Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells.

OBJECTIVE: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. METHODS: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). RESULTS: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). CONCLUSION: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells / Própolis vermelha e L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256

ABSTRACT Objective: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. Methods: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). Results: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). Conclusion: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

RESUMO Objetivo: Avaliar o efeito da própolis vermelha e da L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256. Métodos: O estudo consistiu em dois experimentos com quatro grupos cada (total: 57 hamsters). No experimento 1, os animais foram inoculados com células de tumor de Walker, tendo em seguida administradas as substâncias teste (própolis vermelha 200mg/5mL/kg ou L-lisina 150mg/kg) ou controle (goma arábica 5mL/kg ou água 5mL/kg) por 10 dias. Os animais do experimento 2 receberam própolis vermelha, L-lisina, goma arábica ou água nas mesmas doses, por 33 dias antes do inóculo das células de tumor de Walker, seguido por 10 dias de tratamento com as mesmas substâncias. Baseado em imagens em plano único, foram quantificados a angiogênese (área vascular média), em termos percentuais, e a área (mm2) e o perímetro (mm) do tumor. Resultados: Comparada aos animais que receberam água, a área vascular média, expressa em percentagem, foi significativamente menor nos animais tratados com própolis (p<0,05) e com L-lisina (p<0,001). Conclusão: Tanto a própolis vermelha quanto a L-lisina inibiram a angiogênese no novo modelo de bolsa jugal de hamsters, quando administradas após a inoculação do tumor.

Blueberry inhibits invasion and angiogenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters via suppression of TGF-ß and NF-κB signaling pathways.

Aberrant activation of oncogenic signaling pathways plays a pivotal role in tumor initiation and progression. The purpose of the present study was to investigate the chemopreventive and therapeutic efficacy of blueberry in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to target TGF-ß, PI3K/Akt, MAPK and NF-κB signaling and its impact on invasion and angiogenesis. Squamous cell carcinomas were induced in the HBP by 7,12-dimethylbenz[a]anthracene (DMBA). The effect of blueberry on the oncogenic signaling pathways and downstream events was analyzed by quantitative real-time PCR and immunoblotting. Experiments with the ECV304 cell line were performed to explore the mechanism by which blueberry regulates angiogenesis. Blueberry supplementation inhibited the development and progression of HBP carcinomas by abrogating TGF-ß and PI3K/Akt pathways. Although blueberry failed to influence MAPK, it suppressed NF-κB activation by preventing nuclear translocation of NF-κB p65. Blueberry also modulated the expression of the oncomiR miR-21 and the tumor suppressor let-7. Collectively, these changes induced a shift to an anti-invasive and anti-angiogenic phenotype as evidenced by downregulating matrix metalloproteinases and vascular endothelial growth factor. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation. The results of the present study suggest that targeting oncogenic signaling pathways that influence acquisition of cancer hallmarks is an effective strategy for chemointervention. Identification of modulatory effects on phosphorylation, intracellular localization of oncogenic transcription factors and microRNAs unraveled by the present study as key mechanisms of action of blueberry is critical from a therapeutic perspective.

Tumor blood vessel "normalization" improves the therapeutic efficacy of boron neutron capture therapy (BNCT) in experimental oral cancer.

We previously demonstrated the efficacy of BNCT mediated by boronophenylalanine (BPA) to treat tumors in a hamster cheek pouch model of oral cancer with no normal tissue radiotoxicity and moderate, albeit reversible, mucositis in precancerous tissue around treated tumors. It is known that boron targeting of the largest possible proportion of tumor cells contributes to the success of BNCT and that tumor blood vessel normalization improves drug delivery to the tumor. Within this context, the aim of the present study was to evaluate the effect of blood vessel normalization on the therapeutic efficacy and potential radiotoxicity of BNCT in the hamster cheek pouch model of oral cancer. Blood vessel normalization was induced by two doses of thalidomide in tumor-bearing hamsters on 2 consecutive days. All studies in thalidomide-treated animals were performed 48 h after the first dose of thalidomide, previously established as the window of normalization. Biodistribution studies were performed with BPA at a dose of 15.5 mg (10)B/kg in thalidomide-treated (Th+) and untreated (Th-) tumor-bearing hamsters. The effect of blood vessel normalization prior to BPA administration on the efficacy of BNCT was assessed in in vivo BNCT studies at the RA-3 Nuclear Reactor in tumor-bearing hamsters. Group I was treated with BPA-BNCT after treatment with thalidomide (Th+ BPA-BNCT). Group II was treated with BPA-BNCT alone (Th- BPA-BNCT). Group III was treated with the beam only after treatment with thalidomide (Th+ BO), and Group IV was treated with the beam only (Th- BO). Groups I and II were given the same dose of BPA (15.5 mg (10)B/kg), and all groups (I-IV) were exposed to the same neutron fluence. Two additional groups were treated with the beam only at a higher dose to exacerbate mucositis in precancerous tissue and to explore the potential direct protective effect of thalidomide on radiation-induced mucositis in a scenario of more severe toxicity, i.e. Group V (Th+ hdBO) and Group VI (Th- hdBO). The animals were followed for 28 days. Biodistribution studies revealed no statistically significant differences in gross boron content between Th+ and Th- animals. Overall tumor control (complete response + partial response) at 28 days post-treatment was significantly higher for Group I (Th+ BPA-BNCT) than for Group II (Th- BPA-BNCT): 84 ± 3% compared to 67 ± 5%. Pretreatment with thalidomide did not induce statistically significant changes in overall tumor control induced by the beam only, i.e. 15 ± 5% in Group III (Th+ BO) and 18 ± 5% in Group IV (Th- BO), or in overall tumor control induced by the high-dose beam only, i.e. 60 ± 7% in Group V (Th+ hdBO) and 47 ± 10% in Group VI (Th- hdBO). BPA-BNCT alone (Group II) induced mucositis in precancerous tissue that reached Grades 3-4 in 80% of the animals, whereas pretreatment with thalidomide (Group I) prevented mucositis Grades 3 and 4 completely. Beam-only Group III (Th+ BO) exhibited only Grade 1 mucositis in precancerous tissue, whereas 17% of the animals in beam-only Group IV (Th- BO) reached Grade 2 mucositis. High-dose beam-only group V (Th+ hdBO) exhibited only Grade 2 mucositis, whereas high-dose beam-only group VI (Th- hdBO) reached Grade 3 mucositis in 83% of the animals. In all cases mucositis in precancerous tissue was reversible. No normal tissue radiotoxicity was observed with any of the protocols. Pretreatment with thalidomide enhanced the therapeutic efficacy of BNCT and reduced precancerous tissue toxicity.

Metabolic transformation of DMBA-induced carcinogenesis and inhibitory effect of salvianolic acid b and breviscapine treatment.

Oral cancer typically develops from hyperplasia through dysplasia to carcinoma with a multistep process of carcinogenesis involving genetic alterations resulting in aberrant cellular appearance, deregulated cell growth, and carcinoma. The metabolic transformation during the process of oral carcinogenesis and its implications for cancer therapy have not been extensively investigated. Here, we report a metabonomic study on a classical model of 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral carcinogenesis in hamsters to delineate characteristic metabolic transformation during the carcinogenesis using gas chromatography time-of-flight mass spectrometry (GC-TOF MS). Salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, and Breviscapine, a flavonoid isolated from Herba Erigerontis, were used to treat the hamsters exposed to DMBA to investigate the molecular mechanism of the inhibitory effect of the two agents on oral carcinogenesis. The dynamic changes of serum metabolic profiles indicated that both Sal-B and Breviscapine were able to attenuate DMBA-induced metabolic perturbation, which is consistent with the histopathological findings that Sal-B and Breviscapine significantly decreased the squamous cell carcinoma (SCC) incidence in the two treatment groups. Significant alterations of key metabolic pathways, including elevated glutaminolysis and glycolysis, and decreased cholesterol and myo-inositol metabolism, were observed in the DMBA-induced model group, which were attenuated or normalized by Sal-B or Breviscapine treatment. Elevated inflammation and tumor angiogenesis at gene and metabolite expression levels were also observed in DMBA-induced oral dysplasia and SCC but were attenuated or normalized by Sal-B and Breviscapine along with significantly decreased incidences of SCC formation.

Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell carcinoma.

Blocking angiogenesis may enhance conventional anticancer treatments such as radiation therapy. In this study, we examined the effects of the angiogenesis inhibitor TNP-470 on human OSCC cell lines HSC2 and KB, with combining radiation therapy in the nude mouse. We evaluated cell-induced neovascularization with dorsal air sac assay, and selected two cells (HSC2: low, KB: high) with different level of cell-induced angiogenesis. The angiogenesis inhibitor TNP-470 was given 30 mg/kg s.c. daily on day 1-5, and irradiation, 8 Gy x 1, was administered on day 1 each week for 3 weeks. Significant inhibition of tumor growth relative to untreated controls was achieved in KB cells showing high induced angiogenesis with both TNP-470 (P < 0.01) and radiation (P < 0.01) and combining TNP-470 and radiation (P < 0.01). We saw little effect of TNP-470 either alone or in addition to the effect of radiation on the HSC2 cells showing low induced angiogenesis. These results suggested that TNP-470 significantly enhanced the effect of radiation on the cells with high neovascularization. These findings indicated that individual evaluation of each tumor neovascularization potential will be important before deciding the anti-angiogenesis treatment.

Leukoplakia and angiopoiesis resistance of Herba Erigerontis.

OBJECTIVE: To evaluate the effects of Herba Erigerontis on angiopoiesis and carcinogenesis; to seek an effective drug for prevention of leukoplakia progression to carcinoma and to stop the mechanism of angiopoiesis. METHODS: Carcinomatous conversions of golden hamster cheek pouches induced by Salley way and inhibited by Herba Erigerontis. Ink perfusion, analysis of images, resin cast form of micro-vessel, a-SMA detection and histopathological examination were applied to observe their changing patterns. RESULTS: The rate of leukoplakia conversion to tumor of the Herba Erigerontis group was half that of model group. The proportion of normal cells was approximately 4 times greater than that of model. The a-SMA levels of the Herba Erigerontis treated group was higher than that of model (P < 0.001). There was no significant difference between angeion vessel area and density (P > 0.05) in these 2 groups, which were elevated compared to the control. Nevertheless, the resin casts of the microvasculature in the Herba Erigerontis group showed that its configuration and spatial arrangement was similar to the control group. CONCLUSIONS: Herba Erigerontis has the function of inhibiting leukoplakia progression to tumor. This may be due to its ability to upregulate the expression of a-SMA. It has no obvious effect on angio-hyperplasia and expansion in the course of leukoplakia progression to tumor. However, it may preserve the angio-configuration and spatial arrangement and keep intact the angeion vessel wall. Its mechanism of promoting blood circulation may be through removing blood stasis and benign angiopoiesis so as to resist carcinogenesis.

[Tumor oxygenation in combined whole body hyperthermia and polychemotherapy. Studies exemplified by recurrent carcinoma of the mouth cavity]. / Tumoroxygenierung unter kombinierter Ganzkörperhyperthermie und Polychemotherapie. Untersuchungen am Beispiel eines Rezidivkarzinoms der Mundhöhle.

BACKGROUND AND OBJECTIVE: Previous studies have reported synergistic effects of combined hyperthermia and chemotherapy and/or irradiation. The response to irradiation and chemotherapy of well-oxygenated and vascularized tumors generally is better than that of hypoxic tumors. Therefore, tumor oxygenation is recognized as an important predictive factor in the therapy of malignant tumors. In practice, the head and neck area remains outside of the hyperthermia chamber during whole-body hyperthermia. It was the aim of this study to evaluate if the head and neck region receives sufficient warmth and, if so, if tumor oxygenation increases accordingly. PATIENTS/METHODS: Whole-body hyperthermia, as heat radiation (Enthermics Medical Systems RHS-7500), was applied to the narcotised 60-year-old male patient with a local recurrence tumor pT3 pN2b M0 squamous cell carcinoma of the oral cavity. Tumor oxygenation and temperature were measured by LICOX catheters via one-point measurement during the entire hyperthermia treatment (3.5 h). Parallelly, chemotherapy (ifosfamide/Carboplatin) was given in four cycles (one cycle/month). RESULTS: With a latency of 10 min the increase of intratumoral temperature was comparable to temperatures achieved in the esophagus. The maximum intratumoral temperature was 41.8 degrees C. The average increase in tumor oxygenation was more than 100%. The clinical outcome in the case presented was a partial tumor remission (PR). CONCLUSIONS: During combined whole-body hyperthermia and polychemotherapy, tumor oxygenation is also significantly improved in the head and neck area, despite the fact that the head and neck area remained outside the hyperthermia chamber. The intratumoral temperature was comparable to esophageal and rectal temperatures.

Angiogenic inhibition for the treatment of head and neck cancer.

Tumor angiogenesis is a fundamental step in tumor growth and proliferation. Fumagillin is an anti-angiogenic agent which is secreted by Aspergillus, but is also toxic. A fumagillin analogue, TNP-470, has been developed which is a potent angiogenic inhibitor with few side effects. TNP-470 has inhibited tumor growth in Lewis lung cancer and melanoma in animal models. This study was designed to test this proven anti-angiogenic agent's effects on head and neck cancer growth. Fort,v Harlan nude mice were injected subcutaneously with cancer cells from a human oral squamous cell carcinoma. After 3 weeks of tumor growth 25 mice were injected with TNP-470 subcutaneously at a distant site every other day for 30 days while 10 control mice received saline injections. Five mice began TNP-470 injections at the time of tumor injection to determine if TNP-470 can prevent tumor development. The tumor growth and development was unaffected by TNP-470 as compared to the control group. Therefore, the use of an angiogenic inhibitor had no effect on oral cancer growth. Analysis of the cell line utilized found abnormal mRNA expression, which included high p53 expression and low cyclin Dl expression. These results suggest that oral cancers are less dependent on angiogenesis than other tumor types. The genetic abnormalities may explain the angiogenesis independence that was demonstrated. Results found in other tumor types with angiogenic inhibitors cannot be extrapolated to oral cancer since genetic mutations may allow oral tumors to grow without neovascularization.

Expression of platelet-derived endothelial cell growth factor in oral and oropharyngeal carcinoma.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial cell mitogen from platelets. In this study, we investigated the expression of PD-ECGF and counted microvessels in 58 oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique to examine their prognostic significance and performed tumor in vitro sensitivity to 5-fluorouracil (5-FU) and cisplatin as determined by a bioluminescence assay of the ATP values of tumor cells after continuous exposure. The percentage of PD-ECGF-positive tumor cells (PD-ECGF score) was correlated with the frequency of the recurrence of disease (P=0.0043) but not with sex, tumor size, metastasis, or clinical stage. Overall survival of the high PD-ECGF expression group (>40% PD-ECGF score) was shorter than the low expression (<40%) group (P=0.0365). Vessel count was correlated with lymph node metastasis and clinical stage. The survival of patients with hypervascularity (more than the median of intratumor vessel counts, >82) was shorter than that of those with hypovascularity (vessel count <81, P=0.0446). However, there was no association between PD-ECGF expression and vessel count. Cox proportional multivariate analysis showed that PD-ECGF expression was the most significant independent prognostic indicator for overall survival. The susceptibility to 5-FU cytotoxicity in the extremely high PD-ECGF expression groups (>70% of PD-ECGF score) was significantly higher than that in the low group, whereas there was no difference in their sensitivity to cisplatin. These results showed that carcinoma cells with high PD-ECGF expression were sensitive to 5-FU in spite of poor prognosis. These data provide further information when deciding on adjuvant therapy for oral and oropharyngeal SCCs.