| Background:Cancer is a prominent public health problem worldwide.The main malignancies in China include ten kinds like thyroid cancer,gastric cancer,and lung cancer etc,accounting for about 75% of all new cases that have been diagnosed with cancer.The most common endocrine malignancy is thyroid cancer,and the incidence rate is increasing every year.The pathogenesis of thyroid cancer has not yet been fully elucidated.For example,intake of iodine,genetic factors,radiation exposure,and other factors like sex hormones may all be related to the occurrence of thyroid cancer,and human health and safety has been seriously affected.Especially for women's,more and more malignant tumors affecting their health.Therefore it is imperative to be able to find and implement positive,correct and effective treatment strategies.There are four histological types of thyroid cancer.Papillary thyroid cancer and follicular thyroid cancer are differentiated thyroid cancers,accounting for more than 90% of thyroid malignancies.Differentiated thyroidcancer is generally curable and it is more common.Undifferentiated thyroid cancer has the highest level of malignancy and the mortality rate is extremely high.Therefore,an in-depth study and research on the pathogenesis of thyroid cancer can help us to diagnose thyroid cancer in an earlier stage and develop a more comprehensive and effective novel treatment based on its pathogenesis to further improve the quality of life for cancer patients.Ultimately,the treatment can lead to a more stable and excellent prognosis of the follow-up results,reducing the mortality rate of thyroid cancer.For most cases,gene mutations are crucial for the occurrence and development of tumors.The relationship between genes and tumors is built through the cell's internal molecular signaling pathways which impact cells function,affect and alter the functional proteins.As a consequence,cell proliferation pattern changes and biological characteristics and morphological of cells change,and ultimately those factors leads to the occurrence of tumor.Studies have shown that mi R-21 is involved in the occurrence and development of a variety of tumors.The tumor suppressor gene phosphatase and tensin homologue gene(PTEN)inhibits tumors through Akt dephosphorylation,and PTEN is a target gene of mi R-21.The evidence from modern pharmacological experiments conducted by many scholars shows that the natural drug matrine has the common anti-viral,anti-inflammatory and anti-arrhythmic effects,and it also strongly inhibits tumorigenesis.Studies have shown that matrine can inhibit the proliferation of tumorcells.Clinically,matrine has a good therapeutic effect on liver cancer,gastric cancer and leukemia,while the toxic side effects of matrine ingredients are weak.Therefore,matrine is very likely to be used in anticancer field in the future where it will be made to a plant active drug with good anticancer effect.It may become a promising replacement drug for human papillary thyroid cancer cells(TPC-1).The most effective drug categories for targeted therapy of anaplastic thyroid cancer(ATC)are paclitaxel(paclitaxel or docetaxel),quinones,benzoquinones and platinum(cisplatin or carboplatin).Natural product phenylhydrazone compound--tetrafluoro-benzoquinone has very good anti-tumor activity and low toxicity.The mechanism involves the production of helium-oxygen free radicals which participate in the methylation of molecules involved in important cellular processes,and DNA.Aim:This study aimed to investigate the therapeutic effect and potential molecular mechanism of matrine on TPC-1 cells which includes studying the effect of matrine on the proliferation and cell cycle of cultured TPC-1 human thyroid carcinoma papillary cells in vitro,and also studying the effect of matrine on the expression of mi R-21 and target gene PTEN/Akt pathways in TPC-1 human papillary thyroid carcinoma cells.In addition,this paper investigates the effect of tetrafluoro-benzoquinone on ATC in vivo and in vitro,providing laboratory and theoretical basis for further study of matrine and tetrafluoro-benzoquinone in the treatment of thyroid cancer and exploring the application prospects of matrine and tetrafluoro-benzoquinone in the treatment of thyroid cancer which can provide research idea for developing new drugs for the treatment of thyroid cancer.Method:(1)The effect of matrine on the survival rate of TPC-1 cells was detected by MTT assay.(2)The expression alteration of micro RNA-21 gene after the treatment of matrine in TPC-1 cells was detected by q RT-PCR.(3)Micro RNA-21 mimics plasmid and micro RNA-21 inhibitor plasmid were transfected respectively,and apoptosis was detected by AnnexinV-PI.(4)Western blot was used to detect the changes of mitochondrial apoptosis-related proteins PTEN protein and Akt protein.(5)The growth of HOTHC and ATC cell lines was analyzed by MTT assay.(6)Protein band intensity for Western blot was quantified and nuclear fission was analyzed by staining with DAPI.(7)The tube formation trend of HOTHC cells was analyzed and HOTHC cell migration ability was determined.(8)The mouse model was used to determine tumor size and weight to determine tumor inhibition efficiency.Result:(1).Matrine inhibited the growth of TPC-1 cells dependent on the concentration and time base on MTT method,it can have inhibit rate up to 95.8%(20 mg/ml matrine),the IC50 was 3.54 mg/ml,48 h.(2).Apoptosis of TPC-1 thyroid cancer cells was detected by FITC-A/PI staining flow cytometry.Matrine induced apoptosis of TPC-1cells significantly.The M0 control group was found to be 51.36±2.07%in the G1 phase,23.37±3.25% in the S phase,and 18.49±2.46% in the G2/M phase.After treatment with matrine at 10 mg/ml for 48 h,the proportion of cells in G1 phase increased to 74.49±3.65%,and decreased to 2.42±1.63% in S phase.No significant change in percentage of cells was observed in the G2/M phase.These results indicate that matrine induces cell cycle arrest in the G1/G0 phase of thyroid cancer cells.(3).The level of mi R-21 was determined by real-time q PCR,and TPC-1 cells were treated with matrine at 0,2,5 mg/ml(M0,M2 and M5)for 48 h.Mir-21 was significantly decreased in tpc-1 cells treated with matrine.PTEN results up-regulated M5/M0 to 1.66 times,M2/M0 to1.21 times.Akt levels were reduced by 0.34 times M5/M0 and 0.61 times M2/M0.Experiments showed that mir-21 levels of tpc-1 cells treated with matrine were down-regulated,and subsequently,their target PTEN levels were increased in both m RNA and protein levels,and Akt levels were decreased.On the other hand,mir-21 mimic sequences were transfected into tpc-1 cells,and excessive expression of mir-21 was found to lower PTEN protein levels.These results suggest that matrine can modulate mir-21 to improve PTEN's inhibition of tpc-1 thyroid cancer cells.(4).Western blot was used to detect PTEN protein and phosphorylated Akt(p Akt)protein levels.PTEN was significantly up-regulated compared to the M0 control(M5/M0 1.64-fold,M2/M01.21-fold).The p Akt levels were down-regulated(M2/M0 was 0.61,M5/M0 was 0.24).Mi R-21 mimic significantly reduced the PTEN protein(ratio to M0 blot)and increased p Akt by 1.24 fold(compared to the M0 blot value).These results shed light on the role of matrine in regulating mi R-21/PTEN/Akt pathway-induced apoptosis and cycle arrest in TPC-1 cells.(5).MTT assay results showed that the growth of HOTHC and ATC cell lines decreased significantly within 48 hours of treatment with tetrafluoroparaben.The cell growth was reduced from 98% to 17%,and the cell growth rate was 96% and 21%,respectively.(6).HOTHC cell died.Western blot analysis showed that Bcl-2expression was significantly reduced in the HOTHC cell line.DAPI staining showed the presence of DNA fragmentation and nuclear apoptosis was found in tetrafluoropben-quinone treated HOTHC cells.(7).Treatment of HOTHC cells with tetrafluoro-benzoquinone led to inhibition of hypoxia inducible factor-1(HIF-1)and vascular endothelial growth factor(VEGF)expression.(8).In the treatment with tetrafluoroparaben,the angiogenesis and migration potential of HOTHC cells were inhibited.(9).Treatment with tetrafluoro-benzoquinone inhibits tumor growth in mouse xenograft models.Conclusion:(1).Matrine inhibits TPC-1 human papillary thyroid cancer cell proliferation and induces apoptosis and cell cycle arrest;(2).Matrine reduced the expression of mi R-21 in TPC-1 human thyroid carcinoma papillary cells,induced PTEN up-regulation and p Akt signal down-regulation.(3).The mi R-21/PTEN/Akt pathway may be one of the mechanisms by which matrine inhibits TPC-1 thyroid cancer cells.(4).The growth of HOTHC and ATC cell lines was significantly decreased in the treatment with tetrafluoro-benzoquinone,and the expression of B-cell lymphoma/leukemia-2 gene(Bcl-2)was significantly decreased.Bax,caspase-3(caspase-3)and expression cells for cell apoptosis indicating protein PARP's degradation product(cleaved-PARP)increased.(5).Treatment of HOTHC cells resulted in inhibition of the expression of HIF-1 and VEGF,and the angiogenesis and migration potential of HOTHC cells were inhibited.(6).In mice treated with tetrafluoro-benzoquinone.,there was significant decrease in tumor sizw and weight(7).Matrine and tetrafluorobenzoquinone may be effective alternatives for the treatment of thyroid cancer. |
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