| Laryngeal cancer is one of the most common in head and neck malignancies.To date,the incidence rate of laryngeal cancer is increasing gradually,accounting for 5.7% to 7.6% of all malignant tumors of the head and neck in the northeast areas of China.The treatment of laryngeal cancer includes surgery,chemoradiotherapy and biotherapy,as well as combination of various treatments.Even though,there is no singificant improvement in long-term survival in laryngeal cancer.Recurrence and metastasis are the major causes of treatmen failure.Since the emergence of CSCs theory,CSCs have become a new target for cancer treatment.In fact,CSCs are a small subpopulation of cancer cells and the origin of tumor,serving the roles as stem cells in cancer,with the property of self-renewal,multilineage differentiation,and highly tumorigenic and metastatic ability.Great progresses in studies on CSCs have been made.To date,CSCs have been isolated from variety malignancies including leukemia and assorted solid tumors.In our study,we analysed the possible source of tumor metastasis in laryngeal squamous carcinoma(LSC)cells.Chemotherapeutics for laryngeal cancer is not ideal due to drug resistence of CSCs.It is essential to search for more powerful new antitumor drugs.Dihydroartemisinin(DHA),a semisynthetic derivative of a Chinese medicinal herb,Artemisia annua,possessing the advantages of high safety with great efficiency and few side effects,can suppress multiple tumors including head and neck cancers.Whether DHA can prevent invasion and metastasis of tumor is not clear.Aiming at two afo mentioned problems,we conducted the present investigation to explore whether CSCs are the major cells mediating invasion and metastasis,and the effects of DHA on invasionand metastasis of LSC cells as well as its possible mechanisms by in vitro and vivo experiments.Part one: Comparison of invasiveness and metastasis capability between CD133~+ and CD133~-cells from LSC cellsObjactive: To compare the differences of invasion and migration in CD133~+ and CD133~-cells from LSC cells,and study the possible underlying mechanisms.Methods:1 Isolation of CD133~+ and CD133~-cells from Hep-2 cells of LSC was performed,and purity of sorted cells was detected by flow cytometry before and after culturing expansion.2 The difference of invasion and migration between CD133~+ and CD133~-subpopulations from Hep-2 cells was assessed by transwell chamber test.3 The expression of MMP-9 and E-cadherin was detected by Western blot,both of which are related to migration and invasion of cancer cells.Results:1 Results of flow cytometryThe rate of positive expression for PE-conjugated CD133 in Hep-2 cells was 3.4±0.559% after cells were stained by CD133 antibody.2 Results of transwell chamber assay.CD133~+ subpopulation exhibited stronger potency for migration and invasion than CD133~-cells after incubated in upper chamber for 24 h.3 Results of Western blot.The protein level of MMP-9 is 0.87±0.016 in CD133~+ cells,which is higher than that in CD133~-(0.643±0.011).The differences between the two groups were significant(t=20.08,P<0.01).On the contrary,the level of E-cadherin,another protein related to invasion and migration,is decreased.The expression of E-cadherin in CD133~+ cells(0.376±0.0200)is lower than that in CD133~-cells(0.791±0.015),and the difference between the two groups was significant(t=28.49,P<0.01).Conclusions:1 CD133~+ cells from LSC have a stronger ability in invasion and metastasis than CD133~-cells.CD133~+ cells may be the main source for invasion and metastasis of LSC.2 High invassiveness of CD133~+ cells from LSC is attributed to expressing more protein of MMP-9 and losing E-cadherin,all of which are associated with invasion and metastasis.Part two: Inhibition effects of DHA on invasion and migration of CSCs from LSC through blocking STAT3 signal ing pathwayObjective: To observe the inhibitory effect of DHA on invasion and metastasis of CSCs,and its association with blockade of STAT3 activation by DHA.Methods:1 The CD133~+ cells isolated through flow cytometer were divided into four groups: control group(without any treatment),experiment group 1(treated with IL-6,20ng/ml for 1h),experiment group 2(treated with IL-6 and DHA),and experiment group 3(treated with DHA only).Similar another four groups were categorized while switching to hypoxia exposure.Four groups include control group,hypoxia group,hypoxia plus DHA group and DHA group.2 Changes of invasive and migrated cell number from CD133~+ cells under above-mentioned conditions were assessed by transwell chamber assay.3 The expression of STAT3,p-STAT3,HIF-1,MMP-9 as well as E-cadherin under different conditions was detected by Western blot.Results1 Results of transwell chamber experiment.(1)The number of migrated CSCs in control group,IL-6 group,IL-6 plus DHA group and the DHA group was 162±13/HP,310±13/HP,57±9/HP,48±6/HP,respectively.The differences among the four groups(F=1184,P<0.01)and the comparison between two interclass groups were statistically significant(control vs.IL-6 group,q=41.92,P<0.01;IL-6 vs.IL-6+DHA group,F=71.28,P<0.01;control vs.DHA group,F=32.06,P<0.01).Theresults of invasion assay demonstrated that the number of invaded cells was107±3/HP in control group,204±7/HP in IL-6 group,44±7/HP in IL-6+DHA group,and 30±7/HP in DHA group.Statistical analysis showed that the number of invasion in four groups were remarkably different(F=1453,P<0.01)and the comparison between two interclass groups were statistically significant(control vs.IL-6 group,q=46.68,P<0.01;IL-6 vs.IL-6+DHA group,q=76.79,P<0.01;control vs.DHA group,q=37.11,P<0.01).(2)The transwell testing on migration was also conducted in another four experimental groups treated by hypoxia in the presence or abscense of DHA.It was observed that the migrated CSCs were numbered 137±9/HP in normoxia(control)group,229±7/HP in hypoxia group,59±4/HP in hypoxia plus DHA,and 39±5/HP normoxia plus DHA,respectively.The differences among the four groups are significant(F=1675,P<0.01)and the comparison between two interclass groups were statistically significant(normoxia vs.hypoxia group,q=43.68,P<0.01;hypoxia vs.hypoxia plus DHA group,q=80.57,P<0.01;normoxia vs.normoxia plus DHA group,q=46.32,P<0.01).The number of invaded CSCs was 122±10/HP(normoxia group),190±7/HP(hypoxia group),58±6/HP(hypoxia plus DHA group),and 39±7/HP(normoxia plus DHA group).The data are analysised by one-way analysis of variance and the differences among the four groups are significant(F=705.9,P<0.01).Comparison between two interclass groups were statistically significant(normoxia vs.hypoxia group,q=26.35,P<0.01;hypoxia vs.hypoxia plus DHA group,q=51.35,P<0.01;normoxia vs.normoxia plus DHA group,q=32.19,P<0.01).2 Results of Western blot.(1)The protein level of p-STAT3,STAT3 as well as invasive and metastatic related proteins such as MMP-9 and E-cadherin from CSCs were tested by Western blot in the four groups(treated with DHA with or without IL-6).There was no significant difference among the four groups in the level of STAT3(P>0.05).The differences are significant in the expression of p-STAT3 in four groups(F=10190,P<0.01).The level of p-STAT3 increasedmarkedly when IL-6 was added(control vs IL-6 group,q=20.87,P<0.01).However,it decreased dramatically when DHA was added(IL-6 vs.IL-6 plus DHA,q=183.5,P<0.01,control vs.DHA,q=165,P<0.01).Similarly,variation trend of MMP-9 was consistent with that of p-STAT3.The comparison among the four groups was ananysed by One-way ANOVA(F=7830,P<0.01).The comparison between two groups with interclass was as following: control vs.IL-6 group(q =51.5,P<0.01),IL-6 vs IL-6 plus DHA group(q =143.7,P<0.01),control vs DHA group(q=232.9,P<0.01).Another protein E-cadherin is associated with metastasis whose variation tendency is contrary to that of MMP-9 and p-STAT3.The differences of E-cadherin expression are significant different among the four groups(F=1219,P<0.01).When IL-6 was added,E-cadherin reduced significantly(control vs IL-6,q =9.845,P<0.01).While E-cadherin increased obviously by treatment with DHA(control vs.DHA,q= 59.53,P<0.01;IL-6vs.IL-6+DHA,q=59.85,P<0.01).(2)The expression of related proteins(HIF-1,STAT3,p-STAT3,MMP-9,E-cadherin)in another four groups treated with hypoxia and/or DHA were tested by Western blot.Results were analysed by variance analysis.The difference of protein level of HIF-1?among four groups was significantly different(F=350.9,P<0.01).The level of HIF-1?of CSCs from Hep-2 cell line increased notably when cells was cultured under hypoxia(normoxia vs.hypoxia group,q=28.32,P<0.01).DHA inhibitded HIF-1? expression significantly(normoxia vs normoxia plus DHA,q=12.80,P<0.01;hypoxia vs hypoxia plus DHA,q=8.26,P<0.01).The viaration tendencies of protein level in p-STAT3 and MMP-9 were consistent with that of HIF-1?.The protein level of p-STAT3,MMP-9 and E-cadherin in the four groups are statistically different(p-STAT3,F=2187,P<0.01;MMP-9,F=4833,P<0.01;E-cadherin,F=17730,P<0.01).They increased significantly under the conditon of hypoxia compared with normoxia(p-STAT3,q=34.68,P<0.01;MMP-9,q=45.31,P<0.01).Likewise,when the cells were treated with DHA,the expression of p-STAT3 and MMP-9 proteins decreased distinctly,which were induced byhypoxia(normoxia vs.normoxia plus DHA group,p-STAT3,F=63.68,P<0.01;MMP-9,F=115.7,P<0.01;hypoxia vs.hypoxia plus DHA group,p-STAT3,q=90.30,P<0.01;MMP-9,q=99.39,P<0.01).However,the expression of E-cadherin increased under the condition of hypoxia and difference is significant(q=8.848,P<0.01).The treatment with DHA increased the expression of E-cadherin(normoxia vs.normoxia plus group DHA,q=258.7,P<0.01;hypoxia vs.hypoxia plus DHA,q=196.1,P<0.01).Conclusions:1 The either IL-6 or hypoxia could promote invasion and metastasis in CSCs fromLSC.2 Invasion and metastasis of CSCs from LSC could be inhibited by DHA.The underlying mechanism may reside on inactivation of p-STAT3 specifically by DHA,leading to reduction of invasion and metastasis-related proteins downsteam of p-STAT3.Part three: Comparison of metastasis ability between CD133~-and CD133~+ cells from LSC cells in vivoObjective: To compare the difference of CD133~-and CD133~+ cells from LSC in invasion and metastasis,and explore its possible underlying mechanism by detecting proteins related to invasion and metastasis.Methods:1 To establish the tumor metastasis animal models,nude mices were randomly divided into two groups and injected with CD133~-and CD133~+ cells through the tail vein,respectively.The general condition as well as survival time of each mice was observed and recorded during the whole process of experiment.2 The lung metastasis foci were observed and counted after confirmation by HE staining of suspected metastatic lung tissues from animals of each experimental group.3 The expression of proteins related to invasion and metastasis in pulmonary metastatic foci was detected by Western blot in order to analyse and speculate possible mechanism.Results1 The general condition of mice: the estabolished invasive and metastatic model has a great impact on the general condition of the animals.Part loss of the bodyweight in mice was observed after injection of tumor cells through the tail vein.There were 2 mice were confiremed to have bodyweight loss on the31 st day,and three mice on the 33 rd day in the CD133~-cell group.In mice injected with CD133~+ cells,there were 3 mice were confirmed to have bodyweight loss on the 19 th day,2 mice on the 21 st day,and 1 mice on the29 th day.A progressive deterioration of the mice activities and spirit began to emerge after weight loss.There was no statistically significant difference of bodyweight between the two groups.2 Comparison of differences in pulmonary metastasis between CD133~+and CD133~-groupPulmonary organ in CD133~-group was with better looking than in CD133~+ group.The gross specimen of the lung in CD133~-group with ruddy appearance and smooth surface was seen.However,pulmonary organ in CD133~+ group with pale apperance and rough surface was observed.The number of pulmonary metastasis foci in CD133~+ group is higher than that in CD133~-group,as evidenced by HE staining and microscopic observation.The differences between the two group were statistically significant as analysed by student's T test(t=2.615,P<0.05).3 Comparison of survival time between CD133~-and CD133~+ groupsSudden death occurred in 2 mice on the 34 th day and 36 th day,and 3mice on the 39 th day in CD133~-group,respectively.The survival rate decline gradually,85.71%,71.43% respectively.3 mice died in 39 th day.The final survival rate is 28.57%.In CD133~+ group,three mice had a death on the 29 th,31th and 34 th day,respectively.The survival rate decline gradually,85.71%,71.43% and 57.14% respectively.Three mice had a death in 39 th day.The final survival rate is 28.57%.These data were sorted and analysed by Kaplan-Meier.Log-rank test was used to determine survival and differences between CD133~-and CD133~+group.A p value >0.05 suggests the differencebetween the two groups is not statistically significant.4 The differences of proteins associated with invasion and metastasis in metastatic tumors in two groups.Proteins associated with invasion and mirgration in metastatic tumors were detected by Western blot after the experiment.The expression of p-STAT3 was obviously different in two the groups.The relative protein level of p-STAT3 in CD133~+ group(1.70±0.05)was higher than that in CD133~-group(0.83±0.30).The difference between the two groups had statistical significance as analysed by student's T test(t=4.879,P<0.01).However,there was no significant difference of STAT3 expression between the two groups.The relative level of MMP-9 was obviously different.The relative protein level in CD133~+ group was 1.74±0.51,which was higher than that in CD133~-group(0.47±0.15).The difference of two groups was statistically significant(t=4.168,P<0.05).The relative expression level of E-cadherin in CD133~+group(0.86±0.14)was lower than that in CD133~-group(1.22±0.04).Difference was statistically significant(t=4.241,P<0.05).Conclusions:1 Compared with CD133~-cells,CD133~+ cells from LSC are more likely to metastasize in vivo.CD133~+ cells may be the key cell subpopulation,mediating invasion and metastasis in LSC.2 Constitutive activation of STAT3 leads to changes in protein expression levles of downsteam proteins,such as increased MMP-9 and decreased E-cadherin,a prerequisite of tumor invasion and migration,which makes tumor metastasis more likely to happen.Part four: The influence of DHA on metastasis of CSCs of LSC in vivoand its possible mechanism.Objective: To observe the effect of DHA on invasion and metastasis of tumor and study possible mechanism through detecting proteins related invasion and metastasis by Western blot.Methods:1 Fourteen BALb/c mice were all injected with CD133~+ cells to establishtumor metastatic models.These mice were randomly divided into two groups as control or experiment group.Mice in the experiment group were treated with DHA,and with DMSO in control group.2 Pulmonary metastasis foci were observed and counted after confirmation of the suspected lesions picked from lung tissues of each group by HE staining.3 The expression of proteins related to invasion and metastasis in the pulmonary metastasis tissues were detected by Western blot.Results:1 The general condition: 3 mice in control group were confirmed to have bodyweight loss on the 9th day,the 17 th day and 25 th day,respectively.Another two mices in the control group were confirmed to have loss in bodyweight on the 53 th day,one of had 25% bodyweight loss,another of which had less than 20% bodyweitght loss but with lag in response,limp and flinch etc.One mouse in the experiment group began to lose weight with bad psychosis on the 23 th day.There is no statistically significant difference of bodyweight between the two groups.2 DHA prevented dissemination and metastasis of CSCs of LSC to lung.Gross specimen of lungs had pale apperance with rough surface in control group,while ruddy appearance and smooth surface in DHA-treated group was observed.The pulmonary metastasis foci were counted and analysed.The difference in numbers of lung metastatic lesions between the two groups was statistically significant(t=2.940,P<0.05).3 The influence of DHA on survival of mice.Three mice died on the 12 th day,24 th day and 29 th day,respectively.The survival rates were 85.71%,71.43%,57.14%,respectively.Two mice died on the 59 th day.The final survival rate is 28.57%.In the experiment group,there was only one mouse dead on the 29 th day.There is no obvious change in mental status and diet of the rest of the mice in experiment group.The overall survival time exceeded 59 days.The overall survival rate is85.71%.These data were sorted and analysed by Kaplan-Meier with alog-rank test to determine survival difference between the two groups.Survival rate of experiment group was higher than that of control group as analysed statistically(P<0.05).4 The changes of proteins related to invasion and metastasis after application of DHA.Proteins associated with invasion and metastasis in pulmonary metastasis lesions were detected by Western blot.The relative expression level of p-STAT3 in experiment group(0.235±0.059)was lower than that in control group(1.063±0.222).The difference between two groups had statistical significance as analysed by student's T test(t=6.248,P<0.01).There was no difference in the expression of STAT3 between the two groups.The relative expression level of MMP-9 in DHA-treated group was 0.300±0.033,which was lower than that in the contol group(0.926±0.289).The difference of two groups were statistically significant(t=3.731,P<0.01).However,the relative expression of E-cadherin was 1.305±0.319 in DHA treatment group,which was higher than that in control group(0.737±0.138).The difference of two groups were statistically significant(t=2.830,P<0.05).Conclusions:1 DHA prevent dissemination and formation of lung metastasis of CSCs in vivo.Meanwhile,application of DHA could prolong survival time of mice,compared to control group.2 DHA may suppress the expression of MMP-9 and increase expre3 ssion of E-cadherin by inhibiting phoshorylated STAT3,and thus prevent dissemination and lung metastasis of LSC. |
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