Triptolide induces apoptosis in endometrial cancer via a p53­independent mitochondrial pathway.

Triptolide (TP), the primary active component purified from the traditional Chinese herbal medicine Tripterygium wilfordii Hook. F (TWHF), has been shown to possess antitumor activity in several types of solid tumors. In the present study, we investigated the antitumor effect of TP in human endometrial cancer cells (HEC-1B) and elucidated its possible underlying mechanisms. HEC-1B cells were treated with various doses of TP (10, 20, 40, 80, 160 and 320 nM), and the cell viability was assessed by Cell Counting Kit-8 (CCK-8) and flow cytometric analysis. Results indicated that TP inhibited the proliferation of HEC-1B cells in a dose- and time­dependent manner. To further investigate its mechanisms, the levels of apoptosis and the changes in caspase-3/9 expression in HEC-1B cells by pretreatment with z-VAD-fmk, a pan-caspase inhibitor, were detected by CCK-8 and western blotting. The cytotoxic effects of TP were significantly inhibited by z-VAD­fmk. At the molecular level, TP did not effectively activate the p53 signaling pathway, but upregulated caspase-3/9 and downregulated bcl-2 without changing the bax level. Our studies revealed that TP has an effect on the apoptotic ability of endometrial cancer cells via a p53-independent mitochondrial pathway, presenting a novel strategy to evade drug resistance in tumorigenesis. The ability of TP to be a potential chemotherapeutic agent for endometrial cancer should be considered.

Mechanistic analysis of taxol-induced multidrug resistance in an ovarian cancer cell line.

OBJECTIVES: To establish a taxol-resistant cell line of human ovarian carcinoma (A2780/Taxol) and investigate its biological features. METHODS: The drug-resistant cell line (A2780/Taxol) was established by continuous stepwise selection with increasing concentrations of Taxol. Cell morphology was assessed by microscopy and growth curves were generated with in vitro and in vivo tumor xenograft models. With rhodamine123 (Rh123) assays, cell cycle distribution and the apoptotic rate were analyzed by flow cytometry (FCM). Drug resistance-related and signal associated proteins, including P-gp, MRPs, caveolin-1, PKC-α, Akt, ERK1/2, were detected by Western blotting. RESULTS: A2780/Taxol cells were established with stable resistance to taxol. The drug resistance index (RI) was 430.7. Cross-resistance to other drugs was also shown, but there was no significant change to radioresistance. Compared with parental cells, A2780/Taxol cells were significantly heteromorphous, with a significant delay in population doubling time and reduced uptake of Rh123 (p < 0.01). In vivo, tumor take by A2780 cells was 80%, and tumor volume increased gradually. In contrast, with A2780/Taxol cells in xenograft models there was no tumor development. FCM analysis revealed that A2780/Taxol cells had a higher percentage of G0/G1 and lower S phase, but no changes of G2 phase and the apoptosis rate. Expression of P-gp, MRP1, MRP2, BCRP, LRP, caveolin-1, PKC-α, Phospho-ERK1/2 and Phospho-JNK protein was significantly up-regulated, while Akt and p38 MARK protein expression was not changed in A2780/Taxol cells. CONCLUSION: The A2780/Taxol cell line is an ideal model to investigate the mechanism of muti-drug resistance related to overexpression of drug-resistance associated proteins and activation of the PKC-α/ERK (JNK) signaling pathway.

Clinical application of endoscopic inguinal lymph node resection after lipolysis and liposuction for vulvar cancer.

AIM: To examine lymph nodes obtained after lipolysis and liposuction of subcutaneous fat of the inguinal region of female vulvar cancer patients to explore the feasibility of clinical application. METHODS: The field of operation was on the basis of the range of the conventional resection of inguinal lymph nodes. We injected lipolysis liquid fanwise, started liposuction after 15-20 minutes; then the subcutaneous fatty tissue was sucked out clearly by suction tube. We selected the first puncture holes located on 2-3 cm part below anterior superior spine, the others respectively being located 3cm and 6cm below the first for puncturing into the skin, imbedding a trocar to intorduce CO2 gas and the specular body, and excise the lymph nodes by ultrasonic scalpel. The surgical field chamber was set with negative pressure drainage and was pressured with a soft saline bag after surgery. RESULTS: A lacuna emerged from subcutaneous of the inguinal region after lipolysis and liposuction, with a wide fascia easily exposed at the bottom where lymph nodes could be readily excised. The number of lymph nodes of ten patients excised within the inguinal region on each side was 4-18. The excised average number of lymph nodes was 11 when we had mature technology. CONCLUSION: Most of adipose tissue was removed after lipolysis and liposuction of subcutaneous tissue of inguinal region, so that the included lymph nodes were exposed and easy to excise by endoscope. This surgery avoided the large incision of regular surgery of inguinal region, the results indicating that this approach is feasible and safe for used as an alternative technology.

Modulation of drug resistance in ovarian cancer cells by inhibition of protein kinase C-alpha (PKC-α) with small interference RNA (siRNA) agents.

OBJECTIVE: To determine whether silence of PKC-α expression by small interference RNA (siRNA) might regulate MDR1 expression and reverse chemoresistance of ovarian cancer. METHODS: We measured gene and protein expression of MDR1 and PKC-α in ovarian cancer cells and assessed their correlation with cell drug resistance. We also examined whether blocking PKC-α by RNA interference (RNAi) affected MDR1 expression and reversed drug resistance in drug sensitivity tests. RESULTS: The drug resistance cell lines, OV1228/DDP and OV1228/Taxol, had higher gene and protein expression of MDR1 and PKC-α than their counterpart sensitive cell line, OV1228. SiRNA depressed PKC-α gene protein expression, as well as MDR1 and protein expression and improved the drug sensitivity in OV1228/DDP and OV1228/Taxol cells. CONCLUSION: These results indicated that decreasing PKC-α expression with siRNA might be an effective method to improve drug sensitivity in drug resistant cells with elevated levels of PKC-α and MDR1. A new siRNA-based therapeutic strategy targeting PKC-α gene could be designed to overcome the chemoresistance of ovarian cancer.

Values of three different preoperative regimens in comprehensive treatment for young patients with stage Ib2 cervical cancer.

OBJECTIVE: To compare the clinical efficacy of concurrent chemoradiotherapy, neoadjuvant chemotherapy, and intracavity brachytherapy in comprehensive treatment for young patients with stage Ib2 cervical cancer. METHODS: One hundred and twelve young patients with stage Ib2 cervical cancer were enrolled retrospectively in our hospital from January 2003 to June 2005. They were categorized into three groups according to preoperative regimens, including the concurrent chemoradiotherapy group (Group 1, n=38), the neoadjuvant chemotherapy (Group 2, n=49), and the intracavity brachytherapy group (Group 3, n=25). Radical hysterectomy was performed following these regimens. Chemotherapy and radiotherapy were given according to pelvic lymph node metastasis, deep cervical stromal invasion, intravascular cancer emboli, histological grading, vaginal stump and positive surgical margin. RESULTS: The cancer disappearance and superficial muscle invasion rates were statistically significantly better in the concurrent chemoradiotherapy group than in the other two groups (P<0.01). No statistically significant difference was noted in the deep muscle invasion rate, surgical time and intraoperative blood loss among three groups, but significantly more postoperative complications occurred in the concurrent chemoradiotherapy group. The 2-year pelvic recurrence was statistically significantly lower in the concurrent chemoradiotherapy group compared to other two groups, while the 5-year survival was higher. CONCLUSION: Concurrent chemoradiotherapy is efficacious for young patients with stage Ib2 cervical cancer.

Changes of four proinflammatory proteins in whole saliva during experimental gingivitis.

OBJECTIVE: To examine changes of four proinflammatory proteins in whole saliva in the early stage of plaque-induced experimental gingivitis. METHODS: Eleven young male volunteers were recruited following the cessation of all oral hygiene measures for a period of 21 days. The levels of Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), calprotectin in saliva were determined with enzyme linked immunosorbent assay. The activity of elastase in saliva was examined. RESULTS: IL-1ß, IL-6 and calprotectin in saliva increased gradually as plaque accumulated and peaked on the 14th and 21st day respectively. Moreover, the three proinflammatory proteins showed good correlations with clinical parameters, with IL-1ß correlating with clinical parameters more closely in particular. The activity of elastase in saliva elevated rapidly and peaked on the second day (P < 0.01). However, after the seventh day, elastase activity declined slowly continuously. The change of IL-6 and IL-1ß in saliva showed a similar tendency throughout the experiment, the correlation coefficient was r = 0.687 (P < 0.01), but there was no obvious correlation between calprotectin and elastase, even though both mainly come from neutrophils. CONCLUSION: Our study demonstrated that IL-6, IL-1ß and calprotectin concentrations in saliva could reflect the degree of gingival inflammation. The longitudinal change of elastase activity in saliva during the experimental gingivitis period was quite different from that of other pro-inflammatory proteins; reasons for the decrease of elastase activity in the late gingivitis period need further study.

[The relationship between two kinds of human herpesviruses and experimental gingivitis].

OBJECTIVE: To investigate human cytomegalovirus (HCMV) and Epstein-Barr virus-type 1 (EBV-1) in GCF and saliva during experimental gingivitis in Chinese young subjects and to evaluate the effect of the virus in the initial stage of gingival inflammation. METHODS: GCF of 14 and 45 and saliva without stimulating in 11 Chinese young males with healthy gingiva were collected at baseline (day 0), day 7, 14 and 21 after stopping oral hygiene and day7 after reestablishing oral hygiene (day 28). DNA of HCMV and EBV-1 were detected by nested-polymerase chain reaction (n-PCR) at the times mentioned above. RESULTS: HCMV was detected in GCF of 4 subjects at baseline, 4 subjects at day 7, 3 subjects at day 14 and 2 subjects at day 21 while the subjects were different. At day 28 HCMV could not be detected. EBV-1 was not detectable in GCF during the experimental gingivitis. HCMV was detected in saliva in 4 subjects and EBV-1 was in 3 subjects. And there is no relationship between the detection of the herpesviruses and the clinical parameters as well. CONCLUSION: We suggest that HCMV and EBV-1 are not the important factors during the initial stage of gingival inflammation.

[Effect of periodontal mechanical treatment on herpesviruses in gingival crevicular fluid of patients with chronic periodontitis].

OBJECTIVE: To evaluate the subgingival prevalence of human cytomegalovirus (HCMV), Epstein-Barr virus-1 (EBV-1) in chronic periodontitis (CP) patients before and after treatment and to analyze the relationship between the prevalent variance and periodontal clinical parameters. METHODS: Gingival crevicular fluids of 13 CP patients were collected at baseline, 2 weeks, 2 months and 4 months after periodontal mechanical treatment. HCMV and EBV-1 were detected using nested polymerase chain reaction (n-PCR). RESULTS: The plaque index (PLI), probing depth (PD) and bleeding index (BI) of CP patients at 2 months, 4 months after periodontal mechanical treatment were evidently lower than before treatment, P < 0.01. These parameters at 4 months after treatment were higher than at 2 months, the differences were significant, P < 0.05. The prevalence of HCMV and EBV in CP patients was 42% (33/78), 14% (11/78). EBV and HCMV were mostly coexistent in the same site [9 sites HCMV(+) in 11 EBV positive sites]. The sites of HCMV(+) and EBV(+) were almost deep pockets. Thirteen of 14 sites with deep pockets were HCMV(+), 9 sites were deep pockets in 11 sites EBV(+). The prevalence of HCMV and EBV (8% and 0 respectively) at 2 weeks was the lowest in all four time points. The prevalence of HCMV and EBV at 2 weeks, 2 months and 4 months following treatment was significantly lower than baseline (P < 0.01), but the prevalence of HCMV (15%) at 2 months after treatment was higher than at 2 weeks (8%), the difference was not significant (P = 0.133). CONCLUSIONS: Herpesviruses may play a role in the development of CP. The changes of the prevalence of herpesviruses before the changes of clinical parameters could be detected after periodontal mechanical treatment. The patients should be re-evaluated and re-treated within 2 months after treatment.

[Effect of periodontal mechanical treatment on periodontal pathogenic bacteria in gingival crevicular fluid of chronic periodontitis patients].

OBJECTIVE: To evaluate the subgingival prevalent rates of 6 periodontal pathogenic bacteria in gingival crevicular fluids of CP patients before and after treatment, to analyze the relationship between the prevalent variance and periodontal clinical parameters, and to provide a microbiologic method of evaluating curative effect and estimating the prognosis. METHODS: Gingival crevicular fluids of 13 CP patients were collected at baseline, 2 weeks, 2 months and 4 months after periodontal mechanical treatment. Also, gingival crevicular fluids were collected from 11 healthy subjects. Six periodontal pathogenic bacteria including Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis(Pg), Tannerella forsythensis (Tf), Prevotella intermedia (Pi), Fusobacterium nucleatum(Fn), Prevotella nigrescens (Pn) were detected by 16S rRNA based PCR. RESULTS: The PLI, PD, BI of the CP patients 2 months and 4 months after periodontal mechanical treatment were evidently less than those before treatment. These 4 months after treatment were a little more than those 2 months after. The six bacteria were more frequently detected in the CP patients at baseline than in healthy controls. The prevalent rates of Tf (42.1%, 73.7%, 70.2%), Pg (47.4%, 68.4%, 77.2%), Aa (15.8%, 22.8%, 7.0%), Pn (38.6%, 57.9%, 64.9%), Pi(15.8%, 38.6%, 42.1%) 2 weeks, 2 months and 4 months following treatment were significantly lower than those at baseline (Tf 96.5%, Pg 93.0%, Aa 36.8%, Pn 86.0%, Pi 84.2%), but the prevalent rates of all the detected bacteria 2 months after treatment were higher than those at 2 weeks after. CONCLUSION: Tf, Pg, Aa, Pn and Pi may cooperate in the development of CP. The changes of periodontal pathogenic bacteria could be detected before the changes of clinical parameters and the patients should be re-evaluated and re-treated regularly within 2 months after treatment.

[Comparison of response evaluation methods of neoadjuvant chemotherapy in breast cancer patients].

OBJECTIVE: To compare the efficiency of response evaluation by clinical examination, ultrasonograghy and mammography in neoadjuvant chemotherapy (NAC) for breast cancer. METHODS: A retrospective cohort study was conducted to analyze the data of 141 patients treated with neoadjuvant chemotherapy. Response evaluation was performed by clinical palpation, ultrasound and mammography. RESULTS: Only 12 (8.5%) among the 141 patients presented with a stage I tumor. The tumor size determined by palpation was often larger than that by ultrasound before therapy (P < 0.01). Among patients with suspicions axillary nodes checked by ultrasound, 88.3% (53/60) of them had positive nodes by pathology before NAC, and 34.5% (10/29) of patients with negative nodes determined by ultrasound had positive nodes by pathology. In all the 141 patients, 21(14.9%) showed pathological complete remission in both the primary tumor and lymph node. For response evaluation, the false complete remission rate judged by clinical examination was 46.8% (22/47), and the false tumor residual rate by ultrasound was 84.0% (21/25). In 53.5% (23/43) of patients the response could not be assessed by mammography due to that the tumors were undistinguishable in size. The range of microcalcification was not reduced in 5 patients with a partial response of the tumor. 25 patients experienced needle puncture during therapy. Among them, in the 9 pathologically negative patients, only 3 achieved pCR, and the other 16 positive patients didn't achieve pCR. CONCLUSION: Using the puncture or sentinel lymph node biopsy, clinicians should pay enough emphasis on the pathological determination of the node status before chemotherapy. Clinicians will make a quite of false judgment of the tumor by clinical examination, ultrasound or mammography. They may use needle puncture during therapy to evaluate the response of neoadjuvant chemotherapy, and the result should be analyzed synthetically.

[Relationship between herpesviruses and periodontal pathogenic bacteria in subgingival plaque].

OBJECTIVE: To evaluate the prevalence rates of human cytomegalovirus(HCMV) and Epstein-Barr virus-1(EBV-1) in subgingival plaque and analyze the relationship between herpesviruses, periodontal pathogenic bacteria and periodontal clinical parameters in Chinese patients with aggressive periodontitis(AgP). METHODS: A total of one hundred and twenty subgingival plaque samples were collected from 89 AgP patients and 31 healthy subjects. HCMV and EBV-1 were detected using nested polymerase chain reaction(PCR).Contemporaneously, 8 periodontal pathogenic bacteria including Actinobacillus actinomycetemcomitans(Aa), Porphyromonas gingivalis(Pg), Tannerella forsythensis(Tf), Prevotella intermedia(Pi), Campylobacter rectus(Cr),Fusobacterium nucleatum(Fn), Treponema denticola(Td), Prevotella nigrescens(Pn) were detected by 16S rRNA based PCR. RESULTS: HCMV was more frequently detected in AgP patients (43.8%) than in healthy controls (12.9%, P<0.01). The prevalence rates of HCMV and EBV-1 in AgP patients with 6-8 kinds of bacteria detected were 54.4% and 17.4%, respectively, significantly higher than those with 3-5 kinds of bacteria detected (P<0.05). CONCLUSION: The prevalence rates of HCMV and EBV was higher in AgP patients than in healthy controls. Herpesviruses and periodontal pathogenic bacteria may cooperate synergistically in the development of AgP, which could be considered as a pathogenetic consortium in future investigation of periodontaltitis.

Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb(3+)-tiron complex as a fluorescence probe.

A sensitive method based on the fluorescence quenching effect of the Tb(3+)-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus (ALP) released from fish plasma. The detection limit was 5.4 ng/ml (S/N = 2), and the relative standard deviation of the quenching effect (6 replicates) was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.

CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe- ions in aqueous solution.

Water-soluble cadmium sulfide (CdS) quantum dots (QDs) capped by mercaptoacetic acid were synthesized by aqueous-phase arrested precipitation, and characterized by transmission electron microscopy, spectrofluorometry, and UV-Vis spectrophotometry. The prepared luminescent water-soluble CdS QDs were evaluated as fluorescence probes for the detection of highly reactive hydrogen selenide ions (HSe(-) ions). The quenching of the fluorescence emission of CdS QDs with the addition of HSe(-) ions is due to the elimination of the S(2-) vacancies which are luminescence centers. Quantitative analysis based on chemical interaction between HSe(-) ions and the surface of CdS QDs is very simple, easy to develop, and has demonstrated very high sensitivity and selectivity features. The effect of foreign ions (common anions and biologically relevant cations) on the fluorescence of the CdS QDs was examined to evaluate the selectivity. Only Cu(2+) and S(2-) ions exhibit significant effects on the fluorescence of CdS QDs. With the developed method, we are able to determine the concentration of HSe(-) ions in the range from 0.10 to 4.80 micromol L(-1), and the limit of detection is 0.087 micromol L(-1). The proposed method was successfully applied to monitor the obtained HSe(-) ions from the reaction of glutathione with selenite. To the best of our knowledge, this is the first report on fluorescence analysis of HSe(-) ions in aqueous solution.

[Determination of tetracyclines by a new spectrum technique].

Tetracyclines are light-fluorescence substances, which cannot be detected directly by fluorometry. Herein a new spectrum method was proposed to detect tetracyclines directly by fluorometry. Under optimal conditions, the calibration graph is linear over the range 0.10-9.00 microg x mL(-1) for tetracyclines, and the detection limits of tetracycline, oxytetracycline, chlortetracycline and doxycycline are 0.065, 0.067, 0.068 and 0.070 microg x mL(-2), respectively.

Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance.

AIM: To investigate the mutation in D-loop region of mitochondrial DNA in gastric cancer and its influence on the changes of reactive oxygen species (ROS) and cell cycle. METHODS: The D-loop region was amplified by PCR and sequenced. Reactive oxygen species and cell cycle were detected by flow cytometry in 20 specimens from gastric cancer and adjacent normal tissues. According to the sequence results, gastric cancer tissue was divided into mutation group and control group. Reactive oxygen species, apoptosis and proliferation in the two groups were compared. RESULTS: Among the 20 gastric cancer specimens, 18 mutations were identified in 7 patients, the mutation rate being 35%. There were four microsatellite instabilities in the mutations. No mutation was found in the adjacent tissues. Reactive oxygen species, apoptosis, and proliferation in the mutation group were all significantly higher than those in control group. CONCLUSION: Mutation in D-loop region plays a role in the genesis and development of gastric cancer.

Probing the hydroxyl radical-mediated reactivity of peroxynitrite by a spin-labeling fluorophore.

The decomposition of peroxynitrite at physiological pH yielded a hydroxyl radical, which reacted rapidly with dimethyl sulfoxide (DMSO) to produce a methyl radical (*CH3), which was then trapped by a spin-label fluorophore nitroxide-linked naphthalene (NTEMPO), a carbon-centered radical probe with a low fluorescence intensity, and transformed to a stable diamagnetic O-alkoxyamine, a high-fluorescence compound. The fluorescence increment was proportional to the concentration of the hydroxyl radical, and then to the concentration of peroxynitrite. NTEMPO therefore was demonstrated to be capable of detecting hydroxyl radicals generated from peroxynitrite, and the method was proved to be simple and sensitive. The hydroxyl radical-mediated reactivities of peroxynitrite to several amino acids, such as tyrosine, phenylalanine and histidine, were then evaluated by the spin-labeling fluorophore NTEMPO at pH 7.4 and, 37 degrees C. The obtained data were in good agreement with the reference values, respectively.

[The clinical observations and the measurement of gingival crevicular fluid volume during the experimental gingivitis].

OBJECTIVE: To observe the changes of clinical conditions, and measure the GCF volume during the experimental gingivitis in Chinese for studying the relationship between the GCF volume and the development of gingival inflammation. METHODS: 11 young male subjects with healthy gingiva, who had no systemic diseases, were selected for this study. GCF samples (18 teeth/person) were collected with strips of filter paper and the clinical parameters were recorded at the baseline (0 day), the 7th, 14th, 21st day (without oral hygiene), and 28th day (7 days after reestablishing oral hygiene) during experimental gingivitis. The GCF volume was measured by weighting. RESULTS: During the experimental gingivitis, all of the clinical parameters plaque index (PLI), bleeding index (BI), gingival index (GI) and probing index (PD) increased gradually following the plaque assembling, there were significant differences comparing the data of baseline with the data of afterwards without oral hygiene. On the 28th day, the data reduced to the level of baseline rapidly. The amount of GCF had the same tendency with the clinical parameters during the experimental gingivitis. There was positive correlation between the amount of GCF and clinical parameters. CONCLUSION: The amount of GCF can reflect the development of gingival inflammation.

Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite.

A novel method for the determination of peroxynitrite using rhodamine B hydrazide as a fluorogenic probe is described. The method is based on the oxidation of rhodamine B hydrazide, a colorless, non-fluorescent substance, by peroxynitrite to give rhodamine B-like fluorescence emission. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 7.5x10(-8)-3.0x10(-6) mol l(-1) with a detection limit of 2.4x10(-8) mol l(-1) (3sigma). The optimal conditions for the detection of peroxynitrite were evaluated and the possible detection mechanism was also discussed in this paper.