| Cancer,also known as malignant tumor,is caused by the abnormal cell proliferation.It destroys the normal physiological functions of the human body and has seriously threatened human health and life safety,as well as brought a heavy economic burden to the people.At present,the clinical treatment methods of cancer mainly include surgery,chemotherapy,radiotherapy,etc.,but also face many problems such as drug resistance,poor treatment effect and poor prognosis.Therefore,it is more urgent to look for the new therapeutic drugs and strategies.The orchid herb Dendrobium officinale Kimura et Mi go is a valuable traditional Chinese medicinal with a long history of medicinal use.The highest content of components in Dendrobium officinale are polysaccharides(DOPs),which mainly composed of mannose and glucose.Studies have shown that mannose inhibit tumor growth and promote apoptosis.Dendrobium officinale polysaccharides have also been shown to have effects of inhibiting proliferation and promoting apoptosis on virous types of tumor cells,and the antitumor activity of polysaccharides with different molecular weights is significantly different.However,it has not been reported whether the antitumor activity of carbohydrates mainly composed of mannose including monosaccharides,oligosaccharides and polysaccharides with different molecular weights is different.Based on the research above,in this study,DOPs with different molecular weights were obtained through extraction and oxidative degradation.Osteosarcoma U20S and Saos-2 cells were used as the experimental objects to explore and compare the proliferation inhibition of mannose,mannohexaose,DOPs alone and combined with the traditional antitumor drug cisplatin(DDP).Then,seeking the best anti-osteosarcoma drug to study the effect of combining it with cisplatin on cell apoptosis,and the mechanism of action was explored by molecular biology approaches.The results of this study will provide a certain experimental basis and research directions for the future development of Dendrobium officinale and carbohydrate drugs in t.he treatment of tumors.ObjectiveThe influences of mannose,mannohexaose,and Dendrobium officinale polysaccharides with different molecular weights used alone or in combination with cisplatin on two common osteosarcoma U20S and Saos-2 cells proliferation and apoptosis were tested and compared,and the mechanism of action was explored.Methods(1)The stems of Dendrobium officinale were used as raw material.After degreasing with petroleum ether and removing small molecular compounds with 80%ethanol,the crude Dendrobium officinale polysaccharide DOP122 was obtained by water-extraction and alcohol-precipitation method.Then,the degraded fragments DOP20,DOP8,and DOP2 that with different molecular weights were obtained by the oxidative degradation of Fe2+-Vc-H202 method,and their yields were calculated.(2)The contents of carbohydrate in DOPs were determined by the phenol-sulfuric acid method,and the protein contents were determined by the Bradford method.The molecular weights of DOPs were measured by high-performance gel permeation chromatography(HPGPC),and the monosaccharide compositions were detected by PMP pre-column derivatization method using the high-performance liquid chromatography(HPLC).(3)MTT assay was used to measure the effects of different concentrations(12.5,25,50,100,200 a g/mL)of mannose(Ml),mannohexaose(M6)and DOPs(DOP2,DOP8,DOP20,DOP122)on the proliferation activities of osteosarcoma U20S and Saos-2 cells after intervention for 24 and 48 hours.The appropriate dose concentration was selected for the subsequent experiments.(4)Clonogenic assay was used to measure the effects of Ml,M6,DOP2,DOP8,DOP20,and DOP122(25 μg/mL)on the clonogenic capacity of osteosarcoma U20S and Saos-2 cells.(5)Lactate dehydrogenase(LDH)assay kit was applied to detect the effects of M1,M6,DOP2,DOP8,DOP20,and DOP122(100 μg/mL)on the lactic acid production of osteosarcoma U20S and Saos-2 cells.(6)MTT assay was used to detect the effects of DDP alone on the proliferation of osteosarcoma U20S and Saos-2 cells after intervention for 24 and 48 hours.The appropriate concentration(10 μ M)of DDP was selected for combination with M1,M6,DOP2,DOP8,DOP20,DOP122(100 μg/mL)to detect their effects on the proliferation of osteosarcoma U20S and Saos-2 cells after intervention for 24 and 48 hours.The most effective carbohydrate drug was selected for subsequent experiments that in combination with DDP.(7)MTT assay was used to detect the effects of 10 μM DDP combined with different concentrations of DOP20(12.5,25,50,100,200 μg/mL)on the proliferation of U20S and Saos-2 cells after intervention for 24 hours.The combined effect was evaluated using the Isobologram method.(8)The effects of 10 μM DPP and 100 μg/mL DOP20 alone and combined intervention for 24 hours on the morphology of osteosarcoma U20S and Saos-2 cells were observed under the common inverted microscope and fluorescence microscope after Hoechst 33258 staining.(9)Flow cytometry was used to detect the effects of 10 μM DPP and 100 μg/mL DOP20 alone and combined intervention for 24 hours on-the apoptosis of osteosarcoma U20S and Saos-2 cells stained with Annexin V-FITC/PI.(10)The western blot assay was used to detect the effects of 10 μM DPP and 100 μg/mL DOP20 alone and combined intervention for 24 hours on the expression of P53,Bax,Bak,Bcl-2,Mcl-1,Caspase3,Cleaved caspase3,Caspase9,Cleaved caspase9,PARP,and Cleaved PARP that associated with cell apoptosis signaling pathway in osteosarcoma U20S and Saos-2 cells.Results(1)Four polysaccharide fragments DOP122,DOP20,DOP8,and DOP2 were obtained after extraction and degradation.The yields of them were 19.87%,82.57%,76.19%,and 54.17%,respectively;the carbohydrate contents were 70.73%、87.81%、84.82%、87.57%,respectively;the protein contents were 5.71%、3.32%、2.87%、4.03%,respectively;the molecular weights were 1224.54 kDa,200.99 kDa,80.32 kDa,and 24.89 kDa,respectively;the monosaccharide compositions(mannose:glucose)were 3.29:1,4.12:1,4.47:1,4.28:1,respectively.(2)Ml,M6,DOP2,DOP8,and DOP122 have no significant effect on the proliferation of osteosarcoma U20S and Saos-2 cells,and the cell survival rate was maintained above 80%.However,DOP20 inhibited cell proliferation in a dose-and time-dependent manner.(3)DOP20 significantly inhibited the colony formation of osteosarcoma U20S and Saos-2 cells(P<0.01),but opposite to M1,M6,DOP2,DOP8,and DOP122.(4)M1,M6,DOP2,DOP8,and DOP122 had no significant effect on the production of lactic acid of osteosarcoma U20S and Saos-2 cells(P>0.05),while DOP20 significantly reduced the production of lactic acid of cells(P<0.01).(5)The proliferation activities of osteosarcoma U20S and Saos-2 cells were significantly inhibited by DOP8 alone for 48h(P<0.01),while there is no enhanced effect when combined with DDP(P>0.05).M6 and DOP2 showed an enhanced inhibition of Saos-2 cell proliferation only when in combination with DDP for 24h(P<0.05).However,DOP20 alone for 24h and 48h can inhibit the proliferation activity of U20S and Saos-2 cells(P<0.05 or P<0.01).When combined with DDP,it also showed an enhanced inhibitory effect on cell proliferation than the DDP monotherapy group(P<0.05 or P<0.01),but other carbohydrate drugs had no such effect.(6)Combination of DOP20 and DDP inhibited the proliferation of osteosarcoma U20S and Saos-2 cells in a dose-and time-dependent manner.The combined treatment of the two drugs was shown as synergistic effect.(7)Compared with the blank group of osteosarcoma U20S and Saos-2 cells under a common inverted microscope,a few cells of DOP20 and DDP monotherapy groups appear to be poor adherence,cell shrinkage and floated,while the effect of the combination group was the most obvious.After Hoechst 33258 staining,the nucleus of DOP20 and DDP monotherapy group were observed under a fluorescence microscope with slightly stronger blue fluorescence than the blank group,and there was a small amount of dense staining.The nucleus of the combined treatment group of DOP20 and DDP appeared the phenomena such as shrinkage and rupture.Some fragment-like dense staining was seen,and the apoptosis characteristics were more obvious than the DDP monotherapy group.(8)Compared with the blank group,DOP20 alone can not increase the apoptosis rate of osteosarcoma U20S cells(P>0.05),but increase the apoptosis rate of Saos-2 cells(P<0.01).DDP alone and the combination of DOP20 and DDP significantly increased the apoptosis rate of U20S and Saos-2 cells(P<0.01).Furthermore,there proved a statistical significance when compared the combination group of DOP20 and DDP with DDP monotherapy group(P<0.01).(9)Compared with the blank group,the combination of DOP20 and DDP upregulated expression levels of tumor suppressor protein P53,pro-apoptotic proteins Bax and Bak(P<0.01),down-regulated expression levels of anti-apoptotic proteins Bcl-2 and Mcl-1(P<0.01),and up-regulated the proportions of apoptotic promoter Cleaved caspase9/Caspase9,apoptotic executor Cleaved caspase3/Caspase3,and apoptotic marker Cleaved PARP/PARP(P<0.01)in osteosarcoma U20S and Saos-2 cells.And the effect of the combination group was stronger than DDP monotherapy group in varying degrees(P<0.5 or P<0.01).ConclusionThis study confirmed for that among mannose,mannohexaose and Dendrobium officinale polysaccharides with different molecular weights,Dendrobium officinale polysaccharide DOP20 with a molecular weight of 200.99 kDa alone has the best ability to inhibit the proliferation of osteosarcoma U20S and Saos-2 cells,and the mechanism of action may be associated with blocking glycolysis of cells.When combined with cisplatin,DOP20 can play a synergistic role in inhibiting cell proliferation,and showed an enhanced effect on inducing apoptosis of U20S and Saos-2 cells through the mitochondrial pathway.The results preliminarily indicated that in the vitro study of treatment of osteosarcoma by carbohydrate drugs that mainly composed of mannose,the effects of Dendrobium officinale polysaccharides are better than mannose and mannohexaose.And it is suggested to seek the relatively suitable molecular weight or molecular weight range,but not recommended to use crude polysaccharides. |
PDF Full Text Request