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.

Therapeutic targeting of the endothelin a receptor in human nasopharyngeal carcinoma.

The endothelin A receptor (ET(A)R) autocrine pathway is overexpressed in many malignancies, including nasopharyngeal carcinoma (NPC). In this tumor, ET(A)R expression is an independent determinant of survival and a robust independent predictor of distant metastasis. To evaluate whether ET(A)R represents a new target in NPC treatment, we tested the therapeutic role of ET(A)R in NPC. Cell proliferation was inhibited by the ET(A)R-selective antagonist ABT-627 in two ET(A)R-positive NPC cells in a dose-dependent manner. Proliferation of ET(A)R-negative NPC cells was not decreased. ET(A)R blockade also resulted in sensitization to cisplatin and 5-fluorouracil-induced apoptosis. In nude mice, ABT-627 inhibited the growth of NPC cell xenografts. Combined treatment of ABT-627 with the cytotoxic drug cisplatin or 5-fluorouracil produced additive antitumor effects. The antitumor activity of ABT-627 was demonstrated finally on an experimental lung metastasis by a reduction in the number of tumors. These results support the rationale of combining ABT-627 with current standard chemotherapy to further improve the therapeutic ratio in the treatment of NPC.

[Inhibitory effect of angiogenesis inhibitor YH-16 on liver metastases from colorectal cancer].

BACKGROUND & OBJECTIVE: YH-16, a new recombinant angiogenesis inhibitor, has demonstrated synergetic effects with chemotherapy in non-small-cell lung cancer treatment in stage II clinical trial. This study was to investigate the effect of YH-16 on liver metastases from colon cancer. METHODS: Inhibitory concentration 50% (IC(50)) of YH-16 on vascular endothelial cells and colon cancer cell line CT26 was determined by MTT assay. Furthermore, the mouse mode of colon cancer liver metastases was established by inoculating CT26 cells into the subcapsule of spleen. 60 mice were randomly divided into four groups: control group (0 mg/kg), low-dose YH-16 group (0.40 mg/kg), medium-dose YH-16 group (0.75 mg/kg) and high-dose YH-16 group (1.5 mg/kg). The numbers of liver metastases were examined 2 weeks after drug injection. The expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical method in liver metastases, and tumor microvessel density (MVD) was measured by immunostaining using factor VIII monocolonal antibody. RESULTS: Proliferation of CT26 and vascular endothelial cells was inhibited by YH-16, which the IC(50) was (2.16+/-0.28) microg/ml and (0.64+/-0.10) microg /ml, respectively. In vivo, the liver metastasis rates in control, low-dose, medium-dose and high-dose groups were 100%, 92.3%, 80% and 73.3%, respectively (P<0.05). However, YH-16 did not inhibit the growth of spleen tumors of which median volumes were 1.180 cm(3), 1.201 cm(3), 0.887 cm(3) and 0.781 cm(3), respectively (P>0.05). There was no difference of VEGF expression in liver metastases among the four groups. Moreover, MVD was 65.00+/-9.58, 58.15+/-8.81, 51.60+/-7.10 and 44.53+/-11.47 in the four groups. MVD in medium-dose and high-dose YH-16 groups was lower than that in control group and MVD was lower in high-dose group than that in medium-dose and low-dose groups (P<0.05). CONCLUSION: Angiogenesis inhibitor YH-16 can inhibit liver metastases from colorectal cancer.

[Inhibitory effect of angiogenesis inhibitor YH-16 in combination with 5-FU on liver metastasis of colorectal cancer].

OBJECTIVE: To study the effect of angiogenesis inhibitor YH-16 in combination with 5-FU on liver metastasis of colorectal cancer. METHODS: In vitro, the inhibitory effects of YH-16 and 5-FU on the growth of vascular endothelial cells and colorectal cancer cells were examined by MTT assay. In vivo, colorectal cancer cells were transplanted into BALB/c mice, and the mice were divided into six groups randomly:control group, low-dose YH-16 group, middle-dose YH-16 group, high-dose YH-16 group, 5-FU group and combination group. The number of liver metastases, the size of primary tumor and the toxicity were examined after 2 weeks postoperatively. The expression of vascular endothelial growth factor (VEGF) in liver metastases was detected by immunohistochemistry, and tumor microvessel density (MVD) was measured by immunostaining with CD34 and factor VIII (monoclonal antibodies. RESULTS: In vitro, YH-16 inhibited the growth of colon cancer cells and vascular endothelial cells, with the IC50 at (2.16+/-0.28) microg/ml and (0.64+/-0.10) microg/ml respectively. In vivo high-dose YH-16 and 5-FU had a remarkable inhibitory effect on liver metastasis, and the combination group showed significant enhancement on this effect (P< 0.05). The combination group and 5-FU group could inhibit the growth of primary tumor, but not found in YH-16 group. The toxicity of YH-16 was lower than that of 5-FU (P< 0.05), and the difference was not found in the toxicity between combination group and 5-FU group (P > 0.05). Expression of VEGF in liver metastases was clearly inhibited by YH-16 in combination with 5-FU or 5-FU alone compared to the control group, and MVD in middle-dose and high-dose YH-16 group, 5-FU group and combination group was lower than that in control group (P< 0.05). CONCLUSIONS: The angiogenesis inhibitor YH-16 can inhibit liver metastasis of colorectal cancer through inhibiting the growth of vascular endothelial cells. YH-16 in combination with 5-FU has additive effect on inhibitory activity against liver metastasis.

[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.

[Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA transcripts].

BACKGROUND & OBJECTIVES: The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression. METHODS: Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated. RESULTS: LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines. CONCLUSION: AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.

[Tea polyphenol inhibit on growth of human nasopharyngeal carcinoma cell and xenograft in nude mice].

BACKGROUND AND OBJECTIVE: It was reported that tea polyphenol(TP) possess preventive and anticancer effects on various human cancers. However the report about TP against human nasopharyngeal carcinoma(NPC) was very rare. Our previous studies have also suggested that TP has antiproliferative effect and may induce apoptosis in human NPC cell. In order to further explore the antitumor effect, the authors investigated the growth inhibition effect of TP on various human NPC cell lines and xenograft tumors of human NPC in nude mice. METHOD: Antiproliferative effect of TP against seven human NPC cell lines was tested by MTT method. Antitumor effect of TP was determined using the xenografts models of human NPC cell (CNE2) in nude mice. RESULTS: The fifty percent inhibition concentration (IC50) of TP against NPC cell lines (NPC/HK1, CNE1, CNE2, HNE1, HNE2, SUNE1, and Fadu cells) were calculated to be 55.83, 98.43, 119.21, 127.74, 158.07, 160.72, and 163.59 micrograms/ml, respectively. Average inhibitory rates of TP against xenograft tumors of human NPC in nude mice were 12.7% (P > 0.05), under the dosage of 5 mg.(kg..d)-1, ig x 18 d. Under the dosages of 10 mg.(kg..d)-1, ig x 18 d and 20 mg.(kg..d)-1, ig x 18 average inhibitory rates were 31.0% and 38.5%, (P < 0.01), respectively. CONCLUSION: The results indicated that TP possessed different antiproliferative effect on seven human NPC cell lines in vitro and on xenograft tumor of human NPC in nude mice.

Variable sensitivity of endothelial cells to epirubicin in xenografts of human nasopharyngeal carcinoma CNE-2 cells.

Conventional anticancer drugs show non-specific vascular toxicity, and using anticancer drugs as angiogenesis inhibitors was suggested. However, our previous study suggested that vascular endothelial growth factor (VEGF) protected endothelial cells against chemotherapy drugs in vitro. To further test whether the vascular toxicity of anticancer drugs is active in vivo, epirubicin was i.p. injected into nude mice with s.c. xenografts of human nasopharyngeal carcinoma CNE-2 once (one-day schedule) or once a day from day 1 to day 7 (seven-day schedule). At 48 hours after the single injection or the 7th injection tumors were removed for detection of apoptosis of vascular endothelial cells vessels and the content of VEGF in tumor tissues. The results showed that epirubicin damaged tumor microvessels when the drug was given as a single dose, whereas epirubicin lost its vascular toxicity when the drug was given continuously for seven days, accompanied by higher levels of VEGF in tumor tissues. These results suggest the sensitivity of endothelial cells lining tumor vessels is variable during chemotherapy, and the protective effect of VEGF on endothelial cells might be related to the schedule of administration.

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12h of light and 12h of darkness (LD12:12) for at least 3wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of approximately 2 x 2 mm2). Ten days later, a tumor sample (area of approximately 5 mm2) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G1, S or G2-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G2-M phase cells. The approximate average distribution patterns of G1 and G2-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G1, 21 HALO for S and 15 HALO for G2-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.