantly increased the expression of PTEN in DU145 cells.In addition,the western blot analysis showed that combination treatment markedly inhibited the expression of Bcl-xl and up-regulated caspase-9,caspase-3 expression in DU145 cells.Conclusion:Tangerine extracts significantly inhibited PC3 and DU145 cells survival.the four main PMFs all can inhibite cell survival of PC3 and DU145 cells,tangeretin and 5DN were proved to exhibit stronger effect to the two cell lines.5DN can induce the apoptosis of prostate cancer cells and inhibit its proliferation and migration,and can promote the apoptosis of prostate cancer cells through mitochondrial induction pathway.The combination of tangeretin and mitoxantrone can effectively enhance the toxicity of low-dose mitoxantrone to prostate cancer cells,and they have synergistic effects on the apoptosis of prostate cancer cells.This study provides new ideas for the utilization of tangerine peels and the treatment of prostate cancer and combined chemotherapy.