| Background and Objectives:Head and neck cancer(HNC)is the 7th most common cancers in the world with high prevalence and mortality.Most target-specific anticancer drugs have not achieved the expected results.It is important to find new cytotoxic agents that can delay the development of HNC or even cure it without severely side effects.Because of the action of regulating microenvironment and various cell signal transduction,natural compounds play a key role in cancer therapies.Moringa oleifera Lam.(MO)has been used to deal with several diseases for the high nutritional value and biologically active substances in it.MO has been reported to have inhibitory effects on variety cancers.But so far,few reports about MO and HNC were reported.Whether MO has an inhibitory effect on HNC and the possible mechanism,and the key compounds playing the role of against HNC are the problems need to be explored.Methods:(1)Explore the antioxidant activities and the effects to HNC of the extracts from MO.Samples were extracted with different ethanol solvents(water,50,70,and 90%)from the 4 parts of Moringa oleifera Lam.(seed,root,bark,and leaf).The total phenolic and flavonoid content(TPC,TFC),and antioxidant activity of the 16extracts were compared using Folin Ciocalteu method,aluminum chloride colorim-etric method,DPPH scavenging capacity,Fe3+reduction ability determination,and ABTS.+scavenging ability.Selected one in the 4 extracts from the same parts of MO,analyzed the inhibitory effects with HNC cells of the 4 selected extracts using CCK-8,colony formation,flow cytometry,DAPI stain,and western blot.Then selected one having highest effect against the HNC to the further experiments.(2)The changes in the biological behaviors of HNC cells by the 70%ethanol MO bark extracts(MOB)and the possible mechanism.The MOB was the extract selected in the second chapter having the highest inhibitory effects with HNC cells.After treatment of MOB for 24 h,the proliferation,apoptosis,metastasis capacities of HNC cells(5-8F,CNE-1,CAL27)were investigated using CCK-8,colony formation,flow cytometry,RT-PCR,DAPI stain,EDU stain,wound healing assay,Transwell assay,and Western blot.And the effects of MO to the cisplatin resistance were also explored.The ROS and p38phosphorylation levels in cells were analyzed with active oxygen scavenger(NAC),specific inhibitors(SB203580 and SB202190)and potent activator(anisomycin)of p38.Last,analyzed the proposed compounds in MOB using UHPLC-QE-MS.(3)The proliferation and metastasis capacities of HNC in vivo affected with the purified compounds of 70%ethanol MO bark extracts(MOB).Fractions of MOB were harvested using chloroform,ethyl acetate,n-butanol.The antioxidant activities and the inhibitory effects to HNC cells of these fractions using DPPH scavenging capacity,ABTS.+scavenging ability,CCK-8,and flow cytometry.Constructed xenograft tumor and lung metastasis models using 5-8F cells.Treated the mice with the fraction with higher inhibitory effects for 3 weeks before model construction and still 4 weeks after construction.The fraction then purified with the macroporous adsorption resin and silica gel column.The suppressive abilities of these purified compounds to 5-8F cells were detected using CCK-8.Results:(1)The aqueous extracts of MO leaves and the 70%ethanol extracts of the other3 parts(seed,root,and bark)had the highest antioxidant activities in the extracts from the same parts.The 4 extracts had significant anti-proliferation and pro-apoptosis effects with HNC cells,and the ability against HNC of the 70%ethanol extracts of bark(MOB)was the strongest.There are 16 extracts,and the yields of crude extracts were 2.20 to 27.0%.The yield of the aqueous leaves extracts(MOL)was highest and the MOB is the lowest.The TPC was 4.72-69.0 GAE/g DM,leaves showed the highest TPC value with the90%ethanol leaf extracts having the lowest in the 4 leaf extracts.No statistical difference was detected between 50 and 70%ethanol root extracts,and the TPC in them were higher than other root extracts.But 70%ethanol seed extracts(MOS)and MOB showed the highest TPC in seed and bark extracts.The TFC was 2.74-123 mg RE/g DM,and showed a similar trend as the TPC in seed,root,and leaves.But the 50and 90%ethanol bark extracts were higher than others,while there was no statistical difference between the two.MOS yielded stronger antioxidant activities in the 4 seed extracts(p<0.05),although there was no significant difference between 50 and 70%when using the ABTS.+assays.The 70%ethanol root extracts(MOR)also showed stronger antioxidant activities in the 4 root extracts(p<0.05),but no difference between 50 and70%when using the FRAP assays.MOB showed better antioxidant activities than the other 3 bark extracts(p<0.05),but no difference among 50,70,and 90%when using the FRAP assays.And the MOL showed better antioxidant activities than the other 3leaf extracts when using the ABTS.+assays(p<0.05),while no difference among the aqueous,50,and 70%when using the other assays.In conclusion,MOS,MOR,MOB,and MOL showed the highest antioxidant activities in each part.CAL27 and CNE-1 cells were incubated with the 4 MO extracts(MOS,MOR,MOB,and MOL,0.005–8 mg/m L)for 12,24,and 48 h,the inhibition appeared to be time-and concentration-dependent.During the first 24 h,the proliferation of HNC cells was significantly inhibited by the 4 extracts.Compared to the control,the anti-proliferative effects and the decreasing numbers of colonies were ranked as follows:MOB>MOL>MOR>MOS.After treatment with MO extracts for 24 h,apoptotic profiles were revealed.Compared to the control group,early apoptotic cells showed deepened staining,and crescent-shaped nuclear chromatin aggregated on one side of the nuclear membrane.In the late apoptotic cells,nuclei showed fragmented.The expression of BCL-2 was downregulated,while BAX expression was upregulated.Additionally,the ratio of BCL-2/BAX was downregulated as the following order:MOB>MOL>MOR>MOS.In conclusion,MOB showed the strongest against HNC cells,followed by MOL.(2)The MOB induced reactive oxygen species(ROS)generation and modulated the p38 phosphorylation in HNC cells(5-8F,CNE-1,CAL27,CAL27/CDDP).The MOB significantly inhibited the proliferation and metastasis,and induced apoptosis and reversed the cisplatin resistance in HNC cells,and the MOB can improve the cytotoxic of cisplatin to the cisplatin-resistant cells.MOB significantly inhibited the proliferation of the 3 HNC cells(5-8F,CNE-1,CAL27),and 5-8F was more sensitive to MOB among them.After MOB treatment,the number of cells into S phase and the colonies numbers were significantly reduced in the experiment groups.The apoptotic rates were increased and the nucleus showed apoptotic.The expression of pro-apoptotic protein BAX,Bid increased and the anti-apoptotic protein BCL-2 decreased significantly.And the expression of pro-caspase 3 decreased with the increasing of cleaved caspase 3.MOB reduced the rapid of wound healing,as well as the cell numbers in the Transwell assay.The metastasis related factors slug and vimentin were significantly down-regulated and E-cadherin was up-regulated in the m RNA and protein levels.MOB significantly inhibited the proliferation of cisplatin resistance cells(CAL27/CDDP)with the IC50 293.4?g/ml.And the expression of BAX,Bid,cleaved caspase 3 were significantly up-regulated,BCL-2 and pro-caspase 3 were down-regulated in CAL27/CDDP cells.Compared with cisplatin or MOB used alone,the combination of MOB with cisplatin could increase the cytotoxic and apoptosis.MOB significantly activated reactive oxygen species(ROS)generation but the increase were reversed by NAC in the 4 cells.MOB also decreased the expressions of oxidoreductase SOD2 but increased GPX1.The ROS level in cisplatin resistance cells was significantly higher than in normal HNC cells.After MOB treatment,the expression of p-p38 was significantly upregulated in the normal HNC cell lines(5-8F,CNE-1,CAL27),and significantly suppressed in the cisplatin-resistant cell line CAL27/CDDP cells.Cisplatin was found to significantly increase the expression of p-p38 in CAL27 and CAL27/CDDP cells.Compared to cisplatin or MOB used alone,MOB combined with cisplatin increased p-p38 expression in CAL27 cells,but suppressed p-p38 expression in CAL27/CDDP cells.After intervention with p38specific inhibitors and potent activator,it was found that regulating p-p38 expression could significantly regulate the expressions of apoptosis,migration-related proteins BAX,BCL-2,Caspase 3,E-cadherin,and vimentin.And NAC was also found to simultaneously regulate the expression of p-p38,apoptosis-and migration-related proteins.Using UHPLC-QE-MS technology to analyze MOB,it was found that most of the components contained were flavonoids,and there may be more than 30components.(3)The fraction(MOB-EAF)of MOB extracted by ethyl acetate had the strongest antioxidant activities and suppression effects with 5-8f cells among the 4fractions.And the MOB-EAF inhibited the growth of xenograft tumor and lung metastasis in vivo.Three purified compounds with significant inhibition with 5-8F cells were harvested from MOB-EAF after isolated and purified.Four fractions(MOB-CF,MOB-EAF,MOB-NBF,and MOB-WF)were obtained after MOB was extracted with chloroform,ethyl acetate and n-butanol in turn.MOB-EAF showed the strongest free radical scavenging ability and anti-cancer effect,followed by MOB-NBF.Seven weeks being fed with MOB-EAF,the growth of the tumors significant slower than the control group,and the numbers of lung cancer nodules were also lower than control group.The tumors growth speed in the high concentration group was slower than that of the low concentration group,and the number of lung cancer nodules in the high concentration group also lower than the low concentration group.After isolated and purified the MOB-EAF with macroporous adsorption resin,5sub-components were obtained.The 20%ethanol component had the stronger tumor suppressive properties with the IC50 of 68.28?g/ml.Followed by the 40%ethanol component with the IC50 of 100.5?g/ml.IC50 of the MOB-EAF was 117.6?g/ml,and IC50 of the MOB was 136?g/ml.Nine sub-extracted components were obtained after 20%ethanol component further purified with a silica gel column.Compound 4,compound 5,and compound 6 were found to have strong tumor suppressive properties among the 9 compounds,with IC50 of 55.88?g/ml,95.28?g/ml,79.77?g/ml,respectively.Compound 4 had significantly improved the anti-cancer ability compared with 20%ethanol component.TLC showed the numbers of components in the Compound 4,compound 5,and compound 6 were 6,3,and 3,and the weights were 2.48g,1.48g,and 2.88g.Conclusion:(1)All the extracts from the 4 parts of MO have strong antioxidant activities and suppression properties to HNC cells.(2)MOB regulated the proliferation,apoptosis,metastasis and reversed cisplatin resistance of HNC cells via modulating the ROS/p38 signaling pathway.(3)The purified components of MOB inhibited the growth of xenograft tumor and lung metastasis in vivo. |
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