| Background and objectives:Cancer is a major public health problem all over the world,which has caused enormous burden.Although progress has been made in cancer prevention and treatment,new cancer cases will reach 22.2 million and death cases will reach 13.1 million by2030.Thyroid cancer as a malignant tumor of endocrine system has attracted worldwide attention because of its rising incidence.Thyroid cancer(TC)is the most common endocrine malignancy,which mainly includes four pathological types:papillary thyroid carcinoma(PTC),follicular thyroid carcinoma(FTC),anaplastic thyroid carcinoma(ATC)and medullary thyroid carcinoma(MTC).At present,the etiology of thyroid cancer is unclear,and the medical community is still exploring.Most patients with thyroid cancer have good prognosis,but they need systemic drug intake.Surgery often causes related complications,which seriously affect the quality of life of patients.The increased risk of secondary malignant tumors and the diagnosis of cancer recurrence also cause heavy economic and psychological burden.In addition,ATC is the most invasive thyroid cancer which incidence is only 2%of all thyroid cancer,but the mortality rate is 50%.There is no effective treatment so far.Therefore,it is of great significance to look for chemopreventive agents that reduce the incidence of TC and new chemotherapy drugs against ATC.Resveratrol is known as a polyphenolic plant compound that has an inhibitory effect on many stages of cancer and has no toxic or side effects on normal tissue cells.It is an ideal chemopreventive agent and anticancer drug.We recently found that some ATC cell lines including those with retinoic acid resistance(THJ-16T and THJ-21T)were sensitive to resveratrol in terms of distinct growth arrest and extensive apoptosis,indicating the potential therapeutic values of this nontoxic polyphenol compound in the practical treatment of ATCs.However,THJ-11T ATC cell line had little response to resveratrol treatment due to certain unknown reason(s).Therefore,studying the intrinsic mechanism of individual differences in resveratrol sensitivity has far-reaching implications for clinical individualized medication significance.In view of this,this topic has carried on the concrete research to the following content:(1)Establish a rat multiorgan tumorigenesis model induced by DEN/MNU/DHPN.The incidence and progress of precancerous lesions of multiple organs in rats were analyzed by intragastric or intraperitoneal injection administration of resveratrol.(2)Explore the chemopreventive effect of resveratrol through a series of experimental methods on the occurrence and development of thyroid cancer as well as its mechanism using the DMD model.(3)The difference sensitivity and possible molecular mechanism of resveratrol against ATC cells were discussed by a series of experimental methods on THJ-16T and THJ-11T cells with resveratrol treatment for different time.Materials and Methods:The SD rats used in the experiment were provided by the Laboratory Animal Center of Dalian Medical University.THJ-16T and THJ-11T cells from patients with anaplastic thyroid cancer tissues were established at Copeland Laboratories and were presented by Professor Liu Qiang,Cancer Stem Cell Research Institute,Dalian Medical University.First,the multiorgan tumorigenesis model was established by DEN/MNU/DHPN and IG(intragastric)and IP(intraperitoneal injection)of resveratrol were used to prevent and observe the state of rats.After 30 weeks,the body and main organs weights were recorded.The tissues and organs were observed and photographed.The tissues and organs of each rat were soaked in 10%neutral formalin,embedded in paraffin and prepared into tissue section.The pathological changes of the multiple organs of the rats were observed under the microscope.The prevention mechanism of thyroid precancerous lesions was researched:The content of carcinoembryonic antigen(CEA)and thyroid globulin(Tg)in serum of rats were measured by enzyme linked immunosorbent assay(ELISA),and the expression of TTF-1,Tg,Ki67 and PCNA in the thyroid tissues of each group was measured by immunohistochemical method(IHC);and IHC and protein immunoblotting(WB)were used for detection of NF-κB signal pathway.In the study of Inhibitory effect of resveratrol on human undifferentiated thyroid carcinoma and its mechanism of action,THJ-16T and THJ-11T cells were cultured in 1640 medium containing 5%and 10%fetal bovine serum with 100μM resveratrol treatment for different time.Resveratrol induced apoptosis of ATC cells were detected by MTT assay,HE staining,DAPI staining,TUNEL staining and Annexin V-PI staining;the molecular mechanisms(reactive oxygen species,antioxidant enzymes,Caspase proteins and SULTs)were analyzed by flow cytometry,immunocytochemistry(ICC)and WB methods.Result:Resveratrol Efficacies on Chemoprevention of Carcinogen-induced Rat multiple organ Tumorgenesis.1.Safety of long-term resveratrol treatmentNo rat is dead during the 30 week resveratrol treatment(2 day/time).Final body weights and relative organ weights in chemoprevention study are shown in Table 1.The average final body weights of the resveratrol IP group(591.3±38.4 g)and resveratrol IG group(580.5±37.7 g)are higher than that of Group-2(549.1±42.1 g)with statistically significance(p<0.05).No significant difference of the final body weights and relative organ weights(the liver,lungs,kidneys and spleen)among the groups(p>0.05).2.The analysis results of multi-organ tumorgenesis preventive effectsNo histological alteration was observed in the specimens of normally fed rats,while carcinomas or preneoplastic lesions could be found in the DMD alone group,resveratrol IG group and resveratrol IP group.The incidences of thyroid preneoplastic lesion were 53.3%,33.3%and 26.7%with statistical significance(p<0.05).The incidences of hepatocellularcarcinomas 26.7%,6.7%and 6.7%with statistical significance(p>0.05),the incidences of colon lymphadenosis were 46.7%,26.7%and26.7%with statistical significance(p>0.05)and the frequencies of lung fibrous hyperplasia were 20.0%,13.3%and 6.7%with statistical significance(p>0.05).Resveratrol Efficacies on Chemoprevention of Carcinogen-induced Rat Thyroid Tumorgenesis and Its Mechanisms.1.Sufficient resveratrol availability in thyroidsThe average resveratrol concentration of thyroid tissues was 1.278±0.419 nmol/g(0.256μM)in IG group and 1.752±0.398 nmol/g(0.351μM)in IP group.The intracellular concentration of resveratrol in 5×10~6 THJ-16T cells was 0.362±0.126μM after 60 minutes treatments with 100μM.Only one peak corresponding to trans-resveratrol was detected in both the thyroid tissues and THJ-16T cells extracts.Cellular resveratrol uptake of 100μM resveratrol-treated THJ-16T cells was about 41.4%and 3.1%higher than that of thyroid tissues treated by resveratrol through IG and IP routes,respectively.2.Resveratrol alleviated thyroid tissue lesionsPreneoplastic lesions(hyperplasia and/or adenomas)could be found in the DMD alone group,resveratrol IG group and resveratrol IP group in the incidences of 53.3%,33.3%and 26.7%,respectively.Single and multiple papillary adenomas was found in13.3%(2/15)and 13.3%(2/15)of DMD alone group,6.7%(1/15)and 0%in the resveratrol IG group and 6.7%(1/15)and 0%in resveratrol IP group,respectively.The average area of lesions(hyperplasia/adenomas)of DMD alone group(0.599±0.037 mm~2)was 50.9%larger than that of resveratrol IG(0.397±0.062 mm~2)and 87.8%larger than resveratrol IP group(0.319±0.040 mm~2)with statistically significance.3.Lower serum Tg and CEA levels in resveratrol-treated ratsThe average serum Tg levels in resveratrol IP group(3.327±0.304μg/L)and resveratrol IG group(3.512±0.377μg/L)were lower than that of DMD alone group(4.139±0.628μg/L)(p<0.05)but 9.0%and 15.0%higher than the control group(3.053±0.364μg/L;p>0.05).The average serum CEA levels in resveratrol IP(8.111±0.604μg/L)and IG group(8.280±0.541μg/L)were also lower than that of DMD alone group(9.306±1.049μg/L;p<0.05)and 10.0%(p>0.05)and 12.3%(p>0.05)higher than the control group(7.373±0.225μg/L).In the same experimental groups,the CEA and TG levels of adenoma-bearing rats were higher than that of the tumor-free rats(p<0.05)and the rats with hyperplasia only(p<0.05).4.Variable levels of growth-related factors in the experimental groupsThe levels of Ki67 and PCNA in DMD alone group were higher than that of the resveratrol-treated groups and their levels in the hyperplasia and adenoma tissues were stronger than the surrounding noncancerous tissues.TTF-1 was detected in nuclei of thyroid epithelial cells of all groups and its labeling density,especially in the regions with pathological alterations,was stronger in the DMD alone group in comparison with other groups.Tg is expressed in thyroid epithelial cells of the control group and becomes elevated in the thyroid specimens of resveratrol-treated and,especially DMD alone groups.5.Resveratrol inhibited NF-κB/p65,IL-6 and COX-2 expressionThe results of immunohistochemical staining and Western blotting showed that NF-κB/p65,IL-6 and COX-2 expression levels were relatively low in the control group and distinctly upregulated in the thyroid tissues of DMD alone group.NF-κB/p65,IL-6and COX-2 levels in Res IG group and Res IP group were lower than that in DMD alone but higher than that in the control group.Reversely,Ik Bαwas expressed in high level in the thyroid tissues of the control group and became downregulated in the experimental groups,especially in DMD alone one.Correlation of Reactive Oxygen Species Levels with Resveratrol Sensitivities of Anaplastic Thyroid Cancer Cells1.Different resveratrol sensitivities of THJ-16T and THJ-11T cellsThe data from the MTT assay indicated that resveratrol inhibited the proliferation of THJ-16T cells in a time-dependent manner(12h,P<0.05;24h and 48h,P<0.01).On the other hand,100μM resveratrol-treated THJ-11T cells for 48 hour was no changed in comparison with that of its untreated counterpart(P>0.05).HE staining results revealed distinct less cells,morphological changes and apoptotic phenotype of resveratrol-treated THJ-16T but not THJ-11T cells.2.Extensive apoptosis of resveratrol-treated THJ-16T cellsTUNEL staining demonstrated a higher percentage of nuclei with DNA damage of resveratrol-treated THJ-16T cells.Annexin V-PI staining revealed that the proportion of apoptotic cells in normal cultured THJ-16T cells were 2.67±0.49%,which increased to21.27±2.76%after resveratrol treatment for 48 hours(P<0.05).In contrast,the proportion of apoptotic cells in THJ-11T cells without and with resveratrol treatment were 0.99±0.42%and 1.07±0.41%(P>0.05).3.Resveratrol increased ROS generation in THJ-16T but not THJ-11T cellsIt was found that ROS generation in THJ-16T cells was evaluated at 6h,12h,24h(P<0.01)and 48h(P<0.05)time points in comparison with the basal level at 0h.In the case of THJ-11T cells,no obvious change of ROS levels was found between the cell populations without and with resveratrol treatment(P>0.05).The fluorescent microscopic findings were in accordance with flow cytometry results in terms of time-related increase of ROS levels in resveratrol-treated THJ-16T rather than THJ-11T cells.4.Mitochondrial structural alteration of resveratrol-treated THJ-16T cellsThe ultrastructural changes of mitochondria in THJ-16T cells and THJ-11T cells without and with resveratrol treatment were examined by transmission electron microscope.THJ-16T cells treated with resveratrol displayed typical apoptotic morphological changes such as chromatin condensation and fragmentation(red arrow).In comparison with the intact mitochondria of the control cells,mitochondria in the resveratrol-treated THJ-16T cells showed disappeared cristae and distinct swelling phenotype(white arrow).In contrast,the ultrastructure of THJ-11T cells as well as the mitochondria in them remained unchanged after 48 hour resveratrol treatment.5.Decreased SOD2 and CAT in THJ-16T cellsThe results revealed that decreased levels of SOD2 and CAT were found in resveratrol-treated THJ-16T cells in time-related fashion,while no obvious reduction of SOD2 and CAT levels was observed in THJ-11T cells irrespective to resveratrol treatment.6.Resveratrol caused caspase-9 and-3 activation in THJ-16T cellsThe statuses of pro-caspase-9 and-3,active-caspase-9 and-3 in THJ-16T and THJ-11T cells were checked by Western blotting(Figure 6).The levels of pro-caspase-9 and-3 were decreased 15.3%and 20.8%,meanwhile,active-caspase-9 and-3increased 2.3-fold and 2.4-fold after 48h 100μM resveratrol treatment in comparison with that of normally cultured THJ-16T cells.The levels of the above parameters remained almost unchanged in THJ-11T cells irrespective to resveratrol treatment.7.Downregulated SULTs in ATC cellsThe results revealed that the total SULT1A1 and SULT1C2 levels of THJ-11T cells and,especially,THJ-16T cells were 17.1%and 56.5%lower than their levels in rat normal thyroid tissues.SULT1A1 and SULT1C2 in THJ-11T cells were 16.1%and 5.0%enhanced after resveratrol treatment,while they remained unchanged in resveratrol-treat THJ-16T cells.Conclusion:1.Establish a model of carcinogen-induced rat medium-term multiorgan tumorgenesis.2.Resveratrol administration improves the health status of rats after DMD induction.3.Resveratrol can effectively inhibit the development of liver,lung,colorectal and thyroid tumorgenesis.4.Resveratrol can be detected in rat thyroid tissue after 20mg/kg intragastric administration and intraperitoneal injection.Their concentrations were similar to that of resveratrol in THJ-16T cells treated with 100μM,indicating that resveratrol could prevent the development of thyroid cancer.5.Systemically administered resveratrol efficiently reduces the frequency and severity of thyroid cancer-related lesions in DEN/MNU/DHPN-induced carcinogenic model through inhibiting proliferation and suppressing NF-k B mediated inflammatory reaction.6.Oral administration can achieve the same therapeutic effect as intraperitoneal injection,so it is more suitable for the long-term prevention of thyroid cancer.7.Different undifferentiated thyroid cancer cell lines have different sensitivity to resveratrol:THJ-11T is insensitive,while THJ-16T is sensitive.8.Resveratrol down-regulated the expression of SOD2 and CAT in its sensitive cell line THJ-16T.9.The mechanism of resveratrol inducing apoptosis of ATC cells may be through the increase of ROS production,resulting in mitochondrial oxidative damage,causing caspase-9 and caspase-3 activation,eventually leading to apoptosis.10.The reason for the difference in resveratrol sensitivity may be negatively correlated with the expression level of SULTs,that is,the higher the expression of metabolic enzymes,the worse the sensitivity of resveratrol. |
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