| Aims:1. To investigated HS synthesis of MCF-10A Inhibition the breast cancer cells.2. To investigated HS on the inhibition of breast cancer tumors in mice transplanted.3. In endoplasmic reticulum (ER) stress state, the expression of Hpa in breast cancercells.4. In endoplasmic reticulum (ER) stress state, the effect of HS Inhibited the breastcancer cells.Methords:Part Ⅰ Cell experiment1. Extraction of MCF-10A cell culture medium in the convergence of the HS chain,the concentration of HS (200,400,800,1600mg.L-1),treated the breast cancer cellsMDA-MB-231and SK-BR-3after24、36、72h, then detected the breast cancer cellsproliferation inhibition rate by MTT，and according to this experiment calculated theIC50of HS; And through colony cloning assay detected the effection of HS on theMDA-MB-231and SK-BR-3cell proliferation.2. Different concentrations of2-DG（1.25，2.5，5，10and20mM）and TM（1.5,3，6，9and12μM）treated the breast cancer cells SK-BR-3after24,36,72h, thendetected the breast cancer cells prolifer ation inhibition rate by MTT；Differentconcentrations of HS (200,400,800,1600mg.L-1) combined with2-DG (10mM) orTM (3μM) acting on the SK-BR-3cells after24,36and72h, then detected thebreast cancer cells proliferation inhibition rate by MTT; And through colony cloningassay detected the effection of HS combined with2-DG or TM on SK-BR-3cellproliferation.3. In breast cancer cells MDA-MB-231, SK-BR-3as the research object，With2-DG,TM, HS, and HS combination with2-DG and TM acted on cells at different times(0,6,12,24,36h). Through Western blotting detected the expression of GRP78andHpa. As the same treatment. By FCM detected the apoptosis of breast cancer cells and through Hochest33258observated of morphological changes in nuclei of tumorcells by fluorescence microscopy.4. With2-DG, TM, HS, and HS combination with2-DG and TM acted on breastcancer cells, then detected the activity of caspase-3.5. With2-DG, TM, HS, and HS combination with2-DG and TM acted on breastcancer cells，then detected the expression of BcL-2family proteins.PartⅡ Animals experiment一、HS inhibited the tumor of C3H mice1. The C3H mice with spontaneous breast cancer cells were made to the original tumorthen regulated the concentration of tumor cells as2×107/ml and inoculated intumor-free C3H mice and randomized. After9days, each group injected withnormal saline (NS) and HS through intraperitoneal every day. Measurement thetumor diameter and length every three days and then mapped tumor growth curve.After18days, killed mice, weighed the tumor weight, calculated inhibitory rate andmeasured the provision of weight index.2. Routine paraffin-embedded tumor tissue, sliced and then HE staining，Under themicroscope to observe the pathological changes. Using the red fluorescent one stepTunnel kits detected the apoptosis of tumor.二、 HS inhibited the human breast cancer cells in nude mice1. Breast cancer xenograft model SK-BR-3cells cultured with10%fetal bovine serumDMEM culture medium, under the conditions of in5%CO2,37℃. When75%-85%of the cells fused, Tyrosine digested and centrifuged, washed twice with PBS, andthen suspended of its Army into a final cell concentration of approximately1×107/ml. Taked SK-BR-3cells in0.2ml (about2×106), After local disinfection,inoculated into subcutaneous in nude mice.2. Animal groups Observed the growth of tumors in nude mice the next day,Measurement the tumor diameter and length every three days through calipers,When the tumor volume reached300-500mm3, randomly divided into2groups (n=10), one group was were injected NS, the aother group were injected HS.Measurement the tumor diameter and length every three days and then mappedtumor growth curve. After28days, killed mice, weighed the tumor weight,calculated inhibitory rate and measured the provision of weight index.3. Routine paraffin-embedded tumor tissue, sliced and then HE staining, Under themicroscope to observe the pathological changes. Using the red fluorescent one stepTunnel kits detected the apoptosis of tumorResults:Part Ⅰ Cell experimentHS,2-DG, TM, and HS combination with2-DG or TM effected the breast cancerMDA-MA-231and SK-BR-3proliferation.1. MTT assay showed the results that different concentrations of HS could inhibit theproliferation of breast cancer cells. Compared with the control group, the differencewas significant（P﹤0.05）. With the increasing concentration of HS and treatmenttime, inhibition rate increased. HS (1600μg/ml) had the strongest inhibitionrate.After72h, the maximal inhibition rates were75.43%and79.59%. And thecolony cloning experiments further confirmed the results that HS could inhibited thegrowth of SK-BR-3and MDA-MB-231.2. MTT assay showed the results that breast cancer cell SK-BR-3was not sensitive toER stress inducer2-DG and TM.2-DG (20mM) and TM (12μM) had the strongestinhibited,After72h, the maximum inhibition rate was25.19%and30.53%; UnderTM or2-DG in the induction of breast cancer cells SK-BR-3produced endoplasmicreticulum stress, significantly enhanced HS on the sensitivity to SK-BR-3throughMTT, after72h, the maximum inhibition rate reached80.21%and81.40%. And thecolony cloning experiments further confirmed the results that HS could inhibited thegrowth of SK-BR-3and MDA-MB-231.二、 HS induced apoptosis of MDA-MB-231and SK-BR-31. PI HS induced apoptotic rate of MDA-MB-231and SK-BR-3that reached25.67%and23.3%. Compared with the control group, the difference was significant (P <0.05).2. Using Hochest33258fluorescence staining，HS could induce breast cancer cellnucleus concentration,bright blue, or divided nuclear into leaves debris-like andedge set observation by fluorescence microscopy，In the control group did not showabnormal nuclear morphology, a clear nuclear profile sleek, light blue and uniformstructure.3. Through caspase-3activity detection kit found, HS could induce the expression ofcaspase-3activity in MDA-MB-231and SK-BR-3. Compared with the controlgroup, the difference was significant (P <0.05).4. Through western blotting detected the expression of Bcl-2family proteins,compared with the control group, HS could decrease the expression ofanti-apoptotic protein BcL-2, BcL-xL while the apoptotic protein Bax, Badexpression was increased in MDA-MB-231and SK-BR-3, the difference wassignificant (P <0.01)三、The induction of ER stress could introduce the expression Hpa and apoptosis inMDA-MB-231and SK-BR-3cells.Given to the classic ER stress inducer TM and2-DG could also increaseexpression of GRP78and Hpa, compared with the control group, the difference weresignificantly (P<0.01). PI staining trough flow cytometry analysis, after48h, Thehighest rates were16.7%and15.3%of SK-BR-3, compared with the control group of0h, the difference of48h was significantly (P <0.05).四, The endoplasmic reticulum stress inducer TM and2-DG could increased HSinducing apotosis of SK-BR-3.1. PI staining trough flow cytometry analysis, TM and2-DG induced the highest rateswere16.7%and15.3%of SK-BR-3; The apoptotic rate indued by HS combinationwith TM or2-DG could reached59.85%and58.11%in SK-BR-3, and comparedwith HS group, the difference was significant (P <0.05);2. Using Hochest33258fluorescence staining, Under endoplasmic reticulum stressstate induced by TM and2-DG in SK-BR-3, HS induced more pronounced changesin the nucleus of breast cancer, nucleus was concentrated light blue, or nuclear was divided leaves, debris-like and edge set, in the control group did not show abnormalnuclear morphology and showed a clear smooth nuclear contour, light blue anduniform structure.3. Through caspase-3activity detection kit found, Under endoplasmic reticulum stressstate induced by TM and2-DG in SK-BR-3, HS could significantly enhance theexpression of the caspase-3in SK-BR-3.compared with HS group, the differencewas significant (P <0.01).4. Through western blot, Under endoplasmic reticulum stress state induced by TM and2-DG in SK-BR-3, HS could significantly decrease expression of anti-apoptoticprotein BcL-2, BcL-xL,while the expression of pro-apoptotic proteins Bax, Badwere increased,compared with HS group, the difference was significant (P <0.01).PartⅡ Animals experiment一, HS inhibited tumor of C3H mice1.HS significantly inhibited C3H mice on the growth of breast cancer. After18days,weighted tumor: NS group (0.967±0.15) g, HS group (0.55±0.14) g. Comparedwith the NS group,HS group significantly reduced tumor volume and the tumorinhibition rate reached43.12%,the difference was significant (P＜0.01); The spleenweight index of C3H mice were detected compared with NS group, the differencewas not statistically significant (P＞0.05)2.HE staining showed that the cells in the group of HS had different sizes, deeplystained nucleus, cytoplasm and large areas of punctate necrosis and necrosis of thenon-performance of the structure of hair.3.Through one step Tunel apoptosis detection kit (red fluorescence) detected theapoptosis in tumor tissues, the red fluorescence intensity of HS group weresignificantly higher than NS group by fluorescence microscopy.二, HS inhibited the human breast cancer cells in nude mice1. HS significantly inhibited C3H mice on the growth of breast cancer. After18days,weighted tumor: NS group（5.27±0.37）g, HS group （3.03±0.17）g. Compared with the NS group,HS group significantly reduced tumor volume and the tumorinhibition rate reached42.55%,the difference was significant (P＜0.01); The spleenweight index of C3H mice were detected,compared with NS group, the differencewas not statistically significant (P＞0.05)2. HE staining showed that the cells in the group of HS had different sizes, deeplystained nucleus, cytoplasm and large areas of punctate necrosis and necrosis of thenon-performance of the structure of hair.3. Through one step Tunel apoptosis detection kit (red fluorescence) detected theapoptosis in tumor tissues, the red fluorescence intensity of HS group weresignificantly higher than NS group by fluorescence microscopy.Conclusion:1． The HS synthesized by Normal human breast epithelial cells, has anti-cancer effectin vivo and invro, and its mechanism may be related to the role of caspases andapoptosis family of Bcl-2family.2． Under endoplasmic reticulum stress state, HS can regulate expression ofheparanase.3． Under endoplasmic reticulum stress state, the apoptosis of breast cancer cell linesinducing by HS was increased. |
PDF Full Text Request