OBJECTIVE: To explore the possibility mechanism of non-side population cells (NSP) of Hep-2 be induced into stem-like cancer cells by chemotherapy drug--cisplatin. METHOD: Hep-2 cell lines were sorted by fluorescence-actived cell sorting. The acquired NSP cells in trail group were co-cultured with cisplatin for more than 48 hours,while the control group with normal saline(NS). Then identified the percentage of the side population (SP) cells by flow cytometer. The β-catenin, notch-1 mRNA in trial and control group were detected using quantitative realtime PCR, and the β-catenin, notch-1 protein in two groups were compared by Western blot. RESULT: The percentage of side population cells in two groups were (17.16 ± 0.18)%, (10.05 ± 1.20)%, respectively. There was significant difference between two groups (t = 5.844, P < 0.01). The expression of β-catenin, notch-1 was higher in trail group by qRT-PCR; the protein levels of β- catenin, notch-1 was found to inceased in the trail group by Western blot (t = 5.155, P = 0.031; t = 5.977, P = 0.004). Statistical analysis showed significant difference between two groups (P < 0.05). CONCLUSION NSP cells can be differentiated into stem-like cancer cells after being treating with cisplatin. The supposed mechanism is maybe through wnt/β-catenin, notch signaling transduction pathway abnormalities.
Antineoplastic Agents ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Separation ; Cisplatin ; pharmacology ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; drug effects ; RNA, Messenger ; Signal Transduction ; beta Catenin

Objective To study the transcription and expression of excision repair cross complementing 1(ERCC1) in the peripheral blood and the skin tissue in coal-burning borne endemic arsenism, and to explore the role of arsenism in its pathogenic or carcinogenesis mechanism. Methods According to "Endemic arsenism diagnostic criteria" (WS/T 211-2001), 110 arsenism patients were chosen as case group in Xingren county,Guizhou province and they were divided into 3 groups according to their hnir arsenic: ＜ 2(31 cases),2 ～＜ 4(31 cases),≥4 mg/kg(48 cases), respectively. Another 36 healthy residents about 13 km away from the endemic area were chosen as healthy control group. Under the principle of informed consent, hair samples were collected for arsenic analysis by Ag-DDC and blood samples were collected to determine mRNA expression levels of ERCCI by real-time fluorescence quantitative PCR. At the same time, skin tissue samples were collected from the voluntary surgical treatment of 62 patients with endemic arsenism as case group which were divided into 3 groups according to their hair arsenic: ＜ 2(16 cases), 2 ～＜ 4(20 cases) and ≥4 mg/kg(26 cases), respectively, and these patients were also divided into general pathological changes (32 cases), precancerous (19 cases) and cancerous groups( 11cases), respectively, according to their skin pathologic diagnosis of skin lesions. Another 13 cases pathologically normal without skin cancer surgery from a certain hospital were chosen as control group. Skin samples were collected to detect the ERCC1 protein by immunohistochemical method. Results The mRNA levels of ERCC1 were 0.7156(0.2158 ～ 1.2405),0.5772(0.0843 ～ 1.1234) and 0.5490(0.1895 ～ 0.8431 ), respectively, among ＜ 2, 2 ～＜ 4and ≥4 mg/kg groups, which were lower than the mRNA levels of ERCC1 in the control group[1.5128(1.0000 ～2.1295)], and the difference was statistically significant(all P ＜ 0.05). The expression rate of ERCC1 protein were 87.5%, 80.0% and 77.0%, respectively, among ＜ 2, 2 ～＜ 4 and ≥4 mg/kg groups. The expression rate of ERCC1 protein in 2 ～＜ 4 and ≥4 mg/kg groups were lower than the rate in the control group(100.0%), and the difference was statistically significant (all P ＜ 0.05). The expression rate of ERCC1 protein were 84.4%, 79.0%and 72.8%, respectively, among general pathological changes, precancerous and cancerous groups compared with the control group( 100.0% ), and the difference was statistically significant(all P ＜ 0.05). Conclusions Arsenic from coal-burning can lead to abnormal ERCC1 gene transcription and protein expression, which may inhibit DNA repair through influencing the removal of damaged DNA and promoting the incidence of arsenism development and even skin carcinogenesis.

Objective To investigate the transcription and expression of DNA methyltransferase 1 (DNMT1) mRNA in endemic arsenism patients by burning coal usage,to probe its effects on the development and carcinogenesis of arsenism. Methods In 2008,68 arsenism patients(including 24 mild cases,28 moderate cases and 16 severe cases) were selected in the areas with endemic arsenism according to Standarding of Diagnosis for Endemic Arsenism from Xingren county,Guizhou province. Among the subjects,40 cases were diagnosed by pathological methods,and they were divided into general pathological changes(20),precancerous(14) and cancerous group(6). Tweleve kilometer away from the endemic arsenism area,23 controls were selected in Daguoduo village (non-arsenism exposure). Under the principle of informed consent,blood samples were collected from individuals. The mRNA expression of DNMTI was detected by real-time quantitative reverse transcription polymerase chain reaction(FQ-PCR). At the same time,skin tissue samples were collected from the voluntary surgical treatment patients with endemic arsenism (total 61 cases,including 34 general pathological changes cases,21 precancerous cases and 6 cancerous cases) and from the control(15 cases). DNMT1 protein was detected by immunohistochemical method.Results Average level of DNMT1 mRNA were 0.221 83±0.595 09,0.246 11±0.509 79 and 0.389 27±0.411 33 respectively among mild,moderate and severe arsenism group. DNMT1 mRNA level of mild and moderate group were obviously lower than the control group(0.695 95±0.463 98,all P < 0.01). The mRNA average level of DNMT1 were 0.320 64±0.547 46,0.313 09±0.529 13 and 0.159 07±0.342 56 individually among general pathological changes,precancerous and cancerous group,which were obviously lower than the control group(0.695 95±0.463 98,all P < 0.05). The expression rates of DNMT1 protein in skin were 88.24%(30/34),100%(21/21) and 100% (6/6) among general pathological changes,precancerous and cancerous group were higher than the control group [0(0/15),all P < 0.01],and the extent of expression gradually increased with the aggravation of skin damage(r,= 0.740,P < 0.01). Conclusions DNMT1 participated in the development of the arsenism. High expression of its protein was an early event during the process of the arsenism. DNMT1 may be the new target markers for early diagnosis and treatment of arsenism.

OBJECTIVE: To investigate the changes of laryngeal cancer Hep-2 cells to cisplatin chemosensitivity after the interference of siRNA of β-catenin gene expression. METHOD: Using a small interference RNA (siRNA) technology interfere β-catenin gene of Hep-2 cells . The mRNA and protein levels of β-catenin in the Hep-2 cells of different groups were detected by qPCR and Western blot. It was divided into siRNA-β-catenin-Hep-2 siRNA group, β-catenin-Neg negative control group and blank control group. Cell proliferation inhibition rate of different concentrations of cisplatin on three groups was detected by MTT assay. Calculate the 50% inhibitory effective concentration IC50 value. Check the change of three groups of cells' apoptosis rate by flow cytometry after the same concentrations of cisplatin stimulation. RESULT: β-catenin-siRNA interference fragment can specifically reduce the expression levels of β-catenin mRNA and protein. qPCR illustrated the expression of mRNA in β-catenin-siR-NA-Hep-2 interference group decreased 70% (P < 0.05) compared with the control group, Western blot results showed that the β-catenin protein expression of interference group (0. 545 ± 0.111) decreased significantly compared with blank control group (1.507 ± 0.139) and negative control group (1.429 ± 0.089), P < 0.05. The IC50 calculation software showed that IC50 of cisplatin on β-catenin-siRNA IC50 interference group is (5.81 ± 0.46)μg/ml, the blank control group is (10.10 ± 1.01) μg/ml, the difference between the two groups has statistical signifi- cance (P < 0.01). Cell apoptosis rate of β-catenin-siRNA interference group was (26.15 ± 0.60)%, significantly higher than the control group (14.16 ± 0.05)%, P < 0.05. CONCLUSION To interfere the expression of β-catenin can effectively enhance the sensitivity of laryngeal cancer cells to chemotherapeutic drugs cisplatin. It provides a theoretical support for the reduction of laryngeal cancer chemotherapy drug cisplatin dosage.
Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Laryngeal Neoplasms ; genetics ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; beta Catenin ; genetics

Objective To investigate DNA methylation in the promoter region,mRNA transcription and protein expression of glutathione-S-transferases-P1 (GSTP1) gene and their relation with arsenism.Methods In endemic coal-pollution-borne arsenism area,Jiaole village of Xinren county,Guizhou province,according to the diagnostic criteria of endemic arsenism(WS/T 211-2001),123 cases with endemic arsenism were selected and divided into three groups (mild arsenism group:42 cases,moderate arsenism group:41 cases and severe arsenism group:40 cases).Forty seven residents were selected as controls in a village about 12 km away from the endemic arsenism area.With the informed consent principle,peripheral blood of all respondents was collected in order to analyze DNA methylation and check mRNA.DNA methylation of GSTP1 gene promoter region in peripheral blood was assayed by PCR,and GSTP1 mRNA expression was assayed using real-time quantitative PCR.In addition,other cutaneous specimens originated from 53 cases with arsenism that accepted surgical treatment voluntarily were taken.Of these specimens,general pathological changes were 28 cases,precancerous 20 cases and cancerous 5 cases.Skin tissues of 15 cases of non-tumor surgery patients without abnormal pathological changes were as control group.GSTP1 protein expression in the skin tissue was detected using immunohistochemistry (IHC).Results Among different groups of arsenic poisoning,the positive rate of DNA methylation of GSTP1 gene was 28.57％(12/42) in the mild group,57.10％ (23/41) in the moderate group and 65.00％ (26/40) in the severe group.Compared with the control group (6.38％,3/47),the difference was statistically significant (x2 =7.792,26.000,33.412,all P ＜ 0.01).Among different groups of arsenic poisoning diagnosed by dermapathology,the positive rate of DNA methylation of GSTP1 gene was 21.43％(6/28) in the general pathological change group,50.00％(10/20) in the precancerous group and 80.00％(4/5) in the cancerous group.Compared with the control group(6.67％,1/15),the difference was statistically significant (x2 =3.562,7.468,10.756,all P ＜ 0.05).It showed that the positive rate of DNA methylation of GSTP1 gene increased with aggravation of the disease and dermatic lesion of arsenism (tendency x2 =38.239,x2 =13.659,all P ＜ 0.01).Compared with the control group(0.184 26),the expressions of GSTP1 mRNA in peripheral blood in moderate (0.087 77) and severe arsenic poisoning groups (0.056 93) were significantly reduced(all P ＜0.01),and that of severe group was significantly lower than that of the moderate group (P ＜ 0.01) ; compared with the control group(0.338 45) and the general lesion group(0.276 74),GSTP1 mRNA expression was significantly reduced in precancerous lesion group(0.104 81) and cancerous group(0.043 70),in which the cancerous group was significantly lower than that of the precancerous lesions.The difference of skin tissue GSTP1 protein expression rate between groups was statistically significant (x2 =20.948,P ＜ 0.05),in which the difference between the precancerous lesion group(65.00％,13/20),the cancer group (40.00％,2/5) and the control group(100.00％,15/15)was statistically significant (x2 =12.183,11.778,P ＜ 0.01).Spearman correlation analysis showed that the degree of skin lesion and the level of GSTP1 protein expression was negatively correlated (r =-0.520,P ＜ 0.05).Groups were divided according to DNA methylation of GSTP1 gene,and the mRNA and protein expression of GSTP1 in methylation group(0.038 40,57.14％) was significantly lower compared with that of unmethylated group(0.187 07,95.74％; Z =9.032,x2 =23.134,all P ＜ 0.01).Conclusions Arsenism may lead to DNA methylation of human GSTP1 gene promoter region,thereby inhibiting expression of mRNA and protein.GSTP1 gene plays an important role in arsenism or carcinogenic process.

The inundation of Cordyceps sinensis counterfeits in the market makes it difficult to identify. In this study, 21 batches of wild C. sinensis from 3 different regions, 30 batches of naturally cultured C. sinensis and 4 kinds of counterfeits extracted by methanol and water were analyzed using NMR technology. 9 characteristic peaks were defined as quantitative criterion after comparison, and NMR fingerprints of C. sinensis were established. According to the result it is highly similar between naturally cultured C. sinensis and wild ones by comparing their NMR fingerprints. However, NMR spectra of four kinds of adulterants showed differences with C. sinensis. The result also showed that NMR fingerprint of C. sinensis are highly characteristic and specific. The NMR characteristic fingerprint of wild C. sinensis was consistent with the naturally cultured C. sinensis, and it indicated that the chemical constituents of wild C. sinensis and naturally cultured C. sinensis are nearly the same.

A plan of constructing a platform of excavating and protecting characteristic technology of diagnosis and treatment in research-based hospital of TCM was put forward by Shuguang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine and Research Institution of Characteristic Technology of Diagnosis and Treatment Affiliated with Shanghai Academy of Traditional Chinese Medicine. The platform has begun to take shape on the basis of previous theoretical thought and preliminary practice. It has its own management system and working standards and contents with a clear and definite developing direction. Its working contents include excavating and protecting clinical experience of famous and aged practitioner of Chinese medicine, folk proved recipe and technology of diagnosis and treatment of TCM, intangible cultural heritage of TCM. Some problems appeared during the work, such as the evaluation criterion of the folk proved recipe and technology of diagnosis and treatment, the management of clinical proving projects, the issue of intellectual property protection and so on, which must be further solved.

Objective To explore the influence of coal-arsenic exposure on human T cells proliferation and its mechanism.Methods Blood samples colleoted from individuals which lived in arsenism area of coal-burning type and non-arsenism area in Guizhou Province were divided into exposed group(17),mild(35),moderate(38) and severe arsenism group(19)and control group(35)according to Diagnosis Smndard for Endemic Arsenism (WS/T 211-2001).T cell stimulation index wag determined by methyl thiazolyl tetrazolium(MTT)colorimetric method.The intracellular Ca2+ exponential(IECa2+)in peripheral blood mononuclear cell(PBMC)was analyzed by Fho-3/AM dye and flow cytometry.DNA binding activity of actively T cells nuclear factor(NF-AT)in PBMC was evaluated by electrophoretie mobility shift assay(EMSA).Results Concanavalin A(ConA)stimulation decreased the T cells stimulation indexes in exposed group,mild,moderate and severe arsenism groups(1.315±0.962, 1.611±1.224,1.114±0.545,1.289±0.875)compared with control group(2.322±1.241),all the differences being statistically significant(P＜0.01).After stimulated by anti-CD3 monoclonal antibody(McAb),the T cells stimulation index in exposed group,mild,moderate and severe arsenism group(0.997±0.177,1.103±0.291,1.007±0.221, 0.957±0.205) were lower than that of control group(1.842±0.429,P ＜ 0.01 ). IECa2+ of PBMC after treated by anti-CD3 McAb in mild,moderate and severe arsenism group( 110.130±49.637,92.429±31.191,77.640± 35.372) were lower compared with control group(145.986±59.450,P ＜0.01 ). Moreover,IECa2+ in moderat and severe arsenism group were lower than exposed group(121.337±46.410,P ＜ 0.05). DNA binding activity of PBMC NF-AT in mild,moderate and severe arsenism group(1.354±0.446,1.290±0.291,1.159±0.411 ) were lowered than that of control group(1.722±0.291,P ＜ 0.01) and exposed group(1.611±0.294,P ＜ 0.05). Conclusions The coal-arsenic exposure can reduce the human T cells stimulation indexes,IECa2+ in PBMC and the DNA binding activity of NF-AT. It suggest that arsenic may suppress the proliferation ability of human T cells,which may be partly related to the influence of arsenic on T cell receptor(TCR)/CD3 signal transduetion pathway.

Objective To investigate the activation of T lymphocytes in human peripheral blood and the signaling molecules in protein kinase C/nuclear factor KB(PKC/NF-κB) pathway expressivity or activity changes in human peripheral blood mononuclear cells(PBMCs) exposed to coal-arsenic,to explore the role of PKC/NF-κB signal pathway in activation of T cells in human exposed to coal-arsenic. Methods Blood samples were collected from individuals who lived in arsenism area of coal-burning in Guizhou province, and were divided into asymptomatically exposed group (12),mild arsenism group (33),moderate arsenism group (34) and severe arscnism group (15) according to Diagnosis Standard for Endemic Arsenism (WS/T 211-2001). The individuals who lived in non-arsenism area were control group(27). The ratio of activated T ceils was analyzed by flow cytometry. DNA binding activity of NF-κB in PBMCs was evaluated by electrophoretic mobility shift assay(EMSA). The expression of PKCθ and phospho-PKCθ(pPKCθ) in PBMCs were detected with western blotting analysis. Results The ratio of activating T cells in asymptomatically exposed group[(21.76±15.31)%],mild arsenism group[(18.41±11.36)%],moderate arsenism group[(17.78±11.93)%]and severe arsenism group[(18.79±13.38)%]were all higher than that of control group[(3.19±2.12)%],the difference among all groups being statistically significant(F = 7.893,P < 0.05). DNA binding activity of PBMCs NF-κB in asymptomatically exposed group,mild arsenism group,moderate arsenism group and severe arsenism group(1.49±0.24,1.58±0.30,1.57±0.34,1.51±0.16) were higher than that of the control group(1.30±0.17),the difference being statistically sign/ficant(P < 0.05 or < 0.01). The expression of PBMCs pPKCθ in mild arsenism group,moderate arsenism group and severe arsenism group(0.64± 0.14,0.64±0.27,0.62±0.12) were all lower than that of the control group(0.93±0.20),the difference being statistically significant(P < 0.05). There were significant negative correlations between the expression of pPKCθ and the activity of NF-κB(r =-0.565,P < 0.01). There were significant positive correlations between the activity of NF-κB and the ratio of activating T cells(r = 0.546,P < 0.01). Conclusion Coal-arsenic enhances the DNA binding activity of NF-κB,reduces the expression of PBMCs pPKCθ in human PBMCs and up-regulates the activity of T cells. It suggests that the PKC/NF-κB signal might be one of transduction pathway via activating of T cells by coal-arsenic.

Objective To determine the protein expression of P14ARF,MDM2 and mutant type P53 (P53mt) in skin specimens of coal-burning-type of endemic arseniasis patients and to reveal the molecular mechanism of the disease.Methods Sixty skin specimens from 60 endemic arseniasis patients including 35 of skin lesions patients,19 of precancerous lesion and 6 of skin cancer and 9 normal skin specimens from non-cancer patients were studied.Expression of P14~,MDM2 and P53mt was evaluated by immunohistochemistry using corresponding monoclonal antibodies.Results There was significant difference in the positive rates of P14ARF,MDM2 and P53mt among the 4 groups(x2 =9.39,6.21,20.64,all P ＜ 0.05).The positive rates of P14ARF in precancerous lesion and skin cancer specimens were 46.1％ (6/19) and 33.3％ (2/6),respectively,which were significantly lower than that of the normal skin specimens [88.9％(8/9),all P ＜ 0.05].Decreased expression of P14ARF was correlated with the development of dermopathy (P ＜ 0.05).The positive rates of MDM2 and P53mt in skin lesions,precancerous lesion and skin cancer specimens were 54.2％ ( 19/35 ),63.2％ (10/19),66.7％ (4/6) and 25.7％(9/35),73.7％(14/19),83.3％(5/6),respectively,which were significantly higher than those of the control (0,0,all P＜ 0.05).The expression of MDM2 and P53mt increased with the development of dermopathy(all P ＜ 0.05).Conclusions P53mt protein in skin tissue of coal-burning-type of endemic arseniasis patients is over expressed.Abnormal expression of P14ARF and MDM2 may be one of the reasons lead to abnormal cell cycle control disorders and may play a role in the development of endemic arseniasis.