Randomized study of sinusoidal chronomodulated versus flat intermittent induction chemotherapy with cisplatin and 5-fluorouracil followed by traditional radiotherapy for locoregionally advanced nasopharyngeal carcinoma.

Neoadjuvant chemotherapy plus radiotherapy is the most common treatment regimen for advanced nasopharyngeal carcinoma (NPC). Whether chronomodulated infusion of chemotherapy can reduce its toxicity is unclear. This study aimed to evaluate the toxic and therapeutic effects of sinusoidal chronomodulated infusion versus flat intermittent infusion of cisplatin (DDP) and 5-fluorouracil (5-FU) followed by radiotherapy in patients with locoregionally advanced NPC. Patients with biopsy-diagnosed untreated stages III and IV NPC (according to the 2002 UICC staging system) were randomized to undergo 2 cycles of sinusoidal chronomodulated infusion (Arm A) or flat intermittent constant rate infusion (Arm B) of DDP and 5-FU followed by radical radiotherapy. Using a "MELODIE" multi-channel programmed pump, the patients were given 12-hour continuous infusions of DDP (20 mg/m2) and 5-FU (750 mg/m2) for 5 days, repeated every 3 weeks for 2 cycles. DDP was administered from 10:00 am to 10:00 pm, and 5-FU was administered from 10:00 pm to 10:00 am each day. Chronomodulated infusion was performed in Arm A, with the peak deliveries of 5-FU at 4:00 am and DDP at 4:00 pm. The patients in Arm B underwent a constant rate of infusion. Radiotherapy was initiated in the fifth week, and both arms were treated with the same radiotherapy techniques and dose fractions. Between June 2004 and June 2006, 125 patients were registered, and 124 were eligible for analysis of response and toxicity. The major toxicity observed during neoadjuvant chemotherapy was neutropenia. The incidence of acute toxicity was similar in both arms. During radiotherapy, the incidence of stomatitis was significantly lower in Arm A than in Arm B (38.1% vs. 59.0%, P = 0.020). No significant differences were observed for other toxicities. The 1-, 3-, and 5-year overall survival rates were 88.9%, 82.4%, and 74.8% for Arm A and 91.8%, 90.2%, and 82.1% for Arm B. The 1-, 3-, and 5-year progression-free survival rates were 91.7%, 88.1%, and 85.2% for Arm A and 100%, 94.5%, and 86.9% for Arm B. The 1-, 3-, and 5-year distant metastasis-free survival rates were 82.5%, 79.1%, and 79.1% for Arm A and 90.2%, 85.2%, and 81.7% for Arm B. Chronochemotherapy significantly reduced stomatitis but was not superior to standard chemotherapy in terms of hematologic toxicities and therapeutic response.

Anti-tumor activity and mechanisms of a novel vascular disrupting agent, (Z)-3,4',5-trimethoxylstilbene-3'-O-phosphate disodium (M410).

Vascular disrupting agents (VDAs) have emerged as a new kind of anti-cancer drug in recent years. Structural modification of an active parent compound is an effective approach to developing new agents with more activity and fewer adverse reactions. In our study, six synthesized stilbene derivatives were screened for their cytotoxic activity against human tumor cells, and their mechanisms of action were investigated. The MTT assay was used to determine the anti-proliferative activity of these compounds. Polymerization of tubulin was detected by a tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical analyses. Cell-cycle distribution was detected by flow cytometry. A nude mouse model with xenografted colon cancer was used to demonstrate the in vivo anti-tumor activity, and microvessel density (MVD) was determined by immunohistochemistry. The expression levels of protein and mRNA were detected by Western blot and RT-PCR, respectively. Among the six newly synthesized compounds, (Z)-3,4',5-trimethoxylstilbene-3'-O-phosphate disodium (M410) showed potent cytotoxic activity toward proliferating tumor cells and exhibited a similar cytotoxicity against multi-drug resistant (MDR) tumor cells. M410 inhibited bovine brain tubulin polymerization in a way similar to that of colchicine. In proliferating human umbilical vein endothelial cells (HUVECs), 20 nM of M410 induced cellular tubulin depolymerization within 4 h, which led to M phase arrest. Systemic administration of M410 at nontoxic doses in nude mice resulted in inhibition of tumor growth of human colon cancer LoVo xenografts. The tumor vessel density also decreased after M410 treatment, as determined by immunohistochemical staining for CD31. M410 downregulated hypoxia-inducible factor-1 alpha (HIF-1α) expression, reduced nuclear HIF-1α, and downregulated vascular endothelial cell growth factor (VEGF) mRNA. Our results indicate that M410 is a potent microtubule inhibitor that is cytotoxic, angiogenesis inhibiting and vascular targeting.

Anti-tumour effects of xanthone derivatives and the possible mechanisms of action.

To explore the potential anti-tumour activities of xanthone derivatives, 26 hydroxylxanthones and benzoxanthones and their structurally modified analogues were examined for potential cytotoxic activities against eight human cancer cell lines. Most of the xanthone derivatives exhibited a higher degree of cytotoxicity on HepG2 cells than on the other seven cancer cell lines. Compound 24 (1,3,7-Trihydroxy-12H-benzo[b] xanthen-12-one) showed the highest degree of cytotoxicity of the tested compounds against HepG2 cells and demonstrated good tumour specificity by exhibiting a much higher degree of cytotoxicity against HepG2 cells than against normal liver cells (L02). Several valuable structure-activity relationships were derived from the cytotoxicity data. In addition, we found that compound 24 could downregulate the expression of the Mcl-1 protein, induce changes in the mitochondrial membrane potential and induce apoptosis in HepG2 cells via the mitochondrial pathway. Compound 24 was also shown to inhibit topoisomerase (topo) II activity and downregulate the levels of both topo II mRNA and protein in HepG2 cells. The present results suggest that due to its potent cytotoxicity and good tumour selectivity, compound 24 may be exploited as a potential lead compound in the development of a new anti-tumour agent with specific activity against liver cancer.

Effects of the biological clock gene Bmal1 on tumour growth and anti-cancer drug activity.

The Bmal1 gene plays a key role in controlling circadian rhythms. To better understand how the Bmal1 gene affects tumour growth and the response to anti-cancer drugs, we examined the effect of knockdown of Bmal1 by RNAi both in vitro and in vivo. Down-regulation of Bmal1 gene expression accelerated cell proliferation in vitro and promoted tumour growth in mice. Suppressing Bmal1 expression in murine colon cancer cells (C26) and fibroblast cells (L929) decreased apoptosis induced by Etoposid, reduced the distribution of cells in the G2/M phases treated by Docetaxel and decreased DNA damage induced by Cisplatin. Loss of Bmal1 reduced the expression of per1, per2, per3, wee1 and p53. The expression of p21 and c-myc was also altered in certain cell lines. However, Bmal1 deficiency increased the protein levels of cdc2, cyclin B1, cyclin D1 and cyclin E. Wee1 and cyclin A expression was minimally altered. Thus, the circadian clock gene Bmal1 plays a role in regulating tumour cell apoptosis, cell-cycle progression and DNA damage response and in homoeostasis regulation. Down-regulation of Bmal1 accelerates the development of tumours and may influence the response to anti-cancer drugs.

[Advances in the study of the anti-tumor activity of small molecule vascular disrupting agents].

Vascular disrupting agents (VDAs) have presented a new kind of anti-cancer drug in recent years. VDAs take advantage of the weakness of established tumor endothelial cells and their supporting structures. In contrast to anti-angiogenic therapy, which inhibits the outgrowth of new blood vessels, vascular targeting treatments selectively attack the existing tumor vasculature. Here we summarized the anti-tumor activities, mechanisms and clinical applications of small molecule VDAs.

ApoG2 inhibits antiapoptotic Bcl-2 family proteins and induces mitochondria-dependent apoptosis in human lymphoma U937 cells.

Lymphoma is one of the most common types of hematological malignancies and proteins from the Bcl-2 family are highly expressed in human lymphomas. Apogossypolone (ApoG2), the most potent gossypol derivative, has been classified as a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins. Here, we assessed the in-vitro cytotoxicity of ApoG2 on human U937 lymphoma cells, and explored the underlying intracellular molecular mechanisms of ApoG2. Using the WST-8 assay, we found that ApoG2 inhibited growth of U937 cells in a dose-dependent and time-dependent manner, and the IC50 values were 30.08, 14.81, and 9.26 mumol/l for 24, 48, and 72 h treatments, respectively. ApoG2 also induced apoptosis in U937 cells, as noted through changes in morphological characteristics, including cellular internucleosomal DNA fragmentation and the appearance of a sub-G1 apoptotic peak. Treatment with ApoG2 downregulated Bcl-xL and Mcl-1 protein expression and blocked the binding of Bcl-2 with Bax protein. Furthermore, ApoG2 led to an abundant release of cytochrome c from mitochondria and a five-fold increase in the activity of caspase-3 and caspase-9. Taken together, our results suggest that ApoG2 could effectively suppress the growth of human lymphoma cell line U937 through the inhibition of the antiapoptotic Bcl-2 family proteins and the induction of mitochondria-dependent apoptotic cell death.

[Induction of G2 /M phase arrest and apoptosis of MCF-7 cells by novel benzofuran lignan via suppressing cell cycle proteins].

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.

Circadian variation of plasma cortisol and whole blood reduced glutathione levels in nasopharyngeal carcinoma patients.

BACKGROUND & OBJECTIVE: Glutathione is involved in cellular protection against radiation damage and drug detoxification. This study was to investigate the circadian variation of plasma cortisol and whole blood reduced glutathione (GSH) levels in nasopharyngeal carcinoma (NPC) patients to provide references for chronotherapy for NPC. METHODS: A total of 13 NPC patients and 14 healthy volunteers were involved. Peripheral venous blood was sampled every 4 h during one 24-hour period starting at 12:00 am. The plasma cortisol concentration was determined by radioimmunoassay; the GSH concentration was determined by high performance liquid chromatography. RESULTS: Plasma cortisol levels of both groups displayed clear and similar circadian rhythms. The cortisol level peaked in both groups in the morning and was the lowest at mid-night. In both groups, GSH concentrations showed significant differences according to sampling time (ANOVA for Repeated Measures, F=5.18, P=0.02). By cosinor analysis, the circadian variation of the GSH level in NPC group was marginally statistically significant (Cosinor analysis, P=0.06) with the acrophase appeared at 05:02; the GSH level in control group displayed an obvious circadian rhythm and the acrophase appeared at 07:44+/-01:56 (P<0.01). The rhythm-adjust mean value of glutathione was (19.60+/-1.11) nmol/mg protein in NPC group and (8.95+/-0.46) nmol/mg protein in control group. CONCLUSIONS: The circadian rhythm of the plasma cortisol level is maintained in NPC patients, even in some patients at advanced stages. In NPC patients, GSH shows a trend of circadian variation which is similar to that in healthy controls. These results could be used to select special schedules for chrono-radiotherapy and chrono-chemotherapy for NPC patients.

Reversal effect of BM-cyclin 1 on multidrug resistance in C-A120 cells.

In this study, multidrug-resistant human epidermoid C-A120 cells and the sensitive parental KB cells were used as experimental models. BM-cyclin 1, a traditional antimycoplasma drug, was tested to explore the reversal effect of multidrug resistance and its mechanisms in these cell lines. The MTT analysis showed that BM-cyclin 1 could reverse multidrug resistance effectively in C-A120 cells; the sensitivity of C-A120 cells to adriamycin, etoposide and cisplatin was enhanced by 6.0, 8.2 and 1.7 times, respectively. Immunoblotting analysis and reverse transcription-polymerase chain reaction were used to study the BM-cyclin 1-induced changes in topoisomerase IIalpha. The results showed that the expression of topoisomerase IIalpha in treated C-A120 cells increased significantly. Topoisomerase II catalytic activity increased by 30% compared with the untreated cells, as measured by decatenation of kinetopolast DNA. Immunoblotting analysis also indicated the transcription factor levels of specificity: those of protein 1 (Sp1) and nuclear factor-YA increased after treatment with BM-cyclin 1, whereas the mRNA and protein expression of multidrug resistance protein 2 was significantly downregulated. These results demonstrated that BM-cyclin 1 could effectively reverse the multidrug resistance of C-A120 cells by increasing the expression of topoisomerase IIalpha and by suppressing the expression of multidrug resistance protein 2, strongly suggesting that BM-cyclin 1 is a potential multidrug resistance reversal agent.

[Cytokine-induced killer cell fusion to lower recurrence of hepatocellular carcinoma after transcatheter arterial chemoembolization sequentially combined with radiofrequency ablation: a randomized trial].

OBJECTIVE: To ed evaluate the clinical effects of autologous cytokine-induced killer cell (CIK) fusion to lower recurrence of primary hepatocellular carcinoma (HCC) and the anti-hepatitis B virus (HBV) effect after transcatheter arterial chemoembolization (TACE) sequentially combined with radiofrequency ablation (RFA). METHODS: Sixty-four HCC patients underwent TACE sequentially combined with RFA without residual tumor or extrahepatic metastasis were randomly divided into 2 groups: study group (n = 33), receiving autologous CIK fusion of the dose of (1.1 - 1.5) x 10(10) via the peripheral vein or hepatic artery, firstly once every 3 - 4 weeks for 4 times, and then once every 4 weeks for 4 times; and control group (n = 31). All patients were followed up for 1 year. RESULTS: In the study group, 29 patients (29/33) were recurrence-free during the 1 year follow-up, 3 had recurrence in the liver 5, 6, and 7 months later respectively, and 1 patient had lymphoadenopathy in the hepatic portal 9 months later. In the control group, 23 patients (68.01%) were recurrence-free, 7 had recurrence in the liver within 1 year, and 1 had lung metastasis 11 months later. In the study group, the number of the patients with the HBV DNA content < 1 x 10(3) before treatment was 19, and increased to 29 after the treatment; 2 of the 19 patients who were HBsAg positive before the treatment became HBsAg negative after the treatment, and number of the patients with the HBV DNA content of 10(3) - 10(4) was 3 before the treatment, and became 13 after the treatment among which 1 patient had his HBV DNA content dropping from 1.6 x 10(7) to 1.6 x 10(4). In the control group, only 1 patient showed his HBV DNA content dropping from 1.1 x 10(5) to below 10(3). CONCLUSION: Capable of reducing recurrence, prolonging the recurrence-free span, and attacking HBV, autologous CIK fusion after TACE sequentially combined with RFA is an effective novel therapeutic strategy for HCC.

Cytotoxic activity and cytostatic mechanism of novel 2-arylbenzo[b]furans.

The aims of this study were to screen cytotoxic compounds from 14 newly-synthesized 2-arylbenzo[b]furans and explore their mechanisms of action. Cytotoxicity was determined by the MTT method. Cell-cycle distribution was detected by flow cytometry. Wright-Giemsa staining was performed to demonstrate the morphological features of cells in mitotic phase. Polymerization of tubulin was detected by tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical study. Among the 14 compounds screened, 4-formyl-2-(4-hydroxy-3-methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo[b]furan (ERJT-12) showed significant cytotoxicity. Our results demonstrated that ERJT-12 exhibited anti-cancer activity in a variety of tumour cell lines with an IC50 value (concentration resulting in 50% inhibition of cell growth) of 5.75 approximately 17.29 microM. Cell cycle analysis showed a concentration-dependent accumulation of tumour cells in G2/M phase after treatment with ERJT-12. Further investigation indicated that ERJT-12 blocked the cell cycle in M phase, with separation and dispersion of chromosomes. ERJT-12 inhibited tubulin polymerization in-vitro. Changes of the cellular microtubule network caused by ERJT-12 were also detected, which were similar to the changes caused by colchicine. These results suggested that the anti-cancer activity of ERJT-12 is worth further investigation.

Cytotoxicity of cantharidin analogues targeting protein phosphatase 2A.

Cantharidin is a natural toxin that possesses potent anti-tumor properties. Its clinical application, however, is limited due to severe side-effects. Its cytotoxicity is believed to be mediated by the inhibition of serine/threonine protein phosphatase 2A. In order to identify new compounds with potential clinical therapeutic use, a series of cantharidin analogues, including those with skeletal modifications at 1-C position (analogues 1-6) and those with anhydride modifications (analogues 7-13), were synthesized, and tested for their inhibitory effects on protein phosphatase 2A and their cytotoxicity to a panel of cancer cell lines. In addition, the mode of inhibition of cantharidin and analogue 13 on protein phosphatase 2A was determined by enzymatic kinetics assay. The data indicated that analogue 13 exhibited potent cytotoxicity to all cancer cell lines, and analogues 9, 11 and 12 showed relatively weak cytotoxicity to one or more cell lines, while other analogues showed little cytotoxicity. Accordingly, analogue 13 exhibited potent inhibitory activity on protein phosphatase 2A, and analogues 9, 11 and 12 showed weak inhibitory activity, while other analogues did not show any inhibitory activity. The findings indicate that the cytotoxicity of synthetic cantharidin analogues is likely to be associated with their protein phosphatase 2A inhibitory activity. The mode of inhibition of cantharidin and analogue 13 on protein phosphatase 2A is identified as noncompetitive inhibition by the Lineweaver-Burk plot.

Norcantharidin inhibits DNA replication and induces apoptosis with the cleavage of initiation protein Cdc6 in HL-60 cells.

Norcantharidin (NCTD), a demethylated form of cantharidin, is currently used as an anti-cancer drug in China. However, the exact anti-cancer mechanism of NCTD on human cancer cells remains poorly understood. In the present study, NCTD inhibited proliferation and DNA replication effectively in HL-60 cells. DNA replication-initiation protein Cdc6 was cleaved after 12 h treatment with NCTD. This cleavage generated a truncated Cdc6 fragment with a relative molecular weight of 49 kDa and elongated treatment with NCTD resulted in a complete loss of Cdc6. In addition, we found that Cdc6 was present in both non-chromatin- and chromatin-bound fractions in the untreated HL-60 cells, and NCTD treatment led to the cleavage of Cdc6 in both fractions. NCTD-induced cleavage of Cdc6 was prevented by pre-treatment with caspase-3 inhibitor, suggesting the involvement of caspase-3 activity in the process. Furthermore, NCTD treatment resulted in apoptotic changes including granular nuclear morphology, DNA laddering and sub-G1 arrest in HL-60 cells. In conclusion, our study reveals that NCTD can inhibit DNA replication, and induce apoptosis and caspase-3-dependent cleavage of Cdc6. The anti-cancer effect of NCTD may be closely associated with the dysfunction of Cdc6 and our report is the first to put forward this point of view.

[In vitro inhibitory effect of dibenzoxanthene CY-B12 on proliferation of tumor cell lines and its possible mechanisms].

BACKGROUND & OBJECTIVE: CY-B12, a new dibenzon-xanthene, has been synthesized recently. This study was to investigate the in vitro antiproliferative activity of CY-B12 and its possible mechanisms. METHODS: The inhibitory effects of CY-B12 on proliferation of gastric carcinoma cell line MGC803, nasopharyngeal carcinoma cell line CNE-2, oral epithelial carcinoma cell line KB-3-1, and lung cancer cell line Glc82 were assessed by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry (FCM). Apoptotic morphology of CNE-2 cells was observed under fluorescent microscope after Hoechst33258 staining. The expression of cell cycle-related proteins Cdc25C, Cdc2, and Cyclin B1 were measured by Western blot. DNA damage was detected by single cell gel electrophoresis (SCGE). RESULTS: CY-B12 obviously inhibited the proliferation of MGC803, CNE-2, KB-3-1, and Glc82 cells; the IC(50) values were 7.51, 9.58, 8.84, and 15.99 micromol/L, respectively. After treatment of CY-B12, CNE-2 cells were arrested at G(2)/M phase; chromatin condensation, apoptotic bodies, and sub-G1 peak were observed in CNE-2 cells. The protein expression of Cdc25C in CNE-2 cells was down-regulated by CY-B12 in a dose-dependent mannerû whereas Cyclin B1 and Cdc2 were up-regulated by low dose of CY-B12, and down-regulated by high dose of CY-B12. CY-B12 induced DNA damage in CNE-2 cells in a dose-dependent manner. CONCLUSION: CY-B12 has potent in vitro antiproliferative activity, which may be exerted through breaking DNA, down-regulating cell cycle-related protein Cdc25C, and inducing cell cycle arrest and apoptosis.

Circadian rhythm in dihydropyrimidine dehydrogenase activity and reduced glutathione content in peripheral blood of nasopharyngeal carcinoma patients.

Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme of 5-fluorouracil (5-FU) catabolism. Glutathione (GSH) is a tripeptide involved in platinum complex detoxification. This study explored the circadian rhythms of DPD activity and GSH concentration in the peripheral blood of 16 patients with histologically proven nasopharyngeal carcinoma (NPC) in order to guide the establishment of chronotherapeutic schedules for this cancer. DPD activity and GSH concentration were determined by high performance liquid chromatography (HPLC). Both variables displayed significant circadian rhythms (Cosinor analysis: p = 0.009 and 0.012, respectively). Peak DPD activity occurred at about 02:30 h; whereas, peak GSH concentration occurred around 12:40 h. The differences between the peak and nadir mean values were 25.5% and 38.7%, respectively. The study showed that the circadian rhythms in DPD activity and GSH concentration in Chinese NPC are similar to those reported for western patients with colorectal cancer, despite the differences in race and kinds of cancer. These findings imply that the chronotherapeutic schedule of 5-FU and platinum used to treat European colorectal cancer patients probably is applicable to Chinese NPC patients.

[Reversing effect of brassinolide on multidrug resistance of-CCRF-VCR1000 cells and a preliminary investigation on its mechanisms].

AIM: To investigate the effect of brassinolide, a plant growth modulator, on multidrug resistance (MDR) of human T lymphoblastoid cell line CCRF-VCR 1000 which was obtained by progressively addition of vincristine (VCR) to sensitive CCRF-CEM cells, and to explore preliminarily the mechanism of reversing action. METHODS: MTT method was used to detect the resistant factor of resistant cell line and the reversing fold after addition of brassinolide. The intracellular accumulation of rhodamine 123, a fluorescent dye transported by P-glycoprotein was detected by flow cytometry, the catalytic activity of topoisomerase II was assessed by Sulliven method to find the effect of brassinolide on resistance. The protein expression of p53 was measured using Western blotting in the sensitive cells and resistant cells to explore the effect of brassinolide. RESULTS: The resistant factors of CCRF-VCR cells on adriamycin, VP-16 and VCR are respectively as 153.1, 55.9 and 8123.1 folds comparing to the sensitive cell line CCRF-CEM. After treatment of brassinolide under the concentration of 0.001 - 10.0 microg x mL(-1), the resistance of CCRF-VCR was reversed partly with the reversing folds respectively as 4.4 - 11.6. The intracellular accumulation of rhodamine 123 was significantly reduced in the resistant cells. After treatment of brassinolide, the accumulation increased, the level of fluorescent dye was situated between resistant cells and sensitive cells. No alteration of the catalytic activity of topoisomerase II was found among three groups. The level of protein expression of p53 in resistant cells was higher than that of sensitive cells. After brassinolide treatment, the expression of p53 in CCRF-VCR cells restored to the level of sensitive cells. CONCLUSION: Brassinolide could effectively reverse the resistance of CCRF-VCR cells by inhibiting the effusion of drug transported by P-glucoprotein. To down regulate the abnormal expression of p53 maybe one of the mechanisms of reversing MDR for brassinolide.

Effects of light and food schedules on liver and tumor molecular clocks in mice.

BACKGROUND: Disrupted circadian coordination accelerates malignant growth, but the molecular mechanism is unclear. METHODS: Healthy or Glasgow osteosarcoma-bearing mice (n = 162) were synchronized with light and darkness over 2-3 weeks, submitted to an 8-hour advance onset of light every 2 days (chronic jet lag) to disrupt circadian coordination, or submitted to chronic jet lag and meal timing to prevent molecular clock alteration. The expression of molecular clock genes and of the cell cycle genes c-Myc and p53 in liver and tumor was determined with quantitative reverse transcription-polymerase chain reaction at six circadian times over a 24-hour period of light and darkness and analyzed with analysis of variance and cosinor. Tumor weight was measured daily over the course of the experiment. All statistical tests were two-sided. RESULTS: In synchronized mice, mean mRNA levels of clock genes Rev-erbalpha, Per2, and Bmal1 varied by 206-, four-, and 26-fold, respectively, over the 24 hours in healthy mouse liver; by 36-, 35-, and 32-fold in the livers of tumor-bearing mice; and by 9.4-, 5.5-, and sixfold in tumor tissue (P = .046 to <.001). In mice subjected to chronic jet lag, the periodic changes were dampened and the clock gene rhythms were temporally shifted in liver and ablated in tumor, and tumor growth was accelerated. Meal timing reversed the chronic jet lag-induced alterations in Rev-erbalpha and Per2 expression in liver and of all three clock genes in tumor and slowed tumor growth. Tumor growth differed as a function of light and feeding schedules (P = .04). No obvious rhythm was detected for p53 or c-Myc in liver or tumor tissues of synchronized mice. In healthy mice subjected to chronic jet lag, the mean level of p53 expression was cut in half (P = .002), and a 12-fold circadian variation in c-Myc mRNA level (P = .03) was induced in the liver of healthy mice, whereas complex expression patterns were found in the liver and tumor of tumor-bearing mice. CONCLUSIONS: Altered light-dark or feeding schedules modified the expression of molecular clock genes and genes involved in carcinogenesis and tumor progression.

Circadian expression of dihydropyrimidine dehydrogenase, thymidylate synthase, c-myc and p53 mRNA in mouse liver tissue.

BACKGROUND & OBJECTIVE: Anti-cancer effect of 5-Fluorouracil (5-FU) is mediated mainly by inhibition of the thymidylate synthase (TS), while dihydropyrimidine dehydrogenase (DPD) is an initial and a rate-limiting catabolic enzyme of 5-FU. In this study, the mRNA expression profiles of TS, DPD, p53 and c-myc were investigated in mouse liver. METHODS: A total of 24 male B6D2F1 mice were involved in this study. All the mice were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD12:12) for 4 weeks. Body temperature and rest-activity were monitored with an intra-peritoneal sensor. All the mice were sacrificed at 3, 7, 11, 15, 19, 23 HALO (hours after light onset) respectively and liver samples were obtained and immediately frozen in liquid nitrogen. Total RNA was extracted from the frozen liver samples and one-step real-time quantitive RT-PCR was performed using LightCycler - RNA Amplification Kit SYBR Green I system. RESULTS: Both body temperature and rest-activity displayed similarly rhythmic patterns with peak times located in darkness, while the trough time was located in the light span. DPD showed a circadian expression in mRNA level with a peak at about 16 HALO (P=0.0012). TS showed a trend for a circadian rhythm, with a peak during light (P=0.079). Neither c-myc nor p53 displayed significant circadian rhythm. CONCLUSION: The 24-h patterns in DPD and TS expression were approximately 12 h out of phase, supporting a coordinated regulation of both transcriptional pathways relevant for 5-FU chronopharmacology.

[Circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing human nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: No report was found on research of circadian rhythms of nasopharyngeal carcinoma(NPC). This study was designed to investigate the circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing NPC and to collect necessary data for making clinical chronochemotherapy schedule of NPC. METHODS: Sixty-nine BALB/C nude mice were synchronized with an alternative lighting regimen with 12 hours in light and 12 hours in dark (LD 12:12) for at least 3 weeks. Human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells were implanted into each mouse. Ten days after transplantation, the mice were sacrificed, bone marrow cells were collected in the 3, 7, 11, 15, 19, and 23 hours after light onset (HALO). Single cell suspension was obtained and stained with propidium iodide. The cellular DNA content was measured by flow cytometry. Data were analyzed with analysis of variance(ANOVA) and Cosinor analysis. Proteins were extracted from bone marrow cells and determined; Bcl-2, p53, and p21 expression were tested using Western blot analysis. RESULTS: The proportion of bone marrow cells in phase G1, S, and G2/M varied according to circadian sampling time with statistical significance (ANOVA). Moreover, such variation in G1 and G2/M was statistically valid by Cosinor analysis with an acrophase located at 10.8 HALO and 1.8 HALO, respectively. The distribution curves of phase G1 and G2/M fit to Cosinor changes but not for S phase cells. The expression of p53 and Bcl-2 protein level in bone marrow cells varied in 24 h time scale. No p21 protein expression was found in this experiment. CONCLUSION: DNA synthesis of bone marrow cells in nude mice bearing human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells varies according to circadian rhythm. The expression of p53 and Bcl-2 protein level in bone marrow cells varies in 24 h time scale.

[Application of dendritic cells in clinical tumor therapy].

The active immunotherapy of dendritic cells is hot in tumor therapy research area. This article is a review of the source of dendritic cells, loading antigen, immunotherapy pathway, clinical application, choice of patients, and so on. It makes preparation for further research of dendritic cells.